Features Of Prostate Cancer Cells Research Articles

Role of Kindlin 2 in prostate cancer

Kindlin-2 is a cytoskeletal adapter protein that is present in many different cell types. By virtue of its interaction with multiple binding partners, Kindlin-2 intercalates into numerous signaling pathways and cytoskeletal nodes. A specific interaction of Kindlin-2 that is of paramount importance in many cellular responses is its direct binding to the cytoplasmic tails of integrins, an interaction that controls many of the adhesive, migratory and signaling responses mediated by members of the integrin family of cell-surface heterodimers. Kindlin-2 is highly expressed in many cancers and is particularly prominent in prostate cancer cells. CRISPR/cas9 was used as a primary approach to knockout expression of Kindlin-2 in both androgen-independent and dependent prostate cancer cell lines, and the effects of Kindlin-2 suppression on oncogenic properties of these prostate cancer cell lines was examined. Adhesion to extracellular matrix proteins was markedly blunted, consistent with the control of integrin function by Kindlin-2. Migration across matrices was also affected. Anchorage independent growth was markedly suppressed. These observations indicate that Kindlin-2 regulates hallmark features of prostate cancer cells. In androgen expressing cells, testosterone-stimulated adhesion was Kindlin-2-dependent. Furthermore, tumor growth of a prostate cancer cell line lacking Kindlin-2 and implanted into the prostate gland of immunocompromised mice was markedly blunted and was associated with suppression of angiogenesis in the developing tumor. These results establish a key role of Kindlin-2 in prostate cancer progression and suggest that Kindlin-2 represents an interesting therapeutic target for treatment of prostate cancer.

Estrogens drive the endoplasmic reticulum-associated degradation and promote proto-oncogene c-Myc expression in prostate cancer cellsby androgen receptor/estrogen receptor signaling.

The tumorigenic properties of prostate cancer are regulated by advanced hormonal regulation-mediated complex molecular signals. Therefore, characterizing the regulation of these signal transduction systems is crucial for understanding prostate cancer biology. Recent studies have shown that endoplasmic reticulum (ER)-localized protein quality control mechanisms, including ER-associated degradation (ERAD) andunfolded protein response (UPR) signaling contribute to prostate carcinogenesis and to the development of drug resistance. It has also been determined that these systems are tightly regulated by androgens. However, the role of estrogenic signaling in prostate cancer and its effects on protein quality control mechanisms is not fully understood. Herein, we investigated the regulatory effects of estrogens on ERAD and UPR and their impacts on prostate carcinogenesis. We found that estrogens strongly regulated the ERAD components and IRE1⍺ branch of UPR by Er⍺/β/ARaxis. Besides,estrogenic signaling rigorously regulatedthe tumorigenicity of prostate cancer cells by promoting c-Myc expression and epithelial-mesenchymal transition (EMT). Moreover, estrogenic signal blockage significantly decreased the tumorigenic features of prostate cancer cells. Additionally, simultaneous inhibition of androgenic/estrogenic signals more efficiently inhibited tumorigenicity of prostate cancer cells, including proliferation, migration, invasion and colonial growth. Furthermore, computational-based molecular docking, molecular dynamics simulations and MMPBSA calculations supported the estrogenic stimulation of AR. Present findings suggested that ERAD components and IRE1⍺ signaling are tightlyregulated by estrogen-stimulated AR and Er⍺/β. Our data suggest that treatment approaches targeting the co-inhibition of androgenic/estrogenic signals may pave the way for new treatment approaches to be developed for prostate cancer. The present model of the impact of estrogens on ERAD and UPR signaling in androgen-sensitive prostate cancer cells.

Novel plasma exosome biomarkers for prostate cancer progression in co-morbid metabolic disease

Comorbid Type 2 diabetes (T2D), a metabolic complication of obesity, associates with worse cancer outcomes for prostate, breast, head and neck, colorectal and several other solid tumors. However, the molecular mechanisms remain poorly understood. Emerging evidence shows that exosomes carry miRNAs in blood that encode the metabolic status of originating tissues and deliver their cargo to target tissues to modulate expression of critical genes. Exosomal communication potentially connects abnormal metabolism to cancer progression. Here, we hypothesized that T2D plasma exosomes induce epithelial-mesenchymal transition (EMT) and immune checkpoints in prostate cancer cells. We demonstrate that plasma exosomes from subjects with T2D induce EMT features in prostate cancer cells and upregulate the checkpoint genes CD274 and CD155. We demonstrate that specific exosomal miRNAs that are differentially abundant in plasma of T2D adults compared to nondiabetic controls (miR374a-5p, miR-93-5p and let-7b-3p) are delivered to cancer cells, thereby regulating critical target genes. We build on our previous reports showing BRD4 controls migration and dissemination of castration-resistant prostate cancer, and transcription of key EMT genes, to show that T2D exosomes require BRD4 to drive EMT and immune ligand expression. We validate our findings with gene set enrichment analysis of human prostate tumor tissue in TGCA genomic data. These results suggest novel, non-invasive approaches to evaluate and potentially block progression of prostate and other cancers in patients with comorbid T2D.

