Regulator Of Prostate Cancer Progression Research Articles

Somatostatin, Cortistatin and Their Receptors Exert Antitumor Actions in Androgen-Independent Prostate Cancer Cells: Critical Role of Endogenous Cortistatin.

Somatostatin (SST), cortistatin (CORT), and their receptors (SSTR1-5/sst5TMD4-TMD5) comprise a multifactorial hormonal system involved in the regulation of numerous pathophysiological processes. Certain components of this system are dysregulated and play critical roles in the development/progression of different endocrine-related cancers. However, the presence and therapeutic role of this regulatory system in prostate cancer (PCa) remain poorly explored. Accordingly, we performed functional (proliferation/migration/colonies-formation) and mechanistic (Western-blot/qPCR/microfluidic-based qPCR-array) assays in response to SST and CORT treatments and CORT-silencing (using specific siRNA) in different PCa cell models [androgen-dependent (AD): LNCaP; androgen-independent (AI)/castration-resistant PCa (CRPC): 22Rv1 and PC-3], and/or in the normal-like prostate cell-line RWPE-1. Moreover, the expression of SST/CORT system components was analyzed in PCa samples from two different patient cohorts [internal (n = 69); external (Grasso, n = 88)]. SST and CORT treatment inhibited key functional/aggressiveness parameters only in AI-PCa cells. Mechanistically, antitumor capacity of SST/CORT was associated with the modulation of oncogenic signaling pathways (AKT/JNK), and with the significant down-regulation of critical genes involved in proliferation/migration and PCa-aggressiveness (e.g., MKI67/MMP9/EGF). Interestingly, CORT was highly expressed, while SST was not detected, in all prostate cell-lines analyzed. Consistently, endogenous CORT was overexpressed in PCa samples (compared with benign-prostatic-hyperplasia) and correlated with key clinical (i.e., metastasis) and molecular (i.e., SSTR2/SSTR5 expression) parameters. Remarkably, CORT-silencing drastically enhanced proliferation rate and blunted the antitumor activity of SST-analogues (octreotide/pasireotide) in AI-PCa cells. Altogether, we provide evidence that SST/CORT system and SST-analogues could represent a potential therapeutic option for PCa, especially for CRPC, and that endogenous CORT could act as an autocrine/paracrine regulator of PCa progression.

HOXD13 suppresses prostate cancer metastasis and BMP4-induced epithelial-mesenchymal transition by inhibiting SMAD1.

The HOX genes are a group of highly conserved Homeobox-containing genes that control the body plan organization during development. However, their contributions to tumorigenesis and tumor progression remain uncertain and controversial. Here we provided evidence of tumor-suppressive activity of HOXD13 in prostate cancer. HOXD13 depletion contributes to more aggressiveness of prostate cancer cells in vitro and in vivo. These effects were corroborated in a metastatic mice model, where we observed more bone metastatic lesions formed by prostate cancer cells with HOXD13 ablation. Mechanistically, HOXD13 prevents BMP4-induced epithelial-mesenchymal transition (EMT) by inhibiting mothers against decapentaplegic homolog 1 (SMAD1) transcription. Both bioinformation and our tissue microarray cohort data show that HOXD13 expression inversely correlated in advanced prostate cancer patient specimens. Our findings establish HOXD13 as a negative regulator of prostate cancer progression and metastasis by preventing BMP4/SMAD1 signaling, and potentially suggest new strategies for targeting metastatic prostate cancer.

Abstract 3466: Can grape powder supplemented diet prevent prostate cancer progression

