Epithelial-mesenchymal Transition In Prostate Cancer Cells Research Articles

Juglone suppresses epithelial-mesenchymal transition in prostate cancer cells via the protein kinase B/glycogen synthase kinase-3β/Snail signaling pathway.

Epithelial-mesenchymal transition (EMT) serves an important role in the metastasis of prostate cancer. Juglone is a natural compound isolated from plants that is reported to possess potent cytotoxic properties. However, there are no studies on the anti-EMT effect of juglone in prostate cancer, or its potential underlying mechanisms of action. In the present study, the effect of juglone on the EMT of prostate cancer cells was investigated. Transwell assays were used to demonstrate that juglone inhibits the migration and invasion of the prostate cancer (PC) LNCaP and LNCaP-AI cell lines. Results from western blot analysis demonstrated that juglone increases the expression of the epithelial marker E-cadherin while decreasing the expression of mesenchymal markers (N-cadherin and Vimentin) in a dose-dependent manner. The data from the present study also revealed that juglone downregulates the expression of Snail, a repressor of E-cadherin and an inducer of EMT. Furthermore, juglone prevented inactivation of glycogen synthase kinase-3β (GSK-3β), an endogenous inhibitor of Snail in a dose-dependent manner. Lithium chloride (LiCl), a GSK-3β inhibitor, prevented juglone-mediated downregulation of Snail expression and upregulation of E-cadherin. In addition, phosphorylation and subsequent activation of protein kinase B (Akt), which is known to phosphorylate GSK-3β at serine 9 (Ser9), leading to its inhibition, were significantly decreased by juglone in LNCaP and LNCaP-AI cells. Inhibition of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt pathway by LY294002 augmented juglone-mediated GSK-3β activity by inhibiting Ser9 phosphorylation. These findings indicated that juglone suppresses EMT via the Akt/GSK-3β/Snail pathway, consequently decreasing the invasiveness of PC cells.

Propofol Reversed Hypoxia-Induced Docetaxel Resistance in Prostate Cancer Cells by Preventing Epithelial-Mesenchymal Transition by Inhibiting Hypoxia-Inducible Factor 1α.

Prostate cancer is the second most frequently diagnosed cancer worldwide. Hypoxia-induced epithelial–mesenchymal transition (EMT), driven by hypoxia-inducible factor 1α (HIF-1α), is involved in cancer progression and metastasis. The present study was designed to explore the role of propofol in hypoxia-induced resistance of prostate cancer cells to docetaxel. We used the Cell Counting Kit-8 and 5-ethynyl-2′-deoxyuridine incorporation assay to measure cell viability and cell proliferation, respectively, in prostate cancer cell lines. Then, we detected HIF-1α, E-cadherin, and vimentin expression using western blotting. Propofol reversed the hypoxia-induced docetaxel resistance in the prostate cancer cell lines. Propofol not only decreased hypoxia-induced HIF-1α expression, but also reversed hypoxia-induced EMT by suppressing HIF-1α. Furthermore, small interfering RNA–mediated silencing of HIF-1α reversed the hypoxia-induced docetaxel resistance, although there was little change in docetaxel sensitivity between the hypoxia group and propofol group. The induction of hypoxia did not affect E-cadherin and vimentin expression, and under the siRNA knockdown conditions, the effects of propofol were obviated. These data support a role for propofol in regulating EMT in prostate cancer cells. Taken together, our findings demonstrate that propofol plays an important role in hypoxia-induced docetaxel sensitivity and EMT in prostate cancer cells and that it is a potential drug for overcoming drug resistance in prostate cancer cells via HIF-1α suppression.

