WO2019094613A1 - Method for treating breast cancer and chronic diseases - Google Patents

Abstract

The disclosure provides a method of treating breast cancer, the method comprising administering to mammalian subject in need thereof an inhibitor of Receptor for Advanced Glycation End-product (RAGE). The disclosure further provides a method of inhibiting breast cancer metastasis, the method comprising administering to mammalian subject in need thereof an inhibitor of RAGE.

Description

METHOD FOR TREATING BREAST CANCER AND CHRONIC DISEASES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.

62/583,910, filed on November 9, 2017, the disclosure of which is hereby incorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF TO MATERIALS SUBMITTED

ELECTRONICALLY

[0002] This application contains, as a separate part of the disclosure, a Sequence Listing in computer readable form (Filename: 52280A_Seqlisting.txt; Size:842 bytes; Created:

November 8, 2018), which is incorporated by reference in its entirety.

FIELD OF DISCLOSURE

[0003] The invention relates to materials and methods for treating breast cancer, obesity, nonalcoholic steatohepatitis (NASH), and chronic diseases.

BACKGROUND

[0004] Breast cancer is the most common non-skin cancer and the second leading cause of cancer mortality amongst women in the United States. Siegel et al. (2013). CA Cancer J Clin, 63, 11-30. Although mortality rates from breast cancer have decreased due to surveillance, early detection and use of adjuvant therapies, the five-year survival of metastatic disease is only 22%. Siegel, supra. A better understanding of the mechanisms underlying breast cancer metastasis is needed to develop new and more effective therapies.

SUMMARY

[0005] Disclosed herein is a method of treating breast cancer, the method comprising administering to a mammalian subject in need thereof an inhibitor of Receptor for Advanced Glycation End-product (RAGE). The disclosure further provides a method of inhibiting breast cancer metastasis and/or inhibiting the onset of breast cancer, comprising

administering to a mammalian subject in need thereof an inhibitor of RAGE. The disclosure further provides a method of treating obesity, nonalcoholic steatohepatitis (NASH), or chronic disease, the method comprising administering to a mammalian subject in need thereof an inhibitor of RAGE. BRIEF DESCRIPTION OF THE FIGURES

[0006] Figures 1A-1J. Increased RAGE expression promotes tumor metastasis. Western blot analysis of RAGE in (Fig.lA) human highly lung (4175) and bone (1833) metastatic variants of parental human 231 cells and (Fig. IB) in murine highly metastatic 4T-1 & E0771 compared to non-metastatic 67NR cells. Western blots were performed using anti-RAGE and anti-β actin antibodies. (Fig.lC) Western blot analysis of 231 control & RAGE overexpressing cells (using anti-RAGE and anti-β actin antibodies). (Fig. ID) Scratch/wound assay of 231 control & RAGE transfected cells at Oh / 16h post wounding. (E) Relative cell number (y-axis) invading through matrigel in transwell chamber after 24h to 1% FBS stimuli. (Fig. IF) Proliferation quantified by crystal violet staining after 48h (y-axis OD; white bar=vector; black bar=RAGE). (Fig. lG) Soft agar colony formation assays of 231 control & RAGE cells. Representative cell colonies in soft agar are shown. Colony number per field (y-axs) is shown in the bar graph (white bar=vector; black bar=RAGE). (Fig. lH) 231 vector and 231-RAGE cells (lxlO⁶) were injected into mammary fat pad of NSG mice. Tumor size (y-axis) was measured over the course of 35 days (x-axis). (Fig. II) Tumor weight (y-axis) was measured at time of sacrifice. Data shown are from 10 mice per group. (Fig.lJ)

Immunohistochemistry of lung tissue from 231-vector and 231-RAGE mice stained with human anti-CK7 to visualize tumor cell metastasis. Bar graph illustrates CK7 staining (y- axis; pixel/field) for tested samples.

[0007] Figures 2A-2C. RAGE signaling through MAP kinase and EMT drives MDA-MB- 231 cell invasive gene expression and function. (Fig.2A) Western blot analysis shows EMT markers are increased with RAGE overexpression. (Fig.2B) Western blot of total and phospho-proteins in 231 & 231-RAGE expressing cells. (Fig.2C) Matrigel invasion in transwell chambers after 24h to 1% FBS. Assays performed with control (DMSO), and inhibitors for MEK (U0126, 10 μΜ), Akt (LY294002, 50 μΜ) and p38 (SB203580, 10 μΜ). Data are means + SEM, n=3. *, P<0.05.

[0008] Figures 3A-3E. RAGE knockdown in human highly metastatic breast cancer cells downregulates cell invasion, anchorage-independent growth in soft agar, and downstream signaling. (Fig.3A) Western blot analysis of RAGE in shControl and RAGE sh66 231 and

4175 cells (using anti-RAGE and anti-β actin antibodies). (Fig.3B) 231 & 4175 cells with

RAGE shRNA (and shControl) were quantified for matrigel invasion in transwell chambers after 24h to 1% FBS stimuli, were quantified. (Fig.3C) Proliferation quantified by crystal violet staining after 48h. (Fig.3D) Soft agar colony formation assays of 231 & 4175 cells. Representative cell colonies in soft agar are shown. Colony number per field is shown in the lower panel. For all experiments, data are means + SEM, n=3. *, P<0.05. (Fig.3E) Western blot of total and phospho-proteins in 4175 cells with RAGE shRNA and shControl. For all experiments, data are means + SEM, n=3. *, P<0.05.

[0009] Figures 4A-4F. Knockdown of RAGE inhibitors tumor progression. (Fig.4A) 231 parental or (Fig.4B) 4175 cells (lxlO⁶) with RAGE sh66 or shControl were injected into mammary fat pad of NSG mice (top line=control; bottom line=treated). Tumor size was measured over the course of 35 days, and at time of sacrifice (35 days), tumor weight was measured. Data shown are from 10 mice per group. (Fig.4C-4F) Immunohistochemical analysis of tumors for proliferation (Fig.4C. Ki67), angiogenesis (Fig.4D. CD34), and leukocyte (Fig.4E. CD45) and macrophage (Fig.4F. F4/80) infiltration from 4175-shControl and 4175 RAGE sh66 tumors.

[0010] Figures 5A-5F. RAGE expression in tumor cells is required for breast cancer metastasis in vivo: xenograft models. Lung and liver tissue from 4175 or 231 tumor bearing RAGE sh66 or shControl mice were analyzed for metastasis by immunohistochemistry with anti-human CK7 antibodies. Representative images are shown from tissue for 231 (A&B), 4175 time (Fig.5C and5D) and size (Fig.5E and 5F) -matched mice.

[0011] Figures 6A-6F. RAGE knockdown in mouse highly metastatic breast cancer cells downregulates cell invasion and breast cancer metastasis in vivo: syngeneic models. (Fig.6A) Western blot shows knockdown by different RAGE shRNAs (shlO and shl2) compared to shControl in 4T-1 cells, and RAGE overexpression in 67NR cells compared to vector control (using anti-RAGE and anti-β actin antibodies). (Fig.6B) 4T-1 cells with RAGE shRNA (and shControl) were quantified for matrigel invasion in transwell chambers after 24h to 1% FBS stimuli, were quantified. (Fig.6C and 6D) Soft agar colony formation assays of (Fig.6C) 4T- 1 cells and (Fig.6D) 67NR cells. Representative cell colonies in soft agar are shown. Colony number per field is shown in the lower panel. For all experiments, data are means + SEM, n=3. *, P<0.05. (Fig.6E) 4T-1 cells (lxlO⁶) with RAGE shlO, RAGE shl2 or shControl were injected into mammary fat pad of BALBc mice. Tumor size was measured over the course of 35 days, and at time of sacrifice (35 days), tumor weight was measured. Data shown are from 8 mice per group. (Fig.6F) Lung tissue from 4T-1 tumor bearing RAGE shlO, shl2 or shControl mice were analyzed for metastasis by immunohistochemistry with H&E.

Representative images are shown. [0012] Figures 7A-7D. RAGE knockout in mice impairs tumor growth in vivo. AT-3 murine mammary tumor cells (0.5xl0⁶) were injected into mammary fat pad of C57BL6 wild-type and RAGE knockout mice, and (Fig.7A) tumor progression monitored. (Fig.7B and 7C) Immunohistochemical analysis of tumors for angiogenesis (Fig.7B, CD34), and leukocyte (Fig.7C, CD45) infiltration from wild-type and RAGE -/- tumors. (Fig.7D)

Western blot of total and phospho-ERK 1/2 in tumors from wild-type and RAGE -/- mice.

[0013] Figures 8A-8H. The RAGE inhibitor FPS-ZMl impairs cell invasion and anchorage-independent growth in soft agar. (Fig.8 A) 231 control & RAGE transfected cells were quantified for Matrigel invasion in transwell chambers after 24h to 1% FBS stimuli in the presence of FPS-ZMl (1 μΜ) or DMSO control. (Fig.8B) 4175 cells with RAGE shRNA (and shControl) were quantified for Matrigel invasion in transwell chambers after 24h to 1% FBS stimuli in the presence of FPS-ZMl (1 μΜ) or DMSO control, were quantified.