Dysregulation of the miRNome unveils a crosstalk between obesity and prostate cancer: miR-107 asa personalized diagnostic and therapeutic tool

Prostate-specific antigen (PSA) is the gold-standard marker to screen prostate cancer (PCa) nowadays. Unfortunately, its lack of specificity and sensitivity makes the identification of novel tools to diagnose PCa an urgent medical need. In this context, microRNAs (miRNAs) have emerged as potential sources of non-invasive diagnostic biomarkers in several pathologies. Therefore, this study was aimed at assessing for the first time the dysregulation of the whole plasma miRNome in PCa patients and its putative implication in PCa from a personalized perspective (i.e., obesity condition). Plasma miRNome from a discovery cohort (18 controls and 19 PCa patients) was determined using an Affymetrix-miRNA array, showing that the expression of 104 miRNAs was significantly altered, wherein six exhibited a significant receiver operating characteristic (ROC) curve to distinguish between control and PCa patients (area under the curve [AUC] = 1). Then, a systematic validation using an independent cohort (135 controls and 160 PCa patients) demonstrated that miR-107 was the most profoundly altered miRNA in PCa (AUC = 0.75). Moreover, miR-107 levels significantly outperformed the ability of PSA to distinguish between control and PCa patients and correlated with relevant clinical parameters (i.e., PSA). These differences were more pronounced when considering only obese patients (BMI > 30). Interestingly, miR-107 levels were reduced in PCa tissues versus non-tumor tissues (n = 84) and in PCa cell lines versus non-tumor cells. In vitro miR-107 overexpression altered key aggressiveness features in PCa cells (i.e., proliferation, migration, and tumorospheres formation) and modulated the expression of important genes involved in PCa pathophysiology (i.e., lipid metabolism [i.e., FASN] and splicing process). Altogether, miR-107 might represent a novel and useful personalized diagnostic and prognostic biomarker and a potential therapeutic tool in PCa, especially in obese patients.

Distinct roles for the RNA-binding protein Staufen1 in prostate cancer

BackgroundProstate cancer is one of the most common malignant cancers with the second highest global rate of mortality in men. During the early stages of disease progression, tumour growth is local and androgen-dependent. Despite treatment, a large percentage of patients develop androgen-independent prostate cancer, which often results in metastases, a leading cause of mortality in these patients. Our previous work on the RNA-binding protein Staufen1 demonstrated its novel role in cancer biology, and in particular rhabdomyosarcoma tumorigenesis. To build upon this work, we have focused on the role of Staufen1 in other forms of cancer and describe here the novel and differential roles of Staufen1 in prostate cancer.MethodsUsing a cell-based approach, three independent prostate cancer cell lines with different characteristics were used to evaluate the expression of Staufen1 in human prostate cancer relative to control prostate cells. The functional impact of Staufen1 on several key oncogenic features of prostate cancer cells including proliferation, apoptosis, migration and invasion were systematically investigated.ResultsWe show that Staufen1 levels are increased in all human prostate cancer cells examined in comparison to normal prostate epithelial cells. Furthermore, Staufen1 differentially regulates growth, migration, and invasion in the various prostate cancer cells assessed. In LNCaP prostate cancer cells, Staufen1 regulates cell proliferation through mTOR activation. Conversely, Staufen1 regulates migration and invasion of the highly invasive, bone metastatic-derived, PC3 prostate cells via the activation of focal adhesion kinase.ConclusionsCollectively, these results show that Staufen1 has a direct impact in prostate cancer development and further demonstrate that its functions vary amongst the prostate cancer cell types. Accordingly, Staufen1 represents a novel target for the development of much-needed therapeutic strategies for prostate cancer.

Post-transcriptional Gene Regulation by MicroRNA-194 Promotes Neuroendocrine Transdifferentiation in Prostate Cancer

Potent therapeutic inhibition of the androgen receptor (AR) in prostate adenocarcinoma can lead to the emergence of neuroendocrine prostate cancer (NEPC), a phenomenon associated with enhanced cell plasticity. Here, we show that microRNA-194 (miR-194) is a regulator of epithelial-neuroendocrine transdifferentiation. In clinical prostate cancer samples, miR-194 expression and activity were elevated in NEPC and inversely correlated with AR signaling. miR-194 facilitated the emergence of neuroendocrine features in prostate cancer cells, a process mediated by its ability to directly target a suite of genes involved in cell plasticity. One such target was FOXA1, which encodes a transcription factor with a vital role in maintaining the prostate epithelial lineage. Importantly, a miR-194 inhibitor blocked epithelial-neuroendocrine transdifferentiation and inhibited the growth of cell lines and patient-derived organoids possessing neuroendocrine features. Overall, our study reveals a post-transcriptional mechanism regulating the plasticity of prostate cancer cells and provides a rationale for targeting miR-194 in NEPC.

