WO2016133860A1 - Cancer biomarkers and methods of use thereof - Google Patents

Abstract

Methods and compositions for the prognosis and diagnosis of cancer, particularly prostate cancer or HPV-associated cancer, are disclosed. In accordance with one aspect of the instant invention, methods of providing a diagnosis and/or prognosis for cancer, particularly prostate cancer or HPV-associated cancers, in a subject are provided. In a particular embodiment, the method comprises determining the cellular localization and/or expression of Ecd (e.g., Ecd protein) in a biological sample obtained from the subject, wherein overexpression of Ecd (e.g., compared to a healthy biological sample (i.e., non-cancerous)), particularly cytoplasmic Ecd, is indicative of a poor prognosis.

Description

CANCER BIOMARKERS AND METHODS OF USE THEREOF

This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62/116,599, filed on February 16, 2015 and U.S. Provisional Patent Application No. 62/251,207, filed on November 5, 2015. The foregoing applications are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the field of cancer. More specifically, the invention provides compositions and methods for the identification, prognosis, diagnosis, the selection of treatment modalities, and/or treatment of cancer.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

Precisely regulated cell proliferation is essential for embryonic development as well as homeostasis in adult organs and tissues, whereas uncontrolled cell proliferation is a hallmark of cancer (Hanahan et al. (2000) Cell 100:57-70). A more in-depth understanding of the regulatory controls of cell cycle progression is therefore of great interest.

The Ecd gene was originally inferred from studies of Drosophila

melanogaster ecdysoneless (or ecd) mutants that exhibit defective development due to reduced production of the steroid hormone ecdysone (Garen et al. (1977) Proc. Natl. Acad. Sci., 74:5099-5103). Subsequent cloning of drosophila ecd helped identify a cell autonomous role of the Ecd protein in cell survival aside from its non-cell autonomous role in ecdysis (molting) (Gaziova et al. (2004) Development 131 :2715- 2725). However, the molecular basis of how Ecd functions remains unknown (Gaziova et al. (2004) Development 131 :2715-2725). The human Ecd homologue was initially identified in a screen of human open reading frames that complemented the S. cerevisiae mutants lacking Gcr2 (Glycolysis regulation 2) gene, and it rescued the growth defect caused by reduced glycolytic enzyme activity in Gcr2 mutants. The human gene was initially designated as HSGT1 (human suppressor of Gcr2), and was suggested to function as a co-activator of glycolytic gene transcription (Sato et al. (1999) Mol. Gen. Genet., 260:535-540). However, Ecd protein bears no structural homology to Gcr2 and a true Ecd orthologue is absent in S. cerevisiae, suggesting that Ecd likely functions by distinct mechanisms.

Human Ecd was identified in a yeast two-hybrid screen of human mammary epithelial cell cDNA-encoded proteins for novel binding partners of the Human Papilloma virus 16 (HPV16) E6 oncogene (Zhang et al. (2006) Cancer Res., 66:7167- 7175). Deletion of the Ecd gene in mice causes embryonic lethality, thereby identifying an essential role of Ecd during early embryonic development (Kim et al. (2009) J. Biol. Chem, 284:26402-26410). Notably, Cre-mediated conditional deletion of Ecd in Ecd^a mouse embryonic fibroblasts (MEFs) led to a Gl/S cell cycle arrest. This phenotype can be rescued by ectopic expression of human Ecd (Kim et al. (2009) J. Biol. Chem., 284:26402-26410), indicating an essential role of Ecd in promoting cell cycle progression. Ecd can interact with the retinoblastoma

(RB) protein and reduces the repression of RB on E2F transcription factors, providing a novel mechanism by which Ecd functions as a positive factor of mammalian cell cycle progression (Kim et al. (2009) J. Biol. Chem., 284:26402-26410). Recently, Ecd was shown to play a vital role in pre-mRNA splicing by interacting with the splicing factor pre-mRNA-processing-splicing factor 8 (PRPF8) (Claudius et al.

(2014) PLoS Genet., 10:el004287). Ecd shuttles between nucleus and the cytoplasm, with a predominantly cytoplasmic steady-state localization due to rapid nuclear export (Kim et al. (2010) Biol. Chem., 391 :9-19; Claudius et al. (2014) PLoS Genet, 10:el004287). Consistent with these key cellular roles of Ecd, Ecd has been found to be significantly overexpressed in breast and pancreatic cancers, and its overexpression correlates positively with poor prognostic factors and poor patient survival (Zhao et al. (2012) Breast Cancer Res. Treat, 134: 171-781; Dey et al. (2012) Clin. Cancer Res., 18:6188-6198). Further understanding of the mechanism of Ecd function and its role in other cancers is important for developing further diagnostic and treatment methods.

SUMMARY OF THE INVENTION

In accordance with one aspect of the instant invention, methods of providing a diagnosis and/or prognosis for cancer, particularly prostate cancer or HPV-associated cancers, in a subject are provided. In a particular embodiment, the method comprises determining the cellular localization and/or expression of Ecd (e.g., Ecd protein) in a biological sample obtained from the subject, wherein overexpression of Ecd (e.g., compared to a healthy biological sample (i.e., non-cancerous)), particularly cytoplasmic Ecd, is indicative of a poor prognosis. The method may further comprise detecting at least one other cancer marker (e.g., in the same biological sample, in an adjacent or analogous biological sample, and/or blood or blood-derived sample (e.g., serum)).

In accordance with another aspect of the instant invention, kits are provided. The kits may be used for the diagnosis and/or prognosis of cancer, particularly prostate cancer or HPV-associated cancers, in a subj ect. In a particular embodiment, the kit comprises a first composition comprising at least one Ecd antibody and at least one second composition comprising at least one agent for detecting another cancer marker. In a particular embodiment, the agent of the second composition is an antibody immunologically specific for another cancer marker. In a particular embodiment, the Ecd antibody is immunologically specific for modified (e.g., post- translationally modified (e.g., phosphorylated)) or unmodified forms of Ecd.

In accordance with one aspect of the instant invention, methods for identifying and/or selecting optimal anti-cancer treatments for cancer, particularly prostate cancer or HPV-associated cancers, in a subj ect are provided. In a particular embodiment, the method comprises determining the cellular localization and/or the level of expression of Ecd in a biological sample obtained from the subject and determining an appropriate anti-cancer therapy to administer. The methods may further comprise detecting at least one other cancer marker in the same biological sample or in an adjacent or analogous biological sample. The methods may further comprise administering the selected anti-cancer therapy to the subject. Examples of therapies include, without limitation, the administration of at least one chemotherapeutic agent, treating the subject with radiation, and/or resecting cancerous cells/tissue from the subject. In a particular embodiment, the biological sample is a tumor biopsy. In a particular embodiment, the biological sample is a blood or fraction thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 provides images of immunohistochemical analyses of Ecd in prostate tissue. Tissue sections were stained with primary monoclonal antibodies against Ecd. Samples are hyperplastic normal prostate tissue (left), prostatic intraepithelial neoplasia (center), and prostate cancer (right). Magnification 20X.