(Fig.8C) 4T-1 cells were quantified for Matrigel invasion in transwell chambers after 24h to 1% FBS stimuli in the presence of FPS-ZMl (ΙμΜ) or DMSO control. (Fig.8D) Primary human dissociated tumor (DT28) cells were quantified for Matrigel invasion in transwell chambers after 24h to 1% FBS stimuli in the presence of FPS-ZMl (1 μΜ) or DMSO control. (Fig.8E) Proliferation of 4175 cell treated with FPS-ZMl (1, 10 and 25 μΜ) and DMSO control quantified by crystal violet staining after 72h. (Fig.8F) Proliferation of 4T-1 cell treated with FPS-ZMl (10 μΜ) and DMSO control quantified by crystal violet staining after 72h. (Fig.8G-8H) Soft agar colony formation assays of 4175 (Fig.8G.) and 4T-1 (Fig.8H.) cells treated with FPS-ZMl (1, 10 and 25 μΜ) and DMSO control.

[0014] Figures 9A-9G. The RAGE inhibitor FPS-ZMl reduces tumor progression and metastasis of highly metastatic 4175 cells. 4175 cells were injected into mammary fat pad of NSG mice, and mice treated injected IP with lmg/kg FPS-ZMl or vehicle control twice per week. Tumor size was measured over the course of 35 days, and shown as (Fig.9A) representative mice and (Fig.9B) for tumor size. Data shown are from 5 mice per group. (Fig.9C-9F) Immunohistochemical analysis of tumors for proliferation (Fig.9C, Ki67), angiogenesis (Fig.9D. CD34), and leukocyte (Fig.9E, CD45) and macrophage (Fig.9F, F4/80) infiltration from 4175-control and 4175 FPS-ZMl treated tumors. (Fig.9G) Representative images of lung and liver tissues from control (DMSO) and FPS-ZMl treated mice stained with anti-human CK7 antibodies to visualize metastasis. Non-tumor bearing (NTB) controls are shown. [0015] Figures 10A-10E. RAGE gene expression and clinical outcomes in human breast cancer. Relative expression of RAGE mRNA normalized to beta-actin examined using breast cancer datasets from Oncomine™. Breast cancer stromal datasets (Fig.lOA-lOC) and from metastatic datasets (Fig.lOD and 10E) were compared.

[0016] Figure 11. RAGE inhibitors impair tumor growth of 231-4175 human breast cancer cells in NSG immunocompromised mice. 4175 cells were injected into mammary fat pad of NSG mice and mice injected IP with 1 mg/kg FPS-ZMl, TTP488, or vehicle control (DMSO) twice per week. Tumor size (volume, x-axis) was measured over the course of 35 days (y- axis).

[0017] Figure 12. Tumor growth of 4T-1 breast cancer cells in BALBc mice. 4T-1 cells were injected into mammary fat pad of BALBc mice, and mice injected IP with 1 mg/kg FPS-ZMl, TTP488, or vehicle control (DMSO) twice per week. Tumor size (volume, x-axis) was measured over the course of 35 days (y-axis).

[0018] Figure 13. Mouse weight changes in type 2 diabetic mice on RAGE inhibition. Control (db/m) and diabetic (db/db) mice (5 mice per group) were treated by intraperitoneal (I.P.) injection with 1 mg/kg FPS-ZMl, TTP488, or vehicle control (DMSO) twice per week for 30 days. Mice were weighed weekly. Line graph illustrates weight (g, y-axis) and day (x-axis).

[0019] Figure 14. Combination therapy with FPS-ZMl and doxorubicin impairs tumor growth in syngeneic breast cancer models. BALBc mice were injected with 4T-1 cells and treated with control (DMSO), doxorubicin (5 mg/kg), FPS-ZMl (1 mg/kg), or a combination of doxorubicin and FPS-ZMl. Doxorubicin was administered I.P. on days 3 and 7 (post tumor implantation). FPS-ZMl was administered I.P. on days 2, 6, 10, and 13. N=6 mice per group for all experiments.

[0020] Figure 15. RAGE inhibition with FPS-ZMl reduces liver inflammation in obese mice. Dbdb mice were treated with FPS-ZMl (1 mg/kg, twice per week I.P.) or vehicle (control). RNA was extracted from liver tissue and QPCR performed to determine gene expression levels. Four sets of bars are provided in the bar graph denoting RNA of four targets (from left to right, x-axis), CDE3, f480, IL6, and TNF, with relative normalized expression denoted on the y-axis. Within each set of bars, the left bar corresponds to treatment with vehicle; the right bar corresponds to treatment with FPS-ZMl. [0021] Figure 16. RAGE inhibition with FPS-ZMl reduces liver expression of RAGE and RAGE ligands in obese mice. Dbdb mice were treated with FPS-ZMl (1 mg/kg, twice per week I.P.) or vehicle (control). RNA was extracted from liver tissue and QPCR performed to determine gene expression levels. Three sets of bars are provided in the bar graph denoting RNA of three targets (from left to right, x-axis), RAGE, S 10048, and S 10049, with relative normalized expression denoted on the y-axis. Within each set of bars, the left bar

corresponds to treatment with vehicle; the right bar corresponds to treatment with FPS-ZMl.

[0022] Figures 17A-17B. The RAGE inhibitor FPS-ZMl displays a dose dependent effect on tumor metastasis. Tumor growth (Fig. l7A; y-axis=tumor size, x-axis=day) and metastasis (Fig.l7B; y-axis=luminoscore, x-axis=DMSO, FPS l, FPS2) was measured in tumor bearing mice treated with either DMSO or FPS-ZMl (1 or 2 mg/kg, twice per week).

[0023] Figures 18A-18B. Frequency and dose effects of the RAGE inhibitor FPS-ZMl on tumor metastasis. Tumor growth (Fig.l8A; y-axis=tumor size, x-axis=day) and metastasis (Fig.l8B; y-axis=luminoscore, x-axis=DMSO, FPS dosing) was measured in tumor bearing mice treated with either DMSO or FPS-ZMl. FPS-ZMl was given to mice either twice per week (lmg/kg), or every day (lmg/kg or 2mg/kg).

[0024] Figures 19A-19B. The RAGE inhibitor TTP488 displays a dose dependent effect on tumor metastasis. Tumor growth (Fig.l9A; y-axis=tumor size, x-axis=day) and metastasis (Fig.l9B; y-axis=luminoscore, x-axis=DMSO, TTP dosing) was measured in tumor bearing mice treated with either DMSO or TTP488 (1, 2.5, 5 mg/kg, twice per week).

[0025] Figure 20. FPS-ZMl impairs tumor cell invasion. Cell invasion assays were performed using a Matrigel transwell invasion system. X-axis=cell line and treatment; y- axis= relative invasion (%).

[0026] Figure 21. RAGE inhibitors (FPS-ZMl and TTP488) impair tumor cell invasion. Cell invasion assays were performed as in Figure 20. Cells (4T-1) were treated with either FPS-ZMl (5 μΜ), TTP488 (5 μΜ) or DMSO control.

[0027] Figure 22. RAGE inhibitors (FPS-ZMl and TTP488) impair tumor cell

inflammation. Cytokine and chemokine release from cells was assessed using Proteome Profiler Mouse XL Cytokine arrays with condition media from either control (DMSO), FPS- ZMl (2 μΜ) or TTP488 (2 μΜ). Cytokine levels were quantified by western blot and densitometry. Data shows heat maps where protein levels are decreased. Data is expressed as a percentage of decrease for each cytokine. [0028] Figure 23. RAGE knockout in mice impairs tumor progression. The tumor size of wild-type (WT) and RAGE knockout mice (RKO) Py8119 breast cancer cells were compared over a period of 33 days.

[0029] Figures 24A-24B. RAGE knockout in MMTV-PyMT mice impairs tumor initiation and metastasis. Tumor latency (Fig. 24A) and tumor metastasis (Fig. 24B) were measured for MMTV-WT and MMTV-PyMT RAGE KO (RKO) mice.

[0030] Figure 25. RAGE inhibitors FPS-ZM1 impairs NASH in db/db mice. Db/db mice were treated with FPS-ZM1 (lmg/kg, twice per week) or DMSO control. NASH was assessed by histology of liver and serum analysis of the liver enzyme ALT.

