WO2015158809A1 - Methods and pharmaceutical compositions for the treatment of cancer - Google Patents

Methods and pharmaceutical compositions for the treatment of cancer Download PDF

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Publication number
WO2015158809A1
WO2015158809A1 PCT/EP2015/058243 EP2015058243W WO2015158809A1 WO 2015158809 A1 WO2015158809 A1 WO 2015158809A1 EP 2015058243 W EP2015058243 W EP 2015058243W WO 2015158809 A1 WO2015158809 A1 WO 2015158809A1
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Prior art keywords
nts
cancer
cells
inhibitor
ntsr1
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French (fr)
Inventor
Patricia Forgez
Marco Alifano
Anne Gompel
Jean TREDANIEL
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Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Descartes
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Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Descartes
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the methods of the present invention relates to methods and pharmaceutical compositions for the treatment of cancer.
  • Lung and breast cancers are the most common malignancies in the world, and are the leading cause of cancer-related deaths in western countries (1, 2).
  • the progression of tumors to a metastatic disease is the primary cause of death in most patients and the main target of cancer research.
  • the stage of the disease is the most useful parameter in deciding management and defining prognosis.
  • survival is heterogeneous even within a single stage. Therefore, the identification of biological parameters allowing characterization, and validation of tumor subsets possessing aggressive phenotypes, is an ongoing challenge for cancer biological studies.
  • HER family of proteins EGFR, HER2, HER3, and HER4, share several structural and functional features, despite notably different molecular and cellular functions (3).
  • these functions can be abnormally exacerbated because of genetic defaults, protein overexpression, or over-activation of one or several of these receptors.
  • these regulations are not mutually exclusive and confer a large magnitude of oncogenic activities which are difficult to detect and categorize within tumoral specimens.
  • HERs activate a multiplicity of intracellular pathways via their ability to interact with numerous signal transducers.
  • the clinical criteria used to propose these therapies are based on the detection of genetic defaults in the tumor (HER2 amplification, EGFR mutation). Nevertheless, it was also observed that other patient subsets could benefit from these therapies.
  • the challenge is find a criteria to categorize them.
  • NTS neurotensin
  • NTSRl neurotensin receptor
  • the inventors highlight the cellular mechanisms activated by Neurotensin (NTS) and its high affinity receptor (NTSRl) contributing to cancer cell aggressiveness in 2 models (i.e. lung and breast cancer). They show that the NTS autocrine and/or paracrine regulation causes HER over-expression and activation in tumor cells. Said observations prompt the inventors to investigate whether the NTS/NTSRl complex could be used as a marker to identify subsets of human cancers eligible to treatment with HER inhibitors. They validate the hypothesis by showing that treatment of tumors having a positive expression for the NTS/NTRS1 complex with a HER inhibitor show a dramatic decrease in tumor growth in comparison with the treatment of tumors wherein the expression of NTSRl is absent.
  • one aspect of the present invention relates to a method of the treatment of cancer in a patient in need thereof comprising the steps of i) determining the expression level of NTS and/or NTSRl in tumor sample obtained from the patient, ii) comparing the expression level determined at step i) with a predetermined reference value and iii) administering the patient with a therapeutically effective amount of at least one HER inhibitor when the expression level determined at step i) is higher than the predetermined reference level.
  • the patient suffers from a cancer deriving from epithelial origin.
  • cancer types include, but are not limited to, carcinoma, lymphoma, blastoma (including meduUoblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g.
  • lung cancer including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophagael cancer, tumors of the biliary tract, as well as head and neck cancer, as well as subtypes of any of such cancers, including, but not limited to chemotherapy-resistant, platinum-resistant, advanced, refractory, and/or recurrent types
  • tissue sample means any tissue sample derived from the tumor of the patient.
  • the tissue sample is obtained for the purpose of the in vitro evaluation.
  • the sample can be fresh, frozen, fixed (e.g., formalin fixed), or embedded (e.g., paraffin embedded).
  • the sample results from biopsy performed in a tumour sample of the patient.
  • NTS has its general meaning in the art and refers to neurotensin.
  • An exemplary amino sequence of NTS is SEQ ID NO: l, 2 or 3 and an exemplary nucleic acid sequence of NTS is SEQ ID NO:4.
  • SEQ ID NO: 1 is an exemplary amino sequence of NTS.
  • MMAGMKIQLVCMLLLAFSSWSLCSDSEEEMKALEADFLTNMH SKI SKAHVPSWKMTLLNVC SLVNNLNSPAEE GEVHEEELVARRKLPTALDGFSLEAML IYQLHKICHSRAFQHWELIQE DILDTGNDKNGKEEVIKRKI PYILKRQLYENKPRRPYILKRDSYYY
  • NTSR1 has its general meaning in the art and refers to neurotensin receptor 1.
  • An exemplary amino sequence of NTSR1 is SEQ ID NO:5 and an exemplary nucleic acid sequence of NTSR1 is SEQ ID NO:6.
  • SEQ ID NO:5 An exemplary amino sequence of NTSR1 is SEQ ID NO:5 and an exemplary nucleic acid sequence of NTSR1 is SEQ ID NO:6.
  • Determining an expression level of a gene e.g. NTS or NTSR1 in a tumor sample obtained from a patient can be implemented by a panel of techniques well known in the art. Typically, an expression level of a gene is assessed by determining the quantity of mRNA produced by this gene.
  • nucleic acid contained in the samples e.g., cell or tissue prepared from the patient
  • the samples e.g., cell or tissue prepared from the patient
  • the extracted mRNA is then detected by hybridization (e. g., Northern blot analysis) and/or amplification (e.g., RT-PCR).
  • hybridization e. g., Northern blot analysis
  • amplification e.g., RT-PCR
  • quantitative or semi-quantitative RT-PCR is preferred.
  • Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous.
  • Other methods of Amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA), quantitative new generation sequencing of RNA (NGS).
  • LCR ligase chain reaction
  • TMA transcription-mediated amplification
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based
  • Nucleic acids comprising at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be completely identical, but are typically at least about 80% identical to the homologous region of comparable size, more typically 85% identical and even more typically 90-95% identical. In some embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin).
  • Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more typically of between 15 and 700, typically of between 20 and 500 nucleotides.
  • Primers typically are shorter single- stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified.
  • the probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they typically hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC.
  • SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
  • Nucleic acids which may be used as primers or probes in the above amplification and detection method may be assembled as a kit.
  • a kit includes consensus primers and molecular probes.
  • a preferred kit also includes the components necessary to determine if amplification has occurred.
  • a kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
  • the methods of the invention comprise the steps of providing total R As extracted from cancer cells and subjecting the R As to amplification and hybridization to specific probes, more particularly by means of a quantitative or semiquantitative RT-PCR.
  • Probes made using the disclosed methods can also be used for nucleic acid detection, such as in situ hybridization (ISH) procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).
  • ISH in situ hybridization
  • FISH fluorescence in situ hybridization
  • CISH chromogenic in situ hybridization
  • SISH silver in situ hybridization
  • CGH comparative genomic hybridization
  • in situ hybridization involves contacting a sample containing target nucleic acid sequence (e.g., genomic target nucleic acid sequence) in the context of a metaphase or interphase chromosome preparation (such as a cell or tissue sample mounted on a slide) with a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence).
  • the slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization.
  • the sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids.
  • the probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium).
  • the chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques. Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Pat. Nos.
  • Probes are typically labelled with a detectable molecule.
  • a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase.
  • fluorescein-labeled avidin or avidin-alkaline phosphatase For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)-conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin- conjugated goat antiavidin antibodies, washing and a second incubation with FITC- conjugated avidin.
  • FITC fluorescein isothiocyanate
  • samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer).
  • AP alkaline phosphatase
  • in situ hybridization procedures see, e.g., U.S. Pat. No. 4,888,278.
  • Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties.
  • probes labeled with fluorophores can be directly optically detected when performing FISH.
  • the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following non- limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety.
  • a hapten such as the following non- limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin,
  • Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • a labeled detection reagent such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • the detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.
  • the probe is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH).
  • the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publications Nos. 2006/0246524; 2006/0246523, and 2007/0117153.
  • multiplex detection schemes can be produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample).
  • a first probe that corresponds to a first target sequence can be labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP.
  • the bound probes can be detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 mn).
  • a first specific binding agent in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn
  • a second specific binding agent in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®,
  • Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more typically of between 15 and 700, typically of between 20 and 500.
  • Primers typically are shorter single- stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified.
  • the probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they typically hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC.
  • SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
  • the nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit.
  • a kit includes consensus primers and molecular probes.
  • a preferred kit also includes the components necessary to determine if amplification has occurred.
  • the kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
  • the expression level is determined by DNA chip analysis.
  • DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere- sized bead.
  • a microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose.
  • Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs.
  • a sample from a test subject optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface.
  • the labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling.
  • Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).
  • the expression level of a gene may be expressed as absolute expression level or normalized expression level. Both types of values may be used in the present method.
  • the expression level of a gene is typically expressed as normalized expression level when quantitative PCR is used as method of assessment of the expression level because small differences at the beginning of an experiment could provide huge differences after a number of cycles.
  • expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not relevant for determining the cancer stage of the patient, e.g., a housekeeping gene that is constitutively expressed.
  • Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene.... This normalization allows comparing the expression level of one sample, e.g., a patient sample, with the expression level of another sample, or comparing samples from different sources.
  • the expression level of a gene may be determined at the protein level.
  • the tumor sample of the patient may be contacting with a binding partner specific for the protein of interest (i.e. NTS or NTSR1).
  • the binding partner is an antibody or an aptamer.
  • Polyclonal antibodies of the invention or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • Various adjuvants known in the art can be used to enhance antibody production.
  • antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.
  • Monoclonal antibodies of the invention or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture.
  • the binding partner may be an aptamer.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library.
  • the binding partners of the invention such as antibodies or aptamers, may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art.
  • Labels are known in the art that generally provide (either directly or indirectly) a signal.
  • the term "labelled" with regard to the antibody or aptamer is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g.
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • Cy5 Indocyanine
  • An antibody or aptamer of the invention may be labelled with a radioactive molecule by any method known in the art.
  • radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, Inl l l, Rel86, Rel88.
  • the antibodies against the surface markers are already conjugated to a fluorophore (e.g. FITC-conjugated and/or PE- conjugated).
  • immunostained slices of the tumor tissue sample may be obtained with an automated slide-staining system by using a labelled binding partner as above described (e.g. an antibody).
  • Immuno chemistry is a suitable method for quantifying the expression level of a marker in a tissue sample.
  • the tumor sample is typically fixed in formalin and embedded in a rigid fixative, such as paraffin (wax) or epoxy, which is placed in a mould and later hardened to produce a block which is readily cut.
  • Thin slices of material are prepared using a microtome, placed on a glass slide and submitted to immunohistochemistry, for example using an IHC automate such as BenchMark® XT allowing automatic stained slide preparation for implementing the immunohistochemical staining.
  • digitalisation of the slices may be used to quantify the level of the marker.
  • Digitalisation of the slices may be made by scan capture, for example with a high resolution Hamamatsu NanoZoomer® 2.0-HT scanner.
  • the mean, median, min and max of the relevant staining intensity of all positive stained cells detected in the tumour sample may be provided.
  • the values and the distribution of the staining intensity can be compared to the predetermined reference value.
  • the predetermined reference value is a threshold value or a cut-off value.
  • a “threshold value” or “cut-off value” can be determined experimentally, empirically, or theoretically.
  • a threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of the expression level of NTS or/and NTSR1 in properly banked historical subject samples may be used in establishing the predetermined reference value.
  • the threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative).
  • the optimal sensitivity and specificity can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
  • ROC Receiver Operating Characteristic
  • the optimal sensitivity and specificity can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
  • ROC Receiver Operating Characteristic
  • receiver operator characteristic curve which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests.
  • ROC curve is a comprehensive indicator the reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method.
  • a series of different cut-off values are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis.
  • AUC area under the curve
  • the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values.
  • the AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate.
  • the predetermined reference value ("cut-off) is determined by carrying out a method comprising the steps of:
  • tumour tissue samples in pairs of subsets of increasing, respectively decreasing, number of members ranked according to their expression level
  • step e providing, for each tumour tissue sample provided at step a), information relating to the actual clinical outcome for the corresponding cancer patient (i.e. the duration of the disease-free survival (DFS) or the overall survival (OS) or both);
  • information relating to the actual clinical outcome for the corresponding cancer patient i.e. the duration of the disease-free survival (DFS) or the overall survival (OS) or both;
  • the expression level of NTS or/and NTSR1 has been assessed for 100 cancer samples of 100 patients.
  • the 100 samples are ranked according to the expression level of NTS or/and NTSR1.
  • Sample 1 has the highest expression level and sample 100 has the lowest expression level.
  • a first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples.
  • the next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100.
  • Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated.
  • the predetermined reference value is selected such as the discrimination based on the criterion of the minimum p value is the strongest.
  • the expression level corresponding to the boundary between both subsets for which the p value is minimum is considered as the reference value. It should be noted that according to the experiments made by the inventors, the reference value is not necessarily the median value of expression levels.
  • Kaplan-Meier curves of percentage of survival as a function of time are commonly used to measure the fraction of patients living for a certain amount of time after treatment and are well known by the man skilled in the art. P value is conventionally used in statistical significance testing.
  • high statistical significance values are obtained for a range of successive arbitrary quantification values, and not only for a single arbitrary quantification value.
  • a range of predetermined reference values is provided instead of using a definite predetermined reference value.
  • a cut-off value thus consists of a range of quantification values, e.g. centered on the quantification value for which the highest statistical significance value is found (e.g. generally the minimum P value which is found). For example, on a hypothetical scale of 1 to 10, if the ideal cut-off value (the value with the highest statistical significance) is 5, a suitable (exemplary) range may be from 4-6.
  • a patient may be assessed by comparing values obtained by measuring the expression level of NTS or/and NTSR1, where values greater than 5 reveal that the patient is eligible to a treatment with a HER inhibitor and values less than 5 reveal that the patient is not eligible to said treatment .
  • a patient may be assessed by comparing values obtained by measuring the expression level of NTS or/and NTSR1 and comparing the values on a scale, where values above the range of 4-6 indicate that the patient is eligible to the treatment and values below the range of 4-6 indicate that the patient is not eligible, with values falling within the range of 4-6 indicate that there are still doubts that the patient is eligible to the treatment.
  • HER has its general meaning in the art and refers to a receptor protein tyrosine kinase which belongs to the HER receptor family and includes EGFR, HER2, HER3 and HER4 receptors.
  • ErbBl HER1
  • EGFR epidermal growth factor receptor
  • ErbB2 and "HER2” are used interchangeably herein and refer to human HER2 protein described, for example, in Semba et al, PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al. Nature 319:230-234 (1986) (Genebank accession number X03363).
  • ErbB3 and "HER3” refer to the receptor polypeptide as disclosed, for example, in U.S. Pat. Nos. 5,183,884 and 5,480,968 as well as Kraus et al. PNAS (USA) 86:9193-9197 (1989).
  • ErbB4 and HER4 refer to the receptor polypeptide as disclosed, for example, in EP Pat Appln No 599,274; Plowman et al, Proc. Natl. Acad. Sci. USA, 90: 1746-1750 (1993); and Plowman et al, Nature, 366:473-475 (1993).
  • HER ligand is meant a polypeptide which binds to and/or activates a HER receptor.
  • HER inhibitor refers to an agent which interferes with HER activation or function.
  • HER inhibitors include HER antibodies (e.g. EGFR, HER2, HER3, or HER4 antibodies); small organic molecule HER antagonists; HER tyrosine kinase inhibitors; HER2 and EGFR dual tyrosine kinase inhibitors such as lapatinib/GW572016; antisense molecules (see, for example, WO2004/87207); and/or agents that bind to, or interfere with function of, downstream signaling molecules, such as MAPK or Akt.
  • the HER inhibitor is an antibody or small organic molecule which binds to a HER receptor.
  • the HER inhibitor is a "HER dimerization inhibitor” which is an agent which inhibits formation of a HER dimer or HER heterodimer.
  • the HER inhibitor is an "anti-HER antibody” which is an antibody that binds to a HER receptor.
  • antibody herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
  • the term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical” scFv-Fc dimer; DART (destabilized diabody "Dual Affinity ReTargeting"); small
  • Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments.
  • Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al, 2006; Holliger & Hudson, 2005; Le Gall et al, 2004; Reff & Heard, 2001 ; Reiter et al, 1996; and Young et al, 1995 further describe and enable the production of effective antibody fragments.
  • the anti-HER monoclonal antibody of the invention is used to induce antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) against HER-expressing cells.
  • ADCC antibody dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • the anti- HER antibody may be suitable for disturbing the expression of HER at the cell surface (e.g. by provoking internalization of HER) so that cell migration, cell proliferation and tumour growth of tumor cells will be limited or inhibited.
  • the anti-HER antibody is a "chimeric" antibody in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)).
  • Chimeric antibodies include PRIMATTZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest.
  • the anti-HER antibody is a humanized antibody.
  • "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a HVR of the recipient are replaced by residues from a HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • donor antibody such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • FR residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • humanized HER2 antibodies include huMAb4D5-l, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 or trastuzumab (HERCEPTIN®) as described in Table 3 of U.S. Pat. No. 5,821,337 expressly incorporated herein by reference; humanized 520C9 (W093/21319); and humanized 2C4 antibodies such as pertuzumab.
  • trastuzumab HERCEPTIN®
  • the anti-HER antibody is a human antibody.
  • a "human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage- display libraries. Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al, J. Mol. Biol, 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R.
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSETM technology). See also, for example, Li et al, Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.
  • the anti-HER antibody is a single domain antibody.
  • the term "single domain antibody” (sdAb) or “VHH” refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.
  • the anti-HER antibody is an anti-HER monoclonal antibody- drug conjugate.
  • An "anti-HER monoclonal antibody-drug conjugate" as used herein refers to an anti-HER monoclonal antibody according to the invention conjugated to a therapeutic agent. Such anti-HER monoclonal antibody-drug conjugates produce clinically beneficial effects on HER-expressing tumor cells when administered to a subject.
  • an anti-HER monoclonal antibody is conjugated to a cytotoxic agent, such that the resulting antibody-drug conjugate exerts a cytotoxic or cytostatic effect on a HER- expressing tumor cell when taken up or internalized by the cell. Any cytotoxic agent well known by the skilled person may used.
  • the cytotoxic or cytostatic agent is auristatin E (also known in the art as dolastatin-10) or a derivative thereof.
  • the auristatin E derivative is, e.g., an ester formed between auristatin E and a keto acid.
  • auristatin E can be reacted with paraacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively.
  • auristatin derivatives include AFP (dimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylenediamine), MMAF (dovaline-valine-dolaisoleunine-dolaproine-phenylalanine), and MAE (monomethyl auristatin E).
  • AFP dimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylenediamine
  • MMAF dovaline-valine-dolaisoleunine-dolaproine-phenylalanine
  • MAE monomethyl auristatin E
  • an anti-HER monoclonal antibody of the invention is used to induce antibody dependent cellular cytotoxicity (ADCC).
  • ADCC antibody dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • Methods for inducing ADCC generally include contacting the HER-expressing cell with an effective amount an anti-HER monoclonal antibody comprising an Fc region having ADCC activity, wherein the contacting step is in the presence of a cytolytic immune effector cell expressing an Fc receptor having cytolytic activity.
  • Immune effector cells expressing cytolytic Fc receptors include, for example, NK cells as well certain CD8+ T cells.
  • Methods for inducing CDC generally include contacting the HER-expressing cell with an effective amount an anti- HER monoclonal antibody comprising an Fc region having CDC activity, wherein the contacting step is in the presence of complement.
  • the anti-HER antibody is monospecific, bispecific, trispecific, or of greater multispecificity.
  • Multispecific antibodies, including bispecific and trispecific antibodies, useful for practicing the methods described herein are antibodies that immunospecifically bind to both HER and a second cell surface receptor or receptor complex that mediates ADCC, phagocytosis, and/or CDC, such as CD16/FcgRIII, CD64/FcgRI, killer inhibitory or activating receptors, or the complement control protein CD59.
  • the binding of the portion of the multispecific antibody to the second cell surface molecule or receptor complex enhances the effector functions of the anti-HER antibody.
  • the anti-HER antibody is a bispecific antibody.
  • bispecific antibody has its general meaning in the art and refers to any molecule consisting of one binding site for a target antigen on tumor cells (i.e. a HER receptor) and a second binding side for an activating trigger molecule on an effector cell, such as CD3 on T-cells, CD 16 (FcyRlll) on natural killer (NK) cells, monocytes and macrophages, CD89 (FcaRI) and CD64 (FcyRI) on neutrophils and monocytes/macrophages, and DEC-205 on dendritic cells.
  • the bispecific antibody comprises a binding site for HER.
  • bispecific antibodies avoid competition with endogenous immunoglobulin G (IgG) when the selected binding site for the trigger molecule on the effector cell does not overlap with Fc-binding epitopes.
  • IgG immunoglobulin G
  • single-chain Fv fragments instead of full-length immunoglobulin prevents the molecules from binding to Fc-receptors on non-cytotoxic cells, such as FcyRII on platelets and B-cells, to Fc-receptors that do not activate cytotoxic cells, including FcyRlllb on polymorphonuclear leukocytes (PMN), and to inhibitory Fc-receptors, such as FcyRllb on monocytes/macrophages.
  • FcyRlllb on polymorphonuclear leukocytes (PMN)
  • PMN polymorphonuclear leukocytes
  • inhibitory Fc-receptors such as FcyRllb on monocytes/macrophages.
  • bispecific antibodies Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (see, e.g., Milstein et al, 1983, Nature 305:537-39). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Similar procedures are disclosed in International Publication No. WO 93/08829, and in Traunecker et al, 1991, EMBO J. 10:3655-59. Other examples of bispecific antibodies include Bi-specific T-cell engagers (BiTEs) that are a class of artificial bispecific monoclonal antibodies.
  • BiTEs Bi-specific T-cell engagers
  • BiTEs are fusion proteins consisting of two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain of about 55 kilodaltons.
  • scFvs single-chain variable fragments
  • One of the scFvs binds to tumor antigen (i.e. HER) and the other generally to the a n effector cell (e.g. a T cell via the CD3 receptor.
  • tumor antigen i.e. HER
  • n effector cell e.g. a T cell via the CD3 receptor.
  • Other bispecific antibodies those described in WO2006064136.
  • the bispecific antibody is a Fab format described in WO2006064136 comprising one VH or VHH specific for HER and one VH or VHH specific for an effector cell.