Figure 2 provides a Kaplan-Meier survival curve relating to Ecd expression. The graph shows the survival of male patients within prostate cancer cohort is dependent on the level of Ecd expression. High level of cytoplasmic Ecd correlates with poor overall survival.

Figures 3A-3E: Ecd expression in cervical cancer cell lines and tissue array. Figure 3 A: Western blots of ly sates of two independent primary human foreskin keratinocytes (4FKC and lOFKC) and cervical cancer cell lines, β Actin serves as a loading control. Figure 3B: Densitometric analysis of Figure 3 A using Image J software. Figure 3C: ECD immunohistochemistry of normal (1), adenocarcinoma (2), squamous cell carcinoma (3), and adeno-squamous cell carcinoma (4) of cervix at 10X and insert 40X. Figures 3D and 3E: ECD KD decreases invasion and migration of cervical cancer cell lines. Figure 3D: Western blot showing ECD knockdown. Figure 3E: Boy den chambers assays. Bar diagrams represent number of cells migrated or invaded. Mean+/- S.D. of three independent experiments, done in triplicate.

DETAILED DESCRIPTION OF THE INVENTION

Precise regulation of the entry into, progression though and exit from cell cycle is fundamental to developmental programs and maintenance of adult tissues in multicellular organisms. Notably, components of the cell cycle machinery and the pathways that regulate their functions are commonly altered in cancer and other diseases (Hanahan et al. (2000) Cell 100:57-70). Thus, elucidating how the cell cycle machinery is controlled is an important area of research in cell and cancer biology.

A pull-down screen using the phospho-peptide-binding domain of PIH1D1, the adaptor component of the evolutionarily-conserved prefoldin-like co-chaperone complex R2TP, identified Ecd as one of its binding partners (Horejsi et al. (2014) Cell Rep., 7: 19-26). This interaction was shown to require dual phosphorylation of Ser-505 and Ser-518 on Ecd (Horejsi et al. (2014) Cell Rep., 7: 19-26), suggesting that Ecd phosphorylation may mediate its interaction with the R2TP complex. This interaction has not been demonstrated in the context of endogenous Ecd nor has a functional role of this interaction been determined. The core R2TP complex is composed of four proteins: PIH1D1 (also called NOP17), RPAP3 (also known as hSPAGH), RUVBL1 (also known as Pontin, RVB1, TIP49A, TAP54alpha, ECP-54, TIH1, p50) and RUVBL2 (also known as Reptin, RVB2, TIP49B, TAP54 beta, ECP-51, TIH2, p47) (Kakihara et al. (2012) Biochim. Biophys. Acta 1823: 101-107). Several components of the R2TP/prefoldin complex, including PIH1D1, RUVBL1 and RUVBL2, are also overexpressed in various cancers and are predicted to play important roles in oncogenesis (Kim et al. (2013) Mol. Cell, 49: 172-185; Kakihara et al. (2014) Biomol. Concepts 5:513-520). The R2TP complex is involved in the assembly of multi- subunit complexes, including the small nucleolar ribonucleoproteins (snoRNPs), RNA polymerase II, and phosphatidylinositol 3-kinase-related kinases (PIKKs) and their complexes (Boulon et al. (2012) RNA Biol, 9: 148-154; Horejsi et al. (2010) Mol.

Cell, 39:839-850; Zhao et al. (2008) J. Cell. Biol., 180:563-578). As such, the R2TP complex is involved in a number of essential cellular processes. A comprehensive meta-analysis of The Cancer Genome Atlas (TCGA) datasets (Kim et al. (2013) Mol. Cell., 49: 172-185) revealed that expression of many RUVBL complex genes was significantly higher in breast and colorectal carcinomas when compared to their normal tissue controls. These investigations suggested a correlation between RUVBL complex component overexpression and increased mTORCl signaling and metabolic processes necessary for tumor cell growth (Kim et al. (2013) Mol. Cell, 49: 172-185). PIH1D1 is overexpressed in various breast cancer cell lines where it plays a major role in rRNA transcription (Kamano et al. (2013) FEBS Lett., 587:3303-3308). A co- oncogenic role of ECD with Ras has been shown when introduced into immortal human mammary epithelial cells (Bele et al. (2015) Cell Cycle 14:990-1000), further indicating a collaborative role of ECD and the R2TP or other RUVBL-containing complexes in cell cycle regulation and oncogenesis.

The closely-related RUVBL 1 and RUVBL2 proteins are AAA+ (ATPases associated with diverse cellular activities) that are essential for R2TP function (Matias et al. (2015) Front. Mol. Biosci., 2: 17). RUVBL1 (Pontin) plays an important role in cell cycle regulation (Breig et al. (2014) Leukemia 28: 1271-1279; Boo et al. (2015) Nat. Commun., 6:6810). Germline deletion of Ruvbll was shown to be early embryonic lethal (Boo et al. (2015) Nat. Commun., 6:6810; Rajendra et al. (2014) Nucleic Acids Res., 42: 13736-13748). Depletion of RUVBL1 in AML1-ETO fusion oncogene-expressing leukemic cells was shown to cause cell cycle arrest (Breig et al. (2014) Leukemia 28: 1271-1279) and Cre mediated deletion of Ruvbll in Ruvbll^a/a cells also led to Gl/S cell cycle arrest (Boo et al. (2015) Nat. Commun., 6:6810). The apparent similarities in the embryonic lethality and cell cycle arrest phenotypes imparted by the loss of Ecd or RUVBL1 expression suggested the likelihood that the interaction with the R2TP complex may underlie the functional requirement of Ecd in cell cycle progression.

The mechanism of Ecd-R2TP interaction was analyzed and this interaction was disabled by mutations in Ecd, affecting the latter' s role in cell cycle progression (Mir et al. (2015) Mol. Cell. Biol, pii: MCB.00594-15. [Epub ahead of print];

incorporated herein by reference). Ecd levels and localization do not vary during cell cycle progression. Casein kinase 2 (CK2) phosphorylates Ecd in cells at 6 major sites (503, 505, 508, 572, 579, 584) and a mutant Ecd (6S/A) disabled for CK2-mediated phosphorylation exhibits reduced ability to rescue the cell cycle arrest caused by Ecd gene deletion. Notably, while Ecd can interact with PIH1D1, loss of this interaction by mutating CK2 phosphorylation sites did not impact the Ecd-R2TP association in cells. A novel interaction of Ecd with RUVBL1, independent of Ecd' s interaction with PIH1D1, was identified and is shown to be essential for Ecd's cell cycle progression function. Notably, a phospho-mimetic mutant (6S/D) of Ecd failed to bind PIH1D1 and was incompetent at rescuing the cell cycle arrest caused by Ecd gene deletion, indicating a potential accessory role for PIHIDI-Ecd interaction. Taken together, these results demonstrate that while CK2-mediated phosphorylation of Ecd is important for its role in cell cycle progression, Ecd's interaction with PIH1D1 is dispensable, indicating that the novel RUVBLl-Ecd interaction is particularly critical for Ecd's function in cell cycle.