DETAILED DESCRIPTION

[0031] The Receptor for Advanced Glycation End-product (RAGE) is a multiligand cell surface molecule of the immunoglobulin superfamily. RAGE binds multiple ligands including, e.g., the non-enzymatic protein- adducts (AGEs) that form in the hyperglycemic state of diabetes (Siegel et al. (2013). CA Cancer J Clin, 63, 11-30; Taguchi et al. (2000). Nature, 405, 354-360)), various members of the S lOO/calgranulins (S 100A4, A6-9, S 100B and S 100P) (Kalea et al. (2010). Cancer Res, 70, 5628-5638; Kang et al. (2010). Cell Death Differ, 17, 666-676; Liao et al. (2011). Asian Pac J Cancer Prev, 12, 1061-1065)), amyloid beta (Αβ), LPA, collagen I/IV, and the high mobility group box-1 (HMGB 1) protein (Larsson et al. (2007). Int J Cancer, 121, 856-862). RAGE is able to bind broad ligand classes due to its highly positively charged ligand binding domain, which forms an electrostatic trap for these largely negatively charged ligands. Chavakis et al. (2003). J Exp Med, 198, 1507-1515; Kock et al. (2010). Structure, 18, 1342-1352.

[0032] As described herein, RAGE-signaling in both the tumor and tumor

microenvironment is required for invasion and metastasis of highly metastatic murine and human breast cancer cells. Targeting RAGE with novel small molecule inhibitors

significantly impairs metastasis in vivo. The data described herein demonstrate that therapeutic blockade of RAGE-ligand signaling is a powerful approach to treat invasive and metastatic breast cancer. The disclosure provides a method of treating breast cancer, the method comprising administering to mammalian subject in need thereof an inhibitor of Receptor for Advanced Glycation End-product (RAGE). The disclosure also provides a method of inhibiting breast cancer metastasis, the method comprising administering to mammalian subject in need thereof an inhibitor of RAGE. In various aspects, the subject is a human.

[0033] In various aspects, the RAGE inhibitor is a small molecule. For example, a RAGE inhibitor suitable for use in the context of the disclosure is TTP-488 (chemical name 3-(4-{2- butyl- 1 -[4-(4-chlorophenoxy)-phenyl] - lH-imidazole-4-yl } -phenoxy)-propyl] -diethylamine), also known as azeliragon or PF-04494700). The structure of TTP-488 is provided below.

[0034] TTP-488 is an orally active, antagonist of RAGE-RAGE ligand interaction, which has shown reduction of amyloid accumulation in the brains of mice. In human clinical trials, TTP-488 did not display any adverse effects in both in Phase I and II trials. Low dose treatment with TTP-488 in Alzheimer patients demonstrated slower decline in cognitive function compared to controls.

[0035] Alternatively, the RAGE inhibitor is FPS-ZM1, the structure of which is provided below.

[0036] The disclosure provides a method of treating breast cancer or inhibiting breast cancer metastasis in a subject in need thereof. "Treating" breast cancer does not require a 100% abolition of cancer in the subject. Any decrease in tumor load, tumor burden, or tumor volume; inhibition of tumor cell proliferation; eradication of tumor cells; and the like constitutes a beneficial biological effect in a subject. The progress of the method in treating breast cancer (e.g., reducing tumor size or eradicating cancerous cells) can be ascertained using any suitable method, such as those methods currently used in the clinic to track tumor size and cancer progress. Tumor size can be figured using any suitable technique, such as measurement of dimensions or estimation of tumor volume. Tumor size can be determined by tumor visualization using, for example, CT, ultrasound, SPECT, spiral CT, MRI, photographs, and the like. Measurement of tumor size, detection of new tumors, biopsy, surgical downstaging, PET scans, and the like can point to the overall progression (or regression) of cancer in a human. Similarly, "inhibiting metastasis" does not require a complete blockage of metastasis; any degree of preventing, suppressing, delaying the onset, or slowing metastasis in the subject is contemplated.

[0037] The disclosure further provides a method of inhibiting the onset of breast cancer. The method comprises administering to a mammalian subject in need thereof an inhibitor of Receptor for Advanced Glycation End-product (RAGE). "Inhibiting the onset of breast cancer" does not require 100% prevention of the disease, although complete prevention is contemplated. "Inhibiting the onset" when used in the context of a disease or disorder (for example, breast cancer) also includes lessening the likelihood of the disease or disorder onset or slowing the onset of the disease or disorder.

[0038] The disclosure also provides a method of treating chronic disease, optionally associated with inflammation, comprising administration to a subject in need thereof a RAGE inhibitor, such as a RAGE inhibitor described herein. In various aspects, the chronic disease is obesity, non-alcoholic fatty liver disease (NAFLD), or nonalcoholic steatohepatitis (NASH). In various aspects, the subject has a body mass index of 30 or greater. NAFLD is characterized by the presence of steatosis, whereas NASH is characterized by the histologic presence of steatosis, cytological ballooning, inflammation, and fibrosis. "Treating" the chronic disease, such as obesity or NASH does not require a 100% abolition of the disorder in the subject. Any reduction in, e.g., weight, body mass index, liver inflammation, or accumulation of fatty deposits in the liver, is contemplated. For example, in various aspects, the method reduces the risk of developing the chronic disease (e.g., NAFLD or NASH), arrests or slows the development of the disease or clinical symptoms thereof, or ameliorates the chronic disease (e.g., promotes regression or reversal of the disease state or symptoms thereof). In this regard, the disclosure contemplates a method of reducing liver inflammation comprising administering to a subject in need thereof a RAGE inhibitor, such as a RAGE inhibitor described herein. Liver inflammation and steatosis are detected using any of a number of techniques including, but not limited to, blood tests (to detect, e.g., elevated liver enzymes), ultrasound, computerized tomography (CT) scans, Magnetic resonance imaging (MRI), and biopsy.

[0039] In various aspects, the RAGE inhibitor is provided in a composition (e.g., a pharmaceutical composition) comprising a physiologically-acceptable (i.e.,

pharmacologically-acceptable) carrier, buffer, excipient, or diluent. Any suitable

physiologically-acceptable (e.g., pharmaceutically acceptable) carrier can be used within the context of the disclosure, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition is to be

administered and the particular method used to administer the composition. Suitable composition formulations include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The composition can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.

[0040] A particular administration regimen for a particular subject will depend, in part, upon the amount of RAGE inhibitor administered, the route of administration, and the cause and extent of any side effects. The amount administered to a subject (e.g., a mammal, such as a human) in accordance with the disclosure should be sufficient to affect the desired response over a reasonable time frame. For example, the administration regimen for TTP488 may, in various aspects, comprise daily administration of about 10 mg to about 60 mg to a subject in need thereof.

[0041] The disclosure further contemplates administering the RAGE inhibitor in combination with one or more additional therapeutics. For instance, in the context of cancer, the therapeutic regimen for the subject may include administration of one or more cytotoxic agents or chemotherapeutic agents. Representative additional therapeutic agents include, but not limited to, 5-azacytidine, actinomycin D, amanitin, aminopterin, anguidine, anthracycline, anthramycin (AMC), auristatin, bevacizumab, bleomycin, busulfan, butyric acid,

camptothecin, carboplatin, carmustine, cemadotin, cisplatin, colchicin, a combretastatin, cyclophosphamide, cytarabine, cytochalasin B, dactinomycin, daunorubicin, decarbazine, diacetoxypentyldoxorubicin, dibromomannitol, dihydroxy anthracin dione, a disorazole, docetaxel, dolastatin (e.g., dolastatin 10), doxorubicin, daunorubicin, duocarmycin, echinomycin, emetine, epothilones, esperamicin, ethidium bromide, etoposide, fluorouracil, gemcitabine, geldanamycin, glucocorticoid, irinotecan, lapatinib, melphalan, mercatopurine, methopterin, methotrexate, mithramycin, mitomycin, mitoxantrone, paclitaxel, pertuzumab, propranolol, pteridine, puromycin, taxol, tamoxifen, tenoposide, tetracaine, teniposide, topotecan, trastuzumab, vinblastine, vincristine, vindesine, vinorelbine, or a derivative of any of the foregoing. In various aspects, the RAGE inhibitor is administered in combination with doxorubicin. It will be appreciated that "in combination" does not restrict the timing or order in which the RAGE inhibitor and one or more additional therapies are administered to the subject. The administration of different therapeutic agents can occur simultaneously or sequentially, and by the same or different routes of administration.

[0042] The invention, thus generally described, will be understood more readily by reference to the following example, which is provided by way of illustration and is not intended to limit the invention.

EXAMPLES

Example 1

[0043] This example demonstrates inhibition of breast cancer cell proliferation and metastasis in vivo using RAGE inhibitors.

Materials and Methods

[0044] Cell lines: The MDA-MB-231 breast cancer line and its highly metastatic derivatives (4175 and 1833) are described in Minn et al. (2005). Nature, 436, 518-524;

Vernon et al. (2013). J Immunol, 190, 1372-1379. Cells were cultured in Dulbecco's Modified Eagle Medium (Lifetech, Carlsbad, CA, USA) and 10% fetal calf serum (FCS, Atlanta Biologicals, USA). Murine breast cancer cells 4T1 and 67NR were obtained from ATCC and Karmanos Cancer Institute respectively. AT-3 cells were previously isolated from a spontaneous mammary tumor from the MMTV-PyMT/B6 mouse model. The dissociated primary human tumor cell line (DT28) established from an ER-negative primary breast tumors was cultured as previously described. Drews-Elger et al. (2014). Breast Cancer Res Treat, 144, 503-517. All cell lines were routinely tested for mycoplasma using the MycoAlert™ Mycoplasma Detection Kit (Lonza) and with PCR prior to injection into animals.

