WO2010026565A1 - PKCeta AS A MARKER FOR CANCER PROGNOSIS AND TREATMENT - Google Patents

Abstract

This invention provides a method and kit for the assessment of prognosis of a subject having or suspected of having caner, by determination of the cellular localization of PKCη. This invention further describes a method for the determination of a subject's responsiveness to a chemotherapeutic regimen, wherein little or no change of cellular localization of PKCη to be cytoplasmic, following chemotherapy treatment is indicative of a lack of responsiveness to the chemotherapeutic regimen. This invention further provides treatment methods for invasive breast or lung cancer via the administration of an agent, which inhibits PKCη translocation to membranous compartments of the cell.

Description

PKCeta AS A MARKER FOR CANCER PROGNOSIS AND TREATMENT BACKGROUND OF THE INVENTION

[001] A carcinoma is any malignant cancer that arises from epithelial cells. Carcinomas invade surrounding tissues and organs and may metastasize, or spread, to lymph nodes and other sites. Carcinoma in situ (CIS) is a pre-malignant condition, in which some cytological signs of malignancy are present, but there is no histological evidence of invasion through the epithelial basement membrane. Carcinoma, like all neoplasia, is classified by its histopathological appearance. Adenocarcinoma and squamous cell carcinoma, two common descriptive terms for tumors, reflect the fact that these cells may have glandular or squamous cell appearances respectively. Severely anaplastic tumors might be so undifferentiated that they do not have a distinct histological appearance (undifferentiated carcinoma).

[002] Breast cancer is a carcinoma that starts in the cells of the breast. Worldwide, breast cancer is the second most common type of cancer after lung cancer (10.4% of all cancer incidence, both sexes counted) and the fifth most common cause of cancer death.

Worldwide, breast cancer is by far the most common cancer amongst women, with an incidence rate more than twice that of colorectal cancer and cervical cancer and about three times that of lung cancer. However breast cancer mortality worldwide is just 25% greater than that of lung cancer in women. In 2005, breast cancer caused 502,000 deaths worldwide (7% of cancer deaths; almost 1% of all deaths). The number of cases worldwide has significantly increased since the 1970s, a phenomenon partly blamed on modern lifestyles in the Western world.

[003] The incidence of breast cancer varies greatly around the world being lower in less developed countries and greatest in the more developed countries. The annual age standardized incidence per 100,000 women are in Eastern Asia 18, South Central Asia 22 and sub-Saharan Africa 22, while in the United States the incidence is 141 among white women and 122 among African American women.

[004] North American women have the highest incidence of breast cancer in the world. Among women in the U.S., breast cancer is the most common cancer and the second-most common cause of cancer death (after lung cancer). Women in the U.S. have a 1 in 8

(12.5%) lifetime chance of developing invasive breast cancer and a 1 in 35 (3%) chance of breast cancer causing their death. In 2007, breast cancer was expected to cause 40,910 deaths in the U.S. (7% of cancer deaths; almost 2% of all deaths). A U.S. study conducted in 2005 by the Society for Women's Health Research indicated that breast cancer remains the most feared disease, even though heart disease is a more common cause of death among women.

[005] Because the breast is composed of identical tissues in males and females, breast cancer also occurs in males. Incidences of breast cancer in men are approximately 100 times less common than in women, but men with breast cancer are considered to have the same statistical survival rates as women.

[006] There are many prognostic factors associated with breast cancer: staging, tumor size and location, grade, whether disease is systemic (has metastasized, or traveled to other parts of the body), recurrence of the disease, and age of patient. All of these prognostic parameters are based on previous case statistics gathered over time.

[007] Stage is the most important, as it takes into consideration size, local involvement, lymph node status and whether metastatic disease is present. The higher the stage at diagnosis, the worse the prognosis. Larger tumors, invasiveness of disease to lymph nodes, chest wall, skin or beyond, and aggressiveness of the cancer cells raise the stage, while smaller tumors, cancer-free zones, and close to normal cell behavior (grading) lower it.

[008] Grading is based on how cultured biopsy originated cells behave. The closer to normal these cancer cells are, the slower their growth and a better prognosis. If cells are not well differentiated, they appear immature, divide more rapidly, and tend to spread. Well differentiated is given a grade of 1, moderate is grade 2, while poor or undifferentiated is given a higher grade of 3 or 4 (depending upon the scale used).

[009] Younger women tend to have a poorer prognosis than post-menopausal women due to several factors. Their breasts are active with their cycles, they may be nursing infants, and may be unaware of changes in their breasts. Therefore, younger women are usually at a more advanced stage when diagnosed. [0010] The diagnosis of tumors has evolved during the last years from observation methodology to a more scientific and less biased detection of molecular markers indicating the possibility of tumor development, aggressiveness, prognosis and response to treatment. [0011] The presence of estrogen and progesterone receptors in the cancer cell, while not prognostic, is important in guiding treatment. Those who do not test positive for these specific receptors will not respond to hormone therapy.

[0012] HER2/neu (also known as ErbB-2, ERBB2) is a protein giving higher aggressiveness in breast cancers. It is a member of the ErbB protein family, more commonly known as the epidermal growth factor receptor family. [0013] HER2/neu is notable for its role in the pathogenesis of breast cancer and as a target of treatment. It is a cell membrane surface-bound receptor tyrosine kinase and is normally involved in the signal transduction pathways leading to cell growth and differentiation. HER2 is thought to be an orphan receptor, with none of the EGF family of ligands able to activate it. However, ErbB receptors dimerize on ligand binding, and HER2 is the preferential dimerization partner of other members of the ErbB family. [0014] Approximately 25-35 percent of breast cancers have an amplification of the HER2/neu gene or overexpression of its protein product. Overexpression of this receptor in breast cancer is associated with increased disease recurrence and worse prognosis. Because of its prognostic role as well as its ability to predict response to trastuzumab, a monoclonal antibody that targets this protein, breast tumors are routinely checked for overexpression of HER2/neu. Overexpression also occurs in other cancer such as ovarian cancer and stomach cancer. [0015] BRCAl (breast cancer 1, early onset) is a human gene that belongs to a class of genes known as tumor suppressors, which maintains genomic integrity to prevent uncontrolled proliferation. The multifactorial BRCAl protein product is involved in DNA damage repair, ubiquitination, transcriptional regulation as well as other functions. Variations in the gene have been implicated in a number of hereditary cancers, namely breast, ovarian and prostate. The BRCAl protein is directly involved in the repair of damaged DNA. In the nucleus of many types of normal cells, the BRCAl protein is thought to interact with RAD51 to mend breaks in DNA, though the details and significance of this interaction is the subject of debate. These breaks can be caused by natural radiation or other exposures, but also occur when chromosomes exchange genetic material during meiosis. The BRCA2 protein, which has a function similar to that of BRCAl, also interacts with the RAD51 protein. By repairing DNA, these three proteins play a role in maintaining the stability of the human genome.

[0016] Certain variations of the BRCAl gene lead to an increased risk for breast cancer. Researchers have identified more than 600 mutations in the BRCAl gene, many of which are associated with an increased risk of cancer. These mutations can be changes in one or a small number of DNA base pairs. A mutated BRCAl gene usually makes a protein that does not function properly because it is abnormally short. Researchers believe that the defective BRCAl protein is unable to help fix mutations that occur in other genes. These defects accumulate and may allow cells to grow and divide uncontrollably to form a tumor. In addition to breast cancer, mutations in the BRCAl gene also increase the risk on ovarian, fallopian tube and prostate cancers. Moreover, precancerous lesions (dysplasia) within the fallopian tube have been linked to BRCAl gene mutations.

[0017] Unlike traditional diagnostics, whose role is largely confined to diagnosis and monitoring of a restricted range of diseases, molecular markers supply more effective tools for all aspects of disease management, especially in areas of unmet clinical need. Based on sensitive detection of disease- and often individual-specific biomarkers, they provide an opportunity to intercept the disease process early, stratify patients for treatment and/or predict treatment success. Therefore, the discovery and development of new molecular markers is in high demand in the ever growing field of cancer diagnosis and treatment. SUMMARY OF THE INVENTION

[0018] In one embodiment, this invention provides a method for assessing the prognosis of a subject having or suspected of having cancer, the method comprising:

• obtaining a patient biological sample;

• assessing cellular localization of PKC eta (PKCη) in said sample; and • predicting cancer prognosis of the patient based on said cellular localization, wherein PKCη localization at cellular membranes, as opposed to cytoplasmic localization in said sample is indicative of a poor prognosis for the subject having or suspected of having cancer.