A positive role of Ecd in pre-mRNA splicing has been found based on rescue of splicing defects in the prothoracic gland of Ecd deficient flies by human ECD and interaction of Ecd with a complex containing the spliceosome component PRP8 (Claudius et al. (2014) PLoS Genet., 10:el004287; Grainger et al. (2005) RNA 11:533-557). The affinity purification/mass spectrometry analyses confirmed the interaction of Ecd with PRPF8. The R2TP complex regulates mRNA and ribosome biogenesis by facilitating the assembly of small nucleolar ribonucleoproteins (snoRNPs), which are known to be involved in splicosome modification (Bizarro et al. (2014) J. Cell. Biol, 207:463-480; Bizarro et al. (2015) Nucleic Acids Res., 43:8973-8989). Upregulation of R2TP and snoRNP components is thought to promote ribosome synthesis in cancer cells (Kakihara et al. (2014) Biomol. Concepts 5:513-520). Thus, overexpressed Ecd in tumors may function in concert with R2TP and other RUVBL1 -containing complexes to promote oncogenesis. Taken together, studies presented here demonstrate that CK2-mediated phosphorylation and interaction with RUVBL1 are essential for Ecd's ability to regulate cell cycle progression.

In addition to the above, Ecd functions as a key negative regulator of the unfolded protein response (UPR). Oncogenesis is associated with endoplasmic reticulum (ER) stress as a result of increased bioenergetic and biosynthetic demands of increased cell proliferation and other hallmarks of cancer. The cell reacts to ER stress by initiating a defensive process, UPR which is comprised of cellular mechanisms aimed at adaptation and safeguarding cellular survival or, in cases of excessively severe stress, at initiation of apoptosis and elimination of the irreparable cells. UPR involves three proximal stress sensors, activating transcription factor 6 (ATF6), PKR-like ER kinase (PERK) and inositol-requiring kinase 1 (IRE-1), that are kept in an inactive state through constitutive interaction with ER chaperone

BiP/GRP78. Activated PERK phosphorylates and inactivates eukaryotic initiation factor 2 alpha (eIF2a), which inhibits general protein synthesis to reduce ER stress, but increases the translation of specific mRNAs such as ATF4, which induces expression of the CCAAT/enhancer-binding protein-homologous protein (CHOP), and NERF-2, which induces antioxidant targets. Termination of PERK signaling and de-phosphorylation of eIF2a in the later stages of UPR are required for new protein synthesis. UPR in mammals mediates a coordinated program of cellular protection and mitigation of stress. The response to ER stress, includes suppression of new protein synthesis, exit from cell cycle and increased apoptosis, processes that are not pro-oncogenic. Thus, cancer cells require adaptive mechanisms to eliminate the inhibitory outcomes of UPR while relying on its protective aspects, such as increased anti-apoptotic and autophagic mechanism to support the oncogenic drive. Thus, pathways that mitigate the ill effects of UPR are fundamentally critical to cancer pathogenesis.

Ecd interacts with the PERK/GRP78 complex. Increases in ER stress leads to a reduction in Ecd protein levels in a PERK-dependent manner. However, Ecd mRNA is elevated. Ecd downregulation is associated with elevated phospho-PERK and phospho-eIF2a, a key mediator of PERK-dependent mRNA translation block. Conversely, Ecd overexpression reduces the levels of p-eIF2a in response to ER stress inducers. Thus, Ecd also functions as a novel negative regulator of PERK-mediated arm of UPR.

In accordance with one aspect of the instant invention, biomarkers useful for determining the diagnosis and/or prognosis of cancer are provided. In a particular embodiment, the cancer is, without limitation: prostate cancer, breast cancer, pancreatic cancer, or human papilloma virus (HPV)-associated cancers (cancer cells/tumors infected with HPV (e.g., HPV 16 or HPV 18); e.g., cervical cancer, oral cancer (e.g., cancer of the mouth and/or tongue), oropharyngeal cancer, anogenital cancer, vulvar cancer, vaginal cancer, penile cancer, anal cancer, head and neck cancers, etc.). As described herein, Ecdysoneless (Ecd) has been identified as a novel biomarker for predicting disease diagnosis, prognosis, survival, and therapeutic strategies for various forms of cancer. In a particular embodiment, the expression of unmodified and/or post-translationally modified (e.g., phosphorylated) Ecd is used to predict and/or determine disease diagnosis, prognosis, survival, and therapeutic strategy. In a particular embodiment, cytoplasmic expression of Ecd is used to predict and/or determine disease diagnosis, prognosis, survival, and therapeutic strategy.

Ecd is a novel cell cycle regulator that is essential for mammalian

development and cell cycle progression. The studies presented herein indicate that that the overexpression of Ecd protein serves as a biomarker for various forms of cancer. Particular types of cancer that can be diagnosed and/or treated with the instant invention are described below.

Prostate Cancer

Prostate Cancer (PCa) is the most frequently diagnosed and second most lethal cancer in men. Prostate Specific Antigen (PSA) screenings remains as the current standard of practice to diagnose PCa at potentially early stages. However, the effectiveness of PSA screenings has recently come under scrutiny as to not provide men with any survival advantage. Furthermore, the measurement of PSA can be inconsistent and be affected by other various factors such as prostatitis, urinary tract infection, and benign prostate hyperplasia. The current prognostic factor used for PCa is the Gleason score of biopsied tissue. The score is measured by comparing the morphology of a tissue specimen to normal prostate tissue so as to determine the likelihood that the malady will spread to local or regional tissue. The score strongly correlates with the patient's disease free status and overall survival. Together PSA screening and measuring the Gleason score represents the current standard practice for PCa diagnosis and prognosis.

Through analyzing the expression of cytoplasmic Ecdysoneless (Ecd) from a 600 member cohort of men diagnosed with prostate cancer, it was determined that overexpression of Ecd in prostate tissue specimen highly correlated with prostate cancer progression along with reduced overall survival (see, e.g., Example 1). Further analysis supported the prognostic potential of cytoplasmic Ecd expression for PCa patients and indicates a clinical application for this novel biomarker (see, e.g., Table 1). Ecd emerged as the most significant marker for predicting recurrence of prostate cancer as well as an indicator of therapy resistance in patients.

