WO2016094458A1

WO2016094458A1 - Methods for diagnosis and treatment of epithelial-to-mesenchymal transition of cancer cells and metastatic breast cancer

Info

Publication number: WO2016094458A1
Application number: PCT/US2015/064574
Authority: WO
Inventors: Partha S. RAY
Original assignee: Ray Partha S
Priority date: 2014-12-08
Filing date: 2015-12-08
Publication date: 2016-06-16

Abstract

Disclosed herein are methods of diagnosing and treating metastatic breast cancer. The methods entail detecting an expression level of FOXC1 in a population of breast cancer tumor cells from the subject, detecting an expression level of FOXA1 in the population of breast cancer tumor cells, and diagnosing a patient as having metastatic breast cancer if the expression ratio of FOXC1/FOXA1 in the population of breast cancer tumor cells is elevated as compared to a control. The methods also entail administering a treatment for metastatic breast cancer if the expression ratio of FOXC1/FOXA1 in the population of breast cancer tumor cells is elevated as compared to a control. Methods of predicting overall survival and early (pre-symptomatic) diagnosis of early breast cancer metastasis are also provided herein.

Description

METHODS FOR DIAGNOSIS AND TREATMENT OF EPITHELIAL-TO-MESENCHYMAL

TRANSITION OF CANCER CELLS AND METASTATIC BREAST CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the priority date of United States Provisional Application No. 62/089,228, filed December 8, 2014, and United States Provisional Application No. 62/089,816, filed December 9, 2014. The contents of each of these referenced applications are incorporated herein by reference in their entirety, as if fully set forth herein.

BACKGROUND

[0002] Distant metastatic spread of cancer cells to other organs from the primary site of origin currently constitutes the most significant contributor to cancer-related morbidity and mortality. Epithelial-to-mesenchymal transition (EMT) is a biologic transformation of cancer cells from a non-migratory phenotype to a migratory one, and is thought to initiate the metastatic cascade in cancer. EMT has also been reported to trigger acquisition of stem cell traits in breast cancer. There is a need to develop new approaches to effectively diagnose and treat epithelial-to-mesenchymal transition of cancer cells and breast cancer metastasis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0003] Figure 1 shows the HRAS-transformed MCF10A cell series (M1 -M4) gene expression profiles and normal, basal, luminal A (lumA), luminal B (lumB), and Her2 molecular subtypes. M1 -M4 gene expression profiles were most reflective of the basal- like breast cancer molecular subtype.

[0004] Figure 2 shows the 3D culture RNA-Seq and 2D culture qRT-PCR results for FOXC1 in HRAS-transformed MCF10A cell series (M1 -M4). Figure 2A shows the 3D culture RNA-Seq results for FOXC1 and Figure 2B shows the 2D culture qRT-PCR results for FOXC1 .

[0005] Figure 3 shows the 3D culture RNA-Seq and 2D culture qRT-PCR results for FOXA1 in HRAS-transformed MCF10A cell series (M1 -M4). Figure 2A shows the 3D culture RNA-Seq results for FOXA1 and Figure 2B shows the 2D culture qRT-PCR results for FOXA1 .

[0006] Figure 4 shows the 2D culture qRT-PCR results for various genes in HRAS-transformed MCF10A cell series (M1 -M4). Figure 4A shows the 2D culture qRT- PCR results for CDH1 . Figure 4B shows the 2D culture qRT-PCR results for CDH2. Figure 4C shows the 2D culture qRT-PCR results for Fibronectin (FN1 ). Figure 4D shows the 2D culture qRT-PCR results for Vimentin (VIM). Figure 4E shows the 2D culture qRT-PCR results for SERPINE1 . Figure 4F shows the 2D culture qRT-PCR results for MMP2. Figure 4G shows the 2D culture qRT-PCR results for SNAI2. Figure 4H shows the 2D culture qRT-PCR results for FOXC2.

[0007] Figure 5 shows the 2D culture qRT-PCR results for mammospheres.

[0008] Figure 6 shows immunofluorescent images of FOXC1 counterstained with nuclear DAPI. Figure 6A shows the immunofluorescent image of FOXC1 counterstained with nuclear DAPI in M1 , parental MCF10A cells. Figure 6B shows the immunofluorescent image of FOXC1 counterstained with nuclear DAPI in M2 H-RAS transformed MCF10A cells. Figure 6C shows the immunofluorescent image of FOXC1 counterstained with nuclear DAPI in M3 tumorigenic and metastagenic cells.

[0009] Figure 7 shows mammosphere formation. Figure 7A shows M1 , parental MCF10A cells. Figure 7B shows mammosphere formation in M2 H-RAS transformed MCF10A cells. Figure 7C shows mammosphere formation in M3 tumorigenic and metastagenic cells.

[0010] Figure 8 shows the ratio of FOXC1 to FOXA1 in all patients, the top 50%, top 20%, top 10%, and top 5% of patients with an elevated FOXC1/FOXA1 expression ratio. According to one embodiment, these categories correspond to a cutoff at the 50th percentile line, the 80th percentile line, the 90th percentile line, and the 95th percentile line, respectively.

