Epithelial-to-mesenchymal Transition In Prostate Cancer Cells Research Articles

Abstract 2504: Antiarrhythmic drugs repositioning to counteract the activated phenotype of prostate cancer-associated fibroblasts

Abstract The interplay between cancer cells and adjacent stroma is fundamental to sustain carcinogenesis and promote the acquisition of additional malignant features. Cancer-associated fibroblasts (CAFs) have been shown to fuel prostate cancer (PCa) development and metastasis by mutually interacting with tumor cells. Comparative gene expression profile analysis of CAFs and normal fibroblasts, showed that CAFs are characterized by a positive enrichment of gene sets that regulate cytoskeleton remodeling and muscle contractility, including genes coding for calcium, potassium and sodium channels. Here, for the first time, we investigated the effects of cation channel inhibitors, currently used as antiarrhythmic drugs, on CAF-activated state and CAF-PCa cell interplay. Our results showed that antiarrhythmic drugs are able to interfere with crucial features of reactive prostate CAFs, as indicated by changes in cell morphology and modulation of specific fibroblast activation markers, such as α-SMA, fibroblast activation protein and collagen type I. In addition, although to a variable extent, antiarrhythmics reduced CAF motility and hindered CAF capability to remodel extracellular matrix, as assessed by 3D gel contraction and scratch assays. Interestingly, conditioned medium and co-culture experiments reveled that CAFs were able to promote cell growth and induce epithelial to mesenchymal transition (EMT) in PCa cells, as indicated by E-cadherin and β-catenin down-regulation and Snail and Vimentin up-regulation. Interestingly, antiarrhythmics partially reverted CAF-mediated protumoral effects, abrogating their ability to foster PCa cell proliferation and plasticity. Moreover, based on previous evidence indicating the involvement of matrix metalloproteinase (MMP) in CAF-induced EMT on PCa cells, we found that antiarrhythmics impaired the secretory phenotype of CAFs, as indicated by the reduction of MMP-2 releasing in the medium. Finally, gene expression analysis highlighted that antiarrhythmic-treated CAFs showed a gene expression pattern resembling that of normal fibroblasts. Consistent with our in vitro findings, in vivo experiments confirmed that intra-tumoral administration of medium form antiarrhythmic-treated CAF reduced tumor growth rate of PCa xenografts compared to medium form untreated CAFs. Overall, cation channels inhibition interferes with crucial features of prostate CAFs, affecting their activated state. Moreover, co-culture experiments indicate that antiarrhythmics are able to impair CAF-induced proliferative spur and EMT in PCa cells, and also induce tumor growth delay in vivo. Collectively, such data suggest new opportunities for the therapeutic repositioning of antiarrhythmic drugs with the aim of targeting CAFs and hence interrupting their supportive boost in PCa. Citation Format: Valentina Doldi, Monica Tortoreto, Stefano Percio, Paolo Gandellini, Nadia Zaffaroni. Antiarrhythmic drugs repositioning to counteract the activated phenotype of prostate cancer-associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2504.

Combination Treatment of a Phytochemical and a Histone Demethylase Inhibitor-A Novel Approach towards Targeting TGFβ-Induced EMT, Invasion, and Migration in Prostate Cancer.

Minimizing side effects, overcoming cancer drug resistance, and preventing metastasis of cancer cells are of growing interest in current cancer therapeutics. Phytochemicals are being researched in depth as they are protective to normal cells and have fewer side effects. Hesperetin is a citrus bioflavonoid known to inhibit TGFβ-induced epithelial-to-mesenchymal transition (EMT), migration, and invasion of prostate cancer cells. Targeting epigenetic modifications that cause cancer is another class of upcoming therapeutics, as these changes are reversible. Global H3K27me3 levels have been found to be reduced in invasive prostate adenocarcinomas. Combining a demethylase inhibitor and a known anti-cancer phytochemical is a unique approach to targeting cancer to attain the aforementioned objectives. In the current study, we used an H3K27 demethylase (JMJD3/KDM6B) inhibitor to study its effects on TGFβ-induced EMT in prostate cancer cells. We then gave a combined hesperetin and GSK-J4 treatment to the PC-3 and LNCaP cells. There was a dose-dependent increase in cytotoxicity and inhibition of TGFβ-induced migration and invasion of prostate cancer cells after GSK-J4 treatment. GSK-J4 not only induced trimethylation of H3K27 but also induced the trimethylation of H3K4. Surprisingly, there was a reduction in the H3K9me3 levels. GSK-J4 alone and a combination of hesperetin and GSK-J4 treatment effectively inhibit the important hallmarks of cancer, such as cell proliferation, migration, and invasion, by altering the epigenetic landscape of cancer cells.