  • Patent publications related to HER antibodies include: U.S. Pat. No. 5,677,171, U.S. Pat. No. 5,720,937, U.S. Pat. No. 5,720,954, U.S. Pat. No. 5,725,856, U.S. Pat. No. 5,770,195, U.S. Pat. No. 5,772,997, U.S. Pat. No. 6,165,464, U.S. Pat. No. 6,387,371, U.S. Pat. No. 6,399,063, US2002/0192211A1, U.S. Pat. No. 6,015,567, U.S. Pat. No. 6,333,169, U.S. Pat. No. 4,968,603, U.S. Pat. No.
  • the HER inhibitor is a small organic molecule.
  • small organic molecule refers to a molecule of size comparable to those organic molecules generally sued in pharmaceuticals. The term excludes biological macromolecules (e.g.; proteins, nucleic acids, etc.); preferred small organic molecules range in size up to 2000da, and most preferably up to about 1000 Da.
  • the HER inhibitor is tyrosine kinase inhibitor.
  • a "tyrosine kinase inhibitor” is a molecule which inhibits tyrosine kinase activity of the HER receptor. Examples of such inhibitors include the small organic molecule HER2 tyrosine kinase inhibitor such as TAK165 available from Takeda; CP-724,714, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both HER2 and EGFR- overexpressing cells; GW572016 (available from Glaxo) an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (CI- 1033; Pharmacia); non selective HER inhibitors such as Imatinib me
  • the HER inhibitor is an EGFR inhibitor.
  • GFR inhibitors are well known in the art (Inhibitors of erbB-1 kinase ;Expert Opinion on Therapeutic Patents Dec 2002, Vol. 12, No. 12, Pages 1903-1907, Susan E Kane. Cancer therapies targeted to the epidermal growth factor receptor and its family members. Expert Opinion on Therapeutic Patents Feb 2006, Vol. 16, No. 2, Pages 147-164. Peter Traxler Tyrosine kinase inhibitors in cancer treatment (Part II). Expert Opinion on Therapeutic Patents Dec 1998, Vol. 8, No. 12, Pages 1599-1625). Examples of such agents include antibodies and small organic molecules that bind to EGFR.
  • antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab; ERBUTIX®) and reshaped human 225 (H225) (see, WO 96/40210, Imclone Systems Inc.); IMC-1 1F8, a fully human, EGFR-targeted antibody (Imclone); antibodies that bind type II mutant EGFR (U.S.
  • the anti-EGFR antibody may be conjugated with a cytotoxic agent, thus generating an immunoconjugate (see, e.g., EP659,439A2, Merck Patent GmbH).
  • a cytotoxic agent see, e.g., EP659,439A2, Merck Patent GmbH.
  • small organic molecules that bind to EGFR include ZD 1839 or Gefitinib (IRESSATM; Astra Zeneca); CP-358774 or erlotinib (TARCEVATM; Genentech/OSI); and AG1478, AG1571 (SU 5271 ; Sugen); EMD-7200.
  • the HER inhibitor is a small organic molecule pan-HER inhibitor such as dacomitinib (PF-00299804).
  • the HER inhibitor is selected from the group consisting of cetuximab, panitumumab, zalutumumab, nimotuzumab, erlotinib, gefitinib, lapatinib, neratinib, canertinib, vandetanib, afatinib, TAK-285 (dual HER2 and EGFR inhibitor), ARRY334543 (dual HER2 and EGFR inhibitor), Dacomitinib (pan-ErbB inhibitor), OSI-420 (Desmethyl Erlotinib) (EGFR inhibitor), AZD8931 (EGFR, HER2 and HER3 inhibitor), AEE788 (NVP-AEE788) (EGFR, HER2 and VEGFR 1 12 inhibitor), Pelitinib (EKB-569) (pan-ErbB inhibitor), CUDC-101 (EGFR, HER2 and HDAC inhibitor), XL647 (dual HER2 and
  • the inhibitors cetuximab, panitumumab, zalutumumab, nimotuzumab are monoclonal antibodies, erlotinib, gefitinib, lapatinib, neratinib, canertinib, vandetanib and afatinib are tyrosine kinase inhibitors.
  • the HER inhibitor is an inhibitor of expression.
  • An “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. Therefore, an “inhibitor of HER expression” denotes a natural or synthetic compound that has a biological effect to inhibit the expression of one HER receptor.
  • the inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. Inhibitors of gene expression for use in the present invention may be based on antisense oligonucleotide constructs.
  • Anti-sense oligonucleotides including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the HER receptor mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the HER receptor, and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding the HER receptor can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion.
  • Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).
  • Small inhibitory RNAs can also function as inhibitors of gene expression for use in the present invention.
  • Gene expression can be reduced by contacting the tumor, subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschi, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al.
  • Ribozymes can also function as inhibitors of gene expression for use in the present invention.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleo lytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleo lytic cleavage of the HER receptor mRNA sequences are thereby useful within the scope of the present invention.
  • Specific ribozyme cleavage sites within any potential R A target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
  • antisense oligonucleotides and ribozymes useful as inhibitors of gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
  • Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno
  • Non-cytopathic viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with the gene of interest.
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.
  • Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle).
  • retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • Typical viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • the adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hematopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions.
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also function in an extrachromosomal fashion.
  • Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g., SANBROOK et al, "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
  • the HER inhibitor is administered to the patient in combination with an inhibitor of the neurotensin activation of NTSRl .
  • the expression "inhibitors of the neurotensin activation of NTSRl” should be understood broadly, this expression refers to agents down-regulating the expression of neurotensin or of neurotensin receptor 1, compounds that bind to neurotensin (NTS) or NTSRl and inhibit the neurotensin activation of NTSRl, or a protease that can degrade NTS.
  • inhibitors of the neurotensin activation of NTSRl may be selected from the group consisting of an agent down-regulating the expression of neurotensin or of neurotensin receptor 1, an antibody against neurotensin or a fragment thereof which binds to neurotensin, an antibody against the neurotensin receptor 1 or a fragment thereof which binds to the neurotensin receptor 1, an antagonist of the neurotensin receptor 1, and a protease that can degrade NTS or an inhibitor of NTS or NTSRl expression.
  • the HER inhibitor is administered to the patient in combination with metformin ( ⁇ , ⁇ -Dimethylimidodicarbonimidic diamide).
  • metformin ⁇ , ⁇ -Dimethylimidodicarbonimidic diamide.
  • therapeutically effective amount refers to an amount of a drug (e.g.
  • the effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer.
  • the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
  • the effective amount may extend progression free survival (e.g.
  • the therapeutically effective amount of the drug is effective to improve progression free survival (PFS) and/or overall survival (OS).
  • PFS progression free survival
  • OS overall survival
  • extending survival is meant increasing overall or progression free survival in a treated patient relative to an untreated patient (i.e. relative to a patient not treated with a HER inhibitor), or relative to a patient who does not express a HER receptor at the designated level.
  • the therapeutically effective amount is the amount which leads to an objective response, a partial response or a complete response.
  • An "objective response” refers to a measurable response, including complete response (CR) or partial response (PR).
  • complete response or “CR” is intended the disappearance of all signs of cancer in response to treatment. This does not always mean the cancer has been cured.
  • Partial response or “PR” refers to a decrease in the size of one or more tumors or lesions, or in the extent of cancer in the body, in response to treatment. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the compounds of the invention are administered as a formulation in association with one or more pharmaceutically acceptable excipients to form pharmaceutical composition.
  • pharmaceutically acceptable excipient refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • Pharmaceutical compositions suitable for the delivery of compounds of the present invention and methods for their preparation will be readily apparent to those skilled in the art.
  • the active principle i.e. a compound of the invention
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganis
  • Solutions comprising compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • a surfactant such as hydroxypropylcellulose
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • Compounds of the invention can be formulated into a composition in a neutral or salt form as above described.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the compounds of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • Compounds of the invention may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.
  • other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules ; and any other form currently used.
  • the antibodies or fragment of antibodies are directed to all or a portion of the extracellular domain of HER. In one embodiment, the antibodies or fragment of antibodies are directed to an extracellular domain of HER.
  • FIG. 1 NTS/NTSR1 complex enhanced experimental tumor growth generated in human breast cancer cell lines.
  • Figure 2 NTS autocrine and paracrine regulation enhanced EGFR, HER2, and HER3 basal expression and activation in human breast cancer cell lines.
  • FIG. 3 EGF like ligands and metalloprotease released by NTS.
  • FIG. 4 NTS/NTSR1 expressing tumors response to EGFR/HER2 inhibitors treatment.
  • Figure 7 NTS, NTSR1, HER2, and HER3 immunohistochemistry on breast and lung cancer tumors. Two examples of positive labeling scored 2 or 3 for NTS, NTSR1, HER2, HER3 from breast tumor TMA, labeling was performed on consecutive slides. 100X magnification.
  • Figure 8 NTS autocrine and paracrine regulation enhanced cellular growth in human lung cancer cell lines.
  • LNM-F, R-SI NTS and R-SI NTSR1 were grown in media containing 0 % FCS at low concentration and treated every day with 10 "8 M NTS or JMV 449 for 6 days. The ratio of the number of cells at Day 6/Day 0 was calculated. The result is expressed as the % of fold induction.
  • LNM-R and LNM-F were seeded alone or at the ratio of 20/80 LNM-R LNM-F and grown in 0.1% FCS for 72h. The results are expressed as the ratio of the number of cells at 72h to TO was calculated, and are the mean ⁇ SEM of 7 independent experiments.
  • C to F LNM-R and LNM-F were seeded alone or at the ratio of 20/80 LNM-R/LNM-F and grown in 0.1% FCS for 72h, The ratio of the number of cells at 72h to TO was calculated. The results are expressed as the percentage of the growth induction compared to LNM-F. Results are the mean ⁇ SEM of 2 to 5 independent experiments.
  • DMSO 10 "7 M BIM 46174, 10 "6 M SR 48692, rabbit IgG or anti NTS antibody.
  • Figure 9 NTS regulation enhanced HER2, and HER3 basal expression in human lung cancer cell lines.
  • A) The mixture of cells R/F 20/80 lung cancer cells cultured for 72h, with the histograms representing intensity-based quantification of Western blot bands of basal total protein, EGFR, HER2, and HER3, Values are expressed as the percentage of the control LNM-F cells (which are the population more representative of the mixture) and are the mean ⁇ SEM of 5 to 8 independent experiments.
  • the blots were revealed with EGFR, HER2 or HER3 antibodies.
  • the actin shown is to the protein control for the HER3 Blot.
  • the histograms represent intensity-based quantification of Western blot bands of basal total protein, EGFR, HER2, and HER3. Values are expressed as the percentage of the non-treated cells (control), and are the mean ⁇ SEM of 3 to 6 independent experiments. .
  • Figure 10 NTS regulation enhanced EGFR HER2, and HER3 activation in human lung cancer cell lines.
  • FIG. 11 NTS autocrine and paracrine regulation activate EGF "like" ligands and MMP1 in lung cancer cell lines.
  • R-SI NTS cells were treated or not for 24h with 10 "8 M JMV449.
  • FIG. 12 NTS/NTSR1 complex enhanced experimental tumor growth.
  • A Tumor growth generated by LNM35, LNM-R and LMN-F cells xenografted into nude mice. One million cells from LNM35, LNM-R, LNM-F, or a mixture of LNM-R and LNM-F (50/50) were subcutaneously injected in 24, 36, 34, or 12 nude mice, respectively.
  • B Typical immunohistochemistry for NTSR1 (left) or NTS (right) for tumors generated from LNM-R (top) or LNM-F (bottom) cells. Significant differences at *** P ⁇ 0.001 or ** P ⁇ 0.01 using analysis of variance and Student-Neuman-Keuls test.
  • NTS/NTSR1 expressing tumors are the target for EGFR inhibitors treatment.
  • LNM-R or R-SI NTSR1 cells (LNM-R expressing sh-RNA for NTSR1) were injected into the left and the right flank of the mice, respectively.
  • LNM-R or R-SI NTSR1 cells (LNM-R expressing sh-RNA for NTSR1) were injected into the left and the right flank of the mice, respectively.
  • FIG 14 Immunohistochemistry of NTSR1, lung cancer tumors.
  • FIG. 6 NTS/NTSR1 complex enhanced experimental tumor growth in HCC cells lines.
  • A Experimental tumors were generated from the HCC cancer cell line, HEP 3B and PLC/RF5 and the NTSRl-overexpressing subclones. Comparative growth curves of HEP 3B, HEP-Rla, PLC/RF5, PLC/R1 in 8, 7, 10 and 7 mice, respectively. Tumor volumes were measured twice a week.
  • B Tumor weight at 42 days.
  • C Typical IHC performed on paraffin sections for NTS, NTSR1 EGFR and phosphorylated EGFR labelling, 200X magnification
  • Figure 7 Neurotensin regulation restores response to erlotinib.
  • mice were treated per os with 75 mg/kg erlotinib every day for 22 days. Tumor size was measured every two days. Results shown include the ratio of tumors as compared to day 1. A control group force feed with H20, and three treated groups, force feed every day with 15mg/kg sorafenib, lmg/kg SR 48692, or both. Results shown are the ratio of tumor compare to day 1.
  • EXAMPLE 1 EGFR, HER2, AND HER3 EXPRESSION REMODELING BY THE NEUROTENSIN/NEUROTENSIN RECEPTOR 1 PATHWAY CONTRIBUTES TO BREAST CANCER PROGRESSION.
  • NTS-h high level of NTS
  • NTS-1 low level of NTS
  • Tumor xenografts 3x10 6 cells (MCF-7, NTS-h, NTS-1) resuspended in Matrigel (BD Biosciences) were then inoculated in the right mammary gland of the mice. Tumor growth was induced by a daily intra-peritoneal injection of 2 ⁇ g estradiol per mouse. Institutional Review Board approval was obtained by «Le Comite d'Ethique pour l'Experimentation Animale Charles Darwin # Ce5/2010/049 Officer For drug treatments, 5x10 6 of NTS-h resuspended in Matrigel (BD Biosciences) were then metformininoculated in the right mammary gland of the mice.
  • mice 51 days after injection, 4 groups of 7 mice were randomized as follows : 95.9 ⁇ 14.57 mm 3 for control group, 94.5 ⁇ 15.0 mm 3 for lapatinib group, 91.5 ⁇ 14.09 mm 3 for metformin group and 95.6 ⁇ 12.20 mm 3 for lapatinib and metformin group. Mice were treated for 21 days per os, with sesame oil containing 6% DMSO, or 75 mg/kg lapatinib, or 200 mg/kg metformin or both.
  • Adhesion assays The assay was performed in 96-wells plates coated or not with 50 ⁇ g/mL of type I collagen (Sigma®) overnight at 4 °C or with 1 : 10 diluted-growth factor reduced Matrigel (BD Biosciences®) for 1 hour at 37 °C. Cells were harvested and suspended in adhesion buffer (DMEM, 15 mM HEPES, 1.2g/L sodium bicarbonate, 0.2% BSA). 5x10 4 cells per well were seeded and incubated at 37 °C for 1 or 48 hours. Adherent cells fixed with 5 % paraformaldehyde for 45 min, then colored with 0.1 % cristal violet during 30 min at room temperature. Cells were subjected to 30 min lysis in 1 % SDS under agitation. Absorbance was determined at 570.
  • Anchorage-independent growth assay in soft agar Colony growth assays were performed by seeding on the top of a 0.6 % low gelling temperature agarose layer, 5x10 3 cells in 0.5 mL of cultured medium containing 0.3 % agarose solution ⁇ EGF (10 ng/mL). Culture medium and EGF were replenished every 2-3 days. Cells were incubated at 37°C for 12 days and colonies > 50 ⁇ were counted in the whole well.
  • Cell migration assays 12 wells-culture plates were coated with type I collagen or Matrigel as mentioned in adhesion assay. lxlO 5 cells were plated for 4 hours in an 8 mm- cloning ring placed in the center of the well to form a confluent circle of monolayer cells. The cylinder was removed and cells grow for 16 hours. Cells were then treated in serum- free medium ⁇ EGF (10 ng/mL) in the presence or absence of various cell signaling inhibitors. Four pictures were acquired per well at the initial time of treatment and 48 hours later for comparison with an inverted microscope at 200X magnification. Migration speed was determined using the Morpho Expert software (Explora Nova) and was expressed as the average cells covered distance divided by the experimental duration ( ⁇ /h). Presented results are expressed in percentage of the non-treated condition.
  • Invasion potential was evaluated by a single-cell collagen invasion model, extensively described (1). Briefly, 6-wells plates were coated with 1.25 mL of a 1 mg/mL collagen type I solution, allowed to gel for at least 1 hour at 37 °C in a humidified atmosphere containing 10 % of C02. 2x10 5 viable single-cells, obtained by mild enzymatic dissociation with trypsin/EDTA solution and filtration, are seeded on the top of the gel in presence or not of EGF (100 ng/mL) and/or G66976 (4xl0 8 M) for 24h. Invasion index (cells with invasive extensions versus total number of cells x 100) was determined by counting the number of invading and non- invading cells present in 10 to 15 random fields of an inverted phase-contrast microscope.
  • ELISA Enzyme-linked immunosorbent assay
  • RNA extraction and RT-PCR Total RNA was extracted by the acidic phenol/chloroform guanidine thiocyanate method as documented by (2, 3).
  • the NTS-NTSR1 complex enhances tumor growth and metastasis emergence from breast experimental tumor.
  • MCF-7 cells which constitutively express NTSR1
  • NTSR1 neurotensin full length coding sequence
  • two showed a differential NTS expression based on transcription levels the NTS high expressing clone, NTS-h, and the NTS low expressing clone, NTS-1 ( Figure 1A inset).
  • the NTS expression levels were confirmed by quantitative RT-PCR in both clones, showing a 500-fold and a 4-fold NTS transcript's induction for NTS-h and NTS-1, respectively as compared to the MCF-7 parental cells. Immuno fluorescent staining experiments confirmed this result.
  • NTS-1 cells In wild type cells, no or an extremely weak NTS labeling was noted. In the NTS-1 cells, NTS immunoreactivity was dispersed in small dots throughout the cytosol. The same pattern was observed in NTS-h cells with larger dots of stronger intensity due to the higher NTS expression level. In parallel, in MCF-7 cells, NTSR1 labeling was localized at the cell membrane, whereas an intense intracellular granular labeling of an endocytosed receptor was seen in NTS-h cell. NTS-1 cells showed a dual pattern of NTSR1 localized both at the membrane and inside the cytoplasm.
  • the MCF-7 parental line and the two NTS-overexpressing clones, NTS-1 and NTS-h were xenografted in the mammary fat pad of female athymic mice. Tumor growth rose with NTS expression levels (Figure 1A).
  • the group bearing the MCF-7 cells began to develop tumors after 77 days to reach a small size of 108 ⁇ 56 mm 3 at day 105.
  • mice bearing NTS-overexpressing cells developed much bigger tumors, with a volume of 483 ⁇ 102 mm 3 for NTS-1 at day 105 and 1239 ⁇ 251 mm 3 for NTS-h at 91 days.
  • the NTS/NTSRl complex enhances EGFR, HER2, and HER3 expression and activation in breast cancer cells.
  • NTS-1, NTS-h, and MCF-7 cellular protein content showed an increase of 150 - 275 % of EGFR, HER2, and HER3 ( Figures 2B and C). This effect was confirmed by the stronger labeling of all three HER receptors by immuno cytochemistry in NTS-1 cells as compared to MCF-7 cells ( Figure 2D).
  • NTS-1 cells EGFR is accumulated in clusters close to the nucleus, while in MCF-7, EGFR is at the membrane, HER2 membrane labeling, and HER3 cytosol labeling are more intense in NTS-1 than in MCF-7 cells (Figure 2D see white arrow).
  • NTS-1 cells In parallel, an increase in the activation states for all three receptors was observed in NTS-1 cells. The phosphorylation levels were enhanced up to 400 and 500% for EGFR and HER3, respectively ( Figures 2E and F). Similar observations were made with NTS-h cells and a 200% increase was seen for the three receptors (not shown).
  • MMP9 transcripts were found increased (Figure 3 C) in NTS-1 cells as compared to MCF-7, as was the case for MDA-MB 231 cells (4).
  • MMP9 anchored at the membrane was activated, as a 180% increase of MMP9 release in culture media was detected in NTS-1 as compared to MCF-7 cells and abolished by SR 48692 ( Figure 3 D).
  • metformin was shown to disrupt the crosstalk between insulin receptor and NTS receptor in pancreatic cancer cells (6). In addition to inhibiting the mTOR pathway, metformin prevents ER activation induced by NTS and insulin (7). In a breast cancer cellular model with NTS overexpression, metformin reduced the tumor growth with the same efficiency as lapatinib. No additional effects were detected when both drugs were employed. The final volumes were 160.9 ⁇ 39.84 mm 3 and 150.9 ⁇ 46.05 mm3 when animals were treated with metformin or both drugs, respectively (Fig 4 A and B). The tumor doubling time was in agreement with tumor volume with 13.0 ⁇ 1.28, for controls. For lapatinib, metformin, and metformin + lapatinib double time cannot be calculated for because within the group 2 to 3 tumors shrank, the others grow very slowly The absence of additional response suggested a common signaling cascade was targeted by both drugs.
  • the NTS/NTSR1 complex enhances pro-metastatic cellular effects.
  • NTS-overexpressing cells The invasiveness properties of NTS-overexpressing cells, was studied using a 3 dimensional collagen invasion assay. Results are expressed as the invasion index corresponding to the number of invading cells related to the number of total seeded cells.
  • the introduction of NTS expression into MCF-7 cells induced a small increase in invasiveness properties (Figure 4 E).
  • EGF-induced invasion doubled in NTS-overexpressing cells (20 % invasion index) as compared to MCF-7 (10 % invasion index).
  • the induction of invasiveness was inhibited by PKC inhibitors only in NTS-overexpressing clones, suggesting dependence of this effect on GPCR activation.
  • NTS and NTSR1 correlates with HER2 and HER3 overexpression in breast human tumors.
  • TMA breast cancer tissue microarrays
  • Table 1 Contingent analysis for NTS, NTSR1, HER2 and HER3 expression on samples from breast cancer tissue microarray
  • NTS and NTSR1 were found in 23 % of the samples, in agreement with our previous studies (8), whereas HER2 and HER3 were found in 28 % and 53 % of the samples, respectively.
  • no significant relation between HER2 and NTS/NTSR1 was found.
  • the organization of tumor cell signaling networks evolves with accumulation of genetic and epigenetic alterations in connection with the local stroma and immune system. Identifying factors which contribute and predict tumor aggressiveness are means to understand how the progression of the disease evolves across individuals. Unfortunately, the biological and clinical validation of these factors is difficult because tumors are often heterogeneous and their molecular characteristics change over time. In addition to the clinical parameters, genetic alterations on key genes provide additional information on disease outcome. These genetic alterations can be modulated by regulatory mechanism which may cause changes to the amplitude of cell aggressiveness within the tumor. While genetic alterations acquired by tumors are irreversible, their regulatory process can be deprogrammed to restore a tumor phenotype to a less aggressive and more differentiated form.
