Matthew Tanner

Androgenic regulation of hedgehog signaling pathway components in prostate cancer cells

Hedgehog signaling is thought to play a role in several human cancers including prostate cancer. Although prostate cancer cells express many of the gene products involved in hedgehog signaling, these cells are refractory to the canonical signaling effects of exogenous hedgehog ligands or to activated Smoothened, the hedgehog-regulated mediator of Gli transcriptional activation. Here, we show that the expression of hedgehog ligands and some hedgehog target genes are regulated by androgen in the human prostate cancer cell line, LNCaP and its more metastatic variants (C4-2 and C4-2B). Androgen (R1881) strongly suppressed the expression of hedgehog ligands in these cells and their prolonged maintenance in androgen-deficient medium upregulated Sonic and Indian hedgehog mRNA and protein levels by up to 30,000-fold. Hedgehogs were released into the conditioned medium of androgen-deprived LNCaP cells and this medium was able to increase hedgehog target gene expression in hedgehog-responsive mouse fibroblasts (MC3T3-E1). Moreover, this activity was accompanied by increased expression of Gli target genes, Patched 1 and Gli2, in LNCaP that could be suppressed by cyclopamine, indicating that chronic androgen-deprivation also re-awakens the autocrine responsiveness of the cancer cells to hedgehog. In contrast to the suppressive effects of R1881 on hedgehog ligand and Gli2 expression, we found that Gli1 expression in LNCaP cells was induced by R1881. Given the ability of androgen to modulate the expression and release of hedgehog ligands and the activity of the autocrine hedgehog signaling pathway in these prostate cancer cells, our results imply that chronic androgen deprivation therapy (ADT) for prostate cancer might create a hedgehog signaling environment in the region of the tumor that could ultimately impact on the long term effectiveness of this treatment. This consideration supports the idea of clinically testing hedgehog-blocking drugs in conjunction with ADT in patients with advanced prostate cancer.

Hedgehog/Gli supports androgen signaling in androgen deprived and androgen independent prostate cancer cells

BackgroundCastration resistant prostate cancer (CRPC) develops as a consequence of hormone therapies used to deplete androgens in advanced prostate cancer patients. CRPC cells are able to grow in a low androgen environment and this is associated with anomalous activity of their endogenous androgen receptor (AR) despite the low systemic androgen levels in the patients. Therefore, the reactivated tumor cell androgen signaling pathway is thought to provide a target for control of CRPC. Previously, we reported that Hedgehog (Hh) signaling was conditionally activated by androgen deprivation in androgen sensitive prostate cancer cells and here we studied the potential for cross-talk between Hh and androgen signaling activities in androgen deprived and androgen independent (AI) prostate cancer cells.ResultsTreatment of a variety of androgen-deprived or AI prostate cancer cells with the Hh inhibitor, cyclopamine, resulted in dose-dependent modulation of the expression of genes that are regulated by androgen. The effect of cyclopamine on endogenous androgen-regulated gene expression in androgen deprived and AI prostate cancer cells was consistent with the suppressive effects of cyclopamine on the expression of a reporter gene (luciferase) from two different androgen-dependent promoters. Similarly, reduction of smoothened (Smo) expression with siRNA co-suppressed expression of androgen-inducible KLK2 and KLK3 in androgen deprived cells without affecting the expression of androgen receptor (AR) mRNA or protein. Cyclopamine also prevented the outgrowth of AI cells from androgen growth-dependent parental LNCaP cells and suppressed the growth of an overt AI-LNCaP variant whereas supplemental androgen (R1881) restored growth to the AI cells in the presence of cyclopamine. Conversely, overexpression of Gli1 or Gli2 in LNCaP cells enhanced AR-specific gene expression in the absence of androgen. Overexpressed Gli1/Gli2 also enabled parental LNCaP cells to grow in androgen depleted medium. AR protein co-immunoprecipitates with Gli2 protein from transfected 293T cell lysates.ConclusionsCollectively, our results indicate that Hh/Gli signaling supports androgen signaling and AI growth in prostate cancer cells in a low androgen environment. The finding that Gli2 co-immunoprecipitates with AR protein suggests that an interaction between these proteins might be the basis for Hedgehog/Gli support of androgen signaling under this condition.

Effects of Androgen Receptor and Androgen on Gene Expression in Prostate Stromal Fibroblasts and Paracrine Signaling to Prostate Cancer Cells

The androgen receptor (AR) is expressed in a subset of prostate stromal cells and functional stromal cell AR is required for normal prostate developmental and influences the growth of prostate tumors. Although we are broadly aware of the specifics of the genomic actions of AR in prostate cancer cells, relatively little is known regarding the gene targets of functional AR in prostate stromal cells. Here, we describe a novel human prostate stromal cell model that enabled us to study the effects of AR on gene expression in these cells. The model involves a genetically manipulated variant of immortalized human WPMY-1 prostate stromal cells that overexpresses wildtype AR (WPMY-AR) at a level comparable to LNCaP cells and is responsive to dihydrotestosterone (DHT) stimulation. Use of WPMY-AR cells for gene expression profiling showed that the presence of AR, even in the absence of DHT, significantly altered the gene expression pattern of the cells compared to control (WPMY-Vec) cells. Treatment of WPMY-AR cells, but not WPMY-Vec control cells, with DHT resulted in further changes that affected the expression of 141 genes by 2-fold or greater compared to vehicle treated WPMY-AR cells. Remarkably, DHT significantly downregulated more genes than were upregulated but many of these changes reversed the initial effects of AR overexpression alone on individual genes. The genes most highly effected by DHT treatment were categorized based upon their role in cancer pathways or in cell signaling pathways (transforming growth factor-β, Wnt, Hedgehog and MAP Kinase) thought to be involved in stromal-epithelial crosstalk during prostate or prostate cancer development. DHT treatment of WPMY-AR cells was also sufficient to alter their paracrine potential for prostate cancer cells as conditioned medium from DHT-treated WPMY-AR significantly increased growth of LNCaP cells compared to DHT-treated WPMY-Vec cell conditioned medium.

