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

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Publications (15)


Figure 1: Mammary glands from MMTV-Ron HGFL−/− mice exhibit delayed hyperplasia and invasive ductal carcinoma. A. Quantification of mammary hyperplasia observed in inguinal mammary glands by whole mount and H&E analyses at 2.5, 4, 6, 8 and 10 months in MMTV-Ron HGFL+/+ and MMTV-Ron HGFL−/− mice (n = 28-34/genotype). MMTV-Ron HGFL−/− mice have a significant delay in the development of mammary hyperplasia, with a median time to hyperplasia at 240 days compared to 120 days in the MMTV-Ron HGFL+/+ mice, *P < 0.05. B. Representative mammary whole mounts and H&E sections from 8-month-old MMTVRon HGFL+/+ and MMTV-Ron HGFL−/− mice. Scale Bar 100 μM. C. Quantification of invasive ductal carcinoma at 4 and 6 months. MMTVRon HGFL−/− mice have significantly reduced local tumor invasion compared to MMTV-Ron HGFL+/+ mice (n = 20-30 sections per genotype per time point from 4-6 mice each). *P < 0.05. 
Figure 2: Mammary tumorigenesis is significantly delayed and metastasis reduced in MMTV-Ron HGFL−/− mice. A. MMTV-Ron HGFL−/− mice (n = 19) have a significant delay in palpable tumor formation and the median time to palpable tumors (inset) compared to MMTV-Ron HGFL+/+ mice (n = 29). B. Representative histological images of mammary tumors from MMTV-Ron HGFL+/+ and MMTV-Ron HGFL−/− mice are shown and were taken when tumors represented approximately 10% of body weight in each genotype. C. Mammary tumors from MMTV-Ron HGFL+/+ express HGFL protein while MMTV-Ron HGFL−/− mice do not as depicted by Western analyses of tumor lysates. Tubulin is shown as a loading control. Both processed (active) and pro-HGFL are present in the mammary tumors of HGFL expressing mice. D. HGFL mRNA is expressed in MMTV-Ron HGFL+/+ mammary tumors while tumors from HGFL deficient animals do not express HGFL. (n = 5-6/glands from independent mice were examined/genotype). E. Oncomine data shows that HGFL expression is increased in ductal breast carcinoma compared to normal mammary gland. *P < 0.05. ND, not detectable. 
Figure 3: Lung and liver metastases are reduced in MMTV-Ron HGFL−/− mice. Lung and liver metastases were quantified by histological analysis at 6, 8, and 10 months of age. A. Lung tumor metastasis was significantly reduced in MMTV-Ron HGFL−/− mice with the size of metastatic foci in MMTV-Ron HGFL−/− mice significantly smaller compared to controls. The images show a representative metastatic lesion in the lungs of a MMTV-Ron HGFL+/+ mouse at 6 months while the image for MMTV-Ron HGFL−/− is from 8 months (n = 5-10/group and time point) Scale bar = 100 μM. B. The incidence of liver metastases was significantly reduced in MMTV-Ron HGFL−/− mice at 6 and 10 months of age (n = 5-10/group and time point). C. Mammary tumor tissue was examined by qRT-PCR for expression of EMT genes. N-cadherin and vimentin were significantly down regulated in MMTV-Ron HGFL−/− mice (n = 3-4/grp). *P < 0.05. 
Figure 4: Mammary tumors from MMTV-Ron HGFL−/− mice exhibit significantly reduced proliferation, increased cell death and reduced vessel staining. Quantification of mammary tumor sections and representative images of BrdU, TUNEL and CD31stained sections. A. MMTV-Ron HGFL−/− mice have significantly reduced number of proliferating tumor cells compared to MMTVRon HGFL+/+ tumors (n = 3 fields per mouse, 4 mice/group). B., Mammary tumors from MMTV-Ron HGFL−/− mice exhibit significantly more cell death than tumors from MMTV-Ron HGFL+/+ mice (n = 3 fields per mouse, 4 mice/group). C. Quantification of CD31 staining shows a significant reduction in vessel density in MMTV-Ron HGFL−/− tumors compared to MMTV-Ron HGFL+/+ tumors. (n = 3 fields per mouse, 4 mice/ group). *P < 0.05, Scale bar = 100 μM. 
Figure 5: MMTV-Ron HGFL−/− mice have an altered tumor microenvironment. A. Quantification and representative images of F4/80, Arginase I and iNOS expression in MMTV-Ron mammary tumors by immunohistochemistry. Loss of HGFL increases macrophage infiltration and polarization toward a M1 phenotype, as shown by decreased Arginase I and increased iNOS staining. (n = 3 sections per mouse, 4 mice/group). B. Whole tumor tissue was examined by qRT-PCR for select cytokine and chemokine expression. An increase in inflammatory mediators within the tumor microenvironment of MMTV-Ron HGFL−/− mice compared to control mice was apparent while the expression of T-cell co-stimulatory molecules are significantly increased. (n = 6 per group). *P < 0.05, Scale bar = 100 μM. 