[0019] In one embodiment, this invention further provides a kit for determining the prognosis of a subject having or suspected of having cancer, the kit comprising:

• a first reagent for the detection of PKCη;

• a second reagent for labeling membranous structures in a cell;

• optionally at least two detectable markers, each of which specifically interacts with said first and said second reagent, and wherein overlap between said first and said second reagent is discernible; and

• optionally reagents for isolating and processing biological samples.

[0020] In one embodiment this invention further provides a method for determining the responsiveness of a subject to a particular chemotherapeutic regimen, the method comprising: • obtaining a cancerous or precancerous biological sample from a subject prior to and following treatment of the subject with a chemotherapeutic regimen; and

• detecting PKCη expression and cellular localization in said biological samples; whereby predominant cytoplasmic localization of PKCη in cells from said sample is indicative of responsiveness to said chemotherapeutic regimen, while localization of PKCη at cellular membranes or nuclear membranes in said sample is indicative of lack of responsiveness to said chemotherapeutic regimen.

[0021] In one embodiment this invention further provides a method for treating invasive breast cancer, reducing the incidence of invasive breast cancer, or prolonging remission of invasive breast cancer in a subject, the method comprising contacting a neoplastic or preneoplastic cell in a subject having invasive breast cancer with an agent, which inhibits PKCη translocation from the cytoplasm to membranous structures in said cell. [0022] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of a conflict between the specification and an incorporated reference, the specification shall control. Where number ranges are given in this document, endpoints are included within the range. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges, optionally including or excluding either or both endpoints, in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Where a percentage is recited in reference to a value that intrinsically has units that are whole numbers, any resulting fraction may be rounded to the nearest whole number.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Various embodiments of the PKCη as a marker for cancer prognosis and treatment method are described herein with reference to the figures wherein: [0024] Figure 1 depicts examples of Hematoxylin and eosin staining of normal breast tissue.

[0025] Figure 2 shows high PKCη staining of normal breast tissue; sample is tissue adjacent to a breast tumor.

[0026] Figure 3 presents PKCη staining of various tumor biopsies. Staining of in-situ tumor where magnification of x20 is shown in (A), and magnification of x40 is shown in (B), cytoplasmic staining of invasive ductal carcinoma is shown in (C₅D), staining of invasive ductal carcinoma, where detection of membrane-associated staining is shown in (E) and staining of a tumor with cytoplasmatic and nuclear positive staining for PKCη is shown in

(F). [0027] Figure 4 shows that PKCη over expression in the breast cancer adenocarcinoma

MCF-7 cells (MCF21.5 cells) results in enhanced cell survival after UVC irradiation or camptothecin drug treatment (CPT).

[0028] Figure 5 shows that PARP-I cleavage, which is indicative of apoptosis, is inhibited by PKCη expression in MCF-7 cells (MCF21.5) in response to UVC irradiation or

CPT treatment.

[0029] Figure 6 shows that PKCη expression in MCF-7 cells interferes with caspase 7 activation, cleavage of pro-caspase 9 and release of cytochrome C from mitochondria in response to UVC irradiation or CPT treatment. [0030] Figure 7 indicates that PKCη translocates to the nuclear envelope in response to etoposide drug treatment. Translocation to the cellular membranes occurred after longer time points (3 h), data not shown.

DETAILED DESCRIPTION OF THE INVENTION

[0031] This invention provides, in some embodiments, for the use of PKCη as a marker for cancer prognosis and treatment.

[0032] As exemplified herein, determining the cellular localization of PKCη before and after cancer treatment provided a means for determining the efficacy of the cancer treatment, and furthermore provides a basis for change in treatment of the subject and/or prognosis of the subject, representing embodiments of the invention. [0033] The invention provides, in one embodiment, a method for assessing the prognosis of a subject having or suspected of having cancer, the method comprising:

• obtaining a patient biological sample;

• assessing cellular localization of PKCη in said sample; and

• predicting cancer prognosis of the patient based on said cellular localization, wherein PKCη localization at cellular membranes, as opposed to cytoplasmic localization in said sample is indicative of a poor prognosis for the subject having or suspected of having cancer.

[0034] In one embodiment, prognosis is defined as a medical term denoting a doctor's prediction of how a patient's disease will progress, and whether there is chance of recovery. Prognosis is important in predicting how a patient may respond to treatment.

Symptoms and tests may indicate favorable treatment with standard therapies. Likewise, a number of symptoms, health factors, and tests may indicate a less favorable treatment result with standard treatment, this may indicate that a more aggressive treatment plan may be desired.

[0035] In one embodiment, the subject has or is at risk for a carcinoma. In one embodiment, the subject has or is at risk for breast cancer. In one embodiment, the subject has or is at risk for lung cancer.

[0036] In one embodiment, the method and kits of this invention are for use in establishing the prognosis of patients having cancer. Cancers are classified by the type of cell that resembles the tumor and, therefore, the tissue presumed to be the origin of the tumor. In one embodiment, an example of a type of cancer in which PKCη may serve as a prognostic marker is Carcinoma. Carcinomas are malignant tumors derived from epithelial cells. This group represents the most common cancers, including the common forms of breast, prostate, lung and colon cancer. In another embodiment, this invention may be used in prognosis of Sarcoma. Sarcomas are malignant tumors derived from connective tissue, or mesenchymal cells. Examples of sarcomas are osteosarcoma which arises from bone, chondrosarcoma arises from cartilage, and leiomyosarcoma arises from smooth muscle. In some embodiments, prognosis as embodied by the present invention includes determination of prognosis with subjects presenting with carcinomas or sarcomas. In yet another embodiment, this invention may provide methods and kits for prognosis of lymphoma and leukemia which are malignancies derived from hematopoietic, blood- forming cells. In another embodiment, this invention provides methods and kits for prognosis of germ cell tumors. Germ cell tumors derived from totipotent cells and in adults most often are found in the testicle and ovary, in fetuses, babies, and young children most often found on the body midline, particularly at the tip of the tailbone and blastic tumor. In general germ cell tumor is a tumor which resembles an immature or embryonic tissue. [0037] In one embodiment, the type of cancer to be analyzed in this invention is a carcinoma. Carcinoma is any malignant cancer that arises from epithelial cells. Carcinomas invade surrounding tissues and organs and may metastasize, or spread, to lymph nodes and other sites. Carcinoma in situ (CIS) is a pre-malignant condition, in which some cytological signs of malignancy are present, but there is no histological evidence of invasion through the epithelial basement membrane.

[0038] Carcinomas can be subdivided into several types: in one embodiment, this invention may be used in determining the prognosis and treatment of Adenocarcinoma which is a malignant tumor originating in the epithelial cells of glandular tissue and forming glandular structures. Adenocarcinoma is common in the lung and breast tissue. In another embodiment, this invention relates to prognosis and treatment of squamous cell carcinoma due to squamous metaplasia. In yet another embodiment, this invention may be beneficial for prognosis of small cell carcinoma, including aggressive forms of the disease. In another embodiment, this invention provides methods and kits for prognosis of large cell undifferentiated carcinomas.

[0039] In one embodiment this invention is applied to the assesment of the prognosis of a subject having cancer of a reproductive organ in a subject. In one embodiment, the major organs of the human reproductive system include the external genitalia (penis and vulva) as well as a number of internal organs including the gamete producing gonads (testicles and ovaries). Examples of cancers of the reproductive system include prostate cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, gestational trophoblastic tumors, uterine sarcomas, vaginal cancer, vulvar cancer or penile cancer. In one embodiment, the prognosis is determined for a subject having breast cancer. In one embodiment, the subject is male or female. These cancers may be treated, or in some embodiments, the severity of the disease may be predicted, or in some embodiments, a subject's responsiveness to therapy may be predicted, in some embodiments, by the methods/using the kits of this invention.