Abstract Epidemiological and experimental studies suggest a correlation between diet, particularly fat consumption and the risk of developing prostate cancer (PCa). In previous studies, we reported that stilbenes, namely resveratrol and pterostilbene, restrained prostatic intraepithelial neoplasia (PIN) formation and reduced tumor development and progression in mice models of PCa with high expression of metastasis-associated protein 1 (MTA1), an epigenetic modifier involved in the regulation of prostate cancer progression and metastasis. In this report, we evaluated three major aspects: 1) whether altering diet [low fat diet (LFD) versus high fat diet (HFD)] affects PCa progression; 2) whether grape powder (GP) supplementation to the diets protects against PCa progression, and 3) whether MTA1 targeting is the main mechanism of action. We addressed these questions by utilizing the prostate-specific (PB-Cre4) Pten heterozygous transgenic mice (Pten+/f), which mimic slow-growing PCa progression in humans. Upon accumulating 56 mice of desired genotype, we randomized mice in two major groups and fed them ad libitum with either LFD or HFD. Each of these diets contained either 0% GP or 10% GP supplementation. The same groups were assigned for control wild-type (WT) mice (Pten+/f; Cre-). After 33 weeks on corresponding diets, mice were sacrificed and urogenital system was collected for histological and immunochemical analysis. In addition, protein and RNA was isolated from the prostate tissues for molecular analysis. Blood was also collected for cytokines evaluation and pharmacokinetic analysis. The results demonstrated the following beneficial effects of GP supplementation: LFD and HFD - fed Pten+/f mice showed added favorable histopathology (H&E), intact basal layer (SMA) and luminal cells (CK-8); significant reduction of the proliferative rate of prostate epithelial cells (Ki67); non-significant trend for reducing HFD-induced MTA1 expression in Pten+/f and WT mice; protective effects against “bad” genotype and HFD-induced activation of Akt pathway; attenuation of HFD-associated increase in AR levels in both Pten+/f and WT mice; reduction of HFD-provoked increased inflammation marker IL-1β levels; non-significant trend for reducing angiogenesis (CD31; VEGF-C) in Pten+/f mice; and trend towards decreasing inflammatory TGFβ1 expression in Pten+/f mice. Curious results were obtained concerning the effect of GP on apoptosis. There were no significant changes in body weight, food intake or fat intake in GP supplemented diets groups. Citation Format: Tanvi Joshi, Ishani Patel, Avinash Kumar, Virginia Donovan, Anait S. Levenson. Can grape powder supplemented diet prevent prostate cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3466.

Role of nitric oxide-based immunotherapy in augmenting prostate cancer progression by targeting androgen receptor heterogeneity.

e17537 Background: A significant proportion of men with Prostate Cancer (PCa) develop castration resistant prostate cancer (CRPC) and do not respond to hormonal agents that decrease androgens. In trying to understand the causes of androgen resistance that develop in CRPC, it is considered most relevant to study the role of Androgen receptor (AR) in the development and progression of PCa from androgen dependent to androgen independent state. Recent studies have highlighted the significance of tumor microenvironment (TME) in regulation of PCa progression in addition to AR. A key molecule in the regulation of TME interactions is nitric oxide (NO). We have shown in our recent study, the critical association of NO with the TME in CRPC. However, the effects of NO to modulate the progression of PCa to CRPC with respect to AR still remains largely unexplored. Methods: 22RV1, LNCaP, LNCaPAPIPC(cells expressing no AR), and LNCaPshAR/pATK (cells expressing low AR), cells were used for the study. Cell proliferation was first assessed by MTT assay. The castrated SCID mice were grafted with 22RV1 cells and were treated with GSNO at the dosage of 10mg/kg/day IP. After treatment, animals were humanely sacrificing. Tumor RNA and proteins were analysed for markers that are important for PCa progression using qPCR, western blot and cytokine antibody array. Animal experiments were carried out in compliance with the IACUC of University of Miami. GraphPad Prism (GraphPad Software) was used for statistical analysis. Results: In addition to reducing the tumor burden, the expression of anti-inflammatory (M2) macrophages (CD206 and Arginase1) is decreased and that of the pro-inflammatory (M1) macrophage (iNOS) is increased in mice which received increased NO levels. Furthermore, to study the effects of NO on progression of PCa from androgen dependent to androgen independent stage, we characterized the LNCAP cell models with differential extent of AR knockdown (LNCaP, LNCaPshAR/pATK and LNCaPAPIPC) for the effects of increased NO levels. Results showed that NO had significant impact on cell proliferation on androgen dependent PCa cells however the effects were negligible in cells expressing low or no AR, suggesting that effects of NO on PCa cell proliferation are AR dependent. Conclusions: Our results suggest that during PCa progression, NO suppresses TAMs to target the TME in an AR dependent manner. Further studies are undergoing to establish the impacts of NO in PCa progression.

