WO2007116923A2

WO2007116923A2 - Sez6l2 oncogene as a therapeutic target and prognostic indicator for lung cancer

Info

Publication number: WO2007116923A2
Application number: PCT/JP2007/057620
Authority: WO
Inventors: Yusuke Nakamura; Yataro Daigo; Shuichi Nakatsuru
Original assignee: Oncotherapy Science, Inc.
Priority date: 2006-03-31
Filing date: 2007-03-29
Publication date: 2007-10-18
Also published as: WO2007116923A3

Abstract

The present invention provides a method for assessing the prognosis of non-small cell lung cancer (NSCLC) using SEZ6L2, as well as methods for evaluating the efficacy of a particular therapy for NSCLC. In addition, the present invention provides kits for prognosing NSCLC. Furthermore, treatment of NSCLC cells with vector-based small interfering RNAs (siRNAs) against the SEZ6L2 gene suppressed its expression and resulted in growth suppression of the NSCLC cells. These results indicate that SEZ6L2 may be useful as a diagnostic marker and as a target for development of new molecular therapies for lung cancer.

Description

DESCRIPTION

SEZ6L2 ONCOGENE AS A THERAPEUTIC TARGET AND PROGNOSTIC INDICATOR

FOR LUNG CANCER

This application claims the benefit of U.S. Provisional Application Serial No.60/787,781, filed March 31, 2006, the contents of which are hereby incorporated by reference in its entirety.

Technical Field

The present invention relates to the field of biological science, more specifically to the field of cancer diagnosis and therapy. In particular, the invention relates to a method for diagnosing and prognosing lung cancer and compositions and methods for inhibiting cancer cell proliferation.

Background Art

Lung cancer is the leading cause of cancer deaths worldwide, and non-small cell lung cancer (NSCLC) accounts for nearly 80% of those cases (Jemal A, et al, (2004) CA Cancer J Clin; 54: 8-29.). Regardless of histological subtype, the 5-year survival rate for lung-cancer patients hovers at about 10-15% (Jemal A, et al, (2004) CA Cancer J Clin; 54: 8-29. Naruke T, et al, (1998) JThorac Cardiovasc Surg; 96: 440-7.). In fact, even those patients diagnosed at stage IA have a 5-year survival rate of less than 80% (Naruke T, et al, (1998) J Thorac Cardiovasc Surg; 96: 440-7. Chang MY and Sugarbaker DJ. (2003) Semin Surg Oncol; 21: 74-84.). Within the last decade, several newly-developed cytotoxic agents, such as paclitaxel, docetaxel, gemcitabine, and vinorelbine, have begun to offer multiple treatment choices for patients with advanced lung cancer; unfortunately, each of these regimens confers only a modest survival benefit as compared to cisplatin-based therapies (Schiller JH, et al, (2002) N Engl J Med; 346: 92-8. Smit EF, et al, (2003) J Clin Oncol 2003; 21 : 3909-17.). Accordingly, novel therapeutic strategies, such as molecular-targeted drugs, siRNAs and immunotherapies (antibodies and cancer vaccines) are eagerly anticipated. Although the precise pathways involved in lung tumorigenesis still remain unclear (Huber RM and Stratakis DF. (2004) Lung Cancer; 45: S209-13.), some evidence indicates that tumor cells express cell-surface markers unique to each histological type at a particular stage of differentiation. Since cell-surface proteins or secretory autocrine-growth factors are considered to be more accessible to immune mechanisms and drug-delivery systems, identification of cancer-specific cell-surface and/or secretory proteins is likely to play a role in the development of novel diagnostic markers and therapeutic strategies.

The present inventors have previously screened for genes encoding molecules that are up-regulated in lung cancers, using cDNA microarrays and tumor cells purified by laser- capture microdissection (See WO2004/31413, incorporated by reference herein; see also Kikuchi T, et al, (2003) Oncogene; 22: 2192-205. Kakiuchi S₃ et al (2003) MoI Cancer- Res; 1: 485-99. Kakiuchi S, et al. (2004) Hum MoI Genet; 13: 3029-43. Suzuki C, et al, (2003) Cancer Res; 63: 7038-41. Ishikawa N, et al, (2004) Clin Cancer Res; 10: 8363-70. Kato T, et al, (2005) Cancer Res; 65: 5638-46. Furukawa C, et al., (2005) Cancer Res; 65: 7102-10. Ishikawa N, et al., (2005) Cancer Res; 65: 9176-84. Suzuki C, et al, (2005) Cancer Res 2005; 65: 11314-25). To verify the biological and clinicopathological significance of the respective gene-products, the present inventors performed tissue microarray analysis of clinical lung-cancer materials (Ishikawa N, et al, (2004) Clin Cancer Res; 10: 8363-70. Kato T, et al, (2005) Cancer Res; 65: 5638-46. Furukawa C, et al. (2005) Cancer Res; 65 : 7102- 10. Ishikawa N, et al. , (2005) Cancer Res; 65: 9176-84. Suzuki C, et al, (2005) Cancer Res; 65: 11314-25.). This systematic approach, coupled with a search of cell-surface and/or secretory proteins using bioinformatics tools, led to the identification of SEZ6L2, seizure related 6 homolog (mouse)-like 2 (also referred to as "PSK- 1", UniGene Hs.6314; SEQ ID NOs.1, 2), a gene frequently transactivated in a large population of lung cancers. Though its physiological significance in carcinogenesis or its clinicopathological importance remained unclear, a subsequent study using cDNA microarray combined with bioinformatics analysis demonstrated that SEZ6L2 was one of the 703 genes that was highly expressed in human hepatocellular carcinoma, (Patil MA, et al, (2005) Oncogene; 24: 3737-47.).

Application of multiple strategies for the identification of genes that encode secreted and transmembrane molecules, termed the Secreted Protein Discovery Initiative (SPDI), suggested that SEZ6L2 was a novel transmembrane protein (Clark HF, et al, (2003) Genome Res; 13: 2265-70.). SEZ6L2 was also identified as a gene highly homologous to mouse SEZ6, a gene first identified in the course of differential screening of mRNA from cortical neurons treated with pentylentetrazole (PTZ), a drug known to induce epileptic seizures (Shimizu-Nishikawa K, et al, (1995) Brain Res MoI Brain Res; 28: 201-10.). The SEZ6L2 gene encodes a 92.5-kDa protein with an N-terminal signal peptide, five SUSHI domains (SCR repeat), three CUB (initials of the first three identified proteins containing such domains: complement factor Clr/Cls, embryonic sea urchin protein uEGF, and bone morphogenetic protein 1) domains, and a C-terminal transmembrane domain. Although little is known about the function of proteins possessing such SUSHI and CUB domains, they have been postulated to be primarily involved in the developmental process, cell-cell interaction, and cell adhesion.

Summary of the Invention

The present invention provides a composition composed of an SEZ6L2 siRNA. In a preferred embodiment, the SEZ6L2 siRNA includes a nucleotide sequence of SEQ ID NOs: 12 to 14 as the target sequence. Such siRNAs are demonstrated herein to be effective for inhibiting cell growth of NSCLC cell lines.

Accordingly, the present invention provides a method for treating or preventing lung cancer, particularly NSCLC, using such compositions. An exemplary therapeutic method includes a method of inhibiting cancer cell growth by contacting the cancer cell, either in vitro or in vivo, with a composition containing an SEZ6L2 siRNA that reduces the expression of the SEZ6L2 gene. In a preferred embodiment, the cancer cell is an NSCLC cell. Alternatively, the therapeutic method may involve treating or preventing NSCLC in a subject by administering to the subject a composition composed of an SEZ6L2 siRNA that reduces the expression of SEZ6L2.