[0011] Figure 9 shows the ratio of FOXC1 to FOXA1 in all patients, the top 50%, top 20%, top 10%, and top 5% of patients with an elevated FOXC1/FOXA1 expression ratio. According to one embodiment, these categories correspond to a cutoff at the 50th percentile line, the 80th percentile line, the 90th percentile line, and the 95th percentile line, respectively.

[0012] Figure 10 shows the ratio of FOXC1 to FOXA1 in lymph negative patients, including the bottom 80% and the top 20% of patients with an elevated FOXC1/FOXA1 expression ratio. According to one embodiment, these categories correspond to a cutoff at the 80th percentile line.

[0013] Figure 1 1 shows the Human FOXC1 Amino Acid Sequence (SEQ ID NO: 1 ).

[0014] Figure 12 shows the Human FOXA1 Amino Acid Sequence (SEQ ID NO:4).

SUMMARY

[0015] Provided herein in certain embodiments are methods of treating metastatic breast cancer in a subject. In certain embodiments, the methods may include detecting an expression level of FOXC1 in a population of breast cancer tumor cells from the subject, detecting an expression level of FOXA1 in the population of breast cancer tumor cells, and administering a treatment for metastatic breast cancer to the subject if the expression ratio of FOXC1/FOXA1 in the population of breast cancer tumor cells is elevated as compared to a control. In certain embodiments, determining the expression level of FOXC1 and FOXA1 in the breast cancer tumor cells may be performed via quantitative RT-PCR (qRT-PCR) or RNA sequencing (RNA-Seq). In certain embodiments, the control may be a cutoff expression ratio which is established using a set of FOXC1/FOXA1 expression ratios from a population of patients having a cross-section of all types of breast cancer and the cutoff expression ratio may fall at the 75th percentile line, the 80th percentile line, the 85th percentile line, the 90th percentile line, the 95th percentile line, or at higher than the 95th percentile line. In certain embodiments, the control may be a cutoff expression ratio which is established using a set of FOXC1/FOXA1 expression ratios from a population of patients having node- negative breast cancer and the cutoff expression ratio may fall at the 80th percentile line. In certain embodiments, the expression ratio of FOXC1/FOXA1 in the population of breast cancer tumor cells may be detected at a pre-symptomatic stage of early breast cancer metastasis. In certain embodiments, the treatment may be a therapeutically effective amount of one or more therapeutic agents. In certain embodiments, the one or more therapeutic agents may be selected from chemotherapeutic agents, therapeutic antibodies and fragments thereof, toxins, radioisotopes, enzymes, nucleases, hormones, immunomodulators, antisense oligonucleotides, nucleic acid molecules, chelators, boron compounds, photoactive agents and dyes.

[0016] Also provided herein in certain embodiments are methods of diagnosing metastatic breast cancer in a subject. In certain embodiments, the methods may include detecting an expression level of FOXC1 in a population of breast cancer tumor cells from the subject, detecting an expression level of FOXA1 in the population of breast cancer tumor cells, and diagnosing the subject as having metastatic breast cancer if the expression ratio of FOXC1/FOXA1 in the population of breast cancer tumor cells is elevated as compared to a control. In certain embodiments, determining the expression level of FOXC1 and FOXA1 in the breast cancer tumor cells may be performed via quantitative RT-PCR (qRT-PCR) or RNA sequencing (RNA-Seq). In certain embodiments, the control may be a cutoff expression ratio which is established using a set of FOXC1/FOXA1 expression ratios from a population of patients having a cross-section of all types of breast cancer and the cutoff expression ratio may fall at the 75th percentile line, the 80th percentile line, the 85th percentile line, the 90th percentile line, the 95th percentile line, or at higher than the 95th percentile line. In certain embodiments, the control may be a cutoff expression ratio which is established using a set of FOXC1/FOXA1 expression ratios from a population of patients having node- negative breast cancer and the cutoff expression ratio may fall at the 80th percentile line. In certain embodiments, the expression ratio of FOXC1/FOXA1 in the population of breast cancer tumor cells may be detected at a pre-symptomatic stage of early breast cancer metastasis. In certain embodiments, the methods may further include administering a treatment for metastatic breast cancer if the expression ratio of FOXC1/FOXA1 in the population of breast cancer tumor cells is elevated as compared to the control. In certain embodiments, the treatment may be a therapeutically effective amount of one or more therapeutic agents. In certain embodiments, the one or more therapeutic agents may be selected from chemotherapeutic agents, therapeutic antibodies and fragments thereof, toxins, radioisotopes, enzymes, nucleases, hormones, immunomodulators, antisense oligonucleotides, nucleic acid molecules, chelators, boron compounds, photoactive agents and dyes.

DETAILED DESCRIPTION

[0017] The following description provides specific details for a thorough understanding of, and enabling description for, embodiments of the disclosure. However, one skilled in the art will understand that the disclosure may be practiced without these details. In other instances, well-known structures and functions have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments of the disclosure.