[0045] Generation of RAGE overexpression and gene knockdown cells: Lentiviral vectors encoding human RAGE cDNA (Precision LentiORF; Thermo Scientific) and human / mouse RAGE shRNA (pGIPZ; Thermo Scientific) were used for RAGE overexpression and silencing. Human RAGE cDNA (PLOHS_100006205) and empty vector pLOC control were used. Indicated shRNAs are as follows: shControl (RHS4346), human RAGE sh65

(V3LHS_316665; TGGACTTGGTCTCCTTTCC (SEQ ID NO: 1)), human RAGE sh66 (V3LHS_316666; TACACTTCAGCACCAGTGG (SEQ ID NO: 2)), mouse RAGE shlO (V3LMM_430610; TGACCTCCTTCCCTCGCCT (SEQ ID NO: 3)) and mouse RAGE shl2 (V3LMM_430612; TATTAGGGACACTGGCTGT (SEQ ID NO: 4)). Human RAGE cDNA (PLOHS_100006205) and empty vector control. To generate lentivirus, lentiviral vectors were co-transfected with psPAX2 and pMD2.G (Addgene) into HEK-293T cells (ATCC) with Lipofectamine 2000 (Lifetech). Supernatants were collected at 48 hours and cell debris pelleted. MDA-MB-231, 4175. 4T-1, and 67NR were infected with viral supernatant with 4 μg/ml polybrene and stable expression selected with blasticidin (pLOC) or puromycin (pGIPZ). Stably transduced cells were tested for RAGE overexpression or knockdown by Western blotting (see below).

[0046] Western Blots: Cells were lysed with RIPA buffer with protease (Sigma) and phosphatase inhibitors (Sigma) according to manufacturer's instructions. Western blotting was performed using Invitrogen NuPAGE system as previously described. Jules et al.

(2013). PLoS ONE, 8, e78267. Antibodies used were as follows; human RAGE monoclonal antibody (Millipore; MAB5328), RAGE polyclonal (Santa Cruz; H300), β-actin (Millipore; MAB 1501), E-cadherin (BD Biosciences; 610181), ZO-1 (Cell Signaling; 8193), Vimentin (Cell Signaling; 5741), MMP2 (Millipore), MMP9 (Millipore), Slug (Cell Signaling; 9585), Snail (Cell Signaling; 3879), Twist (Santa Cruz Biotechnology), and Zebl (Cell Signaling; 3396). All phospho-status and total antibodies (MEK 1/2, ERK 1/2, p38, SAPK/JNK, AKT) were obtained from Cell Signaling. All antibody conditions are listed in Table 1.

TABLE 1

Zebl Cell Signaling 3396 WB 1000

Phospho-Akt (Thr308) Cell Signaling 2965 WB 1000

Phospho-Akt (Ser473) Cell Signaling 4060 WB 1000

Akt Cell Signaling 4691 WB 1000

Phospho-MEKl/2 Cell Signaling 2338 WB 1000

MEK1/2 Cell Signaling 9126 WB 1000

Phospho-p44/42 MAPK Cell Signaling 4370 WB 1000

(Erkl/2)

p44/42 MAPK (Erkl/2) Cell Signaling 9102 WB 1000

Phospho-p38 MAPK Cell Signaling 4511 WB 1000 p38 MAPK Cell Signaling 8690 WB 1000

Phospho-SAPK/JNK Cell Signaling 4668 WB 1000

SAPK/JNK Cell Signaling 9258 WB 1000 mDial BD Biosciences 610848 WB 500

Ki67 Abeam abl5580 IHC 100

CD34 eBioscience 14-0341-82 IHC 100

CD45 BD Biosciences 550539 IHC 100

F4/80 Abeam abl6911 IHC 100

CK7 Dako M701801-2 IHC

[0047] Wound healing assays: To assess cell migration, wound-healing (scratch) assays were used. Cells were plated in 6 well plates at 2.5 x 105 cells / well and grown to confluence to form a monolayer and serum starved overnight. A single scratch was made per well with a 200 μΐ pipette tip, and the cell media changed to 1% FBS. Cells were fixed at 0 and 16 h after wounding and images acquired with a light microscope at lOx magnification.

[0048] Cell invasion assay: Cell invasion assays were performed using transwell migration chambers as previously described; 17. 5 x 10 cells were seeded in the upper chamber of 8-μιη porous transwell inserts (ThinCerts, Greiner) coated with 12.5 μg of Growth Factor Reduced Matrigel (BD Biosciences) in serum-free DMEM, and incubated in 24 well plates with 1% FBS as a chemoattractant for 24 hours (48 hours for DT28 cells). Following incubation cells were fixed with methanol for 10 minutes and stained with 2% crystal violet in 2% ethanol solution. Non-migrated cells were removed from transwell chambers with a cotton swab. To quantify the cells, the cell stain was extracted with 10% acetic acid, transferred to a 96 well plate and measured at 595 nm using an iMark Microplate Reader (Biorad). For invasion assays with RAGE inhibitors, cells were pre- treated for lh prior to assays and re-added during invasion assays (both upper and lower chambers) with either FPS-ZM1 (1 μΜ; Millipore) or equal volume DMSO control. [0049] Proliferation assays: Cells were counted and plated in triplicate at 35,000 cells per well of a 12 well plate and grown for 48-72 hours. Cells were fixed with 4%

paraformaldehyde and stained with 0.1% crystal violet for 20 min. Cell number was quantified by extracting crystal violet stain with 10% acetic acid and transferred to a 96 well plate and measured at 595 nm using an iMark Microplate Reader (Biorad).

[0050] Soft agar assay: Cells (5 x 10 cells/well) were resuspended in 0.4% agarose (Sigma) in Iscove's Modified Dulbecco's Medium (IMDM) with 10% FBS and seeded on top of a 0.8% agarose layer (IMDM with 10% FBS) in 6 well plates and cultured for 14 days (4T-1, 67NR cells) or 21 days (231, 4175 cells). Tumor colonies were stained with 0.05% INT (Iodonitrotetrazolium chloride) in PBS overnight at 37 °C and representative images (5 per well) were acquired using a Nikon Eclipse TS 100 microscope. The assay was performed in triplicate and repeated independently three times.

[0051] Animal studies: All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Miami. NOD scid gamma (NOO.Cg-Prkdc^scld Il2rg^tmlWjI/Sz , NSG), BALBc and C57BL6 wild-type mice were purchased from Jackson Laboratory. C57BL6 RAGE knockout mice (RAGE -/-) were previously described. Myint et al. (2006). Diabetes, 55, 2510-2522. As < 95% of the animals inoculated with tumor cells develop tumors and -90% develop lung metastasis (for NSG and BALBc mice); 10 mice per group were used for all studies. Further, mice with tumor growth in exceeding 5% of body weight or exhibiting 20% weight loss were terminated early and excluded from the study. Animals were randomized to each experimental group (RAGE shRNA vs. scramble shRNA; FPS-ZMl vs. vehicle). Injections and measurements were performed by different investigators, and mice injected/measured in a random manner.

[0052] 231 (vector/RAGE, shControl/RAGE sh66) and 4175 (shControl and RAGE shRNA66) were injected in 100 μΐ of Matrigel (lxlO⁶ cells) into the third inguinal mammary fat pad of 8-week old female NSG mice (10 mice per group) as previously described. Drews-

Elger et al. (2014). Breast Cancer Res Treat, 148, 41-59. 4T-1 (shControl, RAGE shRNA 10 and RAGE shRNA12) cells were injected into the third inguinal mammary fat pad of 8-week old female BALBc mice. For AT3 cells, cells were injected into the third inguinal mammary fat pad of 8-week old female C57BL6 wild-type or RAGE knockout mice (10 mice per group). For experiments involving the RAGE inhibitor, NSG mice were injected ip with either DMSO (vehicle control) or with FPS-ZMl (RAGE inhibitor) at a dose of lmg/kg twice per week. Tumor growth was monitored using calipers every 5 days and animals were sacrificed when tumors reached 10% of body weight. Primary tumors and organs were harvested and fixed in 10% formalin and paraffin embedded for pathological analysis.