The present invention also provides pharmaceutical compositions for treating or preventing NSCLC containing as the active ingredient an effective amount of an SEZ6L2 siRNA.

Herein, evidence is presented that SEZ6L2 functions a prognostic indicator of lung cancer. Specifically, a high level of SEZ6L2 expression was associated with poor survival as well as disease stage and node status for patients with lung adenocarcinoma (ADC), suggesting an important role for the SEZ6L2 protein in the development and progression of this disease. As the data herein suggest that up-regulation of SEZ6L2 is a frequent and important feature of lung carcinogenesis, the present inventors accordingly propose that targeting the SEZ6L2 molecule holds promise for development of new diagnostic strategies for clinical management of lung cancers. - A -

Accordingly, it is an object of the present invention to provide a method for assessing or determining the prognosis of a patient with non-small cell lung cancer by comparing an SEZ6L2 level in a patient-derived biological sample with that of a control sample. An elevated expression level is indicative of poor survival. In particular, the higher the expression level of SEZ6L2 measured in the patient derived sample, the poorer the prognosis for post-treatment remission, recovery and/or survival and the higher the likelihood of poor clinical outcome. It is a further object of the present invention to provide kits for assessing an NSCLC prognosis, such kits including SEZ6L2 -detection reagents.

These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples. However, it is to be understood that both the foregoing summary of the invention and the following detailed description are of a preferred embodiment, and not restrictive of the invention or other alternate embodiments of the invention.

Brief Description of the Drawings Fig. 1 is composed of a series of photographs depicting the validation of SEZ6L2 expression and localization in lung cancers. Panel (A) depicts the expression OΪSEZ6L2 in 15 clinical lung-cancer samples, examined by semi-quantitative RT-PCR. Panel (B) depicts the expression OΪSEZ6L2 in 23 lung-cancer cell lines, examined by semi-quantitative RT- PCR. Panel (C) depicts the expression of the SEZ6L2 protein in 6 lung-cancer cell lines, examined by western-blot analysis. Panel (D) depicts the expression of the SEZ6L2 protein on cell surfaces in lung-cancer lines A549, EBC-I, and NCI-H647, evaluated by flow- cytometric analysis. Signal intensity values (Y-axis) of cells treated with anti-human SEZ6L2 polyclonal antibody (gray) or cells treated with rabbit IgG (control; black) were shown. Fig. 2 is composed of a series of photographs depicting the expression and localization of SEZ6L2 in clinical lung cancer tissues. Panel (A) depicts the results of western-blot analysis of the SEZ6L2 protein in two representative pairs of lung adenocarcinoma samples. Panels (B-G) constitutes representative images of immunohistochemical analysis of the SEZ6L2 protein in lung adenocarcinoma tissues. Magnification, X 100 (B, C, D) and X200 (E₅ F₃ G).

Fig. 3 is composed of a series of photographs depicting the expression of the SEZ6L2 protein in normal organ tissues. Panels (A-F) depict the results of immunohistochemical evaluation of the SEZ6L2 protein in representative normal tissues, namely adult heart (A), liver (B), lung (C), kidney (D), and pancreas (E), as well as lung adenocarcinoma tissues (F). Magnification, X 200. Fig. 4 depicts the association of increased SEZ6L2 expression with poorer clinical outcomes among NSCLC patients. Kaplan-Meier analysis of tumor-specific survival in 420 patients with NSCLCs according to the level of SEZ6L2 expression (P = 0.0209; log- rank test).

Detailed Description of the Invention The words "a", "an", and "the" as used herein mean "at least one" unless otherwise specifically indicated.

The SEZ6L2 gene, seizure related 6 homolog (mouse)-like 2 (also referred to as "PSK- 1", UniGene Hs.6314; SEQ ID NOs.1, 2), encodes a 92.5-kDatype I transmembrane protein with an N-terminal signal peptide, five SUSHI domains (SCR repeat), three CUB (initials of the first three identified proteins containing such domains: complement factor Clr/Cls, embryonic sea urchin protein uEGF, and bone morphogenetic protein 1) domains, and a C- terminal transmembrane domain. SEZ6L2 was also identified as a gene highly homologous to mouse SEZ6, a gene first identified in the course of differential screening of mRNA from cortical neurons treated with pentylentetrazole (PTZ), a drug known to induce epileptic seizures (Shimizu-Nishikawa K, et al, (1995) Brain Res MoI Brain Res; 28: 201-10.).

According to a previous study by the present inventors, using a genome-wide cDNA microarray, SEZ6L2 (PSK-I) was identified as a specifically up-regulated gene in non-small cell lung cancer (NSCLC) (WO 2004/031413, e.g. NSC1000/AB105376 of Table 2). Treating and preventing NSCLC: The present invention provides a method for treating, alleviating or preventing

NSCLC in a subject. Therapeutic compounds or compositions may be administered prophylactically or therapeutically to subjects suffering from or at risk of (or susceptible to) developing NSCLC. Such subjects may be identified using standard clinical methods or by detecting an elevated level of SEZ6L2. In the context of the present invention, prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or alternatively delayed in its progression. An exemplary therapeutic method includes a method of inhibiting cancer cell growth by contacting the cancer cell, either in vitro or in vivo, with a composition composed of an SEZ6L2 siRNA that reduces the expression of the SEZ6L2 gene. Alternatively, the therapeutic method may involve treating or preventing NSCLC in a subject by administering to the subject a composition containing an SEZ6L2 siRNA that reduces the expression of SEZ6L2. Small interfering RKAs (siRNA) are composed of a combination of sense strand nucleic acid and antisense strand nucleic acid of the nucleotide sequence encoding SEZ6L2. As used herein, the term "siRNA" refers to a double stranded RNA molecule which prevents translation of a target mRNA. Standard techniques for introducing siRNA into the cell can be used in the treatment or prevention of the present invention, including those in which DNA is a template from which RNA is transcribed. The siRNA may optionally be constructed such that a single transcript has both the sense and complementary antisense sequences from the target gene, e.g., a hairpin.

The therapeutic method of the present invention may be used to suppress expression of the SEZ6L2 gene. Binding of the siRNA to the SEZ6L2 gene transcript in the target cell results in a reduction in the production of the SEZ6L2 protein by the cell. Preferred siRNA of the present invention include the polynucleotides having the nucleotide sequence of SEQ ID NOs: 12 to 14 as the target sequence, both which have been demonstrated to be effective for inhibiting cell growth in NSCLC cell lines. Specifically, a preferred siRNA used in the present invention has the general formula:

5'-[A]-[B]-[A]-3' wherein [A] is a ribonucleotide sequence corresponding to a target sequence of SEZ6L2; [B] is a ribonucleotide sequence consisting of 3 to 23 nucleotides; and [A'] is a ribonucleotide sequence complementary to [A]. Herein, the phrase a "target sequence of SEZ6L2 gene" refers to a sequence that, when introduced into NSCLC cell lines, is effective in suppressing cell viability. Preferred target sequences of the SEZ6L2 gene include the nucleotide sequences of SEQ ID NOs: 12 to 14. The complementary sequence [A'] and [A] hybridize to each other to form a double strand, and the whole siRNA molecule with the general formula 5'-[A]-[B]-[A']-3' forms a hairpin loop structure. As used herein, the term "complementary" refers to a Watson-Crick or Hoogsteen base pairing between nucleotide units of a polynucleotide, and hybridization or binding of nucleotide units indicates physical or chemical interaction between the units under appropriate conditions to form a stable duplex (double-stranded configuration) containing few or no mismatches. In a preferred embodiment, such duplexes contain no more than 1 mismatch for every 10 base pairs. Particularly preferred duplexes are fully complementary and contain no mismatch. The siRNA against the mRNA of the SEZ6L2 gene to be used in the present invention preferably contain a target sequence shorter than the whole mRNA of the SEZ6L2 gene (2855nt), and have a sequence length of 500, 200, or 75 nucleotides. Also included in the present invention is a vector containing one or more of the nucleic acids described herein, and a cell containing such vectors. The isolated nucleic acids of the present invention are useful for siRNA against SEZ6L2 or DNA encoding the siRNA. When the nucleic acids are used for siRNA or coding DNA thereof, the sense strand is preferably longer than 19 nucleotides, and more preferably longer than 21 nucleotides.