CCN3 promotes epithelial-mesenchymal transition in prostate cancer via FAK/Akt/HIF-1α-induced twist expression

Epithelial-mesenchymal transition (EMT) has received considerable attention as a conceptual paradigm for explaining metastatic behavior during cancer progression. NOV/CCN3 is a matrix-associated protein involved in many cellular functions. Previous studies have shown that CCN3 expression is upregulated in prostate cancer (PCa) cells and in PCa patients. In this study, we have provided evidence of tumor promoting effects of CCN3, which includes induction of epithelial-to-mesenchymal transition (EMT) and tumor metastasis. We used an orthotopic in vivo model to demonstrate the prometastatic effects of CCN3. Overexpression or knockdown of CCN3 changed the EMT phenotype in PCa cells. Moreover, treatment with recombinant CCN3 promoted EMT in PCa cells. We also found that CCN3 may promote EMT by activating the FAK/Akt/HIF-1α pathway and this activation is responsible for Twist expression. IHC staining confirmed a positive correlation between the expression of CCN3, Twist, and tumor stage in PCa tissue. Our findings provide insight into the involvement of CCN3 in the EMT regulation of prostate cancer. CCN3 is a promising molecular target that may contribute to a novel therapeutic strategy against metastatic PCa.

Upregulation of long non-coding RNA PlncRNA-1 promotes proliferation and induces epithelial-mesenchymal transition in prostate cancer.

ObjectiveTo confirm that PlncRNA-1 regulates the cell cycle in prostate cancer cells and induces epithelial-mesenchymal transition (EMT) in prostate cancer through the TGF-β1 pathway.ResultsPlncRNA-1 and TGF-β1 expression levels were significantly higher in prostate cancer tissues than in normal prostate tissues (P < 0.05) and were significantly positively correlated. TGF-β1, N-cadherin and Cyclin-D1 were downregulated and E-Cadherin was upregulated in LNCAP cells after silencing of PlncRNA-1, as determined by real-time PCR and Western blot. TGF-β1, N-cadherin and Cyclin-D1 were upregulated and E-cadherin was downregulated in C4-2 cells, as determined by real-time PCR and Western blot. Overexpression of PlncRNA-1 in C4-2 cells was observed when TGF-β1 inhibitor LY2109761 was added. Western blot analysis showed that compared with their expression when TGF-β1 inhibitor LY2109761 was not added, N-Cadherin and CyclinD1 expression decreased and E-Cadherin expression increased. Transwell results showed that the invasive ability of C4-2 cells was enhanced after overexpression of PlncRNA-1, and the invasion ability was decreased after addition of TGF-β1 inhibitor LY2109761. The cell cycle was blocked by overexpression of PlncRNA-1 in C4-2 and by the addition of TGF-β1 inhibitor LY2109761, as determined by flow cytometry. In vitro experiments showed that PlncRNA-1 can regulate the growth of prostate cancer cells and EMT through the TGF-β1 pathway. In vivo experiments also confirmed the above results. Tumor growth was significantly blocked by overexpressing PlncRNA-1 in C4-2 cells and by the TGF-β1 inhibitor LY2109761 in animal experiments.Materials and MethodsThe expression levels of PlncRNA-1 and TGF-β1 were analyzed in 19 prostate cancer tissue samples and in adjacent normal tissue samples, 4 Pca cell lines, including LNCaP, C4-2, DU145, and PC3, and 1 normal prostate epithelial cell line RWPE-1. LNCAP cells were divided into the LNCAP control group and the LNCAP-PlncRNA-1-siRNA group. Cells from the prostate cancer cell line C4-2 were divided into the C4-2 control group and the C4-2-PlncRNA-1 experimental group. Changes in TGF-β1, E-cadherin and N-cadherin were detected by qPCR and Western Blot assay after silencing and overexpression of PlncRNA-1. The cell cycle, cell invasion, and levels of Cyclin-D1, E-Cadherin, and N-Cadherin were observed after adding TGF-β1 inhibitor LY2109761 in the C4-2-PlncRNA-1 group. The effects of TGF-β1 inhibitor LY2109761 on the tumorigenicity of C4-2 cells after overexpression of PlncRNA-1 was investigated in vivo.ConclusionsPlncRNA-1 is an oncogene that regulates the cell cycle, cyclin-D1 and EMT in prostate cancer cells through the TGF-β1 pathway.