Primary tumor and organs were stained with hematoxylin and eosin. Primary tumors were stained with antibodies against CD34 (EBioscience, 14-0341), Ki67 (Abeam, abl5580), CD45 (BD Biosciences, 550566) and F4/80 (Abeam). To quantitate metastasis of tumor cells in tissue, organs were stained with antibodies against human cytokeratin 7 (Leica

Biosystems). Slides were analyzed by a pathologist to confirm the presence of metastases. For both tumor and tissue sections, five view fields were selected randomly from each section (with 2 sections per tumor / tissue used) for each animal. To analyze and quantify antibody staining, percentage of area that stained positive was calculated with the use of ImageJ1.34n software (National Institutes of Health [NIH], Bethesda, MD) as previously described.

Kurozumi et al. (2007). J Natl Cancer Inst, 99, 1768-1781.

[0053] Syngeneic drug models: 100,000 luciferase labelled 4T1 cells, were injected into the 4th mammary fat pad (anatomical right) of BALBc mice. Drug treatments (FPS-ZM1 or TTP488) during the study were given LP. and varied on dosage schedule and amount of drug administered. Tumor size after initial palpability was measured twice weekly by calipers. In vivo imaging was performed using the Xenogen IVIS-200, which allowed for quantitative primary tumor and metastasis measurements. A luminoscore based on average radiance and total photon flux (photons/sec) was generated by drawing regions of interest (ROIs) around the lungs of each mouse.

[0054] Spontaneous models: MMTV-PyMT male mice were bred with either C57BL6 wild-type or RAGE knockout mice. Mouse genotype was confirmed by PCR for both MMTV transgene and knockout of the RAGE gene. Tumor palpability was monitored in mice and at time of sacrifice, lungs were isolated to identify metastasis.

[0055] Experimental metastasis assays: 25,000 luciferase labelled 4T-1 cells were injected into the tail vein of BALBc mice. RAGE inhibitor FPS-ZM1 (2mg/kg) or vehicle control (DMSO) were given IP every day. IVIS was used as above to quantify tumor metastasis.

[0056] Chemotherapy and RAGE inhibitor experiments: 4T-1 cells / BALBc mice were used as described for syngeneic studies above. Mice were treated with either vehicle control (DMSO), doxorubicin (5mg/kg on days 3, 7 and 14 post-tumor injection), FPS-ZM1

(lmg/kg, 2x / week) or doxorubicin and FPS-ZM1 (same as single dose). Tumor size was measured as above. Metastasis was assessed by H&E ex vivo. [0057] In vitro assays: Invasion assays: cellular invasion was tested using transwell Matrigel invasion assays. Cells (50,000) were seeded in the upper chamber of a transwell insert coated with Matrigel. Cells were allowed to invade toward a lower chamber with 1% FBS as stimulant for 24 hours. For inhibitor studies, cells were incubated with either FPS- ZM1, TTP488 or DMSO control. Cytokine arrays: cell were grown on 100mm dishes and treated with either FPS-ZM1, TTP488 or DMSO control for 24 hours. Conditioned media was collected and cytokine release assessed using the Proteome Profiler Mouse XL Cytokine array.

[0058] Oncomine™ analysis of RAGE mRNA expression: The Oncomine™ database was used for initial analysis and extraction of RAGE expression from three breast cancer stromal datasets (Finak, Ma-4, and Karnoub) and two invasive breast cancer datasets (Sorlie-2 and TCGA). Finak et al. (2008). Nat Med, 14, 518-527; Karnoub et al. (2007). Nature, 449, 557- 563; Ma et al. (2009). Breast Cancer Res, 11, R7; Sorlie et al. (2003). Proc Natl Acad Sci U S A, 100, 8418-8423; (2012). Nature, 490, 61-70. Log2 median-centered intensity values for RAGE gene expression were compared between normal breast stroma and stroma from primary breast lesions: invasive breast cancer (Finak), invasive ductal carcinoma (Karnoub), and DCIS (Ma-4). Log2 median-centered intensity values for RAGE gene expression were also compared between primary breast lesions and metastatic breast lesions (Sorlie-2 and TCGA). Comparisons of RAGE expression between groups were performed using one-sided Student's t-test.

[0059] Statistical analysis: All statistical analyses and visualization were performed using GraphPad Prism version 6.00 for Windows, (GraphPad Software, San Diego California USA) or R statistical software for Mac (R 3.0.3, R Foundation for Statistical Computing, Vienna, Austria). Power analysis was performed using Statmate 2.0 (GraphPad Software, San Diego California USA). Data are expressed as means (+ SEM), with significance considered as P<0.05. In all experiments data was normally distributed and variance similar between groups.

Results

[0060] Metastatic human and mouse breast cancer cells show increased RAGE expression: To investigate the role of RAGE in driving breast cancer metastasis, RAGE protein levels were compared by immunoblotting in breast cancer lines with low and high metastatic ability; MDA-MB-231 (hereafter 231) and in highly metastatic variants of 231, MDA-MB- 231-4175 and MDA-MB-231-1833 (hereafter 4175 and 1833), previously selected for increased metastasis to lung and bone in xenograft models, respectively. Minn et al. (2005). Nature, 436, 518-524; Kang et al. (2003). Cancer Cell, 3, 537-549. The highly metastatic 4175 and 1833 cells displayed greater RAGE protein than parental 231 cells (Figure 1A). The murine mammary metastatic 4T1 model, which spontaneously metastasizes from the primary site in vivo in syngeneic hosts, and an isogenic variant thereof, 67NR, with reduced metastatic potential was studied. Aslakson et al. (1992). Cancer Res, 52, 1399-1405;

Sirotnak et al. (1984). Cancer Chemother Pharmacol, 12, 26-30; Stewart et al. (2007). J Immunol, 179, 2851-2859. RAGE levels were higher in the highly metastatic 4T1 compared to their isogenic non-metastatic variants 67NR (Figure IB). Thus in both human and murine breast cancer models, RAGE levels were increased in highly metastatic lines compared to those with reduced metastatic ability.

[0061] RAGE increases breast cancer cell malignancy in vitro and metastasis in vivo: To further investigate whether RAGE mediates human breast cancer metastasis, the effects of RAGE on migration, invasion and soft agar colony formation in 231 cells were determined. 1% FBS (1%) is a rich source of numerous RAGE ligands and was used to activate RAGE dependent effects. RAGE overexpression in 231 cells (Figure 1C) increased cell migration (Figure ID) and invasion through Matrigel (Figure IE). The increased cell migration and invasion did not result from an increase in cell number due to increased proliferation, as no change in cell number was seen between 231 versus 231-RAGE cells after 48 hours of cell growth (Figure IF). To test further RAGE effects on the tumorigenic properties of 231 cells, soft agar assays were performed. RAGE overexpression increased both number and size of 231 cell colonies in soft agar, compared to controls (Figure 1G). Thus, RAGE over- expression increases migration and invasion, and promotes soft agar colony formation of 231 cells in vitro.

[0062] To extend these data in vivo, 231 cells, with or without RAGE overexpression, were implanted into the mammary fat pad in immunocompromised NOD scid gamma (NSG) mice. RAGE overexpression in 231 cells did not affect tumor growth (Figure 1H) or final tumor weight (Figure II). In marked contrast, RAGE overexpression in 231 cells significantly increased the number and size of metastases in lung (Figure 1J).

[0063] RAGE signaling drives cellular invasion through a MEK-dependent expression of EMT regulators: Since RAGE expression is linked to poor breast cancer survival, and since RAGE is overexpressed in metastatic lines and mediates increased tumor cell invasion and soft agar colony formation, the effects of RAGE on signaling pathways and epithelial to mesenchymal transition (EMT) mediators implicated in metastasis was studied. RAGE overexpression in 231 cells increased expression of EMT markers including matrix metalloproteins, MMP-2, MMP-9, and vimentin, whilst concurrently reducing expression of epithelial markers, E-cadherin and ZO-1 (Figure 2A). Furthermore, RAGE overexpression increased expression of EMT transcription factors Slug and Twistl and increased β-catenin, while Snail and Zebl levels were unchanged (Figure 2B).

[0064] RAGE signals through diverse pathways, including various mitogen activated protein kinase (MAP) kinases (ERK 1/2, p38, SAPK/JNK), PDK/Akt and JAK/STAT. Taguchi et al. (2000). Nature, 405, 354-360; Hudson et al. (2008). J Biol Chem, 283, 34457- 34468; Kislinger et al.. (1999). J Biol Chem, 274, 31740-31749; Huang et al. (2001). J Cell Biochem, 81, 102-113; Yeh et al. (2001). Diabetes, 50, 1495-1504; Lander et al. (1997). J Biol Chem, 272, 17810-17814; Huttunen et al. (1999). J Biol Chem, 274, 19919-19924. To test which pathway(s) mediate RAGE dependent migration and invasion in breast cancer cells, the activated phosphorylation status of signaling proteins was compared by Western blot in parental 231-controls and 231-RAGE cells. RAGE overexpression increased activation of Akt, p38 and MEK/ERK pathways, but not of SAPK/JNK. To test which RAGE activated pathways mediate the increased invasive phenotype, invasion assays were carried out with and without inhibitors of MEK (U0126), p38 (SB203580) or PDK/Akt (LY294002). Inhibition of MEK, but not Akt decreased Matrigel invasion in 231 -RAGE (Figure 2C). These data demonstrate that the increase in cell invasion occurs through a RAGE-MAP kinase signaling cascade.