Transient Sox9 Expression Facilitates Resistance to Androgen-Targeted Therapy in Prostate Cancer.

Patients with metastatic prostate cancer are increasingly presenting with treatment-resistant, androgen receptor-negative/low (AR-/Low) tumors, with or without neuroendocrine characteristics, in processes attributed to tumor cell plasticity. This plasticity has been modeled by Rb1/p53 knockdown/knockout and is accompanied by overexpression of the pluripotency factor, Sox2. Here, we explore the role of the developmental transcription factor Sox9 in the process of prostate cancer therapy response and tumor progression. Unique prostate cancer cell models that capture AR-/Low stem cell-like intermediates were analyzed for features of plasticity and the functional role of Sox9. Human prostate cancer xenografts and tissue microarrays were evaluated for temporal alterations in Sox9 expression. The role of NF-κB pathway activity in Sox9 overexpression was explored. Prostate cancer stem cell-like intermediates have reduced Rb1 and p53 protein expression and overexpress Sox2 as well as Sox9. Sox9 was required for spheroid growth, and overexpression increased invasiveness and neural features of prostate cancer cells. Sox9 was transiently upregulated in castration-induced progression of prostate cancer xenografts and was specifically overexpressed in neoadjuvant hormone therapy (NHT)-treated patient tumors. High Sox9 expression in NHT-treated patients predicts biochemical recurrence. Finally, we link Sox9 induction to NF-κB dimer activation in prostate cancer cells. Developmentally reprogrammed prostate cancer cell models recapitulate features of clinically advanced prostate tumors, including downregulated Rb1/p53 and overexpression of Sox2 with Sox9. Sox9 is a marker of a transitional state that identifies prostate cancer cells under the stress of therapeutic assault and facilitates progression to therapy resistance. Its expression may index the relative activity of the NF-κB pathway.

Abstract B007: Stemness features of prostate cancer cells induced by carcinoma-associated fibroblasts

Abstract Introduction: Carcinoma-associated fibroblasts (CAFs) play a key role during cancer progression through several mechanisms, including the induction of the epithelial-mesenchymal transition (EMT) program, giving cancer cells the ability to initiate metastasis. As the cells progress to the mesenchymal state they acquire properties of cancer stem cells (CSC) or mesenchymal stem cell (MSC). However, the contribution of the CAFs in the acquisition of the differential stemness is largely unknown. Here, we studied the stemness features of prostate cancer cells (PC3) during the EMT program induced by fibroblasts obtained from patients with nonmetastatic prostate cancer (CAF) and bone metastatic prostate cancer (mCAF). Materials and Methods: PC3 cells were incubated with conditioned medium (CM) of CAF and mCAF for 7,14, and 21 days to induced EMT. The presence of putative CSCs and MSCs was determined by qPCR, immunocytochemistry and Western blot for the expression of CSCs and MSCs markers, respectively, and functionally evaluated by the ability to perform adipogenic, chondrogrenic, and osteogenic differentiation (MSCs) and in vitro spheroids formation and clonogenic assay (CSCs). Results and Discussion: Our results demonstrate that cancer cells treated with CM-CAF undergo to a partial EMT program, exhibiting certain mesenchymal traits and enriched for cancer stem cells (CSCs). On the other hand, tumor cells exposed to CM-mCAF, exhibited a full EMT conversion with the greater abilities to migrate and invade in vitro and in vivo. This suggests that CAFs and mCAFs induce differential EMT programs in prostate cancer cells, which leads them to acquire CSCs or MSC features enhancing the metastatic capabilities. (FONDECYT 1150397.) Citation Format: Muriel A. Nuñez, Javier Cerda-Infante, Marianela Sánchez, Paula Sotomayor, Alejandro S. Godoy, Viviana P. Montecinos. Stemness features of prostate cancer cells induced by carcinoma-associated fibroblasts [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B007.

