Purnima K. Wagh’s research while affiliated with Cincinnati Children's Hospital Medical Center and other places

The Ron receptor is overexpressed in human breast cancers and is associated with heightened metastasis and poor survival. Ron overexpression in the mammary epithelium of mice is sufficient to induce aggressive mammary tumors with a high degree of metastasis. Despite the well-documented role of Ron in breast cancer, few studies have examined the necessity of the endogenous Ron ligand, hepatocyte growth factor-like protein (HGFL) in mammary tumorigenesis. Herein, mammary tumor growth and metastasis were examined in mice overexpressing Ron in the mammary epithelium with or without HGFL. HGFL ablation decreased oncogenic Ron activation and delayed mammary tumor initiation. HGFL was important for tumor cell proliferation and survival. HGFL loss resulted in increased numbers of macrophages and T-cells within the tumor. T-cell proliferation and cytotoxicity dramatically increased in HGFL deficient mice. Biochemical analysis of HGFL proficient tumors showed increased local HGFL production, with HGFL loss decreasing β-catenin expression and NF-κB activation. Re-expression of HGFL in HGFL deficient tumor cells stimulated cell migration and invasion with coordinate activation of NF-κB and reduced apoptosis. Together, these results demonstrate critical in vivo functions for HGFL in promoting breast tumorigenesis and suggest that targeting HGFL may inhibit tumor growth and reactivate anti-tumor immune responses.

Inherited syndromes provide unique opportunities to identify key regulatory mechanisms governing human disease. We previously identified germline loss-of-function DICER1 mutations in a human syndrome defined by the childhood lung neoplasm, pleuropulmonary blastoma (PPB), which arises during lung development. DICER1 regulates many biological processes critical in development and disease pathogenesis. Significant challenges in defining the role of DICER1 in human disease are identifying cause-effect relationships and generating manipulatable systems that model the complexity of organ development and disease pathogenesis. Herein we report generation of a murine model for PPB and demonstrate that precise temporal and cell type-specific Dicer1 ablation is necessary and sufficient for development of cystic lungs that histologically and phenotypically model PPB. Dicer1 ablation in the distal airway epithelium during early stages of lung development resulted in a cystic lung phenotype indistinguishable from PPB whereas DICER1 function was not required for development of the proximal airway epithelium or during later stages of organogenesis. Mechanistic studies demonstrate that Dicer1 loss results in epithelial cell death, followed by cystic airway dilation, accompanied by epithelial and mesenchymal proliferation. These studies define precise temporal and epithelial cell type-specific DICER1 functions in the developing lung and demonstrate that loss of these DICER1 functions is sufficient for development of cystic PPB. These results also provide evidence that PPB arises through a novel mechanism of non-cell-autonomous tumor initiation, wherein the genetic abnormality initiating the neoplasm does not occur in the cells that ultimately transform, but rather occurs in a benign-appearing epithelial cell component that predisposes underlying mesenchymal cells to malignant transformation. This article is protected by copyright. All rights reserved.

Disease progression and recurrence are major barriers to survival for breast cancer patients. Understanding the etiology of recurrent or metastatic breast cancer and underlying mechanisms is critical for the development of new treatments and improved survival. Here, we report that two commonly overexpressed breast cancer oncogenes, Ron (Recepteur d'Origine Nantaise) and DEK, cooperate to promote advanced disease through multipronged effects on β-catenin signaling. The Ron receptor is commonly activated in breast cancers, and Ron overexpression in human disease stimulates β-catenin nuclear translocation and is an independent predictor of metastatic dissemination. Dek is a chromatin-associated oncogene whose expression has been linked to cancer through multiple mechanisms, including β-catenin activity. We demonstrate here that Dek is a downstream target of Ron receptor activation in murine and human models. The absence of Dek in the MMTV-Ron mouse model led to a significant delay in tumor development, characterized by decreased cell proliferation, diminished metastasis and fewer cells expressing mammary cancer stem cell markers. Dek complementation of cell lines established from this model was sufficient to promote cellular growth and invasion. Mechanistically, Dek expression stimulated the production and secretion of Wnt ligands to sustain an autocrine/paracrine canonical β-catenin signaling loop. Finally, we show that Dek overexpression promotes tumorigenic phenotypes in immortalized human mammary epithelial MCF10A cells and, in the context of Ron receptor activation, correlates with disease recurrence and metastasis in patients. Overall, our studies demonstrate that DEK overexpression, due in part to Ron receptor activation, drives breast cancer progression through the induction of Wnt/β-catenin signaling.Oncogene advance online publication, 23 June 2014; doi:10.1038/onc.2014.173.