  • NTS/NTSR1 complex enhances tumor aggressiveness (tumor growth and metastasis emergence) by enhancing HERs expression, and their activation by the release of specific EGFR and HER3 ligands.
  • This concept can be extended to other tumors, as we observed similar results in lung cancer cells and subsequent experimental tumors (9).
  • NTSR1 activation alters many cellular effects having oncogenic characteristics including proliferation, survival, adherence, migration and invasion, with a synergic effect between NTS and EGF on cellular migration and invasiveness. This latter point may explain the exacerbation of the metastasis process seen due to NTSR1 activation.
  • HERs internalization pathways depend on the expression of ligands and/or their receptors (12, 13). Under physiological conditions (low concentration of ligands and moderate EGFR expression ⁇ 200 000/cell), EGFR internalization is dependent on clathrin coated pits, with a half- life of 6-10 hours.
  • the clathrin pathway becomes saturated, and the complex (ligand-receptor) is internalized with a slow kinetics in a clathrin independent pathway. Under these conditions, the half-life can reach 24 hours (14, 15).
  • HER2 In cells overexpressing HER2, receptors are mainly localized at the cell membrane, indicating that overexpression of HER2 does not lead to accelerating its endocytosis (16).
  • the HER2 heterodimerization with EGFR influences the endocytosis pathway for both receptors.
  • EGFR activation in cells overexpressing HER2 does not affect the membrane expression of HER2 or its degradation (16, 18).
  • overexpression of HER2 had a dominant-negative effect on the down regulation of stimulated EGFR, and stabilized both receptors by altering autophosphorylation patterns (16, 19).
  • HER2 overexpression may also prevent EGFR internalization by clathrin-dependent endocytosis, and it's re-directed from the degradation to the recycling pathway (18, 20, 21).
  • HER3 The activation of HER3 leads to its internalization and its down regulation.
  • HER3 internalization is slower than EGFR internalization (22).
  • HER3 is inefficiently sorted to the degradation pathway, apparently due to the lack of the C-terminal domain, which contains patterns used for targeting to lysosomes (23 , 24) .
  • neuregulins do not guide HER3 to degradation due to the early dissociation of complexes (ligand-receptor) in endosomes (23 ) . In these situations the stabilization of HERs cells are independent of gene transcription, activation and amplification, as in the case for Neurotensin.
  • HER2 overexpressing cells In both cases, deleterious effects of growth factors are often in HER2 overexpressing cells, indicating that ERFG/HER2 and/or HER3/HER2 dimers are related to biological aggressiveness (1 1).
  • the overexpression of both HER2 and HER3 participates in the stabilization of the HERs dimer, and subsequently the sustained activation of the HERs, and the survival pathway.
  • the NS/NTSR1 complex becomes an upstream factor that modulates this regulatory mechanism.
  • NTS/NTSR1 tumor growth induced by NTS/NTSR1 can be restrained by a specific tyrosine kinase inhibitor EGFR and HER2, lapatinib.
  • EGFR tyrosine kinase inhibitor
  • HER2 lapatinib
  • NTSRl was expressed in at least 80 % of tumoral cells, and only 20 % of patients express high level of NTSRl and NTS (8).
  • NTS and NTSRl are expressed in cells clusters with diverse sizes. Tumoral cells with potential aggressiveness characteristics could be detected with NTS/NTSR1 labeling, and specific treatment could be proposed accordingly.
  • Lapatinib is proposed in a second line of treatments, in combination with other drug in advanced or metastatic breast cancers whose tumors overexpress HER2. Our results suggest that a more restrained (or targeted) population, can be determined by taking into account NTS and NTSRl co expression. The resulting subpopulation will provide a significantly better performance for this drug.
  • the activation of the neurotensinergic system in breast tumors induces the overexpression of the EGFR, HER2 and HER3 receptors and their concomitant autocrine activation.
  • the presence of this regulatory mechanism would have a significant impact on cancer progression in tumor cells by accelerating the process of metastasis. It may also modulate the response to HER2 or EGFR immunotherapy, as incomplete blockades and an increase in PI3k/AKT signaling are proposed to be the mechanisms involved in drug resistances (29).
  • EXAMPLE 2 EGFR, HER2 AND HER3 ACTIVATION, CONTROLLED BY
  • LNM35 cell line was sub-cloned by limiting dilution, after few days of culture, clones containing exclusively flat or rounded cells were saved and were named LNM-F for Flat, LNM-R for Rounded. All cells were grown at 37 °C, in a humidified atmosphere of 5% C0 2 .
  • Cell proliferation assays 20 000 cells/well of lung cancer cells were seeded in 24- well culture plates. Medium was replaced by FCS-free medium in presence or absence of NTS or JMV449 10 "8 M. Cells were counted after 5 days of treatment with a particle count and size analyzer (Zl Coulter Particle Counter, Beckman Coulter). For LNM-F/LNM-R (20/80 %) cell mixture: Cells were seeded in 48-well culture plates at a concentration of 40 000 cells/well, media containing 10% FCS. Media is changed 24h after for a media containing 0.1% FCS cells are counted after 48 hours.
  • Western blots 2xl0 6 cells were grown for 72h then serum-starved for 48h in a phenol red- free medium in presence or absence of different concentrations of 510 "6 M SR 48692 and 25 10 "9 M MMP inhibitor (Calbiochem), and lysed (20 mM Tris pH 8.0, 150 mM NaCl, 5 mM MgC12, 0,5 % NP40, 0,5 % glycerol, 1 mM PMSF, protease and phosphatase inhibitor cocktail) at 4°C for 30 min. Primary antibodies were incubated overnight at 4°C.
  • Total anti- EGFR (1 :500), anti-phospho-EGFR (1 :500), anti-phospho-HER2 (1 :500), anti-HER3 (1 :2000), anti-phospho-HER3 (1 :1000) were from Cell Signaling Technology.
  • Total anti- HER2 (1 :2000) was purchased from Neomarkers and anti-Pactin (1 :50000) from Sigma.
  • Secondary anti-rabbit (Santa Cruz Biotechnology) or anti-mouse (Sigma) antibodies, conjugated to HRP, were used at 1 :2000 dilutions for lh at room temperature and visualised by enhanced chemiluminescence (GE Healthcare).
  • Tumor xenografts Lung cancer cells, xenografts were initiated in nude mice by subcutaneous injection of 10 6 cells of LNM35, LNM-F, or LNM-R, and derivative cell clones. For tumors generated from a cell mixture, 10 6 cells from each clone were plated together 72 hours prior to injection. Four to six series were performed; each series included 5- 8 mice. All the procedure were in accordance with the "Guide of the Care and Use of laboratory Animals".
  • Tumor xenografts Lung cancer cells, xenografts were initiated in nude mice by subcutaneous injection of 10 6 cells of LNM35, LNM-F, or LNM-R, and derivative cell clones. For tumors generated from a cell mixture, 10 6 cells from each clone were plated together 72 hours prior to injection. Four to six series were performed, each series included 5- 8 mice. All procedures were in accordance with the "Guide of the Care and Use of laboratory Animals".
  • LNM-R or R-SI NTSRl cells were injected into nude mice by subcutaneous injection, R-SI NTSRl cells in the right flank and LNM-R cells in the left flank.
  • R-SI NTSRl cells were injected 10 days before LNM-R cells.
  • 22 days after R-SI NTSRl cells injection 2 groups of 5 mice were randomized on the size of R-SI NTSRl as follows : 229 ⁇ 40 mm 3 for control group and 242 ⁇ 37 mm 3 for erlotinib group. Mice were treated for 20 days per os.
  • mice were randomized on the size of LNM-R as follows: 19.79 ⁇ 3.00 mm 3 for control group, 18.66 ⁇ 2.21 mm 3 for erlotinib group, 16.82 ⁇ 3.32 mm 3 for metformin group and 18.82 ⁇ 3.00 mm 3 for metformin and erlotinib group. Mice were treated for 17 days per os, with water, or 25 mg/kg erlotinib, or 200 mg/kg metformin or both.
  • Adjuvant radiotherapy or chemotherapy was performed under the care of referring physicians, so no uniform protocol was employed. Long-term outcome was assessed by direct telephone interviews with patient or family (in case of deceased patients). When no clinical follow-up was available, information on vital status was obtained through the municipality of birth of the patient. Informed consent was obtained from all patients. The research was conducted according to recommendations outlined in the Helsinki declaration. Institutional Review Board approval was obtained (CPP He de France II, 2012). Immunohistochemistry: Procedure is detailed in SI. For all cases histologic slides of primary tumors were obtained from paraffin wax embedded tissues. Standard H&E staining was used to ensure the tumoral character of the specimen.
  • Deparaffinized tissue sections (4 ⁇ ) were incubated at 4°C overnight with primary antibody included anti-NTS (1 :200, SC- 20806, Santa Cruz biotechnology®), anti-NTSRl (1 :100; C-20, Santa Cruz Biotechnology®) and anti-ErbB3 (1 :50, NCL-c-erbB-3, NovocastraTM), and anti-ErbB2 (1 :400, A0485, Dako) was incubated at room temperature for 30 minutes.
  • primary antibody included anti-NTS (1 :200, SC- 20806, Santa Cruz biotechnology®), anti-NTSRl (1 :100; C-20, Santa Cruz Biotechnology®) and anti-ErbB3 (1 :50, NCL-c-erbB-3, NovocastraTM), and anti-ErbB2 (1 :400, A0485, Dako) was incubated at room temperature for 30 minutes.
  • NTSR1 staining of cancer cells was scored as positive in the presence of staining cells > 10 %.
  • Semi-quantitative evaluation was also performed: 0: no staining; 2: more than 50% of tumor cell showing a positive stain of high intensity; 1 : intermediate cases.
  • Statistical analysis was carried out using test student T test or Student-Newman-Keuls Multiple Comparisons Test : ***P ⁇ 0.001,**P ⁇ 0.01, and *P ⁇ 0.05.
  • data processing and analysis were performed with the statistical software system SEM (SILEX Development, Mireffleurs, France). Correlations were carried out by the Spearman rank correlation or H-test, as appropriate.
  • Survival analysis was carried out by the Kaplan-Meier method, and univariate comparisons of curves were performed using log rank tests. Risk factors associated with outcomes in univariate analysis with a p value ⁇ 0.05 were entered into a multivariate Cox model analysis, to identify independent predictors of survival. A p value of less than 0.05 was considered significant.
  • the NTS/NTSR1 complex enhances cellular growth
  • NTS and NSTR1 are concomitantly expressed in human lung tumors. NTS actions, possibly occurring in tumor, are therefore mediated through autocrine and/or paracrine regulation (1).
  • LNM-35 LNM-R cells (expressing NTS and NTSR1) and LNM-F cells (expressing mainly NTSR1) were isolated from the parental LNM 35 cells and the observed pheno types remained with cultured passages (Figure 8 A inset).
  • the LNM-R culture media contained large amounts of NTS, which accumulated with time (75 to 625 fmol/ml), whereas the media of LNM-F cells contained 20 fold less NTS.
  • the NTS/NTSR1 complex enhances EGFR, HER2 and HER3 expression and activation.
  • NTSR1 activation in experimental tumors In order to apprehend the contribution of NTSRl in lung tumorigenesis, we developed experimental tumors bearing NTS autocrine, and/or paracrine, or endocrine regulation. We established the growth rate of LNM35 tumor xenografts in the nude mice, in comparison with the two derived sub-clones, LNM-R (NTS+) and LNM-F (NTS-). As shown in figure. 12A, LNM35 xenografts displayed the more drastic tumorigenesis profile with a final tumor volume of 4122 mm 3 . The sub-clones LNM-R, and LNM-F generated smaller tumors with a final volume of 2582 and 1858 mm 3 , respectively.
  • the tumor size is 38 and 55% smaller than LNM35 when generated by LNM-R and LMN-F, respectively.
  • the difference in the tumor growth rates between the parental cells and the two subclones suggested a positive cooperativity between these two cellular populations.
  • NTS and NTSRl immunohistochemistry was performed on tumors. The presence of NTSRl was seen in both LNM-R and LNM-F tumors ( Figure 12B), but with a granular and irregular intensity of labeling.
  • we used an antibody, which detected the presence of NTS precursor in LMN-R and its absence in LNM-F tumors Figure 12B).
  • Tumors expressing NTS/NTSR1 are responsive to EGFR inhibitors
  • mice were xenografted with LNM-R cells expressing NTS and NTSRl on the right side and with a derived clone R Si-NTSRl (1) deleted for NTSRl expression with a stable expression of sh-NSTRl plasmid on the left side (Fig 13 A).
  • a first experiment consisting on 5 animals randomized on R Si-NTSRl tumor with an initial tumor volume of 158 ⁇ 40 mm 3 and 144.07 ⁇ 26 mm 3 for control and erlotinib group, respectively, were selected.
  • the final volume was 318.23 ⁇ 31.56 mm 3 (Fig 13 D).
  • the tumor doubling time was in agreement with the tumor volume, with 2.87 ⁇ 0.13 day, 3.85 ⁇ 0.24 day, 3.09 ⁇ 0.07 day, and 4.03 ⁇ 0.28 day for the control, metformin, erlotinib and metformin + erlotinib treated animals, respectively.
  • the tumors void of NTSRl expression had no detectable response NTSRl to metformin or erlotinib (Fig 13E). This lack of response is consistent with the presence of a NTS autocrine loop, leading to the sustained activation of EGFR and responsible for cancer aggressiveness
  • Overexpression of NTSRl in lung adenocarcinomas correlates with pejorative prognosis
  • NTSRl expression is a negative prognostic marker in a selected population of stage I lung adenocarcinoma treated by surgery alone (1).
  • stage I-III NSCLC all histotypes
  • adenocarcinoma patients operated on between June 15, 2001 and December 31, 2005 were analyzed.
  • NTSRl positive staining was detected in 59 % of cases (160/271), but it was never detected in normal tissues adjacent to the tumor area.
  • 111, 126, and 34 patients were scored as 0, 1 and 2, respectively.
  • NTSRl staining of cancer cells was granular, intracellular, heterogeneous and rarely localized at the plasma membrane (figure 14A left).
  • NTSRl staining in the squamous carcinoma cells was often localized at the membrane level (figure 14A right).
  • NTSRl positive staining was not detected in lepidic adenocarcinomas or even in the lepidic component of invasive adenocarcinomas.
  • NTSRl score 1 was detected in 48 % of adenocarcinomas (57/119), 43% of SCC (46/107), and 39% of LCC (14/36).
  • NTSRl score 2 was detected in 22% of adenocarcinomas (26/119), 7% of SCC (8/ 107), and 3% (1/36) of LCC.
  • NTS oncogenic action is boosted with a sustained NTSRl state of activation.
  • both NTS and NTSRl are expressed in 40%), 60%), 65%, and 80 % of breast, lung, mesothelioma, and head neck squamous carcinomas, respectively, and suggesting that autocrine and/or paracrine NTS regulation occurs in tumors (7-9).
  • Sustained activation of NTSRl induced the overexpression of the two receptors HER2, and HER3, as well as an autocrine activation of EGFR, HER2 and HER3.
  • EGF concomitantly with neuregulin 1. Consequently, both EGFR and HER3 autocrine activities are boosted and the tumoral aggressiveness is potentiated.
  • MMPs and EGF "like" ligands to carcinogenesis and cancer progression are well known.
  • Therapeutical strategies targeting these factors have been largely attempted. However, these factors are often important for the human metabolism and major physiological functions, such as healing, angiogenesis, and gonadogenesis (14).
  • One approach is to specifically antagonize their effects in cancer cells and to target an upstream regulatory factor.
  • the NTS/NTSR1 complex would appear to be convenient, because, it is specifically over- expressed in tumors and its inhibition should only impair the function of these factors where they are deleterious.
  • Metformin is an antidiabetic drug, and has recently been proposed as a potential anticancer compound (17). Metformin was shown to disrupt the crosstalk between insulin receptor and NTS receptor in pancreatic cancer cells (18). Furthermore, in addition to inhibiting the mTOR pathway, metformin prevents ER activation induced by NTS and insulin (19). In our cellular model, metformin reduced tumor growth only in those tumors expressing NTSRl, and thus supporting the significance of the NTS/NTSR1 signaling pathway in tumor growth.
  • NTSRl expression was associated with adenocarcinomas prognosis. This result was confirmed by multivariate analysis, which showed that among the available clinical and pathologic factors, the NTSRl score 2, T, and N were independent predictors of worse prognosis. High expression of NTSRl has been found to be associated to poor survival also in other cancers. Dupouy et al found that NTSRl expression involving > 80% tumor cells was associated with worse survival in breast cancer (16). Similarly, in head and neck cancers, patients with high NTS and NTSRl expression had a higher rate of distant metastasis (9). Therefore, the prognostic role of neurotensin system is probably correlated with its activation rate.
  • NTSRl staining was never detected in broncho lo alveolar subtypes or in the bronchoalveolar component of mixed adenocarcinoma but it was often detected in its invasive compartment, suggesting a role favoring tumor invasion and migration: Also in SCC the staining was found primarily at the membrane as in the non- stimulated cells. In vitro studies have shown that NTS is capable of modulating the migratory ability of adherent cancer cells of different origins (colon, ductal pancreatic, head and neck squamous cell, breast).
  • NTS/NTSR1 complex could be used as a marker to identify subsets of human cancers, and thus make eligible new drugs, kinase inhibitors, or immunotherapy, targeting HERs protein or their downstream pathways.
  • the clinical criteria used to propose these therapies are based on the detection of genetic defaults in the tumor (HER2 amplification, EGFR mutation). Nevertheless, it was also observed that other patient subsets could benefit from these therapies.
  • the challenge is find a criteria to categorize them. For example, cells with neuregulin 1 high expression in association with HER3 autocrine activation and without HER2 amplification, are good responders to lapatinib or HER2 kinase inhibitors (22).
  • NTS/NTSR1 complex contributes to cancer aggressiveness by enhancing concomitantly expression and activation of the three receptors EGFR, HER2, HER3.
  • patients bearing this complex should be responders to kinase inhibitors, and that inhibition of NTS/NTSR1 complex should reduce the rate of tumor progression, providing a longer therapeutic window for the practitioners to treat their patients.
  • NTS/NTSRl enhance experimental HCC tumor progression through EGFR activation
  • mice were implanted with HCC cell lines and NTSR1 overexpressing clones xenografted.
  • HEP3B cells the tumors were measurable 21 days after cells injection, whereas for PLC/PRF5, the tumors were measurable at day 17. Both parental cell lines showed similar growth rates.
  • the tumor burden for both clones overexpressing NTSR1 was measurable earlier at day 13, and the growth rate at day 42 was 2.9 and 2.08 fold higher for HEP-Rla and PLC-Rla as compared to respective parental cells (Figure 15 A).
  • the tumor weights is in correspondence with the tumor size 4.3 and 2.8 fold time higher for HEP- Rla and PLC-Rla as compared to their respective parental cells.
  • the strong labeling was often localized at the front of the tumor or close to the blood vessel. Examples of a thin or strong labelling are shown in figure 15C c and d, respectively. In contrast, in HEP3B tumors, phosphorylated EGFR labeling was absent or very weak as shown in figure 15C g and h.
  • NTS/NTSR1 restores responses to tyrosine kinase inhibitor.
  • HCC cell lines HEP 3B and C were xenografted on the same mice.
  • the mice were randomly distributed in two groups based on the size of the HEP 3B tumors. Since HEP-Rla tumors grow faster than HEP 3B tumors, HEP 3B cells were injected a few days before HEP-Rla cells.
  • the average HEP3B tumor size was 168.2 ⁇ 38.1 mm 3 and the HEP-Rla tumor size was 79.7 ⁇ 16.4 mm 3 at day 1.

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Abstract

The present invention relates to methods and pharmaceutical compositions for the treatment of cancer. In particular one aspect of the present invention relates to a method of the treatment of cancer in a patient in need thereof comprising the steps of i) determining the expression level of NTS and/or NTSR1 in tumor sample obtained from the patient, ii) comparing the expression level determined at step i) with a predetermined reference value and iii) administering the patient with a therapeutically effective amount of at least one HER inhibitor when the expression level determined at step i) is higher than the predetermined reference level.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT
OF CANCER
FIELD OF THE INVENTION:
The methods of the present invention relates to methods and pharmaceutical compositions for the treatment of cancer.
BACKGROUND OF THE INVENTION:
Lung and breast cancers are the most common malignancies in the world, and are the leading cause of cancer-related deaths in western countries (1, 2). The progression of tumors to a metastatic disease is the primary cause of death in most patients and the main target of cancer research. The stage of the disease is the most useful parameter in deciding management and defining prognosis. However, survival is heterogeneous even within a single stage. Therefore, the identification of biological parameters allowing characterization, and validation of tumor subsets possessing aggressive phenotypes, is an ongoing challenge for cancer biological studies.
The HER family of proteins, EGFR, HER2, HER3, and HER4, share several structural and functional features, despite notably different molecular and cellular functions (3). In tumoral cells, these functions can be abnormally exacerbated because of genetic defaults, protein overexpression, or over-activation of one or several of these receptors. In tumors, these regulations are not mutually exclusive and confer a large magnitude of oncogenic activities which are difficult to detect and categorize within tumoral specimens. HERs activate a multiplicity of intracellular pathways via their ability to interact with numerous signal transducers. Genetic alterations, which constitutively activate ErbB2 and EGFR, stimulate many intracellular signaling proteins and pathways, such as the MAPK, PI3K/Akt, and mTOR pathways, Src kinase, and STAT transcription factors (4).
The success of therapies employing HERs immunotherapy or tyrosine kinase inhibitors, while limiting the progression of the disease and extending the disease free survival time, demonstrates the contribution of HERs at the clinical level. For instance in breast cancer, where human epidermal growth factor receptor 2 (HER2) gene amplication is detected in 25% of patients, a monoclonal antibody targeting HER2, Trastuzumab, (Herceptin) significantly improves the survival of these patients. Nevertheless, contribution of the HERs in cancer progression is complex, and some therapeutic investigation with HER inhibitors in the treatment of cancer (e.g. lung cancer) has failed. Thus theses results prompt the skilled persons to identify bio markers that will allow identifications of patients eligible for the treatment of their cancer with HER inhibitors. For example in breast cancer, the clinical criteria used to propose these therapies are based on the detection of genetic defaults in the tumor (HER2 amplification, EGFR mutation). Nevertheless, it was also observed that other patient subsets could benefit from these therapies. The challenge; is find a criteria to categorize them.
