Drive Prostate Cancer Progression Research Articles

The polyphenols (-)-epigallocatechin-3-gallate and luteolin synergistically inhibit TGF-β-induced myofibroblast phenotypes through RhoA and ERK inhibition.

The presence of reactive stroma, predominantly composed of myofibroblasts, is directly associated with and drives prostate cancer progression. We have previously shown that (−)-Epigallocatechin-3-gallate (EGCG), in the form of Polyphenon E, significantly decreases serum levels of HGF and VEGF in prostate cancer patients. Given that HGF and VEGF are secreted from surrounding tumor myofibroblasts, these observations suggested that EGCG may inhibit prostate cancer-associated myofibroblast differentiation. Herein, we demonstrate that micromolar combinations of EGCG and a second polyphenol, luteolin, synergistically inhibit TGF-β-induced myofibroblast phenotypes in prostate fibroblast cell lines, as observed primarily by potentiation of fibronectin expression. Functionally, EGCG and luteolin inhibited TGF-β-induced extracellular matrix contraction, an enhancer of tumor cell invasion. EGCG and luteolin inhibited downstream TGF-β-induced signaling, including activation of ERK and AKT, respectively, but mechanistically, only ERK appeared to be necessary for TGF-β-induced fibronectin expression. Furthermore, neither EGCG nor luteolin affected Smad signaling or nuclear translocation. Rho signaling was found to be necessary for TGF-β-induced fibronectin expression and EGCG and luteolin each reduced RhoA activation. Finally, EGCG and luteolin were shown to reverse TGF-β-induced fibronectin expression, implicating that these natural compounds may be useful not only in preventing but also in treating already activated myofibroblasts and the diseases they cause, including cancer. The ability of EGCG and luteolin to synergistically target myofibroblasts suggests that combined clinical use of these compounds could prevent or reverse cancer progression through targeting the tumor microenvironment, in addition to the tumor itself.

Complex Genomic Rearrangements Contribute to Prostate Cancer Evolution

Abstract Interdependent genomic restructuring, or “chromoplexy,” drives prostate cancer progression.

Abstract 3062: Transcriptional repressor Kaiso promotes cancer progression by targeting miR-31 in prostate cancer.

Abstract Metastatic spread of prostate cancer (PCa) is responsible for the majority of prostate cancer-related deaths. Whereas socioeconomic factors, different geographic areas and ethnicity contribute to the incidence and mortality rates of this disease, they cannot fully explain the tumor biology. Our understanding of the mechanisms that drive PCa progression and metastasis is still limited. Kaiso belongs to a BTB/POZ zinc finger protein family and is known as a transcriptional repressor by binding to sequence-specific Kaiso binding sites or methyl-CpG dinucleotide pairs. Kaiso expression and localization have been reported to correlate with the prognosis and metastatic potential in several human malignancies. Previous studies from our lab showed that the progression of PCa is closely associated with the over-expression and nuclear translocation of Kaiso. Our objective here is to explore the potential molecular mechanisms underlying Kaiso mediated increase in PCa progression. MicroRNAs play an emerging role in cancer initiation and progression. Therefore we proposed to investigate Kaiso targeting microRNAs and their roles in PCa. PC-3 cells, a highly metastatic PCa cell line, were stably transfected with a shRNA plasmid against human Kaiso or a scramble control. Two clones exhibited >80% downregulation of Kaiso in PC-3 cells at either the transcriptional or translational levels were used for further studies. We determined miRNA expression profiles correlated with Kaiso knock down and DNMT1 inhibitor 5-Aza de-methylation treatment using microRNA arrays. Comparative analysis revealed 11 miRNAs were significantly altered by Kaiso knock down and 32 miRNAs were significantly altered by 5-Aza treatment (> 2 fold, p<0.05). Real-time PCR confirmed that miR-31 expression was up-regulated in both Kaiso knock down cells and 5-Aza treated cells. From the analysis of differentially expressed miRNAs, miR-31 expression was also negatively correlated with kaiso expression in a set of prostate cell lines. ChIP assay revealed that kaiso directly binds to miR-31 promoter. The restoration of miR-31 suppressed cell proliferation; induced apoptosis and inhibited tumor cell invasion and metastasis in vitro. Inhibition of miR-31 in PC-3 shKaiso cells restored kaiso knock down-inhibited cell proliferation; migration and metastasis. Xenograft model indicated that miR-31 overexpression significant suppressed tumor formation in vivo. Taken together, our studies suggest that Kaiso regulates specific miRNA expression in PCa cells, indicating that Kaiso could promote cancer progression through regulating miR-31 expression in a methylation dependent manner. Citation Format: Honghe Wang, ShaNekkia Black, Wei Liu, Fu Zhao, Timothy Turner, Clayton Yates. Transcriptional repressor Kaiso promotes cancer progression by targeting miR-31 in prostate cancer. [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 3062. doi:10.1158/1538-7445.AM2013-3062 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Prostate cancer stem cells: molecular characterization for targeted therapy

