Prostate Cancer Cell Migration Research Articles

Systemic Ablation of Camkk2 Impairs Metastatic Colonization and Improves Insulin Sensitivity in TRAMP Mice: Evidence for Cancer Cell-Extrinsic CAMKK2 Functions in Prostate Cancer.

Despite early studies linking calcium-calmodulin protein kinase kinase 2 (CAMKK2) to prostate cancer cell migration and invasion, the role of CAMKK2 in metastasis in vivo remains unclear. Moreover, while CAMKK2 is known to regulate systemic metabolism, whether CAMKK2’s effects on whole-body metabolism would impact prostate cancer progression and/or related comorbidities is not known. Here, we demonstrate that germline ablation of Camkk2 slows, but does not stop, primary prostate tumorigenesis in the TRansgenic Adenocarcinoma Mouse Prostate (TRAMP) genetic mouse model. Consistent with prior epidemiological reports supporting a link between obesity and prostate cancer aggressiveness, TRAMP mice fed a high-fat diet exhibited a pronounced increase in the colonization of lung metastases. We demonstrated that this effect on the metastatic spread was dependent on CAMKK2. Notably, diet-induced lung metastases exhibited a highly aggressive neuroendocrine phenotype. Concurrently, Camkk2 deletion improved insulin sensitivity in the same mice. Histological analyses revealed that cancer cells were smaller in the TRAMP;Camkk2−/− mice compared to TRAMP;Camkk2+/+ controls. Given the differences in circulating insulin levels, a known regulator of cell growth, we hypothesized that systemic CAMKK2 could promote prostate cancer cell growth and disease progression in part through cancer cell-extrinsic mechanisms. Accordingly, host deletion of Camkk2 impaired the growth of syngeneic murine prostate tumors in vivo, confirming nonautonomous roles for CAMKK2 in prostate cancer. Cancer cell size and mTOR signaling was diminished in tumors propagated in Camkk2-null mice. Together, these data indicate that, in addition to cancer cell-intrinsic roles, CAMKK2 mediates prostate cancer progression via tumor-extrinsic mechanisms. Further, we propose that CAMKK2 inhibition may also help combat common metabolic comorbidities in men with advanced prostate cancer.

Autophagy Induced by Muscarinic Acetylcholine Receptor 1 Mediates Migration and Invasion Targeting Atg5 via AMPK/mTOR Pathway in Prostate Cancer.

Increasing numbers of researchers discovered the expression of muscarinic acetylcholine receptor 1 in human cancers, while its function in human prostate cancer is still unclear. Our present study focused on CHRM1 to clarify its role in mediating autophagy in prostate cancer. We used immunohistochemistry, western blotting, and immunofluorescence experiments to observe the expression of muscarinic acetylcholine receptor 1 both in nude mice with subcutaneous tumors and in prostate cancer cells. The autophagy was observed through transmission electron microscopy, western blotting, quantitative real-time PCR, and immunofluorescence. After that, we used lentivirus to establish CHRM1 and Atg5 knockdown models. Then, the migration and invasion abilities after knocking down muscarinic acetylcholine receptor 1 and Atg5 were detected by transwell assays. In addition, the AMPK/mTOR pathway-related targets were detected by western blotting. We found that muscarinic acetylcholine receptor 1 was abundantly expressed both in vitro and in vivo in prostate cancer. The overexpression of muscarinic acetylcholine receptor 1 positively regulated migration and invasion in tumor cells as well as the activation of autophagy. Muscarinic acetylcholine receptor 1 was highly correlated with Atg5 and activated the AMPK/mTOR signaling pathway. Downregulation of Atg5 inhibited cell autophagy in prostate cancer cells and the migration and invasion of prostate cancer cells. Meanwhile, abnormal expressions of AMPK/mTOR pathway-related proteins were found. In conclusion, the present findings indicated that muscarinic acetylcholine receptor 1 is highly expressed in prostate cancer cells and promotes cell invasion and migration of prostate cancer. Autophagy is activated in prostate cancer cells and the activation of muscarinic acetylcholine receptor 1 positively regulates autophagy in prostate cancer cells. Moreover, muscarinic acetylcholine receptor 1 induces autophagy-mediated cell migration and invasion by targeting Atg5 in prostate cancer cells via AMPK/mTOR pathway, which uncovered that regulating muscarinic acetylcholine receptor 1, identified in this study, can be a promising solution for treating prostate cancer.