A standard therapy for breast cancer is the use of receptor tyrosine kinase (RTK) inhibitors. RTKs efficiently target multiple receptors, including the Ron transmembrane receptor, whose activation leads to increased survival, migration, and angiogenesis through signaling pathways such as PI3K/Akt and NF-κB. Ron overexpression is correlated with increased metastasis and poor outcomes in patients. The only ligand for Ron is hepatocyte growth factor-like protein (HGFL), which is an endocrine factor secreted into the circulation from the liver. Published reports demonstrated that ectopic overexpression of HGFL in cancer cells leads to increased metastasis. However, no studies have examined the role of endogenous HGFL in Ron activation during tumor development and metastasis. We sought to test the hypothesis that HGFL is required for Ron receptor activation and mammary tumorigenesis. We utilized mice with mammary-specific Ron overexpression (MMTV-Ron), which develop mammary tumors with 100% incidence. MMTV-Ron mice were crossed to HGFL deficient (HGFL-/-) mice to generate MMTV-Ron X HGFL-/- mice. We compared mammary hyperplasia and tumor latency in MMTV-Ron mice with and without HGFL serially at 2.5, 4, 6, 8 and 10 months. Additional mice were followed to established tumor kinetics and growth and to examine metastatic dissemination. MMTV-Ron X HGFL-/- mice displayed decreased mammary hyperplasia and a significant delay in the time to tumor formation compared to control mice. Kinase assays show that Ron activation is reduced in mice with HGFL loss. Tumor cell proliferation was different between the groups while markers of apoptosis (TUNEL IHC and cleaved caspase 3 expression) were significantly increased in MMTV-Ron X HGFL-/- mice compared to controls. Western blot analysis of tumor lysates showed diminished activation of NF-κB signaling associated with HGFL loss. In conclusion, our data show that HGFL promotes the development of mammary hyperplasia and mammary tumor formation by enhancing tumor cell survival, Ron activation and NF-κB signaling. These data suggest that HGFL represents an excellent target for inhibiting Ron mediated tumor growth in human disease. Citation Format: Nancy M. Benight, Purnima Wagh, Susan E. Waltz. HGFL promotes the development and progression mammary tumors and represents an attractive therapeutic target. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C04.

The Ron receptor tyrosine kinase (macrophage stimulating 1 receptor) is overexpressed in approximately 50% of human breast cancers. Transgenic mice overexpressing Ron in the mammary epithelium [mouse mammary tumor virus driven (MMTV)-Ron expressing mice] develop mammary tumors that exhibit up-regulation of β-catenin and β-catenin target genes. β-Catenin has been shown to be a mediator of mammary tumorigenesis in various breast cancer models, including downstream of Ron. However, the in vivo impact of a conditional loss of β-catenin downstream of Ron receptor overexpression on the onset, growth, turnover, and metastasis of mammary tumors has not been addressed. To determine the significance of β-catenin in the context of Ron overexpression, we conditionally deleted β-catenin in mammary epithelial cells of MMTV-Ron mice. Conditional deletion of β-catenin in the mammary epithelium, through the use of whey acidic protein (WAP)-Cre transgenic mice, significantly delayed the onset of mammary hyperplastic nodules, the presence of palpable mammary tumors, and ultimately decreased liver metastasis. β-Catenin loss in this model was also associated with decreased expression of cyclin D1. In total, these studies support an important role for β-catenin downstream of Ron receptor signaling during the development of mammary tumorigenesis.