Abstract LB-096: Chromatin alterations in human prostate cancer

Abstract Whole human genome is packed into chromatin, which is dynamically remodeled. Chromatin structure has been extensively studied with cell lines, but information about chromatin structure in tissue context is lacking. We present genome-wide chromatin accessibility analysis of clinical tissue samples using transposase-accessible chromatin sequencing (ATAC-seq). Our sample cohort consist 11 benign prostatic hyperplasia (BPH), 16 primary prostate cancer (PC), and 11 castration resistant prostate cancer (CRPC) samples. We identified 23,307 to 136,104 regions of accessible chromatin per sample using MACS2 peak calling. Utilizing a peak unification method resulted in a unified set of 178,333 high confidence peaks across sample set. To find out which loci are differentially accessible during disease progression, we further compared normalized ATAC-seq signal between sample groups across the genome. We identified 4747 and 9445 differentially accessible regions (DARs) for BPH to PC and PC to CRPC comparison, respectively. Out of these, in 2961 and 6652 chromatin was opening and in 1786 and 2793 chromatin was closing in respective comparison. Using DARs, we observe clear separation of the sample groups. Earlier, we have characterized this cohort using DNA, RNA and DNA methylation sequencing as well as SWATH proteomics. Using these data and the same analysis approach as with DARs, we identified 2061 and 2723 differentially methylated regions (DMRs) in BPH to PC and PC to CRPC comparisons, respectively. We compared locations of DARs and DMRs and found out that these occur in different loci overlapping only in 27 and 35 loci in respective comparisons. When integrated with gene expression data, the chromatin accessibility correlated (|coefficient| &gt0.5) with the expression of at least one gene located in the same topologically associating domain (TAD) in altogether 2713 DARs. Next, we examined which transcription factors (TFs) are binding to DARs and thus putatively regulating gene-expression. Using HOMER database, we found several TFs with binding motif enrichment in our DARs. In BPH to PC comparison, opening DARs contain binding sites e.g. for AR and FOXA1 and, in PC to CRPC comparison, opening DARs contain binding sites e.g. for HOXB13, as expected. Interestingly, in PC to CRPC comparison closing DARs contain binding sites for AR and FOXA1 indicating that these TFs have smaller role or alternative regulatory programs when disease progresses. We utilized publicly available CHIP-seq datasets to study this more closely in DARs where ATAC-seq signal correlates with gene-expression within a TAD. Here, while the total number of AR binding sites doubles, the number of AR binding sites in closing DARs is ten times higher in CRPC to PC comparison than in PC to BPH comparison. These results suggest that chromatin accessibility is an important regulator of prostate cancer progression and changes occur in specific loci to where several relevant prostate cancer TFs can bind. Citation Format: Joonas I. Tuominen, Ebrahim Afyounian, Francesco Tabaro, Tomi Häkkinen, Anastasia Shcherban, Matti Annala, Kati Kivinummi, Teuvo Tammela, Juha Kesseli, Leena Latonen, Kirsi Granberg, Tapio Visakorpi, Matti Nykter. Chromatin alterations in human prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-096.

Abstract 2526: Modulation of metastatic factors in prostate cancer cells by DAX-1 (NR0B1), an androgen-induced orphan nuclear receptor

Abstract DAX-1, a member of the nuclear hormone receptor superfamily, has a pivotal role in adrenal and gonadal development. However, recent studies have shown that DAX-1 acts as a transcriptional repressor influencing the progression of many types of cancers. We hypothesize that DAX-1 has key roles in regulating metastatic genes during prostate cancer progression. We found that introducing DAX-1 into prostate cancer cells lacking endogenous expression of DAX-1 leads to a decrease in metastatic rates. Consequently, we analyzed DAX-1 transcriptional regulation over different markers associated with metastasis. Since the androgen receptor is a key regulator of prostate cancer progression, we used CRISPR to knockout DAX-1 expression in hormone-dependent prostate cancer cells, allowing us to understand the role DAX-1 has as a negative regulator of metastatic genes after androgen receptor activation. We have shown that treatment of hormone-dependent prostate cancer cells with a non-aromatizable androgen leads to upregulation of DAX-1 expression. Here we use the same mechanism to induce DAX-1 expression and analyze the effects on metastatic markers. Results have shown that DAX-1 is sufficient to decrease the expression of metastatic markers and inhibit metastatic rates in prostate cancer cells. These results further explain some of the intrinsic mechanisms involved in prostate cancer metastasis and could reveal new therapeutic routes in the treatment of metastatic prostate cancer. Citation Format: Roxxana Valeria Beltran Valencia, Christina Tzagarakis-Foster. Modulation of metastatic factors in prostate cancer cells by DAX-1 (NR0B1), an androgen-induced orphan nuclear receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2526.

