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Targeted Overexpression of Vav3 Oncogene in Prostatic Epithelium Induces Nonbacterial Prostatitis and Prostate Cancer

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Targeted Overexpression of Vav3 Oncogene in Prostatic Epithelium Induces Nonbacterial Prostatitis and Prostate Cancer Research Article Targeted Overexpression of Vav3 Oncogene in Prostatic Epithelium Induces Nonbacterial Prostatitis and Prostate Cancer Yin Liu,1 Jun Qin Mo,1 Qiande Hu,2 Gregory Boivin,1 Linda Levin,3 Shan Lu,2 Dianer Yang,2 Zhongyun Dong,2 and Shan Lu1 Departments of 1Pathology, 2Medicine, and 3Environmental Heath, University of Cincinnati College of Medicine, Cincinnati, Ohio Abstract bute to inflammation in the prostate gland. Chronic inflammation Our previous study revealed that Vav3 oncogene is over- results in a sustained innate immune response, which creates a expressed in human prostate cancer, activates androgen microenvironment rich in cytokines, chemokines, growth factors, receptor (AR), and stimulates growth in prostate cancer cells. and angiogenesis factors, and fosters proliferation and survival, a n n The purpose ofthis study is to further determine the critical step for carcinogenesis (1, 2). Nuclear factor B (NF- B) is a potential role ofVav3 in prostate cancer development in key linking molecule in inflammation and immunity to cancer genetically engineered mouse model. We generated Vav3 development and progression (4, 5). Various carcinogens, onco- transgenic mice by targeted overexpression ofa constitutive genes, and cell signaling pathways, such as phosphatidylinositol n active Vav3 in the prostatic epithelium. We found that over- 3-kinase (PI3K)-Akt signaling (6–8), activate NF- B, which in turn expression ofVav3 led to development ofmouse prostatic leads to expression of inflammatory cytokines and growth factors; intraepithelial neoplasia and prostate cancer at the age ofas blocking of apoptosis; and promotion of proliferation, angiogenesis, early as 3 months. The AR signaling axis and phosphatidy- and tumor invasion process. linositol 3-kinase-Akt signaling were elevated in the prostate Vav3 family oncogenes have three members (Vav1, Vav2, and glands ofVav3 transgenic mice. In addition to prostate Vav3) and are guanine nucleotide exchange factors for Rho family cancer, Vav3 transgenic mice developed significant nonbac- GTPases. They differ in their tissue distributions. Vav1 is primarily terial chronic prostatitis in the prostate gland with notable expressed in hematopoietic cells, whereas Vav2 and Vav3 are more infiltration of lymphomononuclear cells (monocytes, lym- ubiquitously expressed (9, 10). Vav proteins contain multiple phocytes, and plasma cells), which was associated with function motifs and are involved in various cellular signaling elevated incidence ofprostate cancer. DNA microarray and processes including cytoskeleton organization, calcium influx, signaling pathway analysis revealed that the top diseases and phagocytosis, and cell transformation (11). Vav proteins are directly disorders were inflammatory diseases and cancer of the or indirectly activated by receptor protein tyrosine kinase in prostate gland in Vav3 transgenic mice. In vitro analysis various signal transduction pathways. For instance, Vav2 and Vav3 showed that overexpression ofVav3 in prostate cancer cells can be directly activated by the receptor protein tyrosine kinase enhanced nuclear factor-KB (NF-KB) activity, implicating an activity of EphA receptor and epidermal growth factor receptor underlying mechanism ofinnate inflammatory response (EGFR; refs. 12, 13). Vav3, a downstream signal transducer of EGFR/ induced by elevated Vav3 activity. These data showed that HER2, was shown to bind to several partners, including Rac1, Vav3 overexpression in the prostate epithelium enhanced Cdc42, PI3K, Grb2, and phospholipase Cg, leading to alteration in both the AR signaling axis and NF-KB–mediated pathway, cell morphology and cell transformation (14). Overexpression of which potentially contributed to the development ofnon- Vav3 leads to PI3K activation and focus formation in NH3T3 cells, bacterial prostatitis and prostate cancer. [Cancer Res and blocking PI3K activity by PTEN and LY294002 efficiently 2008;68(15):6396–406] inhibits Vav3-induced cell transformation activity (15). Androgen receptor (AR) hypersensitivity plays a critical role in Introduction prostate cancer development and progression to an androgen- independent disease. ARmutation and gene amplification, as well It was widely accepted that inflammation contributes to cancer as overexpression of coactivators, contribute to ARhypersensitivity development (1, 2). Many cancers, such as hepatocellular (16–18). AR, as other nuclear receptors, can also be activated carcinoma, stomach cancer, and colorectal carcinoma, arise from independent of its ligand by phosphorylation, which is mediated by chronic