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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–stroma signaling mediates breast cancer initiation, progression, and metastasis remains unclear. Here we demonstrate this paracrine signaling pathway that 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 (PDGFRβD849V) exerted proproliferative signals on adjacent mammary tumor cells. Stromal expression of PDGFRβD849V also promoted brain metastases of mammary tumor cells expressing high PDGFB when injected intravenously. In the brain, expression of PDGFRβD849V was observed within a subset of astrocytes, and aged mice expressing PDGFRβD849V 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 PDGFRβD849V in their stromal cells as a preclinical model of breast cancer–associated brain metastases and support continued investigation into the clinical prognostic and therapeutic use of PDGFB-to-PDGFRβ signaling in women with breast cancer. Significance: These studies reveal a previously unknown role for PDGFB-to-PDGFRβ paracrine signaling in the promotion of breast cancer brain metastases and support the prognostic and therapeutic clinical utility of this pathway for patients. See related article by Wyss and colleagues, p. 594

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 initiation, progression, and metastasis remains unclear. To test this important research question, we developed a mouse model of mesenchymal-specific PDGFRβ hyper-activation. PDGFRβ mutant mammary glands exhibit increased tertiary side-branching and epithelial proliferation confirming a stromal-specific PDGFRβ effect on neighboring epithelium during normal development. To test the effect of hyper-active mesenchymal PDGFRβ on disease progression, experimental tail vein metastasis assays were performed where we observed prominent brain metastases in 50% of the PDGFRβ mutantmice (n=5/10) with no brain lesions seen in controls (n=0/19). There was no difference in the incidence of lung or liver metastases in the mutant mice suggesting a pro-metastatic function for PDGFRβ in the brain metastatic niche. To rule out dysfunction of the blood brain barrier contributing to the observed metastatic spread, we then intracranially injected mammary tumor cells, and as expected based on our metastasis assay, found that larger tumors formed in the brains of PDGFRβ mutant mice versus controls. To our knowledge, these combined findings are the first example where genetic manipulation of the stroma increases breast cancer associated brain metastases (BCBM). Given that these pre-clinical data suggest that primary breast tumors expressing high PDGFB could preferentially metastasize to the brain, we analyzed PDGFB protein expression in a tissue microarray comprised of HER2-positive and triple negative breast cancer (TNBC) primary tumors (total n=425). While high PDGFB did not correlate with site-independent metastatic recurrence, it was prognostic of brain metastasis, mirroring our mouse data. Evaluation of PDGFB in a small cohort of matched primary breast tumors with associated brain (n=5) and lung metastases (n=2) revealed intense PDGFB staining in 100% of the brain metastases, but only 50% of the lung metastases. These findings further suggest that high primary tumor PDGFBexpression defines a subset of breast cancer patients predisposed to brain metastases and that these patients may benefit from therapeutic inhibition 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 to date (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. Ongoing studies are evaluating how the PDGFRβ-expressing mesenchymal cells within the brain promote a pro-metastatic niche. Citation Format: Sizemore GM, Hammer AM, Thies KA, Hildreth BE, Russell LO, Sizemore ST, Trimboli AJ, Kladney RD, Steck SA, Das M, Bolyard CM, Pilarski R, Schoenfield L, Otero J, Chakravarti A, Ringel M, Kaur B, Leone G, Ostrowski MC. Platelet derived growth factor receptor-β signaling: A novel therapeutic target for breast cancer associated brain metastasis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-11.

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., The tumor microenvironment influences tumor progression. Here the authors show that lack of stromal PTEN phosphatase induces DNA repair defects in the neighboring mammary gland epithelial cells via hyperactivation of EGF-receptor signaling, resulting in higher radiation-induced DNA damage and hyperplasia.

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.