Constructing Bayesian networks by integrating gene expression and copy number data identifies NLGN4Y as a novel regulator of prostate cancer progression.

To understand the heterogeneity of prostate cancer (PCa) and identify novel underlying drivers, we constructed integrative molecular Bayesian networks (IMBNs) for PCa by integrating gene expression and copy number alteration data from published datasets. After demonstrating such IMBNs with superior network accuracy, we identified multiple sub-networks within IMBNs related to biochemical recurrence (BCR) of PCa and inferred the corresponding key drivers. The key drivers regulated a set of common effectors including genes preferentially expressed in neuronal cells. NLGN4Y—a protein involved in synaptic adhesion in neurons—was ranked as the top gene closely linked to key drivers of myogenesis subnetworks. Lower expression of NLGN4Y was associated with higher grade PCa and an increased risk of BCR. We show that restoration of the protein expression of NLGN4Y in PC-3 cells leads to decreased cell proliferation, migration and inflammatory cytokine expression. Our results suggest that NLGN4Y is an important negative regulator in prostate cancer progression. More importantly, it highlights the value of IMBNs in generating biologically and clinically relevant hypotheses about prostate cancer that can be validated by independent studies.

Prostate Cancer-Associated Kallikrein-Related Peptidase 4 Activates Matrix Metalloproteinase-1 and Thrombospondin-1.

Prostate cancer metastasis to bone is terminal; thus, novel therapies are required to prevent end-stage disease. Kallikrein-related peptidase 4 (KLK4) is a serine protease that is overproduced in localized prostate cancer and is abundant in prostate cancer bone metastases. In vitro, KLK4 induces tumor-promoting phenotypes; however, the underlying proteolytic mechanism is undefined. The protein topography and migration analysis platform (PROTOMAP) was used for high-depth identification of KLK4 substrates secreted by prostate cancer bone metastasis-derived PC-3 cells to delineate the mechanism of KLK4 action in advanced prostate cancer. Thirty-six putative novel substrates were determined from the PROTOMAP analysis. In addition, KLK4 cleaved the established substrate, urokinase-type plasminogen activator, thus validating the approach. KLK4 activated matrix metalloproteinase-1 (MMP1), a protease that promotes prostate tumor growth and metastasis. MMP1 was produced in the tumor compartment of prostate cancer bone metastases, highlighting its accessibility to KLK4 at this site. KLK4 further liberated an N-terminal product, with purported angiogenic activity, from thrombospondin-1 (TSP1) and cleaved TSP1 in an osteoblast-derived matrix. This is the most comprehensive analysis of the proteolytic action of KLK4 in an advanced prostate cancer model to date, highlighting KLK4 as a potential multifunctional regulator of prostate cancer progression.

Abstract 2801: Arginylation as a novel regulator of prostate cancer progression

Abstract Post-translational arginylation, mediated by Arginyltransferase (Ate1), is an important protein modification involved in stress response yet remains poorly studied. Our lab recently reported that a loss of Ate1 is sufficient to induce tumorigenesis and loss of contact inhibition in fibroblasts. However, the role of Ate1 in cancer initiation and progression is not clear. This study examines the effects of Ate1 loss in prostate cancer models of tumorigenesis and progression. We first show that Ate1 is required for normal cellular responses to several types of stress present in cancer. Preliminary data in fibroblasts shows that Ate1 is essential for cell death following oxidative stress, hypoxia, UV and gamma radiation, and apoptosis-inducing drugs. These results inspired further tests to examine if cancer progression may be directly stimulated by a loss of Ate1 through increased cell survival and invasiveness. To study the effects of lost Ate1, we characterize established prostate cancer cell lines with Ate1 stably knocked down. In PC-3 prostate cancer cells, a reduction of Ate1 corresponded with increased invasiveness in the Boyden Chamber invasion assay, and reduced H2O2-induced cell death. In LnCaP prostate cancer cells, reduced Ate1 induced significantly higher anchorage-independent growth during a soft-agar growth assay. In conclusion, our data suggests that the loss of Ate1 in prostate cancer serves to stimulate cancer progression, and that Ate1 protein expression levels may predict malignancy. Citation Format: Michael Birnbaum, Akhilesh Kumar, Fangliang Zhang. Arginylation as a novel regulator of prostate cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2801.