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HGFL supports mammary tumorigenesis by enhancing tumor cell intrinsic survival and influencing macrophage and T-cell responses
  • Article
  • Full-text available

April 2015

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378 Reads

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15 Citations

Oncotarget

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Purnima K Wagh

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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.

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Cell- and Developmental Stage-Specific Dicer1 Ablation in the Lung Epithelium Models Cystic Pleuropulmonary Blastoma

December 2014

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91 Reads

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25 Citations

The Journal of Pathology

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.


Figure 2: Dek promotes tumor growth and increases cell proliferation in vivo and in vitro. (a) RontgDek−/− (MMTV-Ron and Dek−/−) mice exhibit delayed tumor onset compared with RontgDek+/+ mice. Time to tumor detection for RontgDek−/− mice was 232.1±10.27 days (N=14) compared with 193.6±4.206 days (N=23; mean±s.e.m.; ***P<0.0001, log-rank test). (b) Preneoplastic mammary glands from RontgDek+/+ mice (24.16±2.728%, N=11) are more proliferative than glands from RontgDek−/− mice (9.401±1.562%, N=8; **P=0.0006, as determined by unpaired two-tailed t-test). Mice were intraperitoneally injected with BrdU before being killed. Immunohistochemistry for BrdU incorporation was performed on preneoplastic glands. Representative images are shown. Results are quantified in the graph on the right as the percentage of BrdU-positive cells in the tissue section. (c) Dek expression levels positively correlate with cellular growth rate in Rontg tumor-derived cell lines. Cell lines were generated from three RontgDek+/+ and three RontgDek−/− tumors from independent mice. A Dek shRNA (Deksh) was introduced into RontgDek+/+ cells to decrease Dek expression or an mDek overexpression construct was retrovirally transduced into RontgDek−/− to complement the loss of Dek. Western blots in the inset show changes in Dek expression (D) with α-tubulin (T) as a loading control. Cells were counted over the time periods shown for each cell line. (d) Tumors from RontgDek−/− mice (N=4) have fewer cells with BCSC cell surface markers compared with RontgDek+/+ tumors (N=3). Flow cytometry was used to quantify the percentage of Lin−/CD49f+/CD24+/CD44low cells. (e) Dek expression promotes the growth of mammospheres. RontgDek+/+ cell line (R7) transduced with Deksh (‘+Deksh’) or NTsh control, and RontgDek−/− cell lines (RD147, RD219, RD238 and RD271) transduced with an mDek construct (‘+mDek’) or control R780 vector were plated in non-adherent mammosphere cultures and measured after 9 days to calculate volume. Data are presented as fold change compared with control (NTsh or R780) spheres from triplicate experiments.
Figure 3: Dek expression supports breast cancer metastasis in vivo and in vitro. (a) RontgDek+/+ mice have a greater metastatic lung tumor burden than RontgDek−/−. Lungs were harvested from six RontgDek−/− and five RontgDek+/+ mice with similar primary tumor volumes for the largest tumor and serial sections were microscopically analyzed for tumor metastases. Total tumor burden from all lung sections in wild-type mice was 18.38±1.754 compared with 8.183±1.661 in knockout mice (P=0.0023, unpaired two-tailed t-test). The average number of lung metastases per mouse is quantified and a representative hematoxylin and eosin staining from each genotype is depicted below. Black arrows highlight metastatic tumors within the lungs. (b) Dek expression correlates with cellular invasion in vitro. RontgDek+/+ cell lines (R7 and RD258) transduced with Deksh (‘+Deksh’) or NTsh control, and RontgDek−/− cell lines (RD147, RD219, RD238 and RD271) transduced with an mDek construct (‘+mDek’) or control R780 vector were subjected to Matrigel transwell assays. Data are represented as fold change compared with the respective control for each cell line (NTsh or R780) for triplicate experiments. Significance was calculated with a one-tailed unpaired t-test.