Amongst the factors contributing to tumor aggressiveness, neurotensin (NTS) and its cognate high-affinity receptor (NTSRl) have been shown to contribute to progression of human tumors, both NTS and NTSRl are expressed in 40%, 60%, 65%, and 80 % of breast, lung, mesothelioma, and head neck squamous carcinomas, respectively, and suggesting that autocrine and/or paracrine NTS regulation occurs in tumors However the impact of the NTS/NTSRl on HER overexpression has never been investigated. SUMMARY OF THE INVENTION:
The methods of the present invention relates to methods and pharmaceutical compositions for the treatment of cancer. In particular, the present invention is defined by the claims. DETAILED DESCRIPTION OF THE INVENTION:
The inventors highlight the cellular mechanisms activated by Neurotensin (NTS) and its high affinity receptor (NTSRl) contributing to cancer cell aggressiveness in 2 models (i.e. lung and breast cancer). They show that the NTS autocrine and/or paracrine regulation causes HER over-expression and activation in tumor cells. Said observations prompt the inventors to investigate whether the NTS/NTSRl complex could be used as a marker to identify subsets of human cancers eligible to treatment with HER inhibitors. They validate the hypothesis by showing that treatment of tumors having a positive expression for the NTS/NTRS1 complex with a HER inhibitor show a dramatic decrease in tumor growth in comparison with the treatment of tumors wherein the expression of NTSRl is absent.
Accordingly, one aspect of the present invention relates to a method of the treatment of cancer in a patient in need thereof comprising the steps of i) determining the expression level of NTS and/or NTSRl in tumor sample obtained from the patient, ii) comparing the expression level determined at step i) with a predetermined reference value and iii) administering the patient with a therapeutically effective amount of at least one HER inhibitor when the expression level determined at step i) is higher than the predetermined reference level. In some embodiments, the patient suffers from a cancer deriving from epithelial origin. Examples of cancer types include, but are not limited to, carcinoma, lymphoma, blastoma (including meduUoblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophagael cancer, tumors of the biliary tract, as well as head and neck cancer, as well as subtypes of any of such cancers, including, but not limited to chemotherapy-resistant, platinum-resistant, advanced, refractory, and/or recurrent types thereof.
The term "tumor sample" means any tissue sample derived from the tumor of the patient. The tissue sample is obtained for the purpose of the in vitro evaluation. The sample can be fresh, frozen, fixed (e.g., formalin fixed), or embedded (e.g., paraffin embedded). In some embodiments the sample results from biopsy performed in a tumour sample of the patient. As used herein the term "NTS" has its general meaning in the art and refers to neurotensin. As used herein the term emcompasses all forms of neurotensin, including preproneurotensin and preneurotensin. An exemplary amino sequence of NTS is SEQ ID NO: l, 2 or 3 and an exemplary nucleic acid sequence of NTS is SEQ ID NO:4. SEQ ID NO: 1 :
MMAGMKIQLVCMLLLAFSSWSLCSDSEEEMKALEADFLTNMH SKI SKAHVPSWKMTLLNVC SLVNNLNSPAEE GEVHEEELVARRKLPTALDGFSLEAML IYQLHKICHSRAFQHWELIQE DILDTGNDKNGKEEVIKRKI PYILKRQLYENKPRRPYILKRDSYYY
SEQ ID NO:2
SDSEEEMKALEADFLTNMHTSKI SKAHVPSWKMTLLNVCSLVNNLNSPAEETGEVHEEELVA RRKLPTALDGFSLEAMLTIYQLHKICHSRAFQHWELIQEDILDTGNDKNGKEEVIKRKI PYI LKRQLYENKPRRPYIL
SEQ ID NO:3
QLYENKPRRPYIL
SEQ ID NO:4
gctgaaggaa agaggaagtg ctagagagag cccccttcag tgtgcttctg
acttttacgg acttggcttg ttagaaggct gaaagatgat ggcaggaatg
aaaatccagc ttgtatgcat gctactcctg gctttcagct cctggagtct
gtgctcagat tcagaagagg aaatgaaagc attagaagca gatttcttga
ccaatatgca tacatcaaag attagtaaag cacatgttcc ctcttggaag
atgactctgc taaatgtttg cagtcttgta aataatttga acagcccagc
tgaggaaaca ggagaagttc atgaagagga gcttgttgca agaaggaaac
ttcctactgc tttagatggc tttagcttgg aagcaatgtt gacaatatac
cagctccaca aaatctgtca cagcagggct tttcaacact gggagttaat
ccaggaagat attcttgata ctggaaatga caaaaatgga aaggaagaag
tcataaagag aaaaattcct tatattctga aacggcagct gtatgagaat
aaacccagaa gaccctacat actcaaaaga gattcttact attactgaga
gaataaatca tttatttaca tgtgattgtg attcatcatc ccttaattaa
atatcaaatt atatttgtgt gaaaatgtga caaacacact tatctgtctc
ttctacaatt gtggtttatt gaatgtgatt tttctgcact aatataaatt
agactaagtg ttttcaaata aatctaaatc ttcagcatga tgtgttgtgt
ataattggag tagatattaa ttaagtcacc tgtataatgt tttgtaattt
tgcaaaacat atcttgagtt gtttaaacag tcaaaatgtt tgatatttta
taccagctta tgagctcaaa gtactacagc aaagcctagc ctgcatatca
ttcacccaaa acaaagtaat agcgcctctt ttattatttt gactgaatgt
tttatggaat tgaaagaaac atacgttctt ttcaagactt cctcatgaat
ctctcaatta taggaaaagt tattgtgata aaataggaac agctgaaaga
ttgattaatg aactattgtt aattcttcct attttaatga atgacattga
actgaatttt ttgtctgtta aatgaacttg atagctaata aaaagacaac
t cL C[ C C cL t C cL cL 3.3. t C 3.3.3.3.3.3. cL cL cL cL cL cL cL cL cL cL cL cL
As used herein, the term "NTSR1" has its general meaning in the art and refers to neurotensin receptor 1. An exemplary amino sequence of NTSR1 is SEQ ID NO:5 and an exemplary nucleic acid sequence of NTSR1 is SEQ ID NO:6. SEQ ID NO:5
MRLNSSAPGTPGTPAADPFQRAQAGLEEALLAPGFGNASGNASERVLAAPSSELDVNT DIYSKVLVTAVYLALFVVGTVGNTVTAFTLARKKSLQSLQSTVHYHLGSLALSDLLTL LLAMPVELYNFIWVHHPWAFGDAGCRGYYFLRDACTYATALNVASLSVERYLAICHPF KAKTLMSRSRTKKFI SAIWLASALLAVPMLFTMGEQNRSADGQHAGGLVC P IH AT VKVVIQVN FMSFIFPMVVI SVLN I IANKLTVMVRQAAEQGQVCTVGGEHS FSMAI EPGRVQALRHGVRVLRAVVIAFVVCWLPYHVRRLMFCYI SDEQW PFLYDFYHYFYMV TNALFYVSS INPILYNLVSANFRHIFLATLACLCPVWRRRRKRPAFSRKADSVSSNH TLSSNATRETLY
SEQ ID NO: (
atgcgcctca acagctccgc gccgggaacc ccgggcacgc cggccgccga ccccttccag cgggcgcagg ccggactgga ggaggcgctg ctggccccgg gcttcggcaa cgcttcgggc aacgcgtcgg agcgcgtcct ggcggcaccc agcagcgagc tggacgtgaa caccgacatc tactccaagg tgctggtgac cgccgtgtac ctggcgctct tcgtggtggg cacggtgggc aacacggtga cggcgttcac gctggcgcgg aagaagtcgc tgcagagcct gcagagcacg gtgcattacc acctgggcag cctggcgctg tccgacctgc tcaccctgct gctggccatg cccgtggagc tgtacaactt catctgggtg caccacccct gggccttcgg cgacgccggc tgccgcggct actacttcct gcgcgacgcc tgcacctacg ccacggccct caacgtggcc agcctgagtg tggagcgcta cctggccatc tgccacccct tcaaggccaa gaccctcatg tcccgaagcc gcaccaagaa gttcatcagc gccatctggc tcgcctcggc cctgctggcg gtgcctatgc tgttcaccat gggcgagcag aaccgcagcg ccgacggcca gcacgccggc ggcctggtgt gcacccccac catccacact gccaccgtca aggtcgtcat acaggtcaac accttcatgt ccttcatatt ccccatggtg gtcatctcgg tcctgaacac catcatcgcc aacaagctga ccgtcatggt acgccaggcg gccgagcagg gccaagtgtg cacggtcggg ggcgagcaca gcacattcag catggccatc gagcctggca gggtccaggc cctgcggcac ggcgtgcgcg tcctacgtgc agtggtcatc gcctttgtgg tctgctggct gccctaccac gtgcggcgcc tcatgttctg ctacatctcg gatgagcagt ggactccgtt cctctatgac ttctaccact acttctacat ggtgaccaac gcactcttct acgtcagctc caccatcaac cccatcctgt acaacctcgt ctctgccaac ttccgccaca tcttcctggc cacactggcc tgcctctgcc cggtgtggcg gcgcaggagg aagaggccag ccttctcgag gaaggccgac agcgtgtcca gcaaccacac cctctccagc aatgccaccc gcgagacgct gtactag
Determining an expression level of a gene (e.g. NTS or NTSR1) in a tumor sample obtained from a patient can be implemented by a panel of techniques well known in the art. Typically, an expression level of a gene is assessed by determining the quantity of mRNA produced by this gene.
Methods for determining a quantity of mRNA are well known in the art. For example nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. Thus the extracted mRNA is then detected by hybridization (e. g., Northern blot analysis) and/or amplification (e.g., RT-PCR). Typically quantitative or semi-quantitative RT- PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous. Other methods of Amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA), quantitative new generation sequencing of RNA (NGS).
Nucleic acids (polynucleotides) comprising at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be completely identical, but are typically at least about 80% identical to the homologous region of comparable size, more typically 85% identical and even more typically 90-95% identical. In some embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin).
Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more typically of between 15 and 700, typically of between 20 and 500 nucleotides. Primers typically are shorter single- stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they typically hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
Nucleic acids which may be used as primers or probes in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. A kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences. In some embodiments, the methods of the invention comprise the steps of providing total R As extracted from cancer cells and subjecting the R As to amplification and hybridization to specific probes, more particularly by means of a quantitative or semiquantitative RT-PCR.
Probes made using the disclosed methods can also be used for nucleic acid detection, such as in situ hybridization (ISH) procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH). For instance, in situ hybridization (ISH) involves contacting a sample containing target nucleic acid sequence (e.g., genomic target nucleic acid sequence) in the context of a metaphase or interphase chromosome preparation (such as a cell or tissue sample mounted on a slide) with a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence). The slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization. The sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids. The probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium). The chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques. Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Pat. Nos. 5,447,841; 5,472,842; and 5,427,932; and for example, in Pinkel et al, Proc. Natl. Acad. Sci. 83:2934-2938, 1986; Pinkel et al, Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al, Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described in, e.g., Tanner et al, Am. J. Pathol. 157: 1467-1472, 2000 and U.S. Pat. No. 6,942,970. Additional detection methods are provided in U.S. Pat. No. 6,280,929.
Probes are typically labelled with a detectable molecule. For example, a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase. For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)-conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin- conjugated goat antiavidin antibodies, washing and a second incubation with FITC- conjugated avidin. For detection by enzyme activity, samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer). For a general description of in situ hybridization procedures, see, e.g., U.S. Pat. No. 4,888,278. Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties. As discussed above probes labeled with fluorophores (including fluorescent dyes and QUANTUM DOTS®) can be directly optically detected when performing FISH. Alternatively, the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following non- limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety. Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand. The detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore. In other examples, the probe is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH). As indicated above, the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publications Nos. 2006/0246524; 2006/0246523, and 2007/0117153.
It will be appreciated by those of skill in the art that by appropriately selecting labelled probe-specific binding agent pairs, multiplex detection schemes can be produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample). For example, a first probe that corresponds to a first target sequence can be labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP. Following exposure of the sample to the probes, the bound probes can be detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 mn). Additional probes/binding agent pairs can be added to the multiplex detection scheme using other spectrally distinct fluorophores. Numerous variations of direct, and indirect (one step, two step or more) can be envisioned, all of which are suitable in the context of the disclosed probes and assays.
Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more typically of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single- stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they typically hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
In some embodiments, the expression level is determined by DNA chip analysis. Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere- sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a sample from a test subject, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).
The expression level of a gene may be expressed as absolute expression level or normalized expression level. Both types of values may be used in the present method. The expression level of a gene is typically expressed as normalized expression level when quantitative PCR is used as method of assessment of the expression level because small differences at the beginning of an experiment could provide huge differences after a number of cycles.
Typically, expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not relevant for determining the cancer stage of the patient, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene.... This normalization allows comparing the expression level of one sample, e.g., a patient sample, with the expression level of another sample, or comparing samples from different sources.
Alternatively, the expression level of a gene (e.g. NTS or NTSR1) may be determined at the protein level. For example, the tumor sample of the patient may be contacting with a binding partner specific for the protein of interest (i.e. NTS or NTSR1).
In some embodiments, the binding partner is an antibody or an aptamer. Polyclonal antibodies of the invention or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies of the invention or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique; the human B-cell hybridoma technique; and the EBV-hybridoma technique. In another embodiment, the binding partner may be an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library.
The binding partners of the invention such as antibodies or aptamers, may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal. As used herein, the term "labelled", with regard to the antibody or aptamer, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be labelled with a radioactive molecule by any method known in the art. For example radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, Inl l l, Rel86, Rel88. Typically, the antibodies against the surface markers are already conjugated to a fluorophore (e.g. FITC-conjugated and/or PE- conjugated).
In some embodiments, immunostained slices of the tumor tissue sample may be obtained with an automated slide-staining system by using a labelled binding partner as above described (e.g. an antibody). Immuno chemistry (IHC) is a suitable method for quantifying the expression level of a marker in a tissue sample. Typically, the tumor sample is typically fixed in formalin and embedded in a rigid fixative, such as paraffin (wax) or epoxy, which is placed in a mould and later hardened to produce a block which is readily cut. Thin slices of material are prepared using a microtome, placed on a glass slide and submitted to immunohistochemistry, for example using an IHC automate such as BenchMark® XT allowing automatic stained slide preparation for implementing the immunohistochemical staining. Then after digitalisation of the slices may be used to quantify the level of the marker. Digitalisation of the slices may be made by scan capture, for example with a high resolution Hamamatsu NanoZoomer® 2.0-HT scanner. The mean, median, min and max of the relevant staining intensity of all positive stained cells detected in the tumour sample may be provided. The values and the distribution of the staining intensity can be compared to the predetermined reference value.
Typically, the predetermined reference value is a threshold value or a cut-off value. A "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of the expression level of NTS or/and NTSR1 in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the expression level of NTS or/and NTSR1 in a group of reference, one can use algorithmic analysis for the statistic treatment of the expression levels determined in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator the reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.
In some embodiments, the predetermined reference value ("cut-off) is determined by carrying out a method comprising the steps of:
a) providing a collection of tumor tissue samples from patients;
b) determining the expression level of NTS or/and NTSR1 for each tumour tissue sample contained in the collection provided at step a);
c) ranking the tumor tissue samples according to said expression level
d) classifying said tumour tissue samples in pairs of subsets of increasing, respectively decreasing, number of members ranked according to their expression level,
e) providing, for each tumour tissue sample provided at step a), information relating to the actual clinical outcome for the corresponding cancer patient (i.e. the duration of the disease-free survival (DFS) or the overall survival (OS) or both);
f) for each pair of subsets of tumour tissue samples, obtaining a Kaplan Meier percentage of survival curve;
g) for each pair of subsets of tumour tissue samples calculating the statistical significance (p value) between both subsets
h) selecting as reference value for the expression level, the value of expression level for which the p value is the smallest. For example the expression level of NTS or/and NTSR1 has been assessed for 100 cancer samples of 100 patients. The 100 samples are ranked according to the expression level of NTS or/and NTSR1. Sample 1 has the highest expression level and sample 100 has the lowest expression level. A first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples. The next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100. According to the information relating to the actual clinical outcome for the corresponding cancer patient, Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated. The predetermined reference value is selected such as the discrimination based on the criterion of the minimum p value is the strongest. In other terms, the expression level corresponding to the boundary between both subsets for which the p value is minimum is considered as the reference value. It should be noted that according to the experiments made by the inventors, the reference value is not necessarily the median value of expression levels. Kaplan-Meier curves of percentage of survival as a function of time are commonly used to measure the fraction of patients living for a certain amount of time after treatment and are well known by the man skilled in the art. P value is conventionally used in statistical significance testing.
In some embodiments, high statistical significance values (e.g. low P values) are obtained for a range of successive arbitrary quantification values, and not only for a single arbitrary quantification value. Thus, in some embodiments, instead of using a definite predetermined reference value, a range of predetermined reference values is provided. In some embodiments, a cut-off value thus consists of a range of quantification values, e.g. centered on the quantification value for which the highest statistical significance value is found (e.g. generally the minimum P value which is found). For example, on a hypothetical scale of 1 to 10, if the ideal cut-off value (the value with the highest statistical significance) is 5, a suitable (exemplary) range may be from 4-6. Therefore, a patient may be assessed by comparing values obtained by measuring the expression level of NTS or/and NTSR1, where values greater than 5 reveal that the patient is eligible to a treatment with a HER inhibitor and values less than 5 reveal that the patient is not eligible to said treatment . In a another embodiment, a patient may be assessed by comparing values obtained by measuring the expression level of NTS or/and NTSR1 and comparing the values on a scale, where values above the range of 4-6 indicate that the patient is eligible to the treatment and values below the range of 4-6 indicate that the patient is not eligible, with values falling within the range of 4-6 indicate that there are still doubts that the patient is eligible to the treatment.
As used herein the "HER" has its general meaning in the art and refers to a receptor protein tyrosine kinase which belongs to the HER receptor family and includes EGFR, HER2, HER3 and HER4 receptors. As used herein the terms "ErbBl," "HER1", "epidermal growth factor receptor" and "EGFR" are used interchangeably herein and refer to EGFR as disclosed, for example, in Carpenter et al. Ann. Rev. Biochem. 56:881-914 (1987), As used herein, the terms "ErbB2" and "HER2" are used interchangeably herein and refer to human HER2 protein described, for example, in Semba et al, PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al. Nature 319:230-234 (1986) (Genebank accession number X03363). As used herein, the term "ErbB3" and "HER3" refer to the receptor polypeptide as disclosed, for example, in U.S. Pat. Nos. 5,183,884 and 5,480,968 as well as Kraus et al. PNAS (USA) 86:9193-9197 (1989). As used herein, the terms "ErbB4" and "HER4" refer to the receptor polypeptide as disclosed, for example, in EP Pat Appln No 599,274; Plowman et al, Proc. Natl. Acad. Sci. USA, 90: 1746-1750 (1993); and Plowman et al, Nature, 366:473-475 (1993). By "HER ligand" is meant a polypeptide which binds to and/or activates a HER receptor.
As used herein the term "HER inhibitor" refers to an agent which interferes with HER activation or function. Examples of HER inhibitors include HER antibodies (e.g. EGFR, HER2, HER3, or HER4 antibodies); small organic molecule HER antagonists; HER tyrosine kinase inhibitors; HER2 and EGFR dual tyrosine kinase inhibitors such as lapatinib/GW572016; antisense molecules (see, for example, WO2004/87207); and/or agents that bind to, or interfere with function of, downstream signaling molecules, such as MAPK or Akt. Typically, the HER inhibitor is an antibody or small organic molecule which binds to a HER receptor. In some embodiments, the HER inhibitor is a "HER dimerization inhibitor" which is an agent which inhibits formation of a HER dimer or HER heterodimer. In some embodiments, the HER inhibitor is an "anti-HER antibody" which is an antibody that binds to a HER receptor. The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (destabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991 , specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/1 1 161 ; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al, 2006; Holliger & Hudson, 2005; Le Gall et al, 2004; Reff & Heard, 2001 ; Reiter et al, 1996; and Young et al, 1995 further describe and enable the production of effective antibody fragments.
In some embodiments, the anti-HER monoclonal antibody of the invention is used to induce antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) against HER-expressing cells. In another particular embodiment, the anti- HER antibody may be suitable for disturbing the expression of HER at the cell surface (e.g. by provoking internalization of HER) so that cell migration, cell proliferation and tumour growth of tumor cells will be limited or inhibited.
In some embodiments, the anti-HER antibody is a "chimeric" antibody in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)). Chimeric antibodies include PRIMATTZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest.
In some embodiments, the anti-HER antibody is a humanized antibody. "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a HVR of the recipient are replaced by residues from a HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al, Nature 321 :522-525 (1986); Riechmann et al, Nature 332:323- 329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1 : 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409. For instance, humanized HER2 antibodies include huMAb4D5-l, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 or trastuzumab (HERCEPTIN®) as described in Table 3 of U.S. Pat. No. 5,821,337 expressly incorporated herein by reference; humanized 520C9 (W093/21319); and humanized 2C4 antibodies such as pertuzumab.
In some embodiments, the anti-HER antibody is a human antibody. A "human antibody" is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage- display libraries. Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al, J. Mol. Biol, 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al, J. Immunol, 147(l):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5 : 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, for example, Li et al, Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.
In some embodiments, the anti-HER antibody is a single domain antibody. The term "single domain antibody" (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called "nanobody®". According to the invention, sdAb can particularly be llama sdAb.
In some embodiments, the anti-HER antibody is an anti-HER monoclonal antibody- drug conjugate. An "anti-HER monoclonal antibody-drug conjugate" as used herein refers to an anti-HER monoclonal antibody according to the invention conjugated to a therapeutic agent. Such anti-HER monoclonal antibody-drug conjugates produce clinically beneficial effects on HER-expressing tumor cells when administered to a subject. In typical embodiments, an anti-HER monoclonal antibody is conjugated to a cytotoxic agent, such that the resulting antibody-drug conjugate exerts a cytotoxic or cytostatic effect on a HER- expressing tumor cell when taken up or internalized by the cell. Any cytotoxic agent well known by the skilled person may used. In some embodiments, the cytotoxic or cytostatic agent is auristatin E (also known in the art as dolastatin-10) or a derivative thereof. Typically, the auristatin E derivative is, e.g., an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with paraacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatin derivatives include AFP (dimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylenediamine), MMAF (dovaline-valine-dolaisoleunine-dolaproine-phenylalanine), and MAE (monomethyl auristatin E). The synthesis and structure of auristatin E and its derivatives are described in U.S. Patent Application Publication No. 20030083263; International Patent Publication Nos. WO 2002/088172 and WO 2004/010957; and U.S. Patent Nos. 6,884,869; 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414. In some embodiments, an anti-HER monoclonal antibody of the invention is used to induce antibody dependent cellular cytotoxicity (ADCC). In ADCC, monoclonal antibodies bind to a target cell (e.g., cancer cell) and specific effector cells expressing receptors for the monoclonal antibody (e.g., NK cells, CD8+ T cells, monocytes, granulocytes) bind the monoclonal antibody/target cell complex resulting in target cell death. Accordingly, in some embodiments, an anti-HER monoclonal antibody comprising an Fc region with effector function is used to induce antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) against a HER-expressing cell. Methods for inducing ADCC generally include contacting the HER-expressing cell with an effective amount an anti-HER monoclonal antibody comprising an Fc region having ADCC activity, wherein the contacting step is in the presence of a cytolytic immune effector cell expressing an Fc receptor having cytolytic activity. Immune effector cells expressing cytolytic Fc receptors (e.g., FcyRIIIa or CD 16) include, for example, NK cells as well certain CD8+ T cells. Methods for inducing CDC generally include contacting the HER-expressing cell with an effective amount an anti- HER monoclonal antibody comprising an Fc region having CDC activity, wherein the contacting step is in the presence of complement.