MiR186 suppresses prostate cancer progression by targeting Twist1.

Prostate cancer (PCa) is the second leading cause of cancer-related deaths in north American men, and most its related deaths are due to advanced and metastatic PCa. However, the molecular mechanisms underlying PCa progression are still unclear. Here we use a pair of prostate cell lines P69/M12, which have the same genetic background and the highly metastatic cell line M12 is a subline derived from P69, to identify the pathogenesis of PCa. We find that a key miRNA--miR186 is significantly reduced in M12 compared to that in P69. Further, we validate that miR186 is also downregulated in human PCa specimens, most significantly in the metastatic patient specimens. The low miR186 expression is correlated with poor patient survival. Through knockdown or overexpression of miR186 in PCa cell lines, we discover that miR186 strongly inhibits cell motility, invasive, soft-agar colony formation, 3D culture growth and vasculogenic mimicry (VM) formation capacity, as well as the epithelial-to-mesenchymal transition (EMT) process by downregulation of its target Twist1. Moreover, the inverse relationship between the expression levels of miR186 and Twist1 is confirmed in vivo tumor metastasis experiment and clinical specimens. Taken together, our findings demonstrate an important role of miR186/Twist1 axis in the regulation of PCa progression, suggesting a potential application of miR186/Twist1 in PCa treatment.

12-HETER1/GPR31, a high-affinity 12(S)-hydroxyeicosatetraenoic acid receptor, is significantly up-regulated in prostate cancer and plays a critical role in prostate cancer progression.

Previously we identified and deorphaned G-protein-coupled receptor 31 (GPR31) as the high-affinity 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] receptor (12-HETER1). Here we have determined its distribution in prostate cancer tissue and its role in prostate tumorigenesis using in vitro and in vivo assays. Data-mining studies strongly suggest that 12-HETER1 expression positively correlates with the aggressiveness and progression of prostate tumors. This was corroborated with real-time PCR analysis of human prostate tumor tissue arrays that revealed the expression of 12-HETER1 positively correlates with the clinical stages of prostate cancers and Gleason scores. Immunohistochemistry analysis also proved that the expression of 12-HETER1 is positively correlated with the grades of prostate cancer. Knockdown of 12-HETER1 in prostate cancer cells markedly reduced colony formation and inhibited tumor growth in animals. To discover the regulatory factors, 5 candidate 12-HETER1 promoter cis elements were assayed as luciferase reporter fusions in Chinese hamster ovary (CHO) cells, where the putative cis element required for gene regulation was mapped 2 kb upstream of the 12-HETER1 transcriptional start site. The data implicate 12-HETER1 in a critical new role in the regulation of prostate cancer progression and offer a novel alternative target for therapeutic intervention.-Honn, K. V., Guo, Y., Cai, Y., Lee, M.-J., Dyson, G., Zhang, W., Tucker, S. C. 12-HETER1/GPR31, a high-affinity 12(S)-hydroxyeicosatetraenoic acid receptor, is significantly up-regulated in prostate cancer and plays a critical role in prostate cancer progression.

Regulation of prostate cancer progression by the tumor microenvironment

Prostate cancer remains the most frequently diagnosed cancer in men in North America, and despite recent advances in treatment patients with metastatic disease continue to have poor five-year survival rates. Recent studies in prostate cancer have revealed the critical role of the tumor microenvironment in the initiation and progression to advanced disease. Experimental data have uncovered a reciprocal relationship between the cells in the microenvironment and malignant tumor cells in which early changes in normal tissue microenvironment can promote tumorigenesis and in turn tumor cells can promote further pro-tumor changes in the microenvironment. In the tumor microenvironment, the presence of persistent immune infiltrates contributes to the recruitment and reprogramming of other non-immune stromal cells including cancer-associated fibroblasts and a unique recently identified population of metastasis-initiating cells (MICs). These MICs, which can also be found as part of the circulating tumor cell (CTC) population in PC patients, promote cancer cell transformation, enhance metastatic potential and confer therapeutic resistance. MICs act can on other cells within the tumor microenvironment in part by secreting exosomes that reprogram adjacent stromal cells to create a more favorable tumor microenvironment to support continued cancer growth and progression. We review here the current data on the intricate relationship between inflammation, reactive stroma, tumor cells and disease progression in prostate cancer.