[0018] Methods for diagnosing and treating metastatic breast cancer based on an elevated expression ratio of FOXC1/FOXA1 in a population of breast cancer tumor cells from a subject having breast cancer are provided herein. The findings described herein fulfill the unmet need of diagnosing clinically occult, asymptomatic metastasis, which allows for treatment of metastatic cancer earlier than currently possible, when the tumor burden is still small enough that it can be treated effectively.

[0019] Distant metastatic spread of cancer cells to other organs from the primary site of origin currently represents the leading cause of cancer-related morbidity and mortality (Howlander et al, 1975-2010). According to current oncology practice guidelines, breast cancer patients (who have already undergone initial treatment for breast cancer) are diagnosed with metastatic spread only when clinical suspicion is aroused, either by abnormal values on screening laboratory tests, or appearance of new clinical symptoms, suggestive of dysfunction in distant organs to which the cancer has metastasized. Even in those patients undergoing routine surveillance during follow-up, most recurrent breast cancer (unlike primary breast cancer) is already symptomatic at the time of diagnosis (Ghezzi et al., 1994; Tomin and Donegan, 1987). Metastatic cancer cell conglomerates have finally exceeded the threshold of detection on conventional imaging tests such as Positron Emission Tomography (PET) (greater than 10 million cells or 0.5cm³) or Computer Tomography (CT) scan (greater than 1 billion cells or 1 cm³) (Friberg and Mattson, 1997). Once abnormal or suspicious masses are found in locations suspected to represent metastatic disease, an image-guided biopsy to establish a tissue diagnosis of breast cancer metastasis is obtained. Tissue diagnosis is important to establish, as palliative intent chemotherapy cannot ethically be undertaken without proof of the existence of metastatic disease. Starting treatment following a diagnosis of metastatic cancer is likely to represent "too little too late" as the tumor burden is overwhelmingly large. Such an approach is less likely to meet with a favorable outcome and certainly would not be a curative one. On the other hand, early detection of such a metastasis, if possible, at the pre-symptomatic stage, would allow starting potentially life-prolonging treatments before metastatic tumor burden is allowed to reach overwhelming proportions.

[0020] Currently, it is still not possible to diagnose clinically occult, asymptomatic metastasis early enough, at a point in time when the total metastatic tumor burden is small enough that it can still potentially be treated effectively. Before drugs can be rationally designed to target the metastatic spread of breast cancer, understanding of the molecular mechanisms that are driving this process must be improved and expanded. Such an understanding is also critical to the development of suitable biomarkers that would allow early detection of the metastatic phenotype. Despite the fact that several prospective studies have been conducted to try and identify screening biomarkers for breast cancer metastasis, such as liver function tests, none have been found to have sufficient sensitivity or specificity for this purpose (Crivellari et al., 1995). Studies with circulating tumor markers, while more promising, have not yet led to recommendations supporting their routine use in the ongoing surveillance of patients already treated for primary breast cancer (Guadagni et al., 2001 ; Kokko et al. 2002; Molina et al., 2010; Molina et al., 2003; Nicolini et al., 2006). Several multicenter, randomized cancer surveillance trials evaluating various follow-up strategies (routine blood tests and imaging tests) have neither been successful in demonstrating earlier diagnosis of metastasis nor have achieved any survival advantage as a result of any intensive surveillance measures (Ghezzi et al., 1994; Delturco et al., 1994; Palli et al., 1999).

[0021] A traditional clinical measure of advanced disease predictive of an elevated risk of future distant metastasis is the presence of cancer cells in the lymph nodes. Lymph node status at the time of diagnosis has very important clinical implications. Currently, if a patient diagnosed with breast cancer is found to have lymph node involvement (lymph node "positive"; LNP), it is the basis of recommending adjuvant chemotherapy. However, it is known from historical observations that not all patients who are lymph node positive actually go on to manifest with distant metastatic disease despite the predicted elevated risk, even in the absence of adjuvant treatment. Since there is not a good way to predict who will or will not develop distant metastatic disease in the lymph node positive group, the overtreatment of some to benefit others is currently considered acceptable in clinical practice. By the same token, if a patient diagnosed with breast cancer is found not to have lymph node involvement (lymph node "negative"; LNN), a favorable prognosis is predicted and is the basis of often withholding adjuvant chemotherapy in an attempt to minimize unnecessary side effects of chemotherapy. Some patients who are lymph node negative still go on to manifest with distant metastatic disease despite the predicted low risk. There is clearly a need to improve the prediction of cancer metastasis in both LNP and LNN groups. This would help to reduce the incidence of both overtreatment and undertreatment of such patients.