[0065] RAGE knockdown in highly metastatic breast cancer cells impairs Matrigel invasion and anchorage-independent growth: Since RAGE overexpression increased migration and invasion of 231 cells, the ability of RAGE gene knockdown to impair these effects in highly metastatic lines was tested. RAGE shRNA decreased RAGE protein levels in 231 and 4175 to >75% of shControl (Figure 3A). Cell invasion assays showed shControl highly metastatic 4175 cells showed greater invasion than shControl parental 231 transwell Matrigel invasion assays as expected (Figure 3B). RAGE knockdown dramatically reduced Matrigel invasion by 4175 cells, and impaired that of the less metastatic parental 231 line (Figure 3B). To further confirm these findings, the effect RAGE knockdown by different shRNAs was observed (RAGE shRNA 65 & 66). As shown in Figure 3B, both RAGE shRNAs reduced cell invasion. Whilst the increase in 4175 cells over time was more rapid than 231 cells, RAGE knockdown had little effect on population increase in 4175 cells and only a modest effect (p = 0.057) in 231 cells (Figure 3C). RAGE is a mediator of soft agar colony formation. Control 4175 cells formed more and larger colonies in soft agar than parental 231 cells (Figure 3D). shRNA-mediated RAGE knockdown in both 4175 and 231 cells significantly reduced both the number and size of soft agar colonies formed compared to shControls. These data further support a role for RAGE as a key mediator of invasion in these breast cancer models.

[0066] To test whether RAGE knockdown would impair downstream signaling activated by RAGE overexpression shown in Figure 2, the activated phosphorylation status of downstream signaling proteins was tested. RAGE shRNA in 4175 cells led to decreased activation of Akt, p38 and MEK/ERK pathways, compared to shControl 4175 cells (Figure 3E). To further extend these mechanistic data, how RAGE knockdown affects levels of its signaling partner mDia-1 was explored. Hudson et al. (2008). J Biol Chem, 283, 34457- 34468. RAGE directly interacts with mDia-1 to induce intracellular signaling. Western blot analysis revealed RAGE shRNA 4175 cells had strikingly lower levels of mDia-1 (Figure 3E). Together these data suggest RAGE signaling regulates tumor cell properties linked to metastasis in breast cancer cells.

[0067] Loss of RAGE in metastatic BC cells impairs tumor growth and metastasis in vivo- xenograft models: To test directly whether RAGE mediates metastasis in vivo, 231 and 4175 cells, with or without RAGE knockdown (RAGE sh66 and shControl), were implanted into the mammary fat pad in NSG mice. Primary tumor growth and the emergence and growth of metastases were assayed over time. RAGE knockdown in 231 cell did not significantly affect tumor growth (Figure 4A); however, in 4175 cells RAGE knockdown led to a decrease in final orthotopic mean tumor volume and tumor weight compared to control 4175 cells (Figure 4B).

[0068] To investigate the mechanisms by which RAGE knockdown affects tumor growth in 4175 cells, tumor sections were stained for Ki-67 to assess proliferation (Figure 4C).

Tumors formed from RAGE knockdown 4175 cells displayed a 28% reduction in

proliferation by Ki-67 staining compared to shControl 4175 cells (Figure 4C). Since RAGE can affect constituents of the tumor microenvironment, the effects of RAGE knockdown on tumor angiogenesis (endothelial cell marker, CD34) and inflammatory cell recruitment (leukocyte cell maker (CD45), macrophage marker (F4/80)) were assessed by IHC. Tumors arising from RAGE knockdown 4175 cells displayed decreased vessel formation (Figure 4D) and a significant decrease in leukocyte and macrophage recruitment (Figure 5E&F), compared to shControl 4175 cells.

[0069] Since the effects of RAGE on breast cancer metastasis are not fully known, it was determined whether RAGE knockdown would affect the ability of 231 and 4175 cells to metastasize from the primary mammary tumor site. All NSG mice implanted with 231 and 4175 shControl cells developed extensive metastasis to lung and liver (Figure 5A-F). In marked contrast, RAGE knockdown in 231 and 4175 cells significantly reduced the number and size of metastases in both lung and liver (Figure 5A-D).

[0070] To account for differences in tumor growth resulting from RAGE knockdown in 4175 cells, metastasis arising from tumors of similar size was quantitated. Even from size matched 4175 RAGE shRNA and shControl tumors (5 mice per group), RAGE knockdown cells showed fewer distant metastasis (Figure 5E&F). Whilst 4175 shControl cells resulted in extensive metastasis to both lung and liver (day 35), RAGE shRNA in 4175 cells (day 43) did not display lung or liver metastasis (Figure 5E&F).

[0071] Loss of RAGE in metastatic BC cells impairs tumor growth and metastasis in vivo: syngeneic studies: To validate the data in other metastatic models of breast cancer, the effects of RAGE gene knockdown in murine 4T-1 cells and consequences of RAGE overexpression in the non-metastatic 67NR cells, were assessed. RAGE shRNA in 4T-1 using different murine specific shRNAs (RAGE shlO & 12), decreased RAGE protein levels compared to shControl, whereas RAGE expression was increased in 67NR cells by lentiviral transduction of RAGE (Figure 6A). As in 4175 cells, RAGE knockdown by both RAGE shlO and shl2, impaired 4T-1 cell invasion compared to shControl (Figure 6B). RAGE knockdown in 4T- 1 cells also decreased the number and size of colonies formed in soft agar (Figure 6C). Furthermore, RAGE overexpression in non-metastatic 67NR increased the number and size of colonies formed in soft agar (Figure 6D).

[0072] Next, 4T-1 cells, with or without RAGE knockdown (RAGE shlO, RAGE shl2 and shControl), were implanted into the mammary fat pad in BALBc mice. Primary tumor growth and the emergence and growth of metastases were assayed over time. RAGE knockdown in 4T-1 with either RAGE shlO or shl2 led to a decrease in final orthotopic mean tumor volume compared to control 4T-1 cells (Figure 6E). Whilst 4T-1 cells expressing shControl resulted in extensive metastasis in lung tissue (Figure 6F), RAGE knockdown in 4T-1 cells with both RAGE shlO and RAGE shl2, resulted in little or no lung metastases (Figure 6F). Thus, these data validate that RAGE expression in tumor cells is important for regulating their migration and invasion, and is required to increase their metastatic potential.

[0073] RAGE knockout in mice impairs tumor growth and progression: The role of RAGE expression in non-tumor cells of the tumor microenvironment was tested. Syngeneic studies with AT-3 cells (MMTV-PyMT spontaneous BC cell model) injected into the mammary fat pad of wild-type and RAGE knockout C57BL6 immunocompetent mice were performed. RAGE knockout mice (RAGE -/-) displayed striking impairment of tumor cell growth with AT3 cells compared to wild-type (RAGE +/+) mice (Figure 7A). Furthermore, IHC analysis of tumor tissue revealed that RAGE -/- mice had decreased vessel formation (Figure 7B) and a significant decrease in leukocyte recruitment (Figure 7C) to the tumor. Western blot analysis of total tumor lysate revealed that RAGE -/- mice to have decreased activation of ERK1/2 (Figure 7D) compared to wild-type mice. These data identify RAGE expression in non-tumor cells as an important regulator of tumor progression.

[0074] The RAGE antagonist, FPS-ZM1, impairs breast cancer cell invasion and anchorage-independent growth: FPS-ZM1 is a RAGE antagonist that interacts with the ligand binding domain of the receptor to block RAGE signaling. Deane et al. (2012). J Clin Invest, 122, 1377-1392. This drug has been tested for potential effects in Alzheimer's disease, but has not been explored as an anticancer agent.

[0075] The effects of FPS-ZM1 on breast cancer growth and invasion in vitro and metastasis in vivo was studied. As shown in Figure 8A, FPS-ZM1 (1 μΜ) abrogated the excess invasion caused by RAGE overexpression in 231-RAGE cells, and decreased Matrigel invasion by highly metastatic 4175 (Figure 8B). Importantly, as a test of drug specificity, FPS-ZM1 (1 μΜ) did not affect cell invasion in 4175 RAGE knockdown cells (Figure 8B). To extend these data to other breast cancer models, FPS-ZM1 effects on invasion of murine 4T-1 cells and of the dissociated primary human tumor cell line, DT28, were tested. FPS- ZM1 (1 μΜ) treatment impaired invasion of both 4T-1 and DT28 cells compared to DMSO control (Figure 8C and 8D).