[0040] In one embodiment, this invention provides methods and kits for assessing the prognosis and assessment of responsiveness to the treatment of breast cancer tumors. In one embodiment, breast cancer tumors can be subdivided to benign tumors (non cancers) as well as malignant tumors (cancers). In one embodiment, this invention can be used for prognosis and prediction of chemotherapy of benign breast tumors which can be further sub-classed to be of Lobular neoplasia which includes lobular carcinoma in situ, intraductal proliferate lesions which include Usual ductal hyperplasia, Flat epithelial hyperplasia, Atypical ductal hyperplasia or Ductal carcinoma in situ, Microinvasive carcinoma, Intraductal papillary neoplasms which include Central papilloma, Peripheral papilloma, Atypical papilloma, Intraductal papillary carcinoma or Intracystic papillary carcinoma, Adenosis, including variants Sclerosing adenosis, Apocrine adenosis, Blunt duct adenosis, Microglandular adenosis or Adenomyoepithelial adenosis, Radial scar/complex sclerosing lesion, Adenomas including Tubular adenoma, Lactating adenoma, Apocrine adenoma, Pleomorphic adenoma or Ductal adenoma, Myoepithelial lesions including Myoepitheliosis, Adenomyoepithelial adenosis, Adenomyoepithelioma or Malignant myoepithelioma, Fibroepithelial tumors including Fibroadenoma, Phyllodes tumour, Periductal stromal sarcoma, low grade or Mammary hamartoma, Tumors of the nipple including Nipple adenoma, Syringomatous adenoma or Paget's disease of the nipple, Malignant lymphoma, metastatic tumors and tumors of the male breast including Gynecomastia or Carcinoma. In one embodiment, this invention can be used for determining the prognosis of a subject and/or the prediction of chemotherapy effectiveness as a treatment for malignant breast tumors in a subject, which cancers can be further subclassed to be of Invasive ductal carcinoma, Mixed type carcinoma, Pleomorphic carcinoma, Carcinoma with osteoclastic giant cells, Carcinoma with choriocarcinomatous features or Carcinoma with melanotic features, Invasive lobular carcinoma, Tubular carcinoma, Invasive cribriform carcinoma, Medullary carcinoma, Mucinous carcinoma and other tumors with abundant mucin including Mucinous carcinoma, Cystadenocarcinoma and columnar cell mucinous carcinoma or Signet ring cell carcinoma, Neuroendocrine tumors including Solid neuroendocrine carcinoma (carcinoid of the breast), Atypical carcinoid tumour, Small cell / oat cell carcinoma or Large cell neuroendocrine carcinoma, Invasive papillary carcinoma, Invasive micropapillary carcinoma, Apocrine carcinoma, Metaplastic carcinomas which include Pure epithelial metaplastic carciomas, Squamous cell carcinoma, Adenocarcinoma with spindle cell metaplasia, Adenosquamous carcinoma, Mucoepidermoid carcinoma and Mixed epithelial/mesenchymal metaplastic carcinomas, Lipid-rich carcinoma, Secretory carcinoma, Oncocytic carcinoma, Adenoid cystic carcinoma, Acinic cell carcinoma, Glycogen-rich clear cell carcinoma, Sebaceous carcinoma, Inflammatory carcinoma, Bilateral breast carcinoma, Haemangioma,

Angiomatosis, Haemangiopericytoma, Pseudoangiomatous stromal hyperplasia, Myofibroblastoma, Fibromatosis (aggressive), Inflammatory myofibroblastic tumor, Lipoma, Angiolipoma, Granular cell tumor, Neurofibroma, Schwannoma, Angiosarcoma, Liposarcoma, Rhabdomyosarcoma, Osteosarcoma, Leiomyoma or Leiomysarcoma. [0041] Prognosis is conducted, inter alia, by assessing PKCη cellular localization.

Assessment may include the use of antibodies including IgM and IgG, specifically recognizing PKCη. In one embodiment, specific antibodies for PKCη are available commercially, for example anti-PKCη from Santa Cruz (sc-215).

[0042] In one embodiment, the term "biological sample" is referred to a tissue or cells retrieved from a subject suspected of having or having cancer. One example of a biological

_. sample is a biopsy taken from a subject for analysis of malignancy.

[0043] In one embodiment, the process of detection of a specific protein by the use of an antibody on a biological sample is termed immunostaining or immunohistochemistry

(IHC). Immunohistochemistry procedures are known to those skilled in the art, for example, as described in "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-HI Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); or, for example, U.S. Pat. Nos.

3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521 all of which are incorporated by reference herein. [0044] In one embodiment detection of a contact between an antibody and PKCη in the biological sample is done by the use of linking to a labeling agent. In one embodiment, the term "a labeling agent" refers to a molecule which renders readily detectable that which is contacted with a labeling agent. In one embodiment, the labeling agent is a marker polypeptide. The marker polypeptide may comprise, for example, green fluorescent protein (GFP), DS-Red (red fluorescent protein), secreted alkaline phosphatase (SEAP), beta- galactosidase, luciferase, or any number of other reporter proteins known to one skilled in the art. In another embodiment, the labeling agent may be conjugated to another molecule which provides greater specificity for the target to be labeled. For example, and in one embodiment, the labeling agent is a fluorochrome conjugated to an antibody which specifically binds to a given target molecule, or in another embodiment, which specifically binds another antibody bound to a target molecule, such as will be readily appreciated by one skilled in the art.

[0045] In one embodiment, determining the prognosis of a subject depends on the cellular localization of PKCη. In one embodiment, detection of PKCη localization at cellular membranes as opposed to cytplasmic localization is indicative of poor prognosis for the assessed subject. In one embodiment, the phrase "cellular membrane" refers to inter alia, a plasma membrane, nuclear membrane, mitochondrial membrane, Golgi membrane, endoplasmic reticulum membrane, lysosomal membrane, endosomal membrane, peroxisomal membrane or vesicular membrane. [0046] In one embodiment, the determination of a poor prognosis is reflected by, inter alia, a reduced life expectancy, none or low responsiveness to cancer therapy, metastasis, enhanced cancer progression, rapid cancer cell growth or proliferation or high cancer aggressiveness. [0047] In one embodiment, the method of determining cancer prognosis is applicable to subjects before, during or after cancer therapy regimen. Cancer therapy regimen comprises of surgery or radiation therapy or chemotherapy or any combination thereof. [0048] In one embodiment, this invention further provides a kit for determining the prognosis of a subject having or suspected of having cancer, the kit comprising:

. • a first reagent for the detection of PKCη;

• a second reagent for labeling membranous structures in a cell;

• optionally at least two detectable markers, each of which specifically interacts with said first and said second reagent, and wherein overlap between said first and said , second reagent is discernible; and

• optionally reagents for isolating and processing biological samples.

[0049] In one embodiment a kit is system which allows rapid, portable detection of a biological or chemical substance in a mixture or a biological sample. Examples of biological kits are PSA EIA Kit for detection of the PSA antigen indicative of prostate cancer and Rheumatoid Factor-directed IgA EIA Kit indicative of autoimmunity (Immuno-

Biological Laboratories, Inc.).

[0050] In one embodiment the kit can be manufactured and sold as a kit of parts, a combinations of the agents listed, other reagents may be incorporated, kits can be sold with each single element and instruction or understanding that they may be combined for use in a diagnostic assay.

[0051] In one embodiment, the first agent is an antibody directed against PKCη. In one embodiment, the second agent is a reagent for labeling membranes. Membrane labeling agents such as lipophilic dyes stain live and fixed cells. In one embodiment, and for example, the lipophilic carbocyanines DiI (DiIC₁₈(3), ), DiO (DiOC₁₈(3), ), DiD (DiICi₈(5), ) and DiR (DiICi₈(7), ) are weakly fluorescent in water but highly fluorescent and photostable when incorporated into membranes. DiI, DiO, DiD and DiR exhibit distinct orange, green, red and infrared fluorescence, respectively, thus facilitating multicolor imaging. In another embodiment, membrane labeling can be achieved by using an antibody directed to proteins located at the membrane. Such proteins are, for example, are receptor molecules embedded with in the cytoplasmic or nuclear membrane.

[0052] In one embodiment, both first agent and second agent can be labeled with labeling agents which are distinct to allow differentiation between PKCη and the membrane upon staining. Examples of labeling agent are described above. Distinct labeling is referring to, in the case of fluorescence, to distinct emission spectra. For example, green fluorescent protein (GFP) where its emission peak is at 509 nm in the lower green portion of the visible spectrum and DS-Red (red fluorescent protein) which has bright red fluorescence with emission maxima at 583 nm.

[0053] In another embodiment, the kit may include additional markers used for cancer staging. Cancer staging is a descriptor (usually numbers I to IV) of how much the cancer has spread. The stage often takes into account the size of a tumor, how deep it has penetrated, whether it has invaded adjacent organs, how many lymph nodes it has metastasized to, and whether it has spread to distant organs. Staging of cancer is important because the stage at diagnosis is the most powerful predictor of survival, and treatments are often changed based on the stage. Staging can be determined by molecular markers such as Ki67 staining for proliferation of cells or by usage of a cancer specific antigen staining. Examples for antigenic polypeptide as cancer markers are KS 1/4 pan-carcinoma antigen (Perez and Walker, 1990, J. Immunol. 142:32-37; Bumal, 1988, Hybridoma 7(4):407-415) or ovarian carcinoma antigen (CA125) (Yu et al, 1991, Cancer Res. 51(2):48-475). In one embodiment other cancer related antigens are prostatic acid phosphate (Tailor et al, 1990, Nucl. Acids Res.