Abstract 1595: HMGA2 induces EMT in prostate cancer cells and may be antagonized by camalexin

Abstract Prostate cancer is the most diagnosed cancer in men and the second leading cause of death in the United States. African American men have the highest incidence and mortality rates of prostate cancer compared to any other race. Epithelial-mesenchymal transition (EMT) plays a critical role in cancer progression and metastasis. Mesenchymal cells are migratory, invasive, and more resistant to apoptosis. Reactive Oxygen Species (ROS) has been shown to promote EMT. High mobility group A (HMGA2) is a non-histone protein that is highly expressed during the embryogenesis, whereas the gene expression is very low or absent during adulthood. Recent studies have been reported an overexpression of HMGA2 protein in malignant cancers. Loss of Let-7 miRNA (repressor of HMGA2) has been found to induce EMT via upregulation of HMGA2 in prostate cancer. There has been no link between ROS and HMGA2. We have reported that camalexin, a 3-thizol-2-yl-indole, may be a candidate treatment for aggressive prostate cancer cells by ROS-mediated apoptosis. The study demonstrated that treating the prostate cancer cell with camalexin increased ROS levels which contributed to decreased cell proliferation and increased apoptosis. We hypothesize that HMGA2 may regulate EMT in part by inducing ROS and that camalexin may antagonize HMGA2 signaling. We analysed HMGA2 and EMT marker expression in a panel of prostate cancer cell lines by western blot analysis. We also transiently and stably overexpressed wild-type HMGA2 and mutant HMGA2 (missing Let-7 binding site) in LNCaP cells. We measured ROS levels using DCFDA dye that detects hydrogen peroxide. We treated ARCaP-M (mesenchymal) cells with different concentrations of camalexin to analyse HMGA2 expression. Our results showed that HMGA2 is highly expressed in aggressive prostate cancer cell lines (C4-2, ARCaP, E006AA) as compared to RWPE1 and LNCaP cells. The transient overexpression of HMGA2 in LNCaP cells decreased E-cadherin more markedly than with mutant HMGA2 but did not show any changes in ROS. We will repeat this experiment with stable clones from HMGA2 overexpression. ARCaP-M cells treated with camalexin induced ROS and decreased HMGA2 expression. We are currently performing in vivo studies using ARCaP-M cells injected subcutaneously into nude mice followed by treatment with camalexin after the tumor grows to 50 mm3. In conclusion, HMGA2 promotes EMT, even more markedly if its Let-7 suppressor binding site is eliminated, and camalexin may target HMGA2 to decrease prostate cancer progression. GRANT SUPPORT: 1P20MD002285; 8G12MD007590 Citation Format: Ohuod A. hawsawi, Basil Smith, Jodi Dougan, Liza J. Burton, Valerie A. Odero-Marah. HMGA2 induces EMT in prostate cancer cells and may be antagonized by camalexin. [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 1595.

Collapsin response mediator protein-1 (CRMP1) acts as an invasion and metastasis suppressor of prostate cancer via its suppression of epithelial\u2013mesenchymal transition and remodeling of actin cytoskeleton organization