HOXA1 enhances the cell proliferation, invasion and metastasis of prostate cancer cells.

HOXA1, a member of the HOX gene family, has been implicated in tumor progression. However, the role of HOXA1 in prostate cancer is not well-established. In the present study, we found that HOXA1 was highly expressed in prostate cancer cells. We then repressed the expression of HOXA1 by short hairpin RNA (shRNA) to investigate the function of HOXA1 in prostate cancer cells. Our invitro data showed that knockdown of HOXA1 attenuated the growth, invasion and migration of prostate cancer DU-145 and PC-3 cells. Furthermore, knockdown of HOXA1 resulted in an increased E-cadherin level and decreased Snail and MMP-3 levels in the DU-145 cells. In addition, knockdown of HOXA1 inhibited activation of ERK1/2 and AKT in the DU-145 cells. Our invivo data revealed that knockdown of HOXA1 suppressed the growth and metastasis of prostate cancer cells. Collectively, our findings suggest that HOXA1 is involved in the regulation of prostate cancer progression, including cell growth, migration, invasion and metastasis. Thus, downregulation of HOXA1 may be a novel approach for the treatment of prostate cancer.

Abstract 5124: 12-Lipoxygenase regulation of prostate cancer progression.

Abstract The underlying mechanism by which elevated omega-6 fatty acids, like arachidonic acid, lead to increased prostate cancer (PCa) risk is unclear. However, it is known that the 12-Lipoxygenase (12-LOX) enzyme that metabolizes arachidonic acid, as well as the 12(S)-HETE metabolic end product together with its cognate receptor, 12-HETER1, are strongly associated with PCa. 12(S)-HETE affects many cancer phenotypes including cell migration and metastasis. It causes the disassembly of complex cellular anchors to the substrata called hemidesmosomes that are comprised of numerous proteins, including ITGB4, which we identified as an interacting partner of 12-LOX. ITGB4 also physically interacts with c-Met at the cell membrane in multiple cell lines, in a cell line and stimulus-specific manner. PCa metastases have increased levels of both 12-LOX and c-Met. Therefore we sought to determine if ITGB4, c-MET and 12-LOX comprise a signaling axis. Our findings revealed that HGF-c-Met-induced invasion could be attenuated by enzymatic inhibitors of 12LOX, but that addition of exogenous 12(S)-HETE could not overcome the block. Furthermore, the physical interaction between β4 and c-Met remained unaffected by enzymatic inhibitors in PCa cells. Likewise, LC-MS analyses, showed that HGF stimulation did not lead to increased 12(S)-HETE production, and knockdown of 12-HETER1, by shRNA had no effect on HGF-induced invasion. Therefore, we hypothesize that 12LOX has dual functions whereby its enzymatic activity leads to hemidesmosome dissociation and cell release from the substrata, and its capacity to modulate c-MET/ITGB4 signaling may be attributed to a scaffolding function. Citation Format: Elizabeth Tovar, Stephanie Tucker, Kenneth V. Honn. 12-Lipoxygenase regulation of prostate cancer progression. [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 5124. doi:10.1158/1538-7445.AM2013-5124

Gata3 antagonizes cancer progression in Pten-deficient prostates

Loss of the tumor suppressor PTEN is a common occurrence in prostate cancer. This aberration leads to the ectopic activation of the PI3K-Akt pathway, which promotes tumor growth. Here, we show that the transcription factor Gata3 is progressively lost in Pten-deficient mouse prostate tumors as a result of both transcriptional down-regulation and increased proteasomal degradation. To determine the significance of this loss, we used conditional loss- and gain-of-function approaches to manipulate Gata3 expression levels in prostate tumors. Our results show that Gata3 inactivation in Pten-deficient prostates accelerates tumor invasion. Conversely, enforced expression of GATA3 in Pten-deficient tissues markedly delays tumor progression. In Pten-deficient prostatic ducts, enforced GATA3 prevented Akt activation, which correlated with the down-regulation of Pik3cg and Pik3c2a mRNAs, encoding respectively class I and II PI3K subunits. Remarkably, the majority of human prostate tumors similarly show loss of active GATA3 as they progress to the aggressive castrate-resistant stage. In addition, GATA3 expression levels in hormone-sensitive tumors holds predictive value for tumor recurrence. Together, these data establish Gata3 as an important regulator of prostate cancer progression.