Prostate cancer stem cells: molecular characterization for targeted therapy

Wnt/β-catenin signalling in prostate cancer

The Wnts are secreted cysteine-rich glycoproteins that have important roles in the developing embryo as well as in tissue homeostasis in adults. Dysregulation of Wnt signalling can lead to several types of cancer, including prostate cancer. A hallmark of the signalling pathway is the stabilization of the transcriptional co-activator β-catenin, which not only regulates expression of many genes implicated in cancer but is also an essential component of cadherin cell adhesion complexes. β-catenin regulates gene expression by binding members of the T-cell-specific transcription factor/lymphoid enhancer-binding factor 1 (TCF/LEF-1) family of transcription factors. In addition, β-catenin associates with the androgen receptor, a key regulator of prostate growth that drives prostate cancer progression. Wnt/β-catenin signalling can be controlled by secreted Wnt antagonists, many of which are downregulated in cancer. Activation of the Wnt/β-catenin pathway has effects on prostate cell proliferation, differentiation and the epithelial-mesenchymal transition, which is thought to regulate the invasive behaviour of tumour cells. However, whether targeting Wnt/β-catenin signalling is a good therapeutic option for prostate cancer remains unclear.

Abstract A57: Functional synergy between the KLF6-SV1 and c-MYC oncogenes in driving prostate cancer metastasis in a novel mouse model of the disease

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related death among males in the United States. Many prostate tumors remain indolent and localized, while others result in metastatic and castrate-resistant disease. In order to uncover key molecular drivers that regulate the progression from indolent localized prostate cancer to lethal invasive and metastatic disease, the development of genetically relevant target driven mouse models is critical. Highly penetrant mouse models of this transition in which human disease relevant genes are the molecular drivers of disease progression are lacking. This transition is considered to be highly clinically relevant as our ability to distinguish between indolent disease and lethal metastatic forms of prostate cancer and has lead to either undertreatment of potentially poor behaving tumors or overtreatment of otherwise indolent disease. In addition, the paucity of biologically relevant models of advanced prostate cancer has hampered our ability to accurately and faithfully test novel therapeutic agents in relevant models of disease. To address this problem, we have developed a new murine model of PCa that combines transgenic overexpression of two genes known to be overexpressed in human prostate cancer (KLF6-SV1 and MYC). Overexpression of the c-Myc protein in the prostate of transgenic mice results in the development of both high-grade PIN and moderately differentiated prostate cancer. Interestingly, SV1 overexpression occurs late in disease progression with the highest protein expression occurring in locally invasive and metastatic human prostate cancers. In addition, SV1 overexpression in prostate cancer cell lines results in increased metastasis in vivo with no effect on local tumor growth. Combined, this data provides a strong experimental rationale for the characterization of the interactive and synergistic roles of these two disease relevant genes in the progression of moderately differentiated prostate cancer to poorly differentiated prostate cancer. Here we demonstrate for the first time that transgenic overexpression of MYC and SV1 results in invasive, metastatic prostate cancer with high penetrance in vivo. Pathological and molecular analyses identified a phenotype consistent with epithelial to mesenchymal transition (EMT), invasion and metastasis in this transgenic mouse model. Distant metastasis to several organs were observed and all double transgenic MYC and SV1 mice demonstrated evidence of capsular and stromal invasion by 36 weeks of age (n=14) as compared to none of the SV1 or MYC mice alone. To further understand the mechanism by which KLF6-SV1 and c-MYC regulate EMT, invasion, and metastasis, RWPE-1 cells, a non-tumorigenic prostate cell line that express low levels of KLF6-SV1 and c-MYC, were used to over-express c-MYC, KLF6-SV1 alone and in combination. Over-expression of these genes in RWPE-1, recapitulate our findings in vivo as assessed by a reduction in epithelial markers, such as E-cadherin and an increase in mesenchymal makers such as vimentin. These studies suggest that increased MYC and SV1 expression may drive a gene signature that is a prognostic indicator of aggressive and potentially metastatic human prostate cancer. In conclusion, this mouse model provides a novel tool for the functional evaluation of disease relevant genetic drivers of prostate cancer progression and should provide a roadmap for testing novel targeted molecular therapeutics to treat advanced prostate cancer. Citation Format: Sudeh Izadmehr, Christine H. Lee, Heriberto Fernandez, Raheleh Hatami, Alexander Kirschenbaum, Alice C. Levine, AnaLisa Difeo, Goutham Narl. Functional synergy between the KLF6-SV1 and c-MYC oncogenes in driving prostate cancer metastasis in a novel mouse model of the disease [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A57.