The cancer cells can acquire migration and invasion capacities during the metastasis process through the developmental regulatory program epithelial–mesenchymal-transition (EMT), and through its reverse process mesenchymal–epithelial transition cancer cells can recolonize at distant metastatic sites. Among the multifaceted effects exerted by this program, reorganization of actin cytoskeleton is the key mechanical drive for the invasive properties gained by cancer cells. Collapsin response mediator protein-1 (CRMP1) is a cytosolic phosphoprotein and originally characterized as the mediator of semaphorin 3A signaling involved in axon differentiation during neural development. Here we report that CRMP1 can act as a suppressor of tumorigenicity and metastasis in prostate cancer cells. We demonstrated that CRMP1 exhibited a decreased expression pattern in high-grade prostate cancer tissues and many prostate cancer cell lines, and its downregulation in cancer cells was attributed to histone deacetylation and direct repression of its gene by the EMT regulator Snail. Functional analyses revealed that CRMP1 suppressed EMT in prostate cancer cells, as its knockdown could trigger EMT and enhance in vitro invasion capacity, whereas its overexpression could inhibit EMT and suppress both in vitro invasion and in vivo metastasis capacities of prostate cancer cells. Moreover, CRMP1 overexpression could significantly confer resistance to EMT induced by Snail or transforming growth factor-β1 in prostatic epithelial cells and prostate cancer cells. Finally, we demonstrated that CRMP1 could associate with actin and WAVE1, an activator of actin nucleation complex Arp2/3, and also its knockdown could stabilize F-actin and trigger the formation of stress fibers in prostate cancer cells. Together, our study shows that CRMP1 acts an EMT and metastasis suppressor in prostate cancer cells via its regulation of actin polymerization and also suggests that targeting the CRMP1-actin signaling in actin organization could be a potential strategy for management of prostate cancer metastasis.

Abstract B75: CCL2 promotes epithelial-mesenchymal transition (EMT) in prostate cancer cells via downregulation of dual-specificity phosphatase 22

Abstract CCL2 is a member of the CC chemokine superfamily that plays a crucial role in mediating macrophage recruitment and prostate cancer (PCa) metastasis. We have discovered that inhibiting the function of androgen receptor (AR) in prostate cancer cells results in the upregulation CCL2 and promotes disease progression by recruiting macrophages, activating signal transducer and activator of transcription 3 (STAT3), and by favoring the epithelial-to-mesenchymal transition. However, the molecular switches controlling CCL2 downstream signaling in PCa cells are not well understood. Here, we established an in vitro cell migration screening system using transwell migration chambers, and identified a higher amount of CCL2 in migrating PCa cells, establishing a link between CCL2 and a subpopulation of PCa cells. Importantly, we found that CCL2 expression correlated with the EMT marker, N-cadherin in migrating PCa cells. Importantly, CCL2 stimulation in PCa cells enhanced the expression levels of several EMT markers and induced STAT3 activation. In addition, CCL2 stimulation in PCa cells induced the downregulation of dual-specificity phosphatase 22 (DUSP22), which has been shown to act as a negative regulator of the IL-6-induced STAT3 activation through a direct interaction with STAT3. This supports that CCL2-induced DUSP22 downregulation in PCa cells as a potential mechanism to promote EMT. Therefore, we believe CCL2 may promote PCa metastasis in an autocrine manner through promotion of EMT in PCa cells with more aggressive motility behaviors. Targeting of the CCL2-STAT3 axis may offer new approaches to suppress the multifaceted support (immunosuppression, macrophage recruitment, and angiogenesis) of CCL2 for PCa cells in complex tumor microenvironment. Citation Format: Wen-Jye Lin, Kouji Izumi, Showping Lin, Hui-Min Ho. CCL2 promotes epithelial-mesenchymal transition (EMT) in prostate cancer cells via downregulation of dual-specificity phosphatase 22. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B75.

LEF1 Targeting EMT in Prostate Cancer Invasion Is Regulated by miR-34a.

The microRNA-34a (miR-34a), a tumor-suppressive microRNA (miRNA), is implicated in epithelial-mesenchymal transition (EMT) and cancer stem cells. Lymphoid enhancer-binding factor-1 (LEF1) is a key transcription factor in the Wnt signaling pathway, and has been suggested to be involved in regulation of cell proliferation and invasion. Here, the molecular mechanism of miR-34a and LEF1 in cooperatively regulating prostate cancer cell invasion is described. Molecular profiling analysis of miRNA levels in prostate cancer cells revealed a negative correlation between miR-34a and LEF1 expression, and the downregulation of LEF1 by miR-34a was confirmed by luciferase assays. Furthermore, miR-34a specifically repressed LEF1 expression through direct binding to its 3'-untranslated regions (3'-UTR). miR-34a modulated the levels of LEF1 to regulate EMT in prostate cancer cells. Functionally, miR-34a negatively correlated with the migration and invasion of prostate cancer cells through LEF1. An analysis of miR-34a expression levels in matched human tumor and benign tissues demonstrated consistent and statistically significant downregulation of miR-34a in primary prostate cancer specimens. These data strongly suggest that miR-34a/LEF1 regulation of EMT plays an important role in prostate cancer migration and invasion. The miR-34a-LEF1 axis represents a potential molecular target for novel therapeutic strategies in prostate cancer.