Abstract A61: Hexim-1 protein expression levels modulate TGFβ activity in prostate cancer

Abstract Hexim-1 is a highly conserved protein from human to mouse, and a well-established inhibitor of RNA polymerase II-dependent transcription elongation and cell proliferation. Others and we have proposed and provided evidence that changes in the expression and activity in Hexim-1 significantly influence the progression of cancer. Here we show that changes in expression levels of Hexim-1 modulate TGFβ1 signaling by inducing posttranslational modifications in the SMADs proteins (small mother against decapentaplegic) at the polylinker region of the Smad1 and Smad3 proteins. Cdk9 serine activity not only targets specific serine residues in the carboxyl terminus of RNA polymerase II during transcriptional elongation but also serine residues in the polylinker region of Smad3. Hexim-1 directly inhibits Cdk9 activity, and therefore, transcription elongation and TGFβ1/Smad3 activity. Our results indicate that prostate cancer accompanies significant changes in Hexim-1 expression as well as sub cellular redistribution. Similar observations were obtained in the TRAMP mouse model of prostate cancer. In order to understand the role of Hexim-1 during prostate cancer progression, we generated a TRAMP mouse model heterozygous for Hexim-1. Our studies in the TRAMP mouse model revealed that lowered levels in Hexim-1 protein expression triggered a more aggressive form of prostate cancer. TGFβ1/Smad3 plays a significant role during proliferation, cell migration, as well as in epithelial mesenchymal transition, which is critical during metastasis. To test the role of Hexim-1 as a modulator of TGF/Smad3 in vitro, we use the epithelial mouse prostate cancer cell line TRAMP-C2 and derived the TRAMP-C2 heterozygous for Hexim-1 (HT). We observed increased serine 208 phosphorylation of Smad3 as well as enhanced transcriptional activity of TGF β/Smad3 target genes involved in EMT such as SM22alpha and alpha smooth muscle actin in the HT-TRAMP-C2 cell line. These findings suggest that Hexim-1 is a critical regulator of prostate cancer progression. Citation Format: Manya Dhar-Mascareno, Eduardo Mascareno, Fang Liu. Hexim-1 protein expression levels modulate TGFβ activity in prostate cancer [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 A61.

Gene expression changes in human prostate carcinoma cells exposed to genotoxic and nongenotoxic aryl hydrocarbon receptor ligands

Carcinogenic polycyclic aromatic hydrocarbons (PAHs) are known as efficient mutagens and ligands of the aryl hydrocarbon receptor (AhR), which has been suggested to play an important role in prostate carcinogenesis. In order to evaluate the complex relationship between the genotoxicity and the AhR-mediated activity of PAHs in prostate cells, we selected benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), as model genotoxic and nongenotoxic AhR ligands, respectively, to explore global changes in gene expression in LNCaP cells by microarray analysis. We identified 112 genes that were differentially expressed in cells treated for 24h with BaP, TCDD or both compounds. Our data indicated that the impacts of BaP and TCDD on transcriptome of LNCaP cells significantly overlap, since over 64% of significantly up-regulated genes and 47% of down-regulated genes were similarly affected by both AhR ligands. This suggested that the activation of AhR played a prominent role in the nongenotoxic effects of BaP in the prostate carcinoma cell model LNCaP. Both AhR ligands suppressed expression of genes associated with cell cycle progression, DNA replication, spindle assembly checkpoint or DNA repair, which probably occurred secondary to inhibition of cell cycle progression. In contrast, we identified Wnt5a, an important regulator of prostate cancer progression, to be induced as early as 6h after exposure to both AhR ligands. The AhR ligand-induced Wnt5a upregulation, together with other observed alterations of gene expression, may further contribute to enhanced cell plasticity of prostate carcinoma cells.