Breast Cancer

Breast cancer still remains the most frequent cancer of women with nearly a million new cases worldwide each year with about 400,000 deaths annually (Parkin et al. (2005) CA Cancer J. Clin., 55:74-108). Delineating the molecular pathways that contribute to aggressive behavior of human breast cancers to identify newer prognostic markers and therapeutic targets is therefore a critical research priority. The most important prognostic factor in breast cancer remains the lymph node status, which strongly correlates with disease-free and overall survival. Additional markers that have now become linked to molecular classification of breast cancer subtypes include the expression of hormone receptors (estrogen receptor or ER and

progesterone receptor or PR) that predicts response to endocrine therapy and overexpression of HER2/neu status that predicts response to anti-HER2/neutherapy with trastuzumab. Concurrently, ER+/PR+ tumors have a more favorable prognosis while HER2/neuoverexpression signifies a markedly worse prognosis. Absence of ER, PR and HER2/neu in the so called triple-negative breast cancers is also associated with a poor prognosis. Identification of newer molecular pathways important in oncogenesis is therefore expected to provide additional useful prognostic and predictive markers to help in the selection of appropriate targeted therapies and provide new therapeutic targets.

Uncontrolled proliferation is a hallmark of cancer and it has now become clear that major drivers of breast cancer oncogenesis enhance the expression and/or activity of cell cycle progression-associated genes (Sherr, C.J. (1996) Science 274: 1672- 1677). Extensive research over the past two decades has led to the current model of how quiescent cells enter cell cycle (Sanchez et al. (2005) Seminars Cell Dev. Biol, 16:311-321; Sun et al. (2007) J. Cell. Biochem., 102: 1400-1404). E2F family transcription factors play a critical role to turn on the expression of a large set of genes required for cell cycle progression (Malumbres et al. (2005) Trends Biochem. Sci., 30:630-641). These transcription factors are held in a repressive complex by their association with hypophosphorylated form of the Retinoblastoma (Rb) protein family members (Rb/pl05, pl07 and Rb2/pl30) (Hinds et al. (1992) Cell 70:993- 1006). Phosphorylation of Rb proteins by cell cycle-associated cyclin-dependent kinases (CDKs) helps dissociate the Rb proteins from E2Fs, resulting in the transcription of E2F target genes (Hinds et al. (1992) Cell 70:993-1006; Ewen et al. (1993) Cell 73:487-497; Hwang et al. (2005) Oncogene 24:2776-2786; Resnitzky et al. (1995) Mol. Cell. Biol, 15:4347-4352; Du et al. (2006) Oncogene 25:5190-5200). Consistent with this basic paradigm, genetic alterations of cell-cycle machinery components are frequent in cancer (Burkhart et al. (2008) Nature Rev. Cancer 8:671- 682; Deshpande et al. (2005) Oncogene 24:2909-2915; Mammas et al. (2008) Path. Oncol. Res., 14:345-354; Band, V. (1998) Intl. J. Oncol., 12:499-507; Kim et al. (2009) J. Biol. Chem, 284:26402-26410). Thus, analyses of novel cell cycle regulatory components provide an opportunity to discover new prognostic markers in breast cancer.

The human Ecd protein is a novel promoter of mammalian cell cycle progression, a function related to its ability to remove the repressive effects of Rb- family tumor suppressors on E2F transcription factors. Given the frequent dysregulation of cell cycle regulatory components in human cancer,

immunohistochemistry of paraffin-embedded tissues was used to examine Ecd expression in normal breast tissue vs. tissues representing increasing breast cancer progression. Initial studies of a smaller cohort without outcomes information showed that Ecd expression was barely detectable in normal breast tissue and in hyperplasia of breast, but high levels of Ecd were detected in benign breast hyperplasia, ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDCs) of the breast. In this cohort of 104 IDC patients, Ecd expression levels showed a positive correlation with higher grade (p=0.04). Further analyses of Ecd expression using a larger, independent cohort (954) confirmed these results, with a strong positive correlation of elevated Ecd expression with higher histological grade (p=0.013), mitotic index (p=0.032), and Nottingham Prognostic Index score (p=0.014). Ecd expression was positively associated with HER2/neu (p=0.002) overexpression, a known marker of poor prognosis in breast cancer. Significantly, increased Ecd expression showed a strong positive association with shorter breast cancer specific survival (BCSS) (p=0.008) and disease-free survival (DFS) (p=0.003) in HER2/neu overexpressing patients. Taken together, the results reveal Ecd as a novel marker for breast cancer progression and show that levels of Ecd expression predict poorer survival in

Her2/neu overexpressing breast cancer patients (see, e.g, Zhao et al. (2012) Breast Cancer Res. Treat., 134: 171 -180; incorporated by reference herein). Pancreatic Cancer

Pancreatic cancer accounts for about 3% of all cancers in the U. S. and accounts for about 7% of cancer deaths. The lethality of pancreatic cancer relates to late clinical presentation of symptoms going undetected until reaching advanced stages and, like make many forms of cancer, its symptoms mimic less severe diseases. Its aggressive nature, poor response to chemo and radiotherapy, as well as its tendency for recurrence has contributed to pancreatic cancer's nearly 100% post- diagnosis mortality. If pancreatic cancer is suspected, patients may undergo imaging scans (e.g., CT, MRI, PET, endoscopic ultrasound) and endoscopic retrograde cholangiopancreaticography to visualize any abnormalities within pancreatic tissue or ducts as well as to obtain a tissue biopsy. Tissue or pancreatic juice specimens are evaluated for aberrations in cellular morphology and for known biomarkers.

Identifying biomarkers for early detection of pancreatic cancer has become an urgent and necessary clinical need.

When examining normal and pancreatic cancer tissues, little or no Ecd expression was observed in normal pancreatic ducts. However, Ecd expression is significantly upregulated in pancreatic tissues including premalignant lesions (PanlNs) that are known to precede the development of invasive ductal carcinoma as well as metastatic organs (see, e.g., Dey et al. (2012) Clin. Cancer Res., 18:6188- 6198; incorporated by reference herein). In addition, the overexpression of Ecd promoted tumor cell survival by regulating cancer cell growth through modulation of glucose metabolism. The presence of aberrant expression of Ecd during the progression of pancreatic cancer, especially in the PanIN lesions, indicates a role for Ecd as a novel early diagnostic biomarker in pancreatic cancer tissue. HPV-associated Cancers

Human Papilloma Viruses (HPVs) are directly implicated in 5% of human cancers. Nearly 99% of all cervical cancer cases are the result of a HPV infection. There are 12 different HPV genotypes or subgroups associated with cervical cancer. The "high-risk" sub-groups of HPV-associated cancers are HPV16 and HPV18. These groups have been linked to the development of 70% of cervical cancer cases, other anogenital cancers, and a rising percentage of head and neck cancer. HPV infections can be detected by examining tissue or cellular specimens for the presence of viral DNA or RNA, especially that of the high-risk HPV types. Typically women undergo Pap smear screens for cervical cancer, yet there are no recommended screening methods in anal, vulvar, vaginal, penile, or oropharyngeal tissues.