[0076] FPS-ZM1 did not affect cell proliferation or viability in any of the cells tested even at high doses (data shown for 4175 at 1, 10 and 25 μΜ and for 4T-1 at 10 μΜ, Figure 8E&F). RAGE inhibitor effects on colony formation in soft agar assays also were tested. In both 4175 and 4T-1 cells, FPS-ZM1 significantly impaired colony number and size in a dose- dependent manner (Figure 8G&H). These data support a role for RAGE as a therapeutic target for breast cancer.

[0077] The RAGE inhibitor FPS-ZMl displays a dose dependent effect on tumor metastasis. Treatment of FPS-ZMl led to a significant reduction in tumor growth (Figure 17A). Assessment of metastasis showed a more striking effect of RAGE inhibitors and a dose-dependent effect of FPS-ZMl on lung metastasis (Figure 17B).

[0078] The RAGE antagonist FPS-ZMl impairs in vivo tumor progression and metastasis of highly metastatic 4175 cells: The data in this study show both in vitro and in vivo that targeting RAGE in both tumor cells and non-tumor cells of the tumor microenvironment are critical for tumor progression and metastasis. The effects of FPS-ZMl on 4175 tumor formation and metastasis from orthotopic primary tumors in NSG also were tested.

Treatment of mice with FPS-ZMl (1 mg/kg injection twice per week, intraperitoneally started 1 day after tumor cell implantation) impaired 4175 tumor growth compared to vehicle controls (Figure 9A&B). Next the effects of FPS-ZMl were assessed by tumor

immunohistochemistry. Whilst a modest reduction was seen in proliferation by Ki-67 staining compared to control treated 4175 cells (Figure 9C), FPS-ZMl treatment resulted in tumor with markedly decreased vessel formation (Figure 9D) and a significant decrease in leukocyte and macrophage recruitment (Figure 9E and 9F). All 4175-implanted vehicle control mice developed extensive metastasis to lung and liver. In contrast, FPS-ZMl treated mice displayed significantly fewer or no metastasis in either lung or liver (Figure 9G). These data suggest that RAGE inhibitor therapy impairs both primary and metastatic breast cancer growth. Together, the data suggest that RAGE is a critical regulator of tumor progression and metastasis through tumor cell intrinsic and extrinsic effects. Therefore, therapeutic targeting of RAGE presents a unique approach to inhibit metastasis through a combined effect on the tumor cell and other cells of the tumor microenvironment.

[0079] Frequency and dose effects of the RAGE inhibitor FPS-ZMl on tumor metastasis. While FPS-ZMl impaired tumor growth (Figure 18A and 18B), no dose dependent effect on tumor growth was observed. FPS-ZMl given to mice every day at 2mg/kg, had the greatest effect on reducing tumor metastasis.

[0080] The RAGE inhibitor TTP488 displays a dose dependent effect on tumor metastasis. Mice treated with TTP488 showed impaired tumor growth compared to DMSO treated mice. Metastasis in mice was impaired with TTP488 treatment (Figure 19A and 19B). [0081] FPS-ZMl impair tumor cell invasion. Treatment of cells with FPS-ZMl (2 μΜ) impaired invasion of 4T-1, E0771 and Py8119 mouse breast cancer cell lines, compared to DMSO control (Figure 20). In addition, both FPS-ZMl and TTP488 impaired tumor cell invasion, with TTP488 displaying a greater degree of inhibition than FPS-ZMl (Figure 21).

[0082] RAGE inhibitors (FPS-ZMl and TTP488) impair tumor cell inflammation.

Treatment of cytokine arrays with condition media from either control (DMSO), FPS-ZMl (2 μΜ) or TTP488 (2 μΜ) showed that FPS-ZMl and TTP488) impair tumor cell inflammation (Figure 22).

[0083] RAGE knockout in mice impairs tumor progression. To confirm data in wild type and RAGE knockout C57BL6 mice with AT-3 cells and to test the role of RAGE in non- tumor cells of the tumor microenvironment, studies were performed using Py8119 breast cancer cells. RAGE knockout mice (RKO) displayed striking impairment of tumor cell growth with Py8119 cells compared to wild-type (WT) mice (Figure 23).

[0084] RAGE knockout in MMTV-PyMT mice impairs tumor initiation and metastasis. To determine the role of RAGE in the initiation and progression of breast cancer, wild-type and RAGE knockout (RAGE KO) mice were crossed with the MMTV-PyMT spontaneous BC model. MMTV-PyMT mice are a well-established mouse model that displays widespread transformation of the mammary epithelium resulting in rapidly forming mammary tumors and metastatic lesions primarily in lymph nodes and lung, closely mimicking the human clinical state. MMTV-PyMT RAGE KO displayed impaired tumor initiation and growth compared to MMTV-PyMT wild-type mice (Figure 24 A). Further, MMTV-PyMT RAGE KO mice had little or no metastatic lesions on the surface of the lungs, whereas MMTV- PyMT wild-type mice displayed extensive metastatic lesions (Figure 24B).

[0085] RAGE inhibitors FPS-ZMl impair experimental metastasis. To dissect the effects of RAGE inhibition on metastasis from primary tumor growth, experimental metastasis assays were performed. Tumor cells were tail vein injected and metastasis followed using IVIS. Treatment of mice with FPS-ZMl (2mg/kg, everyday) strongly impaired tumor metastasis compared to DMSO (control) treated mice. 7 mice per group imaged at the same exposure by IVIS (Data not shown).

[0086] RAGE is overexpressed in human breast cancer and is associated with increased metastasis: To determine whether RAGE is differentially expressed in breast cancer tissues from human subjects, microarray data from breast cancer patient samples in the Oncomine™ database was examined. Datasets were assessed that include breast stroma from human subjects. In the Finak, Karnoub and Ma-4 datasets, RAGE overexpression in breast stroma was associated with breast cancer compared to normal breast stroma (Figure lOA-C). To correlate these findings with outcomes in breast cancer, datasets were examined that compared the expression of genes in primary tumors versus distant metastases. In both the Sorlie-2 and TCGA datasets, increased RAGE expression was associated with the metastatic site versus primary tumor (Figures 10D and 10E). These data together demonstrate that increased RAGE expression is associated with invasive breast cancer and at the metastatic site.

[0087] Comparison of FPS-ZMl and TTP-448: The activity of FPS-ZMl and TTP-488 was compared in various animal models including xenograft and syngeneic breast cancer. For these studies, TTP488 and FPS-ZMl were reconstituted in DMSO, and mice were injected twice per week with lmg/kg of FPS-ZMl, TTP-488 or vehicle control (DMSO). TTP-488 impaired tumor growth to a similar degree compared to FPS-ZMl (Figure 11). FPS-ZMl injection into mice strongly impaired tumor cell metastasis to both lung and liver. TTP-488 also impaired metastasis (especially to liver). The activity of TTP-488 compared to FPS- ZMl also was tested in the syngeneic immunocompetent 4T-l/BALBc model. Similar to NSG mice, treatment of tumor bearing mice with either FPS-ZMl or TTP-488 impaired tumor growth (Figure 12).

[0088] Combination treatment of RAGE inhibitor and doxorubicin (DOX): A major issue with DOX is its cytotoxicity and the ability of breast cancer cells to become chemoresistant. Combination therapy with multiple cytotoxic agents have greater anti-tumor effects than DOX alone, but are associated with greater side-effects and reduced quality of life for women with breast cancer. The effect of combination therapy with DOX and FPS-ZMl was examined. Using the BALBc/4T-l syngeneic model, mice were treated with either DMSO (control), doxorubicin (5mg/kg on days 3 and 7), FPS-ZMl (lmg/kg on days 2, 6, 10 and 13) or combination DOX & FPS-ZMl. Compared to control, DOX or FPS-ZMl monotherapy impaired tumor growth (Figure 14). Remarkably, combination therapy with DOX & FPS- ZMl markedly impaired tumor growth to such a degree, that noticeable tumors were only detected on day 14. An additional experiment was performed using the BALBc/4T-l syngeneic model, wherein mice were treated with either DMSO (control), DOX (5mg/kg on days 3, 7 and 14), FPS-ZMl (lmg/kg twice per week) or combination DOX & FPS-ZMl. Analysis of lung metastasis demonstrated FPS-ZM1/DOX combination to have the greatest effect on metastasis compared to other groups.

Discussion

[0089] The data provided herein demonstrate that RAGE drives tumor cell invasiveness and metastasis in human and mouse breast cancer models through tumor intrinsic and non- tumor cell effects, and that the RAGE antagonist, FPS-ZM1, can impair these processes. RAGE protein expression levels were higher in highly metastatic breast cancer cells lines of both human (231) and mouse (4T-1) origin, and RAGE knockdown impaired both Matrigel invasion and soft agar colony formation. Further, RAGE knockdown in both 231 and highly metastatic 4175 breast cancer cells decreased metastasis from the primary orthotopic tumor site to both lung and liver. Finally, the data show for the first time that a novel RAGE antagonist can powerfully suppress breast cancer cell invasiveness in vitro and metastasis in vivo. Taken together, these data provide evidence that RAGE contributes to breast cancer progression and metastasis, through effects on the tumor and its microenvironment, and that targeting of RAGE presents a novel therapeutic approach in breast cancer.