18(1):4928), prostate specific antigen (Henttu and Vihko, 1989, Biochem. Biophys. Res. Comm. 10(2):903-910; Israeli et al, 1993, Cancer Res. 53:227-230), melanoma-associated antigen p97 (Estin et al, 1989, J. Natl. Cancer Instit. 81 (6):445-44), melanoma antigen gp75 (Vijayasardahl et al, 1990, J. Exp. Med. 171(4): 1375-1380), high molecular weight melanoma antigen (HMW-MAA) (Natali et al., 1987, Cancer 59:55-3; Mittelman et al,

1990, J. Clin. Invest. 86:2136-2144)), prostate specific membrane antigen, carcinoembryonic antigen (CEA) (Foon et al, 1994, Proc. Am. Soc. Clin. Oncol. 13:294), polymorphic epithelial mucin antigen, human milk fat globule antigen, colorectal tumor- associated antigens such as: CEA, TAG-72 (Yokata et al, 1992, Cancer Res. 52:3402- 3408), CO17-1A (Ragnhammar et al, 1993, Int. J. Cancer 53:751-758); GICA 19-9

(Herlyn et al, 1982, J. Clin. Immunol. 2:135), CTA-I and LEA, Burkitt's lymphoma antigen-38.13, CD19 (Ghetie et al, 1994, Blood 83:1329-1336), human B-lymphoma antigen-CD20 (Reffe? al, 1994, Blood 83:435-445), CD33 (Sgouros et al, 1993, J. Nucl. Med. 34:422-430), melanoma-specific antigens such as ganglioside GD2 (Saleh et al, 1993, J. Immunol., 151, 3390-3398), ganglioside GD3 (Shitara et al, 1993, Cancer

Immunol. Immunother. 36:373-380), ganglioside GM2 (Livingston et al, 1994, J. Clin. Oncol. 12:1036-1044), ganglioside GM3 (Hoon et al, 1993, Cancer Res. 53:5244-5250), tumor-specific transplantation type of cell-surface antigen (TSTA) such as virally-induced tumor antigens including T-antigen DNA tumor viruses and envelope antigens of RNA tumor viruses, oncofetal antigen-alpha-fetoprotein such as CEA of colon, bladder tumor oncofetal antigen (Hellstrom et al, 1985, Cancer. Res. 45:2210-2188), differentiation antigen such as human lung carcinoma antigen L6, L20 (Hellstrom et al, 1986, Cancer Res. 46:3917-3923), antigens of fibrosarcoma, human leukemia T cell antigen-Gp37 (Bhattacharya-Chatterjee et al, 1988, J. of Immun. 141:1398-1403), neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR (Epidermal growth factor receptor), HER2 antigen (pl85HER2), polymorphic epithelial mucin (PEM) (Hilkens et al, 1992, Trends in Bio. Chem. Sci. 17:359), malignant human lymphocyte antigen-APO-1 (Bernhard et al, 1989, Science 245:301-304), differentiation antigen (Feizi, 1985, Nature 314:53-57) such as I antigen found in fetal erythrocytes and primary endoderm, I(Ma) found in gastric adenocarcinomas, Ml 8 and M39 found in breast epithelium, SSEA-I found in myeloid cells, VEP8, VEP9, MyI, VM-D5, and Dl 56-22 found in colorectal cancer, TRA- 1-85 (blood group H), C14 found in colonic adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastric cancer, Y hapten, Ley found in embryonal carcinoma cells, TL5 (blood group A), EGF receptor found in A431 cells, El series (blood group B) found in pancreatic cancer, FC 10.2 found in embryonal carcinoma cells, gastric adenocarcinoma, CO-514 (blood group Lea) found in adenocarcinoma, NS-10 found in adenocarcinomas, CO-43 (blood group Leb), G49, EGF receptor, (blood group ALeb/Ley) found in colonic adenocarcinoma, 19.9 found in colon cancer, gastric cancer mucins, T5A7 found in myeloid cells, R24 found in melanoma, 4.2, GD3, Dl.1, OFA-I, GM2, OFA-2,

GD2, Ml:22:25:8 found in embryonal carcinoma cells and SSEA-3, SSEA-4 found in 4-8- cell stage embryos. In another embodiment, the antigen is a T cell receptor derived peptide from a cutaneous T cell lymphoma (see Edelson, 1998, The Cancer Journal 4:62). [0054] In another embodiment, the antigenic peptide or protein is derived from HER2/neu or chorio-embryonic antigen (CEA) for suppression/inhibition of cancers of the breast, ovary, pancreas, colon, prostate, and lung, which express these antigens. Similarly, mucin-type antigens such as MUC-I can be used against various carcinomas; the MAGE, BAGE, and Mart-1 antigens can be used against melanomas. In one embodiment, the methods may be tailored to a specific cancer patient, such that the choice of antigenic peptide or protein is based on which antigen(s) are expressed in the patient's cancer cells, which may be predetermined by, in other embodiments, surgical biopsy or blood cell sample followed by immunohistochemistry.

[0055] In one embodiment, the kit may include reagents for isolating and processing of biological samples. Such reagents are well known to the skilled in the art and are described in details in Current Protocols in Cell Biology Copyright © 2000-2008 by John Wiley & Sons, Inc.

[0056] In one embodiment this invention further provides a method for determining the responsiveness of a subject to a particular chemotherapeutic regimen, the method comprising:

• obtaining a cancerous or precancerous biological sample from a subject prior to and following treatment of the subject with a chemotherapeutic regimen; and

• detecting PKCη expression and cellular localization in the biological samples; whereby predominant cytoplasmic localization of PKCη in cells from the sample is indicative of responsiveness to the chemotherapeutic regimen, while localization of

PKCη at cellular membranes or nuclear membranes in the sample is indicative of lack of responsiveness to the chemotherapeutic regimen.

[0057] In one embodiment, the term responsiveness refers to a measurable change in a tumor as a result of an antitumorigenic treatment. For example, reduction in tumor size or detection of necrosis within a tumor due to administration of chemotherapy. In one embodiment, a chemotherapeutic regimen is the use of a combination of chemotherapeutic drugs which is identified with acronyms representing each drugs first letter. For example, and in one embodiment, ABVD represents Adriamycin (doxorubicin), bleomycin, vinblastine, dacarbazine in one treatment for Hodgkin's lymphoma and CAF represents Cyclophosphamide, Adriamycin (doxorubicin), fluorouracil (5-FU) for treatment of breast cancer. In one embodiment, the chemotherapeutic regimen further includes radiation therapy and surgery.

[0058] In one embodiment radiation therapy (or radiotherapy) may precede or coincide with the effecting of the methods of this invention. [0059] In one embodiment, obtaining a cancerous or precancerous biological sample for effecting the methods of this invention is accomplished by surgery and/or via biopsy, which may include incisional biopsy or core biopsy or excisional biopsy. In some embodiments, the sample is procured via needle aspiration biopsy. [0060] In one embodiment, detection of localization of PKCη in the cancerous or precancerous biological sample is done by immunohistochemistry with an anti-PKCη antibody as described above. In one embodiment, detection of PKCη at the cytoplasm in the cancerous or precancerous biological sample is indicative of responsiveness for the chemotherapeutic regimen, meaning that the regimen is beneficial for the patient's recovery. Furthermore, and in another embodiment, changes in cytoplasmic localization of PKCη as described, after the exposure of the subject to a chemotherapeutic regimen is indicative of responsiveness to the chemotherapeutic regimen, where detection of PKCη at a cell or nuclear membrane is indicative of the lack of responsiveness to the current treatment, poor prognosis and is indicative that a change in the chemotherapeutic regimen administered to the patient should be effected. Furthermore, and in another embodiment, detection of movement of PKCη, from the cytoplasm to a cell or nuclear membrane, following chemotherapeutic regimen is indicative of lack of responsiveness to the current treatment, poor prognosis. [0061] In one embodiment, the method of detection of PKCη localization as an indicative assay for prediction of responsiveness for chemotherapeutic regimen may be repeated upon decision to change the chemotherapeutic regimen, thus resulting in determination of the most suitable chemotherapeutic regimen for a specific patient. In one embodiment, and for example, in treatment of breast cancer, several chemotherapy regimens are applicable. In one embodiment, treatment of the cancer with CAF (Cyclophosphamide, Adriamycin (doxorubicin), fiuorouracil (5-FU)) may be followed by analysis of PKCη localization by the methods and kits of this invention, determining the responsiveness to the current treatment. In one embodiment, if there is detection of PKCη localization at a cellular membrane this may be indicative to a change in chemotherapy regimen. For example, in one embodiment, change of chemotherapy regimen may include CMF (Cyclophosphamide, methotrexate, fiuorouracil (5-FU)) or FEC (Fiuorouracil (5-

FU), epirubicin, cyclophosphamide). In one embodiment, after each change of chemotherapy regimen, the methods and kit of this invention may be used to asses the efficacy of the current regimen. In one embodiment, a change in localization of PKCη to a cell or nuclear membrane is indicative to poor prognosis and responsiveness to the current chemotherapy regimen.