Introduction: Multiple epidemiological studies have shown that prolonged breastfeeding is associated with a reduced risk of developing triple-negative/basal-like breast cancers (TN/BLBC). However, no preclinical models that delineate the mechanism of this link exist. This understanding is critical not only to prevent TN/BLBC, but also to address disparity in breast cancer outcomes, as African-American women have a higher incidence of TN/BLBC and lower prevalence of breastfeeding. We present our data using mouse models that provides new insights into this link. Experimental procedure: We modeled gradual involution (GI) and abrupt involution (AI) of mammary glands (MGs) in wild type FVB/N mice. Uniparous mice were assigned to AI or GI cohorts either by removal of all pups on day7 postpartum (AI) or allowing the pups to naturally wean (GI). MGs were harvested for analysis on postpartum day28, day56 and day120. We assessed MG morphology/histology using whole mounts and H&E stained sections, collagen deposition using Trichrome and PicroSirus red staining, inflammatory markers and immune cell infiltration using immunohistochemistry. Mammary epithelial cell hierarchy was analyzed by Fluorescence-Activated Cell Sorting of a single cell suspension prepared from the MGs. Gene expression was analyzed in mouse mammary epithelial cells using Affymatrix Gene ChIP Mouse Transcriptome array 1.0 and Gene Set Enrichment Analysis (GSEA) was used to analyze gene expression data. Summary: Abruptly involuted MGs exhibited altered morphology including denser stroma, increased collagen deposition with higher levels of Type I collagen, increased inflammation (pStat3-Y705), increased immune cell infiltration and proliferation compared to GI glands. The mammary epithelial cell hierarchy was disrupted with marked expansion of the luminal progenitor (LP) population in the AI glands but not in the GI glands, a trend frequently observed in women heterozygous for BRCA1 mutation at higher risk of developing BLBC. Enrichment of an LP gene signature and Notch pathway was observed in mouse LP cells isolated from the AI glands. Most strikingly, the AI glands developed alveolar hyperplasia, and squamous metaplasia within 4 months of removal of the pups. Conclusion: Involution leading to a pro-inflammatory milieu in the MG is well established. Whether involution that happens gradually after prolonged breastfeeding differs in its effect on MGs has not been well studied. Using novel animal modeling to study the differences in MGs following abrupt vs gradual involution, we report a distinct morphology in the mammary tissue favoring pro-carcinogenic changes following AI. Furthermore, we show for the first time, expansion of the LP population in AI glands, which is known to be cell of origin for TN/BLBC potentially linking the risk of TN/BLBC with lack of breastfeeding. Further studies are ongoing. Citation Format: Mustafa M. Basree, Neelam Shinde, Christopher Koivisto, Maria Cuitino, Raleigh Kladney, Allen Zhang, Hee Kyung Kim, Anthony Trimboli, Jianying Zhang, Gustavo W. Leone, Gina M. Sizemore, Sarmila Majumder, Bhuvaneswari Ramaswamy. Breastfeeding protects against pro-tumorigenic changes in the mammary gland by limiting epithelial luminal progenitor cell expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2242.

Stromal 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 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 (PDGFRβ mutant). PDGFRβ mutant mammary glands exhibit increased tertiary side-branching and epithelial proliferation confirming a stromal-specific PDGFRβ effect on neighboring epithelium. To evaluate the functional relevance of PDGFRβ activation on metastatic progression, we performed tail vein injection of PDGFB expressing murine mammary tumor cells and, surprisingly, observed brain metastases in 50% of the PDGFRβ mutant mice while no brain lesions were seen in controls. There was no difference in the incidence of lung, liver or bone metastases. Mammary tumor cells expressing low PDGFB did not exhibit a similar increase in brain metastases in mutant mice. While there is no observable difference in blood brain barrier permeability in the mutant mice, we bypassed this variable by intracranially injecting mammary tumor cells and found that larger tumors formed in the brains of PDGFRβ mutant mice versus controls. To our knowledge, this is the first example where genetic manipulation of the stroma leads to an increased incidence of BCBM. Also, 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 breast cancer (TNBC) primary tumors. While high PDGFB did not correlate with site-independent metastatic recurrence, it was prognostic of brain metastasis, mirroring our mouse data. Evaluation of PDGFB in a small cohort of matched primary breast tumors with associated brain (n=5) and lung metastases (n=2) revealed intense PDGFB staining in 100% of the brain metastases, but only 50% of the lung metastases. Our findings suggest high primary tumor PDGFB expression defines a subset of breast cancer 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. Citation Format: Katie A. Thies, Anisha M. Hammer, B. Eason Hildreth, Luke O. Russell, Steven T. Sizemore, Anthony J. Trimboli, Raleigh D. Kladney, Chelsea M. Bolyard, Robert Pilarski, Lynn Schoenfield, Jose Otero, Arnab Chakravarti, Matthew Ringel, Balveen Kaur, Gustavo Leone, Michael C. Ostrowski, Gina M. Sizemore. Stromal platelet derived growth factor receptor (PDGFRβ) signaling: A novel therapeutic target for breast cancer brain metastasis (BCBM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 49.