Abstract 1526: Alterations in extracellular matrix modulate AR-regulated transcription in prostate cancer cells

Abstract Introduction: The androgen receptor is a key driver of prostate cancer progression. During progression of prostate cancer alterations to the extracellular matrix occur, including changes in expression of the heterotrimeric glycoprotein laminin (LM), which may alter signaling through the androgen receptor. Purpose: To determine if altered expression of laminin chains affects prostate cancer cell behavior and whether, in androgen receptor (AR) positive lines, AR-regulated transcription is affected by different matrices. Methods: We exposed prostate cancer cell lines (LNCaP, LNCaP C4-2, PC3, and DU145) to different extracellular matrix proteins (fibronectin, collagen I and IV, and laminin 111) as well as to mixed matrices (Matrigel™–LM111 and col IV; and LM-rich prostate cancer derived matrices–LM332, 511 and the LM alpha 4, alpha 4 beta 2, or beta 2 chains). We measured adhesion and proliferation on these different matrices and as well as changes in mRNA expression for AR-regulated genes in the LNCaP lines following exposure to the various matrices. Results: Initially we examined what endogenous laminins the prostate cancer cell lines produced and deposited to determine what they might contribute to the exogenous ECMs tested. When grown on uncoated plastic, we found no expression of LM332 by any of the prostate cancer lines used. Cytoplasmic expression of the LM β1γ1 chains was detected in the lines, but deposition of these laminin chains did not occur. We concluded that these lines are not significantly contributing laminins to the ECM. When exposed to the test ECMs, these prostate cancer lines demonstrated increased adhesion to LM-rich PCa-derived matrices, compared with MatrigelTM, fibronectin, or collagen. An inhibitory integrin beta 1 antibody blocked adhesion to LM-rich PCa-derived matrices by <50%, whereas the same antibody blocked adhesion to fibronectin, collagen, LM111, and Matrigel by >90%, suggesting that additional integrins are involved in adhesion to the LM-rich PCa-derived matrices. When these prostate cancer lines were grown on ECM from LM-rich PCa-derived matrices, increased proliferation resulted compared with proliferation on other ECM components. Exposure of the AR positive LNCaP cells to the different ECM components altered expression of AR-regulated genes, including PSA, TMPRSS2, and IGF-IR. The LM-rich PCa-derived matrices increased expression of these genes compared with exposure to fibronectin, collagen, Matrigel™, and uncoated wells. Summary: Alterations in laminins during progression of prostate cancer affect cancer cell adhesion and proliferation. Further, exposure to LM-rich matrices increased expression of growth promoting AR-regulated genes, which may, in part, modulate cancer cell migration and proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1526. doi:10.1158/1538-7445.AM2011-1526

