Steven T. Sizemore

FOXA1 Represses the Molecular Phenotype of Basal Breast Cancer Cells

Breast cancer is a heterogeneous disease that comprises multiple subtypes. Luminal subtype tumors confer a more favorable patient prognosis, which is, in part, attributed to estrogen receptor (ER)-α positivity and antihormone responsiveness. Expression of the forkhead box transcription factor, FOXA1, similarly correlates with the luminal subtype and patient survival, but is also present in a subset of ER-negative tumors. FOXA1 is also consistently expressed in luminal breast cancer cell lines even in the absence of ER. In contrast, breast cancer cell lines representing the basal subtype do not express FOXA1. To delineate an ER-independent role for FOXA1 in maintaining the luminal phenotype, and hence a more favorable prognosis, we performed expression microarray analyses on FOXA1-positive and ER-positive (MCF7, T47D), or FOXA1-positive and ER-negative (MDA-MB-453, SKBR3) luminal cell lines in the presence or absence of transient FOXA1 silencing. This resulted in three FOXA1 transcriptomes: (1) a luminal signature (consistent across cell lines), (2) an ER-positive signature (restricted to MCF7 and T47D) and (3) an ER-negative signature (restricted to MDA-MB-453 and SKBR3). Gene set enrichment analyses revealed FOXA1 silencing causes a partial transcriptome shift from luminal to basal gene expression signatures. FOXA1 binds to a subset of both luminal and basal genes within luminal breast cancer cells, and loss of FOXA1 increases enhancer RNA transcription for a representative basal gene (CD58). These data suggest FOXA1 directly represses a subset of basal signature genes. Functionally, FOXA1 silencing increases migration and invasion of luminal cancer cells, both of which are characteristics of basal subtype cells. We conclude FOXA1 controls plasticity between basal and luminal breast cancer cells, not only by inducing luminal genes but also by repressing the basal phenotype, and thus aggressiveness. Although it has been proposed that FOXA1-targeting agents may be useful for treating luminal tumors, these data suggest that this approach may promote transitions toward more aggressive cancers.

The Forkhead Box Transcription Factor FOXC1 Promotes Breast Cancer Invasion by Inducing Matrix Metalloprotease 7 (MMP7) Expression

Background: FOXC1 is associated with breast cancer aggressiveness and the basal-like breast cancer subtype but the mechanism through which FOXC1 increases aggressiveness has not been elucidated. Results: FOXC1 induces expression of matrix metalloprotease 7 (MMP7). Conclusion: The aggressive cancer phenotype imparted by FOXC1 is due, at least in part, to expression of MMP7. Significance: MMP7 represents a putative target for the treatment of some basal-like breast cancers. Therapeutic options for treatment of basal-like breast cancers are limited and identification of molecular targets for novel therapies to treat this aggressive cancer is urgently needed. Recently, FOXC1, a forkhead box transcription factor, was identified as a functionally important biomarker of breast cancer aggressiveness and the basal-like breast cancer subtype. However, the mechanism through which FOXC1 controls aggressiveness of basal-like breast cancer remains to be elucidated. Here, we identify matrix metalloprotease 7 (MMP7) as a key downstream effector of FOXC1-mediated invasiveness. Expression of FOXC1 and MMP7 is significantly correlated in breast cancer samples and cell lines at both the mRNA and protein levels. Transient expression of FOXC1 in nontransformed mammary epithelial cell lines resulted in significantly increased expression of MMP7 and an MMP7-dependent increase in invasiveness. In reciprocal experiments, silencing endogenous FOXC1 in basal-like breast cancer cell lines resulted in decreased expression of MMP7 without decreased expression of other matrix metalloproteinases. We also demonstrate that elevated co-expression of FOXC1 and MMP7 is an independent predictor of patient outcome in multivariate analyses of two breast cancer patient cohorts. Together, our findings identify MMP7 as a novel mechanism through which FOXC1 may regulate the basal-like breast cancer invasive phenotype and the propensity of these cancers to metastasize. Furthermore, our findings demonstrate for the first time a correlation between MMP7 expression and basal-like breast cancers, suggesting that MMP7 may be a useful therapeutic target for treatment of this disease.