TGF-β and EGF induced HLA-I downregulation is associated with epithelial-mesenchymal transition (EMT) through upregulation of snail in prostate cancer cells

Human leukocyte antigen class I antigens (HLA-I) is essential in immune response by presenting antigenic peptides to cytotoxic T lymphocytes. Downregulation of HLA-I is observed in primary and metastatic prostate cancers, which facilitates them escape from immune surveillance, thereby promotes prostate cancer progression. In addition, elevated level of growth factors like TGF-β or EGF in microenvironment is related to the prostate cancer deterioration. Thus, we wondered whether TGF-β or EGF was involved in the regulation of HLA-I during the development of prostate cancer cells. In this study, we demonstrated that TGF-β and EGF both downregulated the expression of HLA-I, thereby attenuated the cytotoxic T cell mediated lysis of prostate cancer cells. Next, we revealed that TGF-β and EGF induced downregulation of HLA-I is associated with classical epithelial-mesenchymal transition (EMT) morphological changes and expression profiles. We further illustrated that overexpression of Snail is crucial for HLA-I downregulation and its association with EMT. At last, we discussed that NF-κB/p65 is the plausible target for Snail to induce HLA-I downregulation. Taken together, this is the first evidence to reveal that both TGF-β and EGF can induce HLA-I downregulation which is then proven to be associated with EMT in prostate cancer cells. These discoveries provide a deeper understanding of growth factors induced immune escape and introduce potential therapeutic targets for prostate cancers.

Periostin Mediates TGF-β-Induced Epithelial Mesenchymal Transition in Prostate Cancer Cells

Background: In our previous study, we found that periostin was upregulated in prostate cancer, and its expression could be modulated by TGF-β. TGF-β could upregulate periostin expression in some cells, and both TGF-β and periostin could induce epithelial mesenchymal transition (EMT). We aimed to study the effect of periostin in the process of TGF-β-induced EMT in prostate cancer cells. Methods: We constructed a lentivirus vector containing the periostin gene and transduced it into PC3 and DU145 cells. After confirming periostin overexpression by PCR and Western blotting, we used an MTT assay to establish a growth curve to measure cell proliferation. Additionally, we performed transwell and wound healing assays to measure cell invasion and migration, respectively. Lastly, we measured the expression of EMT associated factors using Western blot analysis to test the effect of periostin on EMT in prostate cancer cells. Results: PCR and Western blot analyses confirmed that periostin was upregulated after infection with the periostin lentiviral vector. Periostin overexpression promoted increased cell proliferation, invasion, and migration as measured by MTT, transwell, and wound healing assays, respectively. Western blot analysis illustrated that periostin overexpression increased the expression of EMT associated factors, and periostin overexpression activated Akt and GSK-3β, which could be inhibited using a PI3K inhibitor. Additionally, TGF-β increased the levels of STAT3, Twist1 and periostin, while both STAT3 shRNA and Twist1 shRNA inhibited periostin expression. However, STAT3 shRNA also decreased Twist1 expression. Although reduction of STAT3, Twist1 or periostin levels with shRNA inhibited TGF-β-induced overexpression of EMT associated factors, periostin overexpression could reverse such inhibition by interfering with STAT3 and Twist1. Similarly, periostin overexpression also reversed inhibition of cell invasion induced by interference of STAT3 and Twist1. Conclusion: Our findings indicate that periostin is an important mediator of TGF-β-induced EMT and suggest that periostin is a potential therapeutic target for suppressing the metastatic progression of prostate cancer.