Shepherding AKT and androgen receptor by Ack1 tyrosine kinase

Ack1 (also known as ACK, TNK2, or activated Cdc42 kinase) is a structurally unique non-receptor tyrosine kinase that is expressed in diverse cell types. It integrates signals from plethora of ligand-activated receptor tyrosine kinases (RTKs), for example, MERTK, EGFR, HER2, PDGFR and insulin receptor to initiate intracellular signaling cascades. Ack1 transduces extracellular signals to cytosolic and nuclear effectors such as the protein kinase AKT/PKB and androgen receptor (AR), to promote cell survival and growth. While tyrosine phosphorylation of AR at Tyr267 regulates androgen-independent recruitment of AR to the androgen-responsive enhancers and transcription of AR target genes to drive prostate cancer progression, phosphorylation of an evolutionarily conserved Tyrosine 176 in the kinase domain of AKT is essential for mitotic progression and positively correlates with breast cancer progression. In contrast to AR and AKT, Ack1-mediated phosphorylation of the tumor suppressor Wwox at Tyr287 lead to rapid Wwox polyubiquitination followed by degradation. Thus, by its ability to promote tumor growth by negatively regulating tumor suppressor such as Wwox and positively regulating pro-survival factors such as AKT and AR, Ack1 is emerging as a critical player in cancer biology. In this review, we discuss recent advances in understanding the physiological functions of Ack1 signaling in normal cells and the consequences of its hyperactivation in various cancers.

Snail regulates cell survival and inhibits cellular senescence in human metastatic prostate cancer cell lines

The epithelial-mesenchymal transition (EMT) is regarded as an important step in cancer metastasis. Snail, a master regulator of EMT, has been recently proposed to act additionally as a cell survival factor and inducer of motility. We have investigated the function of Snail (SNAI1) in prostate cancer cells by downregulating its expression via short (21-mer) interfering RNA (siRNA) and measuring the consequences on EMT markers, cell viability, death, cell cycle, senescence, attachment, and invasivity. Of eight carcinoma cell lines, the prostate carcinoma cell lines LNCaP and PC-3 showed the highest and moderate expression of SNAI1 mRNA, respectively, as measured by quantitative RT-PCR. Long-term knockdown of Snail induced a severe decline in cell numbers in LNCaP and PC-3 and caspase activity was accordingly enhanced in both cell lines. In addition, suppression of Snail expression induced senescence in LNCaP cells. SNAI1-siRNA-treated cells did not tolerate detachment from the extracellular matrix, probably due to downregulation of integrin α6. Expression of E-cadherin, vimentin, and fibronectin was also affected. Invasiveness of PC-3 cells was not significantly diminished by Snail knockdown. Our data suggest that Snail acts primarily as a survival factor and inhibitor of cellular senescence in prostate cancer cell lines. We therefore propose that Snail can act as early driver of prostate cancer progression.

The SWI/SNF ATPase Brm Is a Gatekeeper of Proliferative Control in Prostate Cancer

Factors that drive prostate cancer progression remain poorly defined, thus hindering the development of new therapeutic strategies. Disseminated tumors are treated through regimens that ablate androgen signaling, as prostate cancer cells require androgen for growth and survival. However, recurrent, incurable tumors that have bypassed the androgen requirement ultimately arise. This study reveals that the Brm ATPase, a component of selected SWI/SNF complexes, has significant antiproliferative functions in the prostate that protect against these transitions. First, we show that targeted ablation of Brm is causative for the development of prostatic hyperplasia in mice. Second, in vivo challenge revealed that Brm-/- epithelia acquire the capacity for lobe-specific, castration-resistant cellular proliferation. Third, investigation of human specimens revealed that Brm mRNA and protein levels are attenuated in prostate cancer. Fourth, Brm down-regulation was associated with an increased proliferative index, consistent with the mouse model. Lastly, gene expression profiling showed that Brm loss alters factors upstream of E2F1; this was confirmed in murine models, wherein Brm loss induced E2F1 deregulation in a tissue-specific manner. Combined, these data identify Brm as a major effector of serum androgen-induced proliferation in the prostate that is disrupted in human disease, and indicate that loss of Brm confers a proliferative advantage in prostate cancer.