Hesperetin modulates TGFβ induced metastatic potential of prostate cancer cells by altering histone methylation marks

Prostate cancer is the most prevalent cancer in males, and usually, death occurs due to bone metastasis. TGFβ has been shown to play an essential role in the metastasis of prostate cancer cells by promoting epithelial-to-mesenchymal transition (EMT). Hesperetin is known to possess anti-microbial, anti-fungal, antioxidant, and anti-cancer properties and good bioavailability. Therefore, this study investigated the effect of hesperetin on TGFβ-induced cell proliferation and EMT in prostate cancer cells (PC3). Interestingly, we found that hesperetin can significantly inhibit the cell proliferation of PC3 cells and arrest the cells in the S and G2M phases of the cell cycle. The invasion and migration assay results decipher the inhibitory effect of hesperetin on TGFβ-induced invasion and migration of prostate cancer cells. Our results confirmed that hesperetin also acts through the canonical signaling pathway, as we observed a significant decrease in the expression of pSmad3. Hesperetin can inhibit the TGFβ induced EMT by increasing E-cadherin expression and decreasing N-cadherin expression. Hesperetin could modulate the TGFβ induced histone methylation marks. Further investigation is required to understand the role of hesperetin in modulating these marks and thus inhibiting TGFβ-induced EMT. Hence, the results of our study identified the potential of hesperetin to modulate TGFβ-induced cell proliferation and invasion and migration of prostate cancer cells which may help inhibit the metastatic growth of prostate cancer cells.

GATA binding protein 5-mediated transcriptional activation of transmembrane protein 100 suppresses cell proliferation, migration and epithelial-to-mesenchymal transition in prostate cancer DU145 cells

ABSTRACT It has been reported that transmembrane protein 100 (TMEM100) acts as a tumor regulator in several types of cancers. However, whether the expression of TMEM100 is associated with the development and prognosis of prostate cancer (PCa) remains elusive. Therefore, the present study aimed to uncover the role of GATA binding protein 5 (GATA5)-mediated activation of TMEM100 in the proliferation, migration and epithelial-to-mesenchymal transition (EMT) of PCa cells. The expressions of TMEM100 and GATA5 in PCa patients were analyzed by the GEPIA database. The binding site of GATA5 and TMEM100 promoter was predicted by the JASPAR database. Expressions of TMEM100 and GATA5 in PCa cells were detected by qRT-PCR and Western blot analysis. Cell Counting Kit 8 and colony formation assays were performed to measure cell proliferation. In addition, cell migration, invasion and the expression of EMT-associated proteins were evaluated using wound healing, transwell assay and Western blotting assays, respectively. The bioinformatics analysis revealed that TMEM100 was downregulated in PCa and was associated with overall survival of PCa. In addition, TMEM10 overexpression attenuated cell proliferation, migration, invasion and EMT in PCa cells. The interaction between TMEM100 and GATA5 was verified using dual luciferase reporter and chromatin immunoprecipitation assays. Furthermore, the results showed that GATA5 was downregulated and GATA5 silencing reversed the inhibitory effects of TMEM10 on PCa cells. Overall, the current study suggested that the GATA5-mediated transcriptional activation of TMEM100 could affect the behavior of PCa cells and was associated with poor prognosis in PCa.

ZNF24 is upregulated in prostate cancer and facilitates the epithelial-to-mesenchymal transition through the regulation of Twist1.

Zinc finger protein 24 (ZNF24) has been demonstrated to regulate proliferation, differentiation and migration as well as invasion in several types of cells. However, the molecular role and clinical effects of ZNF24 in prostate cancer (PCa) remain unclear. The present study revealed that ZNF24 expression is upregulated in PCa, and associated with tumor volume, Gleason score, pathological grade and metastasis. Wound healing and Transwell invasion assays revealed that ectopic ZNF24 expression facilitated cell migration and invasion through the Twist1-induced epithelial-to-mesenchymal transition (EMT) process. In addition, colony formation and Cell Counting Kit-8 assays were used to determine the regulatory effects of ZNF24 on proliferation. The results suggested that ZNF24 also promoted cell proliferation in PCa. ZNF24 acted as an oncogene and promoted migration, invasion and EMT of PCa cells via the regulation of Twist1.

MiR-20b-5p, TGFBR2, and E2F1 Form a Regulatory Loop to Participate in Epithelial to Mesenchymal Transition in Prostate Cancer.

The transcription factor E2F1 regulates the expression of the miR-20b-5p precursor and is involved in epithelial-to-mesenchymal transition (EMT). Transforming growth factor-β1 (TGF-β1) induces EMT in prostate cancer (PCa) by binding to TGF-beta receptor 2 (TGFBR2) to activate TGF-β signaling. However, the relationship between TGFBR2, E2F1, and miR-20b-5p in the modulation of EMT in PCa cells remains unknown. In this study, we found that the level of miR-20b-5p expression was significantly lower in PC3 and DU145 cells than that in prostate epithelial (RWPE-1) cells, and TGF-β1 treatment further down-regulated miR-20b-5p expression in these two cell lines. Functional studies showed that miR-20b-5p suppressed TGF-β1-induced migration and invasion of PC3 and DU145 cells by up-regulating E-cadherin and down-regulating vimentin, leading to TGF-β1-induced inhibition of EMT. Using gain and loss of function experiments, it was shown that E2F1 mediated TGF-β1 regulation of miR-20b-5p expression. Further, a luciferase activity assay showed that TGFBR2 was a direct target of miR-20b-5p in PCa cells. These results suggest that miR-20b-5p, TGFBR2, and E2F1 form a regulatory loop to modulate EMT induced by TGF-β1. A novel regulatory mechanism underlying the miR-20b-5p/TGFBR2/E2F1 axis is involved in TGF-β1-induced EMT of PCa cells, and miR-20b-5p may be a potential therapeutic target for PCa.