[0062] In one embodiment, this invention further provides a method for treating cancer, in particular, for treating invasive breast or lung cancer, reducing the incidence of cancer, in particular, for treating invasive breast or lung cancer, or prolonging remission of cancer, in particular, for treating invasive breast or lung cancer, in a subject, the method comprising contacting a neoplastic or preneoplastic cell in the subject with an agent, which inhibits PKCη translocation from the cytoplasm to membranous structures in said cell. [0063] As used herein, the term "treating" includes preventative as well as disorder remitative treatment. As used herein, the term "reducing" have its commonly understood meaning of lessening or decreasing. As used herein, the term "prolonging" means increasing the interval of remission time. As used herein, the term "neoplastic cell" means a cell with abnormal proliferation, hence a tumorigenic cell. A "preneoplastic cell" is a cell with a potential to become neoplastic/tumorigenic. [0064] In one embodiment, the term "agent" refers to compound capable of inhibition the translocation of PKCη to a cell or nuclear membrane in the cells. In one embodiment, the agent is a peptide fragment of an activated kinase C eta receptor. In this approach, inhibition of protein activity is achieved by using peptide fragments that act as inhibitors of translocation. Activation and translocation of the PKC isoforms to specific subcellular sites is believed to confer distinct physiological actions for each PKC isoform and is thought to be achieved by the binding of each activated PKC isoform to specific anchoring proteins.

Receptors for activated C kinases (RACKs) are one example of PKC-binding proteins (Mochly-Rosen, D. and A.S. Gordon, Anchoring proteins for protein kinase C: a mean for isozyme selectivity. FASEB, 1998. 12: p. 35-42 and CSUKAI, M. and D. Mochly-Rozen, Pharmacologic Modulation of Protein Kinase C Isozymes: The Role of Racks and Subcellular Localization. Pharmacological Research, 1999. 39(4): p. 253-259).

[0065] By "peptide" or "polypeptide" as used herein is meant any molecule whose expression can be directed by a specific DNA sequence. The polypeptides of this invention may comprise more than one subunit, where each subunit is encoded by a separate DNA sequence. As used herein in the specification and in the examples section which follows the term "peptide" or "polypeptide" includes native polypeptides such as enzymes, antibodies and receptors (either degradation products, synthetically synthesized polypeptides or recombinant polypeptides) and peptidomimetics (typically, synthetically synthesized polypeptides), such as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the polypeptides more stable while in a body or more capable of penetrating into bacterial cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH₂-NH, CH₂-S, CH₂-S=O, O=C-NH, CH₂-O, CH₂-CH₂, S=C-NH, CH=CH or CF=CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, CA. Ramsden Gd., Chapter

17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder. [0066] Peptide bonds (-CO-NH-) within the polypeptide may be substituted, for example, by N-methylated bonds (-N(CHk)-CO-), ester bonds (-C(R)H-C-O-O-C(R)-N-), ketomethylen bonds (-CO-CH₂-), α-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl, e.g., methyl, carba bonds (-CH₂-NH-), hydroxyethylene bonds (-CH(OH)-CH₂-), thioamide bonds (-CS-NH-), olefinic double bonds (-CH=CH-), retro amide bonds (-NH-C0-), peptide derivatives (-N(R)-CH₂-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom.

[0067] These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time.

[0068] Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for synthetic non-natural acid such as TIC, naphthylelanine (NoI), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.

[0069] In addition to the above, the polypeptides of the present invention also include one or more modified amino acids or one or more non-amino acid monomers (e.g. synthetic, fatty acids, complex carbohydrates etc). Methods for incorporating synthetic amino acids into a polypeptide are described in US patents Nos: US 20070009990A1, US 20050136513A1 and US 7,250,267.

[0070] In one embodiment, the peptide is administered to the subject in parallel to adjunct cancer therapy. As used herein "adjunct cancer therapy" includes surgery, radiation or chemotherapy or any combination thereof. In particular treatment of a subject with the PKCη translocation inhibiting peptide will be in parallel to administration of chemotherapeutic compounds/drugs.

[0071] As used herein, the term "administering" refers to bringing a subject in contact with a compound of the present invention. As used herein, administration can be accomplished in vitro, i.e. in a test tube, or in vivo, i.e. in cells or tissues of living organisms, for example humans. In one embodiment, the present invention encompasses administering the compounds of the present invention to a subject.

[0072] In some embodiments, the term "contacting" or "administering" refers to both direct and indirect exposure to the indicated material.

[0073] In one embodiment, the term "drug" refers to a molecule that alleviates a symptom of a disease or disorder when administered to a subject afflicted thereof. In one embodiment, a drug is a synthetic molecule, or in another embodiment, a drug is a naturally occurring compound isolated from a source found in nature. [0074] In one embodiment, drugs may comprise any agent, which is useful in halting or altering the course of neoplasia or metastasis. In some embodiments, the drug is cytotoxic to neoplastic cells or preneoplastic cells selectively, or in some embodiments, preferentially.

[0075] In one embodiment, a drug of this invention may comprise antineoplastic agents such as platinum compounds (e.g., spiroplatin, cisplatin, and carboplatin), methotrexate, fluorouracil, adriamycin, mitomycin, ansamitocin, bleomycin, cytosine arabinoside, arabinosyl adenine, mercaptopolylysine, vincristine, busulfan, chlorambucil, melphalan (e.g., PAM, L-PAM or phenylalanine mustard), mercaptopurine, mitotane, procarbazine hydrochloride dactinomycin (actinomycin D), daunorubicin hydrochloride, doxorubicin hydrochloride, paclitaxel and other taxenes, rapamycin, manumycin A, TNP-470, plicamycin (mithramycin), aminoglutethimide,estramustine phosphate sodium, flutamide, leuprolide acetate, megestrol acetate, tamoxifen citrate, testolactone, trilostane, amsacrine (m-AMSA), asparaginase (L-asparaginase) Erwina asparaginase, interferon .alpha. -2a, interferon .alpha.-2b, teniposide (VM-26), vinblastine sulfate (VLB), vincristine sulfate, bleomycin sulfate, hydroxyurea procarbazine, and dacarbazine; mitotic inhibitors such as etoposide, colchicine, and the vinca alkaloids, radiopharmaceuticals such as radioactive iodine and phosphorus products; hormones such as progestins, estrogens and antiestrogens; anti-helmintics, antimalarials, and antituberculosis drugs; biologicals such as immune serums, antitoxins and antivenoms; rabies prophylaxis products; bacterial vaccines; viral vaccines; biological response modifiers such as muramyldipeptide, muramyltripeptide, microbial cell wall components, lymphokines (e.g., bacterial endotoxin such as lipopolysaccharide, macrophage activation factor), sub-units of bacteria (such as Mycobacteria, Corynebacteria), the synthetic dipeptide N-acetyl-muramyl-L-alanyl-D- isoglutamine; anti-fungal agents such as ketoconazole, nystatin, griseofulvin, flucytosine (5-fc), miconazole, amphotericin B, ricin, cyclosporins, and β-lactam antibiotics (e.g., sulfazecin); hormones such as growth hormone, melanocyte stimulating hormone, estradiol, beclomethasone dipropionate, betamethasone, betamethasone acetate and betamethasone sodium phosphate, vetamethasone disodium phosphate, vetamethasone sodium phosphate, cortisone acetate, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, flunisolide, hydrocortisone, hydrocortisone acetate, hydrocortisone cypionate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, paramethasone acetate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, prednisone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate, triamcinolone hexacetonide, fludrocortisone acetate, oxytocin, vassopressin, and their derivatives; vitamins such as cyanocobalamin neinoic acid, retinoids and derivatives such as retinol palmitate, and .alpha.-tocopherol; peptides, such as manganese super oxide dismutase; enzymes such as alkaline phosphatase; anti-allergic agents such as amelexanox; antituberculars such as para-aminosalicylic acid, isoniazid, capreomycin sulfate cycloserine, ethambutol hydrochloride ethionamide, pyrazinamide, rifampin, and streptomycin sulfate; antivirals such as amantadine azidothymidine (AZT, DDI, Foscarnet, or Zidovudine), ribavirin and vidarabine monohydrate (adenine arabinoside, ara-A); antianginals such as diltiazem, nifedipine, verapamil, erythritol tetranitrate, isosorbide dinitrate, nitroglycerin (glyceryl trinitrate) and pentaerythritol tetranitrate; antiinflammatories such as diflunisal, ibuprofen, indomethacin, meclofenamate, mefenamic acid, naproxen, oxyphenbutazone, phenylbutazone, piroxicam, sulindac, tolmetin, aspirin and salicylates; antiprotozoans such as chloroquine,hydroxychloroquine, metronidazole, quinine and meglumine antimonate; radioactive particles or ions such as strontium, iodide rhenium and yttrium, or any combination of drug or agent as herein described.