The SEZ6L2 siRNAs of the instant invention inhibit the expression of the SEZ6L2 gene and are thereby useful for suppressing the biological activity of the protein and inhibiting cancer cell growth. Therefore, a composition containing an SEZ6L2 siRNA is useful in treating or preventing NSCLC. Pharmaceutical compositions:

The present invention further provides a pharmaceutical composition for treating or preventing NSCLC containing an amount of an active ingredient effective to inhibit the expression of SEZ6L2 or inhibit cancer cell growth. More particularly, the present invention provides compositions containing as the active ingredient an effective amount of an SEZ6L2 siRNA or derivative thereof (e.g., an expression vector).

The active ingredient may be made into an external preparation, such as liniment or a poultice, by mixing with a suitable base material which is inactive against the derivative. Also, as needed, the active ingredient can be formulated into tablets, powders, granules, capsules, liposome capsules, injections, solutions, nose-drops and freeze-drying agents by adding excipients, isotonic agents, solubilizers, preservatives, pain-killers and such. These can be prepared according to conventional methods for preparing nucleic acid containing pharmaceuticals.

Preferably, the siRNA derivative is administered to the patient by direct application to the ailing site or by injection into a blood vessel so that it will, reach the site of ailment. A mounting medium can also be used in the composition to increase durability and membrane- permeability. Examples of suitable mounting mediums include, but are not limited to, liposome, poly-L-lysine, lipid, cholesterol, lipofectin and derivatives thereof.

The precise amount of active ingredient contained within compositions of the present invention will depend upon a number of factors, including the age and sex of the subject, the precise disorder being treated, and its severity. Nevertheless, determination of an optimal effective dose range is well within the capability of those skilled in the art, especially in light of the detailed disclosure provide herein. Accordingly, the dosage of such compositions can be adjusted suitably according to the patient's condition and used in desired amounts. For example, a dose range of 0.1 to 100 mg/kg, preferably 0.1 to 50 mg/kg can be administered. siRNA and vectors encoding it: Transfection of vectors expressing siRNA for SEZ6L2 leads to growth inhibition of

NSCLC cells. Thus, it is another aspect of the present invention to provide a double- stranded molecule composed of a sense-strand and antisense-strand which functions as an siRNA for SEZ6L2, as well as a vector encoding the double-stranded molecule.

A double-stranded molecule of the present invention is composed of a sense strand and an antisense strand, wherein the sense strand is a ribonucleotide sequence corresponding to an SEZ6L2 target sequence and the antisense strand is a ribonucleotide sequence complementary to the sense strand, such that said sense strand and the antisense strand hybridize to each other to form the double-stranded molecule, and wherein the double-stranded molecule, when introduced into a cell expressing an SEZ6L2 gene, inhibits expression of the gene. The double-stranded molecule of the present invention may be a polynucleotide isolated or derived from its original environment (i.e., when it is a naturally occurring molecule, the natural environment), physically or chemically altered from its natural state, or chemically synthesized. According to the present invention, such double-stranded molecules include those composed of DNA, RNA, and derivatives thereof. ADNA is suitably composed of bases such as A, T, C and Q and T is replaced by U in an RNA.

As described above, the term "complementary" refers to a Watson-Crick or Hoogsteen base pairing between nucleotide units of a polynucleotide, and hybridization or binding of nucleotide units indicates physical or chemical interaction between the units under appropriate conditions to form a stable duplex (double-stranded configuration) containing few or no mismatches. In a preferred embodiment, such duplexes contain no more than 1 mismatch for every 10 base pairs. Particularly preferred duplexes are fully complementary and contain no mismatch. A double-stranded molecule of the present invention contains a ribonucleotide sequence corresponding to an SEZ6L2 target sequence, the target sequence being shorter than the whole niRNA of SEZ6L2 gene (2855nt). Herein, the phrase a "target sequence of SEZ6L2 gene" refers to a sequence that, when introduced into NSCLC cell lines, is effective in suppressing cell viability. Specifically, the target sequence is composed of at least about 10, or suitably about 19 to about 25 contiguous nucleotides from the nucleotide sequence of SEQ ID NO: 1. That is, the sense strand of the present double-stranded molecule is composed of at least about 10 nucleotides, suitably is longer than 19 nucleotides, and more preferably longer than 21 nucleotides. Preferred target sequences include the sequences of SEQ ID NOs: 12 to 14. The present double-stranded molecule including the sense strand and the antisense strand is an oligonucleotide shorter than about 100, preferably about 75, more preferably about 50 and most preferably about 25 nucleotides in length. A suitable double-stranded molecule of the present invention is an oligonucleotide having a length ranging from about 19 to about 25 nucleotides. Furthermore, in order to enhance the inhibition activity of the siRNA, the nucleotide "u" can be added to the 3 'end of the antisense strand of the target sequence. The number of "u"s to be added is at least 2, generally 2 to 10, preferably 2 to 5. The added "u"s form single strand at the 3 'end of the antisense strand of the siRNA.

In addition, the double-stranded molecule of the present invention may be a single ribonucleotide transcript composed of the sense strand and the antisense strand linked via a single-stranded ribonucleotide sequence. Namely, the present double-stranded molecule may have the general formula:

5'-[A]-[B]-[A']-3' wherein [A] is a ribonucleotide sequence corresponding to a target sequence of SEZ6L2; [B] is a ribonucleotide sequence (loop sequence) consisting of 3 to 23 nucleotides; and [A'] is a ribonucleotide sequence complementary to [A]. The complementary sequence [A'] and [A] hybridize to each other to form a double strand, and the whole siRNA molecule with the general formula 5'-[A]-[B]-[A']-3' forms a hairpin loop structure.

The region [A] hybridizes to [A], and then a loop consisting of region [B] is formed. The loop sequence can be selected from those described in Ambion's Technical Bulletin #506 entitled "siRNA Design Guidelines" (see http://www.ambion.com/techlib/tb/tb_506.html), or those described in Jacque, JM., eta!., (2002) Nature 418: 435-8. Additional examples of the loop sequence that can be included in the present double-stranded molecules include: CCC, CCACC or CCACACC: Jacque, JM., etal.,(2002) Nature, Vol. 418: 435-8.; UUCG: Lee, NS., et al, (2002) Nature Biotechnology 20: 500-5.; Fruscoloni, P., et al, (2003) Proc. Natl. Acad. Sci. USA 100(4): 1639-44.; and

UUCAAGAGA: Dykxhoorn, DM., et al, (2002) Nature Reviews Molecular Cell Biology 4: 457-467.

