STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos.

5R37GM062534-17, awarded by the National Institutes of Health. The government has certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the priority of US Provisional Patent

Application No. 62/568,672, which application is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Rapidly dividing tumors require a plethora of nutrients and other factors supplied by the blood stream to thrive and spread. Therefore, many tumors release factors that stimulate stimulate angiogenesis ¹ , a process of new host blood vessel growth.. Since the 1970s ¹ ' ² , it was proposed that inhibition of angiogenesis could starve the tumor of essential nutrients and oxygen leading to tumor cell death. Thus, many pharmaceutical companies developed drugs targeting pro-angiogenic factors, such as the vascular endothelial growth factor (VEGF) family of growth factors. See, e.g., Liang ⁴⁰ and references cited therein. However, clinical trials of anti-angiogenic agents, such as Sunitinib (a small molecule inhibitor of the VEGF receptor) or Bevacizumab (an anti- VEGF antibody), have been largely disappointing, with most patients showing transient responses followed by inevitable resistance. ³ This is especially pertinent in metastatic breast cancer where, after initial approval, the FDA has since rescinded its approval of Avastin®/bevacizumab for use in breast cancer.

Since the emergence of the original theory behind targeting critical mediators of angiogenesis to treat cancer, it has been determined that heterogeneous tumors can employ alternative mechanisms of vascularization ⁴ . One such mechanism relies on vasculature formed by tumor cells themselves that differentiate into endothelial-like cells to form extra-cellular matrix (ECM)-rich tubular structures that are essentially pseudo blood vessels, a process termed vascular mimicry or vasculogenic mimicry (VM). VM was first described in the early 1990s ⁵ and has since been seen in a broad spectrum of tumor types, where its presence is almost universally a poor prognostic indicator ⁶ .

Subpopulations of tumor cells that can form VM channels endow tumors with an alternative vascular system for nutrient supply without requiring host vessel growth through angiogenesis (See, e.g., Leslie ⁴³ ) and, as such, have been postulated to underlie poor responses to anti-angiogenic agents. However, an understanding of how tumor cells acquire VM capabilities and whether VM underlies failure of anti-angiogenic therapy, as well as how to use this information enable the development of effective therapeutic interventions for cancer therapy are lacking and no anti-VM therapies exist due to a poor understanding of the details of how VM occurs.

SUMMARY OF THE INVENTION

Through genetic barcoding of individual breast cancer cells, we discovered a critical role for VM in promoting metastasis by facilitating tumor cell entry into the blood stream ⁷ . This analysis uncovered two novel regulators of VM (SerpinE2 and SLPI), and provided a comparative system through which to understand the underlying biology of VM. Utilizing this comparative system, we have now identified additional critical pathways controlling the establishment and maintenance of VM namely, the Forkhead box protein C2 (FOXC2; also known as MFH1) and inositol-requiring enzyme 1 (IREl) pathways, respectively.

In one aspect, a method for increasing the sensitivity of a tumor to anti-angiogenic therapy comprises treating a patient having a tumor with an anti-angiogenic therapeutic composition or compound and substantially simultaneously inhibiting vascular, or vasculogenic, mimicry (VM). In one embodiment, such a method includes the inhibition of VM by administering a therapeutic compound that inhibits the activity or pathway of the transcription factor FOXC2 or inhibits a FOXC2 pathway target. In another embodiment, such a method includes administering a therapeutic compound that activates or enhances the activity or pathway of IREl or inhibits/diminishes the activity of its target genes.

In another aspect, a method for increasing the sensitivity of a tumor to anti- angiogenic therapy comprises treating a patient having a tumor with an anti-angiogenic therapeutic composition or compound, a therapeutic compound that inhibits the activity or pathway of the transcription factor FOXC2, and a therapeutic compound that activates or enhances the activity or pathway of IRE 1 or inhibits/diminishes the activity of its target genes.

In still a further aspect, a therapeutic composition for inhibiting tumor vascularization and vasculogenic mimicry comprises in a suitable pharmaceutical carrier, an anti-angiogenic therapeutic compound and at least one or a combination of (a) a therapeutic compound that inhibits the activity or pathway of the transcription factor FOXC2; and (b) a therapeutic compound that activates or enhances the activity or pathway of IRE1 or inhibits/diminishes the activity of its target genes.

In a further aspect, a therapeutic regimen comprises (a) administering to a subject with a tumor an anti-angiogenic therapeutic composition or compound; and

(b) administering to said subject substantially simultaneously or sequentially, at least one of i) a therapeutic compound that inhibits the activity or pathway of the transcription factor FOXC2, and ii) a therapeutic compound that activates or enhances the activity or pathway of IRE1 or inhibits/diminishes the activity of its target genes.

In still another aspect, a composition or reagent for diagnosing the existence or evaluating the progression of cancer in a mammalian subject are provided, which compris multiple polynucleotides or oligonucleotides. Each polynucleotide or oligonucleotide hybridizes to a different gene, gene fragment, gene transcript or expression product in a sample selected from gene targets that experience changes in expression during vascular mimicry.

Yet another aspect involves a method for diagnosing the existence or evaluating the progression of a cancer in a mammalian subject comprising identifying changes in the expression of multiple genes in the sample of said subject, said genes selected from genes that change expression in response to increasing or decreasing vascular mimicry. Such methods may be used to assess the efficiacy of the treatment methods also described herein.

Other aspects and advantages of these compositions and methods are described further in the following detailed description of the preferred embodiments thereof. BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A-1G demonstrate that vascular mimicry (VM) is a driver of metastasis in the mouse-derived 4T1 model and is particularly prevelant in aggressive Basal/Claudin- low subtypes of breast cancer. FIG. 1A is a schematic of a mouse model of breast cancer heterogeneity, including single cell clone barcoding and isolation from 4T1 parental cells.

FIG. IB is a heatmap showing relative distribution of individual barcoded clones following mammary fat-pad transplantation of the 23 pooled single cell clones. FIG. 1C is a bar chart showing the number of VM channels formed by each of the individual 4T1 clones described in IB. FIG. ID are images showing the results of an in vitro Matrigel assay for VM. Only VM clones 4T1-E and 4T1-T can form tube structures when plated on Matrigel. All data in FIGs 1 A-1D are adapted from Wagonblast et al ⁷ .

FIG. IE is a bar chart showing enrichment of genes up-regulated in VM clones

4T1-T and 4T1-E in different normal tissues from the Genotype-Tissue Expression (GTex) consortium dataset (see the website www.nature.com/collections/dcfzxywzby).

VM clones specifically up-regulate blood vessel-specific genes. Plotted is the negative log ¹⁰ of the FDR corrected p value.

FIG. IF are two box and whisker plots of the expression of vascular-specific (left) or endothelial-specfic ⁴⁵ (right) genes in breast cancer cell lines corresponding to different sub-types of breast cancer. Vascular-/endothelial-specific genes are enriched in aggressive Basal/Claudin-low cell lines. *** p <0.001, **** p O.0001 Mann-Whitney test.

FIG. 1G are two box and whisker plots of the expression of vascular-specific (left) or endothelial-specific ⁴⁵ (right) genes in a larger number of breast cancer patient samples corresponding to different sub-types of breast cancer. Vascular/endothelial-specific genes are enriched in aggressive Basal/Claudin-low tumors. **** p <0.0001 Mann-Whitney test.

FIGs. 2A-2C demonstrate that VM clones up-regulate secreted/extra-cellular factors that are necessary for maintenance of the VM state.

FIG. 2A is a bar chart showing enrichment of extracellular-related genes in genes whose expression is up-regulated in mouse of human cells that can perform vascular mimicry. Dark bars are mouse 4T1 -derived VM clones; light bars are human VM capable cell lines from the Cancer Cell Line Encyclopedia (CCLE). Plotted is the negative log ¹⁰ of the FDR corrected p value.

FIG. 2B are two images showing the results of an in vitro Matrigel assay for VM where cells were deprived of secreted/extracellular factors by exchanging their medium prior to performing the VM assay (lower image) vs. a control (upper image). FIG. 2C. is a bar chart showing quantification of 3 replicates of the experiment shown in FIG 2B. Bars are mean +/- SEM * p <0.05 student's t-test.

FIGs. 3A-3I demonstrate that the endoplasmic reticulum stress sensor IREl restrains VM and metastasis.

FIG. 3A is a graph showing the RNA expression of ER stress/UPR regulators in basal-claudin-low breast cancer patients with or without relapse, n.s. = not significant, * p <0.05, ** <0.01, Mann-Whitney test.

FIG. 3B is a bar graph showing that IREl inhibition augments, and activation represses, VM tubulogenesis. Bars are mean +/- SEM * p <0.05, ** <0.01, student's t- test.

FIG. 3C is a bar graph showing how suppression of IREl with RNAi enhances tube formation in human MDA-MB-231 breast cancer cells. Bars are mean +/- SEM * p O.05, *** O.001, student's t-test.

FIG. 3D is a bar graph showing suppression of IREl, but not XBP1, with RNAi enhances tube formation in 4Tl-T ^VM breast cancer cells. Bars are mean +/- SEM.

FIG. 3E is a bar graph showing that inhibition of IREl promotes VM via secreted factors. Conditioned medium (CM) from IREl inhibited cells enhances VM in naive 4T1- T ^w cells. Bars are mean +/- SEM.

FIG. 3F is a bar graph showing primary tumor volume of tumors derived from 4T1-T ^M cells transplanted orthotopically into the mammary fat-pad (14 days posttransplantation, expressed as mean fold change vs shREN (control hairpin) +/- SEM, n = 5 mice. n.s. = not significant, ** p < 0.01, student's t-test.

FIG. 3G is a bar graph showing quantification of the number of lung metastases from orthotopic 4T1-T ^W tumors by mCHERRY IHC (14 days post-transplantation), expressed as mean fold change vs shREN +/- SEM, n = 10 mice from 2 independent experiments. *p < 0.05, **p < 0.01, student's t-test. This graph shows micrometastases that can be seen only with a microscope.

FIG. 3H shows representative H and E stained lung sections (left) from orthotopic 4T1-T ^VM tumors (21 days post-transplantation). Histogram expressed as mean fold change in lung metastases number vs shREN +/- SEM. n = 7 (shREN, shIREl#l), n = 8 (shIREl#2). *p < 0.05, ***p < 0.001, student's t-test. The right side of FIG. 3H is a bar graph similar to that of 3G but showing macro-mets (macro-metastases, palpable and/or visible to the eye). FIG. 31 shows Kaplan-Meier relapse-free survival curve of patients described in FIG 3A stratified by mean IREla/β (ERN1, ERN2) expression, IRE1 low = bottom 25%, IREl high = top 75%. Log-rank test. HR = Hazard ratio (95% confidence intervals).

FIGs. 4A-4F demonstrate that regulated IREl -dependent mRNA decay (RIDD) restrains critical mediators of vascular mimicry.

FIG. 4A is a Gene Ontology (GO) analysis of genes significantly (DESeq, FDR < 0.05) up-regulated by IREl inhibition and down-regulated by tunicamycin treatment (foreground), all detected genes (background). Shown is the -logio of the Benjamini- Hochberg-corrected p value.