The Ron receptor tyrosine kinase (TK) is overexpressed in many cancers, including prostate cancer. To examine the significance of Ron in prostate cancer in vivo, we utilized a genetically engineered mouse model, referred to as TRAMP mice, that is predisposed to develop prostate tumors. In this model, we show that prostate tumors from 30-week-old TRAMP mice have increased Ron expression compared with age-matched wild-type prostates. Based on the upregulation of Ron in human prostate cancers and in this murine model of prostate tumorigenesis, we hypothesized that this receptor has a functional role in the development of prostate tumors. To test this hypothesis, we crossed TRAMP mice with mice that are deficient in Ron signaling (TK-/-). Interestingly, TK-/- TRAMP+ mice show a significant decrease in prostate tumor mass relative to TRAMP mice containing functional Ron. Moreover, TK-/- TRAMP+ prostate tumors exhibited decreased tumor vascularization relative to TK+/+ TRAMP+ prostate tumors, which correlated with reduced levels of the angiogenic molecules vascular endothelial growth factor and CXCL2. Although Ron loss did not alter tumor cell proliferation, a significant decrease in cell survival was observed. Similarly, murine prostate cancer cell lines containing a Ron deficiency exhibited decreased levels of active nuclear factor-κB, suggesting that Ron may be important in regulating prostate cell survival at least partly through this pathway. In total, our data show for the first time that Ron promotes prostate tumor growth, prostate tumor angiogenesis and prostate cancer cell survival in vivo.

Our previous studies demonstrated that selective overexpression of the Ron receptor tyrosine kinase in the murine mammary epithelium leads to mammary tumor formation. Biochemical analysis of mammary tumor lysates showed that Ron overexpression was associated with increases in β-catenin expression and tyrosine phosphorylation. β-Catenin has also been shown to be regulated through tyrosine phosphorylation by the receptor tyrosine kinases Met, Fer and Fyn. However, the molecular and physiological roles of β-catenin and β-catenin tyrosine phosphorylation downstream of Ron are not known. To investigate this association, we show that Ron and β-catenin are coordinately elevated in human breast cancers. Our data also demonstrate that activation of Ron, through ligand binding by hepatocyte growth factor-like protein (HGFL), induces the tyrosine phosphorylation of β-catenin, primarily on tyrosine residues Tyr 654 and Tyr 670. In addition, HGFL-mediated Ron activation induces both β-catenin nuclear localization and transcriptional activity, with Tyr 654 and Tyr 670 residues of β-catenin being critical for these processes. We also demonstrate that a knockdown of Ron in breast cancer cell lines leads to a loss of HGFL-induced β-catenin-dependent transcriptional activation and cell growth, which can be rescued by activation of canonical Wnt/β-catenin signaling. Moreover, we show that HGFL-dependent Ron activation mediates upregulation of the β-catenin target genes cyclin D1 and c-myc, and that expression of these target genes in breast cancer cells is decreased following inhibition of Ron and/or β-catenin. Finally, we show that genetic ablation of β-catenin in Ron-expressing breast cancer cells decreases cellular proliferation in vitro, as well as mammary tumor growth and metastasis, following orthotopic transplantation into the mammary fat pad. Together, our data suggest that β-catenin is a crucial downstream regulator of Ron receptor activation and is an important mediator of mammary tumorigenesis.

Breast cancer is a major cause of cancer-related deaths in American women; therefore, the identification of novel breast cancer-related molecules for the discovery of new markers and drug targets remains essential. The human DEK gene, which encodes a chromatin-binding protein and DNA topology regulator, is upregulated in many types of cancer. DEK has been implicated as an oncogene in breast cancer based on mRNA expression studies, but its functional significance in breast cancer growth and progression has not yet been tested directly. We demonstrate that DEK is highly expressed in breast cancer cells compared with normal tissue, and functionally important for cellular growth, invasion and mammosphere formation. DEK overexpression in non-tumorigenic MCF10A cells resulted in increased growth and motility, with a concomitant downregulation of E-cadherin. Conversely, DEK knockdown in MCF7 and MDA-MB-468 breast cancer cells resulted in decreased growth and motility with upregulation of E-cadherin. The use of DEK-proficient and -deficient breast cancer cells in orthotopic xenografts provided further in vivo evidence that DEK contributes to tumor growth. Activation of the β-catenin signaling pathway is important for normal and cancer stem cell character, growth and metastasis. We show that DEK expression stimulated, and DEK knockdown repressed β-catenin nuclear translocation and activity. Importantly, the expression of constitutively active β-catenin rescued breast cancer invasion defects of DEK knockdown cells. Together, our data indicate that DEK expression stimulates the growth, stem cell character and motility of breast cancer cells, and that DEK-dependent cellular invasion occurs at least in part via β-catenin activation.