In some embodiments, the anti-HER antibody is monospecific, bispecific, trispecific, or of greater multispecificity. Multispecific antibodies, including bispecific and trispecific antibodies, useful for practicing the methods described herein are antibodies that immunospecifically bind to both HER and a second cell surface receptor or receptor complex that mediates ADCC, phagocytosis, and/or CDC, such as CD16/FcgRIII, CD64/FcgRI, killer inhibitory or activating receptors, or the complement control protein CD59. In a typical embodiment, the binding of the portion of the multispecific antibody to the second cell surface molecule or receptor complex enhances the effector functions of the anti-HER antibody. In some embodiment, the anti-HER antibody is a bispecific antibody. The term "bispecific antibody" has its general meaning in the art and refers to any molecule consisting of one binding site for a target antigen on tumor cells (i.e. a HER receptor) and a second binding side for an activating trigger molecule on an effector cell, such as CD3 on T-cells, CD 16 (FcyRlll) on natural killer (NK) cells, monocytes and macrophages, CD89 (FcaRI) and CD64 (FcyRI) on neutrophils and monocytes/macrophages, and DEC-205 on dendritic cells. According to the invention, the bispecific antibody comprises a binding site for HER. tApart from the specific recruitment of the preferred effector cell population, bispecific antibodies avoid competition with endogenous immunoglobulin G (IgG) when the selected binding site for the trigger molecule on the effector cell does not overlap with Fc-binding epitopes. In addition, the use of single-chain Fv fragments instead of full-length immunoglobulin prevents the molecules from binding to Fc-receptors on non-cytotoxic cells, such as FcyRII on platelets and B-cells, to Fc-receptors that do not activate cytotoxic cells, including FcyRlllb on polymorphonuclear leukocytes (PMN), and to inhibitory Fc-receptors, such as FcyRllb on monocytes/macrophages. Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (see, e.g., Milstein et al, 1983, Nature 305:537-39). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Similar procedures are disclosed in International Publication No. WO 93/08829, and in Traunecker et al, 1991, EMBO J. 10:3655-59. Other examples of bispecific antibodies include Bi-specific T-cell engagers (BiTEs) that are a class of artificial bispecific monoclonal antibodies. BiTEs are fusion proteins consisting of two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain of about 55 kilodaltons. One of the scFvs binds to tumor antigen (i.e. HER) and the other generally to the a n effector cell (e.g. a T cell via the CD3 receptor. Other bispecific antibodies those described in WO2006064136. In particular the bispecific antibody is a Fab format described in WO2006064136 comprising one VH or VHH specific for HER and one VH or VHH specific for an effector cell.
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In some embodiments, the HER inhibitor is a small organic molecule. As used herein, the term "small organic molecule" refers to a molecule of size comparable to those organic molecules generally sued in pharmaceuticals. The term excludes biological macromolecules (e.g.; proteins, nucleic acids, etc.); preferred small organic molecules range in size up to 2000da, and most preferably up to about 1000 Da.
In some embodiments, the HER inhibitor is tyrosine kinase inhibitor. A "tyrosine kinase inhibitor" is a molecule which inhibits tyrosine kinase activity of the HER receptor. Examples of such inhibitors include the small organic molecule HER2 tyrosine kinase inhibitor such as TAK165 available from Takeda; CP-724,714, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both HER2 and EGFR- overexpressing cells; GW572016 (available from Glaxo) an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (CI- 1033; Pharmacia); non selective HER inhibitors such as Imatinib mesylate (Gleevec™); MAPK extracellular regulated kinase I inhibitor CI- 1040 (available from Pharmacia); quinazolines, such as PD 153035, 4-(3-chloroanilino) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines; curcumin (diferuloyl methane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner-Lamber); quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG); pan- HER inhibitors such as CI- 1033 (Pfizer); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI- 1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-lCl l (Imclone); or as described in any of the following patent publications: U.S. Pat. No. 5,804,396; WO99/09016 (American Cyanimid); WO98/43960 (American Cyanamid); W097/38983 (Warner Lambert); WO99/06378 (Warner Lambert); WO99/06396 (Warner Lambert); WO96/30347 (Pfizer, Inc); W096/33978 (Zeneca); W096/3397 (Zeneca); and WO96/33980 (Zeneca).
In some embodiments, the HER inhibitor is an EGFR inhibitor. GFR inhibitors are well known in the art (Inhibitors of erbB-1 kinase ;Expert Opinion on Therapeutic Patents Dec 2002, Vol. 12, No. 12, Pages 1903-1907, Susan E Kane. Cancer therapies targeted to the epidermal growth factor receptor and its family members. Expert Opinion on Therapeutic Patents Feb 2006, Vol. 16, No. 2, Pages 147-164. Peter Traxler Tyrosine kinase inhibitors in cancer treatment (Part II). Expert Opinion on Therapeutic Patents Dec 1998, Vol. 8, No. 12, Pages 1599-1625). Examples of such agents include antibodies and small organic molecules that bind to EGFR. Examples of antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab; ERBUTIX®) and reshaped human 225 (H225) (see, WO 96/40210, Imclone Systems Inc.); IMC-1 1F8, a fully human, EGFR-targeted antibody (Imclone); antibodies that bind type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized and chimeric antibodies that bind EGFR as described in U.S. Pat. No. 5,891 ,996; and human antibodies that bind EGFR, such as ABX-EGF (see WO98/50433, Abgenix); EMD 55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab) a humanized EGFR antibody directed against EGFR that competes with both EGF and TGF- alpha for EGFR binding; and mAb 806 or humanized mAb 806 (Johns et al, J. Biol. Chem. 279(29):30375-30384 (2004)). The anti-EGFR antibody may be conjugated with a cytotoxic agent, thus generating an immunoconjugate (see, e.g., EP659,439A2, Merck Patent GmbH). Examples of small organic molecules that bind to EGFR include ZD 1839 or Gefitinib (IRESSA™; Astra Zeneca); CP-358774 or erlotinib (TARCEVA™; Genentech/OSI); and AG1478, AG1571 (SU 5271 ; Sugen); EMD-7200. In some embodiments, the HER inhibitor is a small organic molecule pan-HER inhibitor such as dacomitinib (PF-00299804).
In some embodiments, the HER inhibitor is selected from the group consisting of cetuximab, panitumumab, zalutumumab, nimotuzumab, erlotinib, gefitinib, lapatinib, neratinib, canertinib, vandetanib, afatinib, TAK-285 (dual HER2 and EGFR inhibitor), ARRY334543 (dual HER2 and EGFR inhibitor), Dacomitinib (pan-ErbB inhibitor), OSI-420 (Desmethyl Erlotinib) (EGFR inhibitor), AZD8931 (EGFR, HER2 and HER3 inhibitor), AEE788 (NVP-AEE788) (EGFR, HER2 and VEGFR 1 12 inhibitor), Pelitinib (EKB-569) (pan-ErbB inhibitor), CUDC-101 (EGFR, HER2 and HDAC inhibitor), XL647 (dual HER2 and EGFR inhibitor), BMS-599626 (AC480) (dual HER2 and EGFR inhibitor), PKC412 (EGFR, PKC, cyclic AMP-dependent protein kinase and S6 kinase inhibitor), BIBX1382 (EGFR inhibitor) and AP261 13 (ALK and EGFR inhibitor). The inhibitors cetuximab, panitumumab, zalutumumab, nimotuzumab are monoclonal antibodies, erlotinib, gefitinib, lapatinib, neratinib, canertinib, vandetanib and afatinib are tyrosine kinase inhibitors.
In some embodiments, the HER inhibitor is an inhibitor of expression. An "inhibitor of expression" refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. Therefore, an "inhibitor of HER expression" denotes a natural or synthetic compound that has a biological effect to inhibit the expression of one HER receptor. Typically, the inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. Inhibitors of gene expression for use in the present invention may be based on antisense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the HER receptor mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the HER receptor, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding the HER receptor can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).
Small inhibitory RNAs (siRNAs) can also function as inhibitors of gene expression for use in the present invention. Gene expression can be reduced by contacting the tumor, subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschi, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).
Ribozymes can also function as inhibitors of gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleo lytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleo lytic cleavage of the HER receptor mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential R A target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
Both antisense oligonucleotides and ribozymes useful as inhibitors of gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art. Typical viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in KRIEGLER (A Laboratory Manual," W.H. Freeman CO., New York, 1990) and in MURRY ("Methods in Molecular Biology," vol.7, Humana Press, Inc., Cliffton, N.J., 1991). Typical viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hematopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion. Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g., SANBROOK et al, "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
In some embodiments, the HER inhibitor is administered to the patient in combination with an inhibitor of the neurotensin activation of NTSRl . As used herein , the expression "inhibitors of the neurotensin activation of NTSRl" should be understood broadly, this expression refers to agents down-regulating the expression of neurotensin or of neurotensin receptor 1, compounds that bind to neurotensin (NTS) or NTSRl and inhibit the neurotensin activation of NTSRl, or a protease that can degrade NTS. Examples of inhibitors of the neurotensin activation of NTSRl may be selected from the group consisting of an agent down-regulating the expression of neurotensin or of neurotensin receptor 1, an antibody against neurotensin or a fragment thereof which binds to neurotensin, an antibody against the neurotensin receptor 1 or a fragment thereof which binds to the neurotensin receptor 1, an antagonist of the neurotensin receptor 1, and a protease that can degrade NTS or an inhibitor of NTS or NTSRl expression.
In some embodiments, the HER inhibitor is administered to the patient in combination with metformin (Ν,Ν-Dimethylimidodicarbonimidic diamide). As used the term "therapeutically effective amount" refers to an amount of a drug (e.g.
HER inhibitor) effective to treat cancer in the patient. The effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. The effective amount may extend progression free survival (e.g. as measured by Response Evaluation Criteria for Solid Tumors, RECIST, or CA-125 changes), result in an objective response (including a partial response, PR, or complete respose, CR), improve survival (including overall survival and progression free survival) and/or improve one or more symptoms of cancer (e.g. as assessed by FOSI). Most preferably, the therapeutically effective amount of the drug is effective to improve progression free survival (PFS) and/or overall survival (OS). By "extending survival" is meant increasing overall or progression free survival in a treated patient relative to an untreated patient (i.e. relative to a patient not treated with a HER inhibitor), or relative to a patient who does not express a HER receptor at the designated level. Typically, the therapeutically effective amount is the amount which leads to an objective response, a partial response or a complete response. An "objective response" refers to a measurable response, including complete response (CR) or partial response (PR). By "complete response" or "CR" is intended the disappearance of all signs of cancer in response to treatment. This does not always mean the cancer has been cured. "Partial response" or "PR" refers to a decrease in the size of one or more tumors or lesions, or in the extent of cancer in the body, in response to treatment. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
The compounds of the invention (i.e. HER inhibitor or inhibitor of the neurotensin activation of NTSR1) are administered as a formulation in association with one or more pharmaceutically acceptable excipients to form pharmaceutical composition. As used herein, the term "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Pharmaceutical compositions suitable for the delivery of compounds of the present invention and methods for their preparation will be readily apparent to those skilled in the art.
In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle (i.e. a compound of the invention), alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
In particular, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
Solutions comprising compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. Compounds of the invention can be formulated into a composition in a neutral or salt form as above described.
The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the compounds of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
Compounds of the invention may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.
In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules ; and any other form currently used.
In one embodiment, the antibodies or fragment of antibodies are directed to all or a portion of the extracellular domain of HER. In one embodiment, the antibodies or fragment of antibodies are directed to an extracellular domain of HER. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:
Figure 1: NTS/NTSR1 complex enhanced experimental tumor growth generated in human breast cancer cell lines. A) Experimental tumors were generated from the breast cancer cell line, MCF-7 and the NTS-overexpressing subclones. Comparative growth curves of MCF-7, NTS-h (high NTS expression) and NTS-1 (low NTS expression) cells xenografted in 10, 20, and 25 mice, respectively. Tumor volumes were measured every week. Inset, NTS and NTSR1 transcript analysis from 200ng of MCF-7, NTS-h, and NTS-1 total RNA. B) Proportion of animals with metastases, and metastases distribution within organs and lymph nodes. C) Typical H&E staining performed on paraffin sections of (left) invaded lymph node at 5 OX magnification, 200X magnification (black square) or 400X magnification (white square) ; (middle) lung metastasis at 400X magnification ; (right) kidney metastasis at 400X magnification.
Figure 2: NTS autocrine and paracrine regulation enhanced EGFR, HER2, and HER3 basal expression and activation in human breast cancer cell lines.
A) HER2 and HER3 immuno histochemistry performed on paraffin embedded tumors from mice xenograph with MCF-7, NTS-1 or NTS-h. 200X magnification and computer enlargement of specific areas. B) Breast cancer cells MCF-7, NTS-1 and NTS-h, with the histograms representing intensity-based quantification of Western blot bands of basal total protein, EGFR, HER2, and HER3, using Morpho Expert software (Explora Nova, France). Values are expressed as the percentage of the control MCF-7 cells and are the mean ± SEM of 5 to 7 independent experiments. C) Representative western blot analyses of EGFR, HER2, HER3 and ERK 1/2 total protein from MCF-7 and NTS-1 cells treated with 5xl0"6 M SR 48692. D) EGFR, HER2, and HER3 immuno labeling in MCF-7 and NTS-1 cells treated after 48h of seeding. E) Breast cancer cells MCF-7 and NTS-1, with the histograms representing intensity- based quantification of Western blot bands of phosphorylated protein, EGFR, HER2, and HER3. Values are expressed as the percentage of the control MCF-7 cells and are the mean ± SEM of 5 to 7 independent experiments. F) Representative western blot analyses of Phospho EGFR, phosphoHER2, and Phospho HER3 protein from MCF-7 and NTS-1 cells treated with DMSO or 5xl0"6 M SR 48692 for 48h.
Figure 3: EGF like ligands and metalloprotease released by NTS. A) Amount of Hb-EGF (pg/ml), assayed in 0% FCS culture media of MCF-7, or NTS- 1, cells. Cells were not treated, or treated for 24h with DMSO, 5xl O"6M, SR 48692. Using Paired t test p = 0.0088 between DMSO and SR 48692 NTS-1 treated cells, n=4; with unpaired test p=0.03 between MCF-7 and NTS-1, n=5. B) Amount of NGR2 (pg/ml) assay in 0%FCS culture media of MCF-7 or NTS-1 cells not treated or treated for 48 h with DMSO, 5xlO~6M SR 48692. Using Paired t test p = 0.005 between DMSO and SR 48692 NTS-1 treated cells, n=5; with unpaired test p=0.016 between MCF-7 and NTS-1, n=4. C) MMP9 transcript analysis of total RNA from MCF-7, and NTS-1 treated with DMSO or 5x10"6 M SR 48692 for 48h. D) Amount of MMP9 (pg/ml) assay in 0%FCS culture media of MCF-7 or NTS-1 cells not treated or treated for 48 h with DMSO, 5xlO"6M SR 48692. Using Paired t test p =0.005 between DMSO and SR 48692 NTS-1 treated cells, n=5; with unpaired test p=0.003 between MCF-7 and NTS-1, n=5.
Figure 4 : NTS/NTSR1 expressing tumors response to EGFR/HER2 inhibitors treatment. A). NTS-h cells were inoculated in the right mammary gland of the mice. Here is shown an example of a mouse from each group after 21 days of treatment. B) Tumor growths generated by NTS-h cells treated for 21 days with sesame oil 6% DMSO, or 75 mg/kg Lapatinib, or 200 mg/kg metformin, or both. At day one, 7 mice per group were randomized on tumors size reaching approximately 95 mm3. Figure 5: NTS autocrine and paracrine regulation enhanced oncogenic cellular effects on EGF-induced migration and invasion. A). Number of colonies formed on semisolid medium after 12 days expressed as the percentage of MCF-7 cells. B) Adhesion assays were performed on type I collagen supports. After lh or 48h of seeding, cells were gently washed and the remaining attached cells were quantified by spectrophotometric analysis of crystal violet staining. Results represent the mean optic density ± SEM of 4 experiments. C) Speed of migration on type I collagen of MCF-7 and NTS-1 cells, control or treated with EGF (10 ng/mL), in the presence or not of SR 48692 (5 10"6 M). Results represent the mean ± SEM of 9 to 10 independent experiments. D) Speed of migration on type I collagen of MCF-7 and NTS-1 cells control or treated with EGF (10 ng/mL), in the presence or not of the PKC-inhibitor G66976 (5xlO~8M) or or the PLC-inhibitor U73122 (5x10"6 M) Results represent the mean ± SEM of 4 independent experiments. Results represent the mean ± SEM of 3 to 4 experiments. Student-Newman-Keuls Multiple Comparisons Test was performed on the data : ***P<0.001,**P<0.01, and *P<0.05. E) Synergism between NTS and EGF on invasion in a type I collagen invasion assay. Cells were seeded on the top of a type I collagen gel and treated with EGF (100 ng/mL) in the presence or absence of G66976 (5xlO~8 M). Results represent the mean ± SEM of 3 to 4 experiments. Figure 6: Synergy between NTS and EGF to activate EGFR, HER2, and HER3.
A) Breast cancer cells NTS-1 or MCF-7, with the histograms representing intensity- based quantification of Western blot bands of phosphorylated protein, EGFR, HER2, and HER3 treated for 10 min with lOng/ml EGF. Values are expressed as the percentage of the EGF treated MCF-7 cells and are the mean ± SEM of 5 independent experiments. B) Representative western blot analyses of phosphoEGFR, phosphoHER2, phosphoHER3 and actin from MCF-7 and NTS-1 cells treated or not with lOng/ml EFG for 10 min.
Figure 7: NTS, NTSR1, HER2, and HER3 immunohistochemistry on breast and lung cancer tumors. Two examples of positive labeling scored 2 or 3 for NTS, NTSR1, HER2, HER3 from breast tumor TMA, labeling was performed on consecutive slides. 100X magnification.
Figure 8: NTS autocrine and paracrine regulation enhanced cellular growth in human lung cancer cell lines. A) Influence of NTS exogenous treatment on lung cancer cell growth. LNM-F, R-SI NTS and R-SI NTSR1 were grown in media containing 0 % FCS at low concentration and treated every day with 10"8 M NTS or JMV 449 for 6 days. The ratio of the number of cells at Day 6/Day 0 was calculated. The result is expressed as the % of fold induction. Inset, NTS and NTSR1 transcripts analysis from a total of 200 ng of LNM-35, LNM-R, LNM-F, R-SI NTSR1 and R-SI NTS total RNA. B) LNM-R and LNM-F were seeded alone or at the ratio of 20/80 LNM-R LNM-F and grown in 0.1% FCS for 72h. The results are expressed as the ratio of the number of cells at 72h to TO was calculated, and are the mean ± SEM of 7 independent experiments. C to F) LNM-R and LNM-F were seeded alone or at the ratio of 20/80 LNM-R/LNM-F and grown in 0.1% FCS for 72h, The ratio of the number of cells at 72h to TO was calculated. The results are expressed as the percentage of the growth induction compared to LNM-F. Results are the mean ± SEM of 2 to 5 independent experiments. Cells were exposed to C) DMSO, 10"7 M BIM 46174, 10"6 M SR 48692, rabbit IgG or anti NTS antibody. D) DMSO, 10"7M M475271, 10"5 M AG1478, PBS, or 50 μg/ml Herceptin. E) 5xl0"6 M G66976, 10"6 M U0126, 10"6 M PD98059, or 10"7 M LY294002. F) DMSO, 10-5 M D-NAME, 10-5 M L-NAME, 10-5 M H7 or 5 10-6 M G66976.
Figure 9: NTS regulation enhanced HER2, and HER3 basal expression in human lung cancer cell lines. A) The mixture of cells R/F 20/80 lung cancer cells cultured for 72h, with the histograms representing intensity-based quantification of Western blot bands of basal total protein, EGFR, HER2, and HER3, Values are expressed as the percentage of the control LNM-F cells (which are the population more representative of the mixture) and are the mean ± SEM of 5 to 8 independent experiments. B) An example of western blot gel of LNM-F, LNM-R and the mixture LNM-F, LNM-R (20/80) cultured for 72h no treated or treated with DMSO or 5xlO"6M SR 48692. The blots were revealed with EGFR, HER2 or HER3 antibodies. The actin shown is to the protein control for the HER3 Blot. C) Lung cancer cells R-SI NTS treated or not with 10"7M JMV 449, DMSO or 5xlO"6M SR 48692 for 48h. The histograms represent intensity-based quantification of Western blot bands of basal total protein, EGFR, HER2, and HER3. Values are expressed as the percentage of the non-treated cells (control), and are the mean ± SEM of 3 to 6 independent experiments. . Inset, An example of western blot gel of R-SI NTS cells treated with 10"7M JMV449, DMSO or 5x10" 6M SR 48692 for 48h. Western blot bands of basal total EGFR, HER2, and HER3 protein. D) EGFR, HER2, and HER3 immuno labeling in R-SI NTS cells treated of not with 10"7 M JMV449 for 48 h. E) Example of two restrictive areas from a patient with lung adenocarcinoma with a positives labeling for NTS, NTSR1, HER2, HER3.
Figure 10: NTS regulation enhanced EGFR HER2, and HER3 activation in human lung cancer cell lines. A) Lung cancer cells R-SI NTS treated or not with 10"7M JMV 449, DMSO, 5xlO"6M SR 48692 or 25 10"9M iMMP for 48h. The histograms representing intensity-based quantification of Western blot bands of phosphorylated protein, EGFR, HER2, and HER3. Values are expressed as the percentage of the non-treated cells (control), and are the mean ± SEM of 3 to 4 independent experiments. B) An example of a western blot gel of R-SI NTS cells treated with 10"7M JMV449, DMSO, 5xlO"6M SR 4869 or 25 xlO"9M iMMP for 48h. Western blot bands of phosphorylated EGFR, HER2, and HER3 protein.