Stromal PDGFR-α Activation Enhances Matrix Stiffness, Impedes Mammary Ductal Development, and Accelerates Tumor Growth

The extracellular matrix (ECM) is critical for mammary ductal development and differentiation, but how mammary fibroblasts regulate ECM remodeling remains to be elucidated. Herein, we used a mouse genetic model to activate platelet derived growth factor receptor-alpha (PDGFRα) specifically in the stroma. Hyperactivation of PDGFRα in the mammary stroma severely hindered pubertal mammary ductal morphogenesis, but did not interrupt the lobuloalveolar differentiation program. Increased stromal PDGFRα signaling induced mammary fat pad fibrosis with a corresponding increase in interstitial hyaluronic acid (HA) and collagen deposition. Mammary fibroblasts with PDGFRα hyperactivation also decreased hydraulic permeability of a collagen substrate in an in vitro microfluidic device assay, which was mitigated by inhibition of either PDGFRα or HA. Fibrosis seen in this model significantly increased the overall stiffness of the mammary gland as measured by atomic force microscopy. Further, mammary tumor cells injected orthotopically in the fat pads of mice with stromal activation of PDGFRα grew larger tumors compared to controls. Taken together, our data establish that aberrant stromal PDGFRα signaling disrupts ECM homeostasis during mammary gland development, resulting in increased mammary stiffness and increased potential for tumor growth.

Stromal PTEN inhibits the expansion of mammary epithelial stem cells through Jagged-1

Fibroblasts within the mammary tumor microenvironment are active participants in carcinogenesis mediating both tumor initiation and progression. Our group has previously demonstrated that genetic loss of phosphatase and tensin homolog (PTEN) in mammary fibroblasts induces an oncogenic secretome that remodels the extracellular milieu accelerating ErbB2-driven mammary tumor progression. While these prior studies highlighted a tumor suppressive role for stromal PTEN, how the adjacent normal epithelium transforms in response to PTEN loss was not previously addressed. To identify these early events, we have evaluated both phenotypic and genetic changes within the pre-neoplastic mammary epithelium of mice with and without stromal PTEN expression. We report that fibroblast-specific PTEN deletion greatly restricts mammary ductal elongation and induces aberrant alveolar side-branching. These mice concomitantly exhibit an expansion of the mammary epithelial stem cell (MaSC) enriched basal/myoepithelial population and an increase in in vitro stem cell activity. Further analysis revealed that NOTCH signaling, specifically through NOTCH3, is diminished in these cells. Mechanistically, JAGGED-1, a transmembrane ligand for the NOTCH receptor, is downregulated in the PTEN-null fibroblasts leading to a loss in the paracrine activation of NOTCH signaling from the surrounding stroma. Reintroduction of JAGGED-1 expression within the PTEN-null fibroblasts was sufficient to abrogate the observed increase in colony forming activity implying a direct role for stromal JAGGED-1 in regulation of MaSC properties. Importantly, breast cancer patients whose tumors express both low stromal JAG1 and low stromal PTEN exhibit a shorter time to recurrence than those whose tumors express low levels of either alone suggesting similar stromal signaling in advanced disease. Combined, these results unveil a novel stromal PTEN-to-JAGGED-1 axis in maintaining the MaSC niche, and subsequently inhibiting breast cancer initiation and disease progression.

Protein Kinase C Beta in the Tumor Microenvironment Promotes Mammary Tumorigenesis