Centrosome amplification (CA) contributes to carcinogenesis by generating aneuploidy. Elevated frequencies of CA in most benign breast lesions and primary tumors suggest a causative role for CA in breast cancers. Clearly, identifying which and how altered signal transduction pathways contribute to CA is crucial to breast cancer control. Although a causative and cooperative role for c-Myc and Ras in mammary tumorigenesis is well documented, their ability to generate CA during mammary tumor initiation remains unexplored. To answer that question, K-Ras(G12D) and c-Myc were induced in mouse mammary glands. Although CA was observed in mammary tumors initiated by c-Myc or K-Ras(G12D), it was detected only in premalignant mammary lesions expressing K-Ras(G12D). CA, both in vivo and in vitro, was associated with increased expression of the centrosome-regulatory proteins, cyclin D1 and Nek2. Abolishing the expression of cyclin D1, Cdk4 or Nek2 in MCF10A human mammary epithelial cells expressing H-Ras(G12V) abrogated Ras-induced CA, whereas silencing cyclin E1 or B2 had no effect. Thus, we conclude that CA precedes mammary tumorigenesis, and interfering with centrosome-regulatory targets suppresses CA.

The tumour stroma is believed to contribute to some of the most malignant characteristics of epithelial tumours. However, signalling between stromal and tumour cells is complex and remains poorly understood. Here we show that the genetic inactivation of Pten in stromal fibroblasts of mouse mammary glands accelerated the initiation, progression and malignant transformation of mammary epithelial tumours. This was associated with the massive remodelling of the extracellular matrix (ECM), innate immune cell infiltration and increased angiogenesis. Loss of Pten in stromal fibroblasts led to increased expression, phosphorylation (T72) and recruitment of Ets2 to target promoters known to be involved in these processes. Remarkably, Ets2 inactivation in Pten stroma-deleted tumours ameliorated disruption of the tumour microenvironment and was sufficient to decrease tumour growth and progression. Global gene expression profiling of mammary stromal cells identified a Pten-specific signature that was highly represented in the tumour stroma of patients with breast cancer. These findings identify the Pten-Ets2 axis as a critical stroma-specific signalling pathway that suppresses mammary epithelial tumours.

Dysregulation of protein kinase A (PKA) activity, caused by loss of function mutations in PRKAR1A, is known to induce tumor formation in the inherited tumor syndrome Carney complex (CNC) and is also associated with sporadic tumors of the thyroid and adrenal. We have previously shown that Prkar1a+/− mice develop schwannomas reminiscent of those seen in CNC and that similar tumors are observed in tissue-specific knockouts (KO) of Prkar1a targeted to the neural crest. Within these tumors, we have previously described the presence of epithelial islands, although the nature of these structures was unclear. In this article, we report that these epithelial structures are derived from KO cells originating in the neural crest. Analysis of the mesenchymal marker vimentin revealed that this protein was markedly down-regulated not only from the epithelial islands, but also from the tumor as a whole, consistent with mesenchymal-to-epithelial transition (MET). In vitro, Prkar1a null primary mouse embryonic fibroblasts, which display constitutive PKA signaling, also showed evidence for MET, with a loss of vimentin and up-regulation of the epithelial marker E-cadherin. Reduction of vimentin protein occurred at the posttranslational level and was rescued by proteasomal inhibition. Finally, this down-regulation of vimentin was recapitulated in the adrenal nodules of CNC patients, confirming an unexpected and previously unrecognized role for PKA in MET. [Cancer Res 2008;68(8):2671–7]