Figure 11: NTS autocrine and paracrine regulation activate EGF "like" ligands and MMP1 in lung cancer cell lines. A) Amount of Hb-EGF (pg/ml), assayed in 0% FCS culture media of LNM-R cells not treated, or treated for 48h with DMSO, 5xlO"6M, SR 48692. R-SI NTS cells were treated or not for 24h with 10"8M JMV449. Using Paired t test p = 0.065 between DMSO and SR 48692 in LNM-R treated cells n=3; p=0.0015 between control and JMV 449 R-SI NTS treated cells n=3. B) Amount of NRG 1 (pg/ml) assay in 0%FCS culture media of LNM-R cells not treated or treated for 48h with DMSO, 5xlO"6M SR 48692, or in R-SI NTS cells treated for 48h with 10"8M JMV449. Paired t test p = 0.04 between DMSO treated and SR 48692 LNM-R treated cells n=8; p=0.001 between control and JMV 449 R-SI NTS treated cells, n=7. C) Amount of MMP1 (pg/ml) assay in 0%FCS culture media of LNM-R cells not treated or treated for 48h with DMSO, 5xlO"6M SR 48692, or in R-SI NTS cells treated for 48h with 10"8M JMV449. Using Paired t test p = 0.03 between DMSO and SR 48692 LNM-R treated cells, n=4; p=0.036 between control and JMV 449 R-SI NTS treated cells, n=7.
Figure 12: NTS/NTSR1 complex enhanced experimental tumor growth. A). Tumor growth generated by LNM35, LNM-R and LMN-F cells xenografted into nude mice. One million cells from LNM35, LNM-R, LNM-F, or a mixture of LNM-R and LNM-F (50/50) were subcutaneously injected in 24, 36, 34, or 12 nude mice, respectively. B) Typical immunohistochemistry for NTSR1 (left) or NTS (right) for tumors generated from LNM-R (top) or LNM-F (bottom) cells. Significant differences at *** P < 0.001 or ** P< 0.01 using analysis of variance and Student-Neuman-Keuls test. C) One million R-SI NTS cells were injected into the right mice flanks, and one million R-SI NTSR1 cells were injected into the left mice flanks of the same mouse (n=18). In a second set, one million LNM-R cells were injected only into the right mice flanks (n=36). D) Tumor growth generated by mixture of cells expressing or not NTS One million LNM35 or a 50/50 mixture of R-SI NTS and LNM- R cells or a 50/50 mixture of LNM-R and LNM-F or a 50/50 mixture of R-SI NTS and LNM- F cells were injected in the right mice flanks, 28, 17, 11 and 14 mice were injected, respectively. For A, C and D Tumor volumes were measured every week. The ellipsoid formula (4/3 PI x (L/2xl/2xh/2) was used to calculate the volume. Significant differences are shown at *** P < 0.001 and ** p < 0.01 using analysis of variance and Student-Neuman- Keuls test.
Figure 13 : NTS/NTSR1 expressing tumors are the target for EGFR inhibitors treatment.
A). LNM-R or R-SI NTSR1 cells (LNM-R expressing sh-RNA for NTSR1) were injected into the left and the right flank of the mice, respectively. Here is shown an example of a mouse from each group after 17 days of treatment.
B and C) Tumor growth generated by LNM-R cells (left flank) and R-SI NTSR1 cells (right flank) xenografted into nude mice and treated for 17 days with water, or 25 mg/kg erlotinib, or 200 mg/kg metformin, or both. At day one, 9 mice per group were randomized on LNM-R tumors size reaching approximately 20 mm3.
D) Tumor growth generated by R-SI NTSR1 cells xenografted into nude mice and treated for 24 days with water, or 25 mg/kg erlotinib, or 200 mg/kg metformin, or both. At day one, 10 mice per group were randomized on tumors size reaching approximately at 150 mm3.
Figure 14 Immunohistochemistry of NTSR1, lung cancer tumors. A) NTSR1 Immuno labeling in patients with primary lung adenocarcinomas (right) top X 50, bottom X400 and Squamous Cell carcinomas (left), top X100, bottom X200. B) Overall survival of patients operated for NSCLC lung adenocarcinoma according to NTSR1 score. Semiquantitative immunohistochemistry evaluation of NTSR1 : NTSR1 + + +: strongly positive expression (number of staining cells > 50% and the labeling intensity is high = score 2), other: the remaining patients (score 0 and 1). left Survival curve for the first cohort, Center Survival curve for lung adenocarcinomas from the first cohort, right Survival curve for SCC and LCC from the first cohort. C) Overall survival of patients operated for lung adenocarcinoma according to NTSR1 score. Semiquantitative immunohistochemistry evaluation of NTSR1 : NTSR1 + + +: strongly positive expression (number of staining cells> 50% and the labeling intensity is high = score 2), other: the remaining patients (score 0 and 1).
Figure 6: NTS/NTSR1 complex enhanced experimental tumor growth in HCC cells lines. (A) Experimental tumors were generated from the HCC cancer cell line, HEP 3B and PLC/RF5 and the NTSRl-overexpressing subclones. Comparative growth curves of HEP 3B, HEP-Rla, PLC/RF5, PLC/R1 in 8, 7, 10 and 7 mice, respectively. Tumor volumes were measured twice a week. (B) Tumor weight at 42 days. (C) Typical IHC performed on paraffin sections for NTS, NTSR1 EGFR and phosphorylated EGFR labelling, 200X magnification Figure 7: Neurotensin regulation restores response to erlotinib. (A and B)
Experimental tumors were generated from HEP 3B and HEP-Rla in the same mice. Mice were treated per os with 75 mg/kg erlotinib every day for 22 days. Tumor size was measured every two days. Results shown include the ratio of tumors as compared to day 1. A control group force feed with H20, and three treated groups, force feed every day with 15mg/kg sorafenib, lmg/kg SR 48692, or both. Results shown are the ratio of tumor compare to day 1.
EXAMPLE 1: EGFR, HER2, AND HER3 EXPRESSION REMODELING BY THE NEUROTENSIN/NEUROTENSIN RECEPTOR 1 PATHWAY CONTRIBUTES TO BREAST CANCER PROGRESSION.
Material & Methods
Cell culture procedure: The human breast adenocarcinoma cell line MCF-7 and the corresponding NTS-overexpressing clones, NTS-h (high level of NTS) and NTS-1 (low level of NTS), were grown at 37 °C, in a humidified atmosphere of 5% C02, in DMEM supplemented with 10 % fetal calf serum, 2 mM glutamine and G418 0.5 mg/mL (Invitrogen™).
Tumor xenografts: 3x106 cells (MCF-7, NTS-h, NTS-1) resuspended in Matrigel (BD Biosciences) were then inoculated in the right mammary gland of the mice. Tumor growth was induced by a daily intra-peritoneal injection of 2μg estradiol per mouse. Institutional Review Board approval was obtained by «Le Comite d'Ethique pour l'Experimentation Animale Charles Darwin # Ce5/2010/049 ». For drug treatments, 5x106 of NTS-h resuspended in Matrigel (BD Biosciences) were then metformininoculated in the right mammary gland of the mice. 51 days after injection, 4 groups of 7 mice were randomized as follows : 95.9 ± 14.57 mm3 for control group, 94.5 ± 15.0 mm3 for lapatinib group, 91.5 ± 14.09 mm3 for metformin group and 95.6 ± 12.20 mm3 for lapatinib and metformin group. Mice were treated for 21 days per os, with sesame oil containing 6% DMSO, or 75 mg/kg lapatinib, or 200 mg/kg metformin or both.
Western blots. Samples in Laemmli buffer were loaded on 10 % SDS-PAGE and transferred to PVDF membranes. Membranes were exposed to primary antibody overnight. Total anti-EGFR (1 :500), anti-phospho-EGFR (1 :500), anti-phospho-HER2 (1 :500), anti- HER3 (1 :2000), anti-phospho-HER3 (1 : 1000), anti-ERK 1/2 (1 :2000) were from Cell Signaling Technology®. Total anti-HER2 (1 :2000) were purchased from Neomarkers and anti-Pactin (1 :50000) from Sigma®. Secondary anti-rabbit (Santa Cruz Biotechnology) or anti-mouse (Sigma®) antibodies conjugated to HRP were used at 1 :2000 dilutions for lh at room temperature and visualized by enhanced chemiluminescence (GE Healthcare®).
Adhesion assays. The assay was performed in 96-wells plates coated or not with 50μg/mL of type I collagen (Sigma®) overnight at 4 °C or with 1 : 10 diluted-growth factor reduced Matrigel (BD Biosciences®) for 1 hour at 37 °C. Cells were harvested and suspended in adhesion buffer (DMEM, 15 mM HEPES, 1.2g/L sodium bicarbonate, 0.2% BSA). 5x104 cells per well were seeded and incubated at 37 °C for 1 or 48 hours. Adherent cells fixed with 5 % paraformaldehyde for 45 min, then colored with 0.1 % cristal violet during 30 min at room temperature. Cells were subjected to 30 min lysis in 1 % SDS under agitation. Absorbance was determined at 570.
Anchorage-independent growth assay in soft agar: Colony growth assays were performed by seeding on the top of a 0.6 % low gelling temperature agarose layer, 5x103 cells in 0.5 mL of cultured medium containing 0.3 % agarose solution ± EGF (10 ng/mL). Culture medium and EGF were replenished every 2-3 days. Cells were incubated at 37°C for 12 days and colonies > 50 μιη were counted in the whole well.
Cell migration assays: 12 wells-culture plates were coated with type I collagen or Matrigel as mentioned in adhesion assay. lxlO5 cells were plated for 4 hours in an 8 mm- cloning ring placed in the center of the well to form a confluent circle of monolayer cells. The cylinder was removed and cells grow for 16 hours. Cells were then treated in serum- free medium ± EGF (10 ng/mL) in the presence or absence of various cell signaling inhibitors. Four pictures were acquired per well at the initial time of treatment and 48 hours later for comparison with an inverted microscope at 200X magnification. Migration speed was determined using the Morpho Expert software (Explora Nova) and was expressed as the average cells covered distance divided by the experimental duration (μητ/h). Presented results are expressed in percentage of the non-treated condition.
Collagen invasion assays. Invasion potential was evaluated by a single-cell collagen invasion model, extensively described (1). Briefly, 6-wells plates were coated with 1.25 mL of a 1 mg/mL collagen type I solution, allowed to gel for at least 1 hour at 37 °C in a humidified atmosphere containing 10 % of C02. 2x105 viable single-cells, obtained by mild enzymatic dissociation with trypsin/EDTA solution and filtration, are seeded on the top of the gel in presence or not of EGF (100 ng/mL) and/or G66976 (4xl0 8 M) for 24h. Invasion index (cells with invasive extensions versus total number of cells x 100) was determined by counting the number of invading and non- invading cells present in 10 to 15 random fields of an inverted phase-contrast microscope.
ELISA (Enzyme-linked immunosorbent assay) Cells were seeded at 4X106 cells in 100 mm dish in culture media. The next day media was changed to 4.5 mL of serum free media for 48h. To assay Hb-EGF and NRG-2, the media were concentrated with dialysis tube (7 Spectra / Por ® Dialysis Membrane). In all samples Protease Inhibitor Cocktail P8340 [1 : 100] were added. MMP9, HB-EGF and NRG2 released in the culture media were assayed by ELISA kits (DuoSet®ELISA Development System), and (USCN Life Science Inc.).
RNA extraction and RT-PCR. Total RNA was extracted by the acidic phenol/chloroform guanidine thiocyanate method as documented by (2, 3).
Immunohistochemistry. The slides were incubated at 4°C overnight with primary antibody included anti-NTS (1 :200, SC-20806, Santa Cruz biotechnology®), and anti-ErbB3 (1 :50, NCL-c-erbB-3, Novocastra™), anti-NTSRl (1 : 100; SC-7596, Santa Cruz Biotechnology®) and anti-ErbB2 (1 :400, A0485, Dako) was incubated at room temperature for 1 hour and 30 minutes, respectively. The levels of staining were scored based on staining intensity within the tumor sample, with weak as 1, moderate as 2, strong as 3.
Patients: We studied 499 specimen from patients operated for breast cancer in 2008 in Algeria. The average patient age was 51.1 ± 11.7 years. The sizes of the tumors were 36.3 ± 21.7 mm. The SBR histopro gnostic grading was 6% grade I, 63% grade II and 31% grade III.
Statistics. Statistical analysis was carried out using test student T test or Student- Newman-Keuls Multiple Comparisons Test : ***P<0.001,**P<0.01, and *P<0.05. For contingency analysis Fisher's Exact Test was applied. The Odds ratio was performed using the approximation of Woolf.
Results
The NTS-NTSR1 complex enhances tumor growth and metastasis emergence from breast experimental tumor.
MCF-7 cells, which constitutively express NTSR1, were transfected with the neurotensin full length coding sequence to evaluate the influence of an autocrine neurotensinergic signaling loop on the tumorigenicity of the ER-positive breast cancer. Amongst the selected clones, two showed a differential NTS expression based on transcription levels the NTS high expressing clone, NTS-h, and the NTS low expressing clone, NTS-1 (Figure 1A inset). The NTS expression levels were confirmed by quantitative RT-PCR in both clones, showing a 500-fold and a 4-fold NTS transcript's induction for NTS-h and NTS-1, respectively as compared to the MCF-7 parental cells. Immuno fluorescent staining experiments confirmed this result. In wild type cells, no or an extremely weak NTS labeling was noted. In the NTS-1 cells, NTS immunoreactivity was dispersed in small dots throughout the cytosol. The same pattern was observed in NTS-h cells with larger dots of stronger intensity due to the higher NTS expression level. In parallel, in MCF-7 cells, NTSR1 labeling was localized at the cell membrane, whereas an intense intracellular granular labeling of an endocytosed receptor was seen in NTS-h cell. NTS-1 cells showed a dual pattern of NTSR1 localized both at the membrane and inside the cytoplasm.
To evaluate the influence of the NTS autocrine signaling loop on tumor growth, the MCF-7 parental line and the two NTS-overexpressing clones, NTS-1 and NTS-h, were xenografted in the mammary fat pad of female athymic mice. Tumor growth rose with NTS expression levels (Figure 1A). The group bearing the MCF-7 cells began to develop tumors after 77 days to reach a small size of 108 ± 56 mm3 at day 105. During the same period of time mice bearing NTS-overexpressing cells developed much bigger tumors, with a volume of 483 ± 102 mm3 for NTS-1 at day 105 and 1239 ± 251 mm3 for NTS-h at 91 days. None of the animals xenografted with MCF-7 cells developed metastases during the experiment (Figure IB). On the contrary, metastases were observed in 41 % of the NTS-1 group and 76 % of the NTS-h group. The metastases were preferentially found in the homolateral lymph nodes (24% and 59 % respectively) and the lungs (35% and 47 % respectively). Furthermore, 18 % of the animals in the NTS-h group also showed a metastatic spread in the contralateral lymph nodes reflecting a more advanced metastatic stage. Immunohistochemical slides representing examples of a lymph node, a lung, and a kidney metastatic lesion are shown in figure 1C.
The NTS/NTSRl complex enhances EGFR, HER2, and HER3 expression and activation in breast cancer cells.
In order to identify the mechanisms underlying NTS induced growth and metastasis, we searched for the possible interrelation between NTS/NTSRl complex and epidermal growth factor receptors (HERs). We first observed the amplification of HER2 and HER3 protein levels in tumors obtained after the xenograft of NTS-h and NTS-1 cells, as compared to MCF-7 cells. In these cases, the membranes of the labeled tumoral cells was often thicker and more intense (Figure 2 A).
Analysis of NTS-1, NTS-h, and MCF-7 cellular protein content, showed an increase of 150 - 275 % of EGFR, HER2, and HER3 (Figures 2B and C). This effect was confirmed by the stronger labeling of all three HER receptors by immuno cytochemistry in NTS-1 cells as compared to MCF-7 cells (Figure 2D). In NTS-1 cells, EGFR is accumulated in clusters close to the nucleus, while in MCF-7, EGFR is at the membrane, HER2 membrane labeling, and HER3 cytosol labeling are more intense in NTS-1 than in MCF-7 cells (Figure 2D see white arrow). The specific NTSR1 antagonist, SR 48692, reduced HERs increases in NTS expressing cells, but not in wild type cells (Figures 2C and 2S A), validating the contribution of NTS/NTSRl complex in this HERs overexpression.
In parallel, an increase in the activation states for all three receptors was observed in NTS-1 cells. The phosphorylation levels were enhanced up to 400 and 500% for EGFR and HER3, respectively (Figures 2E and F). Similar observations were made with NTS-h cells and a 200% increase was seen for the three receptors (not shown).
Subsequently, we searched for a possible EGFR and HER3 autocrine activation by the release of EGF "like" ligands from the cell membrane following a proteolysis process mediated by NTS. In the culture media from NTS-1 cells, the amount of Hb-EGF was twofold higher than MCF-7 cells. The presence of SR 48692 in the culture media abolished this increase (Figure 3 A). In the same vein, a stronger release of neuregulin 2 could be detected in the culture media of NTS-1 cells compared to MCF-7 cells, and again counteracted by SR 48692 (Figure 3 B). The release of these specific ligands for EGFR and HER3 suggests an activation of metalloproteases due to NTS exposure. MMP9 transcripts were found increased (Figure 3 C) in NTS-1 cells as compared to MCF-7, as was the case for MDA-MB 231 cells (4). In addition, MMP9 anchored at the membrane was activated, as a 180% increase of MMP9 release in culture media was detected in NTS-1 as compared to MCF-7 cells and abolished by SR 48692 (Figure 3 D).
We confirmed the contribution of EGFR and HER2 activation in NTS induced tumor growth by treating NTS-h xenografted mice with lapatinib. Lapatinib inhibits the tyrosine kinase activity of both HER2 and EGFR. This property is currently used in breast cancer treatment. Figures 4 A and B show that the tumor growth rate is reduced when mice are treated with lapatinib. The final tumor volume after 21 days of treatment was 281.5± 71.4 mm3 for the control, and 142.1 ± 39.56 mm3 (P= 0.014 vs control) for lapatinib. We also tested metformin on our model. Metformin is suspected to provide anticancer effects in breast cancer and several clinical trials are currently under way (5). In parallel, metformin was shown to disrupt the crosstalk between insulin receptor and NTS receptor in pancreatic cancer cells (6). In addition to inhibiting the mTOR pathway, metformin prevents ER activation induced by NTS and insulin (7). In a breast cancer cellular model with NTS overexpression, metformin reduced the tumor growth with the same efficiency as lapatinib. No additional effects were detected when both drugs were employed. The final volumes were 160.9 ± 39.84 mm3 and 150.9 ± 46.05 mm3 when animals were treated with metformin or both drugs, respectively (Fig 4 A and B). The tumor doubling time was in agreement with tumor volume with 13.0 ± 1.28, for controls. For lapatinib, metformin, and metformin + lapatinib double time cannot be calculated for because within the group 2 to 3 tumors shrank, the others grow very slowly The absence of additional response suggested a common signaling cascade was targeted by both drugs.
The NTS/NTSR1 complex enhances pro-metastatic cellular effects.
We evaluated the oncogenic cellular effects impacted by NTS. The basal growth capacity of MCF-7 in an anchorage-independent context was doubled in the presence of NTS. In MCF-7 (Figure 4 A), the colonies were organized in a spherical conformation around an inside lumen delimited by a single cell monolayer in wild type cells whereas, NTS-h or NTS-1 the colonies were larger and the cells forming compact spheroids, filled up with cells.
Cell detachment is a pre-requisite in metastatic spreading. For this purpose, we studied the adhesion ability of our cellular models on various matrices. Cells were initially detached from their support and the kinetics of reattachment was evaluated. After 1 hour of seeding, the NTS-h and NTS-1 cells displayed only 68 % and 55 % respectively, of the adhesion capacity on type 1 collagen (Figures 4 B), as compared to MCF-7. Interestingly, this decline in cellular adhesion ability stabilizes over time. At 48 hours of post seeding, the percentage of adherent cells is similar to those after 1 hour (Figure 4 B). Similar observation was made when cells were seeded on matrigel. This suggests that NTS alters the basal cell adhesion capacities of the tumor cells.
We performed migration assays on type I collagen, because migration requires cell interactions with the extracellular matrix. We developed an experimental procedure, in order to estimate the migration speed of the cells. As described in detail in the methods section, this procedure allows measuring the average distance covered by the cells within 48h, while preserving the matrix coating on the dishes. NTS expression significantly affects the basal migration speed of the NTS-1 cells on both collagen and matrigel coated dishes. The migration speed on collagen was 2.50 ± 0.07 μητ/h and 3.10 ± 0.09 μητ/h (p < 0.0001; n=10) and on matrigel was 3.99 ± 0.21 μηι/h and 5.31 ± 0.24 μητ/h (p = 0.003; n=12) for MCF-7 and NTS-1, respectively. As EGFR expression was enhanced under NTS autocrine regulation, we inquired on the effect of EGF on cell speed migration. When cells were treated with EGF, a clear synergic effect between NTS and EGF was observed. The NTS-1 cells migrate 38 % (Figure 4 C) and 42 % faster when treated with EGF on collagen and matrigel respectively, compared to EGF-treated MCF-7 cells. In order to confirm the contribution of the NTS-NTSR1 complex in this synergic effect, MCF-7 and NTS-1 were treated with EGF and the NTSR1 specific antagonist SR 48692. As shown in figure 4 C, this treatment had no effect on the EGF-induced migration of MCF-7 on collagen, whereas, SR 48692 inhibited the acceleration of the migration speed in the EGF-treated NTS-1 cells. Specific PKC and PLC inhibitors were applied and clearly abolished the synergic effect of NTS and EGF observed in NTS-1 cells. In the parental cells, EGF's contribution to the migratory effect was insensitive to these inhibitors although the basal migration speed did increase in matrigel matrix (Figures 4 D). As control, a specific inhibitor of EGFR, the AG1478, completely abolished the EGF-induced migration in both MCF-7 and NTS-overexpressing cells (Figure 4 D). Similar inhibition were seen when cells were seeded on matrigel
The invasiveness properties of NTS-overexpressing cells, was studied using a 3 dimensional collagen invasion assay. Results are expressed as the invasion index corresponding to the number of invading cells related to the number of total seeded cells. The introduction of NTS expression into MCF-7 cells induced a small increase in invasiveness properties (Figure 4 E). However, EGF-induced invasion doubled in NTS-overexpressing cells (20 % invasion index) as compared to MCF-7 (10 % invasion index). The induction of invasiveness was inhibited by PKC inhibitors only in NTS-overexpressing clones, suggesting dependence of this effect on GPCR activation.
