GB2496150A - Biomarker and target for responsiveness and resistance to cancer targeting agents - Google Patents
Biomarker and target for responsiveness and resistance to cancer targeting agents Download PDFInfo
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Abstract
A neuropeptide, NeuromedinU, as a cellular marker for responsiveness and/or resistance to cancer targeting agents or as a target for developing cancer targeting agents to target HER expressing cancers. There are also claims to agents that target tumours expressing one or more of HER1, HER2, HER3, HER4, or epidermal growth factor receptor (EGFR), specifically Trastuzumab, Lapatinib, Neratinib and Afatinib.
Description
Title: Marker and Target for Responsiveness and Resistance to Cancer Agents
Field of the Invention
The invention relates to the novel use of a marker, NeuromedinU, that has clinical potential as a target and poor-prognostic biomarker for cancer that is directly associated with responsiveness and/or resistance to certain cancer targeting agents.
Background of Invention:
Herceptin-2 (HER2) -positive cancers: HER2 (also known as ErbB-2) stands for "Human Epidermal growth factor Receptor 2". This family includes 3 other members, HER1 (EGFR); HER3 and HER4.
HER2 is over-expressed, described herein as "HER2-positive", in approximately 25% of breast cancers and is associated with higher aggressiveness and poor prognosis. This over-expression, however, is not limited to breast cancer and has been identified in a variety of cancer types including bladder, pancreas, Non-small cell lung cancer (NSCLC), ovarian, colon, kidney, head & neck, stomach, prostate, gliomas, and biologically aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma. Abnormal levels of other HER family members, especially EGFR are also associate with cancerous states.
Trastuzumab (Herceptin; Genentech/Roche; targeting HER2) and more recently lapatinib (Tykerb; GSK; targeting both HER2+EGFR) have improved prognosis for patients with HER2-positive breast cancer. More recent drugs developed against HER2 include Neratinib (Phase Ill trials; Pfizer/Puma Biotechnology; targeting both HER2I-EGFR) and Afatinib (Tovok; Boehringer Ingelheim; targeting both HER2+EGFR).
Unfortunately, not all HER2-positive patients respond to HER2-targeted or dual HER2/EGFR-targeted agents and others, who initially benefit, relapse due to development of resistance. Therefore, there is a need to identify biomarkers (ideally minimally-invasive i.e. extracellular also, if possible) for improved patient selection and to develop strategic to improve response and overcome resistance in HER2/EGFR-positive cancers.
Neuromedin U (NmU): Neuromedin U is a highly conserved neuropetide present in many species and in humans it is a 25 amino acid peptide (U-25). NniU has potent activity on smooth muscle and was isolated first from porcine spinal cord and later from other species. Peripheral activities of NmU include stimulation of smooth muscle, increase of blood pressure, alteration of ion transport in the gut, control of local blood flow and regulation of adrenocortical function.
In studies of relatively limited sample sizes, NmU has been detected in some cancers; but will differing associations. For example, increased levels of NmU have been associated with AML, with pancreatic cancers, through studies of ovarian cancer cell lines and some lung cancers, but decreased in oral cancers and suggested to be a tumour suppressor gene from studies of oesophageal cancer cell lines.
Page 1 Object of the Invention: It is an object of the invention to provide a biomarker for the prediction of successful responsiveness of specific chemotherapeutic agents for the treatment of cancer. A further object is to meet the increasing demand for improved treatment of cancers and to facilitate personalised medical treatment. A further object is to enable the determination of the success rate of a particular treatment on a patient. Bioniarkers could come at least in part, help to overcome the problem of patients receiving certain chemotherapeutic agents from which they derive no benefit, and also meet the increasing demand for improved patient outcomes. A further object of the invention is to provide a target for NeuromedinU signaling as a useful therapeutic strategy for treatment of cancer.
With increasing numbers of personalised drugs entering the market, is has become important to determine the success rate of a particular treatment on a patient.