Metformin inhibits epithelial–mesenchymal transition in prostate cancer cells: Involvement of the tumor suppressor miR30a and its target gene SOX4

Tumor metastasis is the leading cause of mortality and morbidity of prostate cancer (PCa) patients. Epithelial–mesenchymal transition (EMT) plays a critical role in cancer progression and metastasis. Recent evidence suggested that diabetic patients treated with metformin have lower PCa risk and better prognosis. This study was aimed to investigate the effects of metformin on EMT in PCa cells and the possible microRNA (miRNA)-based mechanisms. MiRNAs have been shown to regulate various processes of cancer metastasis. We herein showed that metformin significantly inhibits proliferation of Vcap and PC-3 cells, induces G0/G1 cell cycle arrest and inhibits invasiveness and motility capacity of Vcap cells. Metformin could inhibit TGF-β-induced EMT in Vcap cells, as manifested by inhibition of the increase of N-cadherin (p=0.013), Vimentin (p=0.002) and the decrease of E-cadherin (p=0.0023) and β-catenin (p=0.034) at mRNA and protein levels. Notably, we demonstrated significant upregulation of miR30a levels by metformin (P<0.05) and further experiments indicated that miR30a significantly inhibits proliferation and EMT process of Vcap cells. Interestingly, we identified that SOX4, a previously reported oncogenic transcriptional factor and modulator of EMT, is a direct target gene of miR30a. Finally, we screened the expression of miR30a and SOX4 in 84 PCa cases with radical prostatectomy. Of note, SOX4 overexpression is significantly associated with decreased levels of miR30a in PCa cases. In all, our study suggested that inhibition of EMT by metformin in PCa cells may involve upregulation of miR30a and downregulation of SOX4.

Muscadine grape skin extract reverts snail-mediated epithelial mesenchymal transition via superoxide species in human prostate cancer cells

BackgroundSnail transcription factor can induce epithelial-mesenchymal transition (EMT), associated with decreased cell adhesion-associated molecules like E-cadherin, increased mesenchymal markers like vimentin, leading to increased motility, invasion and metastasis. Muscadine grape skin extract (MSKE) has been shown to inhibit prostate cancer cell growth and induce apoptosis without affecting normal prostate epithelial cells. We investigated novel molecular mechanisms by which Snail promotes EMT in prostate cancer cells via Reactive Oxygen Species (ROS) and whether it can be antagonized by MSKE.MethodsARCaP and LNCaP cells overexpressing Snail were utilized to examine levels of reactive oxygen species (ROS), specifically, superoxide, in vitro using Dihydroethidium (DHE) or HydroCy3 dyes. Mitosox staining was performed to determine whether the source of ROS was mitochondrial in origin. We also investigated the effect of Muscadine grape skin extract (MSKE) on EMT marker expression by western blot analysis. Migration and cell viability using MTS proliferation assay was performed following MSKE treatments.ResultsSnail overexpression in ARCaP and LNCaP cells was associated with increased concentration of mitochondrial superoxide, in vitro. Interestingly, MSKE decreased superoxide levels in ARCaP and LNCaP cells. Additionally, MSKE and Superoxide Dismutase (SOD) reverted EMT as evidenced by decreased vimentin levels and re-induction of E-cadherin expression in ARCaP-Snail cells after 3 days, concomitant with reduced cell migration. MSKE also decreased Stat-3 activity in ARCaP-Snail cells.ConclusionsThis study shows that superoxide species may play a role in Snail transcription factor-mediated EMT. Therefore, therapeutic targeting of Snail with various antioxidants such as MSKE may prove beneficial in abrogating EMT and ROS-mediated tumor progression in human prostate cancer.