FIG. 4B shows a heatmap of gene set enrichment analysis (GSEA) ¹⁷ -derived normalized enrichment scores for various literature curated vascular/endothelial-specific gene sets. * FDR < 0.05, ** FDR < 0.01, *** FDR < 0.001, **** FDR < 0.0001.

FIG. 4C shows four bar graphs of VM tube assay branching length quantification of 4T1-T ^VM with indicated knockdowns (by RNAi) against IREl-target genes (i.e., MGP, ANKH, LDHB and PLSCR4). Expressed as mean log2 fold change branching length vs (4T1 -T ^w Vehicle treated cells) +/- SEM, n = 3. ** p < 0.01, *** p O.001, student's t- test.

FIG. 4D is a bar graph showing mRNA stabilization of putative Regulated IRE1- Dependent mRNA Decay (or RIDD) ^11"13 -target genes upon IREl inhibition. mRNA stability change as measured by Actinomycin D run-off assays expressed as log2 fold change vs vehicle treatment +/- SEM, n = 3. * p < 0.05, ** p < 0.01, student's t-test.

FIG. 4E shows mean mRNA expression of LDHB, ANKH, MGP and PLSCR4 in human breast cancer samples representing different subtypes of breast cancer, n represents one patient **** p < 0.0001, Mann-Whitney test.

FIG. 4F shows mean mRNA VM score (calculated as the mean expression of

LDHB, ANKH, MGP, PLSCR4 and mean inverse expression of ERNl/2) in breast cancer patients who did or did not relapse at 5 years. ** p O.001, Mann-Whitney test.

FIGs. 5A-5H show that tumors cells co-opt an endothelial transcription factor, FOXC2, to drive vascular mimicry.

FIG. 5A shows mRNA expression levels of all transcription factors (TFs) in mouse 4T1 VM clones vs all clones. Each dot forming the curve represents a distinct TF. FIG. 5B shows the mRNA expression of FOXC2 and its target gene MEF2C in different 4T1 -derived clones as measured by RNA-Seq. Bars represent mean +/- SEM, n = 2 per cell line.

FIG. 5C shows the mRNA expression of FOXC2 and its target gene MEF2C in matched primary tumor- and lung metastasis-derived 4T1 cell lines as measured by RNA- Seq. Bars represent mean +/- SEM, n = 2 per cell line.

FIG. 5D shows that over expression of FOXC2 in nonVM 4T1-L cells confers VM tube formation ability. Quantification of matri-gel VM tube formation assays of 4T1- L ^nonVM cells over expressing FOXC2 or a negative control (vector). Bars represent mean +/- SEM, n = 3.

FIG. 5E shows that suppression of FOXC2, via RNAi, impedes VM in 4T1-T ^VM cells. Quantification of matri-gel VM tube formation assays of 4T1-T ^W cells with knockdown of FOXC2 with two different shRNAs or a negative control (shREN). Bars represent mean +/- SEM, n = 3. *** p O.001, **** p O.0001, student's t-test.

FIG. 5F shows GSEA plots of FOXC2-target genes (defined from dataset

GSE44335 as top 100 upregulated genes upon FOXC2 over expression) in rank lists of VM clones vs all other clones (Left) or lung metastases vs primary tumor (Right).

FIG. 5G shows enrichment of FOXC2 mRNA in aggressive Basal/Claudin-low Breast tumors. **** p <0.0001, Mann-Whitney test.

FIG. 5H is a Kaplan-Meier relapse-free survival curve of patients stratified by

FOXC2 expression. FOXC2 high = top one third, FOXC2 low = bottom two thirds. Log- rank test. HR = Hazard ratio (95% confidence intervals).

FIGs. 6A-6E shows that FOXC2 drives ectopic expression of vascular/endothelial genes in epithelial cells.

FIG. 6A shows FOXC2 mRNA levels in MDA-MB-231 cells expressing two different FOXC2 targetting shRNAs or a negative control (shREN) used for RNA-Seq analysis.

FIG. 6B shows the overlap of significant Gene Ontology (GO) terms from comparisons of mouse VM clones vs. all other clones and genes that go down with FOXC2 knockdown.

FIG. 6C shows individual GO terms that are significantly enriched in genes that go down with both FOXC2 shRNAs. The top bar graph shows Process terms; the middle graph shows Function terms; and the lower graph shows Component terms. Bars represent the -logio of the Benjamini-Hochberg-corrected p value.

FIG. 6D shows the enrichment, or lack thereof, of targets of FOXC2 or epithelial - to-mesenchymal-promoting transcription factors (SLUG, SNAIL, TWIST) in VM clones. Bars represent the GSEA-derived signed (negative = depleted, positive = enriched) FDR- corrected p value.

FIG. 6E shows that FOXC2, but not EMT transcription factors, modulates ectopic expression of endothelial-specific genes in epithelial cells. Bars represent the GSEA- derived signed (negative = enriched in down-regulated genes, positive = enriched in up- regulated genes) FDR-corrected p value.

FIGs. 7A-7F shows that FOXC2 drives hypoxic gene expression programs and promotes survival under oxygen poor conditions.

FIG. 7A shows GSEA of a hypoxia signature in gene expression changes modulated by FOXC2 knockdown. Hypoxia genes are down regulated by FOXC2 suppression.

FIG. 7B shows a cumulative distribution plot of genes that are targets of the master hypoxia transcription factors HIFla/HIF2a vs all genes. X-axis is the mean Log2 fold change in gene expression with both FOXC2 shRNAs. **** p <0.0001,

Kolmogorov-Smirnov test.

FIG. 7C shows HIFla mRNA levels in MDA-MB-231 cells expressing FOXC2 shRNAs. Bars represent mean +/- SEM vs shREN.

FIG. 7D shows that FOXC2 mRNA levels are induced by exposure to hypoxia in parental 4T1 cells. Bars represent mean +/- SEM vs normoxia.

FIG. 7E shows relative cell survival of MDA-MB-231 cells expressing FOXC2 shRNAs. FOXC2 depleted cells are more sensitive to hypoxia-induced cell death. Bars represent mean +/- SEM vs shREN/normoxia.

FIG. 7F shows relative cell survival of 4T1-T ^W cells expressing FOXC2 shRNAs. FOXC2 depleted cells are more sensitive to hypoxia-induced cell death. Bars represent mean +/- SEM vs shREN/normoxia.

FIGs. 8A-8E illustrate that VM gene expression is associated with failure of anti- angiogenic therapy in multiple model systems.

FIG. 8A shows the outline of the clinical trial design described in Mehta et al (2016) ²¹ , incorporated by reference herein. FIG. 8B shows GSEA using our IRE 1 -regulated gene expression changes and a curated signature of Bevacizumab resistance in patients described in FIG. 8A.

FIG. 8C shows GSEA using our FOXC2-regulated gene expression changes and a curated signature of Bevacizumab resistance in patients described in FIG. 8A.

FIG. 8D shows GSEA using our FOXC2-regulated gene expression changes and a curated signature of Bevacizumab resistance in a Glioblastoma (GBM) xenograft.

FIG. 8E shows GSEA using our FOXC2-regulated gene expression changes and a curated signature of Sunitinib resistance in a Renal Cell Carcinoma (RCC) patient- derived xenograft.

FIGs. 9A-9E shows additional data suggesting that VM promotes resistance to anti-angiogenic therapy (AAT) and that suppression of VM augments response to AAT.

FIG. 9A shows that 4T1-T VM tube formation is indifferent to inhibition of the VEGF pathway with Sunitinib. Quantification of matri-gel VM tube formation assays of HUVEC cells (positive control for dependence on the VEGF pathway) or 4T1-T ^VM cells treated with the VEGFR inhibitor Sunitinib. Bars represent mean +/- SEM vs vehicle.

FIG. 9B shows quantification of tumors from mice orthotopically transplanted with luciferase labelled 4T1-T ^W cells treated with vehicle or the anti-VEGF blocking antibody B20-4.1.1 (Genentech). Bars represent mean tumor volume +/- SEM vs vehicle, n.s = not significant, * p <0.05, student's t-test.

FIG. 9C is a bar graph showing the results of treating 4Tl-TVM-derived tumors with Vehicle (Veh:Veh), Tunicamycin alone (Veh:Tunic), Sunitinib alone (SunitVeh, anti-angiogenic kinase inhibitor), or a combination of Tunicamycin and Sunitinib (Sunit: Tunic) using regimens as indicated. Tumor volumes were measured by bioluminescence prior to and after cessation of therapy. Shown are bioluminescent tumor volumes normalized to pre-treatment and vehicle treated tumors at day 6 after initiation of therapy. 4T1-T ^VM tumors are are sensitized to Sunitinib by inhibiting VM in combination by using Tunicamycin as an IREl activator.

FIG. 9D shows that inhibition of VM by IREl activation with tunicamycin augments response to AAT. Mice were orthotopically transplanted with luciferase labelled 4T1-T ^W cells treated with vehicle, the anti-VEGF blocking antibody B20-4.1.1 (Genentech) alone, tunicamycin alone, or the combination of B20 and tunicamycin. All animals were quantified. Bars represent mean tumor volume +/- SEM vs vehicle, n.s = not significant, * p O.05, ** p <0.01, student's t-test. FIG. 9E shows the results of an assay similar to that of FIG. 9D but showing the effect on VM of the suppression of FOXC2, via RNAi when administered with vehicle or treated with the B20-4.1.1 antibody. shREN represents the negative control. Bars represent mean tumor volume +/- SEM vs vehicle, n.s = not significant, * p <0.05, ** p <0.01, student's t-test.

DETAILED DESCRIPTION

Therapeutic compositions and methods are described for coordinating the inhibition of tumor vascularization and the inhibition or repression of vasculogenic mimicry, including for the treatment of cancers. The data provided in the examples below supports small molecule targeting of VM in combination with anti-angiogenic therapy. In another embodiment, a method is provided that uses a VM-based gene signature as a bio- marker for monitoring response to anti-angiogenic therapy, and/or to identify sub-sets of patients for whom combination anti-VM/anti-angiogenic therapy is beneficial.

/. Definitions and Components of the Compositions and Methods

In the descriptions of the compositions and methods discussed herein, the various components are defined by use of technical and scientific terms having the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published texts. Such texts provide one skilled in the art with a general guide to many of the terms used in the present application. The definitions contained in this specification are provided for clarity in describing the components and compositions herein and are not intended to limit the claimed invention.

The terms "subject", "patient", or "mammalian subject", as used herein include primarily humans, but can also be extended to include a veterinary or farm animal, a domestic animal or pet, and animals normally used for clinical research. In one embodiment, the subject of these methods and compositions is a human. Still other suitable mammalian subjects include, without limitation, murine, rat, canine, feline, porcine, bovine, ovine, and others.

The term "neoplastic disease", "cancer" or "proliferative disease" as used herein refers to any disease, condition, trait, genotype, or phenotype characterized by unregulated or abnormal cell growth, proliferation, or replication. The abnormal proliferation of cells may result in a localized lump or tumor, be present in the lymphatic system, or may be systemic. In one embodiment, the neoplastic disease is benign. In another embodiment, the neoplastic disease is pre-malignant, i.e. , potentially malignant neoplastic disease. In a further embodiment, the neoplastic disease is malignant, i.e. , cancer. In still a further embodiment the neoplastic disease may be caused by viral infection. In one embodiment, the neoplastic disease is a cancer, such as an epithelial cancer.