[0076] The compositions or peptides or drugs of this invention may be administered in any effective, convenient manner including, for instance, administration by intravascular (i.v.), intramuscular (i.m.), intranasal (i.n.), subcutaneous (s.c), oral, rectal, intravaginal delivery, or by any means in which the glucan/composition can be delivered to tissue (e.g. needle or catheter). Alternatively, topical administration may be desired for insertion into epithelial cells. Another method of administration is via aspiration or aerosol formulation. In some embodiments the peptide is administered by implanting or introducing into the body of a subject, an implant or other medical or surgical device that comprises the peptide, e.g. as a component of a coating layer. [0077] It is to be understood that repeated use of reference characters in the present specification and drawings is intended to represent the same or analogous features of the invention.

[0078] It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as set forth in the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed in the scope of the claims. [0079] In the claims articles such as "a,", "an" and "the" mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" or "and/or" between members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention provides, in various embodiments, all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, e.g. in

Markush group format or the like, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in haec verba herein. Certain claims are presented in dependent form for the sake of convenience, but Applicant reserves the right to rewrite any dependent claim in independent format to include the elements or limitations of the independent claim and any other claim(s) on which such claim depends, and such rewritten claim is to be considered equivalent in all respects to the dependent claim in whatever form it is in (either amended or unamended) prior to being rewritten in independent format

[0080] The following examples describe certain embodiments of the invention and and should not be construed as limiting the scope of what is encompassed by the invention in any way.

EXAMPLES

MATERIALS AND METHODS Immunohistochemical Sample collection and analysis [0081] Paraffin embedded human tumors derived from breast cancer patients were received from the Soroka hospital pathology department archive. Information concerning the tumor stage, grade and marker status (ER, PR, p53, erb and Ki67) was retrieved from each patient's medical file. Immunohistochemical staining of the paraffin-cut sections with PKCη specific antibodies was preformed for detection of PKCη expression in the primary breast tumors. As a positive control for PKCη endothelial cells were used, which were always stained intensely. When possible, tumor grade, stage, receptor (ER, PR) status, HER2 expression and expression of PKCη were compared.

PKCTI analysis of tissue pre- and post-neoadjuvant therapy

[0082] Primary assessments of breast lesions were performed by core needle biopsy (CNB), which enabled primary histological evaluation of the lesion. Women diagnosed as having advanced breast tumors were treated with a neoadjuvant protocol which included chemotherapy before the resection of the tumor. Breast cancer tissue was examined by collecting biopsies before (CNB) and after chemotherapy for expression of PKCη, and correlation with tumor aggressiveness, treatment effectiveness, resistance to treatment and long term survival was assessed. As a control, PKC epsilon (PKCε) expression was assessed. [0083] 25 patients with advanced breast cancer, who underwent neoadjuvant therapy with a regimen of CAF - Cyclophosphamide, Doxorubicin and Fluorouracil between 1995 and 2000 were evaluated. Following neoadjuvant therapy, the patients underwent surgery to remove tumors and lymph nodes. Core biopsy samples prior to chemotherapy treatment, and surgical specimens including the primary tumor and lymph node metastases were analyzed. PKCη expression was assessed using immunohistochemistry. Routine pathologic assessment of the tumors was determined, including in-situ versus invasion, tumor size, grade, vascular invasion and lymph node invasion. ER, PR, HER2, Ki67 and p53 expression were all routinely assessed. Epidemiologic data including age, country of origin, number of births, age at tumor occurrence, and menopause status were recorded from medical files. [0084] Core biopsies, tumor biopsies and biopsies from lymph nodes were assessed for

PKCη expression. PKCη expression was analyzed independently in the different cellular components: cytoplasm, cellular membranes, nuclear membrane and nucleus. Immunohistochemistry of cytoplasmatic staining was measured semi-quantitively by a scale of 0-3, as specified: 0 - no positive cells; 1 - 10-20% strongly positive cells or at least weakly positive cells; 2 - 20-50% strongly positive cells or more than 50% weakly positive cells; 3 - >50% strongly positive cells. The intensity of membrane and nuclear staining was defined more generally as negative - 0 or positive - 1.

[0085] Cell culture, UVC irradiation and reagents. MCF-7 cells inducibly expressing PKCη, MCF21.5, were previously described (Fima, 2001). MCF21.5 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) containing 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM 1-glutamine and 10% fetal bovine serum in a 5% CO₂ humidified atmosphere at 37°C. The cells were grown in the presence of 100 μg/ml hygromycine and 2 μg/ml tetracycline and the expression of PKCη was induced by the removal of tetracycline from the growth medium. For the irradiation studies, sub-confluent cells were employed. The medium was removed and the cells were exposed to UVC (254 nm), 1.3 J/m² to 4 J/m² per second for the indicated times. Cell survival was determined 24 h following treatments by cell counting using a 22 coulter counter and by trypan blue staining. Tetracycline and Camptothecin (CPT) were purchased from Sigma. [0086] Cell lysis and immunoblot analysis. Cell lysates were prepared using RIPA buffer containing 10 mM Tris pH 8.0, 100 mM NaCl, 5 mM EGTA, 0.1% SDS, 1% NP- 40, 45 mM β-mercaptoethanol, 50 mM NaF. Protease inhibitors (1 mM PMSF, 10 μg/ml aprotinine and 10 μg/ml leupeptin) and phosphatase inhibitors (100 μM sodium orthovanadate, 50 mM βglycerolphosphate and 5 mM sodium pyrophosphate) were added just before cell lysis. Lysates were placed on ice for 30 min and sheared several times through a 21 -gauge needle. For detection of caspase cleavage, cell lysates were prepared using CHAPS buffer containing 50 mM pipes/HCl (pH 6.5), 2 mM EDTA, 0.1% CHAPS, 5 mM DTT and protease inhibitors as above, followed by freezing and thawing for three times. The cell lysates were centrifuged at 14,000 g for 15 min at 4°C, and protein concentrations were determined using the Bio-Rad protein assay. Aliquots of 100-15Pμg protein were separated on SDS-PAGE (10-15%) and blotted onto PVDF membrane (Bio- Rad). Proteins were detected using anti-PKCη (sc-215), purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Anti-caspase-7 (9915) and anti-PARP-1 (9542) were purchased from Cell Signaling Technology. For the detection of primary antibodies blots were incubated with horseradish peroxidase-conjugated to donkey anti-rabbit or anti- mouse immunoglobulin (Amersham Pharmacia) followed by enhanced chemiluminescence ECL reagent analysis (Amersham Pharmacia).

EXAMPLE 1: PKOn Expression in Breast Cancer Cells [0087] In order to compare the staining of PKCη in tumor versus normal tissue, samples were analyzed for the presence of normal tissue. Out of 53 paraffin cut sections,

11 specimens were found to have normal tissue adjacent to the tumor.

[0088] Referring now to figure 1, which shows typical Hematoxylin and Eosin staining of normal breast tissue.

[0089] Figure 2, shows PKCη staining of normal breast tissue, adjacent to tumor tissue. The sample is a tumor sample including some normal tissue adjacent to the tumor.

PKCη staining in normal tissue was always found to be high in any case where normal tissue was analyzed.

[0090] Tumor tissue was found to be heterogenic in staining. Seven biopsies were found to have low PKCη expression, 16 biopsies were found to have moderate PKCη expression and 30 biopsies were found to have high PKCη expression (Table 1).

Table 1 Immunohistochemical analysis of PKCη expression in normal and tumor breast tissue.

[0091] Referring now to Figure 3, which demonstrates PKCη staining of tumor biopsies. Staining of in-situ tumor in different amplification is shown in Figure 3A x20 and Figure 3B x40. Cytoplasmic staining of invasive ductal carcinoma by PKCη is shown in Figures 3C and 3D in an amplification of x40. Figure 3E is staining of invasive ductal carcinoma, where membrane-associated staining of PKCη is seen in magnification of x40, and an example of a tumor with cytoplasmatic and nuclear staining can be seen in Figure 3F, magnification x20.

[0092] The intracellular localization of PKCη was found to be heterogeneous (Figure 3). In most tumors PKCη expression was essentially cytoplasmic, as seen in figure 3 C and D. Some of the tumors showed punctate cytoplasmic staining while other tumors showed homogenous staining. Nuclear (Fig. 3F) and membrane (Fig. 3E) staining were also seen. No correlation was found between PKCη expression and the grade of the tumor, or the ER, PR status (Table 2). 2

Table 2

Expression of ER, PR and HER2 receptors in tumors expressing high versus low expression of PKCη.