The synergic effects of NTS and EGF on cellular migration and invasion suggest that a new pattern of HER heterodimers, with a higher EGF response potency, is generated in the cells. When cells were stimulated with EGF, the amount of phosphorylated proteins EGFR, HER2, and HER3 in the NTS-overexpressing cells is higher than the phosphorylated proteins in wild type cells, with an increase of 290, 190, and 275 %, respectively as compared to MCF-7 EGF treated cells (Figure 5 A and B).
Overexpression of NTS and NTSR1 correlates with HER2 and HER3 overexpression in breast human tumors.
Co expression of NTS, NTSR1, HER2 and HER3 was analyzed in breast cancer tissue microarrays (TMA). Seven TMAs containing 269 samples each, was scored from 0 to 3 according to the labeling intensity and the proportion of stained cells. Correlations between the expression of NTS/NTSRl complex and the expression of HER2 and/or HER3 were evaluated. We considered that a score of 2 and 3 indicate the overexpressed condition, and consequently we performed contingent's analysis on these combined scores. Results are summarized in table 1.
Table 1 : Contingent analysis for NTS, NTSR1, HER2 and HER3 expression on samples from breast cancer tissue microarray
NTS NTSR1 HER2 HER3 n total 1408 1347 1268 1410
Positive n (%) 1029 (73) 375 (28) 361 (28) 753 (53)
NTS, n (%) 301 (23) P<0.0001 274 (22) 599 (43) P= 0.006
NTSRl, n (%) 102 (8) 244 (19) P<0.0001 HER2, n (%) 218 (18)
NTS & NTSRl, n (%) 85 (7) 204 (16) P<0.0001
NTS & NTSR1 & HER2, n (%) 62 (5) p<0.0001 n total= number of sample readable; Positive = number of sample scored 2 or 3; P = Fisher's Exact Test
NTS and NTSR1 were found in 23 % of the samples, in agreement with our previous studies (8), whereas HER2 and HER3 were found in 28 % and 53 % of the samples, respectively. Amongst this NTS-NTSR1 positive population a higher proportion overexpressed HER3 (16%) than HER2 (7%). HER3 expression was positively associated with those for NTS and NTSR1 (OR= 19.073, 95% Confidence Interval [13.633-26.684], p<0.0001). In contrast, no significant relation between HER2 and NTS/NTSR1 was found. Only 5% of the samples overexpressed the four markers, nevertheless HER2 and HER3 expressions remained strongly significantly associated to NTS/NTR1 expression (OR = 12.117 95% Confidence Interval: to [7.121-20.618], p<0.0001). It should be noted that an absence of correlation between high expression of HER2 and HER3 was detected. Two examples of the same tumor sample labeled for the four markers are shown in figure 6. Discussion:
The organization of tumor cell signaling networks evolves with accumulation of genetic and epigenetic alterations in connection with the local stroma and immune system. Identifying factors which contribute and predict tumor aggressiveness are means to understand how the progression of the disease evolves across individuals. Unfortunately, the biological and clinical validation of these factors is difficult because tumors are often heterogeneous and their molecular characteristics change over time. In addition to the clinical parameters, genetic alterations on key genes provide additional information on disease outcome. These genetic alterations can be modulated by regulatory mechanism which may cause changes to the amplitude of cell aggressiveness within the tumor. While genetic alterations acquired by tumors are irreversible, their regulatory process can be deprogrammed to restore a tumor phenotype to a less aggressive and more differentiated form. In this article we show that the NTS/NTSR1 complex enhances tumor aggressiveness (tumor growth and metastasis emergence) by enhancing HERs expression, and their activation by the release of specific EGFR and HER3 ligands. This concept can be extended to other tumors, as we observed similar results in lung cancer cells and subsequent experimental tumors (9). In breast cancer cells, NTSR1 activation alters many cellular effects having oncogenic characteristics including proliferation, survival, adherence, migration and invasion, with a synergic effect between NTS and EGF on cellular migration and invasiveness. This latter point may explain the exacerbation of the metastasis process seen due to NTSR1 activation. Synergy between NTS and EGF was previously described on DNA synthesis in primary adult rats' hepatocytes (10), and the regulation between these two factors appears to be independent of oncogenic characteristics of the cells. EGF was detected in normal and tumoral breast tissues, with a higher expression found in normal tissues adjacent to tumor (11). We previously showed that NTS is expressed in normal breast epithelial cells. Its expression is regulated by estrogen (8). The synergy between NTS and EGF may therefore occur during the breast carcinogenesis.
Our data showed that under NTS stimulation, EGFR, HER2 and HER3 are up- regulated and intrinsically activated. This over expression was not associated with gene transcriptional or post-transcriptional activity, suggesting that NTS induced a new equilibrium in HERs cellular traffic and a new pattern of HERs dimers formed after stimulation. HERs internalization pathways depend on the expression of ligands and/or their receptors (12, 13). Under physiological conditions (low concentration of ligands and moderate EGFR expression <200 000/cell), EGFR internalization is dependent on clathrin coated pits, with a half- life of 6-10 hours. However, under conditions of receptor overexpression or high concentration of ligands, the clathrin pathway becomes saturated, and the complex (ligand-receptor) is internalized with a slow kinetics in a clathrin independent pathway. Under these conditions, the half-life can reach 24 hours (14, 15).
In cells overexpressing HER2, receptors are mainly localized at the cell membrane, indicating that overexpression of HER2 does not lead to accelerating its endocytosis (16). The HER2 heterodimerization with EGFR influences the endocytosis pathway for both receptors. Treatment by EGF of cells expressing low HER2, resulted in HER2 down-regulation (17). In contrast, EGFR activation in cells overexpressing HER2, does not affect the membrane expression of HER2 or its degradation (16, 18). In addition, overexpression of HER2 had a dominant-negative effect on the down regulation of stimulated EGFR, and stabilized both receptors by altering autophosphorylation patterns (16, 19). HER2 overexpression may also prevent EGFR internalization by clathrin-dependent endocytosis, and it's re-directed from the degradation to the recycling pathway (18, 20, 21).
The activation of HER3 leads to its internalization and its down regulation. However, HER3 internalization is slower than EGFR internalization (22). In addition, HER3 is inefficiently sorted to the degradation pathway, apparently due to the lack of the C-terminal domain, which contains patterns used for targeting to lysosomes (23 , 24) . It has also been suggested that neuregulins do not guide HER3 to degradation due to the early dissociation of complexes (ligand-receptor) in endosomes (23 ) . In these situations the stabilization of HERs cells are independent of gene transcription, activation and amplification, as in the case for Neurotensin.
Associated with HERs regulation, we also observed an increase of NRG2 and Hb- EGF secretion by NTS mediated by MMP activation. The release of these growth factors caused the concomitant activation of HER3 and EGFR. EGF "like" ligands are largely implicated in breast cancer progression, yet, most reports have only studied the expression of ligands specific to EGFR, or those specific to HER3. For example, in one case a 10 fold increase of Hb-EGF in cancer tissues was reported, and in another a high proportion of the four neuregulins and major isoforms were expressed in the cytoplasm of DCIS and IDC of the breast (11, 25, 26). In both cases, deleterious effects of growth factors are often in HER2 overexpressing cells, indicating that ERFG/HER2 and/or HER3/HER2 dimers are related to biological aggressiveness (1 1). Globally, the overexpression of both HER2 and HER3 participates in the stabilization of the HERs dimer, and subsequently the sustained activation of the HERs, and the survival pathway. By enhancing the overexpression and activation of EGFR, HER2 and HER3, the NS/NTSR1 complex becomes an upstream factor that modulates this regulatory mechanism.
In agreement with in vitro studies, tumor growth induced by NTS/NTSR1 can be restrained by a specific tyrosine kinase inhibitor EGFR and HER2, lapatinib. The cascade of biological events from the interaction between NTS and NTSR1 to the activation of HERs receptor appears to be a major contributor of rapid cancer cell growth.
This cascade is inhibited by metformin, known to interfere with activated mTOR and ER pathway (7, 27). The lack of additional effects from the combination of the two drugs suggests that consecutive cellular events lead to increase tumor aggressiveness by NTS. We previously showed that sustained NTSR1 activation generated a permanent PKC dependent activation of ERK signaling (28). It is therefore coherent that metformin and other ERK or PKC inhibitors counteract the same NTS oncogenic cellular effect. PKC appears to be a central signaling hub to generate cell aggressiveness by NTS/NTSR1 through the sustained overexpression and activation of HERs. Experimental tumors report the tumorigenic performance of single cell lines or clones. Nevertheless, heterogeneity is an important characteristic of human tumors. For instance, we found that in 35 % of patient with IDC, NTSRl was expressed in at least 80 % of tumoral cells, and only 20 % of patients express high level of NTSRl and NTS (8). In human tumors, NTS and NTSRl are expressed in cells clusters with diverse sizes. Tumoral cells with potential aggressiveness characteristics could be detected with NTS/NTSR1 labeling, and specific treatment could be proposed accordingly. Lapatinib is proposed in a second line of treatments, in combination with other drug in advanced or metastatic breast cancers whose tumors overexpress HER2. Our results suggest that a more restrained (or targeted) population, can be determined by taking into account NTS and NTSRl co expression. The resulting subpopulation will provide a significantly better performance for this drug.
Conclusion:
The activation of the neurotensinergic system in breast tumors induces the overexpression of the EGFR, HER2 and HER3 receptors and their concomitant autocrine activation. The presence of this regulatory mechanism would have a significant impact on cancer progression in tumor cells by accelerating the process of metastasis. It may also modulate the response to HER2 or EGFR immunotherapy, as incomplete blockades and an increase in PI3k/AKT signaling are proposed to be the mechanisms involved in drug resistances (29).
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EXAMPLE 2: EGFR, HER2 AND HER3 ACTIVATION, CONTROLLED BY
NEUROTENSIN, ENHANCES LUNG TUMOR PROGRESSION.
Material & methods: Cell culture procedures: The LNM35 cell line was sub-cloned by limiting dilution, after few days of culture, clones containing exclusively flat or rounded cells were saved and were named LNM-F for Flat, LNM-R for Rounded. All cells were grown at 37 °C, in a humidified atmosphere of 5% C02.
Cell proliferation assays: 20 000 cells/well of lung cancer cells were seeded in 24- well culture plates. Medium was replaced by FCS-free medium in presence or absence of NTS or JMV449 10"8M. Cells were counted after 5 days of treatment with a particle count and size analyzer (Zl Coulter Particle Counter, Beckman Coulter). For LNM-F/LNM-R (20/80 %) cell mixture: Cells were seeded in 48-well culture plates at a concentration of 40 000 cells/well, media containing 10% FCS. Media is changed 24h after for a media containing 0.1% FCS cells are counted after 48 hours.
Western blots: 2xl06 cells were grown for 72h then serum-starved for 48h in a phenol red- free medium in presence or absence of different concentrations of 510"6 M SR 48692 and 25 10"9M MMP inhibitor (Calbiochem), and lysed (20 mM Tris pH 8.0, 150 mM NaCl, 5 mM MgC12, 0,5 % NP40, 0,5 % glycerol, 1 mM PMSF, protease and phosphatase inhibitor cocktail) at 4°C for 30 min. Primary antibodies were incubated overnight at 4°C. Total anti- EGFR (1 :500), anti-phospho-EGFR (1 :500), anti-phospho-HER2 (1 :500), anti-HER3 (1 :2000), anti-phospho-HER3 (1 :1000) were from Cell Signaling Technology. Total anti- HER2 (1 :2000) was purchased from Neomarkers and anti-Pactin (1 :50000) from Sigma. Secondary anti-rabbit (Santa Cruz Biotechnology) or anti-mouse (Sigma) antibodies, conjugated to HRP, were used at 1 :2000 dilutions for lh at room temperature and visualised by enhanced chemiluminescence (GE Healthcare). Tumor xenografts: Lung cancer cells, xenografts were initiated in nude mice by subcutaneous injection of 106 cells of LNM35, LNM-F, or LNM-R, and derivative cell clones. For tumors generated from a cell mixture, 106 cells from each clone were plated together 72 hours prior to injection. Four to six series were performed; each series included 5- 8 mice. All the procedure were in accordance with the "Guide of the Care and Use of laboratory Animals".
Tumor xenografts : Lung cancer cells, xenografts were initiated in nude mice by subcutaneous injection of 106 cells of LNM35, LNM-F, or LNM-R, and derivative cell clones. For tumors generated from a cell mixture, 106 cells from each clone were plated together 72 hours prior to injection. Four to six series were performed, each series included 5- 8 mice. All procedures were in accordance with the "Guide of the Care and Use of laboratory Animals".
For drug treatments, a first experiment was performed accordingly, 106 of LNM-R or R-SI NTSRl cells (LNM-R expressing sh-RNA for NTSRl) were injected into nude mice by subcutaneous injection, R-SI NTSRl cells in the right flank and LNM-R cells in the left flank. R-SI NTSRl cells were injected 10 days before LNM-R cells. 22 days after R-SI NTSRl cells injection 2 groups of 5 mice were randomized on the size of R-SI NTSRl as follows : 229 ± 40 mm3 for control group and 242 ±37 mm3 for erlotinib group. Mice were treated for 20 days per os. A second experiment was performed as follows: 106 R-SI NTSRl or LNM-R cells were injected at the same time in the right flank or the left flank of the mice, respectively. 7 days after injection 4 groups of 9 mice were randomized on the size of LNM-R as follows: 19.79 ± 3.00 mm3for control group, 18.66 ± 2.21 mm3 for erlotinib group, 16.82 ± 3.32 mm3 for metformin group and 18.82 ± 3.00 mm3 for metformin and erlotinib group. Mice were treated for 17 days per os, with water, or 25 mg/kg erlotinib, or 200 mg/kg metformin or both. A third experiment was performed 15 days after injection of 106 R-si NTSRl cells 4 groups of 10 mice were randomized as follow : 161.37 ± 29.13 mm3 for control group, 129.19 ± 20.89 mm3 for erlotinib group, 152.76 ± 27.86 mm3 for metformin group and 145.30 ± 23.4 mm3 for metformin and erlotinib group. Mice were treated for 17 days as described above. Patients and tissue specimens for NTSR1 immunohistochemistry: A two-step procedure was followed. Firstly, we studied a population of consecutive patients operated on for NSCLC (all histotypes, including adenocarcinoma) in the Thoracic Surgery Dpt of the Hotel-Dieu Hospital, Paris, France between June 15, 2001 and June 14, 2002. Secondly, on the basis of initial results, only adenocarcinoma patients operated on between June 15, 2001 and December 31, 2005 were analyzed. Patient characteristics, treatment procedures, and short-term and long-term outcomes were retrospectively collected using a standardized case report form. Furthermore, a centralized pathological blind revision of the samples was performed by two expert pathologists (D.D., A.L.). In this revision, histologic subtype was determined on the basis of the new International Association for the Study of Lung Cancer/ American Thoracic Society/European Respiratory Society classification.
Adjuvant radiotherapy or chemotherapy was performed under the care of referring physicians, so no uniform protocol was employed. Long-term outcome was assessed by direct telephone interviews with patient or family (in case of deceased patients). When no clinical follow-up was available, information on vital status was obtained through the municipality of birth of the patient. Informed consent was obtained from all patients. The research was conducted according to recommendations outlined in the Helsinki declaration. Institutional Review Board approval was obtained (CPP He de France II, 2012). Immunohistochemistry: Procedure is detailed in SI. For all cases histologic slides of primary tumors were obtained from paraffin wax embedded tissues. Standard H&E staining was used to ensure the tumoral character of the specimen. Deparaffinized tissue sections (4 μιη) were incubated at 4°C overnight with primary antibody included anti-NTS (1 :200, SC- 20806, Santa Cruz biotechnology®), anti-NTSRl (1 :100; C-20, Santa Cruz Biotechnology®) and anti-ErbB3 (1 :50, NCL-c-erbB-3, Novocastra™), and anti-ErbB2 (1 :400, A0485, Dako) was incubated at room temperature for 30 minutes.
For prognosis evaluation, all specimens were scored by an anatomopathologist with special interest in pulmonary pathology (DD). NTSR1 staining of cancer cells was scored as positive in the presence of staining cells > 10 %. Semi-quantitative evaluation was also performed: 0: no staining; 2: more than 50% of tumor cell showing a positive stain of high intensity; 1 : intermediate cases.
Statistical analysis: Statistical analysis was carried out using test student T test or Student-Newman-Keuls Multiple Comparisons Test : ***P<0.001,**P<0.01, and *P<0.05. For human studies, data processing and analysis were performed with the statistical software system SEM (SILEX Development, Mireffleurs, France). Correlations were carried out by the Spearman rank correlation or H-test, as appropriate. Survival analysis was carried out by the Kaplan-Meier method, and univariate comparisons of curves were performed using log rank tests. Risk factors associated with outcomes in univariate analysis with a p value <0.05 were entered into a multivariate Cox model analysis, to identify independent predictors of survival. A p value of less than 0.05 was considered significant.
Results:
The NTS/NTSR1 complex enhances cellular growth
In previous studies, we showed that both NTS and NSTR1 are concomitantly expressed in human lung tumors. NTS actions, possibly occurring in tumor, are therefore mediated through autocrine and/or paracrine regulation (1). In order to evaluate the contribution of NTS in the context of autocrine and/or paracrine regulation, we studied cellular subpopulations from the highly metastatic lung carcinoma cell line, LNM-35 (2). LNM-R cells (expressing NTS and NTSR1) and LNM-F cells (expressing mainly NTSR1) were isolated from the parental LNM 35 cells and the observed pheno types remained with cultured passages (Figure 8 A inset).
We confirmed the differential expression of NTS in the two subclones by radioimmunoassay. The LNM-R culture media contained large amounts of NTS, which accumulated with time (75 to 625 fmol/ml), whereas the media of LNM-F cells contained 20 fold less NTS.
We first evaluated the contribution of NTS/NTSR1 complex on cellular growth on the LMN-R cells silenced for NTS or NTSR1. The clones were named R-SI NTS and R-SI NTSR1, respectively (1) (Figure 8A inset). Exogenous chronic treatment (48h) of R-SI NTS cells with NTS or a low degradable NTS agonist, JMV 449, induced a two fold increase in the cellular growth (Figure 8 A). In contrast, R-SI NTSR1 cells are not responsive, as expected, since the NTSR1 was silenced.
To analyze the autocrine/paracrine cooperativity of the NTS/NTSR1 complex, we created an in vitro model, by mixing LNM-F and LNM-R cell subpopulations. Cells were seeded at sub-confluency with a ratio of 20% of LNM-R and 80% of LNM-F, (R/F 20/80), and counted after 72h of culture. This proportion of the cell subpopulation was chosen because it is similar to the proportion of LNM-R and LNM-F cells in the parental cell line, LNM-35. We observed an increase of 60% in the number of cells of the mix R/F 20/80 compared to LNM-F or LNM-R culture alone (Figure 8B). Fluorescence activated cell sorting showed a higher proportion of cells in S phase and a smaller proportion in Gl phase, as compared to LNM-F cells cultured alone (not shown). To confirm the implication of NTSR1 in the observed growth induction in R/F 20/80, cells were exposed to BIM 46174 (3), an inhibitor of heterotrimeric G proteins, SR 48692 (4), a specific NTSR1 antagonist, and NTS neutralizing antibody. These compounds abolished the increase of tumor growth observed in the cell mixture R/F 20/80 (Figure 8C). A contribution of epidermal growth factor receptors (HERs) to induce NTS cellular growth was suggested by the abolishing effect of M475271, a Src kinase inhibitor, AG 1478, a specific inhibitor of EGFR, and herceptin (trastuzumab), an antibody specific to HER2, which abrogates the growth enhancement effect (Figure 8D). Chemical inhibitors confirmed the contribution of NTSR1 and HERs downstream pathways. Cellular growth amplification was abolished by a PKC inhibitor, Go 6976, (Figure 8E), whereas the NO inhibitor, L-NAME, and the PKA inhibitors, H7, had no effect (Figure 8F). The effect was also abolished by MEK Inhibitors, U0126 and PD98059, and the phosphoinositide 3-kinases inhibitor, the LY294002 (Figure 8E).
The NTS/NTSR1 complex enhances EGFR, HER2 and HER3 expression and activation.
The previous results highlighted a specific effect of NTS in oncogenic processes occurring through an interrelation between NTSR1 and receptor tyrosine kinase systems. We therefore measured the HERs cellular protein content in the mixture of R/F 20/80 cells cultured as previously described. An increase of HER2 and HER3 protein levels, and to a minor extent, EGFR protein levels was observed (Figure 9A). This effect was abolished by SR 48692 as shown on gel figure 9B. Surprisingly, similar mRNA levels were seen for the three receptors in LNM-R/LNM-F 20/80, LNM-R and LNM-F cultured alone. The accumulation of the HERs protein without transcriptional regulation suggests the recycling and degradation of these receptors is altered by NTS/NTSR1 interaction. This is in line with our previous findings showing that sustained NTSR1 activation installed a state of permanent recycling of NTSR1, instead of agonist induced lysosomal degradation (5).
Western blot analysis of R-SI NTS cells exposed for 48h to exogenous NTS agonist also showed a marked increase of HER2 and HER3 protein content. These increases were totally abolished by SR 48692 treatment (Figure 9C and inset). No obvious changes were observed, by immunocytochemistry, in EGFR labeling in R-SI NTS cells, treated or not with JMV 449. In contrast, HER2 and HER3 staining were more intense at the membrane and in the cytosol of cell exposed to NTS agonist (Figure 9D). In type of both experiments, continued exposition to NTS in cells expressing NTSR1 induced the remodeling of HER2 and HER3 expression associated with more aggressive phenotype.
To explore if these mechanisms occur in human tumoral cells, we searched the consecutive slides from 27 specimens with lung adenocarcinoma for clusters of cells concomitantly labeled for NTS, NSTR1, HER2 and HER3. Concomitant expression was observed in restrictive areas of 19 specimens, and examples are shown in figure 9E. However, in 8 other specimens' concomitant overexpression could not be observed. These observations suggest the up regulation of HER2 and HER3 by NTS in specific lung tumoral cells.
