Allen Saliganan

Platelet‐Derived Growth Factor‐D Overexpression Contributes to Epithelial‐Mesenchymal Transition of PC3 Prostate Cancer Cells

The majority of human malignancies are believed to have epithelial origin, and the progression of cancer is often associated with a transient process named epithelial‐mesenchymal transition (EMT). EMT is characterized by the loss of epithelial markers and the gain of mesenchymal markers that are typical of “cancer stem‐like cells,” which results in increased cell invasion and metastasis in vivo. Therefore, it is important to uncover the mechanistic role of factors that may induce EMT in cancer progression. Studies have shown that platelet‐derived growth factor (PDGF) signaling contributes to EMT, and more recently, PDGF‐D has been shown to regulate cancer cell invasion and angiogenesis. However, the mechanism by which PDGF‐D promotes invasion and metastases and whether it is due to the acquisition of EMT phenotype remain elusive. For this study, we established stably transfected PC3 cells expressing high levels of PDGF‐D, which resulted in the significant induction of EMT as shown by changes in cellular morphology concomitant with the loss of E‐cadherin and zonula occludens‐1 and gain of vimentin. We also found activation of mammalian target of rapamycin and nuclear factor‐κB, as well as Bcl‐2 overexpression, in PDGF‐D PC3 cells, which was associated with enhanced adhesive and invasive behaviors. More importantly, PDGF‐D‐overexpressing PC3 cells showed tumor growth in SCID mice much more rapidly than PC3 cells. These results provided a novel mechanism by which PDGF‐D promotes EMT, which in turn increases tumor growth, and these results further suggest that PDGF‐D could be a novel therapeutic target for the prevention and/or treatment of prostate cancer.

In vitro and In vivo Molecular Evidence for Better Therapeutic Efficacy of ABT-627 and Taxotere Combination in Prostate Cancer

Bone is the key metastatic site for prostate cancer. Endothelin 1 (ET-1) produced abundantly by prostate cancer cells binds to its receptor present on bone marrow stromal cells and favors osteoblastic response during bone metastases of prostate cancer. This suggests that interrupting ET-1 interaction with its endothelin A (ET(A)) receptor could be useful for inhibiting prostate cancer bone metastasis and, as such, may enhance the therapeutic activity of docetaxel (Taxotere), the most commonly used drug for the treatment of metastatic prostate cancer. Therefore, the goal of our study was to obtain preclinical data supporting our hypothesis that the combined use of ET(A) receptor antagonist (ABT-627; Atrasentan) with Taxotere will be superior in inducing apoptosis in vitro and inhibiting tumor growth in vivo in a SCID-hu model of experimental bone metastasis induced by C4-2b prostate cancer cells. In vitro studies were done on a panel of prostate cancer cell lines to understand the molecular basis of combination therapy, and we found that the combination was more effective in the inhibition of cell viability and induction of apoptosis in LNCaP and C4-2b cells (androgen receptor positive) but not in PC-3 cells. These results were correlated with inactivation of Akt/nuclear factor-kappaB and its target genes. For in vivo studies, the therapeutic regimen was initiated when the tumor began showing signs of growth and treatment was continued for 5 weeks. Tumor volume and serum prostate-specific antigen were used as terminal index to evaluate the therapeutic advantage of combination therapy relative to a single regimen and untreated control. At termination, we found a 90% reduction in tumor volume by combination treatment relative to the untreated control group. Most importantly, the antitumor activity was associated with the down-regulation of molecular markers in tumor tissues that were similar to those observed in vitro.