[0093] In order to detect PKCη membrane staining, 53 samples were analyzed for cellular localization of PKCη. In 8 of the 53 tumor samples evaluated, membrane staining was detected, as seen in Fig. 3E. The pathological data for these tumors found them to be relatively aggressive tumors. Five of these tumors were Her2 positive, while the three other tumors had a low level of differentiation (grade 3).

EXAMPLE 2

PKCn Expression in Breast Cancer Biopsies Before and After Neoadjuvant

Chemotherapic Treatment [0094] To determine if there is a difference in PKCη localization before and after chemotherapy, breast tissue was evaluated prior to and post neoadjuvant treatment with a protocol which included chemotherapy before the resection of the tumor. The effect of chemotherapy on the expression of PKCη was assessed, as was its correlation with tumor aggressiveness, treatment efficacy, resistance to treatment and long term survival. As a control PKCε expression was assessed.

[0095] PKCη expression was initially examined in core biopsies collected prior to chemotherapy, and thus represented the basal expression of PKCη in the tumor cells. For a control group, specimens from example 1 immunohistochemical study were used. These specimens were not characterized as advanced tumors, and thus the patients did not undergo neoadjuvant therapy. Comparison between both groups showed that women in the advanced tumor group had initially lower expression levels of PKCη (Table 3). Table 3

Cytoplasmatic expression of PKCη in advanced breast tumors versus breast tumors that were not cate orized as advanced.

[0096] Table 4 describes PKCη expression before chemotherapy in the core biopsies, and after chemotherapy in the biopsy (from either lumpectomy or mastectomy) and in the lymph node biopsy (Table 4).

Table 4 Cytoplasmic expression of PKCη in core biopsy, biopsy and lymph nodes

[0097] Cytoplasmic expression was assessed as follows: low expression -1, intermediate expression -2, and high expression -3. In the core biopsies (before treatment) 8 biopsies (36%) were found to have low expression, 4 biopsies (18%) had intermediate expression and 10 biopsies (46%) had high expression. In the biopsies after chemotherapeutic treatment, 2 biopsies (9%) were found to have low expression of PKCη, Seven biopsies (30%) had intermediate expression and 14 biopsies (61%) had high expression of PKCη. In the lymph nodes only 1 biopsy (5%) was shown to have low expression of PKCη. Six biopsies were shown to have intermediate staining (30%), and 13 biopsies (65%) showed high expression of PKCη (table 4). Thus, the expression of PKCη increased after chemotherapy, both locally (p=0.026) and in lymph node metastases (p=0.022). Both results were shown to be statistically significant (Wilcoxon test). [0098] Membrane-associated expression of PKCη was defined as either negative (0) or positive (1). In the core biopsies (pre-chemotherapy), 3 biopsies (14%) were shown to have membrane-associated expression of PKCη, as opposed to 10 biopsies in the post- chemotherapeutic primary tumor (43%) and 9 post-chemotherapeutic lymph node biopsies (45%) (Table 5). Thus membrane-associated expression of PKCη was shown to increase after chemotherapeutic treatment both locally (p=0.05), and in lymph node metastases (p=0.108).

Table 5

Expression of PKCη in the cellular membranes prior to and after chemotherapeutic treatment.

[0099] Membrane-associated expression was defined as negative (0) or positive (1). In the core biopsies (before treatment), 5 (23%) biopsies were shown to have nuclear membrane-associated expression of PKCη, compared to 7 (30%) biopsies of the primary tumor and 9 (45%) lymph node biopsies (Table 6). Thus, expression of PKCη on the nuclear membrane also increased after chemotherapeutic treatment, both locally (p=0.593) and in lymph node metastases (p=0.284).

Table 6

Expression of PKCη in the nuclear membrane prior to and after chemotherapeutic treatment.

EXAMPLE 3

Correlation of PKCn Expression Changes After Neoadjuvant Chemotherapic

Treatment with Prognosis

[00100] In order to determine whether a correlation between the 5 year survival criterion and PKCη subcellular expression exists, patients were evaluated for expression and prognosis. Patients were divided into 2 groups, having either a "good prognosis" or a "poor prognosis". The group of "good prognosis" was composed of 8 patients who survived 5 years after tumor discovery, and the "poor prognosis" group which was composed of 17 women who had died 5 years or less after tumor discovery. This low 5 year survival rate (32%) is attributable to the fact that the study group is made of women who were initially categorized as having advanced tumors. [00101] Women in the poor prognosis group had lower ER and PR expression, higher HER2, Ki67 and p53 expression and a higher percent of documented metastases. There was no difference in the cytoplasmic expression of PKCη in both groups. In the primary biopsy (after chemotherapy) women in the good prognosis group had no cellular membrane expression of PKCη, as opposed to 62.5% expression in the poor prognosis group (p=0.07). In addition, women in the good prognosis group had no nuclear membrane expression, as opposed to 44% in the poor prognosis group (p=0.057), being close to statistical significance. In lymph node biopsies a similar correlation was found in cellular membrane expression (Table T). Thus women with poor prognosis showed high cellular membrane-associated expression of PKCη after chemotherapeutic treatment, whereas women with good prognosis showed no cellular membrane-associated expression.

Table 7 Expression of PKCη based on patient prognosis.

[00102] Expression of PKCε was next studied. PKCε was chosen as a control for several reasons. Like PKCη, PKCε is a member of the novel PKC sub-family, sharing several similarities to PKCη. Increasing evidence links PKCε to promotion of metastatic tumor cell phenotype. In addition immunohistochemical studies showed that the more aggressive high grade breast tumors (grade 3) showed higher expression of PKCε than low grade (grade 1) breast tumors. PKCε was also shown to correlate with ER and PR status. [00103] PKCε staining was completely different in staining pattern as compared to PKCη staining. Very high percentage of nuclear staining was detected, and in addition connective tissue was stained intensely, producing high background. Cytoplasmic expression of PKCε was similar in all study groups - pre and post chemotherapy (Table 8). Table 8 C to lasmic ex ression of PKCε in core bio s bio s and l m h nodes

[00104] Membrane-associated expression of PKCε was defined as either negative (0) or positive (1). In the core biopsies (before treatment), 9 (41%) biopsies were shown to have membrane-associated expression of PKCε, as opposed to 13 (57%) biopsies of the primary tumor and 12 (63%) lymph node biopsies (Table 9).

Table 9

Expression of PKCε in cellular membranes prior to and after chemotherapeutic treatment.

[00105] Expression of PKCε on the nuclear membrane was also defined as negative (0) or positive (1). In core biopsies (before treatment), 11 (50%) biopsies were shown to have membrane-associated expression of PKCε, as opposed to 17 positive biopsies of the primary tumor (74%) and 7 positive lymph node biopsies (37%) (Table 10).

Table 10

Expression of PKCε in the nuclear membrane prior to and after chemotherapeutic treatment.

[00106] Expression of PKCε in the nucleus was next defined. In core biopsies (before treatment), ,12 (55%) biopsies were shown to have nuclear expression of PKCε, as opposed to 13 (57%) biopsies of the primary tumor and 3 (16%) lymph node biopsies (Table 11). Table 11 Expression of PKCε in the nucleus prior to and after chemotherapeutic treatment.

[00107] Figure 8 graphically depicts the expression differences between PKCη and

PKCε. Differential PKCη detection was evident, with no appreciable detection on either cell or nuclear membranes of samples from subjects in the "good prognosis" group, and pronounced membranal expression in samples from subjects in the "poor prognosis" group (Figure 8A). In contrast, there was, however, no differential expression of PKCε detected in samples from either group (Figure 8B).

[00108] In summary, there is no trend in the expression of PKCε in the tumor cells before and after chemotherapeutic treatment. In addition no correlation was found between the cytoplasmic, membrane-associated or nuclear expression of PKCε and the prognosis of the patients. EXAMPLE 4

Altering PKCn Subcellular Localization in Cancer Treatment

[00109] In order to determine whether altering subcellular localization of PKCη positively affects cancer, specific peptides, which inhibit PKCη translocation, will be synthesized according to the method described in (Liu, GS 1999). These peptides will be used in an in-vitro breast cancer system (MCF7 cells) in combination with chemotherapy to asses the role of inhibition of PKCη translocation on the efficiency of chemotherapy, cell cycle progression and growth in soft agar as a measurement of metastasis proficiency. The same translocation inhibitors will be used in an in-vivo breast cancer mouse model (MMTV-c-myc [Stewart, TA 1984] or MMTV-Cyclin D [Wang, TC 1994] transgenic mice) by systemic administration of the peptides and assaying the development and aggressiveness of the breast tumors in addition to testing in-vivo the effects of PKCη translocation inhibition on chemotherapeutic response in this mouse breast tumor model.