Environmental microcystin exposure triggers the poor prognosis of prostate cancer: Evidence from case-control, animal, and in vitro studies

Microcystin-leucine-arginine (MC-LR) is positively linked with multiple cancers in humans. However, the association between MC-LR and the risk and prognosis of prostate cancer has not been conducted in epidemiological studies. No reported studies have linked MC-LR exposure to the poor prognosis of prostate cancer by conducting experimental studies. The content of MC-LR was detected in most of the aquatic food in wet markets and supermarkets in Nanjing and posed a health risk for consumers. MC-LR levels in both prostate cancer tissues and serum were significantly higher than controls. The adjusted odds ratio (OR) for prostate cancer risk by serum MC-LR was 1.75 (95%CI: 1.21-2.52) in the whole subjects, and a positive correlation between MC-LR and advanced tumor stage was observed. Survival curve analysis indicated patients with higher MC-LR levels in tissues exhibited poorer overall survival. Human, animal, and cell studies confirmed that MC-LR exposure increases the expression of estrogen receptor-α (ERα) and promotes epithelial-mesenchymal transition (EMT) in prostate cancer. Moreover, MC-LR-induced decreased E-cadherin levels, increased vimentin levels, and increased migratory and invasive capacities of prostate cancer cells were markedly suppressed upon ERα knockdown. MC-LR-induced xenograft tumor growth and lung metastasis in BALB/c nude mice can be effectively alleviated with ERα knockdown. Our data demonstrated that MC-LR upregulated vimentin and downregulated E-cadherin through activating ERα, promoting migration and invasion of prostate cancer cells. Our findings highlight the role of MC-LR in prostate cancer, providing new perspectives to understand MC-LR-induced prostatic toxicity.

CircPDHX promotes prostate cancer cell progression in vitro and tumor growth in vivo via miR-497-5p/ACSL1 axis

BackgroundCircular RNAs (circRNAs) have been proved could regulate many cancers, including prostate cancer (PCa). In this paper, we reconnoitered the roles of circRNA pyruvate dehydrogenase complex component X (circPDHX) in PCa. MethodsThe circPDHX, microRNA (miR)-497-5p and acyl-CoA synthetase long chain family member 1 (ACSL1) contents were detected by quantitative real-time PCR and Western blot analysis. Cell proliferation was measured by cell counting kit-8 assay, 5-Ethynyl-2′-deoxyuridine assay, and colony formation assay. Cell migration was examined by wound healing assay. The apoptosis was detected by flow cytometry assay. The ELISA kits were applied to quantify the fatty acid metabolites. Furthermore, the interplay between miR-497-5p and circPDHX or ACSL1 was detected by dual-luciferase reporter assay and RIP assay. The role of circPDHX in PCa was supplementary substantiated in vivo. ResultsCircPDHX and ACSL1 contents were upregulated, and the miR-497-5p level was downregulated in PCa. CircPDHX deficiency attenuated PCa cell proliferation, migration, and fatty acid metabolites, while intensified cell apoptosis. CircPDHX bound to miR-497-5p to adjust ACSL1. Moreover, miR-497-5p inhibited the PCa progression by regulating ACSL1. In the meantime, circPDHX deficiency repressed PCa tumor growth in vivo. ConclusionCircPDHX stimulated PCa development via miR-497-5p/ACSL1, which presented a new thought for PCa treatment.

