Mengqian Chen

Cyclin-dependent kinase 8 mediates chemotherapy-induced tumor-promoting paracrine activities

Conventional chemotherapy not only kills tumor cells but also changes gene expression in treatment-damaged tissues, inducing production of multiple tumor-supporting secreted factors. This secretory phenotype was found here to be mediated in part by a damage-inducible cell-cycle inhibitor p21 (CDKN1A). We developed small-molecule compounds that inhibit damage-induced transcription downstream of p21. These compounds were identified as selective inhibitors of a transcription-regulating kinase CDK8 and its isoform CDK19. Remarkably, p21 was found to bind to CDK8 and stimulate its kinase activity. p21 and CDK8 also cooperate in the formation of internucleolar bodies, where both proteins accumulate. A CDK8 inhibitor suppresses damage-induced tumor-promoting paracrine activities of tumor cells and normal fibroblasts and reverses the increase in tumor engraftment and serum mitogenic activity in mice pretreated with a chemotherapeutic drug. The inhibitor also increases the efficacy of chemotherapy against xenografts formed by tumor cell/fibroblast mixtures. Microarray data analysis revealed striking correlations between CDK8 expression and poor survival in breast and ovarian cancers. CDK8 inhibition offers a promising approach to increasing the efficacy of cancer chemotherapy.

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.

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.

The Stress Response Mediator ATF3 Represses Androgen Signaling by Binding the Androgen Receptor

ABSTRACT Activating transcription factor 3 (ATF3) is a common mediator of cellular stress response signaling and is often aberrantly expressed in prostate cancer. We report here that ATF3 can directly bind the androgen receptor (AR) and consequently repress AR-mediated gene expression. The ATF3-AR interaction requires the leucine zipper domain of ATF3 that independently binds the DNA-binding and ligand-binding domains of AR, and the interaction prevents AR from binding to cis-acting elements required for expression of androgen-dependent genes while inhibiting the AR N- and C-terminal interaction. The functional consequences of the loss of ATF3 expression include increased transcription of androgen-dependent genes in prostate cancer cells that correlates with increased ability to grow in low-androgen-containing medium and increased proliferative activity of the prostate epithelium in ATF3 knockout mice that is associated with prostatic hyperplasia. Our results thus demonstrate that ATF3 is a novel repressor of androgen signaling that can inhibit AR functions, allowing prostate cells to restore homeostasis and maintain integrity in the face of a broad spectrum of intrinsic and environmental insults.

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.

Paracrine hedgehog increases the steroidogenic potential of prostate stromal cells in a Gli‐dependent manner

Acquired intratumoral steroidogenesis is involved in progression of prostate cancer to castration resistant disease (CRPC) and a target for improved therapeutics. Recent work has shown that prostate cancer cells can acquire steroidogenic activity as they progress to a therapeutic‐resistant state. However, benign prostate stromal cells (PrSCs) also have steroidogenic potential though they are often overlooked as a source of intratumoral androgens. Here, we present preliminary studies showing that the steroidogenic activity of primary human PrSCs is significantly increased by exposure to a Hedgehog agonist (SAG) or by transduction of PrSCs with lentiviruses that expresses active Gli2 (Gli2ΔN), a transcription factor that is triggered by Hh signaling. Comparative gene expression profiling on Chips, that was confirmed by quantitative real‐time PCR, revealed that hedgehog agonist treatment induced in these cells expressions of hedgehog target genes (Gli1, Ptch1, and SCUBE1) plus a specific cadre of genes involved in cholesterol/steroid biosynthesis, metabolism, and transport. Genes involved downstream in steroid hormone generation, including CYP17A1 and CYP19A1 were also induced. Both the hedgehog agonist and the Gli2‐expressing lentivirus significantly increased the output of testosterone (T) from PrSCs that were supplemented with dihydroepiandrosterone (DHEA), an adrenal precursor of T. Finally, knockdown of Gli2 by siRNA suppressed the ability of SAG to induce this response. Collectively, our data indicate that hedgehog/Gli signaling may be a factor in acquired intratumoral steroidogenesis of a prostate tumor through its actions on stromal cells in the tumor microenvironment and an influence for the development of CRPC. Prostate 72:817–824, 2012.

