Josie Ursini-Siegel

Insights from transgenic mouse models of ERBB2-induced breast cancer

One-third of patients with breast cancer overexpress the ERBB2 receptor tyrosine kinase, which is associated not only with a more aggressive phenotype but also reduced responsiveness to hormonal therapies. Over the past two decades, many ERBB2 mouse models for breast cancer have conclusively shown that this receptor has a causal role in breast cancer development. These mouse models have also enabled the mechanisms controlling tumour growth, angiogenesis, metastasis, dormancy and recurrence in ERBB2-positive breast cancer to be elucidated. In addition, a mouse model has recently been described that accurately recapitulates many of the hallmarks associated with the early stages of the human disease.

Integrated molecular profiles of invasive breast tumors and ductal carcinoma in situ (DCIS) reveal differential vascular and interleukin signaling

We use an integrated approach to understand breast cancer heterogeneity by modeling mRNA, copy number alterations, microRNAs, and methylation in a pathway context utilizing the pathway recognition algorithm using data integration on genomic models (PARADIGM). We demonstrate that combining mRNA expression and DNA copy number classified the patients in groups that provide the best predictive value with respect to prognosis and identified key molecular and stromal signatures. A chronic inflammatory signature, which promotes the development and/or progression of various epithelial tumors, is uniformly present in all breast cancers. We further demonstrate that within the adaptive immune lineage, the strongest predictor of good outcome is the acquisition of a gene signature that favors a high T-helper 1 (Th1)/cytotoxic T-lymphocyte response at the expense of Th2-driven humoral immunity. Patients who have breast cancer with a basal HER2-negative molecular profile (PDGM2) are characterized by high expression of protumorigenic Th2/humoral-related genes (24–38%) and a low Th1/Th2 ratio. The luminal molecular subtypes are again differentiated by low or high FOXM1 and ERBB4 signaling. We show that the interleukin signaling profiles observed in invasive cancers are absent or weakly expressed in healthy tissue but already prominent in ductal carcinoma in situ, together with ECM and cell-cell adhesion regulating pathways. The most prominent difference between low and high mammographic density in healthy breast tissue by PARADIGM was that of STAT4 signaling. In conclusion, by means of a pathway-based modeling methodology (PARADIGM) integrating different layers of molecular data from whole-tumor samples, we demonstrate that we can stratify immune signatures that predict patient survival.

ShcA signalling is essential for tumour progression in mouse models of human breast cancer

To explore the in vivo significance of ShcA during mammary tumorigenesis, we used mice expressing several phosphotyrosine‐deficient ShcA alleles under the control of their endogenous promoter. We show that all three ShcA tyrosine phosphorylation sites are involved in the early stages of mammary tumour progression, including loss of the myoepithelial cell layer surrounding hyperplasias and during progression to carcinoma. We have determined that signals emanating from Y313 are important for tumour cell survival, whereas Y239/240 transduce signals promoting tumour vascularization. We further demonstrate that loss of ShcA expression in mammary epithelial cells abrogates tumour development. This study is the first to directly demonstrate that signalling downstream from the ShcA adaptor protein is critical for breast cancer development.

Elevated Expression of DecR1 Impairs ErbB2/Neu-Induced Mammary Tumor Development

ABSTRACT Tumor cells utilize glucose as a primary energy source and require ongoing lipid biosynthesis for growth. Expression of DecR1, an auxiliary enzyme in the fatty acid β-oxidation pathway, is significantly diminished in numerous spontaneous mammary tumor models and in primary human breast cancer. Moreover, ectopic expression of DecR1 in ErbB2/Neu-induced mammary tumor cells is sufficient to reduce levels of ErbB2/Neu expression and impair mammary tumor outgrowth. This correlates with a decreased proliferative index and reduced rates of de novo fatty acid synthesis in DecR1-expressing breast cancer cells. Although DecR1 expression does not affect glucose uptake in ErbB2/Neu-transformed cells, sustained expression of DecR1 protects mammary tumor cells from apoptotic cell death following glucose withdrawal. Moreover, expression of catalytically impaired DecR1 mutants in Neu-transformed breast cancer cells restored Neu expression levels and increased mammary tumorigenesis in vivo. These results argue that DecR1 is sufficient to limit breast cancer cell proliferation through its ability to limit the extent of oncogene expression and reduce steady-state levels of de novo fatty acid synthesis. Furthermore, DecR1-mediated suppression of tumorigenesis can be uncoupled from its effects on Neu expression. Thus, while downregulation of Neu expression may contribute to DecR1-mediated tumor suppression in certain cell types, this is not an obligate event in all Neu-transformed breast cancer cells.