PTEN loss mediated Akt activation promotes prostate tumor growth and metastasis via CXCL12/CXCR4 signaling

IntroductionThe chemokine CXCL12, also known as SDF-1, and its receptor, CXCR4, are overexpressed in prostate cancers and in animal models of prostate-specific PTEN deletion, but their regulation is poorly understood. Loss of the tumor suppressor PTEN (phosphatase and tensin homolog) is frequently observed in cancer, resulting in the deregulation of cell survival, growth, and proliferation. We hypothesize that loss of PTEN and subsequent activation of Akt, frequent occurrences in prostate cancer, regulate the CXCL12/CXCR4 signaling axis in tumor growth and bone metastasis.MethodsMurine prostate epithelial cells from PTEN+/+, PTEN+/−, and PTEN−/− (prostate specific knockdown) mice as well as human prostate cancer cell lines C4-2B, PC3, and DU145 were used in gene expression and invasion studies with Akt inhibition. Additionally, HA-tagged Akt1 was overexpressed in DU145, and tumor growth in subcutaneous and intra-tibia bone metastasis models were analyzed.ResultsLoss of PTEN resulted in increased expression of CXCR4 and CXCL12 and Akt inhibition reversed expression and cellular invasion. These results suggest that loss of PTEN may play a key role in the regulation of this chemokine activity in prostate cancer. Overexpression of Akt1 in DU145 resulted in increased CXCR4 expression, as well as increased proliferation and cell cycle progression. Subcutaneous injection of these cells also resulted in increased tumor growth as compared to neo controls. Akt1 overexpression reversed the osteosclerotic phenotype associated with DU145 cells to an osteolytic phenotype and enhanced intra-osseous tumor growth.ConclusionsThese results suggest the basis for activation of CXCL12 signaling through CXCR4 in prostate cancer driven by the loss of PTEN and subsequent activation of Akt. Akt1-associated CXCL12/CXCR4 signaling promotes tumor growth, suggesting that Akt inhibitors may potentially be employed as anticancer agents to target expansion of PC bone metastases.

Bone marrow stromal cells enhance prostate cancer cell invasion through type I collagen in an MMP‐12 dependent manner

At the cellular level, the process of bone metastasis involves many steps. Circulating cancer cells enter the marrow, proliferate, induce neovascularization, and ultimately expand into a clinically detectable, often symptomatic, metastatic deposit. Although the initial establishment and later expansion of the metastatic deposit in bone require tumor cells to possess invasive capability, the exact proteases responsible for this phenotype are not well known. The objective of our study was to take an unbiased approach to determine which proteases were expressed and functional during the initial interactions between prostate cancer cells and bone marrow stromal (BMS) cells. We found that the combination of human prostate cancer PC3 and BMS cells stimulates the invasive ability of cancer cells through type I collagen. The use of inhibitors for each of the major protease families indicated that 1 or more MMPs was/were responsible for the BMS‐induced invasion. Gene profiling and semiquantitative RT‐PCR analysis revealed an increased expression of several MMP genes because of PC3/BMS cell interaction. However, only MMP‐12 showed an increase in protein expression. Downregulation of MMP‐12 expression in PC3 cells by siRNA inhibited the enhanced invasion induced by PC3/BMS cell interaction. In vivo, MMP‐12 was found to be primarily expressed by prostate cancer cells growing in bone. Our data suggest that BMS cells induce MMP‐12 expression in prostate cancer cells, which results in invasive cells capable of degradation of type I collagen.

The Inactive 44-kDa Processed Form of Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Enhances Proteolytic Activity via Regulation of Endocytosis of Active MT1-MMP

Membrane type 1 (MT1) matrix metalloproteinase (MMP-14) is a membrane-tethered MMP considered to be a major mediator of pericellular proteolysis. MT1-MMP is regulated by a complex array of mechanisms, including processing and endocytosis that determine the pool of active proteases on the plasma membrane. Autocatalytic processing of active MT1-MMP generates an inactive membrane-tethered 44-kDa product (44-MT1) lacking the catalytic domain. This form preserves all other enzyme domains and is retained at the cell surface. Paradoxically, accumulation of the 44-kDa form has been associated with increased enzymatic activity. Here we report that expression of a recombinant 44-MT1 (Gly285–Val582) in HT1080 fibrosarcoma cells results in enhanced pro-MMP-2 activation, proliferation within a three-dimensional collagen I matrix, and tumor growth and lung metastasis in mice. Stimulation of pro-MMP-2 activation and growth in collagen I was also observed in other cell systems. Expression of 44-MT1 in HT1080 cells is associated with a delay in the rate of active MT1-MMP endocytosis resulting in higher levels of active enzyme at the cell surface. Consistently, deletion of the cytosolic domain obliterates the stimulatory effects of 44-MT1 on MT1-MMP activity. In contrast, deletion of the hinge turns the 44-MT1 form into a negative regulator of enzyme function in vitro and in vivo, suggesting a key role for the hinge region in the functional relationship between active and processed MT1-MMP. Together, these results suggest a novel role for the 44-kDa form of MT1-MMP generated during autocatalytic processing in maintaining the pool of active enzyme at the cell surface.