Therapy-induced developmental reprogramming of prostate cancer cells and acquired therapy resistance

Treatment-induced neuroendocrine transdifferentiation (NEtD) complicates therapies for metastatic prostate cancer (PCa). Based on evidence that PCa cells can transdifferentiate to other neuroectodermally-derived cell lineages in vitro, we proposed that NEtD requires first an intermediary reprogramming to metastable cancer stem-like cells (CSCs) of a neural class and we demonstrate that several different AR+/PSA+ PCa cell lines were efficiently reprogrammed to, maintained and propagated as CSCs by growth in androgen-free neural/neural crest (N/NC) stem medium. Such reprogrammed cells lost features of prostate differentiation; gained features of N/NC stem cells and tumor-initiating potential; were resistant to androgen signaling inhibition; and acquired an invasive phenotype in vitro and in vivo. When placed back into serum-containing mediums, reprogrammed cells could be re-differentiated to N-/NC-derived cell lineages or return back to an AR+ prostate-like state. Once returned, the AR+ cells were resistant to androgen signaling inhibition. Acute androgen deprivation or anti-androgen treatment in serum-containing medium led to the transient appearance of a sub-population of cells with similar characteristics. Finally, a 132 gene signature derived from reprogrammed PCa cell lines distinguished tumors from PCa patients with adverse outcomes. This model may explain neural manifestations of PCa associated with lethal disease. The metastable nature of the reprogrammed stem-like PCa cells suggests that cycles of PCa cell reprogramming followed by re-differentiation may support disease progression and therapeutic resistance. The ability of a gene signature from reprogrammed PCa cells to identify tumors from patients with metastasis or PCa-specific mortality implies that developmental reprogramming is linked to aggressive tumor behaviors.

Inhibition of CDK8 mediator kinase suppresses estrogen dependent transcription and the growth of estrogen receptor positive breast cancer

Hormone therapy targeting estrogen receptor (ER) is the principal treatment for ER-positive breast cancers. However, many cancers develop resistance to hormone therapy while retaining ER expression. Identifying new druggable mediators of ER function can help to increase the efficacy of ER-targeting drugs. Cyclin-dependent kinase 8 (CDK8) is a Mediator complex-associated transcriptional regulator with oncogenic activities. Expression of CDK8, its paralog CDK19 and their binding partner Cyclin C are negative prognostic markers in breast cancer. Meta-analysis of transcriptome databases revealed an inverse correlation between CDK8 and ERα expression, suggesting that CDK8 could be functionally associated with ER. We have found that CDK8 inhibition by CDK8/19-selective small-molecule kinase inhibitors, by shRNA knockdown or by CRISPR/CAS9 knockout suppresses estrogen-induced transcription in ER-positive breast cancer cells; this effect was exerted downstream of ER. Estrogen addition stimulated the binding of CDK8 to the ER-responsive GREB1 gene promoter and CDK8/19 inhibition reduced estrogen-stimulated association of an elongation-competent phosphorylated form of RNA Polymerase II with GREB1. CDK8/19 inhibitors abrogated the mitogenic effect of estrogen on ER-positive cells and potentiated the growth-inhibitory effects of ER antagonist fulvestrant. Treatment of estrogen-deprived ER-positive breast cancer cells with CDK8/19 inhibitors strongly impeded the development of estrogen independence. In vivo treatment with a CDK8/19 inhibitor Senexin B suppressed tumor growth and augmented the effects of fulvestrant in ER-positive breast cancer xenografts. These results identify CDK8 as a novel downstream mediator of ER and suggest the utility of CDK8 inhibitors for ER-positive breast cancer therapy.