[0022] The process of cancer metastasis is not random. Rather, it consists of a series of linked, sequential steps by which non-migratory cancer cells of epithelial (E) origin transform into migratory mesenchymal (M) cells (epithelial-to-mesenchymal transition or EMT). They then detach from neighboring cells, move freely through adjacent tissues, enter the bloodstream leaving their tissue site of origin, manage to survive in this new migratory environment, and finally exit into a new destination tissue and colonize it, usually undergoing a reverse change referred to as mesenchymal-to- epithelial transition (MET) (Thiery et al., 2009; Yang and Weinberg, 2008). EMT is a critical precursor event that enables cancer cells to metastasize. Traditionally, E and M cell surface markers have been used to define and follow such cells as they undergo dynamic transition between E and M states. However, recent findings would suggest that cell surface E and M markers may not always be accurate in reflecting the subtle changes along the EMT spectrum. For example, loss of expression of E-cadherin, an epithelial cell surface marker, is widely believed to be requisite for and synonymous with acquisition of a mesenchymal phenotype. Yet, it has been shown that loss of E- cadherin is not necessary for functional EMT to occur (Hollestelle et al., 2013; Shamir et al., 2014). Thus, methods relying solely on cell surface expression of E and M marker expression may not be accurate in capturing the true polarization state of a cell, especially cells that are "poised" for such change and are precursors of the metastatic process, but have not yet manifested with overt changes in cell surface marker expression.

[0023] Transcription factor (TF) Forkhead box C1 (FOXC1 ), strongly associated with the basal-like and claudin-low breast cancer molecular subtypes (see Ray et al.., 2010), is a powerful epithelial-to-mesenchymal transition (EMT) inducer and is also a marker of stem/progenitor cells. In contrast, TF Forkhead box A1 (FOXA1 ), strongly associated with luminal subtypes, is an EMT repressor and a luminal differentiation marker, thus seemingly exerting reciprocally opposite transcriptional effects to that of FOXC1 . It was hypothesized that effective EMT program activation status in breast cancer might be better predicted by examining the expression ratio of an EMT inducer and EMT repressor, such as FOXC1/FOXA1 , theoretically being more reflective of net transcriptional effect than either component alone.

[0024] As shown in Example 1 below, elevated FOXC1/FOXA1 expression ratio indicates EMT program activation in breast cancer, and predicts the associated occurrence of lymph-node-independent distant metastasis and death in human patients. These findings allow for the early (pre-symptomatic) diagnosis of clinically occult (node negative) metastasis by using the FOXC1/FOXA1 ratio as a biomarker of metastasis and permit institution of appropriate therapy earlier than currently possible. The current study provided herein improves the understanding of EMT and highlights the importance of future studies geared towards unraveling mechanisms involved in regulating FOXC1 and FOXA1 expression in breast cancer.

[0025] Provided herein are methods of diagnosing and/or treating EMT of cancer cells. In certain embodiments, a method of treating EMT of cancer cells may comprise detecting an expression level of FOXC1 in a population of breast cancer tumor cells from the subject; detecting an expression level of FOXA1 in the population of breast cancer tumor cells; and administering a treatment for EMT of cancer cells if an expression ratio of FOXC1/FOXA1 is elevated as compared to a control. In certain embodiments, the subject may be lymph node negative.

[0026] Also provided are methods of diagnosing and/or treating metastatic breast cancer. In certain embodiments, a method of treating metastatic breast cancer in a subject may comprise detecting an expression level of FOXC1 in a population of breast cancer tumor cells from the subject; detecting an expression level of FOXA1 in the population of breast cancer tumor cells; and administering a treatment for metastatic breast cancer to the subject if an expression ratio of FOXC1/FOXA1 in the population of breast cancer tumor cells is elevated as compared to a control. In certain embodiments, the subject may be lymph node negative.

[0027] Also provided in certain embodiments are methods of predicting the associated occurrence of lymph-node independent distant metastasis. In certain embodiments, a method of predicting the associated occurrence of lymph-node independent distant metastasis in a subject may comprise detecting an expression level of FOXC1 in a population of breast cancer tumor cells from the subject; detecting an expression level of FOXA1 in the population of breast cancer tumor cells; and predicting that the subject may have an associated occurrence of lymph-node independent distant metastasis if an expression ratio of FOXC1/FOXA1 is elevated in the population of breast cancer tumor cells as compared to a control.

[0028] As provided herein, methods used to determine the expression level of FOXC1 and FOXA1 may include any suitable method, including but not limited to, immunohistochemistry (or other immunoassay), PCR, RT-PCR, qRT-PCR (or any other PCR-based method), and/or the methods, assays and materials described in International Application Nos. PCT/US10/44817 entitled "Methods for Diagnosis, Prognosis, and Treatment of Primary and Metastatic Basal-Like Breast Cancer and Other Cancer Types;" and PCT/US 12/23871 entitled "FOXC1 Antibodies and Methods of Their Use;" the subject matter of both of which are hereby incorporated by reference as if fully set forth herein.