CircPDE5A regulates prostate cancer metastasis via controlling WTAP-dependent N6-methyladenisine methylation of EIF3C mRNA

BackgroundCircular RNA (circRNA) is a novel class noncoding RNA (ncRNA) that plays a critical role in various cancers, including prostate cancer (PCa). However, the clinical significance, biological function, and molecular mechanisms of circRNAs in prostate cancer remain to be elucidated.MethodsA circRNA array was performed to identified the differentially expressed circRNAs. circPDE5A was identified as a novel circRNA which downregulated in clinical samples. Functionally, the in vitro and in vivo assays were applied to explore the role of circPDE5A in PCa metastasis. Mechanistically, the interaction between circPDE5A and WTAP was verified using RNA pulldown followed by mass spectrometry, RNA Immunoprecipitation (RIP) assays. m6A methylated RNA immunoprecipitation sequencing (MeRIP-seq) was then used to identified the downstream target of circPDE5A. Chromatin immunoprecipitation assay (ChIP) and dual-luciferase reporter assay were used to identified transcriptional factor which regulated circPDE5A expression.ResultscircPDE5A was identified downregulated in PCa tissues compared to adjacent normal tissue and was negatively correlated with gleason score of PCa patients. circPDE5A inhibits PCa cells migration and invasion both in vitro and in vivo. circPDE5A blocks the WTAP-dependent N6-methyladenisine (m6A) methylation of eukaryotic translation initiation factor 3c (EIF3C) mRNA by forming the circPDE5A-WTAP complex, and finally disrupts the translation of EIF3C. Moreover, the circPDE5A-dependent decrease in EIF3C expression inactivates the MAPK pathway and then restrains PCa progression.ConclusionsOur findings demonstrate that FOXO4-mediated upregulation of circPDE5A controls PCa metastasis via the circPDE5A-WTAP-EIF3C-MAPK signaling pathway and could serve as a potential therapeutic targer for PCa.

ELOVL2 restrains cell proliferation, migration, and invasion of prostate cancer via regulation of the tumor suppressor INPP4B

BackgroundProstate cancer is one of the most common malignancies in men. Members of the elongation of the very-long-chain fatty acid (ELOVL) gene family have been reported to participate in the occurrence and development of various cancers. However, the function of ELOVL gene family members (ELOVLs) in prostate cancer has not been fully elucidated. MethodsThe mRNA expression and prognostic value of ELOVLs in prostate cancer were analyzed using the GEPIA database. The Oncomine database and PrognoScan database were used to further verify the mRNA expression level and prognostic value of ELOVL2 in prostate cancer. RT–qPCR and Western blotting were used to validate the expression levels of ELOVL2 in four prostate cancer cell lines. Immunohistochemistry was performed to detect the ELOVL2 protein expression levels in prostate cancer tissues. Coexpression analysis in the cBioPortal database and enrichment analysis in Kyoto Encyclopedia of Genes and Genomes (KEGG), CCK8, colony formation, and transwell assays were used to identify the functions and mechanisms of ELOVL2. ResultsELOVL2 expression was upregulated in prostate cancer tissues compared with normal tissues. High expression of ELOVL2 indicated a better prognosis in prostate cancer patients. ELOVL2 expression was negatively correlated with Gleason score. Knockdown of ELOVL2 promoted cell proliferation, colony formation, migration, invasion, and the growth of subcutaneous xenografts and activated the PI3K/Akt signaling pathway by downregulating INPP4B, while overexpression of ELOVL2 reversed these effects. In addition, overexpression of INPP4B blocked the promoting effect of sh-ELOVL2 on cell proliferation, colony formation, migration, invasion, and the PI3K/Akt signaling pathway. ConclusionsOur findings suggest that ELOVL2 might be a prognostic biomarker and therapeutic target for prostate cancer.