Screening and identification of epithelial-to-mesenchymal transition-related circRNA and miRNA in prostate cancer

Epithelial-to-mesenchymal transition (EMT) plays a vital role in the progression and metastasis of prostate cancer. However, the molecular mechanisms underlying prostate cancer metastasis are not fully demonstrated. In this study, EMT was induced by interferon-γ (IFN-γ) in PC-3M IE8 cells. High-throughput sequencing was used to screen the differentially expressed circular RNAs (circRNAs) and miRNAs in the cells with or without IFN-γ treatment. EMT-related circRNAs and miRNAs were further identified by quantitative real-time PCR (qPCR). In addition, the relationships among circRNAs, miRNAs, and mRNA were predicted. After cells were treated with IFN-γ, western blot analysis was conducted to detect the expression levels of EMT markers. E-cadherin expression levels were found to be downregulated, and Twist expression levels were found to be upregulated. Our results also found that IFN-γ promoted PC-3M IE8 cell migration and invasion, indicating that IFN-γ could induce EMT in PC-3M IE8 cells. Furthermore, high-throughput sequencing results revealed 827 upregulated and 1279 downregulated circRNAs and 39 upregulated and 2076 downregulated miRNAs in the IFN-γ group compared with the control group. KEGG analysis showed that both differentially expressed circRNAs and differentially expressed miRNAs were enriched in the MAPK signaling pathway related to EMT. Furthermore, the qPCR results revealed that the expression of hsa_circ_0001085, hsa_circ_0004916, hsa_circ_0001165, hsa-miR-196b-5p, and hsa-miR-187-3p in the IFN-γ group was consistent with the sequencing results. hsa_circ_0001165 and hsa_circ_0001085 were used to construct the network of circRNA-miRNA-mRNA. It was found that hsa_circ_0001165 may regulate TNF expression through hsa-miR-187-3p to induce EMT in prostate cancer cells. In addition, hsa_circ_0001085 may indirectly regulate the PI3K-Akt signaling and TGF-β signaling pathways through hsa-miR-196b-5p and the MAPK signaling pathway through has-miR-451a, which played a regulatory role in prostate cancer cells in the EMT induction model. The results obtained in this study lay the foundation for future study.

The metastasis suppressor CD82/KAI1 represses the TGF-β 1 and Wnt signalings inducing epithelial-to-mesenchymal transition linked to invasiveness of prostate cancer cells.

The epithelial-to-mesenchymal transition (EMT) is closely associated with cancer invasion and metastasis. Since the transforming growth factor β (TGF-β) and Wnt signals induce EMT in various epithelial cell types, we examined whether and how the CD82/KAI1 metastasis suppressor affects the TGF-β and Wnt signal-dependent EMT in human prostate cancer cells. The invasiveness of cancer cells was evaluated by examining their ability to pass through the basement membrane matrigel. The subcellular localizations of Smad4 and β-catenin proteins were respectively examined by confocal microscopy following immunofluorescence antibody staining and immunoblotting analysis following subcellular fractionation. The transcriptional activities of the TGF-β1 -responsive TRE and Wnt-responsive Tcf/Lef promoters were determined by a luciferase reporter assay following transfection of the recombinant reporter vector into the cell. TGF-β1 and Wnt3a treatments of human prostate cancer cells without CD82 expression resulted in not only increased invasiveness but also EMT involving the development of motile structures, downregulation of E-cadherin, and upregulation of the mesenchymal proteins. However, in the cells with high levels of CD82, the TGF-β1 and Wnt3a stimulations neither elevated invasiveness nor induced EMT. Furthermore, the TGF-β1 signaling events occurring in the CD82-deficient cells, such as phosphorylation of Smad2, nuclear translocation of Smad4, and transactivation of the TRE promoter, did not take place in the high CD82-expressing cells. Further, high CD82 expression interfered with the Wnt signal-dependent alterations in the phosphorylation pattern of glycogen synthase kinase 3β (GSK-3β) in prostate cancer cells, which allowed GSK-3β to continue phosphorylating β-catenin, thereby attenuating the Wnt signaling effects on the nuclear translocation of β-catenin and subsequent transactivation of the Tcf/Lef promoter. The results of the present study suggest that CD82/KAI1 functions in suppressing TGF-β1 - and Wnt-induced EMT in prostate cancer cells by inhibiting the TGF-β1 /Smad and Wnt/β-catenin pathways. Therefore, loss or decrease of CD82 expression is likely to render prostate cancer cells prone to respond to the TGF-β1 and Wnt signals with EMT, resulting in the development of a motile and invasive mesenchymal phenotype related to the initiation of the metastatic cascade.