Cooperative Interactions between Androgen Receptor (AR) and Heat-Shock Protein 27 Facilitate AR Transcriptional Activity

Androgen receptor (AR) transactivation is known to enhance prostate cancer cell survival. However, the precise effectors by which the prosurvival effects of androgen and AR drive prostate cancer progression are poorly defined. Here, we identify a novel feed-forward loop involving cooperative interactions between ligand-activated AR and heat-shock protein 27 (Hsp27) phospho-activation that enhance AR stability, shuttling, and transcriptional activity, thereby increasing prostate cancer cell survival. Androgen-bound AR induces rapid Hsp27 phosphorylation on Ser(78) and Ser(82) residues in an AR- and p38 kinase-dependent manner. After this androgen-induced, non-nuclear phospho-activation, Hsp27 displaces Hsp90 from a complex with AR to chaperone AR into the nucleus and interact with its response elements to enhance its genomic activity. Inhibition of Hsp27 phosphorylation, or knockdown using the antisense drug OGX-427, shifted the association of AR with Hsp90 to MDM2, increased proteasome-mediated AR degradation, decreased AR transcriptional activity, and increased prostate cancer LNCaP cell apoptotic rates. OGX-427 treatment of mice bearing LNCaP xenografts transfected with an androgen-regulated, probasin-luciferase reporter construct resulted in decreased bioluminescence and serum PSA levels as pharmacodynamic readouts of AR activity, as well as AR, Hsp27, and Hsp90 protein levels in LNCaP tumor tissue. These data identify novel nongenomic mechanisms involving androgen, AR, and Hsp27 activation that cooperatively interact to regulate the genomic activity of AR and justify further investigation of Hsp27 knockdown as an AR disrupting therapeutic strategy in prostate cancer.

Ras signaling in prostate cancer progression.

When prostate cancer is first detected it generally is dependent on the presence of androgens for growth, and responds to androgen ablation therapies. However, the disease often recurs in a disseminated and apparently androgen independent (AI) form, and in this state is almost invariably fatal. Considerable evidence indicates that the Androgen receptor (AR) continues to be required even in androgen independent (AI) disease. Thus, a key to understanding hormone independent prostate cancer is to determine the mechanism(s) by which the AR can function even in the absence of physiologic levels of androgen. In this article, we argue that growth factors and receptors that utilize Ras family members drive prostate cancer progression to a state of androgen hypersensitivity; and that post-translational modifications (e.g., phosphorylations) of transcriptional cofactors might be responsible for modulating the function of the AR so that it is active even at low concentrations of androgen.

Failure of hormone therapy in prostate cancer involves systematic restoration of androgen responsive genes and activation of rapamycin sensitive signaling.

Androgen deprivation therapy for advanced prostate cancer is often effective, but not curative. Molecular pathways mediating the therapeutic response and those contributing to the subsequent hormone-refractory cell growth remain poorly understood. Here, cDNA microarray analysis of human CWR22 prostate cancer xenografts during the course of androgen deprivation therapy revealed distinct global gene expression profiles in primary, regressing and recurrent tumors. Elucidation of the genes involved in the transition between these states implicated specific molecular mechanisms in therapy failure and tumor progression. First, we identified a set of androgen-responsive genes whose expression decreased during the therapy response, but was then systematically restored in the recurrent tumors. In addition, altered expression of genes that encode known targets of rapamycin or that converge on the PI3K/AKT/FRAP pathway was observed in the recurrent tumors. Further suggestion for the involvement of these genes in hormone-refractory prostate cancer came from the observation that cells established from the recurrent xenografts were strongly inhibited in vitro by rapamycin. The results of this functional genomic analysis suggest that the combined effect of re-expression of androgen-responsive genes as well as the activation of rapamycin-sensitive signaling may drive prostate cancer progression, and contribute to the failure of androgen-deprivation therapy.