Preferable siRNAs having hairpin loop structure of the present invention are shown below. In the following structure, the loop sequence can be selected from the group consisting of: CCC, UUCG, CCACC, CCACACC, and UUCAAGAGA. Among these sequences, a most preferred loop sequence is UUCAAGAGA (corresponding to "ttcaagaga" in a DNA): ccaaccggcugcuucugca -[B]-ugcagaagcagccgguugg (for the target sequence of SEQ ID NO: 12); cuggaagugacccagacca -[B]-uggucugggucacuuccag (for the target sequence of SEQ ID NO: 13); and gcuucagggaaagucccuu -[B]-aagggacuuucccugaagc (for the target sequence of SEQ ID NO: 14);

The present invention further provides a vector encoding a double-stranded molecule of the present invention. The vector encodes a transcript having a secondary structure and is composed of the sense strand and the antisense strand, and which suitably includes a single- stranded ribonucleotide sequence linking the sense strand and the antisense strand. The vector preferably includes a regulatory sequence adjacent to the region encoding the present double-stranded molecule that directs the expression of the molecule in an adequate cell. For example, the double-stranded molecules of the present invention may be intracellularly transcribed by cloning their coding sequence into a vector containing, e.g., a RNApol III transcription unit from the small nuclear RNA (snRNA) U6 or the human Hl RNA promoter. Alternatively, the present vectors may be produced, for example, by cloning the target sequence into an expression vector such that the objective sequence is operatively-linked to a regulatory sequence of the vector in a manner to allow expression thereof (transcription of the DNA molecule) (Lee, NS., et al., (2002) Nature Biotechnology 20: 500-5.). For example, the transcription of an RNA molecule having an antisense sequence to the target sequence may be driven by a first promoter {e.g., a promoter sequence linked to the 3 '-end of the ^■ cloned DNA) and that having the sense strand to the target sequence by a second promoter (e.g., a promoter sequence linked to the 5'-end of the cloned DNA). The expressed sense and antisense strands then hybridize to each other in vivo to generate an siRNA construct that silences a gene containing the target sequence. Furthermore, two constructs (vectors) may be utilized to respectively produce the sense and anti-sense strands of a siRNA construct. For introducing the vectors into a cell, transfection-enhancing agent can be used.

FuGENE (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical) are examples of suitable transfection-enhancing agent.

As used herein, the phrase "control level" refers to an SEZ6L2 expression level associated with a known disease state (e.g., positive prognosis group, early stage, etc.). The control level may correspond to a single measurement associated with a single known sample or to a database of expression patterns identified from previously tested cells.

Herein, in the context of cancer treatment, the term "efficacious" refers to a treatment that leads to a reduction in the expression of SEZ6L2 or a decrease in size, prevalence or metastatic potential of non-small cell lung cancer in a subject. When a treatment is applied prophylactically, "efficacious" means that the treatment retards or prevents occurrence of non-small cell lung cancer or alleviates a clinical symptom of non-small cell lung cancer. The assessment of non-small cell lung cancer can be made using standard clinical protocols. Furthermore, the efficaciousness of a treatment may be determined in association with any known method for diagnosing or treating non-small cell lung cancer. For example, non- small cell lung cancer can be diagnosed histopathologically or by identifying symptomatic anomalies such as chronic cough, hoarseness, coughing up blood, weight loss, loss of appetite, shortness of breath, wheezing, repeated bouts of bronchitis or pneumonia and chest pain.

In the context of assessing the prognosis of a patient with the non-small cell lung cancer, involving the step of comparing the expression level of SEZ6L2 in the patient-derived biological sample with a control level, an increase in the expression level of SEZ6L2 expression indicates a less favorable prognosis. The term "prognosis" refers to a forecast as to the probable outcome of the disease as well as the prospect of recovery from the disease as indicated by the nature and symptoms of the case. Accordingly, a negative or poor prognosis is defined by a lower post-treatment survival term or survival rate. Conversely, a positive or good prognosis is defined by an elevated post-treatment survival term or survival rate.

The present invention is based on the finding that a relatively high expression level of SEZ6L2 (as compared to a control level) is associated with poor prognosis in non-small cell lung cancer (NSCLC) patients. In view of the evidence provided herein, that SEZ6L2 expression is associated with poor prognosis of cancer patients such as lung adenocarcinoma, the present invention provides methods for determining a prognosis for cancer patients. In one embodiment, the method of the present invention includes the steps of: a. detecting SEZ6L2 expression level in a specimen collected from a subject whose NSCLC prognosis is to be predicted, and b. assessing the prognosis of the subject as poor, when the detected SEZ6L2 expression level is elevated as compared to a control level. The present invention provides a method for assessing or determining a prognosis of a lung cancer patient. For the purposes of this invention, the present invention is intended to encompass predictions and likelihood analysis of lung cancer progression, particularly NSCLC recurrence, metastatic spread and disease relapse. The prognostic methods of the present invention are intended to be used clinically in making decisions concerning treatment modalities, including therapeutic intervention, diagnostic criteria such as disease staging, and disease monitoring and surveillance for metastasis or recurrence of neoplastic disease.

NSCLC prognosis and progression of the disease can be predicted by the present invention. In particular, the present invention is useful for predicting or testing the prognosis of lung adenocarcinoma (ADC). In the context of the present invention, NSCLC prognosis is predicted by measuring the expression level of SEZ6L2 in a test population of cells, {i.e., a patient-derived biological sample). Preferably, the test cell population contains an epithelial cell, e.g., a cell obtained from lung tissue. Gene expression can also be measured from blood or other bodily fluids, such as sputum. Other biological samples can be used to determine protein level. For example, the level of protein in blood or serum derived from a subject to be assessed can be measured by immunoassay or other conventional biological assays. These test samples may be obtained from the subject at various points in time, including before, during and/or after the treatment.

In the context of the present invention, expression of SEZ6L2 is determined in the test cell or biological sample and compared to expression level associated with a control sample. In this context, a standard value of SEZ6L2 expression level associated with a good prognosis group may be useful as a control level of the present method. In the present method, when the SEZ6L2 expression level in a sample specimen is high as compared with that of control level, then the sample is deemed to have an elevated level of SEZ6L2 expression. The standard value may be obtained by any method known in the art. For example, a range of mean ± 2 S.D. or mean ± 3 S.D. may be used as standard value. The expression levels of SEZ6L2 in the control samples and the specimen from the subject may be determined at the same time.

Alternatively, a determination of poor prognosis can result when strong staining is observed by immunohistochemical analysis of sample tissue. In order to predict poor prognosis, the strength of staining of the specimen can be assessed by comparing it with a control reagent providing strong staining result. The control reagent may be prepared from SEZ6L2 expressing cells or from tissue whose expression level is controlled to adjust to that of strong staining sample. In the context of the present invention, SEZ6L2 expressing cells may include cells or cell lines derived from tumor. Alternatively, SEZ6L2 expressing cells may be prepared by transfection of suitable host cell with an SEZ6L2 expressing vector. In the context of the present invention, "assessment of prognosis" means that a prognosis of an NSCLC patient is determined. When the SEZ6L2 expression level in a subject sample falls within the range associated with a control sample, the subject is predicted to have good prognosis. Conversely, when the SEZ6L2 expression level in a subject sample exceeds the range associated with a control sample, the subject is predicted to have poor prognosis. For example, an increase in the level of expression of SEZ6L2 in a patient- derived tissue sample as compared to a control sample indicates that the subject has poor prognosis. In other words, when the expression level of SEZ6L2 is closer to the expression level in the good prognosis group, the subject is predicted to have good prognosis.

In the present method, the expression level of SEZ6L2 may be detected by any one of the method selected from the group consisting of: (a) detecting the mRNA encoding the amino acid sequence of SEQ ID NO: 2,

(b) detecting the protein having the amino acid sequence of SEQ ID NO: 2, and

(c) detecting a biological activity of the protein having the amino acid sequence of SEQ ID NO: 2.