Over the past decade it has become evident that the tumor microenvironment (TME) actively participates in carcinogenesis. Tumor-associated fibroblasts, for example, modulate neighboring tumor epithelium through growth factor secretion to initiate and promote tumor growth. The platelet derived growth factor receptors (PDGFRs), PDGFRalpha and PDGFRbeta, are receptor tyrosine kinases activated by PDGF that may be critical and actionable mediators of breast tumor-stromal communication. PDGFRs are predominately expressed in breast tumor stroma while their cognate ligands are specifically expressed in tumor epithelium and associated endothelium. In some cancers, tumor-derived PDGFs act on the TME to recruit tumor associated fibroblasts; however, this role has not been described in breast cancer. To begin to evaluate a role for PDGFRbeta, we utilized publicly available gene expression data to confirm upregulation in tumor stroma compared to tumor epithelium. Importantly, PDGFRB is increased in tumor stroma compared to normal stroma. To directly test whether stromal PDGFR activation promotes tumor growth, we co-injected murine mammary tumor cells with or without PDGFR-expressing mouse mammary fibroblasts (MMFs) orthotopically in FVB/N mice. MMF inclusion increased tumor cell proliferation as well as associated angiogenesis while systemic treatment with imatinib mesylate, a small molecule inhibitor for PDGFR, restored both proliferation and angiogenesis back to baseline. These findings indicated the importance of PDGFR signaling in tumor initiation leading us to develop a mouse model of stromal-specific PDGFRbeta activation using the Fsp-cre transgene previously published by our group (henceforth referred to as “PDGFRbeta mutant”). PDGFRbeta mutant mammary glands exhibit increased tertiary side-branching and epithelial proliferation confirming a stromal-specific PDGFRbeta effect on neighboring epithelium during development. Further, MMFs isolated from the PDGFRbeta mutant mice exhibit increased motility towards PDGF-B expressing tumor cells in vitro, which implies increased response and recruitment of the mutant MMFs towards an expanding tumor. To test whether PDGFRbeta mutant mice harbor a mammary TME supportive of increased tumor growth, we injected murine mammary tumor cells orthotopically into either control or PDGFRbeta mutant mice finding that the time required to meet early removal criteria (tumor &gt1.2cm3) was shorter in the mutant mice compared to controls. Ongoing studies are evaluating whether systemic PDGFR inhibition will abrogate this observed increase in tumorigenesis. In summary, our data suggest that stromal PDGFRbeta signaling is pro-tumorigenic in breast cancer and that inhibition using well-described PDGFR inhibitors could be a valid therapeutic approach for women whose tumors express increased PDGF-to-PDGFR tumor-stromal signaling. Citation Format: Gina M. Sizemore, Anisha M. Hammer, Katie A. Thies, Steven T. Sizemore, Anthony J. Trimboli, B. Eason Hildreth, Raleigh D. Kladney, Arnab Chakravarti, Gustavo Leone, Michael C. Ostrowski. Stromal platelet derived growth factor receptor-beta (PDGFRbeta) promotes breast cancer progression [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 2966. doi:10.1158/1538-7445.AM2017-2966