EXAMPLE 5 PKCTI expression enhances cell survival

[00110] MCF7 cells inducibly expressing PKCη under the control of a tetracycline- responsive promoter (MCF21.5 cells) (Fima 2001) were grown in the presence or absence of 2 μg/ml tetracycline for 48h, were irradiated with increasing dosages of UVC irradiation (Fig. 4A), or were treated with increasing concentrations of CPT (Fig. 4B). Results are expressed as a percentage of viable cells in treated cultures compared to untreated control cultures and are representative of three separate experiments. Irradiation of cells for 20 seconds (1.3 J/m² per sec) led to a decrease in cell survival of about 52%, while the inducible expression of PKCη inhibited this effect and a decrease of only 20% was observed (Fig. 4A). The effect of PKCη expression on the survival of cells treated by CPT was similar. CPT exposure at a concentration of 2.5μM led to a decrease in cell survival of about 47% in control cells compared to a decrease of only 20% in the presence of PKCη expression (Fig. 4B). EXAMPLE 6

PARP-I cleavage is inhibited by PKCη expression in response to UVC irradiation and

CPT treatments

[00111] Sub-confluent MCF21.5 cells were grown in the presence or absence of 2 μg/ml tetracycline for 48 h and were exposed to increasing dosages of UVC irradiation (1.3 J/m² per s), as shown in Fig. 4A, or were treated with increasing concentrations of CPT, as presented in Fig. 4B. After 24 hours, whole cell lysates were prepared and subjected to immunoblotting with PARP-I specific antibodies that recognized the full length (116kD) and the PARP-I cleaved form (89 kD) of the protein. The inducible expression of PKCη protein in response to the presence/absence of tetracycline is also shown (Fig. 5A). As shown in Fig. 5 A, UVC irradiation resulted in the cleavage of PARP-I, as shown by the appearance of the 89 kD fragment. This cleavage was dose-dependent, and was inhibited by the inducible expression of PKCη in these cells (in response to tetracycline removal). A similar effect of PKCη protection against CPT- induced PARP-I cleavage is shown in Fig. 5B. Results represent three independent experiments. EXAMPLE 7

PKCη expression in MCF-7 cells inhibited caspase-7 and caspase-9 activation, and cytochrome C release from mitochondria in response to UVC irradiation or CPT treatment

[00112] MCF21.5 cells were grown in the presence or the absence of 2μg/ml tetracycline for 48h. The cleaved form of caspase-7 and full length form of caspase-9 were examined using specific antibodies, 24h after UVC irradiation at indicated dosages and after CPT treatment at the indicated concentrations (Fig. 6A). Results are representative of three separate experiments. Reduced production of the active form of caspase-7 is observed in cells expressing PKCη. Similarly, PKCη inhibited the cleavage of the upstream initiator pro-caspase-9, as significantly less cleavage of pro-caspase-9 was detected in PKCη -expressing cells. These results demonstrate interference of PKCη in the activation of caspase-7 and caspase-9.

[00113] Sub confluent MCF21.5 cells were grown in the presence or absence of 2μg/ml tetracycline for 48h and were then treated with UVC irradiation or CPT for 4 or 6 hours, respectively. The cells were immunostained with a native monoclonal antibody against cytochrome C (6H2.B4) and the release of cytochrome C from the mitochondria was detected using fluorescence microscope (Fig. 6B). The release of cytochrome C was quantified by counting 200 cells, and the results are expressed as the percentage of cells with mitochondrial staining in treated slides compared to control slides (Fig. 6B). The bars represent the mean values of two independent experiments and the error bars represent standard deviation.

EXAMPLE 8 Translocation of PKOn to the nuclear envelope in response to etoposide treatment

[00114] Cells were transfected with GFP-PKCη-WT, and 48h post transfection the cells were treated with etoposide (an inhibitor of topoisimerase II). Time lapse images of GFP-

PKCη expressing MCF-7 cells treated with 800μM Etoposide (Fig. 7A); HeLa cells treated with lOOμM Etoposide (Fig. 7B) and Cos-7 cells treated with 400μM Etoposide (Fig. 7C) were analyzed live using confocal scanning microscopy (X-100, Carl Zeiss). MCF-7 cells transfected with GFP-PKCα and treated with 800μM Etoposide are shown in Fig. 7D. MCF-21.5 cells, grown for 48h in the absence of tetracycline, thus, inducibly expressing

PKCη, were treated with 800μM Etoposide for different time points. Isolated NE and cytosolic fractions were extracted. 40 μg protein per lane of the cytosolic fraction cell lysate and maximum relative amount of protein from the NE fraction were loaded and separated by 10% SDS/PAGE. Immunoblot analysis of the fractions probed by anti-PKCη, anti- PKCα, anti-Lamin B and anti-Actin is presented in Fig. 7E

Claims

WHAT IS CLAIMED IS:

I. A method for assessing the prognosis of a subject having or suspected of having cancer, the method comprising:

• obtaining a biological sample from a subject; • determining a cellular localization of PKCη in said sample; and

• assessing cancer prognosis of the subject based upon said cellular localization, wherein PKCη localization at a cellular membrane, as opposed to cytoplasmic localization in said sample is indicative of a poor prognosis for the subject having or suspected of having cancer.

2. The method of claim 1, wherein said assessing is accomplished with the use of an antibody specifically interacting with PKCη.

3. The method of claim 1, wherein said cancer is a carcinoma.

4. The method of claim 1, wherein said cancer is a cancer of a reproductive organ in the subject.

5. The method of claim 1 , wherein said cancer is a breast or cervical cancer.

6. The method of claim 1, wherein said subject has been administered a cancer therapy regimen.

7. The method of claim 7, further comprising assessing the subject's responsiveness to said regimen, wherein lack of or minimal PKCη cytoplasmic localization following administration of said regimen is indicative of poor responsiveness to said regimen.

8. A kit for determining the prognosis of a subject having or suspected of having cancer, the kit comprising:

• a first reagent for the detection of PKCη;

• a second reagent for labeling membranous structures in a cell; • optionally at least two detectable markers, each of which specifically interacts with said first and said second reagent, and wherein overlap between said first and said second reagent is discernible; and

• optionally reagents for isolating and processing biological samples.

9. The kit of claim 8, wherein said first reagent, said second reagent or a combination thereof is an antibody.

10. The kit of claim 8, wherein said detectable markers are fluorescent.

I 1. The kit of claim 10, wherein said detectable markers have distinct emission spectra.

12. The kit of claim 8, further comprising reagents for detecting markers, which indicate the staging of a cancer.

13. A method for determining the responsiveness of a subject to a particular chemotherapeutic regimen, the method comprising: • obtaining a cancerous or precancerous biological sample from a subject prior to and following treatment of the subject with a chemotherapeutic regimen; and • detecting PKCη expression and cellular localization in said biological samples; whereby predominant cytoplasmic localization of PKCη in cells from said sample is indicative of responsiveness to said chemotherapeutic regimen, while localization of PKCη at cellular or organelle membranes in said sample is indicative of lack of responsiveness to said chemotherapeutic regimen.

14. The method of claim 13, wherein said determining is accomplished with the use of an antibody specifically interacting with PKCη.

15. The method of claim 13, wherein said cancer is a carcinoma.

16. The method of claim 13, wherein said cancer is a cancer of a reproductive organ in the subject.

17. The method of claim 13, wherein said cancer is a breast or a cervical cancer.

18. The method of claim 13, wherein said chemotherapeutic regimen comprises radiation, surgery, administration of a chemotherapeutic compound or a combination thereof.

19. The method of claim 12, further comprising altering a chemotherapeutic regimen based on a finding of lack of responsiveness.

20. The method of claim 19, further comprising repeating steps (i) and (ii) to determine responsiveness to the altered chemotherapeutic regimen.

21. A method for treating invasive breast cancer, reducing the incidence of invasive breast cancer, or prolonging remission of invasive breast cancer in a subject, the method comprising contacting a neoplastic or preneoplastic cell in a subject with an agent, which inhibits PKCη translocation from the cytoplasm to membranous structures in said cell.

22. The method of claim 21, wherein said subject is further administered a chemotherapeutic compound.

23. The method of claim 21, wherein said method further comprises providing adjunct cancer therapy to said subject.

24. The method of claim 23, wherein said adjunct therapy comprises surgery or radiation therapy.

25. The method of claim 21, wherein said agent, which inhibits PKCη translocation is a peptide fragment of an activated kinase C eta receptor.

26. The method of claim 21, wherein said cancer is a carcinoma.

27. The method of claim 21, wherein said cancer is a cancer of a reproductive organ in the subject.

28. The method of claim 21, wherein said cancer is a breast cancer.