SPARC Induces E-Cadherin Repression and Enhances Cell Migration through Integrin αvβ3 and the Transcription Factor ZEB1 in Prostate Cancer Cells

Secreted protein acidic and rich in cysteine (SPARC), or osteonectin, is a matricellular protein that modulates interactions between cells and their microenvironment. SPARC is expressed during extracellular matrix remodeling and is abundant in bone marrow and high-grade prostate cancer (PCa). In PCa, SPARC induces changes associated with epithelial–mesenchymal transition (EMT), enhancing migration and invasion and increasing the expression of EMT transcriptional factor Zinc finger E-box-binding homeobox 1 (ZEB1), but not Zinc finger protein SNAI1 (Snail) or Zinc finger protein SNAI2 (Slug). It is unknown whether the SPARC-induced downregulation of E-cadherin in PCa cells depends on ZEB1. Several integrins are mediators of SPARC effects in cancer cells. Because integrin signaling can induce EMT programs, we hypothesize that SPARC induces E-cadherin repression through the activation of integrins and ZEB1. Through stable knockdown and the overexpression of SPARC in PCa cells, we demonstrate that SPARC downregulates E-cadherin and increases vimentin, ZEB1, and integrin β3 expression. Knocking down SPARC in PCa cells decreases the tyrosine-925 phosphorylation of FAK and impairs focal adhesion formation. Blocking integrin αvβ3 and silencing ZEB1 revert both the SPARC-induced downregulation of E-cadherin and cell migration enhancement. We conclude that SPARC induces E-cadherin repression and enhances PCa cell migration through the integrin αvβ3/ZEB1 signaling pathway.

Long non-coding RNA AC245100.4 contributes to prostate cancer migration via regulating PAR2 and activating p38-MAPK pathway

Prostate cancer (PCa) is the second most common cause of cancer-related mortality in men. Prostate cancer metastasis usually observed at the last stage is the major cause of prostate cancer-related death. Long non-coding RNAs were reported to be involved in tumorigenesis and progression of prostate cancer. This study aimed to investigate the effects and related mechanisms of AC245100.4 in prostate cancer. Knockdown and overexpression of AC245100.4 was used to detect the effect of AC245100.4 on cell migration. qRT-PCR was used to confirm the downstream target of AC245100.4. RAP-MS was used to find pathways related to AC245100.4. Western blot was performed to detect the expression of p-p38 and p38. We found that AC245100.4 promoted the migration of prostate cancer cells via regulating PAR2. The AC245100.4 or PAR2 knockdown resulted in a decrease in Vimentin but an increase in E-cadherin protein levels, while the AC245100.4 or PAR2 overexpression got the opposite results. Moreover, we discovered that AC245100.4 activated the p38-MAPK via regulating PAR2. In brief, these results have suggested that AC245100.4 and PAR2 served as oncogenic factors in cellular migration in PCa cells.

Role of DKK1 in growth and migration of prostate cancer cells

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

The microRNA-3622 family at the 8p21 locus exerts oncogenic effects by regulating the p53-downstream gene network in prostate cancer progression.

For human prostate cancer, the chromosome 8p21 locus, which contains NKX3.1 and the microRNA (miR)-3622 family (miR-3622a/b), is a frequently deleted region. Thus, miR-3622 is proposed as a suppressor for prostate cancer, but its role remains debatable. In the present study, we found that expression of miR-3622a was lower, whereas expression of miR-3622b-3p was higher in human prostate cancer tissues than in normal prostate tissues. miR-3622a-3p inhibited cell migration and invasion of human prostate cancer cells, whereas miR-3622b-3p facilitated cell proliferation, migration, and invasion. To address the opposing roles of miR-3622 family members in various human prostate cancer cell lines, we knocked out (KO) endogenous miR-3622, including both miR-3622a/b. Our results showed that miR-3622 KO reduced cell proliferation, migration, and invasion in vitro and tumor growth and metastasis in vivo. Functional analyses revealed that miR-3622 regulated the p53-downstream gene network, including AIFM2, c-MYC, and p21, to control apoptosis and the cell cycle. Furthermore, using CRISPR interference, miRNA/mRNA immunoprecipitation assays, and dual-luciferase assays, we established that AIFM2, a direct target of miR-3622b-3p, is responsible for miR-3622 KO-induced apoptosis. We identified an miR-3622-AIFM2 axis that contributes to oncogenic function during tumor progression. In addition, miR-3622 KO inhibited the epithelial-mesenchymal transition involved in prostate cancer metastasis via upregulation of vimentin. The results show that miR-3622b-3p is upregulated in human prostate cancers and has an oncogenic function in tumor progression and metastasis via repression of p53 signaling, especially through an miR-3622-AIFM2 axis. In contrast, for human prostate cancer, deletion of the miR-3622 locus at 8p21 reduced the oncogenic effects on tumor progression and metastasis.