[0029] Detecting the expression level of FOXC1 and/or FOXA1 may be accomplished by an in vitro immunoassay, such as immunocytochemistry (ICC), immunohistochemistry (IHC), Western blot or fluorescent in situ hybridization (FISH). Alternatively, an in vivo imaging modality may be used, such as magnetic resonance imaging (MRI), positron emission tomography (PET) or microPET, computed tomography (CT), PET/CT combination imager, cooled charged coupled device (CCD), camera optical imaging, optical imaging and single photon emission computed tomography (SPECT). When the presence of FOXC1 and/or FOXA1 is determined by an in vivo method, the FOXC1 and/or FOXA1 antibody or functional fragment thereof should be conjugated to an intracellular delivery agent to facilitate deliver of the antibody or functional fragment thereof to the cytoplasm of target cells.

[0030] In certain embodiments, an anti-FOXC1 monoclonal antibody may be used to detect expression level of FOXC1 in a cell. As described in detail in PCT/US12/23871 , the antibody may specifically bind a target antigenic peptide sequence of human FOXC1 (Figure 1 1 ; SEQ ID NO: 1 ). In one aspect, the target antigenic peptide sequence is 5' -AHAEQYPGGMARAYGPYTPQPQPKD-3' (SEQ ID NO:2), which corresponds to amino acids 51 to 75 of SEQ ID NO: 1 (see Figure 1 1 ). A cysteine residue may be added to the N-terminus (i.e., 5'-C- AHAEQYPGGMARAYGPYTPQPQPKD-3' (SEQ ID NO:3)) to assist in conjugation to the carrier protein as necessary. In certain embodiments, an anti-FOXA1 antibody may be used to detect the expression level of FOXA1 in a cell. The antibody may specifically bind a target antigenic peptide sequence of human FOXA1 (Figure 12; SEQ ID NO:4; NCBI Reference Sequence: NP_004487.2).

[0031] In certain embodiments, the methods described herein may be used to treat metastatic breast cancer. For example, determination of the expression levels of FOXC1 and FOXA1 can be used to dictate the administration of a therapeutic agent when the expression ratio of FOXC1 to FOXA1 is elevated as compared to a control. The method of treating metastatic breast cancer includes a step of administering a therapeutically effective amount or dose of a treatment for metastatic breast cancer.

[0032] An "elevated" expression ratio is typically in comparison to a control. In certain embodiments, the control is a cutoff expression ratio. In some aspects, a cutoff expression ratio may be established using a set of FOXC1/FOXA1 expression ratios from a population of relevant subjects. For example, the set of FOXC1/FOXA1 expression ratios are from a population of node-negative breast cancer patients. Alternatively, the FOXC1/FOXA1 expression ratios are from a population of patients having a specific type of breast cancer (e.g., basal-like breast cancer patients) or population of patients having a cross-section of all types of breast cancer. In these embodiments, the cutoff expression ratio is determined by selecting a FOXC1/FOXA1 expression ratio within the population that falls at or higher than the 50th percentile line, at or higher than the 60th percentile line, at or higher than the 70th percentile line, at or higher than the 75th percentile line, at or higher than the 80th percentile line, at or higher than the 85th percentile line, at or higher than the 90th percentile line, or at or higher than the 95th percentile line. In one preferred embodiment, the cutoff expression ratio falls at or above the 80th percentile line. Within these embodiments, an "elevated" FOXC1/FOXA1 expression ratio falls above the cutoff expression ratio.

[0033] In other embodiments, the control is an index of expression ratios. For example, a set of FOXC1/FOXA1 expression ratios from a population of relevant subjects may be used to establish a standard curve or reference index of FOXC1/FOXA1 expression ratios by plotting the FOXC1/FOXA1 expression ratios against a specific clinical outcome measure such as presence of metastatic disease, overall survival, breast cancer specific survival, recurrence free survival, metastasis free survival, or other suitable diagnostic or prognostic outcome measures. Such a standard curve or reference index may be used to categorize or stage a subject's individual FOXC1/FOXA1 expression ratio such that if a FOXC1/FOXA1 expression ratio falls in an upper range of ratios within a standard curve or reference index that correlates to an abnormal condition (e.g., metastatic disease) or outcome (e.g., survival), the subject's ratio is considered to be "elevated".

[0034] In certain embodiments, a node-negative breast cancer patient that has a FOXC1/FOXA1 expression ratio that is in the highest 20% of a population of node- negative breast cancer patients, or is above the 80th percentile of a population of node negative breast cancer patients (i.e., above the control cutoff ratio) indicates or can predict early stages of metastasis before other measurable symptoms occur. [0035] Overall survival of a subject with breast cancer may also be predicted if the subject has an elevated FOXC1/FOXA1 expression ratio as compared to a control. In certain embodiments, when the expression ratio of FOXC1/FOXA1 in a population of breast cancer tumor cells from the subject falls at or higher than the 50th percentile line, at or higher than the 60th percentile line, at or higher than the 70th percentile line, at or higher than the 75th percentile line, at or higher than the 80th percentile line, at or higher than the 85th percentile line, at or higher than the 90th percentile line, or at or higher than the 95th percentile line, the subject may have a significantly decreased 10 year overall survival as compared to the 10 year survival of a population of patients that have all types of breast cancer (see Figure 9). In certain embodiments, when the expression ratio of FOXC1/FOXA1 in the population of breast cancer tumor cells from a node-negative breast cancer patient is in the highest 20% of a population of node- negative breast cancer patients, or is at or above the 80th percentile of a population of node negative breast cancer patients (i.e., above or higher than the control cutoff ratio), the subject may have a significantly decreased 10 year overall survival as compared to a population of node-negative breast cancer patients that have a FOXC1/FOXA1 expression ratio that is lower than the 80th percentile of a population of node negative breast cancer patients (see Figure 10).

