The organization of the extracellular matrix has a profound impact on cancer development and progression. The matrix becomes aligned throughout tumor progression, providing “highways” for tumor cell invasion. Aligned matrix is associated with breast density and is a negative prognostic factor in several cancers; however, the underlying mechanisms regulating this reorganization remain poorly understood. Deletion of the tumor suppressor Pten in the stroma was previously shown to promote extracellular matrix expansion and tumor progression. However, it was unknown if PTEN also regulated matrix organization. To address this question, a murine model with fibroblast-specific Pten deletion was used to examine how PTEN regulates matrix remodeling. Using second harmonic generation microscopy, Pten deletion was found to promote collagen alignment parallel to the mammary duct in the normal gland and further remodeling perpendicular to the tumor edge in tumor-bearing mice. Increased alignment was observed with Pten deletion in vitro using fibroblast-derived matrices. PTEN loss was associated with fibroblast activation and increased cellular contractility, as determined by traction force microscopy. Inhibition of contractility abrogated the increased matrix alignment observed with PTEN loss. Murine mammary adenocarcinoma cells cultured on aligned matrices derived from Pten−/− fibroblasts migrated faster than on matrices from wild-type fibroblasts. Combined, these data demonstrate that PTEN loss in fibroblasts promotes extracellular matrix deposition and alignment independently from cancer cell presence, and this reorganization regulates cancer cell behavior. Importantly, stromal PTEN negatively correlated with collagen alignment and high mammographic density in human breast tissue, suggesting parallel function for PTEN in patients.

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.

Disrupting paracrine Hedgehog signaling in pancreatic cancer stroma through genetic deletion of fibroblast Smoothened leads to proteasomal degradation of fibroblast PTEN and accelerates tumor growth.

The importance of the tumor–associated stroma in cancer progression is clear. However, it remains uncertain whether early events in the stroma are capable of initiating breast tumorigenesis. Here, we show that in the mammary glands of non-tumor bearing mice, stromal-specific phosphatase and tensin homolog (Pten) deletion invokes radiation-induced genomic instability in neighboring epithelium. In these animals, a single dose of whole-body radiation causes focal mammary lobuloalveolar hyperplasia through paracrine epidermal growth factor receptor (EGFR) activation, and EGFR inhibition abrogates these cellular changes. By analyzing human tissue, we discover that stromal PTEN is lost in a subset of normal breast samples obtained from reduction mammoplasty, and is predictive of recurrence in breast cancer patients. Combined, these data indicate that diagnostic or therapeutic chest radiation may predispose patients with decreased stromal PTEN expression to secondary breast cancer, and that prophylactic EGFR inhibition may reduce this risk.

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 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 mammary stem cell 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 mammary epithelial stem cell niche, and subsequently inhibiting breast cancer initiation and disease progression.

Platelet-derived growth factor receptor-beta (PDGFRβ) is a receptor tyrosine kinase found in cells of mesenchymal origin such as fibroblasts and pericytes. Activation of this receptor is dependent on paracrine ligand induction, and its preferred ligand PDGFB is released by neighboring epithelial and endothelial cells. While expression of both PDGFRβ and PDGFB has been noted in patient breast tumors for decades, how PDGFB-to-PDGFRβ tumor-stromal signaling mediates breast cancer (BC) initiation, progression, and metastasis remains unclear. Here we demonstrate this paracrine signaling pathway mediates both primary tumor growth and metastasis; specifically, metastasis to the brain. Elevated levels of PDGFB accelerated orthotopic tumor growth and intracranial growth of mammary tumor cells while mesenchymal-specific expression of an activating mutant PDGFRβ (PDGFRβ D849V) exerted pro-proliferative signals on adjacent mammary tumor cells. Stromal expression of PDGFRβ D849V also promoted brain metastases of mammary tumor cells expressing high PDGFB when injected intravenously. In the brain, expression of PDGFRβ D849V was observed within a subset of astrocytes, and aged mice expressing PDGFRβ D849V exhibited reactive gliosis. Importantly, the PDGFR-specific inhibitor crenolanib significantly reduced intracranial growth of mammary tumor cells. In a tissue microarray comprised of 363 primary human breast tumors, high PDGFB protein expression was prognostic for brain metastases, but not metastases to other sites. Our results advocate the use of mice expressing PDGFRβ D849V in their stromal cells as a preclinical model of BC-associated brain metastases (BCBM) and support continued investigation into the clinical prognostic and therapeutic use of PDGFB-to-PDGFRβ signaling in women with BC.