Figure 5. Dek depletion delays xenograft tumor growth and results in decreased Wnt10b and activated β-catenin expression in vivo (A) Dek depletion (Deksh2) in Ron tg Dek +/+ R7 cells delays xenograft tumor growth. Ron tg Dek +/+ R7 NTsh and Deksh2 cells were injected into inguinal mammary fat pads in nude mice and monitored for tumor growth. (B) Dek depletion inhibits Wnt/β-catenin activity in xenograft tumors. Immunohistochemical staining for Dek, Wnt10b, and activated β-catenin was performed on xenograft tumors from R7 NTsh and Deksh2 cells. (C)
Figure 6: Ron and DEK cooperate in human breast cancer to promote disease progression. (a) DEK is a downstream target gene of activated Ron signaling in T47D human breast cancer cells. T47D cells were treated with HGFL to activate Ron signaling and DEK expression was analyzed by quantitative RT–PCR. Expression was compared with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and normalized to expression in untreated cells. For the 24 h time point, P=0.07 and significance was calculated with a one-tailed unpaired t-test. (b) DEK protein levels are elevated following HGFL-mediated activation of Ron signaling. T47D cells were treated with HGFL for the time periods shown and whole-cell lysates were analyzed by western blotting. (c) DEK overexpression in MCF10A cells induces phenotypes of advanced breast cancer in 3D culture, including increased acinar size, cells present within the lumen and cellular invasion. MCF10A cells transduced with R780 or R780:DEK (hDek) were grown in Matrigel 3D culture (top and middle panels) to test for morphology or Matrigel–collagen 3D cultures to test for invasion (bottom panel). Immunofluorescence was performed on day 20 of culture and visualized with a Zeiss LSM510 scanning confocal microscope; the white size bar represents 10 μm. Phase-contrast images are also shown in the middle and bottom panels, and black arrows indicate invading cells in the bottom panel. The Golgi marker GM130 is used to mark cell apical–basal polarity, and white arrows highlight cells with deregulated polarity. The percentage of cells with mislocalized GM130 per acinar structure is depicted below the images, which was calculated from triplicate experiments. (d) DEK and Ron expression predict β-catenin expression levels in primary human breast cancer. Serial sections from two tissue microarrays of patient-derived breast infiltrating ductal carcinomas were stained by immunohistochemistry for Dek, Ron and β-catenin expression. Correlation was analyzed by χ2 testing. (e and f) Combined DEK and Ron expression predict disease relapse in a cohort of 1000 unmedicated patients (P=0.00018) (e) and progression to distant metastatic disease in 1609 patients treated with systemic therapies (P=0.00098) (f). A meta-analysis of patient and gene expression data archived in Kaplan–Meier Plotter (http://www.kmplot.com) was performed to generate Kaplan–Meier curves. The numbers under each graph represent the number of patients at each time point.
The DEK oncogene promotes cellular proliferation through paracrine Wnt signaling in Ron receptor-positive breast cancers

June 2014

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211 Reads

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61 Citations

Oncogene

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.


Abstract C04: HGFL promotes the development and progression mammary tumors and represents an attractive therapeutic target

May 2014

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15 Reads

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.