[0036] "Treating" or "treatment" of a condition may refer to preventing the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, generating a complete or partial regression of the condition, or some combination thereof. Treatment may also mean a prophylactic or preventative treatment of a condition.

[0037] As used herein, a "subject" refers to a mammal, such as a human. In some embodiments, the subject is a patient.

[0038] In some embodiments, the treatment used in the methods herein may be administered as a combination of one or more therapeutic agents for the treatment of metastatic breast cancer. "A combination" or "in combination with," as used herein, means in the course of treating the same cancer in the same subject using two or more agents, drugs, treatment regimens, treatment modalities or a combination thereof, in any order. This includes simultaneous administration, as well as in a temporally spaced order of up to several days apart. Such combination treatment may also include more than a single administration of any one or more of the agents, drugs, treatment regimens or treatment modalities. Further, the administration of the two or more agents, drugs, treatment regimens, treatment modalities or a combination thereof may be by the same or different routes of administration. In some embodiments, the treatment used in the methods herein may be administered in combination with local therapy, such as surgery or radiation therapy, or a combination thereof.

[0039] Examples of therapeutic agents that may be administered as a treatment include, but are not limited to, chemotherapeutic agents, therapeutic antibodies and fragments thereof, toxins, radioisotopes, enzymes (e.g., enzymes to cleave prodrugs to a cytotoxic agent at the site of the tumor), nucleases, hormones, immunomodulators, antisense oligonucleotides, nucleic acid molecules (e.g., mRNA molecules, cDNA molecules or RNAi molecules such as siRNA or shRNA), chelators, boron compounds, photoactive agents and dyes. The therapeutic agent may also include a metal, metal alloy, intermetallic or core-shell nanoparticle bound to a chelator that acts as a radiosensitizer to render the targeted cells more sensitive to radiation therapy as compared to healthy cells.

[0040] Chemotherapeutic agents that may be used in accordance with the embodiments described herein are often cytotoxic or cytostatic in nature and may include, but are not limited to, alkylating agents, antimetabolites, anti-tumor antibiotics, topoisomerase inhibitors, mitotic inhibitors hormone therapy, targeted therapeutics and immunotherapeutics. In some embodiments the chemotherapeutic agents that may be used as therapeutic agents in accordance with the embodiments of the disclosure include, but are not limited to, 13-cis-Retinoic Acid, 2-Chlorodeoxyadenosine, 5- Azacitidine, 5-Fluorouracil, 6-Mercaptopurine, 6-Thioguanine, actinomycin-D, adriamycin, aldesleukin, alemtuzumab, alitretinoin, all-transretinoic acid, alpha interferon, altretamine, amethopterin, amifostine, anagrelide, anastrozole, arabinosylcytosine, arsenic trioxide, amsacrine, aminocamptothecin, aminoglutethimide, asparaginase, azacytidine, bacillus calmette-guerin (BCG), bendamustine, bevacizumab, bexarotene, bicalutamide, bortezomib, bleomycin, busulfan, calcium leucovorin, citrovorum factor, capecitabine, canertinib, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, cortisone, cyclophosphamide, cytarabine, darbepoetin alfa, dasatinib, daunomycin, decitabine, denileukin diftitox, dexamethasone, dexasone, dexrazoxane, dactinomycin, daunorubicin, decarbazine, docetaxel, doxorubicin, doxifluridine, eniluracil, epirubicin, epoetin alfa, erlotinib, everolimus, exemestane, estramustine, etoposide, filgrastim, fluoxymesterone, fulvestrant, flavopiridol, floxuridine, fludarabine, fluorouracil, flutamide, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin, granulocyte - colony stimulating factor, granulocyte macrophage-colony stimulating factor, hexamethylmelamine, hydrocortisone hydroxyurea, ibritumomab, interferon alpha, interleukin - 2, interleukin-1 1 , isotretinoin, ixabepilone, idarubicin, imatinib mesylate, ifosfamide, irinotecan, lapatinib, lenalidomide, letrozole, leucovorin, leuprolide, liposomal Ara-C, lomustine, mechlorethamine, megestrol, melphalan, mercaptopurine, mesna, methotrexate, methylprednisolone, mitomycin C, mitotane, mitoxantrone, nelarabine, nilutamide, octreotide, oprelvekin, oxaliplatin, paclitaxel, palbociclib, pamidronate, pemetrexed, panitumumab, PEG Interferon, pegaspargase, pegfilgrastim, PEG-L-asparaginase, pentostatin, plicamycin, prednisolone, prednisone, procarbazine, proteasome inhibitors, raloxifene, rituximab, romiplostim, ralitrexed, sapacitabine, sargramostim, satraplatin, sorafenib, sunitinib, semustine, streptozocin, tamoxifen, tegafur, tegafur-uracil, temsirolimus, temozolamide, teniposide, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, trimitrexate, alrubicin, vincristine, vinblastine, vindestine, vinorelbine, vorinostat, Wnt pathway inhibitors (e.g., iCRT3) or zoledronic acid.