Page 2
Description:
Brief Description of Drawings
Fig 1: NMU was found to be up-regulated in (A-i) RCC1954 LapR cells compared sensitive cells by 5-fold (A-il) HCC1954 LapR CM compared sensitive CM by 1.7-fold (p=0.002) (A-ill) SKBR3 LapR cells compared sensitive cells by 11-fold (p=0.0004) (A-iv) SKBR3 LapR CM compared sensitive CM by 2.1-fold (p=0.008) Pulse treatment of HCC19S4 and SKBR3 also showed up-regulation of NmU (B-i) RCC19S4 cells (B-u) NmU was significantly up-regulated (p=0.02) in hiM (1.7-fold) and 10 tiM (3.4-fold) Lapatinib treated HCC19S4 CM. (B-ui) NmU was significantly up-regulated in both ljiM (p=0.0008) and 10 F.tM (p=0.0O1) Lapatinib treated SKBR3 cells by 5.4-and 2.5-fold, respectively. (B-iv) NmU was also found to be significantly up-regulated in both 1pM (p=0.OO5) and 10MM (p= 0.014) Lapatinib treated SKBR3 CM by 1.5-and 1.7-fold, respectively.
Fig 2: (A-i) HCC19S4-NmU transfected cells showed 58.5-fold up-regulation of NmU compared to mock-plasmid transfected cells. (A-il) NmU-transfected SKBR3 cells showed 9900.83-fold up-regulation of NmU compared to mock-plasmid transfected cells (B-i) Significant increase in proliferation was observed in NmU-transfected RCC19S4 cells compared to mock-transfected at each lapatinib dilution (0.4iM =2.6-fold (p=0.0012); 0.8j.iM =2.9-fold (p=O.0008); 1.2MM =2.6-fold (p=O.0013) (B-il) Significant increase in proliferation was also observed in NmU-transfected RCC19S4 cells compared to mock-transfected at each neratinib dilution (0.4MM =1.9-fold (p=0.0006); 0.8pM =2.9-fold (p=0.016); 12pM =4.3-fold (p=0.0001). (B-ill) Herceptin also showed increase in proliferation in NmU-transfected HCC1954 cells compared to mock-transfected at each dilution (7kg =1.1-fold; l4pg =1.2-fold (p=0.039); 28kg =1.1-fold.
(C-i) Significant increase in proliferation was observed in NmU-transfected SKBR3 cells compared to mock-transfected at each lapatinib dilution (5OnM =1.4-fold (p=0.0003); lOOnM =2.1-fold (p=0.0O6); 200nM =2.2-fold (p=0.0007) (B-il) Significant increase in proliferation was also observed in NmU-transfected SKBR3 cells compared to mock-transfected at each neratinib dilution (5OnM =1.6-fold (p=0.014); lOOnM =2.0-fold (p=0.0004); 200nM =1.8-fold (p=0.002). (B-ui) Herceptin also showed significant increase in proliferation in NmU-transfected HCC1954 cells compared to mock-transfected at each dilution (7kg =1.5-fold (p=0.00S); l4pg =1.6-fold (p=O.OO4); 28pg =1.8-fold (p=0.0l7).
Fig 3: NmU-specific siRNA (NmU-1 and NmU-2) showed a significant down-regulation of NmU compared to scrambled transfected cells (A-i) HCC1954 LapR NmU-1 transfected cells showed 62.2% and NmU-2 showed 39.1% down-regulation (p<0.OOl) (A-il) SKBR3 LapR NmU-1 transfected cells showed 73.6% and NmU-2 showed 62.7% down-regulation (p<0.001). Cells were treated with NmU-Page 3 targeted 5iRNA or Scrambled (5CR) 5iRNA for 48 hours followed by Lapatinib for 72 hours. (A-ui) T47D innately resistance cell line showed a knock-down of 68,7% in NmU-1 and 67.7% NmU-2 transfected cells (A-iv) MDA-MB-361 (B-i) Inhibition of NmU in LapR cells and innately resistance cell lines restores or sensitizes not only to lapatiib but also to neratinib and herceptin. Proliferation, followed by 72 hours of 5l.IM Lapatinib treatment, was reduced significantly by 49.2% in NmU-1 (p=0.007) and 34.3% in NmU-2 (p=0.03) transfected HCC1954 LapR cells (B-U) Proliferation, followed by 72 hours of 3j.xM Lapatinib treatment, was reduced significantly by 11.8% in NmU-1 (p=0.007) and 10% in NmU-2 (p=0.03) transfected SKBR3 LapR cells (B-Ui) T47D showed a significant reduction in proliferation in both NmU-1 (15.5%; p=0.002) and NmU-2 (11.8%; p=0.016) (B-iv) (C-i) Proliferation, followed by 72 hours of hiM neratinib treatment, was reduced significantly by 42.6% in NmU-1 (p= 2.11E-05) and 56.1% in NmU-2 (p=O.0O8) transfected RCC1954 LapR cells compared to scrambled. (c-U) SKBR3 LapR cells also showed sensitivity to neratinib by 58.0% in NmU- 1 (p=0.0004) and 20.0% in NmU-2 (p=0.036) transfected compared to scrambled. (C-ui) (D) Trastuzumab also showed growth inhibitory effects when treated with 15ig (D-i) HCC1954 LapR cells showed proliferation of 50.4% (p=3.9E-07) in NmU-1 and 67.2% (p=O.000l) in NmU-2 transfected cells (D-ii) Fig 4: (A-i) NmU-transfected HCC1954 cells showed a significant wound closure at both 24 hours and 48 hours. At 24 hours there was 38.6% wound closure (p=0.02) and at 48 hours there was 49.2% wound closure (p=0.001). (A-u) NmU-transfected RCC1954 cells showed a wound closure of 1.1% and 3.1% at 24 hours and 48 hours, respectively. There was a significant wound closure at 72 hours (6.6%; (p=0.037)) in NmU-transfected SKBR3 cells compared to mock-transfected.