infection and inflammation. Compelling data indicate that a variety of signaling pathways (19–21). Phosphorylation of AR inflammation is also associated with prostate cancer (3). The facilitates the recruitment of coactivators and chromatin remod- source of intraprostatic inflammation is, however, largely unknown, elers and modifiers, resulting in enhanced expression of its target although multiple factors, such as infectious agents, dietary genes (22). Numerous studies have shown that the EGFR/HER2- carcinogens, hormone change, and physical trauma, may contri- PI3K-Akt pathway contributes to ARhypersensitivity and prostate cancer development and progression. Overexpression of HER2 in prostate cancer cells up-regulates ARactivity and stimulates Note: Supplementary data for this article are available at Cancer Research Online androgen-independent growth in prostate cancer cells (23). The (http://cancerres.aacrjournals.org/). elevated ARactivity and PI3K-Akt signaling, such as mediated by Requests for reprints: Shan Lu, Department of Pathology, Genome Research Institute, University of Cincinnati College of Medicine, Building A, Room 259, 2120 East PTEN deletion and mutation, play an important role in prostate Galbraith Road, Cincinnati, OH 45237-0507. Phone: 513-558-5109; Fax: 513-558-1312; cancer (24–28). Prostate-specific deletion of murine PTEN, a tumor E-mail: [email protected]. I2008 American Association for Cancer Research. suppressor gene that shuts off PI3K-Akt signaling, induces prostatic doi:10.1158/0008-5472.CAN-08-0645 intraepithelial neoplasia lesions, which later progress to invasive Cancer Res 2008; 68: (15). August 1, 2008 6396 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2008 American Association for Cancer Research. Vav3 Induces Prostate Cancer in Transgenic Mice prostate cancer in mice (29). Multiple growth factors and cytokines, For genotyping of Vav3 transgenic mice by PCR, the upper primer pS5 including EGF, signal through the PI3K-Akt pathway and activate (5¶-GATTAATTAATAGCAATTCCTCGACGA-3¶) and the lower primer Lw3 ¶ ¶ ARin prostate cancer cells, which can be accomplished by direct (5 -CTGGCCTCTCTGGCCATTATCATCGTGTTT-3 ) are designed from vector ARphosphorylation by active Akt (19–21). IRES sequence. Western blot analysis. Western blot analysis was done as previously Recently, we and others found that Vav3 oncogene is described (34). Briefly, aliquots of samples with the same amount of protein, overexpressed in human prostate cancer and several lines of determined using the Bradford assay (Bio-Rad), were mixed with loading prostate cancer cells (30, 31). Vav3 enhances ARactivity and buffer [final concentrations of 62.5 mmol/L Tris-HCl, pH 6.8, 2.3% SDS, 100 stimulates androgen-independent growth in prostate cancer cells. mmol/L DTT, and 0.005% bromophenol blue], boiled, fractionated in a SDS- We further showed that Vav3, as a signal transducer, up- PAGE, and transferred onto a 0.45-Am nitrocellulose membrane (Bio-Rad). regulates ARactivity partially via PI3K-Akt signaling (30). These The filters were blocked with 2% fat-free milk in PBS and probed with first findings suggest that Vav3 overexpression may contribute to antibody in PBS containing 0.1% Tween 20 (PBST) and 1% fat-free milk. The prostate cancer development and/or progression. The purpose of membranes were then washed four times in PBST and incubated with this study was to further determine the role of Vav3 in prostate horseradish peroxidase–conjugated secondary antibody (Bio-Rad) in PBST cancer in genetically engineered mouse model. We found that containing 1% fat-free milk. After washing four times in PBST, the the targeted overexpression of a constitutive active Vav3 in the membranes were visualized with the enhanced chemiluminescence Western blotting detection system (Amersham Co.). For Western blot analysis of epithelium of the prostate gland induced prostatic intraepithelial Vav3 expression, the first antibody was incubated overnight at 4jC. neoplasia (mPIN) and prostate cancer, which is correlated with Reporter assay. Cells (105 per well) were seeded in 12-well tissue culture elevated ARsignaling axis and PI3K-Akt signaling in the prostate plates. The next day, Optifect-mediated transfection was used for the gland. Moreover, we found that Vav3 overexpression also led to transient transfection assay according to the protocol provided by significant chronic nonbacterial inflammation in the prostate Invitrogen/Life Technologies, Inc. The cells were then treated with hormone gland, which was associated with elevated incidence of prostate or drugs in stripped medium for 24 h. Subsequently, the cell extracts were cancer. DNA microarray and signaling pathway analysis revealed prepared and luciferase activity was assessed in a Berthold Detection that the top diseases and disorders are inflammatory diseases System (Pforzheim) using a kit from Promega
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