In the present invention, the mRNA, the protein, or the biological activity of the protein may be detected using conventional methods. Methods for detecting a given protein, mRNA or biological activity thereof are well known to those skilled in the art. For example, the mRNA may be detected using known PCR or hybridization based technologies. Alternatively, any immunoassay format may be applied to detect the protein. The biological activity of SEZ6L2 can be also determined using any suitable method.

In the present invention, assessment of a poor prognosis may be used to determine further treatment, e.g., to stop further treatments that reduce quality of life, to treat the cancer in a different manner than previously used or to treat the cancer more aggressively.

Accordingly, the assessment of prognosis using SEZ6L2 expression levels as a indicator will enable clinicians to choose, in advance, the most appropriate treatment for each individual NSCLC patient, without requiring the information of conventional clinical staging of the disease and using only routine procedures for tissue-sampling. The present invention also provides kits for assessing an NSCLC prognosis, such kits including SEZ6L2-detection reagents. For example, in the context of the present invention, the SEZ6L2 detecting reagent may include any one or more component selected from the group consisting of:

(a) a reagent for detecting the mRNA encoding the amino acid sequence of SEQ ID NO: 2,

(b) a reagent for detecting the protein having the amino acid sequence of SEQ ID NO: 2, and

(c) a reagent for detecting the biological activity of the protein having the amino acid sequence of SEQ ID NO: 2. Suitable SEZ6L2-detection reagents include nucleic acids that specifically bind to or identify an SEZ6L2 nucleic acid, such as oligonucleotide sequences which are complementary to a portion of the SEZ6L2 nucleic acid sequence or antibodies that bind to proteins encoded by an SEZ6L2 nucleic acid.

The SEZ6L2-detection reagents may be packaged together in the form of a kit. For example, the reagents may be packaged in separate containers, e.g., a nucleic acid or antibody (either bound to a solid matrix or packaged separately with reagents for binding it to the matrix) in one container, a control reagent (positive and/or negative) in a second container, and/or a detectable label in a third container. Tissue samples obtained from normal lung, a lung cancer subject with good prognosis, and a lung cancer subject with poor prognosis are useful as control reagents in the context of the present invention. Furthermore, SEZ6L2 expressing cells may also be prepared by transfecting a suitable host cell with an SEZ6L2 expressing vector. The transformant expressing SEZ6L2 can be used as control reagent. In particular, a transformant showing the same SEZ6L2 expression level as that of a lung cancer sample associated with good prognosis, and a lung cancer sample with poor prognosis are preferable control reagents useful in the comparison step of the present invention. The control instructions (e.g., written, tape, CD-ROM, etc.) for carrying out the assay may also be included in the kit. The assay format of the kit may be a Northern hybridization or a sandwich ELISA, both of which conventional in the art.

For example, an SEZ6L2 detection reagent may be immobilized on a solid matrix, such as a porous strip, to form at least one SEZ6L2 detection site. The measurement or detection region of the porous strip may include a plurality of sites, each containing a nucleic acid. A test strip may also contain sites for negative and/or positive controls. Alternatively, control sites may be located on a strip separate from the test strip. Optionally, the different detection sites may contain different amounts of immobilized nucleic acids, i.e., a higher amount in the first detection site and lesser amounts in subsequent sites. Upon the addition of test sample, the number of sites displaying a detectable signal provides a quantitative indication of the amount of SEZ6L2 present in the sample. The detection sites may be configured in any suitably detectable shape and are typically in the shape of a bar or dot spanning the width of a test strip.

Another a well known technique for evaluating the level of a protein in a tissue sample is immunohistochemical analysis. For example, an SEZ6L2 detection reagent, for example an anti-SEZ6L2 antibody (first antibody), may be labeled with signal generating molecules via direct linkage or an indirect labeling technique. Anti- immunoglobulin antibody recognizing the first antibody can be used as the second antibody for indirect labeling technique of the first antibody. The second antibody may be labeled with suitable signal generating molecule or binding ligand, such as biotin. Any enzymes, chromophore, fluorophore, and luminophore can be used as signal generating molecule for the immunohistochemical analysis. The biotin ligand further may bind avidin-peroxidase.

The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control.

Examples Hereinafter, the present invention is described in detail with reference to Examples.

However, materials, methods and such described therein only illustrate aspects of the invention and in no way are intended to limit the scope of the present invention. As such, materials, methods and such similar or equivalent to those described therein may be used in the practice or testing of the present invention.

[Example 1] General Methods

(1) Cell lines and Clinical Samples

The 23 human lung-cancer cell lines used in the present application included nine adenocarcinomas (ADCs; A427, A549, LC319, NCI-H1373, PC-3, PC-9, PC-14, NCI-H1666, and NCI-H1781), nine squamous-cell carcinomas (SCCs; EBC-I, LU61, NCI-H520, NCI- H1703, NCI-H2170, RERF-LC-AI, SK-MES-I, NCI-H226, and NCI-H647), one large-cell carcinoma (LCC; LXl), and four small-cell lung cancers (SCLCs; DMS 114, DMS273, SBC-3, and SBC-5). A human bronchial epithelial cell line, BEAS2B (American Type Culture Collection; ATCC) was also included in the panel of the cells used in the present application. All cells were grown in monolayers in appropriate media supplemented with 10% fetal calf serum (FCS) and were maintained at 37⁰C in an atmosphere of humidified air with 5% CO₂. Surgically-resected primary NSCLC samples were obtained earlier with informed consent (Kikuchi T, et a/., (2003) Oncogene; 22: 2192-205.). A total of 420 formalin-fixed samples of primary NSCLCs (stage I-IIIA) including 263 ADCs, 116 SCCs, 28 LCCs, 13 adenosquamous carcinomas (ASCs) and adjacent normal lung tissues, had been obtained earlier along with clinicopathological data from patients undergoing surgery. ADCs were also classified into two groups: 129 mixed subtypes with bronchioloalveolar-cell carcinoma (BAC) components and 134 unmixed subtypes without BAC (non-BAC). SCLCs from postmortem materials (20 individuals) were used in the present application. NSCLC specimen and five tissues (heart, liver, lung, kidney, and pancreas) from post-mortem materials (2 individuals with ADC) were also obtained. The present application and the use of all clinical materials obtained with written informed consent were approved by the Institutional Research Ethics Committees. The histological classification of the tumor specimens was performed by the WHO criteria (Travis WD, et al. Histological Typing of Lung and Pleural Tumors: World Health Organization International Histological Classification of Tumors, 3rd edn. Berlin: Springer, 1999,). The postsurgical pathologic tumor-node-metastasis stage was determined according to the guidelines of the American Joint Committee on Cancer (Sobin LH, Wittekind CH, editors. UICC TNM classification of malignant tumors, 5th ed. New York: John Wiley, 1997.).

(2) Semi-quantitative RT-PCR analysis

Total RNA was extracted from cultured cells and clinical tissues using Trizol reagent (Life Technologies, Inc. Gaithersburg, MD, USA) according to the manufacturer's protocol. . Extracted RNAs and normal human-tissue polyA RNAs were treated with DNase I (Roche Diagnostics, Basel, Switzerland) and then reversely-transcribed using oligo (dT)₁₂_₁₈ primer and Superscript II reverse transcriptase (Life Technologies, Inc.). Semi-quantitative RT- PCR experiments were carried out with synthesized SEZ6L2 gene-specific primers (5 '-GGGAGTATGAAGTTTCCATCTG-S ' (SEQ ID NO.3) and

5'-GGATGCTGGTTTATTTACTGTAGG-S ' (SEQ ID NO.4)), or with beta-actin

primers (5'-ATCAAGATCATTGCTCCTCCT-S ' (SEQ ID N0.5) and 5'-CTGCGCAAGTTAGGTTTTGT-S' (SEQ ID NO.6)) as an internal control. All PCR reactions involved initial denaturation at 94⁰C for 2 min followed by 22 (for

ACTB) or 30 cycles (for SEZ6L2) of 94⁰C for 30 s, 54 - 6O⁰C for 30 s, and 72⁰C for 60 s on a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA).