Breast cancer is a leading cause of mortality in women worldwide, in part due to the tumor microenvironment which increases tumor heterogeneity and abets tumor growth. Fibroblasts are cells of mesenchymal origin that are an important component of normal and tumor stroma. Genetic alterations in these cells were shown by several groups including our own to cause fibroblast activation and fuel tumor progression giving rise to more aggressive disease. Platelet-Derived Growth Factor Receptor (PDGFR) alpha is a receptor tyrosine kinase that is chiefly expressed in mesenchymal cells such as fibroblasts. Ligand binding (PDGFAA) activates this receptor. PDGFRα signaling plays critical roles in development and aberrant signaling is seen in several types of cancer, such as lung, pancreas, GI and brain. The central goal of this study was to elucidate the role of stromal PDGFRα in breast cancer development and metastasis, where its role remains largely unknown. To address this goal, we developed a genetic mouse model of stromal activation of PDGFRα in the mammary gland by crossing an auto-activating Pdgfra mutant allele with a mesenchymal specific Cre recombinase. We found that stromal PDGFRα activation completely abrogated postnatal mammary gland ductal formation, with significantly reduced terminal end bud formation. PDGFRα activation also led to progressive fibrosis in the mouse mammary fat pad. As early as four weeks of age, mammary collagen (trichrome staining; second harmonic generation) and hyaluronan deposition (Alcian Blue) was greatly increased in vivo. In fact, this increase in collagen and hyaluronan deposition in mutant animals is believed to be responsible for the observed increased in stiffness of mutant mammary tissue (atomic force microscopy {AFM}). pFAK, which can be activated due to mechanical stress, was increased in mammary epithelia of the mutant mice in vivo corroborating the AFM results. Further, when tumor cells were injected into the mammary glands of the PDGFRα mutants, tumors grew faster as compared to controls. Importantly, we found that mRNA expression of PDGFRA correlates with worsened patient outcomes in HER2+ disease, while expression of both the ligand (PDGFA) and the receptor were found to correlate with increased incidence of lung metastases. We further discovered that in HER2+ patients, PDGFRA levels correlate with breast density. Breast density is the third strongest risk factor for breast cancer, and is directly related to collagen deposition and breast stiffness, thus suggesting a novel predictive role of PDGFRA as a molecular readout of stiffness and density. Studies are underway to utilize mouse models of HER2+ breast cancer to study both primary tumor growth and metastases. Taken together, our mouse studies and paralleling human data analyses suggest that the stromal PDGFRα signaling provides a novel theranostic window in breast cancer treatment and prognosis. Citation Format: Anisha Mathur Hammer, Gina M. Sizemore, Vasudha Shukla, Steven T. Sizemore, Maria Cuitino, Cynthia J. Timmers, Quinn Verfurth, Arnab Chakravarti, Gustavo W. Leone, Samir N. Ghadiali, Michael C. Ostrowski. PDGFR-α induced stiffness abrogates mammary ductal development and enhances tumorigenesis in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4337. doi:10.1158/1538-7445.AM2017-4337

Stromal platelet-derived growth factor receptor-beta (PDGFRβ) has emerged as an actionable mediator of breast tumor-stromal communication. As a receptor tyrosine kinase, PDGFRβ is activated by its ligand, PDGFB, which is released by neighboring tumor epithelium and endothelium. However, how PDGF signaling mediates breast cancer (BC) initiation, progression, and metastasis remains unclear. To evaluate PDGFRβ in this disease, we developed a mouse model of stromal-specific PDGFRβ activation using the Fsp-cre transgene previously published by our group. Mesenchymal-specific activation of PDGFRβ promotes preferential experimental brain metastasis of PDGFB-expressing mammary tumor cells when injected intravenously and accelerates intracranial tumor growth of these cells. Mammary tumor cells expressing low levels of PDGFB do not exhibit a similar increase in brain metastases in PDGFRβ mutant mice. To our knowledge, this is the first example where genetic manipulation of the stroma leads to an increased incidence of BCBM. Our pre-clinical data suggests that primary breast tumors that express high PDGFB could preferentially metastasize to the brain. To test this in patients, we analyzed PDGFB protein expression in a tissue microarray comprised of HER2-positive and triple negative BC primary tumors. While high PDGFB did not correlate with site-independent metastatic recurrence, it was prognostic of brain metastasis, mirroring our mouse data. Our findings suggest that high primary tumor PDGFB expression defines a subset of BC patients predisposed to brain metastases. These patients may benefit from therapeutic intervention of PDGFRβ signaling. To test this pre-clinically, we treated mice harboring intracranial tumors with the PDGFR-specific inhibitor, crenolanib. Excitingly, crenolanib treatment significantly inhibited the brain tumor burden in these mice. Combined, our findings (1) advocate that primary tumor expression of PDGFB is a novel prognostic biomarker for the development of BCBM and (2) support clinical trial evaluation of PDGFR inhibitors for the prevention and treatment of BCBM.