Conditional Deletion of β-Catenin in Mammary Epithelial Cells of Ron Receptor, Mst1r, Overexpressing Mice Alters Mammary Tumorigenesis

April 2012

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27 Reads

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19 Citations

Endocrinology

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.


Figure 1: Ron expression in wild-type and TRAMP mouse prostates. (a) Prostates from 30-week-old wild-type (TK+/+) or TK−/− mice are histologically similar. Representative sections of the anterior prostates of TK+/+ and TK−/− mice are shown. (b) Ron is highly expressed in 30-week-old TK+/+ TRAMP+ prostates compared with TK+/+ prostate tissue. No Ron expression is detected in TK−/− TRAMP+ prostates. Representative tissue sections were stained with a Ron-specific antibody or with an isotype control immunoglobulin G (IgG). TK+/+ prostates have minimal Ron expression, whereas TK+/+ TRAMP+ prostate tumors have significantly elevated Ron expression. (c) Quantitative real-time PCR analysis shows increased Ron mRNA levels in TK+/+ TRAMP+ prostate tumors (n=5) relative to TK+/+ prostates (n=6). Data are expressed as means±s.e. No detectable Ron mRNA expression was observed in the prostates of TK−/− or TK−/− TRAMP+ mice. (d) Top: western analysis of Ron protein levels in a TK+/+ prostate and in a prostate tumor from a TK+/+ TRAMP+ mouse. Bottom: Densitometry analysis of relative Ron protein levels in TK+/+ (n=4) and TK+/+ TRAMP+ (n=5) prostates. Data are expressed as means±s.e. *P<0.05 compared with TK+/+ group.
Figure 2: TRAMP mice deficient in functional Ron have decreased GU complex and prostate tumor size. (a) A 30-week-old TK−/− TRAMP+ mice (n=12) exhibit decreased GU complex and prostate tumor mass relative to TK+/+ TRAMP+ mice (n=16). Data are expressed as means±s.e. (b) Gross examination of GU complexes from TK+/+ TRAMP+ mice and TK−/− TRAMP+ mice shows decreased GU complex size in the TK−/− TRAMP+ mice. (c) Representative histological analysis of TK+/+ TRAMP+ and TK−/− TRAMP+ prostates. The asterisk depicts the large tumor bearing area of the TK+/+ TRAMP+ prostate, whereas the arrow indicates one of the several neoplastic regions of TK−/− TRAMP+ prostate. (d) Representative image of a lung section containing metastatic foci (arrows) from a TK+/+ TRAMP+ mouse and a corresponding section from a TK−/− TRAMP+ lung. Lungs and prostates were stained with hematoxylin and eosin.
Figure 3: Prostate tumors from TK−/− TRAMP+ mice are less vascularized than prostates from TRAMP mice expressing wild-type Ron. (a) TRAMP mice lacking functional Ron (TK−/− TRAMP+) have decreased vascularization as determined by immunohistochemistry with a CD31-specific antibody. (b) Mean vessel density per area was determined with n=4 mice for the TK+/+ TRAMP+ group and n=3 mice for the TK−/− TRAMP+ group. Data are expressed as means±s.e. *P<0.05 compared with TK+/+ TRAMP+ group. (c, d) Quantitative real-time PCR was performed on RNA isolated from prostate tumors from TK+/+ TRAMP+ and TK−/− TRAMP+ mice. Expression of the angiogenic factors VEGF (c) and CXCL2 (d) are shown. Data are expressed as means±s.e. with n=4 prostates per group. *P<0.05 compared with TK+/+ TRAMP+ group.
Figure 4: Prostate cell proliferation and apoptosis in TK+/+ TRAMP+ and TK−/− TRAMP+ mice. (a) BrdU immunostaining was performed on prostates from 30-week TK+/+ TRAMP+ and TK−/− TRAMP+ mice. (b) Detection of TUNEL-positive cells in prostate tissue of TK+/+ TRAMP+ and TK−/− TRAMP+ mice at 30 weeks of age. (c) Prostates from 30-week-old TK+/+ TRAMP+ and TK−/− TRAMP+ mice did not exhibit differences in BrdU staining, but did contain significant differences in the extent of TUNEL-positive cell staining. Data are expressed as means±s.e. Five separate areas were counted from four independent specimens per group, and representative images are shown. *P<0.05 compared with TK+/+ TRAMP+ group. Arrows depict a few of the positive staining cells.
Figure 5: Ron loss in the TRAMP prostate results in decreased levels of active NF-κB. (a) NF-κB reporter assays performed with prostate cells derived from TK+/+ TRAMP+ and TK−/− TRAMP+ prostates. Decreased NF-κB reporter activity was observed in TK−/− TRAMP+ cells compared with TK+/+ TRAMP+ cells. Relative NF-κB activity for each sample is depicted. Data are expressed as the mean±s.e. (b) Nuclear extracts from 30-week-old TK+/+ TRAMP+ and TK−/− TRAMP+ prostates were isolated and examined by western analysis. TK−/− TRAMP+ prostates exhibit decreased levels of nuclear NF-κB p65 relative to TK+/+ TRAMP+ prostates. PARP is shown as a nuclear loading control. *P<0.05.
The Ron receptor promotes prostate tumor growth in the TRAMP mouse model