Roles for CCN proteins in Lysophosphatidic Acid and Sphingosine 1‐Phosphate Signaling in Prostate Cancer Cells

IntroductionCysteine rich angiogenic factor (CCN1/Cyr61) and connective tissue growth factor (CCN2/CTGF) are members of the CCN family of matricellular proteins. These secreted proteins interact with extracellular matrix proteins and integrins to modulate cell signaling. CCN proteins have been implicated in survival and other mitogenic responses in various cancers. CCN1 and CCN2 are the two early inducible genes in this family. Lysophosphatidic acid (LPA) and sphingosine‐1‐phosphate (S1P) are lipid growth factors known to contribute to tumor progression in prostate cancer. In previous work, our group showed that LPA stimulates proliferation and migration in prostate cancer cells via the G protein‐coupled receptor LPAR1. Free fatty acid receptor 4 (FFAR4) agonists, inhibit LPA‐induced responses in prostate cancer cells. We also demonstrated that CCN1 expression is induced by LPA in human prostate cancer cells. However, the roles of CCN1 and CCN2 in cancer cell signaling have not been fully elucidated.HypothesisWe hypothesize that CCN proteins contribute to LPA‐ and S1P‐induced adhesion and mitogenic signaling in prostate cancer cells.Methods and ResultsPC‐3 human prostate cancer cells were used in the current study. For all experiments, cells were serum starved for 24 hours and then treated with and without 10 µM LPA or S1P. Immunoblotting showed that CCN1 and CCN2 were induced by LPA after 2‐5 hours in PC‐3 cells. The response was inhibited by the LPA receptor antagonist Ki1625. TUG‐891, an FFAR4 agonist, inhibited LPA‐induced CCN1 expression in PC‐3 cells. S1P also induced CCN1 expression in PC‐3 cells. In related experiments, whole cell extracts, and extracellular matrix were subjected to immunoblotting for CCN1, and immunofluorescence microscopy was used to localize the induced CCN1. CCN1 protein levels in extracellular matrix were increased 2‐5 hours after LPA treatment, as compared to untreated controls, and CCN1 was detected outside the cells by confocal microscopy. In cell adhesion assays, LPA‐treated PC‐3 cells exhibited an increase in adhesion to fibronectin‐coated 96‐well plates that was detected by 2 hours. Erk MAPK activation in response to LPA was biphasic, with the later phase occurring after CCN1/2 induction.ConclusionThe results of this study suggest that CCN proteins may play roles in LPA‐ and S1P‐induced signaling in prostate cancer cells and are thus potential therapeutic targets.

Isoflurane Promotes Cell Proliferation, Invasion, and Migration by Regulating BACH1 and miR-375 in Prostate Cancer Cells In Vitro.