A critical feature of oncogenesis is the accumulation of key genetic and epigenetic alterations that help tumor cells escape the normal homeostatic controls on cell survival and proliferation. Tumor viruses, such as HPVs, have provided powerful tools to dissect these mechanisms since a limited set of oncogenes trigger and maintain early steps of oncogenic transformation. It is now well-accepted that the "high-risk" HPV subgroup, exemplified by HPV 16, is causally linked to essentially all cases of cervical cancer, the second most common cancer in women. An increasingly large proportion of head and neck squamous cell carcinomas (HNSCCs), the sixth most common cancer worldwide, are linked to HPVs. As the incidence of HNSCC associated with tobacco and alcohol consumption continues to decline, HPV- associated HNSCC is expected to continue to rise. While the availability of HPV - directed vaccines that protect against HPV infection, such as Gardasil®, are expected to reduce the emergence of new HPV-associated cancers, the current vaccines are not effective against all HPVs and will not prevent oncogenesis in previously infected individuals. Thus, new avenues for diagnosing and treating HPV-associated cancers are needed.

Oncogenic transformation by HPVs is mediated by two early genes, E6 and E7. Both genes directly interact with tumor suppressors and several other cellular proteins that contribute to HPV-mediated oncogenesis. Ecd is a novel target of E6 and interacts with high-risk HPV E6 proteins, HPV 16 and 18. It is noteworthy, that while PIH1D1 binding to Ecd requires the phosphorylation of the serine residues, E6 binding occurs in the absence of phosphorylation as demonstrated through use of purified proteins. E6 competes with PIHlDlfor binding to Ecd. Based on these results, Ecd forms a base, or scaffold, to help recruit the R2TP complex to binding partners of Ecd, and that interaction of E6 with the unstructured C-terminal region may mimic the effect of Ecd-PIHIDI interaction in facilitating the R2TP complex function towards specific Ecd partner proteins.

Besides Ecd's involvement in cellular survival mechanism, Ecd is also overexpressed in both cervical cancer cell lines and patient derived tissue specimens. Ecd overexpression significantly correlates with both squamous cell carcinoma as well as adenocarcinoma of the cervix. These studies support the use of Ecd as a novel diagnostic and prognostic biomarker.

Assays

In accordance with the instant invention, methods of identifying, determining an increased risk for, diagnosing, and/or prognosis of a cancer, particularly prostate cancer or an HPV-associated cancer, in a patient are provided. In a particular embodiment, the method comprises determining the localization and/or expression of Ecd (e.g., protein and/or mRNA) (e.g., GenBank Gene ID: 1 1319). More particularly, the method may comprise determining the cellular location and/or expression of Ecd protein (e.g., nuclear and/or cytoplasmic). The methods may further comprise obtaining a biological sample from the subject. In a particular embodiment, the biological sample is tumor tissue (e.g., tumor biopsy). As explained herein, when Ecd is overexpressed (e.g., compared to a healthy or non-cancerous sample (e.g., a sample obtained from a healthy subject or a non-cancerous sample obtained from the same subject (e.g., an adjacent healthy tissue))), the subject has a poor prognosis, optionally with increased risk of metastasis, and higher risk of death.

The method may further comprise detecting the presence of at least one other cancer marker. The other cancer marker may be detected in the same biological sample as the one used for Ecd detection or may be from another biological sample from the patient (e.g., an adjacent or analogous biological sample, blood sample, or serum sample).

For prostate cancer, other cancer markers that can be detected in the subject

(e.g., in a biological sample from the subject) include, without limitation, prostate specific antigen (PSA; detected, for example, in blood, serum, or urine), prostate cancer gene 3 (PCA3; detected, for example, in urine), phosphatase and tensin homolog (PTEN; loss of PTEN is indicative of cancer), transmembrane protease, serine 2 (TMPRSS2)-ETS-related gene (ERG) fusion (TMPRS S2-ERG; detected, for example, in urine or biopsy), RUVBLl, RUVBL2, RAF, BRAF, speckle-type POZ protein (SPOP), Enhancer of zeste homolog 2 (EZH2), and serine protease inhibitor Kazal-type 1 (SPINK1). In a particular embodiment, the other cancer marker is selected from the group consisting of RUVBLl, RUVBL2, PSA, PC A3, and PTEN. In a particular embodiment, at least PSA is detected in addition to Ecd.

For HPV-associated cancers, other cancer markers that can be detected in the subject (e.g., in a biological sample from the subject) include, without limitation, HPV viral genomes (e.g., HPV 16 or HPV 18 viral genotypes), HPV E6 (e.g., protein or mRNA), HPV E7 (e.g., protein or mRNA), pl6, p53, Rb, RUVBLl, RUVBL2, and pl6INK4a/Ki-67. With regard to cervical cancer, other cancer markers include, without limitation, pi 6, p53, minichromosome maintenance deficient 2 (MCM2) and topoisomerase (DNA) II Alpha (TOP2A). In addition to or in place of the use of other cancer markers, the subject may be given a Pap test (Pap smear) for the diagnosis of cervical cancer.

The ability to detect the above markers - either as a nucleic acid molecule or as a protein - is well known in the art. In a particular embodiment, the markers, particularly Ecd, are detected as proteins. For example, the markers may be detected with antibodies which are immunologically specific for the marker (e.g., via immunohistochemistry). Anti-Ecd antibodies of the instant invention include, without limitation, polyclonal antibodies, monoclonal antibodies, and fragments thereof. The anti-Ecd antibodies may also be immunologically specific for modified forms of Ecd such as phosphorylated Ecd. Other detection assays for the cancer markers include, without limitation, PCR amplification (inclusive of RT-PCR), karyotype analysis, and immunoassays (e.g., immunohistochemistry, ELISA, immunoblotting (Western blotting), tissue microarray, and multiplex immunoassay). Ecd and cancer markers may be detected in tissue sample and/or blood or serum samples.

