Anthony J. Trimboli

Pten in stromal fibroblasts suppresses mammary epithelial tumours

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.

Direct Evidence for Epithelial-Mesenchymal Transitions in Breast Cancer

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.

Evidence for a stepwise program of extrathymic T cell development within the human tonsil

The development of a broad repertoire of T cells, which is essential for effective immune function, occurs in the thymus. Although some data suggest that T cell development can occur extrathymically, many researchers remain skeptical that extrathymic T cell development has an important role in generating the T cell repertoire in healthy individuals. However, it may be important in the setting of poor thymic function or congenital deficit and in the context of autoimmunity, cancer, or regenerative medicine. Here, we report evidence that a stepwise program of T cell development occurs within the human tonsil. We identified 5 tonsillar T cell developmental intermediates: (a) CD34⁺CD38dimLin⁻ cells, which resemble multipotent progenitors in the bone marrow and thymus; (b) more mature CD34⁺CD38brightLin⁻ cells; (c) CD34⁺CD1a⁺CD11c⁻ cells, which resemble committed T cell lineage precursors in the thymus; (d) CD34⁻CD1a⁺CD3⁻CD11c⁻ cells, which resemble CD4⁺CD8⁺ double-positive T cells in the thymus; and (e) CD34⁻CD1a⁺CD3⁺CD11c⁻ cells. The phenotype of each subset closely resembled that of its thymic counterpart. The last 4 populations expressed RAG1 and PTCRA, genes required for TCR rearrangement, and all 5 subsets were capable of ex vivo T cell differentiation. TdT⁺ cells found within the tonsillar fibrous scaffold expressed CD34 and/or CD1a, indicating that this distinct anatomic region contributes to pre-T cell development, as does the subcapsular region of the thymus. Thus, we provide evidence of a role for the human tonsil in a comprehensive program of extrathymic T cell development.

Targeted Deletion of Prkar1a Reveals a Role for Protein Kinase A in Mesenchymal-to-Epithelial Transition

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.

Selective roles of E2Fs for ErbB2- and Myc-mediated mammary tumorigenesis

Previous studies have demonstrated that cyclin D1, an upstream regulator of the Rb/E2F pathway, is an essential component of the ErbB2/Ras (but not the Wnt/Myc) oncogenic pathway in the mammary epithelium. However, the role of specific E2fs for ErbB2/Ras-mediated mammary tumorigenesis remains unknown. Here, we show that in the majority of mouse and human primary mammary carcinomas with ErbB2/HER2 overexpression, E2f3a is up-regulated, raising the possibility that E2F3a is a critical effector of the ErbB2 oncogenic signaling pathway in the mammary gland. We examined the consequence of ablating individual E2fs in mice on ErbB2-triggered mammary tumorigenesis in comparison to a comparable Myc-driven mammary tumor model. We found that loss of E2f1 or E2f3 led to a significant delay in tumor onset in both oncogenic models, whereas loss of E2f2 accelerated mammary tumorigenesis driven by Myc-overexpression. Furthermore, southern blot analysis of final tumors derived from conditionally deleted E2f3−/loxP mammary glands revealed that there is a selection against E2f3−/− cells from developing mammary carcinomas, and that such selection pressure is higher in the presence of ErbB2 activation than in the presence of Myc activation. Taken together, our data suggest oncogenic activities of E2F1 and E2F3 in ErbB2- or Myc-triggered mammary tumorigenesis, and a tumor suppressor role of E2F2 in Myc-mediated mammary tumorigenesis.

Ets2 in tumor fibroblasts promotes angiogenesis in breast cancer.

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.

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.

Viral Oncogene Expression in the Stem/Progenitor Cell Compartment of the Mouse Intestine Induces Adenomatous Polyps

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.

Abstract 110: A bioinformatics view of networking in the mouse mammary microenvironment

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC 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.

Abstract C28: Platelet-derived growth factor receptor-β (PDGFRβ) in the breast metastatic tumor microenvironment

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.