In various embodiments of the methods and compositions described herein, the cancer can include, without limitation, breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma (liver cancer), anal cancer, penile cancer, vulvar cancer, vaginal cancer, melanoma, leukemia, myeloma, lymphoma, glioma, and multidrug resistant cancer. In another embodiment, the neoplastic disease is Kaposi's sarcoma, Merkel cell carcinoma, multicentric Castleman's disease, primary effusion lymphoma, tropical spastic paraparesis, adult T-cell leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, posttransplantation lymphoproliferative disease, nasopharyngeal carcinoma, pleural mesothelioma, osteosarcoma, ependymoma and choroid plexus tumors of the brain, and non-Hodgkin's lymphoma. In still other embodiments, the cancer may be a systemic cancer, such as leukemia. In one aspect, as exemplified, the cancer is a human glioblastoma. In another aspect, the cancer is a prostate adenocarcinoma. In still another embodiment, the cancer is a lung adenocarcinoma. In one embodiment, the cancer is non-small cell lung adenocarcinoma (NSCLC). In another embodiment, the cancer is squamous cell carcinoma. In another embodiment, the cancer is liver cancer. In another embodiment, the cancer is a breast cancer, such as, without limitation, breast

adenocarcinoma. In yet a further embodiment, a cancer as referred to herein is a condition in which the subject's cancer or tumor is, or becomes over a period of time, refractory to treatment with anti-angiogenic therapy.

By the term "anti-angiogenic compound", "anti-angiogenic therapy" or "anti- angiogenic therapeutic composition" as described herein is meant treatment with or use of any therapeutic agent that blocks or inhibits angiogenesis, inhibits blood vessel growth, or disrupts or removes angiogenic vessels either in vitro or in vivo. These compounds or compositions can cause tumor regression in various types of neoplasia (including benign neoplasia) or cancer. Known therapeutic candidates include naturally occurring angiogenic inhibitors, including without limitation, angiostatin, endostatin, and platelet factor-4. In another embodiment therapeutic candidates include, without limitation, specific inhibitors of endothelial cell growth, such as TNP-470, thalidomide, and interleukin-12. Still other anti-angiogenic agents include those that neutralize angiogenic molecules, such as including without limitation, antibodies to fibroblast growth factor or antibodies to vascular endothelial growth factor or antibodies to platelet derived growth factor or antibodies or other types of inhibitors of the receptors of EGF, VEGF or PDGF. In other embodiments, antiangiogenic agents include without limitation suramin and its analogs, and tecogalan. In other embodiments, anti-angiogenic agents include without limitation agents that neutralize receptors for angiogenic factors or agents that interfere with vascular basement membrane and extracellular matrix, including, without limitation, metalloprotease inhibitors and angiostatic steroids. Another group of anti-angiogenic compounds includes, without limitation, anti-adhesion molecules, such as antibodies to integrin alpha v beta 3. Still other anti-angiogenic compounds or compositions, include, without limitation, kinase inhibitors, thalidomide, itraconazole, carboxyamidotriazole, CM101, IFN-a, IL-12, SU5416, thrombospondin, cartilage-derived angiogenesis inhibitory factor, 2-methoxyestradiol, tetrathiomolybdate, thrombospondin, prolactin, and linomide. In one particular embodiment, the anti-angiogenic compound is an antibody to VEGF, such as Avastin®/bevacizumab (Genentech).

FOXC2 or Forkhead box protein C2 (also known as MFH1) is a transcriptional activator that belongs to a large family of nuclear transcription factor proteins sharing a common forkhead/winged helix DNA binding domain. The human mRNA sequence for FOXC2 is found in the NCBI database as NM_005251.2; the protein sequence is published as NCBI database accession number NP-005242.1. This gene has been implicated in a wide number of cellular processes, e.g., as a regulator of epithelial to mesenchymal transition (EMT) and stem cell properties, including tumor-initiation capacity, metastatic competence, and chemotherapy resistance, tumor recurrence, triple- negative breast cancer (TNBC) progression and human lymphedemia-distichiasis syndrome, and tumor metastasis, and adipocyte morphogenesis. In addition, the transcriptional activity of FoxC2 influences expression of cytokine receptors such as

CXCR4.See, e.g., Pietila M, et al. FOXC2 regulates the G2/M transition of stem cell-rich breast cancer cells and sensitizes them to PLK1 inhibition. Scientific Reports. Apr 2016;6:23070. doi: 10.1038/srep23070, and references cited therein. By the term "FOXC2 pathway targets" is meant to include, without limitation, any gene or encoded protein, which in cooperation with FOXC2, operates to cause, enhance, or increase VM when FOXC2 is activated or its expression increased. Such targets include, without limitation, one or more of the 25 interacting proteins identified in the STRING Interaction Network, version 10.5, namely, NDAC2, PPP2R2A, FMN1, PPP2R1A, RPS6KB1, PIN1, RPS6KA5, SGK3, RPS6KA6, AKT3, AKT1, STK32B, STK32C, RPS6KB2, PPP2R1A, PPP2R2A, RPS6KA4, FMN1, C5orf24, SGK2, SGK1, ZBTB34, RPS6KA3, AKT2, STK32A, RPS6KA2, HDAC2 and RPS6KA1. Such targets also include one or more of the transcriptional targets of the FOXC2 pathway that play key roles in vasculature development and metastasis, namely, MEF2C, SERPINE2, SLPI, GREM1, TMEM100, SERPINE1, CYP1B1, ANGPTL4, FGF2, PRKCA, PRKD1, ITGA5, GATA6, DDAH1, ADM, HMOX1, HIPK2, CCBE1, IL8, WNT5A, PTK2B, ECMl, HIFIA, SRPX2, TBXA2R, HSPBl, SPHKl, HGF, RAPGEF2, C3AR1, HDAC9, C5AR1, PDGFB, MTDH, RRAS, RHOB, SIRT1, CIB1, CCL5, ERAP1, C19ORF10, BTG1, PIK3R6, PLCG1, EGR1, ITGB2, GATA4, PHACTR1, RCAN2, SOBP, VCAN, FRY, FAM129A, GLIPR1, OSR1, NOV, EPS8, VIM, SDC2, COL6A2, WWTR1, TSC22D1, EN02, ABI3BP, FOXL1, VASN, MYLK, PPP1R3C, DOCK10, KANK2, FN1, ANGPT1, LGALS3BP, CAMK1D, SOD3, CXXC5, CSGALNACT1, PNRC1, HTRA3, SDC3, SPPl, PLSCR4, ICAMl, TSPAN15, OSMR, KDELR3, TRIOBP, GBP4, ANGPTL2, TRIB2, and SLC15A3.

As used herein, the phrase "inhibits FOXC2" means that the expression or activity of the FOXC2 gene or level of RNA molecule encoding it is down-regulated, or less than that observed, in the absence of the selected FOXC2 modulator therapeutic reagent, with the result that vascular mimicry (VM) of a subject's tumor is inhibited, disrupted, or repressed. In one embodiment, this inhibition of VM co-operates with the anti- angiogenic effect of an anti-angiogenic compound, such as an anti-VEGF antibody. In another embodiment, this inhibition of VM synergizes with the anti-angiogenic effect of an anti-angiogenic compound, such as an anti-VEGF antibody. In one embodiment, this inhibition of VM co-operates with both the anti-angiogenic effect of an anti-angiogenic compound and the VM inhibiting effect of the IREl modulator. In another embodiment, this inhibition of VM synergizes with both the anti-angiogenic effect of the anti- angiogenic compound, and the VM inhibitory effect of the IREl modulator. In another embodiment, this inhibition of VM operates to prevent re-vascularization by VM of the tumor after the anti-angiogenic compound, such as an anti-VEGF antibody, reduces the normal vascularization of the tumor.

As used herein, the phrase "inhibits the FOXC2 pathway" or "inhibits a FOXC2 pathway target" means that the effect of a FOXC2 modulator on the expression or activity of the target RNA molecules encoding one or more target protein or protein subunits or peptides of the FOXC2 pathway up-regulates or down-regulates the target, such that the expression, level, or activity is greater than or less than that observed in the absence of the FOXC2 modulator therapeutic reagent, with the result that vascular mimicry (VM) of a subject's tumor is inhibited, disrupted or repressed. It should be understood that in one embodiment, certain FOXC2 pathway targets behave similarly to FOXC2, i.e., one target may be directly inhibited or its activity suppressed to achieve VM inhibition in a manner parallel to that of FOXC2. In another embodiment, depending upon the FOXC2 pathway target and its relationship to FOXC2 (i.e., FOXC2 expression may inhibit the target), the target may be directly activated or its activity enhanced (i.e., in a manner opposite to FOXC2) by the modulator to achieve VM inhibition. In one embodiment, this inhibition of VM co-operates with the anti-angiogenic effect of an anti-angiogenic compound, such as an anti-VEGF antibody. In another embodiment, this inhibition of VM synergizes with the anti-angiogenic effect of an anti-angiogenic compound, such as an anti-VEGF antibody. In another embodiment, this inhibition of VM operates to prevent re- vascularization by VM of the tumor after the anti-angiogenic compound, such as an anti- VEGF antibody, reduces the normal vascularization of the tumor.

As used herein, a "FOXC2 or FOXC2 pathway modulator" or "therapeutic compounds that inhibit FOXC2 or the FOXC2 pathway" refer to a therapeutic reagent, compound or composition that directly inhibits FOXC2 expression or activity so as to inhibit, disrupt or repress vascular mimicry, or directly inhibits a FOXC2 pathway target's expression or activity so as to inhibit, disrupt or repress vascular mimicry. These same phrases are also used to refer to a therapeutic reagent, compound or composition that directly activates or enhances a FOXC2 pathway target's expression or activity so as to inhibit, disrupt or repress vascular mimicry. In certain examples, therefore, FOXC2 modulators are therapeutic compounds that inhibit FOXC2 or the FOXC2 pathway, including without limitation, antibodies for FOXC2 or an associated pathway target, such as those provided by R&D Systems (MAB5044), Novus Biologicals Antibodies for FOXC2 (NB100-1269), ThermoFisher Scientific (MA5-17077), etc. Other inhibitors include shRNA, siRNA or RNAi sequences directed to FOXC2 or one of the "parallel- acting" targets (see, e.g., the FOXC2 directed inhibitors available from, e.g., Origene, Rockville, MD or SantaCruz Biotechnology, Inc.; or ViGene Biosciences) or

CRISPR/Cas guide systems that are commercially available or may be readily developed. Other FOXC2 pathway modulators directly activate certain FOXC2 pathway targets that are normally inhibited by FOXC2 expression or activity.