The platelet derived growth factor (PDGF) pathway is a prime example of tumor-stroma signaling in a number of cancer types. Others have shown that PDGF receptors are expressed in breast fibroblasts and pericytes while PDGF ligands are often expressed in breast cancer cells and tumor-associated endothelium; however, how PDGF signaling mediates breast cancer initiation, progression and metastasis remains unclear. Importantly, our evaluation of publicly available datasets revealed that PDGFB expression correlates with breast cancer patient metastatic recurrence leading to the hypothesis that PDGF-B to PDGFR signaling promotes metastatic progression of breast cancer. Given that PDGF-B preferentially activates PDGFRβ, we established an in vivo system to investigate this pathway during breast cancer progression. We utilized a mesenchymal-specific promoter to drive Cre recombinase and conditionally activate PDGFRβ by way of the endogenous Pdgfrb promoter (hereafter “PDGFRβ mutant”). A murine mammary tumor cell line which expresses high levels of PDGF-B was injected either by tail vein or intracranially to evaluate metastatic seeding and distant tumor growth. Following tail vein injection of tumor cells, we observed 50% incidence of brain metastases in the PDGFRβ mutant mice while no brain lesions were seen in the controls. There was no difference in incidence of lung, liver or bone metastases (other common sites of breast cancer metastasis). Not surprisingly, larger tumors formed in the brains of PDGFRβ mutant mice when cells were injected intracranially. Brains were stained for phospho-PLCγ as a way to confirm activation of PDGFRβ. To our knowledge, this is the first example where genetic manipulation of the stroma leads to an increased incidence of breast brain metastases. Furthermore, this study highlights a role for stromal activation of PDGFRβ in the brain microenvironment and during metastatic progression. For the 20-30% of patients that develop breast cancer brain metastases, the one-year survival rate is sadly less than 20%, and how the brain microenvironment contributes to metastatic seeding and subsequent growth of tumor cells remains poorly understood. To confirm translational relevance, we analyzed a small cohort of matched primary breast tumors and brain metastases for PDGFRβ expression observing strong stromal staining in fibroblasts and pericytes within and around all of the primary tumors similar to previous studies. Importantly, high PDGFRβ expression was found in the perivasculature of all associated brain metastases suggesting a functional role in the establishment or growth at this site. Combined, our findings strongly suggest that high primary tumor expression of PDGF-B/PDGFRβ might define a subset of breast cancer patients predisposed to brain metastases. These patients may benefit from therapeutic targeting of PDGFR signaling as a means to thwart metastatic seeding in the brain. Citation Format: Katie A. Thies, Anisha M. Hammer, Anthony J. Trimboli, B. Eason Hildreth, Luke O. Russell, Chelsea M. Bolyard, Raleigh D. Kladney, Steven T. Sizemore, Robert Pilarski, Lynn Schoenfield, Jose Otero, Arnab Chakravarti, Balveen Kaur, Gustavo Leone, Michael C. Ostrowski, Gina M. Sizemore. Stromal platelet derived growth factor receptor-beta (PDGFRbeta) promotes breast brain metastasis [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 3911. doi:10.1158/1538-7445.AM2017-3911

Genetic and epigenetic events that alter gene expression and/or protein function or localization are thought to be the primary mechanism that drives tumorigenesis and governs the clinical behavior of cancers. Yet, a number of studies have shown that the effects of oncogene expression or tumor suppressor ablation are highly dependent on cell type. The molecular basis for this cell-type specificity and how it contributes to tumorigenesis are unknown. Here, expression of a truncated SV40 large T antigen in murine intestinal crypts promoted the formation of numerous adenomatous polyps in the colon and small intestine. In contrast, when the same T-antigen construct is expressed in villous enterocytes, the consequences are limited to hyperplasia and dysplasia. The T-antigen–induced polyps show high levels of the proto-oncogene c-Myc protein even though there is no transport of β-catenin to the nucleus. Targeting the expression of viral oncogenes to intestinal crypts or villi provides a murine model system for studying cell-type specific effects in tumorigenesis, and is particularly relevant to the study of APC/β-catenin–independent pathways contributing to the generation of intestinal polyps. Implications: This mouse model system describes the formation of colon polyps in the absence of Wnt/β-catenin signaling. Mol Cancer Res; 12(10); 1355–64. ©2014 AACR.