Abstract 1407: Mitogen-activated protein kinase kinase 4 (MEK4) is a key regulator of prostate cancer progression

Abstract Mitogen-activated protein kinase kinase 4 (MEK4) is a dual-specificity kinase that has been implicated in cancer progression in multiple cancer types, including prostate cancer (PCa). MEK4 is upregulated in invasive PCa lesions in human tissue. We hypothesize that increased MEK4 promotes PCa invasion and metastasis. Our group has created PC3-M cells stably transfected with either constitutively-active or increased levels of wild-type MEK4. In a Matrigel Boyden chamber assay, constitutively-active or wild-type MEK4 was shown to increase PCa invasion. Cancer cell invasion requires the coupling of cell migration with increased production of proteases. We hypothesized that MEK4 promoted invasion via production of proteases, but not via cell migration. Using an uncoated boyden chamber, MEK4 was shown not to affect migration. Using quantitative real time polymerase chain reaction (qRT/PCR), we went on to demonstrate that MEK4 increased MMP-2 and MMP-9 transcript expression, but did not affect MMP-10 expression. In addition to these studies, we have also looked at the effect of increased or constitutively-active MEK4 on an orthotopic mouse model of PCa. In this model, PCa cells are injected into the prostates of nude mice and tumor size, circulating tumor cells, and lung metastasis are quantified. Preliminary data shows that constitutively-active MEK4 causes an increase in primary tumor size as well as an increase in circulating tumor cells in both the blood and the bone marrow. In summary, we demonstrated that chronic high expression of MEK4 increases human PCa invasion, that this is not due to increased migration, but is associated with increases in MMP-2 and MMP-9. Further, constitutively-active MEK4 appears to have different biological effects than high levels of wild type MEK4 in vivo. Ongoing studies are evaluating the effect of wild type MEK4 and of constitutively-active MEK4 on the formation of distant soft tissue metastasis in the above murine model. In related studies we are seeking to identify downstream proteins critical for MEK4's effects on cell invasion and metastasis both in vitro and in vivo. We are also seeking to examine the effects of sustained MEK4 knockdown on PCa invasion and metastasis. 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 1407. doi:10.1158/1538-7445.AM2011-1407

Prostate Cancer Gene Expression Marker 1 (PCGEM1): A Patented Prostate- Specific Non-Coding Gene and Regulator of Prostate Cancer Progression

The search for a classic biomarker for prostate cancer has been on for a very long time, and it is still elusive. Despite the extensive and prolonged use of the prostate-specific antigen (PSA) as a screening tool for prostate cancer, many clinicians still identify its limitations in the grading of tumors, and monitoring of response to treatment. These limitations are based on the concealment of cancer by low levels of PSA, and over diagnosis and over treatment that are resultant of excessively high levels of antigens. To overcome these limitations, a plethora of candidate biomarkers have been investigated and identified. Regrettably, none of these putative biomarkers has shown ideal utility for prostate cancer detection and prognostication. In spite of these, various patents have been filed and granted for several of these biomarkers. The discovery of an exclusive marker for prostate cancer may be mired by the heterogeneity of the disease, since the molecular mediators of the disease are linked with its etiopathogenesis. Thus the search for biomarkers of prostate cancer must recognize its etiology and downstream mediators. In this review, PCGEM1, a patented prostatespecific non-coding RNA gene with an upregulated transcription in African American malignancy will be discussed in relationship to the etiology of prostate cancer.

Regulation of Prostate Cancer Progression by Galectin-3

Galectin-3, a beta-galactoside-binding protein, has been implicated in a variety of biological functions including cell proliferation, apoptosis, angiogenesis, tumor progression, and metastasis. The present study was undertaken to understand the role of galectin-3 in the progression of prostate cancer. Immunohistochemical analysis of galectin-3 expression revealed that galectin-3 was cleaved during the progression of prostate cancer. Galectin-3 knockdown by small interfering RNA (siRNA) was associated with reduced cell migration, invasion, cell proliferation, anchorage-independent colony formation, and tumor growth in the prostates of nude mice. Galectin-3 knockdown in human prostate cancer PC3 cells led to cell-cycle arrest at G(1) phase, up-regulation of nuclear p21, and hypophosphorylation of the retinoblastoma tumor suppressor protein (pRb), with no effect on cyclin D1, cyclin E, cyclin-dependent kinases (CDK2 and CDK4), and p27 protein expression levels. The data obtained here implicate galectin-3 in prostate cancer progression and suggest that galectin-3 may serve as both a diagnostic marker and therapeutic target for future disease treatments.