The Prognostic Ease and Difficulty of Invasive Breast Carcinoma

Breast carcinoma (BC) has been extensively profiled by high-throughput technologies for over a decade, and broadly speaking, these studies can be grouped into those that seek to identify patient subtypes (studies of heterogeneity) or those that seek to identify gene signatures with prognostic or predictive capacity. The sheer number of reported signatures has led to speculation that everything is prognostic in BC. Here, we show that this ubiquity is an apparition caused by a poor understanding of the interrelatedness between subtype and the molecular determinants of prognosis. Our approach constructively shows how to avoid confounding due to a patients subtype, clinicopathological profile, or treatment profile. The approach identifies patients who are predicted to have good outcome at time of diagnosis by all available clinical and molecular markers but who experience a distant metastasis within 5 years. These inherently difficult patients (~7% of BC) are prioritized for investigations of intratumoral heterogeneity.

A complex containing LPP and α-actinin mediates TGFβ-induced migration and invasion of ErbB2-expressing breast cancer cells.

Summary Transforming growth factor &bgr; (TGF&bgr;) is a potent modifier of the malignant phenotype in ErbB2-expressing breast cancers. We demonstrate that epithelial-derived breast cancer cells, which undergo a TGF&bgr;-induced epithelial-to-mesenchymal transition (EMT), engage signaling molecules that normally facilitate cellular migration and invasion of mesenchymal cells. We identify lipoma preferred partner (LPP) as an indispensable regulator of TGF&bgr;-induced migration and invasion of ErbB2-expressing breast cancer cells. We show that LPP re-localizes to focal adhesion complexes upon TGF&bgr; stimulation and is a critical determinant in TGF&bgr;-mediated focal adhesion turnover. Finally, we have determined that the interaction between LPP and &agr;-actinin, an actin cross-linking protein, is necessary for TGF&bgr;-induced migration and invasion of ErbB2-expressing breast cancer cells. Thus, our data reveal that LPP, which is normally operative in cells of mesenchymal origin, can be co-opted by breast cancer cells during an EMT to promote their migration and invasion.

The ShcA adaptor protein is a critical regulator of breast cancer progression

Breast cancer affects 1 in 8 North American women throughout their lifetime and is the second leading cause of cancer-related deaths. Breast cancer is a heterogeneous disease whose progression from hyperplasia to ductal carcinoma in situ and invasive carcinoma is regulated by the aberrant expression of multiple mediators; including growth factors, cytokines, chemokines and proteases that are produced both by the mammary tumor itself and the adjacent reactive stroma. These signals promote tumor cell proliferation, survival, establishment of a tumor vasculature, invasion and ultimately metastasis to secondary organs. Moreover, the ability of the tumor to create a state of local immune suppression allows tumor cells to evade clearance by the immune system. ShcA is an adaptor protein that relays extracellular signals downstream of receptor tyrosine kinases. Clinical studies suggest that activation of the ShcA signaling pathway is associated with poor patient prognosis. Moreover, recent studies with transgenic mouse models have clearly demonstrated the importance of tumor autonomous ShcA signaling, as well as signaling in cells comprising the tumor microenvironment, for the regulation of these biological processes, which contribute to breast cancer development and metastasis.