Cholesterol Induces Epithelial-to-Mesenchymal Transition of Prostate Cancer Cells by Suppressing Degradation of EGFR through APMAP.

Cholesterol increases the risk of aggressive prostate cancer and has emerged as a potential therapeutic target for prostate cancer. The functional roles of cholesterol in prostate cancer metastasis are not fully understood. Here, we found that cholesterol induces the epithelial-to-mesenchymal transition (EMT) through extracellular-regulated protein kinases 1/2 pathway activation, which is mediated by EGFR and adipocyte plasma membrane-associated protein (APMAP) accumulation in cholesterol-induced lipid rafts. Mechanistically, APMAP increases the interaction with EGFR substrate 15-related protein (EPS15R) to inhibit the endocytosis of EGFR by cholesterol, thus promoting cholesterol-induced EMT. Both the mRNA and protein levels of APMAP are upregulated in clinical prostate cancer samples. Together, these findings shed light onto an APMAP/EPS15R/EGFR axis that mediates cholesterol-induced EMT of prostate cancer cells. SIGNIFICANCE: This study delineates the molecular mechanisms by which cholesterol increases prostate cancer progression and demonstrates that the binding of cholesterol-induced APMAP with EPS15R inhibits EGFR internalization and activates ERK1/2 to promote EMT. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/12/3063/F1.large.jpg.

TGFβ-induced epithelial-to-mesenchymal transition in prostate cancer cells is mediated via TRPM7 expression.

Growth factors, such as the transforming growth factor beta (TGFβ), play an important role in promoting metastasis of prostate cancer, thus understanding how TGFβ could induce prostate cancer cell migration may enable us to develop targeted strategies for treatment of advanced metastatic prostate cancer. To more clearly define the mechanism(s) involved in prostate cancer cell migration, we undertook a series of studies utilizing non‐malignant prostate epithelial cells RWPE1 and prostate cancer DU145 and PC3 cells. Our studies show that increased cell migration was observed in prostate cancer cells, which was mediated through epithelial‐to‐mesenchymal transition (EMT). Importantly, addition of Mg2+, but not Ca2+, increased cell migration. Furthermore, TRPM7 expression, which functions as an Mg2+ influx channel, was also increased in prostate cancer cells. Inhibition of TRPM7 currents by 2‐APB, significantly blocked cell migration in both DU145 and PC3 cells. Addition of growth factor TGFβ showed a further increase in cell migration, which was again blocked by the addition of 2‐APB. Importantly, TGFβ addition also significantly increased TRPM7 expression and function, and silencing of TRPM7 negated TGFβ‐induced cell migration along with a decrease in EMT markers showing loss of cell adhesion. Furthermore, resveratrol, which decreases prostate cancer cell migration, inhibited TRPM7 expression and function including TGFβ‐induced cell migration and activation of TRPM7 function. Together, these results suggest that Mg2+ influx via TRPM7 promotes cell migration by inducing EMT in prostate cancer cells and resveratrol negatively modulates TRPM7 function thereby inhibiting prostate cancer metastasis.

Thymoquinone inhibits epithelial-mesenchymal transition in prostate cancer cells by negatively regulating the TGF-β/Smad2/3 signaling pathway.

Thymoquinone, a major ingredient of black seed oil (Nigellasativa), has been shown to exhibit anticancer capacity in various types of cancers. However, there are few studies concerning the correlation between thymoquinone and epithelial-to-mesenchymal transition (EMT) in prostate cancer. In the present study, we firstly found that thymoquinone showed antimetastatic capacity in prostate cancer DU145 and PC3 cells. Additionally, thymoquinone reversed EMT by increasing E-cadherin expression and decreasing vimentin and Slug expression in a concentration-dependent manner. Recent studies have shown that the transforming growth factor-β (TGF-β) signaling pathway may be associated with EMT. Intriguingly, the expression of TGF-β, Smad2 and Smad3 at the mRNA and protein levels was notably reduced upon thymoquinone treatment in prostate cancer DU145 and PC3 cells. Subsequently, we confirmed that thymoquinone repressed metastasis and EMT of prostate cancer through downregulation of the TGF-β/Smad2/3 signaling pathway, which may be partially reversed by TGF-β overexpression. In summary, our findings demonstrated that thymoquinone suppressed the metastatic phenotype and reversed EMT of prostate cancer cells by negatively regulating the TGF-β/Smad2/3 signaling pathway. These findings suggest that thymoquinone is a potential therapeutic agent against prostate cancer which functions by targeting TGF-β.

PEBP4 silencing inhibits hypoxia-induced epithelial-to-mesenchymal transition in prostate cancer cells.