Protein kinase C beta (PKCβ) expression in breast cancer is associated with a more aggressive tumor phenotype, yet the mechanism for how PKCβ is pro-tumorigenic in this disease is still unclear. Interestingly, while it is known that PKCβ mediates angiogenesis, immunity, fibroblast function and adipogenesis, all components of the mammary tumor microenvironment (TME), no study to date has investigated whether stromal PKCβ is functionally relevant in breast cancer. Herein, we evaluate mouse mammary tumor virus–polyoma middle T-antigen (MMTV–PyMT) induced mammary tumorigenesis in the presence and absence of PKCβ. We utilize two model systems: one where PKCβ is deleted in both the epithelial and stromal compartments to test the global requirement for PKCβ on tumor formation, and second, where PKCβ is deleted only in the stromal compartment to test its role in the TME. MMTV–PyMT mice globally lacking PKCβ live longer and develop smaller tumors with decreased proliferation and decreased macrophage infiltration. Similarly, when PKCβ is null exclusively in the stroma, PyMT-driven B6 cells form smaller tumors with diminished collagen deposition. These experiments reveal for the first time a tumor promoting role for stromal PKCβ in MMTV–PyMT tumorigenesis. In corroboration with these results, PKCβ mRNA (Prkcb) is increased in fibroblasts isolated from MMTV–PyMT tumors. These data were confirmed in a breast cancer patient cohort. Combined these data suggest the continued investigation of PKCβ in the mammary TME is necessary to elucidate how to effectively target this signaling pathway in breast cancer.

Hypomethylation of the MMP7 promoter and increased expression of MMP7 distinguishes the basal-like breast cancer subtype from other triple-negative tumors

Identification of novel targets for the treatment of basal-like breast cancer is essential for improved outcomes in patients with this disease. This study investigates the association of MMP7 expression and MMP7 promoter methylation with subtype and outcome in breast cancer patient cohorts. Immunohistochemical analysis was performed on a breast cancer tissue microarray and validated in independent histological samples. MMP7 expression significantly correlated with patient age, tumor size, triple-negative (TN) status, and recurrence. Analysis of publically available datasets confirmed MMP7 gene expression as a prognostic marker of breast cancer metastasis, particularly metastasis to the brain and lungs. Methylation of the MMP7 promoter was assessed by methylation-specific PCR in a panel of breast cancer cell lines and patient tumor samples. Hypomethylation of the MMP7 promoter significantly correlated with TN status in DNA from patient tumor samples, and this association was confirmed using The Cancer Genome Atlas (TCGA) dataset. Evaluation of a panel of breast cancer cell lines and data from the Curtis and TCGA breast carcinoma datasets revealed that elevated MMP7 expression and MMP7 promoter hypomethylation are specific biomarkers of the basal-like molecular subtype which shares considerable, but not complete, overlap with the clinical TN subtype. Importantly, MMP7 expression was identified as an independent predictor of pathological complete response in a large breast cancer patient cohort. Combined, these data suggest that MMP7 expression and MMP7 promoter methylation may be useful as prognostic biomarkers. Furthermore, MMP7 expression and promoter methylation analysis may be effective mechanisms to distinguish basal-like breast cancers from other triple-negative subtypes. Finally, these data implicate MMP7 as a potential therapeutic target for the treatment of basal-like breast cancers.

Changes in BAI1 and Nestin Expression Are Prognostic Indicators for Survival and Metastases in Breast Cancer and Provide Opportunities for Dual Targeted Therapies

The 2-year survival rate of patients with breast cancer brain metastases is less than 2%. Treatment options for breast cancer brain metastases are limited, and there is an unmet need to identify novel therapies for this disease. Brain angiogenesis inhibitor 1 (BAI1) is a GPCR involved in tumor angiogenesis, invasion, phagocytosis, and synaptogenesis. For the first time, we identify that BAI1 expression is significantly reduced in breast cancer and higher expression is associated with better patient survival. Nestin is an intermediate filament whose expression is upregulated in several cancers. We found that higher Nestin expression significantly correlated with breast cancer lung and brain metastases, suggesting both BAI1 and Nestin can be therapeutic targets for this disease. Here, we demonstrate the ability of an oncolytic virus, 34.5ENVE, to target and kill high Nestin-expressing cells and deliver Vstat120 (extracellular fragment of BAI1). Finally, we created two orthotopic immune-competent murine models of breast cancer brain metastases and demonstrated 34.5ENVE extended the survival of immune-competent mice bearing intracranial breast cancer tumors. Mol Cancer Ther; 14(1); 307–14. ©2014 AACR.