The aim of this study was to investigate the mechanism of isoflurane in proliferation, invasion, and migration in prostate cancer (PC) cells in vitro by regulating BACH1 and miR-375. The effect of different concentrations of isoflurane (0%, 0.5%, 1%, and 2%) on PC cell proliferation (PC3 and 22RV1) was measured. After PC cells and normal human prostate stromal immortalized WPMY-1 cells were treated with isoflurane, BACH1 and miR-375 expression was measured. Subsequently, PC3 and 22RV1 cells underwent gain- and loss-of-function assays with or without 4-h 2% isoflurane pretreatment. The levels of miR-375, BACH1, and PTEN were assessed. The binding of BACH1 to miR-375 promoter was detected by ChIP assay. Dual-luciferase reporter assay detected the targeting relationship of miR-375 with BACH1 and PTEN. Isoflurane promoted PC3 and 22RV1 cell proliferation. In addition, isoflurane elevated the levels of BACH1 and miR-375 in a dosage-dependent manner in PC cells. Transfection with miR-375 inhibitor or sh-BACH1 repressed PC cell proliferation, invasion, and migration, while exposure to 2% isoflurane for 4 h before transfection counteracted the inhibitory effects of sh-BACH1 or miR-375 inhibitor on PC cells. PTEN expression was suppressed after 2% isoflurane treatment, but the transfection with miR-375 inhibitor partly abrogated this suppressive effect in PC cells. Moreover, BACH1 bound to miR-375 and miR-375 negatively targeted PTEN. miR-375 mimic could partially reverse the inhibitory effects of sh-BACH1 on the proliferation, invasion, and migration of isoflurane-treated PC cells. Isoflurane facilitated PC cell proliferation, migration, and invasion by activating BACH1 to upregulate miR-375.

TLK1‐MK5 Axis Modulates Actin Filaments and Focal Adhesion Components to Promote PCa Cell Migration and Invasion

BackgroundMetastatic dissemination of prostate cancer (PCa) accounts for majority of PCa related deaths. However, the exact mechanism of PCa cell spread is still unknown. We uncovered a novel interaction between two unrelated promotility factors, tousled‐like kinase 1 (TLK1) and MAPK‐activated protein kinase 5 (MK5), which initiates a signaling cascade promoting metastasis. In PCa, TLK1‐MK5 signaling might be crucial as androgen deprivation therapy (ADT) leads to increased expression of both TLK1 and MK5 in metastatic patients. Moreover, TCGA analysis also revealed higher expression of both TLK1 and MK5 in metastatic tumors. Therefore, we hypothesize that TLK1‐MK5 axis promotes PCa cell migration and invasion through actin reorganization as well as focal adhesion proteins modification and disruption of this interaction may help to keep the tumor localized.Methods and ResultsWe conducted scratch wound repair and proliferation assays to determine if TLK1 and MK5 can regulate motility and proliferation. Both genetic depletion and pharmacologic inhibition of TLK1 and MK5 resulted in reduced migration of both non‐malignant MEF and PCa cells without affecting cellular proliferation rate. Trans‐well invasion assays also demonstrated reduced invasion of MK5−/− MEF cells in Matrigel and other ECMs. However, TLK1 overexpression alone in the MK5−/− MEF cells did not increase the wound healing and invasion which suggested that TLK1 cannot enhance cellular migration and invasion in absence of MK5. We confirmed TLK1 binding of MK5 by co‐IP, His‐ and GST‐pull down assays and established that TLK1 phosphorylates MK5 on three residues (S160, S354, S386) that results in the catalytic activation of MK5. While western blotting (WB) detected substantial amount of pMK5 S354 and TLK1 in all major PCa cell lines, anti‐androgen treatment increases pMK5 S354 level in a dose‐dependent manner and pharmacologic inhibition of TLK1 reduces pMK5 S354 level in LNCaP cells, which suggest TLK1 as an authentic pMK5 S354 kinase. IHC analysis of TRAMP mice and human PCa TMA also revealed higher pMK5 S354 level in metastatic lesions compared to the low‐risk tumors. In addition, pMK5 S354 was found to colocalize with neuroendocrine marker chromogranin A, together of which may suggest pMK5 as a potential biomarker of PCa aggressiveness. The motility reduction of PCa cells upon MK5 inhibition is a consequence of impaired actin filamentation as we observed in LNCaP and PC3 cells. Our WB analysis demonstrated that MK5 inhibition by a repurposed inhibitor results in decreased FAK Y861, Paxillin Y118, HSP27 S78/82 phosphorylation, and ERK3 stabilization, which could lead to altered actin filamentation, focal adhesions, morphology, as well as motility and invasion.ConclusionOur data support that TLK1‐MK5 axis is functionally involved in driving PCa cell metastasis and clinical aggressiveness, hence, disruption of this axis may inhibit the metastatic capacity of PCa.