Additionally, small chemical compounds, such as, p38 MAPK inhibitors

(including but not limited to, SB203580, AL 8697, AMG 548, BIRB 796, CMPD-1, DBM 1285 dihydrochloride, EO 1428, JX 401, ML 3403, Org 48762-0, PH 797804, RWJ 67657, SB 202190, SB 203580, SB 203580 hydrochloride, SB 239063, SB 706504, SCIO 469 hydrochloride, SKF 86002 dihydrochloride, SX 011, TA 01, TA 02, TAK 715, VX 702, VX 745 and p38 MAPK Inhibitor Tocriset™. See, e.g., www.tocris.com/ pharmacology/p38-mapk), Cdk/Cdk5 inhibitors (including but not limited to, (i?)-CR8, Aminopurvalanol A, Arcyriaflavin A, AZD 5438, BMS 265246, BS 181 dihydrochloride, CGP 60474, CGP 74514 dihydrochloride, CVT 313, (i?)-DRF053 dihydrochloride,

Flavopiridol hydrochloride, 10Z-Hymenialdisine, Indirubin-3'-oxime, Kenpaullone, NSC 625987, NSC 663284, NSC 693868, NU 2058, NU 6140, Olomoucine, [Ala ⁹² ]-pl6 (84- 103), PD 0332991 isethionate, PHA 767491 hydrochloride, Purvalanol A, Purvalanol B, Ro 3306, Roscovitine, Ryuvidine, Senexin A, SNS 032 and SU 9516. See, e.g., www.tocris.com/pharmacology/cyclin-dependent-protein-kinases ), PDGFR inhibitors (including but not limited to, Imatinib meseylate; Toceranib; Sunitinib malate; SU 6668; SU 16f; PD 166285 dihydrochloride; KG 5; GSK 1363089; DMPQ dihydrochloride; CP 673451; AP 24534; AG 18; and AC 710. See, e.g. , www.tocris.com/pharmacology/ pdgfir), PKA inhibitors (including but not limited to, H89 dichloride; Fasudil

hydrochloride; cGMP Dependent Kinase Inhibitor Peptide; KT 5720; PKA inhibitor fragment (6-22) amide; PKI (5-24); PKI 14-22 amide, myristoylated; and cAMP antagonist, e.g. , cAMPS-Rp, triethylammonium salt. See, e.g.,

www.tocris.com/pharmacology/protein-kinase-a), PKD inhibitors (including but not limited to, CID 755673, CID 2011756, CRT 0066101, and kb NB 142-70. See, e.g., www.tocris.com/pharmacology/protein-kinase-d), PI3K inhibitors (including but not limited to, PI 3-Κβ inhibitor, e.g., AZD 6482; PI 3-kinase inhibitors, e.g. , A66, AS 252424, AS 605240, BAG 956, CZC 24832, ETP 45658, GSK 1059615; LTURM 36, LY 294002 hydrochloride, 3-Methyladenine, PI 103 hydrochloride, PI 3065, PI 828, PP 121, Quercetin, STK16-IN-1, TG 100713, TGX 221, Wortmannin; KU 0060648; LY 303511 ; PF 04691502; and PF 05212384. See, e.g., www.tocris.com/pharmacology/pi-3-kinase), MET inhibitors and MET kinase inhibitors (including but not limited to, Crizotinib, GSK 1363089, K 252a, Norleual, PF 04217903 mesylate, PHA 665752, SGX 523, SU 11274, SU 5416, and XL 184. See, e.g. , www.tocris.com/pharmacology/met-receptors), CAMK inhibitors (including but not limited to, CaM kinase III inhibitors, e.g., A 484954, NH 125; CaM kinase II inhibitors, e.g. , KN93 phosphate, KN 93, Arcyriaflavin A,

Autocamtide-2-related inhibitory peptide, Autocamtide-2-related inhibitory peptide, myristoylated, KN-62; and CaM kinase inhibitor, e.g. , STO-609 acetate. See, e.g., www.tocris.com/pharmacology/cam-kinase), FGFR inhibitors (including but not limited to, PD161570, AP 24534, FUN 1 hydrochloride, PD 166285 dihydrochloride, PD 173074, SU 5402, and SU 6668. See, e.g. , www.tocris.com/pharmacology/fgfr), and/or blocking antibodies against the above targets or their ligands may be useful as modulators of this pathway.

IRE1, the transmembrane protein kinase inositol-requiring enzyme 1, is encoded by ERNl, the endoplasmic reticulum to nucleus signaling). The encoded protein contains two functional catalytic domains, a serine/threonine-protein kinase domain and an endoribonuclease domain. The human mRNA sequence for IREl/ERNl is found in the NCBI database as Gene ID 2081, NM_001433.4. The protein sequence is published as NCBI database accession number NP_001424.3. This protein functions as a sensor of unfolded proteins in the endoplasmic reticulum (ER) and triggers an intracellular signaling pathway termed the unfolded protein response (UPR). The UPR is an ER stress response that is conserved from yeast to mammals and activates genes involved in degrading misfolded proteins, regulating protein synthesis, and activating molecular chaperones. IREl suppresses mRNAs encoding secreted proteins to relieve overloading of the ER by secretory proteins in addition to mediating the splicing and activation of the stress response transcription factor X-box binding protein 1 (XBP1).

By the term "IREl pathway targets" is meant to include, without limitation, any gene or encoded protein, which in cooperation with IREl, operates to decrease or inhibit VM when IREl is activated or its expression increased and/or when the activity of its target genes is inhibited or diminished. Such targets include, without limitation, one or more of the 25 interacting proteins identified in the STRING Interaction Network, version 10.5, namely, RB1CCA, XBP1, CCND1, PYCARD, CDK7, DERL1, MNAT1, CCND2, GTF2H1, GTF2H2, ERCC3, PYDC1, ACADB, ACACA, AKAP4, ERC1, BCCIP, CCND3, CCNY, PHKA2, ERCC2, DERL3, ATG13, GTF2H3, and PHKG2, among others. Such targets may also include without limitation, targets that are repressed upon IREl activation identified by RNA-Seq that play key roles in vasculture development and metastasis, namely, MGP, RBP1, SLPI, SERPINE2, AQP1, SFRP1, ICAM1, ANK, COL6A1, PROS1, PLSCR4, HTRA3, DECR1, NEURL3, ZHX1, PFN2, DMP1, IL1R1, NODI, PADI2, RBP2, GCHFR, SAMSN1, C1QTNF1, ABCG1, TFDP2, PAPLN, TNFRSF9, OAF, PLAT, TSLP, MEGF6, H2AFV, ADD2, PADI3, DUSP27, GSTT1, S100A4, DNAJC12, HSPB1, SCN5A, NOV, CTSH, PRKG2, NGEF, FSD1L, UGDH, FBLIM1, LIX1L, AKR1C13, LPXN, DUSP6, RNF130, PTGR1, TMOD2, CST3, ANKRD6,RTKN2, IL12RB1, LDHB, BEND5, GM10471, SPN, RAET1E, RIN2, PDE6D, GNB4, MCTP1, PER3, LHPP, CALR3, CADM1, ITGB2, GHR, CRIPl, MSRB2, EGR2, PAQR7 DOK1, ACSBG1, LEPROT, FAM131B, GPRIN3, COL16A1, GRAP, FKBP1B, GSTM5, KANK2, PSG17, PIK3CD, INF2, MYLK, EML1, TDRD7, ALDH7A1, FAM219A, SH3BGRL, FAM221A, FAM102B, FN1, MAGED2, NUSAP1, M1AP, CISH, TBC1D2B, ATPIF1, MGST3, CNP, XKR5, NEIL3, RALGPS2, MTCH1, CAND2, MEST, TMEM243, XRCC3, NINJ2, ECM1, CPNE3, RAF1, SEPN1, CHST12, NADSYN1, CX3CL1, CD82, CDHR1, PEAR1, POLD4, NR2F1, FHL2, ATHL1, CDKN2AIPNL, RAET1D, SCARA3, PLSCR2, and CRTAP.

As used herein, the phrase "activates IREl" means that the expression or activity of the IREl gene or level of RNA molecule encoding it is up-regulated or greater than that observed in the absence of a IREl modulator therapeutic reagent, with the result that vascular mimicry (VM) of a subject's tumor is inhibited, disrupted, or repressed. In one embodiment, this inhibition of VM co-operates with the anti-angiogenic effect of an anti- angiogenic compound, such as an anti-VEGF antibody. In another embodiment, this inhibition of VM synergizes with the anti-angiogenic effect of an anti-angiogenic compound, such as an anti-VEGF antibody. In one embodiment, this inhibition of VM co-operates with both the anti-angiogenic effect of an anti-angiogenic compound and the VM inhibiting effect of the FOXC2 modulator. In another embodiment, this inhibition of VM synergizes with both the anti-angiogenic effect of the anti-angiogenic compound, and the VM inhibitory effect of the FOXC2 modulator. In another embodiment, this inhibition of VM operates to prevent re-vascularization by VM of the tumor after the anti- angiogenic compound, such as an anti-VEGF antibody, reduces the normal

vascularization of the tumor.

As used herein, the phrase "activates IREl" or "inhibits an IREl pathway target" means that the effect of an IREl modulator on the expression or activity of the target RNA molecules encoding one or more target protein or protein subunits or peptides of the IREl pathway is up regulated or down regulated such that the expression, level, or activity is greater than or less than that observed in the absence of the IREl modulator therapeutic reagent, with the result that vascular mimicry (VM) of a subject's tumor is inhibited, disrupted or repressed. It should be understood that in one embodiment, certain IREl pathway targets behave similarly to IREl, i.e., the target, like IREl itself, may be directly activated or its activity enhanced to achieve VM inhibition in a manner parallel to that of IREl . In another embodiment, depending upon the IREl pathway target and its relationship to IREl (i.e., IREl expression may inhibit the target), the target may be directly inhibited or its activity suppressed (i.e., in a manner opposite to IREl) by the IREl modulator to achieve VM inhibition. In one embodiment, this inhibition of VM co-operates with the anti-angiogenic effect of an anti-angiogenic compound, such as an anti-VEGF antibody. In another embodiment, this inhibition of VM synergizes with the anti-angiogenic effect of an anti-angiogenic compound, such as an anti-VEGF antibody. In one embodiment, this inhibition of VM co-operates with both the anti-angiogenic effect of an anti-angiogenic compound and the VM inhibiting effect of the FOXC2 modulator. In another embodiment, this inhibition of VM synergizes with both the anti- angiogenic effect of the anti-angiogenic compound, and the VM inhibitory effect of the FOXC2 modulator. In another embodiment, this inhibition of VM operates to prevent revascularization by VM of the tumor after the anti-angiogenic compound, such as an anti- VEGF antibody, reduces the normal vascularization of the tumor.