A role for the tumor microenvironment (TME) in cancer progression is irrefutable and our laboratory has been at the forefront of this field providing evidence for both tumor suppressive and oncogenic roles of the TME. The PDGF pathway is an exemplar for the study of tumor-stroma interaction as PDGF receptors (PDGFR) are frequently expressed in the fibroblasts and pericytes within the tumor-associated stroma of epithelial tumors including breast cancer. In contrast, PDGF ligands are expressed by the epithelial tumor cells themselves. However, beyond a few descriptive studies, the role of interactive PDGFRβ signaling in the TME during breast cancer initiation, progression and metastases is not understood. This can be attributed in part to limited in vivo models to study the complex TME, especially for breast cancer associated metastases. To overcome this limitation, we have established a transgenic knock-in mouse model that expresses constitutively active PDGFRβ in the stroma of the mammary gland as well in the lung and the brain, two common sites of metastatic breast cancer dissemination. We have found that these mice develop mammary gland hyperplasia highlighting the importance of PDGFRβ in the TME in driving mammary epithelial cell growth. To test whether activation of mutant PDGFRβ in either the lung or the brain increases metastatic growth at either site, two experimental metastases assays were performed: (1) tail vein and (2) intracranial injections to test for lung and brain metastatic outgrowth, respectively. Tail vein injection of the non-metastatic murine mammary cancer cell line DB7 cells led to pronounced lung metastases in PDGFRβ knock-in mice in less than 4 weeks. No macrometastases were seen in the control at this same time point. Similar to the surge in lung metastasis, intracranial injection of DB7 cells led to an increase in tumor growth in brains of the mutant versus wild type controls, revealing an important role for PDGFRβ signaling in the breast cancer metastatic microenvironment. In addition, knockdown of PDGF-B in mammary cancer cells represses intracranial growth in wild type animals. Combined these data support a role for PDGFRβ signaling in the breast cancer metastatic microenvironment. Ongoing investigation is aimed to delineate how activated PDGF-B to PDGFRβ signaling primes the metastatic niche. Citation Format: Gina Sizemore, Anisha Mathur, Katie Thies, Chelsea Bolyard, Steven Sizemore, Raleigh Kladney, Anthony Trimboli, Balveen Kaur, Gustavo Leone, Michael Ostrowski. Platelet-derived growth factor receptor-β (PDGFRβ) in the breast metastatic tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C28.

We developed stromal- and epithelial-specific cre-transgenic mice to directly visualize epithelial-mesenchymal transition (EMT) during cancer progression in vivo. Using three different oncogene-driven mouse mammary tumor models and cell-fate mapping strategies, we show in vivo evidence for the existence of EMT in breast cancer and show that myc can specifically elicit this process. Hierarchical cluster analysis of genome-wide loss of heterozygosity reveals that the incidence of EMT in invasive human breast carcinomas is rare, but when it occurs it is associated with the amplification of MYC. These data provide the first direct evidence for EMT in breast cancer and suggest that its development is favored by myc-initiated events. [Cancer Res 2008;68(3):937–45]

One of the major confounding factors in the treatment of breast cancer is the cellular heterogeneity of the breast. An important cellular component of the normal breast as well as tumor stroma is the fibroblast. Fibroblasts are cells of mesenchymal origin that express platelet derived growth factor receptor alpha (PDGFR-α), a pharmaceutically targetable receptor tyrosine kinase. While activation of PDGFR-α was shown to be a poor prognostic factor in glioblastoma and gastro-intestinal stromal tumors, the role of stromal PDGFR-α signaling in mammary development and carcinomas is yet to be explored. To elucidate the role of stromal PDGFR-α signaling in this context, we utilized mice harboring the activating Pdgfra-D842V mutation knocked into the endogenous Pdgfra locus under a Lox-STOP-Lox cassette. To obtain selective activation of PDGFR-α in the fibroblastic stroma, these mice were crossed with fibroblast specific Fsp-cre mice. Whole mount carmine alum staining revealed that the D842V mutants have severely impaired mammary ductal development. Further analyses at the histological level revealed that the D842V glands have increased extra-cellular matrix (ECM) deposition (Masson's trichrome) likely due to the observable increase in fibroblasts (vimentin positivity). Immuno-fluorescent dual staining for keratin 8 and Ki67 demonstrated that stromal PDGFR-α activation increases the proliferation of neighboring mammary ductal cells, potentially making them more tumorigenic. To gain insight into the molecular mechanisms underlying this stromal-epithelial cross-talk, primary mammary epithelial cells and fibroblasts were isolated for downstream assays. Western blot analyses showed that mutant mammary fibroblasts have increased levels of activated PLC-γ, AKT and JNK which are known oncogenic pathways in the stroma. Gene expression arrays followed by global pathway analyses showed that pro-tumorigenic processes such as proliferation and metastasis are enriched in the epithelium from the mutant mice, while pro-tumor pathways such as KRAS and JNK were found to be enriched in the mutant fibroblasts. Interestingly, Pdgfra-D842V mutant mice have significantly increased stiffness as measured by atomic force microscopy of naïve tissue. This result implies that the observed increase in fibroblast number and resulting ECM deposition is sufficient to alter the entire mammary tissue. Since breast density correlates with higher breast cancer risk and tumor invasiveness, this phenotype reveals a novel function for stromal PDGFR-α signaling in breast cancer susceptibility. Studies are underway to evaluate whether these mice are predisposed to mammary tumor formation. Given our present data, we conclude that activation of stromal PDGFR-α signaling not only abrogates mammary development, but also makes the mammary ductal epithelium more tumorigenic. Citation Format: Anisha Mathur, Gina M. Sizemore, Subhasree Balakrishnan, Vasudha C. Shukla, Maria Cuitino, Anthony J. Trimboli, Samir Ghadiali, Gustavo W. Leone, Michael C. Ostrowski. Stromal PDGFR-α activation stalls mammary ductal development and increases tumorigenic potential of mammary epithelia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 985. doi:10.1158/1538-7445.AM2015-985