Downregulation of miR-485-3p promotes proliferation, migration and invasion in prostate cancer through activation of TGF-β signaling.

Prostate cancer (PC) is the second leading cause of cancer-related death among men worldwide. Downregulation of miR-485-3p has been revealed to participate in the tumorigenesis and progression of many types of cancer. However, the clinical and biological role of miR-485-3p in PC remains largely unknown. The expression of miR-485-3p was analyzed in the published databases and detected in our clinical samples and cell lines by RT-qPCR assay. CCK8, transwell invasion and migration, and colony formation assays were performed to investigate the biological function of miR-485-3p. Bioinformatical analysis, RIP, western blotting and luciferase reporter assays were carried out to explore the downstream mechanism of miR-485-3p. The level of miR-485-3p was downregulated in PC tissues, particularly in primary PC tissues with metastasis relative to normal prostate tissues. miR-485-3p downregulation was positively correlated with poor disease-free and overall survival in patients with PC. Functionally, miR-485-3p overexpression dramatically suppressed the proliferation, migration and invasion ability of PC cells in vitro. Mechanistically, miR-485-3p overexpression suppressed the activity of TGF-β signaling by targeting TGFBR2 to play tumor-suppressive roles in PC progression. Our study reports the miR-485-3p/TGFBR2/ TGF-β signaling axis in tumor development of PC, suggesting miR-485-3p may be a potential target to develop therapeutic strategies against PC.