PO-328 Stemness features of prostate cancer cells induced by carcinoma-associated fibroblasts

IntroductionCarcinoma-associated fibroblasts (CAFs) play a key role during cancer progression through several mechanisms including the induction of the epithelial-mesenchymal transition (EMT) program, giving cancer cells the ability to initiate metastasis. As the cells progress to the mesenchymal state acquire properties of Cancer Stem Cells (CSC) or Mesenchymal Stem Cell (MSC). However, the contribution of the CAFs in the acquisition of the differential stemness is largely unknown. Here, we studied the stemness features of prostate cancer cells (PC3) during the EMT program induced by fibroblasts obtained from patients with non-metastatic prostate cancer (CAF) and bone metastatic prostate cancer (mCAF).Material and methodsPC3 cells were incubated with conditioned medium (CM) of CAF and mCAF for 7,14 and 21 days to induced EMT. The presence of putative CSCs and MSCs was determined by qPCR, immunocytochemistry and western blot for the expression of CSCs and MSCs markers respectively and functionally evaluated by the ability to perform adipogenic, chondrogrenic and osteogenic differentiation (MSCs) and in vitro spheroids formation and clonogenic assay (CSCs).Results and discussionsOur results demonstrate that cancer cells treated with CM-CAF undergo to a partial EMT program, exhibiting certain mesenchymal traits and enriched for cancer stem cells (CSCs). On the other hand, tumour cells exposed to CM-mCAF, exhibited a full EMT conversion with the greater abilities to migrate and invade in vitro and in vivo.ConclusionThis suggests that CAFs and mCAFs induce differential EMT programs in prostate cancer cells, which leads them to acquire CSCs or MSC features enhancing the metastatic capabilities.

Abstract 2441: Effects of hypoxia on Src family members' activity in prostate cancer cells

Abstract Metastasis is the major reason of disease progression and poor prognosis in prostate cancer. Src family kinases (SFKs), including c-Src, Lyn and Fyn, are non-receptor tyrosine kinases that have been shown to play an essential role in castration resistance and metastasis in prostate cancer. Hypoxia, a typical hallmark of solid malignancies, is able to promote metastasis-associated functions. However, whether SFK members are upregulated under hypoxia is unclear. The current project will detect the impact of hypoxia on cell functions and molecular features in prostate cancer cells. For hypoxic exposure, prostate cancer cells were exposed to low oxygen tensions (1% O2) for varied durations (0, 2, 6, 24 h). To test cell migration, “wound-healing” assay was used. Cell invasion was detected by seeding cells into Matrigel-coated transwell chamber and cells on the bottom membranes were counted after treatment. Clonogenic assay was performed to test cell survival. At the molecular level, SFKs' phosphorylation in cancer cells and patient tissues was detected by Western blotting, and gene knockdown was accomplished by siRNA transfection. To detect the effects of Src inhibitors, cells were treated with saracatinib for indicated time periods. Phosphorylation of all three typical SFKs, e.g., c-Src, Lyn and Fyn, were highly expressed in prostate cancer patient tissues. At the cellular level, while short term hypoxic exposure (2-6 h) induced greater effects than prolonged hypoxia (24 h) in PC-3ML, these cellular functions were increased with 24 h hypoxic exposure in C4-2B cells. Further, hypoxia enhanced SFK phosphorylation in the pattern that was consistent with cell functions in both cell lines. Knockdown SRC, but not LYN or FYN, abolished hypoxia-induced invasion and p-SFK expression. Lastly, SFK inhibitor saracatinib showed stronger inhibition on functional behaviors facilitated under hypoxia than normal conditions. Our data show that hypoxia is able to enhance metastatic phenotypes by activating SFKs in prostate cancer cells. Interestingly, c-Src may be the most important signaling molecule for hypoxia-mediated behaviors. More importantly, SFK inhibitors are able to impair hypoxia-induced cell functions, suggesting their therapeutic potential by suppressing tumor metastasis that is driven under hypoxia in prostate cancer. Citation Format: Yao Dai, Dietmar Siemann. Effects of hypoxia on Src family members' activity in prostate cancer cells [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 2441.

Abstract 818: Unravelling the role of androgen targeted therapies in epithelial plasticity during prostate cancer progression