[0090] No studies to date have evaluated the role of RAGE on local progression and metastasis from the orthotopic site and the effects of RAGE antagonists. The impact of RAGE on tumor progression and metastasis using the metastatic MDA-MB-231 cells and their highly metastatic derived subline 4175 is described above. The highly metastatic 4175 cells had higher RAGE expression compared to parental 231 cells. Consistent with the increased RAGE expression linked to malignancy, gene knockdown of RAGE by shRNA reduced Matrigel invasion and soft agar colony formation of both 231 and 4175 cells. These data were validated in murine 4T-1 metastatic breast cancer cells and their derived non- metastatic 67NR variants. RAGE knockdown in 4T-1 cells impaired invasion and soft agar colony formation, whereas overexpression of RAGE in 67NR increased colony formation in soft agar. The in vivo studies described herein revealed that RAGE knockdown in the 4175 cells decreased tumor growth at the orthotopic site, but did not affect tumor growth of 231 cells. Further, RAGE knockdown prevented the emergence of distant metastasis of 231 and 4175 cells in both lung and liver, in both a time and tumor size matched manner.

[0091] The data provided herein further show for the first time that the use of small molecule RAGE antagonists is an effective treatment in multiple breast cancer models. As described above, RAGE plays a role in both the tumor cell (shRNA knockdown) and non- tumor cells of the tumor microenvironment (RAGE host knockout). FPS-ZMl is a highly attractive therapeutic approach to target multiple mechanisms that promote progression and metastasis. The data show FPS-ZMl inhibits cell invasiveness and soft agar colony formation, but does not appear to inhibit tumor cell viability or proliferation. These effects were demonstrated not only in the 4175, 231 parental, and 4T-1 cells, but also primary DT28 breast cancer cells dissociated from a patient with triple negative breast cancer. Further, the data show for the first time in vivo that RAGE inhibition (with FPS-ZMl) affects tumor progression and inhibits metastasis of tumor cells to lung and liver. FPS-ZMl was originally developed and tested in murine models of Alzheimer's disease, and was shown to bind and inhibit RAGE with high affinity. Deane et al. (2012). J Clin Invest, 122, 1377-1392. These studies also established that FPS-ZMl had few toxic side effects, even at high doses in toxicity studies (500 mg/kg) in C56BL6j mice.

[0092] The data set forth herein provide strong evidence that RAGE is a key mediator of breast cancer metastasis in the models evaluated and strongly implicate it as a mediator of metastasis in vivo. The Example demonstrates a clear tumor cell intrinsic role of RAGE in affecting breast cancer cell invasion and metastasis. Furthermore, these data from RAGE knockout mice (and with FPS-ZMl) demonstrate a role of RAGE in breast cancer through cells of the breast tumor microenvironment. Further, these data show RAGE knockdown in 4175 cells and treatment with the RAGE inhibitor reduces angiogenesis and recruitment of inflammatory cells to the tumor. The data implicate RAGE as a mediator of breast malignancy through effects on both the tumor cell itself and the associated tumor microenvironment.

Example 2

[0093] This example describes the effect of RAGE inhibition on obesity and NASH. Materials and Methods

[0094] Mouse studies: Db/db mice were obtained from jaxlabs and diabetic status confirmed by blood glucose monitoring. High-fat diet mice: C57BL6 wild-type mice were maintained on a high-fat diet for 32 weeks.

[0095] Ex vivo analysis: Histology: Liver from mice was used for histology. H&E was performed to visualize gross changes in liver. Trichrome staining was performed to visualize changes in collagen / liver fibrosis. Oil-red O staining was performed to assess fat content / accumulation in liver. F4/80 staining by IHC was performed to assess macrophage accumulation / inflammation in liver. Serum: mouse serum was analyzed for the liver enzyme ALT.

Results

[0096] The impact of RAGE inhibitors on diabetes and obesity was examined in db/db mice. Db/db mice develop diabetes in a similar manner to type 2 diabetes in humans.

Obesity develops at 3-4 weeks and hyperglycemia and frank insulin-resistant diabetes is present by 8 weeks in C57BL6 mice. Mice were treated with lmg/kg (twice per week) of FPS-ZMl (compared to db/m control mice), compared to DMSO vehicle control. Significant weight loss was observed in obese dbdb mice, but no change in dbm mice (Figure 13).

[0097] A major consequence of the obesity epidemic is Nonalcoholic Steatohepatitis (NASH). Currently there is no effective standard treatment for NASH once manifested. Mice treated with RAGE inhibitor (FPS-ZMl, lmg/kg, twice per week) were examined to determine if NASH was altered. In db/db control mice, the liver displayed extensive fat accumulation indicative of NASH as detected by H&E staining of liver samples. In contrast, FPS-ZMl treated db/db mice displayed far less fat accumulation in liver. As NASH is characterized by chronic liver inflammation, the effects of RAGE inhibitor FPS-ZMl on inflammatory mediators of NASH was determined. Using RNA isolated from db/db mice described above, QPCR was performed for a range of major inflammatory mediators of NASH. RAGE inhibition led to lower levels of inflammatory macrophages (F4/80), but did not change total T-cell levels (CD3E). RAGE inhibition also led to a reduction in the proinflammatory mediators of NASH, IL-6 and TNF-alpha (Figure 15). The effects of FPS- ZMl on accumulation of RAGE ligands and RAGE expression levels in db/db mouse liver also was examined. As shown in Figure 16, FPS-ZMl decreased liver expression of RAGE and inhibited expression/accumulation of the RAGE ligands S 100A8 and S 100A9. In addition, mice treated with FPS-ZMl demonstrate less fat accumulation in the liver, and less collagen (fibrosis). Analysis of ALT similarly shows a decrease in the serum of db/db mice compared to controls (Figure 25).

[0098] RAGE inhibitor FPS-ZMl impairs NASH in mice on a high-fat diet. Mice were fed a HFD for 32 weeks to induce fat accumulation in the liver and NASH. Mice were treated throughout the study with either FPS-ZMl, TTP488 (lmg/kg / twice per week), or DMSO control. Liver histology demonstrated less fat accumulation in both TTP488 and FPS-ZMl treated mice compared to controls. Trichrome staining revealed less fibrosis in RAGE inhibitor treated mice. F4/80 staining also demonstrated less inflammation in RAGE inhibitor treated mice. Data also showed that TTP488 displayed a greater inhibitory effect than FPS- Zml (data not shown).

[0099] In summary, the results described herein demonstrate that multiple RAGE inhibitors (FPS-ZM1 and TTP-488) inhibit the development of a number of chronic disease states, including obesity and NASH.

[00100] The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document. In addition, the invention includes, as an additional aspect, all

embodiments of the invention narrower in scope in any way than the variations specifically mentioned above. With respect to aspects of the invention described or claimed with "a" or "an," it should be understood that these terms mean "one or more" unless context

unambiguously requires a more restricted meaning. With respect to elements described as one or more within a set, it should be understood that all combinations within the set are contemplated. If aspects of the invention are described as "comprising" a feature, embodiments also are contemplated "consisting of" or "consisting essentially of" the feature.

[00101] Although the applicant(s) invented the full scope of the claims appended hereto, the claims appended hereto are not intended to encompass within their scope the prior art work of others. Therefore, in the event that statutory prior art within the scope of a claim is brought to the attention of the applicants by a Patent Office or other entity or individual, the applicant(s) reserve the right to exercise amendment rights under applicable patent laws to redefine the subject matter of such a claim to specifically exclude such statutory prior art or obvious variations of statutory prior art from the scope of such a claim. Variations of the invention defined by such amended claims also are intended as aspects of the invention. Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, and all such features are intended as aspects of the invention.

[00102] All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED:

1. A method of treating breast cancer, the method comprising administering to a mammalian subject in need thereof an inhibitor of Receptor for Advanced Glycation End- product (RAGE).

2. A method of inhibiting breast cancer metastasis, the method comprising administering to a mammalian subject in need thereof an inhibitor of Receptor for Advanced Glycation End-product (RAGE).

3. A method of inhibiting the onset of breast cancer, the method comprising administering to a mammalian subject in need thereof an inhibitor of Receptor for Advanced Glycation End-product (RAGE).

4. A method of treating obesity, the method comprising administering to a mammalian subject in need thereof an inhibitor of Receptor for Advanced Glycation End- product (RAGE).

5. A method of treating nonalcoholic steatohepatitis (NASH), the method comprising administering to a mammalian subject in need thereof an inhibitor of Receptor for Advanced Glycation End-product (RAGE).

6. The method of any one of claims 1-5, wherein the subject is a human.

7. The method of any one of claims 1-6, wherein the inhibitor of RAGE is FPS- ZM1 or TTP-448.

8. The method of any one of claims 1-7, further comprising administering the subject one or more chemotherapeutic agents.