[0041] Therapeutic antibodies and functional fragments thereof, that may be used as therapeutic agents in accordance with the embodiments of the disclosure include, but are not limited to, alemtuzumab, bevacizumab, cetuximab, edrecolomab, gemtuzumab, ibritumomab tiuxetan, panitumumab, rituximab, tositumomab, and trastuzumab and other antibodies associated with breast cancer.

[0042] Toxins that may be used as therapeutic agents in accordance with the embodiments of the disclosure include, but are not limited to, ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

[0043] Radioisotopes that may be used as therapeutic agents in accordance with the embodiments of the disclosure include, but are not limited to, ³²P, ⁸⁹Sr, ⁹⁰Y, ^99mTc, "Mo, ³ 1, ¹⁵³Sm, ¹⁷⁷Lu, ¹⁸⁶Re, ²¹³Bi, ²²³Ra and ²²⁵Ac.

[0044] The treatment and administration steps described herein may include any suitable treatment used in accordance with standard practice for treatment of breast cancer. The treatment is not limited to any particular treatment. One skilled in the art will appreciate that any United States Food and Drug Administration (FDA) approved therapeutic treatment or off-label treatment may be used in accordance with the methods provided herein.

[0045] As provided herein, a therapeutically effective amount or effective amount is an amount of a treatment that will yield the most effective results in terms of efficacy of treatment in a given subject or population of cells. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the treatment, the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication) or cells, the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. Further, an effective or therapeutically effective amount may vary depending on whether the treatment is administered alone or in combination with another compound, drug, therapy or other therapeutic method or modality. One skilled in the clinical and pharmacological arts will be able to determine an effective amount or therapeutically effective amount through routine experimentation, namely by monitoring a cell's or subject's response to administration of the treatment and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy, 21 ^st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005, which is hereby incorporated by reference as if fully set forth herein.

[0046] The treatment as described herein may be administered by any suitable route of administration, alone or as part of a pharmaceutical composition. A route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, transdermal (e.g., topical cream or ointment, patch), or vaginal. "Transdermal" administration may be accomplished using a topical cream or ointment or by means of a transdermal patch. "Parenteral" refers to a route of administration that is generally associated with injection, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.

[0047] In some embodiments, the pharmaceutical composition may also include a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may be a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other ingredients of the formulation. It also must be suitable for contact with any tissue, organ, or portion of the body that it may encounter, meaning that it must not carry a risk of toxicity, irritation, allergic response,

immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

[0048] The following examples are intended to illustrate various embodiments of the invention. As such, the specific embodiments discussed are not to be construed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of invention, and it is understood that such equivalent embodiments are to be included herein. Further, all references cited in the disclosure are hereby incorporated by reference in their entirety, as if fully set forth herein.

EXAMPLES Example 1 : FOXC1/FOXA1 Transcriptional Balance in Breast Cancer: From Acquisition of Mesenchymal and Stem Cell Traits to Occult Lymph Node Independent Breast Cancer Metastasis.

[0049] RNA-Seq profiling of the Harvey-Ras (HRAS)-transformed MCF10A cell series, a well characterized and widely accepted in vitro model of breast cancer progression and metastasis (see Wang et al., 1997), was used to correlate measured FOXC1/FOXA1 ratios to dynamic shifts in EMT marker expression in 3D matrigel cultures and to stem cell traits observed in primary and secondary mammosphere suspension cultures (see Figures 1 -7). The ability of the FOXC1/FOXA1 expression ratio was further tested to predict lymph node independent breast cancer metastasis and death in independent human breast cancer gene expression datasets (see Figures 8-10).

[0050] RNA-Seq and qRT-PCR profiling confirmed progressive increase in FOXC1/FOXA1 ratio to correlate with a progressive loss of E-cadherin expression and synchronous gain of EMT markers N-cadherin, Fibronectin, and Vimentin (see Figures 2-4). FOXC1/FOXA1 ratio was found to be directly proportional to mammosphere formation efficiency, a surrogate indicator of stem cell enrichment (see Figures 5-7). In patients without any evidence of nodal metastasis, elevated FOXC1/FOXA1 ratio was associated with significantly decreased 10 year Overall Survival (HR 2.58;95%CI 1.39 to 4.80, p = 0.003, 295 patient Van de Vijver dataset; van 't Veer et al., 2002), 10 year Disease-specific Survival (HR 1 .74;95%CI 1 .16 to 2.61 , p = 0.008, 1992 patient Curtis dataset; Curtis et al., 2012)) and predicted the development of lung metastasis.