As used herein, an "IREl or IREl pathway modulator" or "therapeutic compounds that activate IREl or the IREl pathway" refer to a therapeutic reagent, compound or composition that directly activates IREl expression or activity so as to inhibit, disrupt or repress vascular mimicry, or directly activates an IREl pathway target's expression or activity so as to inhibit, disrupt or repress vascular mimicry. Alternatively, an IREl pathway modulator refers to a therapeutic reagent, compound or composition that directly inhibits or reduces an IREl pathway target's expression or activity so as to inhibit, disrupt or repress vascular mimicry. In certain examples, therefore, therapeutic compounds that activate IRE1 include tunicamycin. Additionally, small molecule chemical compounds, such as thapsigagin, DTT, brefaldin A, bortezimib, acetaminophen, amiodarone, arsenic trioxide, Bleomycin, cisplatin, clozapine, olanzapine, cyclosporin, diclofenac, indomethacin, efavirenz, Proteasome inhibitors, zidovudine, sertraline, troglitazone, erlotinib, doxorubicin, and anitbodies directed against targets of the IRE1 pathway listed above, may also be useful as IRE1 modulators of this pathway.

Additionally, therapeutic compounds that inhibit an IRE1 pathway target that is normally inhibited when IRE1 itself is activated can include antibodies for that IRE1 pathway target, such as those provided by the same commercial entities referenced above for FOXC2 antibodies. Other "IRE1 modulators" therefore include shRNA, siRNA or RNAi sequences directed to one of those IRE1 targets that are activated when IRE1 is inhibited or CRISPR/Cas guide systems that are commercially available or may be readily developed.

As used herein for the described methods and compositions, the term "antibody" refers to an intact immunoglobulin having two light and two heavy chains or fragments thereof capable of binding to a FOXC2 protein or suitable FOXC2 pathway target or an IREl pathway target (that is inhibited when IREl is activated). Thus, by reference to an antibody includes a monoclonal antibody, a synthetic antibody, a recombinant antibody, a chimeric antibody, a humanized antibody, a human antibody, or a bi-specific antibody or multi-specific construct. The term "antibody fragment" as used herein for the described methods and compositions refers to less than an intact antibody structure having antigen- binding ability. Such fragments, include, without limitation, an isolated single antibody chain or an scFv fragment, which is a recombinant molecule in which the variable regions of light and heavy immunoglobulin chains encoding antigen-binding domains are engineered into a single polypeptide. Other scFV constructs include diabodies, i.e., paired scFvs or non-covalent dimers of scFvs that bind to one another through complementary regions to form bivalent molecules. Still other scFV constructs include complementary scFvs produced as a single chain (tandem scFvs) or bispecific tandem scFvs. Other antibody fragments include an Fv construct, a Fab construct, an Fc construct, a light chain or heavy chain variable or complementarity determining region (CDR) sequence, etc. Still other antibody fragments include monovalent or bivalent minibodies (miniaturized monoclonal antibodies) which are monoclonal antibodies from which the domains non-essential to function have been removed. In one embodiment, a minibody is composed a single-chain molecule containing one VL, one VH antigen- binding domain, and one or two constant "effector" domains. Linker domains connect these elements. In still another embodiment, the antibody fragments useful in the methods and compositions herein are "unibodies", which are IgG4 molecules from with the hinge region has been removed.

By "pharmaceutically acceptable carrier or excipient" is meant a solid and/or liquid carrier, in in dry or liquid form and pharmaceutically acceptable. The

compositions are typically sterile solutions or suspensions. Examples of excipients which may be combined with the anti-angiogenic compound, the FOXC2 modulator or IRE1 activator include, without limitation, solid carriers, liquid carriers, adjuvants, amino acids (glycine, glutamine, asparagine, arginine, lysine), antioxidants (ascorbic acid, sodium sulfite or sodium hydrogen-sulfite), binders (gum tragacanthin, acacia, starch, gelatin, polygly colic acid, polylactic acid, poly-d,l-lactide/glycolide, polyoxaethylene, polyoxapropylene, polyacrylamides, polymaleic acid, polymaleic esters, polymaleic amides, polyacrylic acid, poly aery lie esters, polyvinylalcohols, polyvinylesters, polyvinylethers, polyvinylimidazole, polyvinylpyrrolidon, or chitosan), buffers (borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids), bulking agents (mannitol or glycine), carbohydrates (such as glucose, mannose, or dextrins), clarifiers, coatings (gelatin, wax, shellac, sugar or other biological degradable polymers), coloring agents, complexing agents (caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl- -cyclodextrin), compression aids, diluents, disintegrants, dyes, emulsifiers, emollients, encapsulating materials, fillers, flavoring agents (peppermint or oil of wintergreen or fruit flavor), glidants, granulating agents, lubricants, metal chelators (ethylenediamine tetraacetic acid (EDTA)), osmo-regulators, pH adjustors, preservatives (benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, hydrogen peroxide, chlorobutanol, phenol or thimerosal), solubilizers, sorbents, stabilizers, sterilizer, suspending agent, sweeteners (mannitol, sorbitol, sucrose, glucose, mannose, dextrins, lactose or aspartame), surfactants, syrup, thickening agents, tonicity enhancing agents (sodium or potassium chloride) or viscosity regulators. See, the excipients in "Handbook of Pharmaceutical Excipients", 5 ^th Edition, Eds. : Rowe, Sheskey, and Owen, APhA Publications (Washington, DC), 2005 and US Patent No. 7,078,053, which are incorporated herein by reference. The selection of the particular excipient is dependent on the nature of the compound selected and the particular form of administration desired.

Solid carriers include, without limitation, starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, calcium carbonate, sodium carbonate, bicarbonate, lactose, calcium phosphate, gelatin, magnesium stearate, stearic acid, or talc. Fluid carriers without limitation, water, e.g. , sterile water, Ringer's solution, isotonic sodium chloride solution, neutral buffered saline, saline mixed with serum albumin, organic solvents (such as ethanol, glycerol, propylene glycol, liquid polyethylene glycol, dimethylsulfoxide (DMSO)), oils (vegetable oils such as fractionated coconut oil, arachis oil, com oil, peanut oil, and sesame oil; oily esters such as ethyl oleate and isopropyl myristate; and any bland fixed oil including synthetic mono- or diglycerides), fats, fatty acids (include, without limitation, oleic acid find use in the preparation of injectables), cellulose derivatives such as sodium carboxymethyl cellulose, and/or surfactants.

By "chemotherapeutic agent or therapy" is meant a drug or therapy designed for using in treating cancers. Examples of chemotherapeutics which may be utilized as described herein include, without limitation, cisplatin, carboplatin, 5-fluorouracil, cyclophosphamide, Oncovin, vincristine, prednisone, rituximab, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, carmustine, lomustine, semustine, thriethylenemelamine, triethylene thiophosphoramide, hexamethylmelamine altretamine, busulfan, triazines dacarbazine, methotrexate, trimetrexate,

fluorodeoxyuridine, gemcitabine, cytosine arabinoside, 5-azacytidine, 2,2'- difluorodeoxycytidine, 6-mercaptopurine, 6-thioguanine, azathioprine, 2'- deoxycoformycin, erythrohydroxynonyladenine, fludarabine phosphate, 2- chlorodeoxyadenosine, camptothecin, topotecan, irinotecan, paclitaxel, vinblastine, vincristine, vinorelbine, docetaxel, estramustine, estramustine phosphate, etoposide, teniposide, mitoxantrone, mitotane, or aminoglutethimide. Other anti-cancer therapies for use with the methods and compositions as described herein include non-chemical therapies. In one embodiment, the additional or adjunctive therapy includes, without limitation, radiation, acupuncture, surgery, chiropractic care, passive or active immunotherapy, X-ray therapy, ultrasound, diagnostic measurements, e.g., blood testing. In one embodiment, these therapies are utilized to treat the patient. In another embodiment, these therapies are utilized to determine or monitor the progress of the disease, the course or status of the disease, relapse or any need for booster administrations of the compounds discussed herein.

By "administering" or "route of administration" is delivery of the anti-angiogenic compound, FOXC2 modulator or IRE1 modulator, with or without a pharmaceutical carrier or excipient, or with or without another chemotherapeutic agent into the subject with cancer, the environment of the cancer cell or the tumor microenvironment of the subject. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, systemic routes, such as intraperitoneal, intravenous, intranasal, intravenous, intramuscular, intratracheal, subcutaneous, and other parenteral routes of administration or intratumoral or intranodal administration. In one embodiment, the route of administration is oral. In another embodiment, the route of administration is intraperitoneal. In another embodiment, the route of administration is intravascular. Routes of administration may be combined, if desired. In some embodiments, the administration is repeated periodically.

By "effective amount" is meant the amount or concentration ( by single dose or in a dosage regimen delivered per day) of the anti-angiogenic compound, FOXC2 modulator and/or IRE1 modulator sufficient to retard, suppress or prevent the occurrence of vascularization to the tumor or cancer cell and simultaneously suppress vascular mimicry, while providing the least negative side effects to the treated subject. The amount of anti- angiogenic compound, FOXC2 modulator and/or IRE1 modulator for administration alone or in combination with an additional reagent, e.g., chemotherapeutic, antibiotic or the like can be determined with regard to the age, physical condition, weight and other considerations. In one embodiment, the effective amount(s)is an amount larger than that required when a anti-angiogenic compound is administered to inhibit angiogenesis of a tumor in a subject. In another embodiment, the effective amount of the anti-angiogenic compound is the same as that reported for its use as a sole therapeutic. In still another embodiment, the effective amount is that required to reduce or suppress vascularization of the tumor when administered in combination with the FOXC2 modulator or IRE1 modulator. In a further embodiment, the combination of the FOXC2 modulator and/or IRE1 modulator with the anti-angiogenic compound permits lower than usual amounts of any one of the three therapeutic reagents alone to achieve the desired therapeutic effect. In another embodiment, the combination of the anti-angiogenic compound with the FOXC2 modulator and/or IRE1 modulator and further with another chemotherapy treatment protocol permits adjustment of the additional protocol regimen to achieve the desired therapeutic effect. In one embodiment, the effective amount of the anti- angiogenic compound with the FOXC2 modulator and/or IRE1 modulator is within the range of 1 mg/kg body weight to 100 mg/kg body weight of each therapeutic agent in humans including all integers or fractional amounts within the range. In certain embodiments, the effective amount is at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg/kg body weight, including all integers or fractional amounts within the range. In one embodiment, the above amounts represent a single dose of each therapeutic agent. In another embodiment, the above amounts define an amount(s) of each therapeutic agent to be delivered to the subject per day. In another embodiment, the above amounts define an amount delivered to the subject per day in multiple doses. In still other embodiments, these amounts represent the amount delivered to the subject over more than a single day.

"Control" or "Control subject" as used herein with reference to diagnostic methods refers to the source of the reference FOXC2 or IRE1 gene expression signatures or profiles to which the gene signature of the subject is being compared, as well as the particular panel of control subjects described herein. In one embodiment, the control or reference level is from a single subject. In another embodiment, the control or reference level is from a population of individuals sharing a specific characteristic, e.g., increasing VM or decreasing VM or no VM. In yet another embodiment, the control or reference level is an assigned value which correlates with the level of a specific control individual or population, although not necessarily measured at the time of assaying the test subject's sample. In one embodiment, the control subject or reference is from a patient (or population) having a non-cancerous nodule. In another embodiment, the control subject or reference is from a patient (or population) having a cancerous tumor.