Background The nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) has become a potential target for the prevention and treatment of breast cancer. However, recent in vitro and in vivo studies have raised the question of whether activation of PPARγ leads to the promotion or reduction of tumor formation. Studies using several cancer cell lines, animal models, and a variety of PPARγ agonists have shown discordant results, including changes in cellular proliferation, differentiation, and apoptosis of cancer cells and tumors. Methods We studied the effects of low-, moderate-, and high-dose treatment of the PPARγ ligands 15-deoxy-Δ12,14 prostaglandin J2 (15dPGJ2) and troglitazone (TGZ) on parameters of cell growth, differentiation, and apoptosis in the epithelial breast cancer cell line MDA-MB-231. Results The biologic effects of these compounds depend largely on ligand concentration and the degree of PPARγ activation. For example, low concentrations of 15dPGJ2 (2 >10 μM and of TGZ at 100 μM blocked cell growth. TGZ (100 μM) slowed cell cycle progression, and 15dPGJ2 (10 μM) caused an S-phase arrest in the cell cycle and induced morphological characteristics consistent with apoptosis. Expression of CD36, a marker of differentiation in these cells, was induced by 2.5 μM 15dPGJ2 or 5 to 100 μM TGZ. However, higher concentrations of 15dPGJ2 did not alter CD36 expression. Transcriptional activation studies demonstrated that 15dPGJ2 is a more potent PPARγ ligand than TGZ. Regardless of the ligand used, though, low transcriptional activation correlated with an increased cellular proliferation, whereas higher levels of activation correlated with cell cycle arrest and apoptosis. Conclusions PPARγ activation induces several important and seemingly opposite changes in neoplastic cells, depending on the magnitude of PPARγ activation. These data may explain, at least in part, some of the discordant results previously reported.

Research over the past three decades has revealed that arachidonic acid (AA) and oxygen-containing derivatives of AA, termed eicosanoids, play pivotal roles in controlling key cellular events that lead to acute and chronic inflammation (for review, see [1]). While it has been suggested for more than 50 years that diets high in certain fatty acids stimulate tumor development in animals, only within the last five years has there been strong mechanistic evidence that links AA metabolism to cell proliferation and apoptosis. Interest in this link began to intensify in the early 1990s as a result of several key observations; (1) Epidemiological studies in humans demonstrated that the use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a reduction in colon cancer deaths [2–5]. (2) Animal studies revealed that NSAIDs attenuate in vivo growth in colon, mammary, esophageal, lung and oral cancers [6–14]. (3) There is increased production of eicosanoids (in particular, prostaglandins) as well as a marked upregulation of certain isoforms of cyclooxygenase in transformed cells and tumors [6, 15–21]. Collectively, these observations emphasized the importance of understanding the mechanisms by which AA metabolism influences cellular events such as mitogenesis and apoptosis. This chapter will review in vitro and in vivo studies that have examined the relationship between AA metabolism and cell proliferation with a special emphasis on apoptosis. While the bulk of the data collected to date on this topic has focused on AA metabolism and cell proliferation in cellular models of cancer, similar relationships are now being observed in cell proliferation and survival during inflammation. It is important to point out that most of the major molecular events that link AA metabolism and cell proliferation currently are poorly understood and many events remain to be elucidated. Moreover, there are several circumstances where AA or its metabolites appear to have one effect on a particular cell type and another (often the opposite effect) in other cell types. Further progress in this area will depend, in large part, on our capacity to resolve these ambiguous links and controversial findings.