Hypoxia induced epithelial-to-mesenchymal transition (EMT) to facilitate the tumor biology. Phosphatidylethanolamine-binding protein 4 (PEBP4) is a member of the PEBP family and has been reported to be upregulated in various cancer types. The definite function of PEBP4 in regulating the EMT of prostate cancer, however, is still unclear. Here, we examined the functional role of PEBP4 and the underlying molecular mechanisms in hypoxia-induced EMT in prostate cancer cells. Our results showed that PEBP4 mRNA and protein expression was markedly increased in the human prostate cancer tissues and cell lines. Knockdown of PEBP4 significantly inhibited hypoxia-induced migration/invasion and EMT program. Furthermore, knockdown of PEBP4 prevented hypoxia-induced the expression of p-Akt and p-mTOR in prostate cancer cells. Taken together, this study reported here provided evidence that knockdown of PEBP4 inhibited hypoxia-induced EMT in prostate cancer cells. Our study uncovered a novel role for PEBP4 in prostate cancer progression, which might support the potential for PEBP4 targeting in prostate cancer therapy.

Jak2-Stat5a/b Signaling Induces Epithelial-to-Mesenchymal Transition and Stem-Like Cell Properties in Prostate Cancer

Active Stat5a/b predicts early recurrence and disease-specific death in prostate cancer (PC), which both typically are caused by development of metastatic disease. Herein, we demonstrate that Stat5a/b induces epithelial-to-mesenchymal transition (EMT) of PC cells, as shown by Stat5a/b regulation of EMT marker expression (Twist1, E-cadherin, N-cadherin, vimentin, and fibronectin) in PC cell lines, xenograft tumors in vivo, and patient-derived PCs ex vivo using organ explant cultures. Jak2-Stat5a/b signaling induced functional end points of EMT as well, indicated by disruption of epithelial cell monolayers and increased migration and adhesion of PC cells to fibronectin. Knockdown of Twist1 suppressed Jak2-Stat5a/b-induced EMT properties of PC cells, which were rescued by re-introduction of Twist1, indicating that Twist1 mediates Stat5a/b-induced EMT in PC cells. While promoting EMT, Jak2-Stat5a/b signaling induced stem-like properties in PC cells, such as sphere formation and expression of cancer stem cell markers, including BMI1. Mechanistically, both Twist1 and BMI1 were critical for Stat5a/b induction of stem-like features, because genetic knockdown of Twist1 suppressed Stat5a/b-induced BMI1 expression and sphere formation in stem cell culture conditions, which were rescued by re-introduction of BMI1. By using human prolactin knock-in mice, we demonstrate that prolactin-Stat5a/b signaling promoted metastases formation of PC cells in vivo. In conclusion, our data support the concept that Jak2-Stat5a/b signaling promotes metastatic progression of PC by inducing EMT and stem cell properties in PC cells.

Abstract 2873: Identification of a new mechanism of microRNA turnover from miR-106a-363 cluster leading to epithelial-to-mesenchymal transition in prostate cancer

Abstract Background: Prostate cancer (PCa) is the second leading cause of cancer mortality in US. The majority of PCa mortality is due to the recurrent of metastatic castration resistant PCa. The acquisition of epithelial-to-mesenchymal transition (EMT) signifies PCa metastasis. In particular, altered microRNAs (miRNAs) expression is closely associated with PCa progression from prostatic intraepithelial neoplasia to metastatic adenocarcinoma. miRNAs are small noncoding RNAs regulating approximately 60% protein-coding genes by post-transcriptional suppression or translational inhibition. miRNA gene expression at post-transcriptional level becomes more complicated when multiple miRNAs derived from the same cluster generate a polycistronic primary transcript, and each individual miRNA displays different expression profile and functional role. miRNA-363 (miR-363) belongs to the miR-106a-363 cluster containing miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92a-2 and miR-363. Unlike the other five miRNAs which are closely resemble to the oncogenic miR-17-92 cluster in their seed sequence and function, miR-363 has been implicated to play a tumor suppressor role in several types of cancer, indicating a different regulatory mechanism of miR-363 from the miR-106a-363 cluster. Interferon-induced tetratricopeptide repeat 5 (IFIT5) is first characterized as a viral RNA binding protein and has been shown to directly bind to cellular tRNA, which partially shared a structural similarity with precursor miRNAs (pre-miRNAs). However, until now, there is no report indicating the RNA recognition role of IFIT5 in miRNA biogenesis machinery. Results: In the present study, miR-363 was clearly identified as a tumor suppressor miRNA by inhibiting EMT in PCa cells via targeting slug/SNAI2. More importantly, we have demonstrated, for the first time, that IFIT5 is able to recognize a unique structure at the 5′ end of precursor miR-363, which facilitates pre-miR-363 degradation by the recruitment of a 5′-3′exoribonuclease, XRN1. Meanwhile, we have also shown that the significant elevation of IFIT5 is detected in several PCa cells undergone EMT leading to highly metastatic potential. In addition, an inverse correlation between miR-363 and IFIT5 mRNA level was found in human PCa specimens. Conclusion: We unveil IFIT5 complex as a new post-transcriptional regulatory mechanism specific for miR-363 turnover at the precursor stage, which determines the stability and unique functional role of miR-363 distinct from other oncogenic miRNA members in the miR-106a-363 cluster. Overall, this study provides an insight of miRNA biogenesis machinery in cancer metastasis and new strategies of therapeutic intervention of PCa. Citation Format: U-Ging Lo, Rey-Chen Pong, Diane Yang, Jiancheng Zhou, Leah Gandee, Shu-Fen Tseng, Jer-Tsong Hsieh. Identification of a new mechanism of microRNA turnover from miR-106a-363 cluster leading to epithelial-to-mesenchymal transition in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2873. doi:10.1158/1538-7445.AM2015-2873