"Sample" as used herein means any biological fluid or tissue that contains immune cells and/or cancer cells. The most suitable sample for use in this invention includes whole blood. Other useful biological samples include, without limitation, peripheral blood mononuclear cells, plasma, saliva, urine, synovial fluid, bone marrow,

cerebrospinal fluid, vaginal mucus, cervical mucus, nasal secretions, sputum, semen, amniotic fluid, bronchoscopy sample, bronchoalveolar lavage fluid, and other cellular exudates from a patient having cancer. Still other samples include tissue from a tumor biopsy. Such samples may further be diluted with saline, buffer or a physiologically acceptable diluent. Alternatively, such samples are concentrated by conventional means.

By "change in expression" is meant an upregulation of one or more selected genes in comparison to the reference or control; a downregulation of one or more selected genes in comparison to the reference or control; or a combination of certain upregulated genes and down regulated genes.

In the context of the diagnostic compositions and methods described herein, reference to multiple gene targets in a gene signature or profile means any one or any and all combinations of the FOX2C or IRI-1 gene targets listed above, and including other genes that change expression during VM. For example, suitable gene expression profiles include profiles containing any number between at least 1 through at least about 500 genes that change expression during VM. In certain embodiment, A VM gene signature or gene profile is formed by at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200, 250, 300, 350, 400, 450 or 500 of the gene targets that change in expression during VM. See e.g., the targets identified herein and in the Figures.

The term "polynucleotide" specifically includes cDNAs. The term includes DNAs (including cDNAs) and RNAs that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritiated bases, are included within the term

"polynucleotides" as defined herein. In general, the term "polynucleotide" embraces all chemically, enzymatically and/or metabolically modified forms of unmodified polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells.

The term "oligonucleotide" refers to a relatively short polynucleotide, including, without limitation, single-stranded deoxyribonucleotides, single- or double-stranded ribonucleotides, RNA:DNA hybrids and double-stranded DNAs. Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using automated oligonucleotide synthesizers that are commercially available. However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms. The terms "a" or "an" refers to one or more. For example, "an expression cassette" is understood to represent one or more such cassettes. As such, the terms "a" or "an"), "one or more," and "at least one" are used interchangeably herein.

As used herein, the term "about" means a variability of plus or minus 10 % from the reference given, unless otherwise specified.

The words "comprise", "comprises", and "comprising" are to be interpreted inclusively rather than exclusively, i.e., to include other unspecified components or

The words "consist", "consisting", and its variants, are to be interpreted exclusively, rather than inclusively, i.e., to exclude components or steps not specifically recited.

As used herein, the phrase "consisting essentially of limits the scope of a described composition or method to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the described or claimed method or composition. Whereever in this specification, a method or composition is described as "comprising" certain steps or features, it is also meant to encompass the same method or composition consisting essentially of those steps or features and consisting of those steps or features.

II. Compositions

In one embodiment, a therapeutic composition for the treatment or inhibition of tumor vacuolization comprises the combination of an anti-angiogenic therapeutic compound and a therapeutic compound that inhibits or prevents vasculogenic mimicry or vascular mimicry (VM). In one embodiment, the therapeutic composition contains, in a suitable pharmaceutical carrier, an anti-angiogenic therapeutic compound, and a FOXC2 modulator therapeutic compound that inhibits the activity or pathway of the transcription factor FOXC2, thereby inhibiting or suppressing VM. As used throughout the specification and for simplicity, the exemplary anti-angiogenic therapeutic compound is referred to as an anti-VEGF antibody. In other embodiments, the term anti-VEGF antibody can be replaced with another anti-angiogenic compound identified above.

In another embodiment, the therapeutic composition contains in a suitable pharmaceutical carrier, an anti-angiogenic therapeutic compound, i.e., anti-VEGF antibody and a therapeutic compound that activates or enhances the activity or pathway of IRE1, i.e., an IRE1 activator. In still another embodiment, the therapeutic composition contains in a suitable pharmaceutical carrier, an anti-angiogenic therapeutic compound, a therapeutic compound (i.e., a FOXC2 modulator) that inhibits the activity or pathway of the transcription factor FOXC2 and a therapeutic compound that activates or enhances the activity or pathway of IRE1 and inhibits the activity of the target genes of IRE1.

These compositions contain the two or three therapeutic components in effective amounts. The anti-VEGF antibody is present in an amount effective to suppress normal vascularization of a tumor present in a subject to be treated with the composition. If, present, the FOXC2 modulator is in an amount effective to inhibit the normal functioning of the FOXC2 pathway and suppress or prevent the occurrence of VM. If present, the IRE1 modulator is present in an amount effective to activate or overexpress the normal functioning of the IRE1 pathway and suppress or prevent the occurrence of VM. In compositions containing both the FOXC2 modulator and the IRI1 activator with the anti- VEGF antibody, the FOXC2 modulator and the IRI1 activator may be employed in effective amounts lower than those used when the inhibitor or activator is used alone (with the anti-VEGF).

The various components of the compositions are prepared for administration by being suspended or dissolved in a pharmaceutically or physiologically acceptable carrier In one embodiment, these at least two or all three components may be present in a pharmaceutical carrier in a single solution for simultaneous administration to the subject having cancer.

It is also anticipated that where desired, a therapeutic kit is provided that contains individually packaged effective amounts of the two (anti-VEGF antibody and at least one of the FOXC2 modulator or IRE1 modulator) or three (anti-VEGF antibody, FOXC2 modulator and IRE1 modulator) components. Such a kit is convenient for administration of each component separately and sequentially and can contain additional "booster" doses of any of the three components, where needed. Conventional kit components, such as packaging, additional pharmaceutical carriers, drug delivery devices and any adjunctive treatment modalities, may be included in the kit.

In yet another aspect, a composition or kit for diagnosing or evaluating the efficacy of cancer treatment in a mammalian subject includes multiple polynucleotides or oligonucleotides, wherein each polynucleotide or oligonucleotide hybridizes to a different gene, gene fragment, gene transcript or expression product in a patient sample, where each gene, gene fragment, gene transcript or expression product is selected from genes that are upregulated or down-regulated in the course of VM or in the course of treatment for VM. Such genes include the gene targets identified herein as FOXC2 pathway targets, including for example, LDHB, ANKH, MGP, PLSCR4 and ERN1/2 or IRE1 pathway targets, identified above. By evaluating the gene targets that change in expression during vascular mimicry in a cancer patient, a physician may assess the severity of the cancer and/or the success of the treatment described herein. In one embodiment of such a diagnostic composition, at least one polynucleotide or

oligonucleotide is attached to a detectable label.

III. The Methods

Any of the above-described compositions and/or kits with individual components may be employed in methods for the treatment or inhibition of tumor vacularization and/or the treatment of cancers. In one embodiment, a method for increasing the sensitivity of a tumor to anti-angiogenic therapy comprises treating a patient having a tumor with an anti-angiogenic therapeutic composition or compound and substantially simultaneously inhibiting vascular, or vasculogenic, mimicry (VM). Inhibition of VM comprises further administering at least one of a therapeutic compound that inhibits the activity or pathway of the transcription factor FOXC2; and a therapeutic compound that activates or enhances the activity or pathway of IREland/or inhibits the activity of its target genes.

Thus in one embodiment, the method involves administering to a subject with a cancer the anti-VEGF antibody in a suitable pharmaceutical carrier. This method also involves administering a therapeutic compound that inhibits the activity or pathway of the transcription factor FOXC2 in an amount effective to suppress VM in a pharmaceutical carrier. Such administration can occur by a suitable route of administration and dosage depending upon the physical condition of the subject, and whether these components are being administered simultaneously in a single composition or sequentially.

Thus in another embodiment, the method involves administering to a subject with a cancer the anti-VEGF antibody in a suitable pharmaceutical carrier. This method also involves administering a therapeutic compound that activates or enhances the activity or pathway of IRE1 in an amount effective to suppress VM in a pharmaceutical carrier. Such administration can occur by a suitable route of administration and dosage depending upon the physical condition of the subject, and whether these components are being administered simultaneously in a single composition or sequentially.

Thus yet another embodiment, the method involves administering to a subject with a cancer the anti-VEGF antibody in a suitable pharmaceutical carrier. This method also involves administering a therapeutic compound that inhibits the activity or pathway of the transcription factor FOXC2 in an amount effective to suppress VM in a pharmaceutical carrier. This method also involves administering a therapeutic compound that activates or enhances the activity or pathway of IREl in an amount effective to suppress VM in a pharmaceutical carrier. Such administration can occur by a suitable route of administration and dosage depending upon the physical condition of the subject, and whether these components are being administered simultaneously in a single composition or sequentially.

Methods for determining the timing of frequency (boosters) of administration on one, two or all three of the components will include an assessment of disease response, including assessments of tumor size. In another embodiment, any of these above-receited methods further comprises administering to the subject along with the therapeutic agents, an adjunctive therapy, such as chemotherapy or radiation, or others as described above directed toward the cancer or tumor being treated.

Additional modifications of these methods includes changing the FOXC2 pathway target being treated with the FOXC2 pathway modulator (inhibitor or activator, as necessary) as defined above with each "booster" treatment or changing the IREl pathway target being treated with the IREl modulator as defined above with each "booster" treatment. In another embodiment, administration of the FOXC2 modulator and IREl modulator are alternated in the regimen. In still another embodiment, treatment steps can involve alternating or repeating the administration of FOXC2 modulators, wherein each treatment step is designed to directly effect a different or alternative FOXC2 pathway target or multiple FOXC2 pathway targets, simultaneously or sequentially. In still another embodiment, treatment steps can involve alternating or repeating the

administration of IREl modulators, wherein each treatment step is designed to directly effect a different or alternative IREl pathway target, or multiple IREl pathway targets, simultaneously or sequentially. One of skill in the art can assemble any number of treatment regimens by alternating the two or three active components of the methods. In still another embodiment, a method for the treatment or inhibition of tumor vacularization and/or for the treatment of a cancer comprises treating a patient having a tumor with an antibody to VEGF and substantially simultaneously inhibiting vascular, or vasculogenic, mimicry (VM). As described above, inhibition of VM comprises administering at least one of a therapeutic compound that inhibits the activity or pathway of the transcription factor FOXC2; and a therapeutic compound that activates or enhances the activity or pathway of IRE1 and/or inhibits the activity of its target genes. In one embodiment, the cancer is a breast cancer. In other embodiments, the cancer is any of those identified above.

In still another aspect, a method for diagnosing or evaluating cancer characterized by VM in a mammalian subject involves identifying changes in the expression of three or more genes in the sample of a subject, said genes selected from the gene targets identified herein; and comparing that subject's gene expression levels with the levels of the same genes in a reference or control. Changes in expression of such gene targets correlates with a diagnosis or evaluation of the progression of a cancer, e.g., breast cancer, characterized by characteristic gene target expression changes that occur with increasing VM. Alternatively, changes in expression of such gene targets correlates with a diagnosis or evaluation of the treatment of a cancer with angiogenic therapy coupled with anti-VM therapy as described herein, wherein successful treatment is characterized by gene target expression changes that occur with decreasing VM. The compositions and methods described herein provide the ability to distinguish the progress of vascular mimicry in a patient, by determining a characteristic RNA expression profile of the genes of the blood of a mammalian, preferably human, subject. The profile of certain genes upregulated or down-regulated during VM is compared with the profile of one or more subjects of the same class (e.g., patients having lung cancer or a non-cancerous nodule) or a control to provide a useful diagnosis.