(B-i) NmU-transfected HCC1954 cells showed a significant (p=0.026) increase in migration (121.4%) compared to mock-transfected cells. (B-u) SKBR3 NmU-transfected cells also showed an increase in migration (38.8%) compared mock-transfected cells.
(C-i) NmU-transfected HCC1954 cells showed a significant (p=0.0006) increase in invasion (60.5%) compared to mock-transfected cells. (C-u) SKBR3 NmU-transfected cells shows a significant p=O.OO1) increase in invasion (49.9%) compared mock transfected cells.
(D-i) NmU over-expression resulted in significant (p=O.OOS) reduction (12.4-fold) in cell death under Anoikis condition in HCC1954 cells compared to mock-transfected cells. (D-ii) SKBR3 NmU over-expression resulted in significant (p=0.02) reduction (2.7-fold) in cell death under Anoikis condition.
FigS: (A-i) Scrambled-transfected HCC19S4 LapR cells showed a significant wound closure at both 48 hours and 72 hours. Wound closure at 24 hours was also observed but was not significant. (A-U) Page 4 Scrambled-transfected HCC1954 LapR cells showed a wound closure of 5.2% NmU-1 3.3% and NmU- 2 2.8% at 24 hours. Significant wound closure was observed at 48 hours in scrambled-transfected cells (20.8%) compared to NmU-1 (p=0.021) and NmU-2 (p=0.034). There was a significant wound closure at 72 hours in scrambled-transfected cells (48.5%) compared to NmU-2 (p=0.009), however compared to NmU-2 significance was reached.
(B-i) HCC19S4 LapR siRNA transfected cells showed a significant reduced invasion in NmU-1 (39.8%;p=0.025) and NmU-2 (33.9%;p=0.028) transfected cells compared to Scr-transfected cells. (B-ii) HCC1954 LapR 51RNA transfected cells showed a significant reduced migration in NmU-1 (%;p=) and NmU-2 (%;p=) transfected cells compared to Scr-transfected cells (C-i) NmU over-expression resulted in significant (p=O.OO5) reduction (12.4-fold) in cell death under Anoikis condition in HCC1Y54 cells compared to mock-transfected cells. (D-ii) SKBR3 NmU over-expression resulted in significant (p=O.O2) reduction (2.7-fold) in cell death under Anoikis condition.
In brief, when we studies our HER2 positive breast cancer cell lines, we found NmU to be at significantly higher levels in HER2-positive cell lines (Figure 1 A-i & A-u) which we had developed, over a 6 months period, to be resistant to HER2-targeted agents (in this case, Lapatinib). This was reflected in the extracellular environment, support the relevance of NmU as a extracellular (minimally-invasive biomarkers) for response to F-11R2-targed agents (Figure 1 A-ui & A-iv). When we exposed the cells to the drug for a short time (48 hrs), we found a similar trend within and extracellular to the cells, indication that this is something that could potentially be picked up early on as an indication of whether or not a patients with a HER2-positive tumour is likely to respond to the available H ER2-targetetd agents.
We subsequently cloned and sequenced human NmU eDNA, introduced it into HER2 cells with limited levels and found that its presence (NmU compared to mock tronsfected) decreased the responsiveness of the cells to a range of HER2-targedt drugs, lapatinib (Figure 2 Bi & Ci), neratinib (Figure 2 Bii & Cii) and Trastuzumab/Herceptin (Figure 2 Biii & Ciii).
