Ralph Buttyan

Increased expression of class III β -tubulin in castration-resistant human prostate cancer

Background:Class III β-tubulin (βIII-tubulin) is expressed in tissues of neuronal lineage and also in several human malignancies, including non-small-cell lung carcinoma, breast and ovarian cancer. Overexpression of βIII-tubulin in these tumours is associated with an unfavourable outcome and resistance to taxane-based therapies. At present, βIII-tubulin expression remains largely uncharacterised in prostate cancer.Methods:In this report, we evaluated the expression of βIII-tubulin in 138 different human prostate tumour specimens by immunohistochemistry from patients with hormone-treated or hormone-untreated prostate cancer. βIII-tubulin expression was also examined in various prostatic cancer cell lines including in androgen-sensitive human prostate cancer cells, LNCaP, grown in androgen-depleted medium in 2D cultures or as tumour xenografts when the host mouse was castrated.Results:Whereas moderate-to-strong βIII-tubulin expression was detected in only 3 out of 74 (4%) hormone-naive tumour specimens obtained from patients who never received hormone therapy, 6 out of 24 tumour specimens (25%) from patients treated for 3 months with neoadjuvant hormone therapy and 24 out of 40 (60%) castration-resistant tumour specimens from chronic hormone-treated patients were found to express significant levels of βIII-tubulin. These findings were supported by in vitro and in vivo settings.Conclusion:Our data indicate that βIII-tubulin expression is augmented in prostate cancer by androgen ablation and that the expression of this β-tubulin isoform is associated with the progression of prostate cancer to the castration-resistant state, a stage largely responsible for mortality from prostate cancer.

Hormone-refractory prostate cancer: a multi-step and multi-event process.

Since the pioneering studies of Huggins in 1941, it has been known that prostate cancer cells, like certain normal epithelial cells, can chronically depend on a critical level of androgenic stimulation for their continuous growth and survival. The entire issue of the development of resistance to androgen ablation therapy for metastatic prostate cancer is based on the fact that a portion of cells can survive without androgen stimulation. The cell mechanism of androgen independent status is unclear. For some authors, a portion of the cells present within a patient with a prostate cancer before therapy is naturally androgen independent (selection hypothesis). However, this hypothesis does not consider gene alteration during prostate cancer natural history and probably hormone-refractory prostate cancer (HRPC) is due to a multi-step and multi-event process. In this literature review, different cell pathways that lead to HRPC are described..

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.

The hedgehog/Gli signaling paradigm in prostate cancer.

Hedgehog is a ligand-activated signaling pathway that regulates Gli-mediated transcription. Although most noted for its role as an embryonic morphogen, hyperactive hedgehog also causes human skin and brain malignancies. The hedgehog-related gene anomalies found in these tumors are rarely found in prostate cancer. Yet surveys of human prostate tumors show concordance of high expression of hedgehog ligands and Gli2 that correlate with the potential for metastasis and therapy-resistant behavior. Likewise, prostate cancer cell lines express hedgehog target genes, and their growth and survival is affected by hedgehog/Gli inhibitors. To date, the preponderance of data supports the idea that prostate tumors benefit from a paracrine hedgehog microenvironment similar to the developing prostate. Uncertainty remains as to whether hedgehog’s influence in prostate cancer also includes aspects of tumor cell autocrine-like signaling. The recent findings that Gli proteins interact with the androgen receptor and affect its transcriptional output have helped to identify a novel pathway through which hedgehog/Gli might affect prostate tumor behavior and raises questions as to whether hedgehog signaling in prostate cancer cells is suitably measured by the expression of Gli target genes alone.

Gli2 Expression and Human Bladder Transitional Carcinoma Cell Invasiveness

PURPOSEnHedgehog 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.nnnMATERIALS AND METHODSnWe 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.nnnRESULTSnCell 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.nnnCONCLUSIONSnWeak 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.