Such methods of gene expression profiling include methods based on

hybridization analysis of polynucleotides, methods based on sequencing of

polynucleotides, and proteomics-based methods. The most commonly used methods known in the art for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization; RNAse protection assays; nCounter® Analysis; and PCR-based methods, such as RT-PCR. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA- RNA hybrid duplexes or DNA-protein duplexes. Representative methods for sequencing- based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS).

It should be understood that other modifications of these methods by selection of the components identified above may be readily performed using knowledge in the art coupled with the teachings in this specification.

The following examples, protocols and methods described in the examples are not considered to be limitations on the scope of the claimed invention. Rather this specification should be construed to encompass any and all variations that become evident as a result of the teaching provided herein. One of skill in the art understand that changes or variations can are made in the disclosed embodiments of the examples, and expected similar results can are obtained. For example, the substitutions of reagents that are chemically or physiologically related for the reagents described herein are anticipated to produce the same or similar results. All such similar substitutes and modifications are apparent to those skilled in the art and fall within the scope of the invention.

EXAMPLE 1 : VM IS A DRIVER OF METASTASIS AND IS PARTICULARLY PREVALENT IN AGGRESSIVE BREAST CANCER SUBTYPES.

Recently, using genetic tracking of clonal lineages derived from the same parental population (FIGs. 1A-1C), we have identified sub-clones of breast cancer cells that are competent to perform VM (4T1-E ^W and 4T1-T ^W , FIG 1B-1C) ⁷ . By comparing gene expression between VM-competent and incompetent sub-clones, we identified several putative mediators of VM two of which we functionally validated in vitro and in vivo. Through manipulation of these novel VM mediators, SerpinE2 and SLPI, we discovered an essential role for VM in metastasis by facilitating tumor cell intravasation (entry into the blood stream) ⁷ . These data not only established the importance of VM for metastasis but also identified and validated the first bone fide modulators of the VM phenotype. Despite these recent advances, a broad overview of the prevalence of VM across diverse breast cancer sub-types is lacking.

To survey the VM landscape in breast cancer we first applied tissue-specific expression analysis (TSEA) (http://genetics.wustl.edu/jdlab/tsea/), where tissue-specific genes across 25 normal tissue types are interrogated for enrichment in a gene list, to genes that are up-regulated in 4T1 -derived VM clones. This analysis demonstrated that VM clones ectopically express genes that are normally vascular-specific (FIG. IE).

Leveraging this cardinal feature of VM revealed that human cell lines derived from aggressive Basal/Claudin-low tumors also up-regulate vascular-specific genes (FIG. IF, left panel) and endothelial-enriched genes (FIG. IF, right panel). While elevated expression of vascular-specific and endothelial genes also seen in patients (FIG. 1G) could reflect the abundance of host vessels in the micro-environment, the fact that these genes are also up-regulated in cell lines grown in culture in the absence of endothelium, supports that there is a cell intrinsic program of vascular mimicry (VM) that is especially prevalent in aggressive Basal/Claudin-low tumors (FIGs. 1E-1G).

EXAMPLE 2: IREl -MEDIATED RESTRAINT OF A SECRETORY PROGRAM THAT MAINTAINS THE VM STATE.

A. 4T1 -derived VM clones and VM capable cell lines from the Cancer Cell Line Encyclopedia (CCLE) up-regulate the expression of genes encoding secreted/extra- cellular factors such as extracellular matrix (ECM) and ECM-regulatory proteins (FIG. 2A). We performed experiments in which we exchanged the medium on VM clones prior to replating the cells on matri-gel for VM tube assays, thus depriving the cells of high concentrations of cell-derived secreted factors. We have shown that VM clones broadly require secretion of extra-cellular factors to maintain their ability to form VM tubes in vitro (FIGs. 2B-2C). Given this striking result and the documented ECM-richness of VM tubular structures formed in vivo*' ⁵ we hypothesized that regulators of the secretory pathway influence the ability of tumor cells to perform VM and thus metastasis.

To prioritize candidates with clinical relevance, in a setting where VM is prevalent (Basal/Claudin-low tumors; see FIGs 3A-3I), we looked at the mRNA expression levels of critical regulators of secretion in patients with aggressive

Basal/Claudin-low breast cancer that either did or did not relapse with metastatic disease ⁹ . We focused our analysis on regulators of the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) as highly secretory cells have an increased load on the ER as the site of synthesis of most secreted proteins. As such the UPR plays a critical role in adaptive responses that help negate increased traffic through the ER by a variety of mechanisms ¹⁰ . Analysis of the three main branches of the UPR: PERK, ATF4 (a critical downstream target of the PERK pathway), ATF6 and IREl, revealed a clear and selective downregulation of the IREl branch of the UPR in patients with metastatic relapse (FIG 3A). Both the IREl a and the IREl/? isoforms were suppressed and when considering their aggregate mean expression, the suppression was even more significant (FIG 3A, p =0.0026).

B. To ascertain whether IRE1 regulation of VM underlies these effects we turned to the in vitro VM tube assay. Inhibition of IRE1 with two independent small molecule inhibitors of its RNAse activity enhanced VM tubulogenesis while activation of IREl with Tunicamycin suppressed VM (FIG. 3B). Plating the remaining treated cells in 2D ruled out any demonstrable effects on cell viability that might account for suppression of VM by Tunicamycin (data not shown). Moreover, knockdown or IREl with two independent shRNAs also enhanced VM tube formation in human Claudin-low MDA- MB-231 cells (FIG 1C).

IREl is a ER trans-membrane kinase/ribonuclease that responds to unfolded protein accumulation in the ER by two main mechanisms: (1) it cleaves a retained intron within the mature XBP1 mRNA which results in a frame shift and an active transcription factor that drives the expression of chaperones and other genes which increase the folding capacity of the ER ¹¹ (2) it directly cleaves mRNAs encoding secreted and membrane proteins leaving free 5' and 3' ends that are substrates for degradation in a process termed Regulated IREl -Dependent mRNA Decay (or RIDD) ^11"13 . To determine whether XBP1 or RIDD were important for IREl 's effects on VM we knockdown IREl or XBP1 in 4T1-TVM cells and assessed tube forming ability, as shown in FIG. 3D, knockdown of IREl but not XBP1 recapitulated the effects seen with the small molecule inhibitors. These data support that an XBP1 -independent function of IREl controls VM, such as RIDD.

C. Given the importance of secretion for VM (FIGs. 2A-2C) and the role that RIDD plays in restraining secretion, we hypothesized that the IREl effects we observed on VM may be mediated by secreted factors. To test this hypothesis, we acutely (4 hrs) treated 4T1-T ^W cells with IREl inhibitors, followed by extensive washes with PBS (to remove remaining drug) and replacement with fresh medium. We then allowed the treated cells to condition the media for 24 hrs, followed by filtration (to remove cells) and addition to naive 4T1-T ^W cells for 24 hrs followed by replating onto matri-gel for VM tube assays. As shown in FIG. 3E, conditioned medium (CM) from IREl inhibitor-treated cells enhanced VM, providing evidence that IREl restrains the expression of secreted VM drivers. D. To determine whether IREl played a causal role in metastasis in vivo we transduced 4T1-TVM cells with two different shRNAs targeting IREl and transplanted them orthotopically into the mammary fat-pad of syngeneic Balb/C mice. Subsequently, we measured primary tumor volume and harvested the lungs to enumerate metastases by IHC (immunohistochemistry) staining for mCherry contained within the shRNA vector. Knockdown of IREl had relatively minor effects on primary tumor burden (FIG. 3F) but increased the number of metastatic lung nodules 3-4 fold with both independent shRNAs (FIG. 3G). Measurment of macro metastases at later time points by H and E staining confirmed enhancement of metastasis by IREl knockdown (FIG. 3H). Furthermore, segregation of Basal/Claudin-low patients based on mean IREl α/β expression demonstrated that those in the bottom quartile had significantly shorter relapse-free survival than patients in the top 75% (FIG. 31). These data provide evidence that suppression of the IREl pathway in breast cancer patients favors metastasis and poor prognosis. Considered together these data indicate that enhancement of VM via suppression of IREl favors metastasis in both mouse models and human patients.

E. To ascertain the effects of IREl inhibition or activation on the transcriptome more broadly we performed RNA-Seq on polyA+ RNA extracted from 4T1-TVM cells treated with an IREl inhibitor or Tunicamycin looking for gene expression changes that were mirrored in the two conditions, since they have opposing effects of IREl activity and VM. Gene Ontology (GO) analysis of genes that were significantly down-regulated by Tunicamycin/IREl activation and significantly up- regulated by IREl inhibition were enriched for regulators of vasculature development and secreted ECM/ECM-affiliated genes (FIG. 4 A), likely reflecting the RIDD arm of the IREl pathway. Moreover, gene set enrichment analysis (GSEA) of various endothelial signatures revealed that genes whose expression is normally restricted to the endothelium were upregulated by IREl inhibition and downregulated by tunicamycin (FIG. 4B) indicating a broad role for IREl in regulating ectopic expression of endothelial genes in VM clones, a hallmark feature of the VM state. Moreover, these same endothelial signatures were upregulated in IREllow cell lines from the CCLE (FIG. 4B) derived from diverse solid tumor-types.

F. To identify critical targets of IREl that may mediate its effects on VM we ranked the genes that went up with IREl inhibition and down with activation by their fold change in VM clones versus all clones and in lung metastases-derived cell lines versus primary tumor-derived cell lines (data not shown). We then systematically knocked down each of the top 30 genes and performed VM tube assays (data not shown). This approach revealed 4 IRE1 -target genes, MGP, ANK (ANKH in human), LDHB and PLSCR4, that suppressed VM tube formation when knocked down with two independent shRNAs (FIG. 4C). Critically we corrected all observed effects on tube formation for viability (measured by cell-titer Glo) to ensure that these IRE1 -target genes represent bone fide regulators of VM. The mRNAs encoding these target genes where significantly stabilized upon IRE1 inhibition (FIG. 4D) providing evidence that these mRNAs are RIDD substrates. Further analysis of these genes in breast cancer patient samples revealed highest expression in aggressive Basal/Claudin-low tumors (FIG. 4E) where VM is particularly prevelant.

Moreover, Basal/Claudin-low patients who had relapsed at 5 years had a higher VM score (caculated as the mean expression of LDHB, ANKH, MGP, PLSCR4 and mean inverse expression of ERN1/2) than those that were disease-free (FIG. 4F), evidencing a role for these genes in metastatic relapse.

EXAMPLE 3: ACQUISITION OF A VM STATE VIA CO-OPTION OF AN

ENDOTHELIAL TRANSCRIPTIONAL FACTOR.