(3) Northern-blot analysis.

Human multiple-tissue blot (16 normal tissues including heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood leukocyte; BD Biosciences Clontech, Palo Alto, CA, USA) was hybridized with a ³²P-labeled PCR product of SEZ6L2. The cDNA probes of SEZ6L2 were prepared by RT-PCR using primers, 5¹-GCTATGAGGGCTTTGAGCTTATC-3^l (SEQ ID NO.7) and S'-AGAAGCAAAGGTGGAGAGACTGT-S' (SEQ ID NO.8). Pre-hybridization, hybridization, and washing were performed according to the supplier's recommendations. The blots were autoradiographed with intensifying screens at -8O⁰C for one week. (4) Preparation of anti-SEZ6L2 polyclonal antibody

Rabbit antibodies specific for the extracellular portion of SEZ6L2 were raised by immunizing rabbits with 6-histidine fused human SEZ6L2 protein (codons 737-787; GenBank Accession No. NM_012410), and purified with standard protocols using affinity columns (AfFi-gel 10; Bio-Rad Laboratories, Hercules, CA, USA) conjugated with the 6-histidine fused protein. On western blots, the antibody was confirmed to be specific to SEZ6L2 using lysates from NSCLC tissues and cell lines as well as normal lung tissues.

(5) Western-blot analysis

Cells and tissues were lysed in lysis buffer; 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% NP-40, 0.5% deoxycholate-Na, 0.1% SDS, plus protease inhibitor (Protease Inhibitor Cocktail Set III; Calbiochem Darmstadt, Germany). An ECL western-blotting analysis system was used (GE Healthcare Bio-sciences, Piscataway, NJ), as previously described (Kato T, et al, (2005) Cancer Res; 65: 5638-46. Furukawa C, et al, (2005) Cancer Res; 65: 7102-10.). SDS-PAGE was performed in 7.5% polyacrylamide gels. PAGE-separated proteins were electroblotted onto nitrocellulose membranes (GE Healthcare Bio-sciences) and incubated with a rabbit polyclonal anti-human SEZ6L2 antibody. A goat anti-rabbit IgG- HRP antibody (GE Healthcare Bio-sciences) was utilized as the secondary antibodies for these experiments.

(6) Flow-cytometric analysis Lung-cancer cells (1 x 10⁶ cells) were incubated with a rabbit polyclonal anti-human

SEZ6L2 antibody for detecting the extracellular domain of the protein (0.34 mg/ml) or control rabbit IgG (0.34 mg/ml; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4⁰C for 1 hour. The cells were washed in PBS and then incubated with AlexaFluor 488-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR, USA) at 4°C for 30 min. The cells were washed in PBS and analyzed on a FACScan flow cytometer (Becton Dickinson Labware, Bedford, MA, USA) and analyzed by ModFit software (Verity Software House, Inc., Topsham, ME, USA).

(7) Immunohistochemistry and tissue microarray

Tumor-tissue microarrays were constructed using 440 formalin-fixed primary lung cancers (420 NSCLCs and 20 SCLCs), according to the method published previously (Callagy G, et al, (2003) Diagn MoI Pathol; 12: 27-34. Callagy G, et al, (2005) J Pathol; 20S: 388-96, Chin SF, et al, (2003) MoI Pathol; 56: 275-9.). The tissue area for sampling was selected by visual alignment with the corresponding HE-stained section on a slide. Three, four, or five tissue cores (diameter 0.6 mm; height 3-4 mm) taken from a donor tumor block were placed into a recipient paraffin block using a tissue microarrayer (Beecher Instruments, Sun Prairie, WI, USA). A core of normal tissue was punched from each case, and 5-μm sections of the resulting microarray block were used for immunohistochemical analysis.

To investigate the presence of SEZ6L2 protein in clinical samples that had been embedded in paraffin blocks, the sections were stained as previously described (Ishikawa N, et al, (2004) Clin Cancer Res; 10: 8363-70. Kato T, et al, (2005) Cancer Res; 65: 5638-46. Furukawa C, et al. (2005) Cancer Res; 65: 7102-10. Ishikawa N, et al, (2005) Cancer Res; 65: 9176-84. Suzuki C, et al, (2005) Cancer Res; 65: 11314-25.). Briefly, 16.25 μg/ml of a rabbit polyclonal anti- human SEZ6L2 antibody was added after blocking of endogenous peroxidase and proteins. The sections were then incubated with HRP-labeled anti-rabbit IgG as the secondary antibody. Substrate-chromogen was added and the specimens were counterstained with hematoxylin.

Three independent investigators assessed SEZ6L2 positivity semi-quantitatively without prior knowledge of clinicopathological data. The intensity of SEZ6L2 staining was evaluated using following criteria: strong positive (2+), dark brown staining in more than 50% of tumor cells completely obscuring membrane and cytoplasm; weak positive (1+), any lesser degree of brown staining appreciable in tumor cell membrane and cytoplasm; absent (scored as 0), no appreciable staining in tumor cells. Cases were accepted only as strongly positive if reviewers independently defined them as such.

(8) Statistical analysis Statistical analyses were performed using the StatView statistical program (SAS, Cary,

NC, USA). Contingency tables were used to analyze the relationship between SEZ6L2 expression and clinicopathological variables in NSCLC patients. Tumor-specific survival curves were calculated from the date of surgery to the time of death related to NSCLC, or to the last follow-up observation. Kaplan-Meier curves were calculated for each relevant variable and for SEZ6L2 expression; differences in survival times among patient subgroups were analyzed using the log-rank test. Univariate and multivariate analyses were performed with the Cox proportional-hazard regression model to determine associations between clinicopathological variables and cancer-related mortality. First, associations between death and possible prognostic factors were analyzed including age, gender, histological type, pT- classification, and pN-classification, taking into consideration one factor at a time. Second, multivariate Cox analysis was applied on backward (stepwise) procedures that always forced strong SEZ6L2 expression into the model, along with any and all variables that satisfied an entry level of a P value less than 0.05 As the model continued to add factors, independent factors did not exceed an exit level of P < 0.05.