NTS induced EGFR, HER2 and HER3 activation mediated by MMPs activation and EFG like ligand release
In parallel, we observed a sustained activation states for all three receptors. R-SI NTS treated by JMV449 for 48h, showed an increase of 250% for the three receptors. This enhancement was completely abolished with treatment by SR 48692 and a metalloproteinase inhibitor, iMMP (Figure 10A and B). Metalloproteinases are known, though proteolysis process, to establish HERs autocrine activation with the shedding or activation of EGF "like" ligand at the cell membrane. We searched for an activation of EGF "like" ligands by NTS. We found a major increase in Hb-EGF levels in R-SI NTS cells media treated with NTS agonist for 24h, and a decrease of Hb-EGF cellular production when LNM-R were exposed to SR 48692 (Figure 11 A). In this cell, EGFR autocrine regulation would be enhanced by the release of HB-EGF under the influence of NTS. Similarly, neuregulin 1 (NRGl) was found more intensively released when R-SI NTS cells were treated with NTS agonist. In LNM-R cells, the NTSR1 antagonist, SR48692 reduced the amount NRGl, a specific ligand for HER3, released in the culture media. Increased amounts of activated NRGl sustained the hypothesis of HER3 autocrine regulation established under NTS exposure (figure 11B). In parallel, MMPl was found to be released in the media of R-SI NTS cells treated with NTS agonist (Figure 11C). In cells bearing NTS autocrine regulation, MMPl released was also decreased in presence of SR 48692 (Figure 11C). Several matrix metalloproteases are regulated in NSCLC including MMPl, which is up regulated in both adenocarcinomas and squamous cell lung cancer (6)
NTSR1 activation in experimental tumors. In order to apprehend the contribution of NTSRl in lung tumorigenesis, we developed experimental tumors bearing NTS autocrine, and/or paracrine, or endocrine regulation. We established the growth rate of LNM35 tumor xenografts in the nude mice, in comparison with the two derived sub-clones, LNM-R (NTS+) and LNM-F (NTS-). As shown in figure. 12A, LNM35 xenografts displayed the more drastic tumorigenesis profile with a final tumor volume of 4122 mm3. The sub-clones LNM-R, and LNM-F generated smaller tumors with a final volume of 2582 and 1858 mm3, respectively. The tumor size is 38 and 55% smaller than LNM35 when generated by LNM-R and LMN-F, respectively. The difference in the tumor growth rates between the parental cells and the two subclones suggested a positive cooperativity between these two cellular populations. To confirm this hypothesis, we mixed the two subclones at the same density before injecting into the mice. The same rate of tumor growth was then observed by injecting LNM35 cells (4122 mm3) or the LNM-F and LNM-R mixture (3782 mm3), as shown in figure 12A. NTS and NTSRl immunohistochemistry was performed on tumors. The presence of NTSRl was seen in both LNM-R and LNM-F tumors (Figure 12B), but with a granular and irregular intensity of labeling. In order to best visualize NTS, we used an antibody, which detected the presence of NTS precursor in LMN-R and its absence in LNM-F tumors (Figure 12B).
We explored the effects of NTS systemic regulation on the tumor growth enhancement, R-SI NTS cells were injected subcutaneously into the right flank and R-SI NTSRl cells in the left flank of the mice. Figure 5C shows that the R-SI NTS tumor xenografts reach the size of the tumors initiated by the corresponding LMN-R parental cells, whereas R-SI NTSRl tumors remain at the same smaller size that was observed in mice bearing only R-SI NTSRl xenografts, suggesting that the circulating NTS produced by the R- SI NTSRl tumor enhanced the tumor growth of R-SI NTS xenografted into the other flank.
We evaluated the relative tumorigenic potential of the NTS autocrine and/or paracrine regulation. R-SI-NTS and LNM-R cells were mixed to generate xenografts bearing autocrine and paracrine NTS regulation as expressed in the original parental cells LNM35 and in the mixture LNM-R and LNM-F. Alternatively, we mixed cell lines not expressing NTS (R-SI NTS and LNM-F cells). When R-SI NTS and LNM-R cells mixture was injected in mice, the size of the tumor generated by this heterologous cell population (4122 mm3) was similar to tumors generated by the parental LNM35 cells (3885 mm3), and the mixture of LNM-R and LNM-F (3782 mm3) (Figure 5D) demonstrating that in all cases, the NTS autocrine regulation participates with paracrine regulation to strongly enhance tumor progression. In contrast, when a mixture of the R-SI NTS and LNM-F cells, not expressing NTS, was xenografted in nude mice, the tumor volume was globally 40% smaller than the xenografts bearing NTS autocrine and paracrine regulation (Figure 12D). When the cells do not release NTS, there is no cooperation between cells, and tumor growth is slower. The overall conclusion of this series of experiments suggested that NTS participates in enhancing tumor growth via autocrine, paracrine and systemic pathways.
Tumors expressing NTS/NTSR1 are responsive to EGFR inhibitors
We evaluate the therapeutic effect of tyrosine inhibitors specific to EFGR, such as erlotinib, on cells expressing both NTS and NTSRl . Mice were xenografted with LNM-R cells expressing NTS and NTSRl on the right side and with a derived clone R Si-NTSRl (1) deleted for NTSRl expression with a stable expression of sh-NSTRl plasmid on the left side (Fig 13 A). A first experiment, consisting on 5 animals randomized on R Si-NTSRl tumor with an initial tumor volume of 158 ± 40 mm3 and 144.07 ± 26 mm3 for control and erlotinib group, respectively, were selected. When treated with erlotinib the final tumor volume of LNM-R tumor is only 30% of the final tumor volume of water treated animals (Fig 13 B). The doubling time is accordingly, with 3.85 ± 0.043 day and 4.41± 0.198 day for animal treated with water and erlotinib respectively. R SI-NTSRl tumors, none-expressing NTSRl, were not responsive erlotinib (figure 13 C).
As these cells are very aggressive, with a very high growth rate, a second experiment was performed on groups of 9 mice, randomized on LNM-R tumors when the volumes reached approximately 20 mm3. LNM-R tumors were sensitive to erlotinib and to a lesser extent with mertfomin. The final tumor volumes after 17 days of treatment were 960.87 ± 146.19 mm3 for the control, 367.18 ± 53.55 mm3 (P= 0.0008 vs control) for erlotinib and 612.41 + 104.97 mm3 (P= 0.05 vs control) for metformin treated animals, respectively. Use in co treatment metformin slightly improves but not significantly the response to erlotinib. The final volume was 318.23 ± 31.56 mm3 (Fig 13 D). The tumor doubling time was in agreement with the tumor volume, with 2.87 ± 0.13 day, 3.85 ± 0.24 day, 3.09 ± 0.07 day, and 4.03 ± 0.28 day for the control, metformin, erlotinib and metformin + erlotinib treated animals, respectively. The tumors void of NTSRl expression had no detectable response NTSRl to metformin or erlotinib (Fig 13E). This lack of response is consistent with the presence of a NTS autocrine loop, leading to the sustained activation of EGFR and responsible for cancer aggressiveness Overexpression of NTSRl in lung adenocarcinomas correlates with pejorative prognosis
A preliminary work of our team, suggested that the NTSRl expression is a negative prognostic marker in a selected population of stage I lung adenocarcinoma treated by surgery alone (1). We aimed therefore at assessing the prognostic significance of expression of NTSRl in a population of consecutive patients with stage I-III NSCLC (all histotypes) referred to our institution for surgery. Firstly, we studied a population of consecutive patients operated on for NSCLC (all histotypes) between June 15, 2001 and June 14, 2002. Secondly, on the basis of initial results, only adenocarcinoma patients operated on between June 15, 2001 and December 31, 2005 were analyzed.
In the first subpopulation (n=271), NTSRl positive staining was detected in 59 % of cases (160/271), but it was never detected in normal tissues adjacent to the tumor area. In the NTSRl semi-quantitative evaluation, 111, 126, and 34 patients were scored as 0, 1 and 2, respectively. In the lung adenocarcinomas, NTSRl staining of cancer cells was granular, intracellular, heterogeneous and rarely localized at the plasma membrane (figure 14A left). On the contrary, NTSRl staining in the squamous carcinoma cells was often localized at the membrane level (figure 14A right). Interestingly, NTSRl positive staining was not detected in lepidic adenocarcinomas or even in the lepidic component of invasive adenocarcinomas.
NTSRl score 1 was detected in 48 % of adenocarcinomas (57/119), 43% of SCC (46/107), and 39% of LCC (14/36). NTSRl score 2 was detected in 22% of adenocarcinomas (26/119), 7% of SCC (8/ 107), and 3% (1/36) of LCC. NTSRl score 2 was correlated with adenocarcinoma histological types (p = 0.013), but not with sex, age, smoking status, stage of disease and presence of vascular or lymphatic emboli. The prognostic significance of NTSRl expression was assessed in 228 of the 271 patients, due to postoperative deaths (n=20) and loss at follow-up (n=23). NTSRl score 2 was associated with poor 5-year overall survival as compared with NTSRl scored 0 or 1 (36.5% [95% CI 22.27% - 53.5]) versus 55.4% [95% CI 48.2%) - 62.31] respectively, p = 0.039) (Figure 14B Left). No difference was observed in survival between patients with NTSRl score 0 and those with score 1. When data were analyzed according to histological type, among patients with adenocarcinomas, NTSRl score 2 was significantly associated with worse 5-year overall survival as compared with NTSRl score 0 or 1 (36.1% [95% CI 20.29% - 55.54] versus 61.2% [95% CI 50.72% - 70.79%], p=0.028) (Figure 14B center). In contrast, among patients with either SCC or LCC, NTSRl score did not predict survival (Figure 14B right). The second populations focused only on adenocarcinoma subtypes. An additional 270 consecutive patients with adenocarcinoma was then included (See patient baseline characteristics in table 1). Together, 389 patients with adenocarcinomas were analyzed. The NTSRl score 2 was observed in 91.5 % of patients (76/389). The correlation between NTSRl and patient survival was determined on 363 of the 389 patients. The 5-year overall survival in this population was 55.3%. 5-year survival was 71.3%; 54.9%>, 38.8%, 32.9% in patients with pTl, pT2, pT3 and pT4 tumors, respectively (p = 0.0000018). These figures were 65.1%, 50.7%, 34.7% for pNO, pNl, pN2 disease, respectively (p = 0.0000001). The NTSRl score 2 was associated with worse 5-year overall survival as compared with NTSRl score 0 and 1 (42.2% [32.42% - 54.74%] versus 58.5% [52.62% - 64.07], p = 0.019) (Figure 14C). Multivariate analysis in all adenocarcinoma patients showed that pN (p=0.0000001), pT (p=0.00004) and NTSRl score 2 (p=0.0069) were independent predictors of worse survival.
Discussion
Genetic defaults carried by tumors, represent specific biological markers which reveal altered regulatory pathways. The most well-known examples include the fusion genes (BCR- ABL and EML4-ALK), the activating mutations (EGFR, K-RAS, Scr, rBRAF), and genomic amplification (HER2, MET). Accordingly, specific therapies employing EGFR and HER2 inhibitors or antibodies have been developed and were shown to improve the outcome of the disease. In parallel, cell signaling networks are evolving with the accumulation of genetic and epigenetic alterations in connection with the local stroma and the immune system. Identification of factors which will be a contributor and a reporter of tumor cell aggressiveness should enable to modulate tumor and disease progression. Here we observe a factor, abnormally expressed in a high proportion of tumors, the NTS/NTSR1 complex, contributed to tumor aggressiveness due to its sustained activation and generated modifications resulting in the stimulation of epidermal growth factor receptors.
Our experimental studies have shown that the NTS oncogenic action is boosted with a sustained NTSRl state of activation. In human tumors, both NTS and NTSRl are expressed in 40%), 60%), 65%, and 80 % of breast, lung, mesothelioma, and head neck squamous carcinomas, respectively, and suggesting that autocrine and/or paracrine NTS regulation occurs in tumors (7-9). Sustained activation of NTSRl induced the overexpression of the two receptors HER2, and HER3, as well as an autocrine activation of EGFR, HER2 and HER3. The transcriptional level of these three receptors was not altered by NTS, suggesting a new equilibrium in the sequence synthesis-activation-degradation-recycling is therefore taking place in the cells under the influence of NTS. The higher expression of the HER2 isoform suggests that a larger proportion of dimers containing HER2 should be activated in the cell. This context was previously described in breast cancer cells with HER2 gene amplification. It was shown that the excess of HER2 intracellular domains impaired EGFR/HER2 endocytosis (10), by preventing the entry of activated EGFR into clathrin-coated vesicles and limiting the action of phosphatases and maintaining EGFR signaling (11). In addition, it was also reported that under HER2 overexpression the rate of lysosomal targeting was significantly reduced, and rapid recycling of activated EGFR back to the cell surface occurred, as well decreased ligand dissociation from the EGFR (12). The necessary threshold of HER2 expression levels to trigger these processes is not known. The therapeutic benefit of Transtuzumab in patients not bearing HER2 gene amplification testify that similar deleterious effects may be induced by other mechanisms regulating HER2 expression (13). Interestingly, we show in this report that NTS chronically activates the release of HB-
EGF concomitantly with neuregulin 1. Consequently, both EGFR and HER3 autocrine activities are boosted and the tumoral aggressiveness is potentiated. The contribution of MMPs and EGF "like" ligands to carcinogenesis and cancer progression is well known. Therapeutical strategies targeting these factors have been largely attempted. However, these factors are often important for the human metabolism and major physiological functions, such as healing, angiogenesis, and gonadogenesis (14). One approach is to specifically antagonize their effects in cancer cells and to target an upstream regulatory factor. In this context, the NTS/NTSR1 complex would appear to be convenient, because, it is specifically over- expressed in tumors and its inhibition should only impair the function of these factors where they are deleterious. The validity of this concept was tested with the use of a specific NTSR1 antagonist which significantly reduced all the NTS induced oncogenic effects in vitro and in certain cases in vivo (15, 16). Nevertheless, the antagonist's ability to cross the blood brain barrier compromised its use for cancer therapy. The use of new pharmacological molecules to antagonize or neutralize intense and constant NTS-NTSR1 activation should reduce the tumor aggressiveness because tumoral cells bearing NTS and NTSR1 are susceptible to incur EGFR and HER3 activated concomitantly, as the example shown in figure 9E.
We attempted to target NTSR1 activation pathway with metformin. Metformin is an antidiabetic drug, and has recently been proposed as a potential anticancer compound (17). Metformin was shown to disrupt the crosstalk between insulin receptor and NTS receptor in pancreatic cancer cells (18). Furthermore, in addition to inhibiting the mTOR pathway, metformin prevents ER activation induced by NTS and insulin (19). In our cellular model, metformin reduced tumor growth only in those tumors expressing NTSRl, and thus supporting the significance of the NTS/NTSR1 signaling pathway in tumor growth.
In a second approach we targeted the end point of the signalization cascade generated by NTS/NTSR1 complex with molecules proposed for lung cancer therapy. The sustained activation of EGFR by NTS/NTSR1 autocrine loop, mines the activating EGFR mutations. As expected, the tumors expressing NTS/NTRS1 complex are responsive to erlotinib, an EGFR inhibitor used for lung cancer patients bearing EGFR mutations (20). In contrast, tumors void for NTSRl expression and bore by the same mice were not responsive to erlotinib. No additional response was seen with the concomitant treatment of metformin and erlotinib, confirming the hypothesis that the signaling events are on the same cascade.
We found that NTSRl expression was associated with adenocarcinomas prognosis. This result was confirmed by multivariate analysis, which showed that among the available clinical and pathologic factors, the NTSRl score 2, T, and N were independent predictors of worse prognosis. High expression of NTSRl has been found to be associated to poor survival also in other cancers. Dupouy et al found that NTSRl expression involving > 80% tumor cells was associated with worse survival in breast cancer (16). Similarly, in head and neck cancers, patients with high NTS and NTSRl expression had a higher rate of distant metastasis (9). Therefore, the prognostic role of neurotensin system is probably correlated with its activation rate. In this context, the difference in staining between the different histological subtypes in our series explains the respective prognostic role of NTSRl . Within adenocarcinomas, NTSRl staining was never detected in broncho lo alveolar subtypes or in the bronchoalveolar component of mixed adenocarcinoma but it was often detected in its invasive compartment, suggesting a role favoring tumor invasion and migration: Also in SCC the staining was found primarily at the membrane as in the non- stimulated cells. In vitro studies have shown that NTS is capable of modulating the migratory ability of adherent cancer cells of different origins (colon, ductal pancreatic, head and neck squamous cell, breast). In addition, it has been showed that NTSRl induces and enhances the invasive phenotype in LNCaP and HNSCC tumor cells. Involved mechanisms remain unclear but metalloproteinases are probably involved (9, 21). The NTS/NTSR1 complex could be used as a marker to identify subsets of human cancers, and thus make eligible new drugs, kinase inhibitors, or immunotherapy, targeting HERs protein or their downstream pathways. The clinical criteria used to propose these therapies are based on the detection of genetic defaults in the tumor (HER2 amplification, EGFR mutation). Nevertheless, it was also observed that other patient subsets could benefit from these therapies. The challenge; is find a criteria to categorize them. For example, cells with neuregulin 1 high expression in association with HER3 autocrine activation and without HER2 amplification, are good responders to lapatinib or HER2 kinase inhibitors (22).
Conclusion
In summary our findings suggest that the NTS/NTSR1 complex contributes to cancer aggressiveness by enhancing concomitantly expression and activation of the three receptors EGFR, HER2, HER3. As an extrapolation of these findings, we propose that patients bearing this complex should be responders to kinase inhibitors, and that inhibition of NTS/NTSR1 complex should reduce the rate of tumor progression, providing a longer therapeutic window for the practitioners to treat their patients.
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EXAMPLE 3:
NTS/NTSRl enhance experimental HCC tumor progression through EGFR activation To determine the contribution of NTS/NTSR1 complex on tumor progression, mice were implanted with HCC cell lines and NTSR1 overexpressing clones xenografted. For HEP3B cells the tumors were measurable 21 days after cells injection, whereas for PLC/PRF5, the tumors were measurable at day 17. Both parental cell lines showed similar growth rates. The tumor burden for both clones overexpressing NTSR1 was measurable earlier at day 13, and the growth rate at day 42 was 2.9 and 2.08 fold higher for HEP-Rla and PLC-Rla as compared to respective parental cells (Figure 15 A). As shown in Figure 15B the tumor weights is in correspondence with the tumor size 4.3 and 2.8 fold time higher for HEP- Rla and PLC-Rla as compared to their respective parental cells.
We confirmed the expression of NTS in the tumors of HEP 3B and HEP-Rla cells with an antibody directed against the long fragment NTS. In both tumors, clusters of strong intra-cytoplasmic labelling were distributed randomly on the slide (Figure 15C a and e). The labelling of NTSR1 in HEP-Rla revealed to be mostly intra-cytoplasmic and weak, with a few exceptional clusters of cells with very strong cytoplasmic and membrane expression (Figure 15C b). As expected, no NTSR1 labelling was seen in HEP3B tumors (Figure 15C f). In HEP-Rla tumors the labelling intensity of phosphorylated EGFR was heterogeneous and very strong with a thick to a thin line around the entire cell membrane. The strong labeling was often localized at the front of the tumor or close to the blood vessel. Examples of a thin or strong labelling are shown in figure 15C c and d, respectively. In contrast, in HEP3B tumors, phosphorylated EGFR labeling was absent or very weak as shown in figure 15C g and h.
NTS/NTSR1 restores responses to tyrosine kinase inhibitor.
As shown above, NTSR1 activation induced a sustained EGFR activation, which we believe, acts as EGFR driver mutation. To explore this hypothesis, the HCC cell lines HEP 3B and C were xenografted on the same mice. The mice were randomly distributed in two groups based on the size of the HEP 3B tumors. Since HEP-Rla tumors grow faster than HEP 3B tumors, HEP 3B cells were injected a few days before HEP-Rla cells. In the control group, the average HEP3B tumor size was 168.2 ± 38.1 mm3 and the HEP-Rla tumor size was 79.7 ± 16.4 mm3 at day 1. The treated group carried HEP 3B tumors of 164.7 ± 31.2 mm3 and HEP-Rla tumors of 87.2 ± 19.13 mm3 at day 1. Mice were daily treated, per os, with 75 mg/kg erlotinib or H20 for 22 days. The growth rate of the HEP-Rla tumor was drastically affected by the EGFR inhibitor, whereas HEP3B tumors did not respond to erlotinib (figure 16 A and B). Over the period of the treatment, the doubling time was 7.0 ± 0.55 days and 10.1 ± 1.1 (p = 0.0016) for HEP-Rla for control and treated with erlotinib, respectively. For the HEP3B tumors the doubling time was similar 7.8 ± 0.52 and 7.7 ± 0.84 days for control and treated group respectively. The contribution of NTS/NTSRl complex to tumor growth is relayed by EGFR activation, suggesting that tumor over expressing NTSR1 may be responsive to EGFR inhibitor.

Claims

CLAIMS:
1. A method of the treatment of cancer in a patient in need thereof comprising the steps of i) determining the expression level of NTS and/or NTSR1 in tumor sample obtained from the patient, ii) comparing the expression level determined at step i) with a predetermined reference value and iii) administering the patient with a therapeutically effective amount of at least one HER inhibitor when the expression level determined at step i) is higher than the predetermined reference level.
2. The method of claim 1 wherein the patient suffers from a cancer deriving from epithelial origin.
3. The method of claim 1 wherein the patient suffers from a carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.
4. The method of claim 1 wherein the patient suffers from a cancer selected from the group consisting of squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophagael cancer, tumors of the biliary tract, and head and neck cancer,.
5. The method of claim 1 which comprises the steps of providing total RNAs extracted from cancer cells and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi-quantitative RT- PCR.
6. The method of claim 1 wherein the expression level of NTS or NTSR1 is determined at the protein level.
7. The method of claim 1 wherein the tumor sample of the patient is contacting with a binding partner specific for NTS or NTSR1.
8. The method of claim 1 wherein the HER inhibitor is selected from the group consisting of EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, and HER4 inhibitors.
9. The method of claim 1 wherein the HER inhibitor is a HER antibody selected from the group consisting of EGFR antibodies, HER2 antibodies, HER3 antibodies, and HER4 antibodies.
10. The method of claim 1 wherein the HER inhibitor is selected from the group consisting of small organic molecule HER antagonists; HER tyrosine kinase inhibitors; HER2 and EGFR dual tyrosine kinase inhibitors, and HER dimerization inhibitors.
11. The method of claim 1 wherein the HER inhibitor is selected from the group consisting of cetuximab, panitumumab, zalutumumab, nimotuzumab, erlotinib, gefitinib, lapatinib, neratinib, canertinib, vandetanib, afatinib, TAK-285, ARRY334543, Dacomitinib, OSI-420, AZD8931, AEE788, Pelitinib, CUDC-101, XL647, BMS-599626, PKC412, BIBX1382 and AP261 13.
12. The method of claim 1 wherein the HER inhibitor is a pan-HER inhibitor.
13. The method of claim 1 wherein the HER inhibitor is a inhibitor of HER expression.
14. The method of claim 1 wherein the HER inhibitor is administered to the patient in combination with an inhibitor of the neurotensin activation of NTSR1 such as an antibody against neurotensin or a fragment thereof which binds to neurotensin, or an antibody against the neurotensin receptor 1 or a fragment thereof which binds to the neurotensin receptor 1.
15. The method of claim 1 wherein the HER inhibitor is administered to the patient in combination with metformin.
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