Inhibition of IREl can enhance VM in clones that can already perform VM but is not able to induce VM in non-VM clones, indicating that the secretory program that IREl restrains is necessary but not sufficient for VM. Therefore, we set out to identify factors that drive otherwise epithelial cells towards a VM state. We reasoned that, like many phenotypic transitions, the acquisition of endothelial-like properties and ectopic vascular- specific gene expression by VM tumor cells may be driven by a master transcription factor (TF). Therefore, we ranked all TFs by their change in mRNA expression between VM clones and all other clones and found that FOXC2 and one of its important target genes MEF2C were the 2 ^nd and 3 ^rd most up-regulated TFs respectively, in VM cells (FIG. 5A and FIG. 5B show individual clones). FOXC2 and MEF2C mRNA levels were also significantly elevated in cell lines derived from lung metastases relative to the primary tumor (FIG. 7C) highlighting a role in metastasis. FOXC2 was of interest because it is critically important in normal endothelial development. Mice lacking both copies die pre- and peri-natally with profound cardiovascular defects ¹⁴ . In collaboration with ETS transcription factors, FOXC2 can specify gene expression to the endothelium ¹⁵ .

Moreover, direct reprogramming of fibroblasts into endothelial-like cells requires up- regulation of FOXC2 ¹⁶ , evidencing a critical role in trans-differentiation of non- endothelial cell types to endothelium.

To determine if FOXC2 up-regulation would be sufficient to drive non-VM cells towards a VM/endothelial-like state, we performed the following experiments. Enforced expression of murine FOXC2 in one of the most VM-deficient clones, 4Ti-L ^nonVM , was sufficient to induce tube-forming potential (FIG. 7D) in a cell culture VM tube assay. Conversely, depletion of FOXC2 by RNAi in VM-competent 4T1-T ^W , dramatically suppressed tubulogenesis (FIG. 7E). To determine whether a broader FOXC2-driven transcriptional program was operative in VM cells, we utilized a publically available gene expression dataset, of Human Mammary Epithelial (HMLE) cells over-expressing FOXC2 (GSE44335), to define a set of FOXC2-target genes. Gene set enrichment analysis (GSEA) ¹⁷ , using these FOXC2-target genes, demonstrated that they were globally up-regulated in VM clones relative to all other clones (FIG 7F, left panel) and in lung metastases relative to primary tumors (FIG 7F, right panel). Analysis of breast cancer patients revealed highest expression of FOXC2 in Basal/Claudin-low tumors (FIG. 7G), precisely the same subtypes that have the highest proclivity for ectopic vascular- specific gene expression.

Moreover, stratification of patients with Basal/Claudin-low tumors by FOXC2 expression revealed significantly shorter relapse-free survival times of patients whose tumors exhibit high FOXC2 expression (FIG. 7H). Together these data demonstrate that FOXC2 is both necessary and sufficient to establish a VM state de novo. These data provide evidence that FOXC2 may be a master regulator of vascular mimicry and metastasis by inducing an epithelial-to-endothelial transition (EET).

To gain an understanding of the genes that FOXC2 regulates in VM-proficient human tumor cells we depleted FOXC2 using two different shRNAs in MDA-MB-231 ^w cells (FIG. 6A) and performed RNA-Seq. Overlaying GO terms of shFOXC2 DOWN genes and VM UP genes across species revealed 45 GO terms that were significant in both settings (FIG. 6B). These GO terms described processes that are known to be important for VM from our data and others such as ECM, hypoxia, and vasculature development (FIG. 6D). Moreover, the top GO terms upon FOXC2 knockdown in MDA- MB-23 l ^w cells related to ECM and secretion. This data evidences that there is interplay between the FOXC2 and IRE1 pathways (FIG. 6C). FOXC2 has been previously shown to promote metastasis in 4T1 breast tumors and has been implicated in another form of trans-differentiation, the epithelial-to-mesenchymal transition (EMT) ¹⁸ . EMT involves the loss of epithelial characteristics and gain of mesenchymal characteristics including increased migratory capacity and loss of cell-cell contacts and is thought to mediate metastasis through these effects ¹⁹ .

However, from our data we demonstrate that FOXC2 promotes metastasis through

VM, raising the critical question of whether FOXC2-driven metastasis is mediated via EMT, EET or both. To address this question we first asked whether VM clones have undergone an EMT at the gene expression level by analyzing the enrichment of target- genes of bone-fide EMT transcription factors (TFs) in VM clones using GSEA and signatures derived from gene expression data of HMLE cells overexpressing the EMT TFs, TWIST, SNAIL, or SLUG (GSE43495). Only FOXC2 targets were significantly enriched in VM clones (FIG. 6D), suggesting that VM clones don't manifest a "typical" EMT signature at the gene expression level. Moreover, while overexpression (OE) of FOXC2 in HMLE cells or knockdown of FOXC2 in MDA-MB-231 ^W cells caused enrichment and depletion respectively, of endothelial-enriched genes, none of the EMT TFs significantly altered this gene set (FIG. 6E). These data support that FOXC2 drives expression of a set of vascular/endothelial genes in non-endothelial cells and that this is not a common feature of the EMT. It is possible that an EMT is a required intermediate of an EET.

A striking observation from our RNA-Seq data was a profound enrichment of genes involved in the cellular response to low-oxygen levels, the hypoxic response. As shown in FIG. 7A, genes characteristically up-regulate during hypoxia were globally down-regulated upon loss of FOXC2. This data supports the conclusion that FOXC2 acts to maintain their expression. Moreover, target-genes of the master hypoxic transcription factors HIFla and HIF2a were also globally down-regulated by suppression of FOXC2 (FIG. 7B). Not only were the target-genes of HIFla reduced by FOXC2 depletion but also the mRNA encoding HIFla itself was down-regulated (FIG. 7C). These data are particularly intriguing in the context of vascular mimicry as it has been suggested that the hypoxic tumor micro-environment may select for clones that are VM competent.

To test whether hypoxic induction of FOXC2 is the molecular mechanism underlying this phenomenon we subjected parental 4T1 cells to hypoxia in vitro for 24 hrs and measured FOXC2 mRNA expression by qPCR. Exposure to hypoxic culture conditions was sufficient to substantially induce FOXC2 mRNA (FIG. 7D). These data demonstrate that low-oxygen conditions, such as those encountered in the core of a tumor or induced by depriving the tumor of blood supply with anti-angiogenic therapy (AAT), promote VM via induction of FOXC2. Without wishing to be bound by theory, the inventors hypothesize that this model would predict that cells that induce FOXC2 survive in hypoxic enrvironments sufficiently long to resupply themselves with blood via VM.

Accordingly, suppression of FOXC2 in MDA-MB-231 (FIG. 7E) or 4T1-T ^W (FIG. 7F) cells enhanced hypoxia-induced cell death in cell culture, providing evidence that FOXC2 expressing cells have a selective advantage under hypoxia. Together these data demonstrate that FOXC2 couples short term survival under low-oxygen conditions with endothelial differentiation to resupply the tumor with blood via VM.

EXAMPLE 4: ASSOCIATION OF VM GENE EXPRESSION WITH FAILURE OF ANTI-VEGF THERAPY IN BREAST CANCER PATIENTS.

Inhibitors of angiogenesis, such as the VEGFR2 inhibitor Sunitinib or the VEGF- blocking antibody Bevacizumab (Bev), have shown disappointing results in clinical trials displaying variable responses in multiple tumor types especially breast cancer

(https://www.cancer.gov/about-cancer/treatment/drugs/fda- bevacizumab).

In view of a recently published study ²¹ in which previously untreated ductal breast cancer patients received neo-adjuvant, single agent, Bev for 2 weeks, we addressed the role of VM in clinical resistance to anti-angiogenic therapy. The authors ²¹ took core biopsies immediately prior and two weeks post-therapy, RNA extracted and subjected to gene expression profiling by microarray. They additionally measured tumor response to Bev via MRI imaging before and after therapy facilitating the correlation of gene expression changes with a quantitative measure of anti -tumor activity (FIG. 8A) ²¹ . The authors identified a set of genes whose levels were significantly elevated in patients who failed to respond.

To assess the role of VM as a resistance mechanism in this clinical cohort we examined whether this Bev resistance gene signature was altered by perturbations that influence VM, i.e. manipulation of the IREl or FOXC2 pathways. Gene set enrichment analysis of the IREl -regulated (FIG. 8B) or FOXC2-regulated (FIG. 8C) gene expression data sets that we have generated revealed that Bev resistance genes are globally up- regulated by IREl inhibition (FIG. 8B) or FOXC2 over-expression (FIG. 8C). These perturbations enhance VM. Analysis of these data sets further revealed that they were globally down-regulated by IRE1 activation (with Tunicamycin) or FOXC2 knockdown. These latter perturbations restrain VM. Moreover, signatures of Bevacizumab resistance from a Glioblastoma (GBM) mouse model (FIG. 8D) and Sunitinib resistance from a Renal Cell Carcinoma (RCC) patient-derived xenograft model (FIG. 8E) were globally up-regulated by FOXC2 over-expression and suppressed by FOXC2 knockdown. To our knowledge, these data provide the first clinically -relevant evidence that failure to respond to anti-angiogenic therapy in patients is associated with increased expression of VM- related genes. Thus, these data support that co-targeting of VM can enhance response to anti-angiogenic cancer therapies.

EXAMPLE 5: SUPRESSION OF VM WITH LOW-DOSE TUNICAMYCIN

AUGMENTS RESPONSE TO AAT.

To empirically determine whether VM drives resistance to AAT in our 4T1 system, we first measured the effect of AAT on tube formation of 4T1-T ^W in cell culture (FIG. 9A). While Sunitinib treatment suppressed tube formation by endothelial cells (HUVEC) as expected, it had no demonstrable effect on 4T1-T ^W tube formation (FIG. 9A), confirming the indifference of VM clones to VEGF signaling. Next we implanted parental-4Tl ^nonVM and 4T1-T ^W cells to generate VM-deficient and proficient tumors respectively. We allowed tumors to form for 6 days after which we intitated treatment with B20-4.1.1 (a VEGF-blocking antibody that recognizes VEGF of both human and murine origin) bi-weekly. After 3 doses of B20, parental-4Tl VM-deficient tumors had significantly shrunk by 75%. In contrast, 4T1-T VM-proficient tumors did not demonstrate a significant response to B20/AAT (FIG. 9B).

We next determined whether suppression of VM via low-dose tunicamycin could enhance response of VM tumors to AAT. Treatment with either Sunitinib and

tunicamycin (FIG. 9C) or B20 and tunicamycin (FIG. 9D) revealed that co-inhibition of VM and angiogenesis produced better responses than suppression of either process alone.

Considered together, our data provide evidence for critical roles for transcriptional control in the initiation of VM, secretory programs in the maintenance of VM, and an association of VM with poor response to anti-angiogenic therapy. These data support small molecule targeting of VM in combination with anti-angiogenic therapy. In another embodiment, we use a minimal VM-based gene signature as a bio-marker of response to anti-angiogenic therapy and as a means to identify sub-sets of patients for whom combination anti-VM/anti-angiogenic therapy is beneficial.

Each patent, patent application, and publication, including websites cited throughout the specification, including provisional US patent application No. 62/568,672, is incorporated herein by reference. While the invention has been described with reference to particular embodiments, it is appreciated that modifications can be made thout departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

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