(9) RNA interference assay Using the vector-based RNA interference (RNAi) system, psiHlBX3.0, which the present inventors had established earlier to direct the synthesis of siRNAs in mammalian cells (Suzuki C, et al. , (2003) Cancer Res; 63 : 7038-41. Kato T, et al., (2005) Cancer Res; 65 : 5638-46. Furukawa C, et al, (2005) Cancer Res; 65: 7102-10. Suzuki C, et a/., (2005) Cancer Res; 65: 11314-25.), 10 μg of siRNA-expression vector with 30 μl ofLipofectamine 2000 (Invitrogen) were transfected into two NSCLC cell lines (A549, LC319) that endogenously over-expressed SEZ6L2. The transfected cells were cultured for five days in the presence of appropriate concentrations of geneticin (G418). Cell numbers and viability were measured by Giemsa staining and MTT assay in triplicate. The target sequences of the synthetic oligonucleotides for RNAi were as follows: control 1 (EGFP: enhanced green fluorescent protein (GFP) gene, a mutant of

Aequorea victoria GFP), 5'-GAAGCAGCACGACTTCTTC-S' (SEQ ID NO.9); control-2 (LUC, luciferase gene from Photinus pyralis), 5'-CGTACGCGGAATACTTCGA-3'(SEQ ID NO lO); control3 (Scramble: Chloroplast Euglena gracilis gene coding for the 5S and 16S rRNA), 5'-GCGCGCTTTGTAGGATTCG-S ' (SEQ ID NO.ll); siRNA-SEZ6L2-l (si-1), 5'-CCAACCGGCTGCTTCTGCA-S ' (SEQ ID NO.12); siRNA-SEZ6L2-2 (si-2), '5-CTGGAAGTGACCCAGACCA-S ' (SEQ ID NO.13); siRNA-SEZ6L2-3 (si-3), '5-GCTTCAGGGAAAGTCCCTT-S ' (SEQ ID NO.14). To validate the instant RNAi system, individual control siRNAs were tested by semi- quantitative RT-PCR to confirm the decrease in expression of the corresponding target genes that had been transiently transfected to A549 or LC319 cells. Down-regulation OΪSEZ6L2 expression by functional siRNA, but not by controls, was also confirmed in the cell lines used for this assay.

[Example 2] SEZ6L2 expression in lung tumors, cell lines, and normal tissues

To search for novel target molecules for development of therapeutic agents and/or diagnostic markers for NSCLC, genes that showed more than a 3 -fold higher level of expression in cancer cells than in normal cells, in half or more of the 37 NSCLCs analyzed by cDNA microarray, were first screened (Kikuchi T, et al, (2003) Oncogene; 22: 2192-205.). Among the 23,040 genes screened, the SEZ6L2 transcript was identified as a good candidate (3-fold or higher expression in 81% of the NSCLC cases), and confirmed its transactivation by semi-quantitative RT-PCR experiments in 12 of 15 additional lung-cancer tissues and in 19 of 23 lung-cancer cell lines (NSCLC and SCLC samples), while its expression in normal lung tissue cells or a human bronchial epithelial cell line, BEAS2B, was hardly detectable (Fig. IA, B)

A rabbit polyclonal antibody specific to human SEZ6L2 was subsequently generated and confirmed by western-blot analysis an expression of SEZ6L2 protein in 4 cancer cell lines of lung, in which the SEZ6L2 transcript had been detected at a high level (Fig. 1C). No band was found in two cell lines, which expressed no SEZ6L2 transcript. As SEZ6L2 was suggested to be a type I membrane protein, SEZ6L2 expression on the surfaces of lung-cancer cells was validated using flow-cytometry with anti-SEZ6L2 polyclonal antibody. This analysis indicated that the antibody bound to A549 and EBC-I cells, in which SEZ6L2 transcript had been detected at a high level, but not to NCI-H647 cells, which had not expressed SEZ6L2 (Fig. ID). The expression of the SEZ6L2 protein in NSCLC tissues was also examined using the same antibody. Western-blot analysis revealed the increased SEZ6L2 protein expression in tumor tissues in representative pairs of ADC samples analyzed (Fig. 2A). Immunohistochemical analysis of tumor tissues detected positive staining for SEZ6L2 specifically in cancer cells in 7 of the ten NSCLC cases examined, but the staining was hardly detectable in surrounding normal lung epithelial cells (Fig. 2B-D). Interestingly, the invasive border of the tumor adjacent to the non-cancerous cells showed the tendency of strong staining. SEZ6L2 localized at the plasma membrane as well as in the cytoplasm of tumor cells (Fig. 2E-G).

Northern-blot analysis using human SEZ6L2 cDNA as a probe detected a 3.2-kb transcript of weak signal only in brain, pancreas, prostate, and testis among the 16 normal human tissues (data not shown). The expression of the SEZ6L2 protein was also examined with anti-SEZ6L2 antibody on five normal tissues (heart, liver, lung, kidney, and pancreas), and it was found to be hardly detectable in these tissues (Fig. 3A-E) while positive SEZ6L2 staining appeared in lung tumor tissues (Fig. 3F).

[Example 3] Association of SEZ6L2 expression with poor prognosis of NSCLC patients

To verify the biological and clinicopathological significance of SEZ6L2, the expression of the SEZ6L2 protein was additionally examined by means of tissue microarrays containing lung-cancer tissues from 440 patients. A pattern of SEZ6L2 expression on the tissue array was classified as ranging from absent (scored as 0) to weak/strong positive

(scored as 1+ ~ 2+). Of the 420 NSCLC cases examined, SEZ6L2 was strongly stained in 31 (7.4%; score 2+), weakly stained in 296 (70.5%; score 1+), and not stained in 93 cases (22.1%; score 0) (details are shown in Table 1). Weak positive staining (score 1+) was observed in 65% (13 of 20) of SCLC cases examined. As shown in Table 1, gender (higher in female; P = 0.007 by Fisher's exact test) and histological type (higher in ADC; P < 0.001 by Fisher's exact test) were significantly associated with the SEZ6L2 positivity (score 1+ ~ 2+). The median survival time of NSCLC patients was significantly related to the expression levels of SEZ6L2 (3172 days in score 0 cases, 2346 days in 1+, and 1134 days in 2+; P = 0.0209 by log-rank test; Fig. 4). By univariate analysis, pT stage (Tl, T2 vs T3, 4), pN stage (NO vs Nl, N2), age (< 65 vs > 65), histological classification (ADC versus other histological types), and strong SEZ6L2 positivity (score 0, 1+ vs 2+) were all significantly related to poor tumor-specific survival among NSCLC patients (P = O.0001, <0.0001, 0.0038, 0.0027, 0.0102, and 0.0138, respectively, Table. 2). In multivariate analysis of the prognostic factors, pT stage, pN stage, age, and strong SEZ6L2 expression were indicated to be an independent prognostic factor (P = 0.0001, < 0.0001, < 0.0001, 0.0144, respectively; Table. 2).

Table 1. Association between SEZ6L2-positivity in NSCLC tissues and patients' characteristics (n=420)

SEZ6L2 SEZ6L2 _SEZ6L2 P-value Total strong weak absent strong/weak positive positive vs absent n = 420 n = 31 n = 296 n = 93

Gender Male 290 20 195 75

0.007 ⁺

Female 130 11 101 18

Age (years)

<65 207 15 147 45

NS

≥65 213 16 149 48

Histological type

ADC 263 24 198 41

SCC 116 3 77 36 <0.001

Others 41 4 21 16 pT factor

T1+T2 301 22 212 67

NS

T3+T4 119 9 84 26 pN factor

NO 259 14 180 65

NS

N1+N2 161 17 116 28

Smoking history

Never smoker 129 10 96 23

NS

Smoker 291 21 200 70

ADC, adenocarcinoma; SCC, squamous-cell carcinoma Others, large-cell carcinoma plus adenosquamous-cell carcinoma *ADC versus other histology ⁺P < 0.05 (Fisher's exact test) NS, no significance

Table 2. Cox's proportional hazards model analysis of prognostic factors in patients with NSCLCs

Hazards

Variables 95% Cl Unfavorable/Favorable P-value ratio

Univariate analysis

SEZ6L2 1.789 1.126-2.841 Strong(+) / Weak(+) or (-) 0.0138 *

Age ( years ) 1.520 1.145-2.018 65 ≥ / <65 0.0038*

Gender 1.640 1.187-2.265 Male / Female 0.0027 *

Histological type 1.444 1.091-1.912 others / ADC¹ 0.0102* pT factor 1.889 1.411-2.528 T3+T4 / T1+T2 <0.0001 * pN factor 2.930 2.197-3.908 N1+N2 / N0 <0.0001 *