Inhibitory effects of Berberine on the migratory and invasive abilities of cancer cells

The Epithelial-to-Mesenchymal Transition (EMT) is the complex program that involve in oncogenic pathways regulating in migratory and invasion of cancer cells, angiogenesis, and metastasis. As such, targeting the critical EMT inducers or EMT pathways represents an important therapeutic strategy for preventing or treating cancer cells metastasis. Berberine, an isoquinoline alkaloid traditional herbal medicine with no organ toxicity. The increasing reports indicated that berberine played anti-angiogenesis, anti-invasion, and anti-metastasis roles in different human cancer cells. However, there is still unknown about the inhibitory effect of berberine on invasive behavior and EMT of prostate cancer cells. Our study shows for the first time to indicate important role played by berberine, in repressing the metastatic process and the invasive ability of prostate cancer cells. We showed that the migratory and invasive abilities of prostate cancer cells were inhibited by berberine through inhibition of TGF-β signaling. Among the TGF-β signaling molecules were inhibited by berberine, high BMP7 and NODAL gene expressions of metastatic prostate cancer tissues were associated with shorter survival of prostate cancer patients and provide potential therapeutic interventions.

P21 activated kinase-1 mediates transforming growth factor β1-induced prostate cancer cell epithelial to mesenchymal transition

Transforming growth factor beta (TGFβ) is believed to play a dual role in prostate cancer. Molecular mechanism by which TGFβ1 suppresses early prostate tumor growth and induces epithelial-to-mesenchymal transition (EMT) in advanced stages is not known. We determined if P21-activated kinase1 (Pak1), which mediates cytoskeletal remodeling is necessary for the TGFβ1 induced prostate cancer EMT. Effects of TGFβ1 on control prostate cancer PC3 and DU145 cells and those with IPA 3 and siRNA mediated Pak1 inhibition were tested for prostate tumor xenograft in vivo and EMT in vitro. TGFβ1 inhibited PC3 tumor xenograft growth via activation of P38-MAPK and caspase-3, 9. Long-term stimulation with TGFβ1 induced PC3 and DU145 cell scattering and increased expression of EMT markers such as Snail and N-cadherin through tumor necrosis factor receptor-associated factor-6 (TRAF6)-mediated activation of Rac1/Pak1 pathway. Selective inhibition of Pak1 using IPA 3 or knockdown using siRNA both significantly inhibited TGFβ1-induced prostate cancer cell EMT and expression of mesenchymal markers. Our study demonstrated that TGFβ1 induces apoptosis and EMT in prostate cancer cells via activation of P38-MAPK and Rac1/Pak1 respectively. Our results reveal the potential therapeutic benefits of targeting TGFβ1-Pak1 pathway for advanced-stage prostate cancer.

Tumor-secreted Hsp90 Subverts Polycomb Function to Drive Prostate Tumor Growth and Invasion

Prostate cancer remains the second highest contributor to male cancer-related lethality. The transition of a subset of tumors from indolent to invasive disease is associated with a poor clinical outcome. Activation of the epithelial to mesenchymal transition (EMT) genetic program is a major risk factor for cancer progression. We recently reported that secreted extracellular Hsp90 (eHsp90) initiates EMT in prostate cancer cells, coincident with its enhanced expression in mesenchymal models. Our current work substantially extended these findings in defining a pathway linking eHsp90 signaling to EZH2 function, a methyltransferase of the Polycomb repressor complex. EZH2 is also implicated in EMT activation, and its up-regulation represents one of the most frequent epigenetic alterations during prostate cancer progression. We have now highlighted a novel epigenetic function for eHsp90 via its modulation of EZH2 expression and activity. Mechanistically, eHsp90 initiated sustained activation of MEK/ERK, a signal critical for facilitating EZH2 transcriptional up-regulation and recruitment to the E-cadherin promoter. We further demonstrated that an eHsp90-EZH2 pathway orchestrates an expanded repertoire of EMT-related events including Snail and Twist expression, tumor cell motility, and anoikis resistance. To evaluate the role of eHsp90 in vivo, eHsp90 secretion was stably enforced in a prostate cancer cell line resembling indolent disease. Remarkably, eHsp90 was sufficient to induce tumor growth, suppress E-cadherin, and initiate localized invasion, events that are exquisitely dependent upon EZH2 function. In summary, our findings illuminate a hitherto unknown epigenetic function for eHsp90 and support a model wherein tumor eHsp90 functions as a rheostat for EZH2 expression and activity to orchestrate mesenchymal properties and coincident aggressive behavior.