STAT3 Establishes an Immunosuppressive Microenvironment during the Early Stages of Breast Carcinogenesis to Promote Tumor Growth and Metastasis

Immunosurveillance constitutes the first step of cancer immunoediting in which developing malignant lesions are eliminated by antitumorigenic immune cells. However, the mechanisms by which neoplastic cells induce an immunosuppressive state to evade the immune response are still unclear. The transcription factor STAT3 has been implicated in breast carcinogenesis and tumor immunosuppression in advanced disease, but its involvement in early disease development has not been established. Here, we genetically ablated Stat3 in the tumor epithelia of the inducible PyVmT mammary tumor model and found that Stat3-deficient mice recapitulated the three phases of immunoediting: elimination, equilibrium, and escape. Pathologic analyses revealed that Stat3-deficient mice initially formed hyperplastic and early adenoma-like lesions that later completely regressed, thereby preventing the emergence of mammary tumors in the majority of animals. Furthermore, tumor regression was correlated with massive immune infiltration into the Stat3-deficient lesions, leading to their elimination. In a minority of animals, focal, nonmetastatic Stat3-deficient mammary tumors escaped immune surveillance after a long latency or equilibrium period. Taken together, our findings suggest that tumor epithelial expression of Stat3 plays a critical role in promoting an immunosuppressive tumor microenvironment during breast tumor initiation and progression, and prompt further investigation of Stat3-inhibitory strategies that may reactivate the immunosurveillance program.

Receptor Tyrosine Kinase Signaling Favors a Protumorigenic State in Breast Cancer Cells by Inhibiting the Adaptive Immune Response

Using transgenic mouse models of breast cancer that ablate Src homology and collagen A (ShcA) expression or oncogene-coupled ShcA signaling, we previously showed that this adaptor is critical for mammary tumor onset and progression. We now provide the first evidence that ShcA regulates mammary tumorigenesis, in part, through its ability to regulate the adaptive immune response. Inactivation of ShcA signaling within tumor cells results in extensive CD4(+) T-cell infiltration and induction of a humoral immune response in mammary tumors. This is associated with a robust CTL response in preneoplastic lesions that are deficient in ShcA signaling. Moreover, mammary tumor progression of ShcA-deficient hyperplasias is accelerated in a T cell-deficient background. We also uncover a clinically relevant correlation between high ShcA expression and low CTL infiltration in human breast cancers. Finally, we define a novel ShcA-regulated immune signature that functions as an independent prognostic marker of survival in human epidermal growth factor receptor 2(+) and basal breast cancers. We reveal a novel role for tumor cell-derived ShcA in the establishment and maintenance of an immunosuppressive state.

The ShcA SH2 domain engages a 14-3-3/PI3'K signaling complex and promotes breast cancer cell survival.

The ShcA adapter protein transmits activating signals downstream of receptor and cytoplasmic tyrosine kinases through the establishment of phosphotyrosine-dependent complexes. In this regard, ShcA possesses both a phosphotyrosine-binding domain (PTB) and Src homology 2 domain (SH2), which bind phosphotyrosine residues in a sequence-specific manner. Although the majority of receptor tyrosine kinases expressed in breast cancer cells bind the PTB domain, very little is known regarding the biological importance of SH2-driven ShcA signaling during mammary tumorigenesis. To address this, we employed transgenic mice expressing a mutant ShcA allele harboring a non-functional SH2 domain (ShcR397K) under the transcriptional control of the endogenous ShcA promoter. Using transplantation approaches, we demonstrate that SH2-dependent ShcA signaling within the mammary epithelial compartment is essential for breast tumor outgrowth, survival and the development of lung metastases. We further show that the ShcA SH2 domain activates the AKT pathway, potentially through a novel SH2-mediated complex between ShcA, 14-3-3ζ and the p85 regulatory subunit of phosphatidylinositol 3 (PI3′) kinase. This study is the first to demonstrate that the SH2 domain of ShcA is critical for tumor survival during mammary tumorigenesis.