Abstract Recent evidence suggests that the use of potent androgen receptor (AR) antagonists have contributed to the increased prevalence of prostate cancer (PCa) tumors exhibiting therapy resistance. Androgen targeted therapies (ATT) inhibit the androgen/AR axis which PCa cells rely on for growth. ATT are initially effective however most men progress to metastatic castration resistant prostate cancer (mCRPC). Mechanisms of resistance include programs of epithelial plasticity (EP) such as epithelial-to-mesenchymal transition (EMT) and neuroendocrine transdifferentiation (NEtD). EMT enables PCa cells to become more motile and invasive. PCa cells that undergo NEtD gain neuroendocrine (NE)-like traits. Patients with NE prostate cancer (NEPC) usually present with visceral metastasis, are refractory to ATT and have short response to chemotherapies. We aim to characterize models of therapy-induced NEtD (tNEPC) and to investigate the role of tumor plasticity in the progression to mCRPC. To recapitulate ATT in vitro, androgen-dependent LNCaP cells were treated with either enzalutamide (Enz) (10μM) or shRNA targeting AR in culture medium containing fetal bovine serum (FBS) or charcoal stripped serum (CSS) with or without 10nM dihydrotestosterone (DHT). To model EMT, LNCaP cells were engineered to express doxycycline (Dox) inducible overexpression of EMT transcription factor Snail. Androgen deprivation in vitro induced expression of NE markers in LNCaP cells compared with FBS treated cells (mean fold increase ± SEM): neuron specific enolase (ENO2) (6.0 ± 2.5), neural cell adhesion molecule (NCAM) (10.4 ± 6.8) and midkine (MDK) (13.2 ± 2.4). Cells co-treated with Enz and DHT had increased expression of NE markers compared to DHT alone: ENO2 (2.7 ± 0.6), NCAM (2.9 ± 0.3) and MDK (2.3 ± 1.8). AR knockdown resulted in a potent induction of NE markers compared with control cells: ENO2 (8.3 ± 1.4), NCAM (8.3 ± 2.4) and MDK (9.0 ± 2.2). Dox-induced Snail enhanced androgen deprivation-mediated upregulation of NE markers compared with no-Dox cells: ENO2 (1.3 ± 0.1), NCAM (8.3 ± 2.4) and MDK (9.0 ± 2.2). To more accurately model a preclinical setting, LNCaP tumors were grown subcutaneously in severe combined immune deficiency mice. Once tumors grew to 200mm3, mice were castrated and upon tumor recurrence mice were treated with Enz (10mg/kg). At the ethical endpoint, expression of EMT and NE markers will be assessed. Patients treated with ATTs show an increase in NE-like traits. Thus, there is a need to identify molecular mechanisms involved in this adaptive response. In vitro, we have modelled the acquisition of NE-like features in PCa cells using ATTs, EMT drivers and AR knockdown. Further investigation using these models may lead to the development of biomarkers and therapeutic targets for the clinical management of advanced PCa. Citation Format: Ellca Ratther, Katrina G. Sweeney, Nataly Stylianou, Qiuhua Hu, Gregor Tevz, Colleen C. Nelson, Elizabeth D. Williams, Brett G. Hollier. Unravelling the role of androgen targeted therapies in epithelial plasticity during prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 818. doi:10.1158/1538-7445.AM2017-818

Loss of miR-449a-caused PrLZ overexpression promotes prostate cancer metastasis.

Distant metastasis is the worst prognostic factor for PCa patients. It has been reported that miR-449a enhances radiosensitivity of prostate cancer cells, but the function of miR449a in metastasis of prostate cancer is mainly unknown. In the present study, we strove to investigate the function and diagnostic value of miR-449a in metastasis of prostate cancer. qRT-PCR was used to quantify the expression of miR449a and PrLZ in PCa cell lines and tissues. We found that miR449a expression was decreased in PCa cell lines. Moreover, miR-449a was downregulated in PCa tissues, especially in primary lesion tissues of metastatic PCa patients. CCK8, FACS, Transwell and tube formation assay were performed to assess growth and metastasis of PCa cells in vitro. Lentivirus mediated miR-449a overexpression suppressed proliferation of LNcap and PC-3, and miR-449a also significantly inhibited invasion and angiogenesis ability of LNcap and PC-3. IHC showed that PrLZ was upregulated in PCa tissues. Luciferase assay and western blotting verified that miR-449a targeted PrLZ expression. Moreover, PrLZ shRNA also significantly suppressed proliferation and metastasis of LNcap and PC-3. In addition, western blotting revealed that miR-449a overexpression and PrLZ shRNA all remarkably inhibited the stemness features in LNcap and PC-3. Furthermore, BALB/c nude mouse subcutaneous xenograft model was uesd to verify the function of miR-449a and PrLZ. Our results showed that miR-449a and PrLZ shRNA significantly suppressed PC-3 tumorigenesis and metastasis in vivo. Our studies suggested that miR-449a decreased in malignant process of PCa and was accompanied by excess expression of PrLZ. The loss of miR-449a caused PrLZ overexpression regulated prostate cancer progression and metastasis via regulating the stemness features of prostate cancer cells. The diagnostic value of miR-449a as a distant metastasis predictor of PCa needs further investigation.

Abstract 5070: CD44 alternative splice variants are associated with prostate cancer cell identity and migration