Abstract 3958: T315, a novel integrin-linked kinase inhibitor, suppresses hypoxia-induced epithelial-to-mesenchymal transition in prostate cancer.

Abstract The epithelial-to-mesenchymal transition (EMT) is an early event in metastasis that involves the loss by epithelial cells of many of their distinctive epithelial characteristics and the acquisition of mesenchymal properties. EMT can be induced in cancer cells by several factors, such as hypoxia and TGF-β1, leading to an aggressive and malignant phenotype. In prostate cancer therapy, EMT can be a major clinical challenge as it can contribute to tumor recurrence, therapy resistance, and metastasis. Recently, the integrin-linked kinase (ILK) has been identified as an important protein involved in the process of EMT by inducing the protein expression and activation of Snail, one of the major EMT markers in various cancer cells. The objective of this study was to evaluate the ability of T315, a novel ILK inhibitor developed in our laboratory, to block hypoxia-induced EMT in prostate cancer cells and to validate the role of ILK in hypoxia-induced EMT. Based on our results, the protein expression level of ILK was induced by hypoxia as well as EMT examined by efficiently decreasing the protein expression level of E-cadherin and increasing those of vimentin, Snail, and Zeb1 in PC-3 cells. This ILK induction was due to transcriptional regulation by HIF1α which also could be induced by positively regulating ILK promoter. To elucidate the mechanism of hypoxia-induced EMT in prostate cancer, we used T315 to show its ability to downregulate PKB/Akt and mTOR activities and decrease HIF1α expression. These results showed that hypoxia-induced EMT is driven by HIF1α/ILK positive loop in prostate cancer. Meanwhile, we also demonstrated that YB-1, a DNA/RNA binding protein, plays as a transcriptional factor to negatively regulate the expression of Foxo3a which has been showed as an EMT-related protein to regulate Snail and E-cadherin expression. Our results showed T315 could increase Foxo3a expression by inhibition of YB-1 protein expression. On the other hand, according to a previous study, GSK3β-mediated Snail phosphorylation altered the nuclear sequestration of Snail and caused Snail to undergo proteasomal degradation. Our results also showed that T315 could cause GSK3β dephosphorylation, increase the phosphorylation status of Snail and promote Snail degradation. More importantly, we performed functional assays to observe the inhibition of hypoxia-induced EMT in prostate cancer. T315 showed its ability to inhibit hypoxia-induced cell motility dose-dependently in migration, invasion and 3D culture assays. In conclusion, these results indicate that the inhibition of ILK can block hypoxia-induced EMT in prostate cancer cells as reflected by changes in molecular markers and cell behavior, and that this inhibition can be achieved by treatment with the novel small molecule agent T315, which may have therapeutic benefits for prostate cancer patients with subsequent metastasis. Citation Format: Chih-Chien Chou, Su-Lin Lee, Samuel K. Kulp, Ching-Shih Chen. T315, a novel integrin-linked kinase inhibitor, suppresses hypoxia-induced epithelial-to-mesenchymal transition in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3958. doi:10.1158/1538-7445.AM2013-3958

Abstract 5551: Role of YB-1, a regulator of epithelial to mesenchymal transition, in prostate cancer: inhibition by a dietary flavonoid fisetin.