A ROS/STAT3/HIF-1α signaling cascade mediates EGF-induced TWIST1 expression and prostate cancer cell invasion

Epidermal growth factor (EGF) has been known to induce epithelial-mesenchymal transition (EMT) and prostate cancer cell progression. However, a detailed underlying mechanism by which EGF induces EMT and prostate cancer cell progression remained to be answered. Hypoxia-inducible factor (HIF)-1α and TWIST1 are transcription factors implicated in EMT and cancer metastasis. The purpose of this study is to determine the underlying mechanism of EGF-induced TWIST1 expression and prostate cancer invasion. siRNAs were used to silence genes. Immunoblotting, quantitative RT-PCR and immunofluorescence analysis were used to examine protein or mRNA expression. Modified Boyden chamber and invasion assay kit with Matrigel-coated inserts were used to determine prostate cancer cell migration and invasion, respectively. We observed that EGF induced HIF-1α expression and morphological change of prostate cancer epithelial cells to mesenchymal cells. Silencing HIF-1α expression dramatically reduced EGF-induced TWIST1 expression and prostate cancer cell EMT. Conversely, transfection of the cells with HIF-1α siRNA reversed the reduced E-cadherin expression by EGF. Pretreatment of the cells with pharmacological inhibitors of reactive oxygen species [ROS, N-acetylcysteine (NAC)] and STAT3 (WP1066) but not p38 MAPK (SB203580) significantly reduced EGF-induced HIF-1α mRNA and protein expression. Further, pretreatment of the cells with NAC attenuated EGF-induced STAT3 phosphorylation. In addition, we showed that TWIST1 mediated EGF-induced N-cadherin expression, leading to prostate cancer invasion. We demonstrate a mechanism by which EGF promotes prostate cancer cell progression through a ROS/STAT3/HIF-1α/TWIST1/N-cadherin signaling cascade, providing novel biomarkers and promising therapeutic targets for prostate cancer cell progression.

Stabilization of Snail through AKT/GSK-3β signaling pathway is required for TNF-α-induced epithelial–mesenchymal transition in prostate cancer PC3 cells

Metastasis induced by chronic inflammation has been considered as a major challenge during cancer therapy. Epithelial–mesenchymal transition (EMT) is associated with cancer invasion and metastasis promoted by pro-inflammatory cytokine TNFα. However, the mechanisms underlying TNFα-induced EMT in prostate cancer cells is not entirely clear. Here we showed that EMT induced by longstanding stimulation with TNFα in prostate cancer PC3 cells is mediated by up-regulation of the transcriptional repressor Snail. TNFα-mediated EMT was characterized by acquiring mesenchymal fusiform morphology, increasing the expression of Vimentin and decreasing the expression of E-cadherin. Exposure to TNFα increased the expression of transcription factor Snail via post-transcriptional regulation process and induced Snail nuclear localization in PC3 cells. Moreover, overexpressed Snail in PC3 cells induced EMT. Conversely, suppressing Snail expression abrogated TNFα-induced EMT, suggesting that Snail plays a crucial role in TNFα-induced EMT in prostate cancer cells. Finally, we showed that TNFα time-dependently activated NF-κB, AKT, ERK, p38 MAPK signaling pathways, and elevated Snail stability by activating AKT pathway that subsequently inhibited GSK-3β activity. Taken together, these results reveal that stabilization of Snail via AKT/GSK-3β signaling pathway is required for TNFα-induced EMT in prostate cancer cells. This study offers a better understanding of TNFα-induced metastasis and provides an effective therapeutic strategy for prostate cancer treatment.

Abstract C15: CRMP1 functions to suppress epithelial-mesenchymal transition and invasion capacity of prostate cancer cells