The methods of the instant invention may further comprise identifying an appropriate treatment modality to administer to the subject and, optionally, treating the diagnosed patient with the selected treatment modality. In general, a patient with a good or excellent prognosis may be treated with a conventional treatment regimen, refrain from treatment, or be monitored for indolent tumors that are slow growin. A patient with a poor prognosis may be treated with an alternative or more aggressive regimen. In other words, upon diagnosing the patient and determining the disease prognosis by the methods of the instant invention, patients can be stratified and categorized based on the disease severity and survivability. By the methods of the instant invention, the poor prognosis patient will not have to wait for the conventional treatment regimen to fail before moving onto the more aggressive treatment. In addition, good prognosis patients with indolent forms of cancer or benign tumors can be monitored for changes in disease progression rather than receiving unnecessary and potentially harmful treatments. Furthermore, knowledge of the likely clinical course of the disease allows the patient to have a more realistic expectation of the prospective course of the cancer treatment.

As explained hereinabove, when Ecd is overexpressed (e.g., in the cytoplasm) in the biological sample, the subject has a poor prognosis, optionally with increased risk of metastasis, and higher risk of death. In a particular embodiment, the level of expression of Ecd in the biological sample obtained from the subject can be compared to the expression of Ecd in a corresponding biological sample from a healthy or non- cancerous subject, and/or the level of expression of Ecd in the biological sample obtained from the subj ect can be compared to the expression of Ecd in a

corresponding biological sample from subject with cancer or corresponding biological samples from subj ects with cancer at different stages, wherein the stage of cancer of the test subject corresponds to the biological sample whose level of expression of Ecd matches the level of expression of Ecd in the biological sample from the test subject.

When Ecd is not overexpressed, a conventional treatment modality may be selected for the subject. For prostate cancer, conventional treatment options include, without limitation, expectant management, surgery (e.g., radical prostatectomy), radiation therapy (e.g., external beam radiation, brachytherapy), cryosurgery, hormone therapy (e.g., androgen deprivation therapy (e.g., luteinizing hormone- releasing hormone (LHRH) analogs (e.g., degarelix, abiraterone), anti-androgens (e.g., flutamide, bicalutamide, nilutamide, enzalutamide))), chemotherapy (e.g., docetaxel, cabazitaxel, mitoxantrone, estramustine, doxorubicin, etoposide, vinblastine, paclitaxel, carboplatin, vinorelbine) and vaccines (e.g., sipuleucel-T). For HPV-associated cancers, conventional treatment options include, without limitation, surgery (e.g., resection of the tumor or cancer cells), radiation therapy, and chemotherapy. For HPV-associated cervical cancer specifically, conventional treatments options include, without limitation, surgery (e.g., cryosurgery, laser surgery, conization, hysterectomy, trachelectomy), radiation therapy (e.g., external beam radiation, brachy therapy), and chemotherapy (e.g., cisplatin, carboplatin, paclitaxel, topotecan, gemcitabine).

When Ecd is overexpressed, a more aggressive treatment modality may be selected for the subject. In a particular embodiment, a treatment modality for a cancer with a higher stage is selected for the subject. For example, if the cancer is otherwise determined to be stage II, but Ecd is determined to be overexpressed, then the treatment modality of stage III or stage IV cancer may be selected (and optionally administered) for the subject. In a particular embodiment, the treatment selected for the patient wherein Ecd is overexpressed comprises administering a CK2 inhibitor (e.g., CX-4945 (silmitasertib), ellagic acid, 4,5,6,7-tetrabromobenzotriazole (TBB), (2E)-3-(2,3,4,5-tetrabromophenyl)-2-propenoic acid (TBCA), 3-[[5-(4- methylphenyl)thieno[2,3-d]pyrimidin-4-yl]thio]propanoic acid (TTP 22), apigenin, 2- (4,5,6,7 etrabromo-2-(dimethylamino)-lH-benzo[d]imidazol-l -yl)acetic acid (TMCB), CK2 siRNA, CK2 antisense, and CK2 inhibitors provided in U. S. Patents 8,324,231 ; 7,741,304; 8,372,851 ; and 8, 101,625), trastuzmab, and/or endoplasmic reticulum (ER) stress inducers (e.g., neflinavir, bortezumib, atazanavir,

verispelostatin, brefeldin A, and breflate; particularly neflinavir or bortezumib). In a particular embodiment, the method comprises the administration of a CK2 inhibitor. In a particular embodiment, the method comprises the administration of a

conventional therapy as described hereinabove. The methods of the instant invention may also comprise the administration of at least one chemotherapeutic agent or anticancer therapy (e.g., radiation and/or surgery to remove cancerous cells or a tumor (e.g., resection)). The agents administered to the subject may be contained with a composition comprising at least one pharmaceutically acceptable carrier. When more than one agent is being administered, the agents may be administered separately or sequentially (before or after) and/or at the same time. The agents may be

administered in the same composition or in separate compositions.

In accordance with another aspect of the present invention, kits for identifying and/or diagnosing cancer are provided. In a particular embodiment, the kit comprises antibodies specific for Ecd. As stated hereinabove, the anti-Ecd antibodies may be monoclonal or polyclonal, or fragments thereof, and can be used in a variety of assys to detect Ecd such as immunoassays, immunohistochemistry, multiplex assys, tissue microarrays, ELISA assays, and the like. In a particular embodiment, the anti-Ecd antibody is a monoclonal antibody. The anti-Ecd antibodies may also be immunologically specific for modified forms of Ecd such as phosphorylated Ecd. The antibodies of the kit may be lyophilized or maintained in a carrier. The kits may further comprise at least one other agent (e.g., nucleic acid probes, antibodies, etc.) for detecting the presence and/or amount of another cancer marker. In a particular embodiment, the kit comprises at least one other antibody immunologically specific for another cancer marker. The antibodies may be contained within the same composition or in separate compositions. For example, the kit may comprise a first composition comprising at least one Ecd antibody (optionally with at least one carrier) and a second composition comprising at least one other cancer marker antibody (optionally with at least one carrier). The kits may further comprise instruction material and/or at least one control (e.g., a sample from a healthy subject (with baseline Ecd expression) and/or a sample with Ecd overexpression). The antibodies and/or agents of the kit may be detectably labeled. In a particular embodiment, the kit comprises secondary antibodies which are detectably labeled.

Definitions

The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, a "biological sample" refers to a sample of biological material obtained from a subject, preferably a human subject, including a tissue, a tissue sample, a cell sample, a tumor sample, and a biological fluid, (e.g., blood, blood fraction, serum, or urine). A biological sample may be obtained in the form of, e.g., a tissue biopsy, such as, an aspiration biopsy, a brush biopsy, a surface biopsy, a needle biopsy, a punch biopsy, an excision biopsy, an open biopsy, an incision biopsy and an endoscopic biopsy. A tumor sample or biopsy may be obtained, for example, by the surgical removal of tissue from within a patient and/or tissue obtained from an excised organ.