FOXC1 Is Enriched in the Mammary Luminal Progenitor Population, but Is Not Necessary for Mouse Mammary Ductal Morphogenesis

ABSTRACT Expression of FOXC1, a forkhead box transcription factor, correlates with the human basal-like breast cancer (BLBC) subtype, and functional analyses have revealed its importance for in vitro invasiveness of BLBC cells. Women diagnosed with this breast tumor subtype have a poorer outcome because of the lack of targeted therapies; thus, continued investigation of factors driving these tumors is critical to uncover novel therapeutic targets. Several processes that dictate normal mammary morphogenesis parallel cancer progression, and enforced expression of FOXC1 can induce a progenitor state in more-differentiated mammary epithelial cells. Consequently, evaluating how FOXC1 functions in the normal gland is critical to further understand BLBC biology. Although FOXC1 is well known to control normal development of a number of tissues, its role in the mammary gland has not yet been investigated. Herein, we describe FOXC1 expression patterning in the normal breast, where it is localized to the basal/myoepithelium, suggesting that FOXC1 would be required for normal development. However, mammary glands lacking Foxc1 have no overt defect in ductal outgrowth, alveologenesis, or lineage specification. Of significant interest, we found that expression of FOXC1 is enriched in the normal luminal progenitor population, which is the postulated cell of origin of BLBC. These results indicate that FOXC1 is unnecessary for mammary morphogenesis and that its role in BLBC likely involves processes that are unrelated to cell lineage specification.

Synthetic Lethality of PARP Inhibition and Ionizing Radiation is p53-dependent

PARP inhibitors (PARPi) are potentially effective therapeutic agents capable of inducing synthetic lethality in tumors with deficiencies in homologous recombination (HR)-mediated DNA repair such as those carrying BRCA1 mutations. However, BRCA mutations are rare, the majority of tumors are proficient in HR repair, and thus most tumors are resistant to PARPi. Previously, we observed that ionizing radiation (IR) initiates cytoplasmic translocation of BRCA1 leading to suppression of HR-mediated DNA repair and induction of synthetic PARPi lethality in wild-type BRCA1 and HR-proficient tumor cells. The tumor suppressor p53 was identified as a key factor that regulates DNA damage–induced BRCA1 cytoplasmic sequestration following IR. However, the role of p53 in IR-induced PARPi sensitization remains unclear. This study elucidates the role of p53 in IR-induced PARPi cytotoxicity in HR-proficient cancer cells and suggests p53 status may help define a patient population that might benefit from this treatment strategy. Sensitization to PARPi following IR was determined in vitro and in vivo utilizing human breast and glioma tumor cells carrying wild-type BRCA1 and p53, and in associated cells in which p53 function was modified by knockdown or mutation. In breast and glioma cells with proficient HR repair, IR-induced BRCA1 cytoplasmic sequestration, HR repair inhibition, and subsequent PARPi sensitization in vitro and in vivo was dependent upon functional p53. Implications: Implications: p53 status determines PARP inhibitor sensitization by ionizing radiation in multiple BRCA1 and HR-proficient tumor types and may predict which patients are most likely to benefit from combination therapy. Mol Cancer Res; 16(7); 1092–102. ©2018 AACR.

Mitotic vulnerability in triple-negative breast cancer associated with LIN9 is targetable with BET inhibitors

Triple-negative breast cancers (TNBC) are highly aggressive, lack FDA-approved targeted therapies, and frequently recur, making the discovery of novel therapeutic targets for this disease imperative. Our previous analysis of the molecular mechanisms of action of bromodomain and extraterminal protein inhibitors (BETi) in TNBC revealed these drugs cause multinucleation, indicating BET proteins are essential for efficient mitosis and cytokinesis. Here, using live cell imaging, we show that BET inhibition prolonged mitotic progression and induced mitotic cell death, both of which are indicative of mitotic catastrophe. Mechanistically, the mitosis regulator LIN9 was a direct target of BET proteins that mediated the effects of BET proteins on mitosis in TNBC. Although BETi have been proposed to function by dismantling super-enhancers (SE), the LIN9 gene lacks an SE but was amplified or overexpressed in the majority of TNBCs. In addition, its mRNA expression predicted poor outcome across breast cancer subtypes. Together, these results provide a mechanism for cancer selectivity of BETi that extends beyond modulation of SE-associated genes and suggest that cancers dependent upon LIN9 overexpression may be particularly vulnerable to BETi. Cancer Res; 77(19); 5395-408. ©2017 AACR.