Abstract The most common signature of aggressive cancers such as prostate cancer (PCa) involves metastasis. Tumor cell migration is a key step for formation of cancer metastasis and recent data suggest that mammalian target of rapamycin (mTOR) controls the metastatic landscape of PCa by regulating the expression of genes known to be involved in PCa metastasis. Also, current estimates suggest that PI3K/Akt/mTOR signaling is upregulated in 30-50% of PCa patients. We observed that the expression of a set of four signature genes YB-1, MTA1, CD-44 and vimentin, regulated by mTOR and known to be involved in metastasis, is significantly higher in PCa PC-3 and DU145 cells compared to normal prostate epithelial cells. siRNA mediated knockdown of YB-1 in both PC-3 and DU-145 cells resulted in inhibition of EMT as seen by a significant decrease in mesenchymal morphology associated with induction of E-cadherin and inhibition of YB-1. We recently showed that fisetin, a dietary flavonoid, is also a specific inhibitor of the mTOR pathway. We hypothesized that fisetin will inhibit epithelial to mesenchymal transition (EMT) in PCa cells by binding to and inhibiting the YB-1. Using in silico docking studies, we observed that fisetin interacts with the cold shock domain (CSD) of the YB-1 molecule. Fisetin binds to YB-1 protein located on residues from β1 and β4 strands of the CSD and shows consistent binding modes and tight affinity within the amino acid pocket. Calculated free binding energy for these conformations ranged from -11.9845 to -9.6273 kcal/mol suggesting strong binding affinity. Treatment of PCa PC-3 and DU145 cells with fisetin (0-80 μM) resulted in inhibition of cell motility that was associated with inhibition of phosphorylation of mTOR at Ser2481 and Ser2448 and YB-1. Using immuno-fluorescence we found that fisetin treatment induced E-cadherin re-expression and inhibited vimentin expression in both DU-145 and PC-3 cells suggesting a reversal of the EMT. This reversal of EMT phenotype was also associated with induction of occludin and claudin-1 and inhibition of fibronectin, N-cadherin, MTA1 and CD-44 expression. We also observed that fisetin treatment inhibited nuclear expression of repressors of EMT such as Snail, Slug and Twist. Because tumor cell migration is a key step for formation of cancer metastasis, we determined the effect of fisetin on cell migration using PC-3 cells. Fisetin treatment inhibited cell migration at both 40 and 80 μM that was associated with decrease in the expression of MMP2 and MMP9. In summary, we observed that YB-1 expression induces EMT in PCa cells and identified fisetin as a novel agent that binds to it and potentially interferes with its activity. Based on our observations we suggest that fisetin could be developed as a clinically relevant inhibitor of mTOR/YB-1 with therapeutic benefit for PCa metastasis and invasion. Citation Format: Vaqar Mustafa Adhami, Mohammad Imran Khan, Rahul K. Lall, Imtiaz A. Siddiqui, Deeba N. Syed, Mario Sechi, Shah-Jahan M. Dodwad, Hasan Mukhtar. Role of YB-1, a regulator of epithelial to mesenchymal transition, in prostate cancer: inhibition by a dietary flavonoid fisetin. [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 5551. doi:10.1158/1538-7445.AM2013-5551

Abstract 281: Hsp27 is an essential effector of EGF-induced epithelial to mesenchymal transition in prostate cancer.

Abstract Introduction: Prostate cancer (PCa) is the most common cancer and the second leading cause cancer death in North American men. An important determinant for metastasis is epithelial to mesenchymal transition (EMT) which is known to be correlated to castrate resistant prostate cancer (CRCP). Previous report demonstrated the overexpression of Hsp27 and its phosphorylated form in poor diagnosis prostate cancer. In addition, both epidermal growth factor (EGF) and its receptor (EGFR) are up–regulated in PCa during progression to CRPC. However, the role of EGF and EGFR in PCa progression remains unclear. Our objective is to establish that Hsp27 is a central “node” for several signaling pathways activated by EGF and known to modulate EMT. Methods: To assess the role of Hsp27 in EMT, gain and loss of Hsp27 function has been conducted in LNCaP cells with or without EGF stimulation. EMT markers expression has been evaluated at the protein (WB) and mRNA level (qRT–PCR). Furthermore, invasion, migration assays in addition with MMP–9 activity have been done to evaluate the migratory capacity of those cells. Also, to better understand the molecular mechanism involved in EMT induction, transactivation and Chip assays have been performed. The in vitro results have been confirmed with an in vivo study using an Hsp27 antisense oligonucleotide (OGX-427) in a murine model. Results: We found that Heat shock protein 27 (Hsp27) is up–regulated with CRPC progression and drives EMT in PCa cells by decreasing epithelial cell markers, up–regulating mesenchymal markers and enhancing cell migration and MMP–9 activity. Hsp27 knockdown induces sequestration of beta–catenin in the cytoplasm by increasing its binding to GSK–3beta and abrogating EGF induced beta–catenin nuclear translocation. Additionally, by decreasing the nuclear translocation of beta-catenin, Hsp27 knockdown decreases the binding of beta–catenin to slug promoter thereby lessening its transcriptional activity. As Slug is known to be a critical key in EMT induction, we confirmed the role of EGF and Hsp27 in EMT induction and our capacity to abrogate the EGF-induced EMT effect by Hsp27 knockdown. Finally, targeting Hsp27 using antisense oligonucleotide OGX-427 in vivo, significantly reduced tumor cell metastasis in a PCa murine model. Conclusion: This study shows that EGF through Hsp27 induces modifications in gene expression and morphology toward a mesenchymal phenotype. The mechanism involving Hsp27, β-catenin, nuclear β-catenin/TCF and EGF signaling cascades described in the present study contribute to the comprehension of mechanisms driving metastasis in aggressive castration-resistant prostate cancer. This study reveals that Hsp27 is a crucial effector of EGF–dependent and independent EMT and suggests that targeting Hsp27 holds promises for a new strategy in metastatic PCa. Citation Format: Thomas D. Cordonnier, Jennifer L. Bishop, Masaki Shiota, Ario Takeuchi, Ka Mun Nip, Martin Gleave, Amina Zoubeidi. Hsp27 is an essential effector of EGF-induced epithelial to mesenchymal transition in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 281. doi:10.1158/1538-7445.AM2013-281