As used herein, "diagnose" refers to detecting and identifying a disease in a subject. The term may also encompass assessing or evaluating the disease status (e.g., progression, regression, stabilization, response to treatment, etc.) in a patient known to have the disease.

As used herein, the term "prognosis" refers to providing information regarding the impact of the presence of cancer (e.g., as determined by the diagnostic methods of the present invention) on a subject's future health (e.g., expected morbidity or mortality, the likelihood of getting cancer, and the risk of metastasis). In other words, the term "prognosis" refers to providing a prediction of the probable course and outcome of a cancer or the likelihood of recovery from the cancer. The term

"prognosis" is recognized in the art and encompasses predictions about the likely course of disease or disease progression, particularly with respect to likelihood of disease remission, disease relapse, tumor recurrence, metastasis, and death. A "good prognosis" may refer to the likelihood that a patient afflicted with cancer will remain cancer-free after therapy. A "poor prognosis" may refer to the likelihood of a relapse or recurrence of the underlying cancer or tumor after treatment, the likelihood of developing metastases, and/or the likelihood of death. In particular embodiments, the time frame for assessing prognosis is, for example, less than one year, one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, or more years.

The term "treat" as used herein refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc.

The phrase "effective amount" refers to that amount of therapeutic agent that results in an improvement in the patient's condition.

A "carrier" refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., Tween® 80, Polysorbate 80), emulsifier, buffer (e.g., Tris HC1, acetate, phosphate), water, aqueous solutions, oils, bulking substance (e.g., lactose, mannitol), excipient, auxilliary agent or vehicle with which an active agent of the present invention is used.

The term "probe" as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains about 10-100, about 10-50, about 15-30, about 15-25, about 20-50, or more nucleotides, although it may contain fewer nucleotides. The probes herein may be selected to be complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to "specifically hybridize" or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target, although they may. For example, a non- complementary nucleotide fragment may be attached to the 5 ' or 3' end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.

An "antibody" or "antibody molecule" is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen. The term includes polyclonal, monoclonal, chimeric, single domain (Dab) and bispecific antibodies. As used herein, antibody or antibody molecule contemplates recombinantly generated intact immunoglobulin molecules and molecules comprising immunologically active portions/fragments of an immunoglobulin molecule such as, without limitation: Fab, Fab', F(ab')₂, F(v), scFv, scFv₂, scFv-Fc, minibody, diabody, tetrabody, and single variable domain (e.g., variable heavy domain, variable light domain).

With respect to antibodies, the term "immunologically specific" refers to antibodies that bind to one or more epitopes of a protein or compound of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.

The term "isolated" may refer to a compound or complex that has been sufficiently separated from other compounds with which it would naturally be associated. "Isolated" is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with fundamental activity or ensuing assays, and that may be present, for example, due to incomplete purification, or the addition of stabilizers.

As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the composition of the invention for performing a method of the invention.

Chemotherapeutic agents are compounds that exhibit anticancer activity and/or are detrimental to a cell (e.g., a toxin). Suitable chemotherapeutic agents include, but are not limited to: toxins (e.g., saporin, ricin, abrin, ethidium bromide, diptheria toxin, and Pseudomonas exotoxin); taxanes; alkylating agents (e.g., temozolomide, nitrogen mustards such as chlorambucil, cyclophosphamide, isofamide, mechlorethamine, melphalan, and uracil mustard; aziridines such as thiotepa; methanesulphonate esters such as busulfan; nitroso ureas such as carmustine, lomustine, and streptozocin; platinum complexes (e.g., cisplatin, carboplatin, tetraplatin, ormaplatin, thioplatin, satraplatin, nedaplatin, oxaliplatin, heptaplatin, iproplatin, transplatin, and lobaplatin); bioreductive alkylators such as mitomycin, procarbazine, dacarbazine and altretamine); DNA strand-breakage agents (e.g., bleomycin); topoisomerase II inhibitors (e.g., amsacrine, menogaril, amonafide, dactinomycin, daunorubicin, Ν,Ν-dibenzyl daunomycin, ellipticine, daunomycin, pyrazoloacridine, idarubicin, mitoxantrone, m-AMSA, bisantrene, doxorubicin (adriamycin), deoxy doxorubicin, etoposide (VP- 16), etoposide phosphate, oxanthrazole, rubidazone, epirubicin, bleomycin, and teniposide); DNA minor groove binding agents (e.g., plicamydin); antimetabolites (e.g., folate antagonists such as methotrexate and trimetrexate); pyrimidine antagonists such as fluorouracil, fluorodeoxyuridine, CB3717, azacitidine, cytarabine, and floxuridine; purine antagonists such as mercaptopurine, 6-thioguanine, fludarabine, pentostatin;

asparginase; and ribonucleotide reductase inhibitors such as hydroxyurea);

anthracyclines; and tubulin interactive agents (e.g., vincristine, vinblastine, and paclitaxel (Taxol®)).

Radiation therapy refers to the use of high-energy radiation from x-rays, gamma rays, neutrons, protons and other sources to target cancer cells. Radiation may be administered externally or it may be administered using radioactive material given internally. Chemoradiation therapy combines chemotherapy and radiation therapy.

The following examples are provided to illustrate certain embodiments of the invention. It is not intended to limit the invention in any way.

EXAMPLE 1

The specific recognition of both the human and mouse Ecd proteins in Western blot analyses by the anti-Ecd monoclonal antibody (mAb) has been shown (Kim et al. (2009) J. Biol. Chem, 284:26402-26410; Kim et al. (2010) Biol. Chem, 391 :9-19; Zhang et al. (2006) Cancer Res., 66:7167-7175). Furthermore, Ecd shuttles between the nucleus and the cytoplasm with a fast nuclear export resulting in a predominantly cytoplasmic localization in cells (Kim et al. (2010) Biol. Chem, 391 :9-19).

As Ecd functions to promote cell cycle progression, the relative Ecd expression was assessed by comparing the intensity of immunohistochemical (IHC) staining with anti-Ecd mAb in tissue specimens in different prostate tissue samples. A clear cytoplasmic staining was observed in tumor tissues, whereas little or no staining was observed in normal tissue. A similar low or no staining was observed in hyperplastic tissue specimens. As seen in Figure 1, the level of cytoplasmic Ecd expression increases with the progression of prostate cancer. Accordingly, these results indicated that the levels of Ecd expression positively correlate with increasing grade of prostate tumors.

It was then assessed if Ecd expression could predict the outcome of disease in prostate cancer patients. For this purpose, the estimated predictive power of Ecd expression was assessed using the Kaplan-Meier survival analysis. Patients with high Ecd expression exhibited a statistically significant reduction in prostate cancer- specific survival as compared to patients with no to moderate Ecd expression (Figure 2). These results indicate that Ecd expression can serve as an independent marker of disease-specific survival outcome in prostate cancer patients.