A novel function for platelet-derived growth factor D: induction of osteoclastic differentiation for intraosseous tumor growth

Although increasing evidence suggests a critical role for platelet-derived growth factor (PDGF) receptor β (β-PDGFR) signaling in prostate cancer (PCa) progression, the precise roles of β-PDGFR and PDGF isoform-specific cell signaling have not been delineated. Recently, we identified the PDGF-D isoform as a ligand for β-PDGFR in PCa and showed that PDGF-D is activated by serine protease-mediated proteolytic removal of the CUB domain in a two-step process, yielding first a hemidimer (HD) and then a growth factor domain dimer. Herein, we demonstrate that the expression of PDGF-D in human PCa LNCaP cells leads to enhanced bone tumor growth and bone responses in immunodeficient mice. Histopathological analyses of bone tumors generated by PDGF-D-expressing LNCaP cells (LNCaP-PDGF-D) revealed osteolytic and osteoblastic responses similar to those observed in human PCa bone metastases. Importantly, we discovered a novel function of PDGF-D in the regulation of osteoclast differentiation, independent of the RANKL/RANK signaling axis. Although both PDGF-B and -D were able to activate β-PDGFR, only PDGF-D was able to induce osteoclastic differentiation in vitro, and upregulate the expression and nuclear translocation of nuclear factor of activated T cells 1, a master transcription factor for osteoclastogenesis. Taken together, these results reveal a new function of PDGF-D as a regulator of osteoclastic differentiation, an activity critical for the establishment of skeletal metastatic deposit in PCa patients.

Cediranib inhibits both the intraosseous growth of PDGF D-positive prostate cancer cells and the associated bone reaction.

The major cause of death in prostate cancer (PCa) cases is due to distant metastatic lesions, with the bone being the most prevalent site for secondary colonization. Utilization of small molecule inhibitors to treat bone metastatic PCa have had limited success either as monotherapies or in combination with other chemotherapeutics due to intolerable toxicities. In the current study, we developed a clinically relevant in vivo intraosseous tumor model overexpressing the platelet‐derived growth factor D (PDGF D) to test the efficacy of a newly characterized vascular endothelial growth factor receptor (VEGFR)/PDGFR inhibitor, cediranib (also called AZD2171).

The mouse prostate: a basic anatomical and histological guideline

Despite substantial similarities in embryological, cellular and molecular biology features, human and mouse prostates differ in organ morphology and tissue architecture. Thus, a clear understanding of the anatomy and histology of the mouse prostate is essential for the identification of urogenital phenotypes in genetically engineered mice, as well as for the study of the etiology, development, and treatment of human prostatic diseases for which mouse models are used. The purpose of this manuscript is to provide a brief guide for the dissection of the mouse prostate and the identification of its different lobes and histology, to both basic researchers and medical pathologists who are unfamiliar with mouse tissues.