Abstract Patient tumors can demonstrate tremendous cell-to-cell genetic heterogeneity while cultured cancer cell lines are often more homogeneous. This fundamental difference can undermine in vitro findings and their application to the more complex in vivo tumor environment. However, it should not be assumed that cultured cells are homogeneous even in well established and widely used cell lines. In my study of CD44 alternative splicing in the prostate cancer cell line PC3, I encountered varying CD44 alternative splicing expression profiles dependent on the origin of the PC3 cell line. CD44 is a cell surface glycoprotein that binds to components of the extracellular matrix, including hyaluronan, and is primarily involved in cell-cell and cell-matrix interactions. CD44 contains 10 variable exons that when combined in particular combinations significantly alter/guide CD44 downstream activities. In the context of cancer biology, alternative splicing of CD44 can control the epithelial to mesenchymal transition (EMT) of cancer cells and is associated with metastasis in many types cancers such as breast and prostate. To determine if cellular heterogeneity was the source of variable CD44 alternative splicing profiles, I isolated PC3 single cell clones. From these clones I identified two cell populations based on colony morphology: a compact colony subset with cells of a epithelial phenotype (PC3-EL), and a diffuse colony subset with cells of a mesenchymal phenotype (PC3-ML). I then performed reverse transcription-PCR (rt-PCR) on cDNA generated from these two populations with primers that specifically amplify the CD44 variable region to generate a fingerprint of CD44 alternative splicing. I found that PC3 cells with an epithelial phenotype express multiple CD44 variant exons (CD44v), including the epithelium-associated CD44v1,v8-10 variant (CD44E). However, phenotypically mesenchymal PC3 cells show a complete loss of CD44 splice variants and primarily express standard CD44v1 (CD44S). These differences in CD44 mRNA alternative spicing are manifested at the protein level as phenotypically epithelial PC3 cells express predominantly high molecular weight CD44 variants. I then monitored the association of CD44v expression on cell migration and determined that PC3 cells that express CD44E are non-motile, while PC3 cells that predominantly express CD44S are highly motile. Acquisition of CD44S over CD44E can signal EMT, an early and critical step in cancer metastasis. These data indicate that the CD44 alternative splice fingerprint may provide a predictive biomarker for EMT and the acquisition of other early pro-metastatic features in prostate cancer cells. Citation Format: David J. Bond, John D. Lewis. CD44 alternative splice variants are associated with prostate cancer cell identity and migration. [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 5070.

Targeting ACLY sensitizes castration-resistant prostate cancer cells to AR antagonism by impinging on an ACLY-AMPK-AR feedback mechanism.

The androgen receptor (AR) plays a central role in prostate tumor growth. Inappropriate reactivation of the AR after androgen deprivation therapy promotes development of incurable castration-resistant prostate cancer (CRPC). In this study, we provide evidence that metabolic features of prostate cancer cells can be exploited to sensitize CRPC cells to AR antagonism. We identify a feedback loop between ATP-citrate lyase (ACLY)-dependent fatty acid synthesis, AMPK, and the AR in prostate cancer cells that could contribute to therapeutic resistance by maintaining AR levels. When combined with an AR antagonist, ACLY inhibition in CRPC cells promotes energetic stress and AMPK activation, resulting in further suppression of AR levels and target gene expression, inhibition of proliferation, and apoptosis. Supplying exogenous fatty acids can restore energetic homeostasis; however, this rescue does not occur through increased β-oxidation to support mitochondrial ATP production. Instead, concurrent inhibition of ACLY and AR may drive excess ATP consumption as cells attempt to cope with endoplasmic reticulum (ER) stress, which is prevented by fatty acid supplementation. Thus, fatty acid metabolism plays a key role in coordinating ER and energetic homeostasis in CRPC cells, thereby sustaining AR action and promoting proliferation. Consistent with a role for fatty acid metabolism in sustaining AR levels in prostate cancer in vivo, AR mRNA levels in human prostate tumors correlate positively with expression of ACLY and other fatty acid synthesis genes. The ACLY-AMPK-AR network can be exploited to sensitize CRPC cells to AR antagonism, suggesting novel therapeutic opportunities for prostate cancer.

1323 RNA-BINDING MOTIF PROTEIN 3 ATTENUATES THE STEM CELL-LIKE PROPERTIES OF PROSTATE CANCER CELLS BY INTERFERING WITH CD44 VARIANT SPLICING