[0051] As shown by the results presented herein, an elevated FOXC1/FOXA1 expression ratio indicated EMT program activation in breast cancer. Elevated FOXC1/FOXA1 expression ratio also indicated the associated occurrence of lymph- node-independent distant metastasis and death in human patients. This discovery allows for the early (pre-symptomatic) diagnosis of clinically occult (node negative metastasis by using the FOX/C1 FOXA1 ratio as a biomarker of early breast cancer metastasis.

REFERENCES [0052] The references, patents and published patent applications listed below, and all references cited in the specification above are hereby incorporated by reference in their entirety, as if fully set forth herein.

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3. van 't Veer LJ, et al. (2002) Gene expression profiling predicts clinical outcome of breast cancer. Nature. Jan 31 ;415(6871 ):530-6.

4. Curtis, C, et al. (2012) The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature 486, 346-352.

5. International Application No. PCT/US10/44817, entitled "Methods for Diagnosis, Prognosis, and Treatment of Primary and Metastatic Basal-Like Breast Cancer and Other Cancer Types," filed August 6, 2010.

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Claims

CLAIMS What is claimed is:

1 . A method of treating metastatic breast cancer in a subject, the method comprising: detecting an expression level of FOXC1 in a population of breast cancer tumor cells from the subject; detecting an expression level of FOXA1 in the population of breast cancer tumor cells; and administering a treatment for metastatic breast cancer to the subject if the expression ratio of FOXC1/FOXA1 in the population of breast cancer tumor cells is an elevated FOXC1/FOXA1 expression ratio as compared to a control.

2. The method of claim 1 , wherein determining the expression level of FOXC1 and FOXA1 in the breast cancer tumor cells is performed via quantitative RT-PCR (qRT- PCR) or RNA sequencing (RNA-Seq).

3. The method of claim 1 , wherein the control is a cutoff expression ratio which is established using a set of FOXC1/FOXA1 expression ratios from a population of patients having a cross-section of all types of breast cancer and wherein the cutoff expression ratio falls at the 75th percentile line, the 80th percentile line, the 85th percentile line, the 90th percentile line, the 95th percentile line, or at higher than the 95th percentile line.

4. The method of claim 1 , wherein the control is a cutoff expression ratio which is established using a set of FOXC1/FOXA1 expression ratios from a population of patients having node-negative breast cancer and wherein the cutoff expression ratio falls at the 80th percentile line.

5. The method of claim 4, wherein the expression ratio of FOXC1/FOXA1 in the population of breast cancer tumor cells is detected at a pre-symptomatic stage of early breast cancer metastasis.

6. The method of claim 1 , wherein the treatment is a therapeutically effective amount of one or more therapeutic agents.

7. The method of claim 6, wherein the one or more therapeutic agents is selected from chemotherapeutic agents, therapeutic antibodies and fragments thereof, toxins, radioisotopes, enzymes, nucleases, hormones, immunomodulators, antisense oligonucleotides, nucleic acid molecules, chelators, boron compounds, photoactive agents and dyes.

8. A method of diagnosing metastatic breast cancer in a subject, the method comprising: detecting an expression level of FOXC1 in a population of breast cancer tumor cells from the subject; detecting an expression level of FOXA1 in the population of breast cancer tumor cells; and diagnosing the subject as having metastatic breast cancer if the expression ratio of FOXC1/FOXA1 in the population of breast cancer tumor cells is elevated as compared to a control.

9. The method of claim 8, wherein determining the expression level of FOXC1 and FOXA1 in the breast cancer tumor cells is performed via quantitative RT-PCR (qRT- PCR) or RNA sequencing (RNA-Seq).

10. The method of claim 8, wherein the control is a cutoff expression ratio which is established using a set of FOXC1/FOXA1 expression ratios from a population of patients having a cross-section of all types of breast cancer and wherein the cutoff expression ratio falls at the 75th percentile line, the 80th percentile line, the 85th percentile line, the 90th percentile line, the 95th percentile line, or at higher than the 95th percentile line.

1 1 . The method of claim 8, wherein the control is a cutoff expression ratio which is established using a set of FOXC1/FOXA1 expression ratios from a population of patients having node-negative breast cancer and wherein the cutoff expression ratio falls at the 80th percentile line.

12. The method of claim 8, wherein the expression ratio of FOXC1/FOXA1 in the population of breast cancer tumor cells is detected at a pre-symptomatic stage of early breast cancer metastasis.

13. The method of claim 8, further comprising administering a treatment for metastatic breast cancer if the expression ratio of FOXC1/FOXA1 in the population of breast cancer tumor cells is elevated as compared to the control.

14. The method of claim 13, wherein the treatment is a therapeutically effective amount of one or more therapeutic agents.

15. The method of claim 14, wherein the one or more therapeutic agents is selected from chemotherapeutic agents, therapeutic antibodies and fragments thereof, toxins, radioisotopes, enzymes, nucleases, hormones, immunomodulators, antisense oligonucleotides, nucleic acid molecules, chelators, boron compounds, photoactive agents and dyes.