Abstract Collapsin response mediator protein 1 (CRMP1) is originally identified and characterized as a cytoplasmic phosphoprotein involved in the Semaphorin 3A (Sema3A)-regulated growth cone collapse during neural development, via its regulation of cytoskeleton dynamics, and then also characterized as an invasion suppressor in lung cancer. Thus, we hypothesize that CRMP1 may be involved in the epithelial-mesenchymal transition (EMT) process and invasion or metastasis via its regulation on cytoskeleton organization in prostate cancer cells. Results of immunohistochemistry study showed that CRMP1 exhibited a reduced expression pattern in high Gleason-scored clinical prostate cancer tissues. In vitro functional studies showed that stable CRMP1-knockdown could induce EMT in prostate cancer cells (DU145), while its ectopic expression could induce the reverse process mesenchymal-epithelial transition (MET) in both DU145 and PC-3 prostate cancer cells, as evidenced by significant change of cellular morphology and altered expression levels of epithelial and mesenchymal markers. Confocal immunofluorescence showed that reorganization of F-actin was seen after shRNA-mediated CRMP1-knockdown in DU145 cells. It was also revealed that CRMP1 overexpression could confer the resistance to TGF-β1- and Snail-induced EMT in prostate cells. Results of CRMP1 promoter-driven luciferase reporter and ChIP assays indicated that Snail was a direct transrepressor of CRMP1 gene, which showed a downregulation pattern in both prostate cancer cells (DU145) and immortalized prostatic epithelial cells (BPH1) with overexpression of Snail. Furthermore, wound healing and Matrigel invasion assays showed that CRMP1 could inhibit cell migration and invasion capacity in DU145 and PC-3 cells. Taken together, our findings suggest that CRMP1 could play a role in prostate cancer invasion and metastasis, via its regulation on EMT and MET process. (This work was supported by a Direct Grant for Research and a RGC-GRF grant 2009/2010) Citation Format: Ganhui Cai, Shan Yu, Yanping Ji, Franky Leung Chan. CRMP1 functions to suppress epithelial-mesenchymal transition and invasion capacity of prostate cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C15.

Another road leads to HIF-1 activation: implications for prostate cancer progression

Another road leads to HIF-1 activation: implications for prostate cancer progression

Abstract 5672: A novel anti-cancer effect of hesperidin: reversion of epithelial mesenchymal transition in human prostate cancer cells

Abstract Prostate cancer is the most common noncutaneous malignancy in men. About one third of prostate cancers invade surrounding tissues, and metastasize to distant organs at the time of diagnosis. Once prostate cancer metastasizes to distant organs, the disease is basically not curable. The median survival for men with metastatic prostate cancer is 1 to 3 years. Epithelial mesenchymal transition (EMT) is a process facilitating cancer cell invasion and metastasis. Hesperidin (3′-5,7-trihydroxy-4′-methoxyflavanone 7-rhamnoglucoside) is a flavonoid found abundantly in citrus fruits. It demonstrates anti-inflammatory, anti-oxidative, and anti-proliferative actions in various cancer types. Its effect on EMT of prostate cancer is not known. We therefore hypothesize that hesperidin inhibits the cell growth and EMT of prostate cancer cells. In our study, we use androgen-sensitive prostate cancer LNCaP cells, androgen-insensitive Du 145 and PC3 cells, and benign prostate BPH-1 cells. These cell lines are treated with increasing concentrations of hesperidin, from 10 µM to 200 µM. We first examine the effects of hesperidin on cell viability by MTT assays. Hesperidin inhibits all three prostate cancer cell viability in a dose dependent manner but it has no significant effects on that of normal prostate cells, BPH-1. We then examine whether hesperidin reverses EMT in these cells by investigating the migration ability and the expression of EMT marker. Hesperidin at 100 µM and 200 µM inhibits the cell migration ability of PC3 and LNCaP as showed by wound scratch assay. Next, we examine expression of E-cadherin, a molecular marker for EMT, by immunohistochemistry. Hesperidin at 100 µM significantly enhances the membranous expression of E-cadherin in LNCaP cells after 48 hours of treatment as compared to the control. There are no significant changes in androgen receptor (AR) protein level or PSA mRNA after hesperidin treatment suggestive that the effects of hesperidin on LNCaP cells are AR-independent. In conclusions, our data provide novel findings that hesperidin can induce the reversion of EMT in prostate cancer cells. Hesperidin may therefore be a potential chemopreventive agent in human prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5672.