You have accessJournal of UrologyProstate Cancer: Basic Research (VI)1 Apr 20131323 RNA-BINDING MOTIF PROTEIN 3 ATTENUATES THE STEM CELL-LIKE PROPERTIES OF PROSTATE CANCER CELLS BY INTERFERING WITH CD44 VARIANT SPLICING Yu Zeng, Dong Gao, Dana Wodzenski, Takumi Shiraishi, Naoki Terada, Jun Luo, Robert Getzenberg, and Prakash Kulkarni Yu ZengYu Zeng Baltimore, MD More articles by this author , Dong GaoDong Gao Baltimore, MD More articles by this author , Dana WodzenskiDana Wodzenski Baltimore, MD More articles by this author , Takumi ShiraishiTakumi Shiraishi Baltimore, MD More articles by this author , Naoki TeradaNaoki Terada Baltimore, MD More articles by this author , Jun LuoJun Luo Baltimore, MD More articles by this author , Robert GetzenbergRobert Getzenberg Baltimore, MD More articles by this author , and Prakash KulkarniPrakash Kulkarni Baltimore, MD More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.2677AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Stress response pathways appear to play an important role in cancer evolution. RNA-binding motif protein 3 (RBM3) is a stress-response protein that is down-regulated under certain micro-environmental stress effects. Further, RBM3 expression in primary tumors is inversely correlated with poorer prognosis suggesting that RBM3 may suppress tumor progression. The goal of this study was to investigate the effects of RBM3 on stem cell-like features of prostate cancer cells. METHODS RBM3 mRNA expression was detected by RT-PCR. The normal prostate cell line, PrEC, was sorted using CD133 by flow cytometry. PC3 cells were transfected with pCMV6-RBM3 vector. Soft agar clonogenic assay and prostasphere assay were performed to evaluate stem cell-like features. The expressions of stem cell-related genes were determined using PCR arrays. PC3-RBM3 and PC3-GFP clones were subcutaneously inoculated in nude mice and tumor growth were compared. RESULTS RBM3 expression was low in human fetal tissues and in the CD133+ PrEC cells. In human prostate cancer, the expression of RBM3 was up-regulated in primary tumor tissues in comparison to adjacent normal glands but was decreased in metastatic tumors. RBM3 mRNA expression was down-regulated when cells were grown in soft agar or exposed to heat or cytotoxic stress. Over-expressing RBM3 in PC3 cells greatly attenuated their stem cell-like properties including the capability to re-colonize in soft agar, the ability to form prostaspheres, as well as tumorgenic potential in nude mice. Further, forced expression of RBM3 or culturing cells at 32∥C suppressed splicing of the CD44 variant v8-v10 but increased the expression of standard CD44 (CD44s) isoform. By contrast, lowering RBM3 expression by siRNA or culturing cells in soft agar that enrich the stem cell-like cell population, increased the ratio of mRNA levels of CD44v8-v10 to CD44s. Importantly, knocking down CD44v8-v10 using variant-specific siRNAs impaired PC3 cells to form colonies in soft agar, suggesting strongly that splicing of CD44v8-v10 that is inhibited by RBM3, contribute to the stem cell-like properties of the PC3 cells. CONCLUSIONS Thus down-regulation of RBM3 in order to maintain ‘stemness' may represent a natural response of cancer cells to adapt to microenvironmental stress, which could be the driving force behind cancer evolution. Accordingly, modulating RBM3 levels pharmacologically may help develop novel therapeutic strategies against the so-called ?cancer stem cells'. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e540-e541 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yu Zeng Baltimore, MD More articles by this author Dong Gao Baltimore, MD More articles by this author Dana Wodzenski Baltimore, MD More articles by this author Takumi Shiraishi Baltimore, MD More articles by this author Naoki Terada Baltimore, MD More articles by this author Jun Luo Baltimore, MD More articles by this author Robert Getzenberg Baltimore, MD More articles by this author Prakash Kulkarni Baltimore, MD More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract B23: Curcumin suppresses the growth and metastasis of prostate cancer cells by downregulation of SKP2.

Abstract Purpose: Prostate Cancer (PCa) is the second leading cause of cancer-related deaths in American men. The morbidity and mortality to PCa are 2.39-fold higher in African American men as compared to Caucasian males. Aberrant overexpression of SKP2 is frequently found in human cancers including prostate cancer, but little is known about pharmacological compounds targeting Skp2 activity. This study investigated inhibitory effects of curcumin (diferuloylmethane) on malignant features of prostate cancer cells in vitro and xenograft mouse models in vivo, and mechanistically on Skp2 level. Design Methods: Prostate cancer cells (PC3) and mouse prostate cancers cells (MPC3) in culture were treated with curcumin solution for indicated periods of time in vitro. Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot analyses were used to determine mRNA and protein expression of Skp2 in cancer cells. Cell proliferation, colony formation, and invasion assay were compared between curcumin-treated and control cells. Mouse prostate cancer cells were injected into immunodeficient mice followed with curcumin administration (200mg/kg/day). Tumorigenesis was analyzed in curcumin-treated and control mice after 20 days. Results: Curcumin treatment of PC3 cells resulted in a significant reduction of cell proliferation, soft agar transformation and invasion as compared to controls. Curcumin treatment led to a decrease of Skp2 level through a reduction of Skp2 mRNA and a shortened half-life of Skp2 protein. As expected, p27 protein, a major downstream target of Skp2, was increased upon curcumin treatment, whereas c-Myc protein level was not affected by curcumin treatment in prostate cancer cells. MPC3 cells, administered into immunodeficient mice via tail vein or orthotopic implantation, developed lung metastasis or enlarged prostate tumors, respectively. Importantly, cancer progression was significantly suppressed by curcumin treatment. A reduced level of Skp2 protein and induction of cell senescence were detected in tumors of curcumin-treated mice, but not in tumors of control mice. Conclusion: Our results indicated that curcumin suppresses both the tumor growth in mouse prostates and distant metastasis of prostate cancer cells in lung by inhibiting Skp2 level. Citation Format: Yuanying Gong, Wenfu Lu, Yingqiu Xie, Qing Yang, Robert J. Matusik, Zhenbang Chen. Curcumin suppresses the growth and metastasis of prostate cancer cells by downregulation of SKP2. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B23.