Bone-induced c-kit expression in prostate cancer: A driver of intraosseous tumor growth

Loss of BRCA2 function stimulates prostate cancer (PCa) cell invasion and is associated with more aggressive and metastatic tumors in PCa patients. Concurrently, the receptor tyrosine kinase c‐kit is highly expressed in skeletal metastases of PCa patients and induced in PCa cells placed into the bone microenvironment in experimental models. However, the precise requirement of c‐kit for intraosseous growth of PCa and its relation to BRCA2 expression remain unexplored. Here, we show that c‐kit expression promotes migration and invasion of PCa cells. Alongside, we found that c‐kit expression in PCa cells parallels BRCA2 downregulation. Gene rescue experiments with human BRCA2 transgene in c‐kit‐transfected PCa cells resulted in reduction of c‐kit protein expression and migration and invasion, suggesting a functional significance of BRCA2 downregulation by c‐kit. The inverse association between c‐kit and BRCA2 gene expressions in PCa cells was confirmed using laser capture microdissection in experimental intraosseous tumors and bone metastases of PCa patients. Inhibition of bone‐induced c‐kit expression in PCa cells transduced with lentiviral short hairpin RNA reduced intraosseous tumor incidence and growth. Overall, our results provide evidence of a novel pathway that links bone‐induced c‐kit expression in PCa cells to BRCA2 downregulation and supports bone metastasis.

Abstract 4088: PTEN loss mediated Akt activation promotes prostate tumor growth via CXCL12/CXCR4 signaling.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: Chemokines are a family of cytokines known to regulate the migration of cells. The chemokine CXCL12, also known as SDF-1, and its receptor CXCR4 are associated with prostate cancer bone metastasis. The tumor suppressor PTEN (phosphatase and tensin homolog) is a critical regulator of growth factors and inhibitor of PI3K. Loss of PTEN is frequently observed in cancer, resulting in the deregulation of cell survival, growth, and proliferation. Previous studies have found that PTEN is lost or mutated in 30-80% of primary prostate cancer, and 50% of prostate cancer bone metastases. In mouse models of prostate cancer, it has been shown that loss of PTEN is critical for tumor initiation, and the level of PTEN expression is inversely associated with prostate tumorigenesis. Murine epithelial cells from PTEN-deficient prostate tumors also display increased expression of CXCR4 and CXCL12. We hypothesize that loss of PTEN and subsequent activation of Akt, frequent occurrences in prostate cancer, regulate the CXCL12/CXCR4 signaling axis in prostate cancer progression. Methods: Prostate-specific deletion of floxed exon 5 of PTEN was achieved by Cre recombinase expressed under the control of an androgen-responsive probasin promoter. Stable cell lines were developed from PTEN+/+, PTEN+/−, and PTEN−/− murine prostate epithelial cells isolated from prostates of corresponding mice at 8 weeks of age. PTEN status in these cell lines was confirmed by genotyping and immunoblot analyses. Human prostate cancer cell lines BPH-1, C4-2B, PC3, and DU145 were also utilized. Gene expression and invasion were analyzed in the presence or absence of Akt inhibitor. Additionally, DU145 with overexpressed HA-tagged Akt1 was also utilized in a subcutaneous injection model. Results: Loss of PTEN resulted in increased expression of CXCR4 and CXCL12, suggesting that loss of PTEN may play a key role in the regulation of these chemokines in prostate cancer. Upon treatment of PTEN-null cells with Akt inhibitor, the induction of CXCL12/CXCR4 was reversed, further demonstrating the role of the PTEN regulated pathway in the expression of these genes. Akt inhibition also resulted in decreased invasion in response to CXCL12. Overexpression of Akt1 in DU145 resulted increased CXCR4 expression, as well as increased proliferation and cell cycle progression. Subcutaneous injection of these cells also resulted in increased tumor growth as compared to neo controls. Conclusions: These results suggest the basis for activation of CXCL12 signaling through CXCR4 in prostate cancer driven by the loss of PTEN and subsequent activation of Akt. Citation Format: Katie Conley-Lacomb, Allen Saliganan, Yong Q. Chen, Hyeong-Reh C. Kim, Michael L. Cher, Sreenivasa R. Chinni. PTEN loss mediated Akt activation promotes prostate tumor growth via CXCL12/CXCR4 signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4088. doi:10.1158/1538-7445.AM2013-4088