Julie A. Wallace

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.

Pten in the breast tumor microenvironment: modeling tumor-stroma coevolution.

Solid human tumors and their surrounding microenvironment are hypothesized to coevolve in a manner that promotes tumor growth, invasiveness, and spread. Mouse models of cancer have focused on genetic changes in the epithelial tumor cells and therefore have not robustly tested this hypothesis. We have recently developed a murine breast cancer model that ablates the PTEN tumor suppressor pathway in stromal fibroblasts. Remarkably, the model resembles human breast tumors both at morphologic and molecular levels. We propose that such models reflect subtypes of tumor-stromal coevolution relevant to human breast cancer, and will therefore be useful in defining the mechanisms that underpin tumor-stroma cross-talk. Additionally, these models should also aid in molecularly classifying human breast tumors on the basis of both the microenvironment subtypes they contain as well as on the tumor subtype.

Transformed epithelial cells and fibroblasts/myofibroblasts interaction in breast tumor: a mathematical model and experiments

It is well known that tumor and its microenvironment, or stroma, interact with each other and that this interaction plays a critical role in tumor initiation, growth, and metastasis. This interaction consists of complex relations between tumor cells, stromal cells such as fibroblasts, epithelial cells and immunocytes, the vascular system, the extracellular matrix, and cytokines secreted by the cells. Understanding these relationships may lead to new therapeutic approaches to cancer. In the present paper, we consider tumor-stroma crosstalk in a simple in vitro situation which involves interaction between tumor epithelial cells from breast cancer and a microenvironment consisting of just fibroblasts. The two populations of cells are separated by a semi-permeable membrane that allows only cytokines to cross over. We develop a mathematical model that includes two critical growth factors: TGF-β, produced by the tumor cells, and EGF, secreted by the fibroblasts. The TGF-β modifies the microenvironment by transforming fibroblasts into myofibroblasts. Myofibroblasts secrete higher concentrations of EGF than fibroblasts, thereby, increasing the proliferation of tumor cells. Thus already in this simple setup one sees a mutual interaction between tumor cells and their microenvironment. We conducted experiments which show good agreement with the model’s simulations, hence confirming the model’s ability to predict aspects of tumor cell behavior in response to signaling from fibroblasts.

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.

Noncatalytic PTEN missense mutation predisposes to organ-selective cancer development in vivo

Inactivation of phosphatase and tensin homology deleted on chromosome 10 (PTEN) is linked to increased PI3K-AKT signaling, enhanced organismal growth, and cancer development. Here we generated and analyzed Pten knock-in mice harboring a C2 domain missense mutation at phenylalanine 341 (Pten(FV)), found in human cancer. Despite having reduced levels of PTEN protein, homozygous Pten(FV/FV) embryos have intact AKT signaling, develop normally, and are carried to term. Heterozygous Pten(FV/+) mice develop carcinoma in the thymus, stomach, adrenal medulla, and mammary gland but not in other organs typically sensitive to Pten deficiency, including the thyroid, prostate, and uterus. Progression to carcinoma in sensitive organs ensues in the absence of overt AKT activation. Carcinoma in the uterus, a cancer-resistant organ, requires a second clonal event associated with the spontaneous activation of AKT and downstream signaling. In summary, this PTEN noncatalytic missense mutation exposes a core tumor suppressor function distinct from inhibition of canonical AKT signaling that predisposes to organ-selective cancer development in vivo.

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.

ETS Transcription Factors in the Tumor Microenvironment

ETS factors are involved in cancer progression through transcriptional regulation of factors mediating cell cycle, cell growth, apoptosis, cell adhesion and migration. The biological processes regulated by ETS factors are important not only in tumor cells, but also in the surrounding cells comprising the tumor microenvironment. Additionally, ETS factors can serve as transcriptional activators and repressors to regulate gene expression in a cell context-specific fashion. Here we discuss recent advances in which the regulatory roles of ETS factors in stromal cells are critical during both development and cancer. These new findings uncover the importance of ETS signaling in the stromal compartment of tumors and shed light on new potential mechanisms of ETS factors.

Cry Protein Crystals: A Novel Platform for Protein Delivery

Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer’s patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues.

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.