403 Mitochondrial superoxide dismutase overexpression changes neuroendocrine, proliferation and apoptosis resistance features in prostate cancer cells

403 Mitochondrial superoxide dismutase overexpression changes neuroendocrine, proliferation and apoptosis resistance features in prostate cancer cells

Deficient histone acetylation and excessive deacetylase activity as epigenomic marks of prostate cancer cells

Aberrant epigenomic alterations include incorrect histone modifications involving altered expression of chromatin-modifying proteins. They contribute to gene silencing and carcinogenesis. The nature of the epigenomic alterations occurring with prostate cancer remains to be fully identified. The acetylation status of histone H3 in human prostate cancer cells was assessed with multiple acetylation sites at N-termini. In contrast to the non-malignant prostatic cell lines RC165N/h and RC170N/h which possess stem cell properties, cancer cell lines LNCaP, DU-145, and PC-3 were either not acetylated or reduced in density (50-70%), at N-termini lysines 9, 14, 18, and 23 of histone H3. Deficient acetylation of histone H3 was similarly detected with clinical prostatic adenocarcinomas as compared to normal tissues. Cancer cell lines and adenocarcinomas exhibited varied acetylation status at particular lysines, indicating the possible presence of deacetylation patterns reflecting individual cancer cell clones. A significantly elevated activity of histone deacetylases (HDACs) was determined in both cancer cell lines and adenocarcinomas. Inhibition of HDACs enhanced histone acetylation and p21 gene expression, indicating that excessive HDAC activity is a requisite for deficient histone acetylation. Deficient histone acetylation involving excessive HDAC activity may represent epigenomic features of prostate cancer cells, and the aberrant enzyme activity is probably an underlying cause of disrupting the epigenomes of normal prostatic cells.

Phytoestrogen modulation of bone-related cytokines and its impact on cell viability in human prostate cancer cells

Aims Prostate cancer (PCa) has a high propensity to metastasize to the bone. PCa cells produce several bone-related factors, namely parathyroid hormone related protein (PTHrP), its PTH type 1 receptor (PTH1R), osteoprotegerin (OPG), and receptor activator of NF-kappa B ligand (RANKL). The effects of these factors might explain, at least in part, the ability of PCa cells to grow in and interact with bone. Main methods We first analyzed the expression of the aforementioned factors (by western blot and flow cytometry), and their modulation by the phytoestrogens genistein and daidzein (as potential anti-tumoral agents), in human PCa cells in vitro. We also assessed the impact of these osteomimetic factors on PCa cell viability (by propidium iodide staining and flow cytometry, and trypan blue staining). Key findings Genistein and daidzein, at nM range, increased both the PTHrP/PTH1R system and the OPG/RANKL protein ratio, while genistein and, to a lesser extent, daidzein, at > μM doses, inhibited cell viability in PCa cells. Both N- and C-terminal domains of PTHrP inhibited genistein-induced cell death by modulating transcription factor Runx-2 and the Bcl-2/Bax protein ratio in PCa cells. Significance Our findings indicate that high doses of genistein and daidzein cause PCa cell death. On the other hand, low doses of these phytoestrogens induce some osteomimetic features in PCa cells with putative impact on PCa development.

S100A8 and S100A9 activate MAP kinase and NF-κB signaling pathways and trigger translocation of RAGE in human prostate cancer cells

S100 proteins, a multigenic family of calcium-binding proteins, have been linked to human pathologies in recent years. Deregulated expression of S100 proteins, including S100A8 and S100A9, was reported in association with neoplastic disorders. In a previous study, we identified enhanced expression of S100A8 and S100A9 in human prostate cancer. To investigate potential functional implications of S100A8 and S100A9 in prostate cancer, we examined the influence of over-expressed and of purified recombinant S100A8 and S100A9 proteins in different prostate epithelial cell lines. S100A8 and S100A9 were secreted by prostate cancer cells, a finding which prompted us to analyze a possible function as extracellular ligands. S100A8/A9 induced the activation of NF-kappaB and an increased phosphorylation of p38 and p44/42 MAP kinases. In addition, extracellular S100A8/A9 stimulated migration of benign prostatic cells in vitro. Furthermore, in immunofluorescence experiments, we found a strong speckled co-localization of intracellular S100A8/A9 with RAGE after stimulating cells with recombinant S100A8/A9 protein or by increasing cytosolic Ca2+ levels. In summary, our findings show that S100A8 and S100A9 are linked to the activation of important features of prostate cancer cells.