May 2011

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158 Reads

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23 Citations

Oncogene

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.


Β-Catenin is required for Ron receptor-induced mammary tumorigenesis

March 2011

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218 Reads

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52 Citations

Oncogene

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.



The human DEK oncogene stimulates Β-catenin signaling, invasion and mammosphere formation in breast cancer

February 2011

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149 Reads

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97 Citations

Oncogene

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.


Citations (10)


... However, overexpression of RON and its ligand by tumor cells can activate RON signaling in the tissue-resident macrophages to promote growth and inhibit inflammation. Thus, the activation of RON in both macrophages and cancerous cells has led to mounting evidence that such signaling promotes both tumor growth and metastasis through tumor microenvironment (TME) signaling [18][19][20]. ...

Reference:

RON Receptor Signaling and the Tumor Microenvironment
HGFL supports mammary tumorigenesis by enhancing tumor cell intrinsic survival and influencing macrophage and T-cell responses

Oncotarget

... Nevertheless, these epithelial areas frequently displayed a botryoid or even cystic appearance. We propose that such cystic changes, commonly observed in tumors from the DICER1 mesenchymal neoplasm spectrum -such as PPB I and CN -are not a result of DICER1-driven neoplastic epithelial growth, as previously suggested 4,13 . Instead, we hypothesize that these cystic transformations are likely a reactive phenomenon, driven by neoplastic mesenchymal proliferation, which may exert pressure on adjacent structures, such as kidney tubules or alveolar spaces, leading to their compression and subsequent cystic dilation due to fluid or air entrapment. ...

Cell- and Developmental Stage-Specific Dicer1 Ablation in the Lung Epithelium Models Cystic Pleuropulmonary Blastoma
  • Citing Article
  • December 2014

The Journal of Pathology

... Supporting this, studies have demonstrated DEK's significant role in tumorigenesis. Han et al. discovered that DEK is significantly involved in the proliferation of serous ovarian cancer cells, with high DEK expression levels correlating with an increased Ki-67 proliferation index (30) , while Privette et al. demonstrated that DEK oncogene stimulates cellular proliferation via Wnt signaling in Ron receptor-positive breast cancers (31) . ...

The DEK oncogene promotes cellular proliferation through paracrine Wnt signaling in Ron receptor-positive breast cancers

Oncogene

... Indeed, reduced expression was evident (Fig. 5B). To further examine transcription factor responses downstream from β-catenin activation, we utilized a cell line derived from a mammary tumor of MMTV-RON (MR) mouse containing a β-catenin floxed allele [28], referred to as MRBC cells. MRBC cells treated with a Crerecombinase expressing adenovirus (Ad-Cre) were stably derived from parental MRBC cells generating β-catenin −/− cells (referred to as MRBC−/− cells, Fig. 5C). ...