When we knocked down natural levels of NmU (using 2 independent siRNA vs. a non-specific scrambled sequencs) we found that this sensitised the celis to a range of HER2-targedt drugs, lapatinib, neratinib and Trastuzumab/Herceptin. Figure 3 Ai, Au & Aiii illustrates knock-down of expression of natural levels of NmU; Figure3 Bi, Bii, Biii & Biv illustrates response to lapatinib; Figure 3 Ci, Cii, Ciii & Civ illustrates response to neratinib; Figure 3 Di, DU, Diii & Div illustrates response to Trastuzuma b/Herceptin.
Introducing NmU into cells caused them to also be more "aggressive" than mock transfected cells i.e. more motile, more invasive (Figure 4 A-C); and to resist a form of apoptosis known as anoikis (Figure 4 D).
Page 5 Knock-down og NmU in these HER2-positive cells caused them to be less "aggressive" i.e. less motile, less invasive; and more sensitive to a form of apoptosis known as anoikis (Figure 5 A-c).
When we assessed NmU expression in a very large cohort of publically-available microarray data sets, we found NmU presence to be particularly associated with poor outcome in HER2-positive breast tumours; as the KM analysis on lOOs of tumour specimens show. P-value = O.0000SHazard ration: 1.8 (Figure 5-clinical Data) Through analysis of mRNA expression in 4607 breast tumours, we identified NmU to be particularly associated with HER2-postive cancers and to be associated with poorer outcome for those patients (ie shorter time to death); Through studies of cell line models which we had developed to being resistant to lapatinib (to "mimic" and help elucidate mechanism of resistance to HER-targeted agents, we observed NmU to be at higher levels in resistant cells -so potentially a cell marker of resistance and actively involved in the resistance).
Interesting, we found the same trend when study medium conditioned by the cells i.e. higher amounts of NmU outside the resistant cell compared to the sensitive parent cell lines (potential as minimally-invasive circulating predictive biomarker); To establish if the increase in NmU might occur early on in resistance development i.e. an early indication of whether or not the tumour cells would respond to HER-targeting (important to know rather than wasting someone's life and expensive drugs) we exposed cells to drug for short time and found the increased levels in exposed cells and externally. Suggesting, yes, has potential as an early cell and extracellular indicator lack of response.
To establish if this would also be the case for other HER-targeted agents, we cloned human NmU cDNA, transfected it into cells and we found that this increased resistance of the cells not only to lapatinib, but also Trastuzumab and neratinib (likely also the case for Afatinib; we just haven't that drug in house yet to test). Suggest NmU to contribute to the mechanism of resistance.
Further supporting a functional role for NmU in resistance to HER-targeted agents, we knocked down its endogenous levels in 4 cell lines -using 2 independent siRNAs-and found that this sensitised the cells to lapatinib, but also Trastuzumab and neratinib (likely also the case for Afatinib; we just haven't that drug in house yet to test).
So not just a potential marker of response, but seems to be actively involved in the resistance mechanism. Potentially co-targeting NmU and HER2 and/or EGFR and/or other HER family members Page 6 Analysis of our NmU over-expressing and knock-down cell lines also indicated NmU to be associated with increased movement of cells; invasion through extracellular matrix; and resistance to anoikis (a form of apoptosis that typically occurs when epithelial cells are forced to exist unattached to a matrix and to exits in suspension). All of these cells are requirements that cancer cells need to be able to break away from primary tumours; get into the bloodstream/lymph vessel and survive there on their way to metastasis. This further supports the relevance of NmU as a new drug target (to target alone or in combination with HER-agents).
Over-expression of NmU is associated with the cells becoming resistant to the HER2-targeted and dual HER2/EGFR-targeted agents. Knocking NmU down sensitises the cancer cells to the F-IER2-targeted and dual HER2/EGFR-targeted agents. The amounts of NmU found outside the cells seems to reflect the sensitive/resistant nature, suggesting it is a cell-based and extracellular predictive biomarker for response and also a new drug target (possibly to co-target with HER-targeted agents).
As HER2 over-expression is not restricted to breast cancer and has been identified in a variety of cancer types including bladder, pancreas, NSCLC, ovarian, colon, kidney, head & neck, stomach, prostate, gliomas, and biologically aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma the potential for NmU as a cell-based and extracellular (minimally-invasive/blood based) biomarker as well as new target/co-target is potentially very broad among a broad range of F-IER2/EGFR-positive cancers Page 7
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