Tumor fibroblasts are active partners in tumor progression, but the genes and pathways that mediate this collaboration are ill-defined. Previous work demonstrates that Ets2 function in stromal cells significantly contributes to breast tumor progression. Conditional mouse models were used to study the function of Ets2 in both mammary stromal fibroblasts and epithelial cells. Conditional inactivation of Ets2 in stromal fibroblasts in PyMT and ErbB2 driven tumors significantly reduced tumor growth, however deletion of Ets2 in epithelial cells in the PyMT model had no significant effect. Analysis of gene expression in fibroblasts revealed a tumor- and Ets2-dependent gene signature that was enriched in genes important for ECM remodeling, cell migration, and angiogenesis in both PyMT and ErbB2 driven-tumors. Consistent with these results, PyMT and ErbB2 tumors lacking Ets2 in fibroblasts had fewer functional blood vessels, and Ets2 in fibroblasts elicited changes in gene expression in tumor endothelial cells consistent with this phenotype. An in vivo angiogenesis assay revealed the ability of Ets2 in fibroblasts to promote blood vessel formation in the absence of tumor cells. Importantly, the Ets2-dependent gene expression signatures from both mouse models were able to distinguish human breast tumor stroma from normal stroma, and correlated with patient outcomes in two whole tumor breast cancer data sets. The data reveals a key function for Ets2 in tumor fibroblasts in signaling to endothelial cells to promote tumor angiogenesis. The results highlight the collaborative networks that orchestrate communication between stromal cells and tumor cells, and suggest that targeting tumor fibroblasts may be an effective strategy for developing novel anti-angiogenic therapies.

Applying a novel bioinformatics strategy, the objective of this study was to identify signaling interactions that occur within cell types of the mouse mammary gland. Mining through typical microarray data is quite a challenge, but it is even more difficult to extract biological relevance within the mammary microenvironment. The approach here involves a combinatorial strategy that effectively integrates basic research design, statistics, and bioinformatics. We hypothesized that cells in the microenvironment potentiate tumorigenesis by signaling with each other through critical pathways sharing common ‘network hubs’. Mammary glands were harvested from 8-week-old mice that were wildtype (WT) or had fibroblast-specific Pten deletion (fPten-/-). Fibroblasts, macrophages, endothelial and epithelial cells were selected from the glands through cell sorting and selective cell culture. Replicate samples of their cDNA were applied onto Mouse Exon Arrays. The raw data were processed through Robust Mean Analysis (RMA), and then subjected to Empirical Bayes Arrays (EBArrays) analysis to generate lists of differentially expressed genes between the fPten-/- and WT mice for the four cell types. Each gene was given a probability value as a measure of its true differential expression, and only genes with a value more than or equal to 0.7 were considered for further analyses. Three bioinformatics tools- Database for Annotation, Visualization and Integrated Discovery (DAVID), Biometric Research Branch (BRB) ArrayTools, and Ingenuity Pathway Analysis® (IPA) - were used to analyze the four gene lists. Analysis by DAVID revealed that for the fibroblasts and the macrophages, the major biological machinery activated in the fPten-/- mice related to extracellular matrix remodeling and immune response. The endothelial cells displayed genes involved in complement activation pathway. Interestingly, the genes expressed in epithelial cells related to various aspects of epithelial-mesenchymal transition. Together, this suggests that even in the absence of tumor, the fPten-/- stromal signaling infuses a tumorigenic potential into the microenvironment. A filter on BRB ArrayTools selected genes that had a 2-fold change. Average hierarchical clustering based on Spearman correlation was done to generate heat maps. This refined the list of significant genes between the genotypes for each cell type. The four fPten-/- derived genelists were then uploaded into IPA. This confirmed the output from DAVID, and further revealed that ERBB2, MMP9, TNFα, TGFβ, and NFκB, β-catenin, and Ets, were key network hubs and transcription factors, respectively, through which signaling occurred in the mammary microenvironment. The top merged networks across cell types displayed shared nodes important for communication. In summary, this analytical approach gave an insight into the ‘network players’ and ‘cellular crosstalk’ critical for a tumorigenic environment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 110.