Conditional Deletion of β-Catenin in Mammary Epithelial Cells of Ron Receptor, Mst1r, Overexpressing Mice Alters Mammary Tumorigenesis
  • Citing Article
  • April 2012

Endocrinology

... RON loss in this model led to decreases in mammary tumor formation and metastasis and established RON signaling as a crucial regulator of mammary tumor formation, growth, and metastasis, as well as in the regulation of angiogenesis. Similar to these studies in breast cancer, the loss of RON in the TRAMP mouse model of prostate cancer led to a reduction in prostate tumor mass and tumor vascularization [25]. ...

The Ron receptor promotes prostate tumor growth in the TRAMP mouse model

Oncogene

... The Wnt/β-catenin signalling pathway is a critical oncogenic pathway for DLBCL progression and facilitates multiple malignant cellular behaviours and characteristics, including proliferation, apoptosis, and stemness [38][39][40]. In addition, c-MYC is a target gene of β-catenin [41]. Our data suggested the existence of a c-MYC/SNHG20/β-catenin positive feedback loop in DLBCL cells. ...

Β-Catenin is required for Ron receptor-induced mammary tumorigenesis

Oncogene

... DEK is an evolutionarily conserved nuclear protein that participates in multifaceted cellular functions [9], including transcriptional regulation [10,11], DNA replication [12], chromatin organization [12,13], DNA damage repair [14], and mRNA processing [15]. In cancer, DEK's overexpression and dysregulation is frequently associated with tumorigenesis and aggressive phenotypes across diverse malignancies, including acute myeloid leukemia [16,17], breast cancer [18,19], colorectal cancer [20,21], and melanoma [21]. Several studies have identified mechanisms driving DEK dysregulation in tumors, which include the formation of DEK fusion proteins and copy number alterations [16,17,22,23]. ...

The human DEK oncogene stimulates Β-catenin signaling, invasion and mammosphere formation in breast cancer

Oncogene

... It is reported that neither c-Met nor HGF expression correlated with ER in primary breast cancers [45], but HGF/c-Met signaling can affect ER signaling through the regulation of H19, Pratima Basak et al. found that combination blocking Notch and c-Met signaling will decrease H19 expression and substantially decrease ERα expression, leading to TR cells sensitize to endocrine therapies [46]. Although the study on the interaction of HGF/c-Met signaling and ER signaling in TR cells is limited, but many previous research have reported that signaling through growth factor signaling pathways, in particular through receptor tyrosine kinases, confer tamoxifen resistance in an estradiol-independent manner, such as the MAPK 1/2 signaling pathway [47,48], PI3K pathway [49]. HGF/c-Met signaling pathway enhance ER + BC cells resistant to tamoxifen may bypass the ER signaling, but it may also exit cross-talk with ER signaling pathway, the details still need more explorations. ...

Ron Receptor Tyrosine Kinase Activation Confers Resistance to Tamoxifen in Breast Cancer Cell Lines 1

Neoplasia

... 3.5. USP39 is positively correlated to CHK2 in clinical lung cancer samples CHK2 has been identified as a tumor suppressor, which is mutated or depleted in various cancers, including breast, colon, bladder, ovary and prostate carcinomas, albeit at low frequencies [49][50][51][52]. Besides mutated in cancer, low level of CHK2 was also observed in lung cancers, which contributes to chemo-radiation resistance [53][54][55]. ...

Chk2*1100delC Acts in Synergy with the Ron Receptor Tyrosine Kinase to Accelerate Mammary Tumorigenesis in Mice
  • Citing Article
  • April 2010

Cancer Letters

... RON is overexpressed in a number of cancers and is naturally expressed in macrophages, specifically those residing in the local tissue [11]. Additionally, tissueresident macrophages expressing RON have been shown to polarize into a pro-tumorigenic state [12][13][14][15]. Monocytes, the precursors to macrophages, do not express this receptor [16]. ...

Met‐Related Receptor Tyrosine Kinase Ron in Tumor Growth and Metastasis

Advances in Cancer Research