Multivariate analysis

SEZ6L2 1.814 1.126-2.922 Strong(+) / Weak(+) or (-) 0.0144*

Age ( years ) 1.930 1.442-2.581 65 ≥ / <65 O.0001*

Gender 1.413 0.989-2.019 Male / Female 0.0572*

Histological type 1.184 0.861-1.628 others / ADC¹ 0.2982 pT factor 1.787 1.329-2.404 T3+T4 / T1+T2 0.0001 * pN factor 2.356 1.761-3.153 N1+N2 / N0 <0.0001 *

¹ ADC, adenocarcinoma ⁺P < 0.05 [Example 4] Inhibition of endogenous SEZ6L2 expression by siRNA in NSCLCs

To assess whether up-regulation of SEZ6L2 plays a role in growth or survival of lung cancer cells, three independent plasmids designed to express siRNA against SEZ6L2 (si-1, si- 2, and si-3) were constructed, along with three different control plasmids (siRNAs for EGFP, LUC and Scramble). The treatment of NSCLC cells with the three effective and specific siRNAs reduced expression of SEZ6L2, but did not suppress cell growth significantly (data not shown), suggesting that up-regulation of SEZ6L2 is not directly related to growth or survival of cancer cells.

Industrial Applicability

The utility of SEZ6L2 as a prognostic indicator of lung cancers is demonstrated herein. As such, the present invention provides method for assessing or determining a non-small cell lung cancer (NSCLC) prognosis in a subject in need thereto. Accordingly, the present invention enables clinicians to choose, in advance, the most appropriate treatment for each individual NSCLC patient, even without the information of conventional clinical staging of the disease and using only routine procedures for tissue-sampling.

The present invention further describes SEZ6L2 siRNAs and method of using same to inhibit cancer cell growth. Accordingly, the present invention provides methods for treating or preventing lung cancer, particularly NSCLC, using such siRNAs, as well as derivatives and pharmaceutical formulations thereof.

All publications, databases, sequences, patents, and patent applications cited herein are herby incorporated by reference.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention, the metes and bounds of which are set by the appended claims.

Claims

1. A method of treating or preventing NSCLC in a subject comprising administering to said subject an SEZ6L2 small interfering RNA (siRNA) composition.

2. The method of claim 1, wherein the SEZ6L2 siRNA composition comprises an siRNA having a nucleotide sequence targeting the sequence selected from any one of SEQ ID

NOs: 12 to 14.

3. The method of claim 2, wherein the siRNA has the general formula

5'-[A]-[B]-[A']-3' wherein [A] is a ribonucleotide sequence corresponding to a nucleotide sequence selected from any one of SEQ ID NOs: 12 to 14; [B] is a ribonucleotide sequence consisting of 3 to 23 nucleotides; and [A] is a ribonucleotide sequence complementary to [A].

4. A double-stranded molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises a ribonucleotide sequence corresponding to an SEZ6L2 target sequence selected from any one of SEQ ID NOs: 12 to 14, and wherein the antisense strand comprises a ribonucleotide sequence which is complementary to said sense strand, wherein said sense strand and said antisense strand hybridize to each other to form said double- stranded molecule, and wherein said double-stranded molecule, when introduced into a cell that expresses the an SEZ6L2 gene, inhibits expression of said gene.

5. The double-stranded molecule of claim 4, wherein said SEZ6L2 target sequence comprises at least about 10 contiguous nucleotides from the nucleotide sequence of SEQ ID NO: 1.

6. The double-stranded molecule of claim 5, wherein said SEZ6L2 target sequence comprises from about 19 to about 25 contiguous nucleotides from the nucleotide sequence of SEQ ID NO: 1.

7. The double-stranded molecule of claim 4, wherein said double-stranded molecule is a single ribonucleotide transcript comprising the sense strand and the antisense strand linked via a single-stranded ribonucleotide sequence.

8. The double-stranded molecule of claim 4, wherein the double- stranded molecule is an oligonucleotide of less than about 100 nucleotides in length.

9. The double-stranded molecule of claim 8, wherein the double-stranded molecule is an oligonucleotide of less than about 75 nucleotides in length.

10. The double-stranded molecule of claim 9, wherein the double-stranded molecule is an oligonucleotide of less than about 50 nucleotides in length.

11. The double-stranded molecule of claim 10, wherein the double-stranded molecule is an oligonucleotide of less than about 25 nucleotides in length.

12. The double-stranded polynucleotide of claim 11, wherein the double stranded molecule is an oligonucleotide of between about 19 and about 25 nucleotides in length.

13. A vector encoding the double-stranded molecule of claim 4.

14. The vector of claim 13, wherein the vector encodes a transcript having a secondary structure and comprises the sense strand and the antisense strand.

15. The vector of claim 14, wherein the transcript further comprises a single-stranded ribonucleotide sequence linking said sense strand and said antisense strand.

16. A vector comprising a polynucleotide comprising a combination of a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises the nucleotide sequence selected from any one of SEQ ID NOs: 12 to 14, and said antisense strand nucleic acid consists of a sequence complementary to the sense strand.

17. The vector of claim 16, wherein said polynucleotide has the general formula 5'-[A]-[B]-[A]-3' wherein [A] is a nucleotide sequence selected from any one of SEQ ID NOs: 12 to 14; [B] is a nucleotide sequence consisting of 3 to 23 nucleotides; and [A'] is a nucleotide sequence complementary to [A].

18. A pharmaceutical composition for treating or preventing NSCLC comprising a pharmaceutically effective amount of an SEZ6L2 small interfering RNA (siRNA) as an active ingredient, and a pharmaceutically acceptable carrier.

19. The pharmaceutical composition of claim 18, wherein the SEZ6L2 siRNA comprises a nucleotide sequence targeting the sequence selected from any one of SEQ ID NOs: 12 to 14.

20. The pharmaceutical composition of claim 19, wherein the siRNA has the general formula 5'-[A]-[B]-[A]-3' wherein [A] is a ribonucleotide sequence corresponding to a nucleotide sequence selected from any one of SEQ ID NOs: 12 to 14; [B] is a ribonucleotide sequence consisting of 3 to 23 nucleotides; and [A'] is a ribonucleotide sequence complementary to [A].

21. A method for assessing non-small cell lung cancer (NSCLC) prognosis in a subject whose NSCLC prognosis is to be predicted, wherein the method comprises the steps of: a. detecting SEZ6L2 expression level in a specimen collected from said subject, and b. assessing the prognosis of the subject as poor when the detected SEZ6L2 expression level is elevated as compared to a control level.

22. The method of claim 21, wherein the NSCLC is lung adenocarcinoma.

23. The method of claim 21, wherein the SEZ6L2 expression level in the specimen is detected by: a. contacting the specimen with an antibody recognizing the SEZ6L2 protein; and b. detecting the antibody bound to the specimen.

24. A kit for assessing the prognosis of non-small cell lung cancer (NSCLC) comprising an SEZ6L2-detection reagent selected from the group consisting of: a. a reagent for detecting mRNA encoding the amino acid sequence of SEQ ID

NO: 2, b. a reagent for detecting the protein comprising the amino acid sequence of SEQ ID NO: 2, and c. a reagent for detecting the biological activity of the protein comprising the amino acid sequence of SEQ ID NO: 2.

25. The kit of claim 24, wherein the reagent for detecting the protein comprising the amino acid sequence of SEQ ID NO: 2 is anti-SEZ6L2 antibody.