Gli2 Expression and Human Bladder Transitional Carcinoma Cell Invasiveness

PURPOSE Hedgehog signaling regulates Gli transcription factors. Aberrant hedgehog signaling can be oncogenic and drugs that block hedgehog are being tested as anticancer agents. We considered whether hedgehog/Gli signaling may be involved in human bladder transitional cell carcinoma proliferative or invasive behavior. MATERIALS AND METHODS We stratified the human bladder transitional cell carcinoma lines RT4 (ATCC), 253JP, 253BV, UMUC6 and UMUC3 for relative growth rate by cell counting and for in vitro invasiveness by Matrigel invasion assay. Cells were tested for growth inhibition by the hedgehog blocking drug cyclopamine or the inactive mimic tomatidine. Cell RNA was characterized for hedgehog signaling component expression, including ligands, receptors and signaling mediators, by quantitative reverse transcriptase-polymerase chain reaction. Gli2 expression or activity was modified by Gli2 expression lentiviruses or the Gli inhibitor GANT61. We measured effects on proliferation and invasiveness. RESULTS Cell growth rates and invasiveness were stratified into an equivalent order (RT4 <243JP <253BV <UMUC6 <UMUC3). All cells were weakly growth inhibited by tomatidine and cyclopamine. Gli2 was the only hedgehog signaling molecule of which expression correlated with stratification. Manipulation of Gli2 expression or activity significantly affected cell invasiveness. CONCLUSIONS Weak growth suppression by cyclopamine suggests that hedgehog signaling is not involved in bladder cancer cell proliferation but Gli2 expression strongly correlated with invasive behavior. Increased Gli2 expression increased low Gli2 cell invasiveness while Gli inhibition by GANT61 decreased high Gli2 cell invasiveness. Results suggest that Gli2 expression by noncanonical signaling contributes to bladder cancer cell invasiveness.

Differential Expression of Vascular Endothelial Growth Factor, and Angiopoietin 1 and 2 in Functionally Divergent Experimental Rabbit Models of Bladder Hypertrophy

PURPOSE Partial bladder outlet obstruction or ovariectomy with subsequent estrogen replenishment induces bladder hypertrophy in rabbits and yet the functional outcomes of these procedures differ. We investigated whether these models might be distinguished by differential expression of the genes controlling angiogenesis. MATERIALS AND METHODS Groups of male rabbits underwent sham surgery or partial bladder outlet obstruction for 1 or 2 weeks. Groups of females underwent sham surgery, ovariectomy or ovariectomy plus estrogen for 1 or 2 weeks. Bladders from each group were weighed and assayed for the contractile response, smooth muscle content and vascular density. Mucosa and muscle layers were separated and RNA from the fractions was assayed by quantitative real-time polymerase chain reaction to measure the relative expression of vascular endothelial growth factor, and angiopoietin 1 and 2 mRNA. RESULTS Male bladders with partial outlet obstruction had attributes that typified hypertrophy with a loss of contractile function. Vascular endothelial growth factor expression was up-regulated in the mucosa and muscle layers but the effect was most pronounced in mucosa. Angiopoietin 1 expression was significantly up-regulated in muscle. Female bladders with ovariectomy plus estrogen had attributes that typified bladder hypertrophy with increased contractile function. Vascular endothelial growth factor expression was up-regulated early in mucosa but more highly and consistently increased in muscle. Angiopoietin 1 and 2 expression was not significantly affected. CONCLUSIONS Although these models have similar outcomes with regard to bladder hypertrophy, they have opposite functional outcomes that coincide with compartmental differences in the expression of genes involved in the regulation of angiogenesis. The disparity in gene expression might explain the difference in the functional outcomes.

Unique Effects of Wnt Signaling on Prostate Cancer Cells: Modulation of the Androgen Signaling Pathway by Interactions of the Androgen Receptor Gene and Protein with Key Components of the Canonical Wnt Signaling Pathway

Canonical Wnt is a ligand-driven signaling pathway that augments the intracellular stability of β-catenin. β-Catenin is transcriptional coactivator protein that is best known for its role in regulating the activity of the Tcf/LEF-1 transcription factor family but it binds and regulates several other types of transcription factors including the androgen receptor (AR). Aberrant Wnt signaling can contribute to oncogenesis and this is often a factor in human colon cancer where mutations in key Wnt regulatory genes promote constitutive β-catenin stabilization in the tumor cells. This chapter reviews the evidence that Wnt signaling might also play some role in prostate cancer through its effects on the expression of androgen-dependent genes. These effects are mechanistically driven through a diverse set of interactions between three key molecules in the Wnt pathway, β-catenin, glycogen synthase kinase-3β, and Tcf/LEF-1, with the human AR gene and protein. For the most part, these interactions enhance AR expression or its functional activity as a transcription factor and they have the potential of augmenting the growth of prostate cancer cells even when androgen levels are within castrate range. Although the common gene mutations that drive abnormal Wnt signaling in other human cancers are not as frequently observed in human prostate tumors, gene expression profiles of prostate tumors often show overexpression of wnt ligands in the cancer cells and this may have a comparable effect on prostate cancer development or progression. Given the important role of the canonical Wnt signaling pathway in promoting oncogenesis in some human tissues as well as the specific involvement of key Wnt molecules in regulating androgen action in prostate cancer cells, effectors of the Wnt signaling pathway likely represent unique targets to bring metastatic and castration-resistant prostate cancers under control.