A cox's regression model of Ecd expression was also used in a survival analysis (Table 1). The model represents an equation for the hazard as a function of survival using explanatory variables. Positive value indicates that the hazard is high (poor prognosis), whereas a negative value indicates that the hazard is low (better prognosis).

Table 1: Multivariate survival analysis using cox's regression model for Ecd overexpression. RR: ratio. CI: confidence interval.

EXAMPLE 2

Ecd expression in cervical cancer cell lines and tissue arrays was studied.

Specifically, Western blots for detecting Ecd expression in two independent primary human foreskin keratinocytes (4FKC and lOFKC) and four cervical cancer cell lysates (HeLa, SiHa, CaSki, and C33A) were performed. As can be seen in Figures 3A and 3B, the four cervical cancer cell lines overexpressed Ecd compared to the primary foreskin keratinocytes. An Ecd immunohistochemistry analysis of normal (1), adenocarcinoma (2), squamous cell carcinoma (3), and adeno-squamous cell carcinoma (4) of cervix was also performed. As seen in Figure 3C, Ecd expression was increased in the cancerous samples.

The effects of reducing the overexpression of Ecd were also studied.

Specifically, Ecd was knocked down by delivering Ecd siRNA to the cervical cancer cell lines SiHa and HeLa. The reduction in Ecd expression was confirmed by Western blot (Figure 3D; compare left column controls with central and right column where siRNA #1 and #2 were delivered, respectively). Boy den chamber assays were also performed and demonstrated that decreasing Ecd expression decreased the invasiveness of the cervical cancer cell lines (Figure 3E).

Consistent with a role of Ecd in HPV oncogenesis, Ecd selectively binds to the high-risk HPV E6 proteins. Mutational analysis of Ecd reveals that E6 binds to a C- terminal region that harbors 3 serine residues. Phosphorylation of these serine residues by Casein Kinase-2 (CK2) creates a motif for binding of PIH1D1, a component of the HSP90-associated co-chaperone R2TP complex.

The data provided herein shows that Ecd is overexpressed in cervical cancer cell lines (Figs. 3A and 3B) and tumor tissues (Fig. 3C). The knockdown of Ecd in cervical cancer cell lines reduces migration and invasion (Figs. 3D and 3E). These results indicate that Ecd overexpression can serve as an independent marker of cervical cancer and that reduction of Ecd overexpression can be therapeutic towards cervical cancer. While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

What is claimed is

1. A method of diagnosing and/or providing a prognosis for cancer in a subject, said method comprising determining the expression of ecdysoneless (Ecd) in a biological sample obtained from said subject;

wherein increased expression of Ecd compared to the expression of Ecd in a biological sample from a healthy control is indicative of a poor prognosis and disease progression.

2. The method of claim 1, wherein said cancer is prostate cancer.

3. The method of claim 1, wherein said cancer is a human papilloma virus (HPV)- associated cancer.

4. The method of claim 2, further comprising detecting at least one other prostate cancer marker in said biological sample.

5. The method of claim 4, wherein said other prostate cancer marker is selected from the group consisting of prostate specific antigen (PSA), prostate cancer gene 3

(PCA3), phosphatase and tensin homolog (PTEN), transmembrane protease, serine 2 (TMPRSS2)-ETS-related gene (ERG) fusion (TMPRSS2-ERG), RUVBLl, RUVBL2, RAF, BRAF, speckle-type POZ protein (SPOP), enhancer of zeste homolog 2 (EZH2), and serine protease inhibitor Kazal-type 1 (SPINK1).

6. The method of claim 3, further comprising detecting at least one other HPV- associated cancer marker in said biological sample.

7. The method of claim 6, wherein said other HPV-associated cancer marker is selected from the group consisting of HPV viral genomes, pi 6, p53, HPV E6, HPV E7, RUVBLl, RUVBL2, and pl6INK4a/Ki-67.

8. The method of claim 1, wherein the cytoplasmic expression of Ecd is determined.

9. The method of claim 1, wherein said biological sample is a tumor sample.

10. The method of claim 1 further comprising identifying and/or selecting a treatment modality for the subject.

11. The method of claim 10, wherein said treatment modality comprises treating the subject with a casein kinase 2 (CK2) inhibitor.

12. A method for treating cancer in a subject, said method comprising:

a) determining the expression of ecdysoneless (Ecd) in a biological sample obtained from said subject; and

b) administering anti-cancer therapy when Ecd is determined to be

overexpressed, thereby treating the cancer in said subject.

13. The method of claim 12, wherein said cancer is prostate cancer.

14. The method of claim 12, wherein said cancer is a human papilloma virus (HPV)- associated cancer.

15. The method of claim 12, further comprising detecting at least one other prostate cancer marker in said biological sample.

16. The method of claim 15, wherein said other prostate cancer marker is selected from the group consisting of prostate specific antigen (PSA), prostate cancer gene 3 (PCA3), phosphatase and tensin homolog (PTEN), transmembrane protease, serine 2 (TMPRSS2)-ETS-related gene (ERG) fusion (TMPRSS2-ERG), RUVBLl, RUVBL2, RAF, BRAF, speckle-type POZ protein (SPOP), enhancer of zeste homolog 2 (EZH2), and serine protease inhibitor Kazal-type 1 (SPINK1).

17. The method of claim 14, further comprising detecting at least one other HPV- associated cancer marker in said biological sample.

18. The method of claim 17, wherein said other HPV-associated cancer marker is selected from the group consisting of HPV viral genomes, HPV E6, HPV E7, pi 6, p53, RUVBL1, RUVBL2, and pl6INK4a/Ki-67.

19. The method of claim 12, wherein the cytoplasmic expression of Ecd is determined.

20. The method of claim 12, wherein said biological sample is a tumor sample.

21. The method of claim 12, wherein step b) comprises administering at least one chemotherapeutic agent.

22. The method of claim 12, wherein step b) comprises treating the subject with radiation or resecting cancerous cells from said subject.

23. The method of claim 12, wherein step b) comprises administering a casein kinase 2 (CK2) inhibitor, trastuzmab, and/or an endoplasmic reticulum (ER) stress inducer.

24. The method of claim 12, wherein step b) comprises administering a casein kinase 2 (CK2) inhibitor.

25. A kit comprising a first composition comprising at least one Ecd antibody and a second composition comprising at least one agent for detecting another cancer marker.

26. The kit of claim 25, wherein said cancer is prostate cancer or human papilloma virus (HPV)-associated cancer.

27. The kit of claim 25, wherein said agent is an antibody immunologically specific for said cancer marker.