PD07-02 TARGETING WNT5A/FZD2 SIGNALING OVERCOMES RESISTANCE TO ENZALUTAMIDE IN ADVANCED PROSTATE CANCER

You have accessJournal of UrologyCME1 May 2022PD07-02 TARGETING WNT5A/FZD2 SIGNALING OVERCOMES RESISTANCE TO ENZALUTAMIDE IN ADVANCED PROSTATE CANCER Shu Ning, Chengfei Liu, Wei Lou, Alan Lombard, Leandro D'Abronzo, Aiming Yu, Christopher Evans, and Allen Gao Shu NingShu Ning More articles by this author , Chengfei LiuChengfei Liu More articles by this author , Wei LouWei Lou More articles by this author , Alan LombardAlan Lombard More articles by this author , Leandro D'AbronzoLeandro D'Abronzo More articles by this author , Aiming YuAiming Yu More articles by this author , Christopher EvansChristopher Evans More articles by this author , and Allen GaoAllen Gao More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002526.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Androgen receptor (AR) blockade using antiandrogens is a mainstay for the treatment of castration resistant prostate cancer (CRPC). Unfortunately, drug resistance occurs frequently due to mechanisms that are not completely understood. Wnt5a, a representative ligand of non-canonical Wnt signaling, is expressed in circulating tumor cells from CRPC patients treated with enzalutamide. FZD2, the cognate frizzled receptor for Wnt5a, is the most commonly co-upregulated non-canonical Wnt receptor in prostate cancer. Here we determine the contribution of non-canonical Wnt5a/FZD2 to enzalutamide treatment resistance, and explore the potential of targeting Wnt5a/FZD2 to overcome antiandrogen resistance in castration resistant prostate cancer. METHODS: Wnt5a/FZD2 expression was examined in enzalutamide resistant C4-2B MDVR cells. Wnt5a and FZD2 expression were modulated using specific siRNAs. Cell growth, colony formation, migration and invasion were determined in vitro. RNA sequencing was analyzed on C4-2B MDVR cells with WNT5a/FZD2 knocked down; gene expression of non-canonical Wnt signaling, AR and AR-V7 signature genes were analyzed. A novel RNA bioengineered Wnt5a siRNA (tRNA-siWnt5A) was developed to target Wnt5a/FZD2 signaling. The effect of tRNA-siWnt5a on tumor growth and sensitivity to enzalutamide was evaluated in vitro and in vivo. RESULTS: Wnt5a and FZD2 are highly co-upregulated in CRPC patients and enzalutamide resistant C4-2B MDVR cells compared to parental C4-2B cells. Knocking down Wnt5a and FZD2 abrogates the increase of full-length AR and AR variant expression and diminishes the enrichment of genes involved in the non-canonical Wnt signaling pathway. Blocking Wnt5a and FZD2 using specific siRNAs suppresses prostate cancer cell growth, colony formation, migration and invasion. Wnt5a and FZD2 knockdown with siRNA resensitized C4-2B MDVR cells to enzalutamide treatment. Targeting Wnt5a using the bioengineered tRNA-siWnt5A inhibited the growth of enzalutamide resistant prostate cancer cells and resensitized cells to enzalutamide in vitro, and resistant CRPC LuCaP35CR PDX tumor growth in vivo. CONCLUSIONS: Our studies suggest that Wnt5a/FZD2 confers enzalutamide resistance and prostate cancer survival and proliferation. Targeting the non-canonical Wnt5a/FZD2 pathway suppresses tumors expressing high level of Wnt5a and FZD2, not only overcoming resistance but potentiating anti-tumor effects of enzalutamide in CRPC patients. Source of Funding: CA250082, CA 225836, DOD PC180180 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e98 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shu Ning More articles by this author Chengfei Liu More articles by this author Wei Lou More articles by this author Alan Lombard More articles by this author Leandro D'Abronzo More articles by this author Aiming Yu More articles by this author Christopher Evans More articles by this author Allen Gao More articles by this author Expand All Advertisement PDF DownloadLoading ...

RP1-59D14.5 triggers autophagy and represses tumorigenesis and progression of prostate cancer via activation of the Hippo signaling pathway

Prostate cancer (PCa) is one of the major malignant tumors among men worldwide. Long noncoding RNAs (lncRNAs) have been documented as important modulators in human cancers, including PCa. In our study, we investigated the role and potential mechanism of RP1-59D14.5 in PCa. RP1-59D14.5 expressed at a low level in PCa cells. Gain-of-function assays including colony formation and transwell assays displayed that RP1-59D14.5 overexpression repressed PCa cell proliferation, migration, and invasion. Besides, RP1-59D14.5 up-regulation induced autophagy in PCa cells. Mechanically, luciferase reporter assays and western blot verified that RP1-59D14.5 activated the Hippo pathway in PCa cells. Through RNA-binding protein immunoprecipitation (RIP) and RNA pull-down assays, we validated that RP1-59D14.5 functioned as a competing endogenous RNA (ceRNA) to regulate large tumor suppressor kinase 1/2 (LATS1/2) via targeting miR-147a. Moreover, RP1-59D14.5 recruited HUR to promote casein kinase 1 (CK1) expression. Collectively, RP1-59D14.5 promoted yes-associated protein (YAP) degradation to activate the Hippo pathway in PCa progression via targeting the miR-147a/LATS1/2 axis and recruiting HUR to promote the interaction of CK1 and β-transducin repeat-containing protein (βTrCP). These results implied that RP1-59D14.5 acted as a tumor suppressor in PCa, which might be a target for PCa treatment.

TRPM8-Rap1A Interaction Sites as Critical Determinants for Adhesion and Migration of Prostate and Other Epithelial Cancer Cells.

Simple SummaryProstate cancer is the second leading cause of cancer death among men. Its poor prognosis is mainly due to metastases, and therefore, it is crucial to understand the mechanisms by which cancer cells can spread throughout the body. In this context, among others, we demonstrated the TRPM8 channel to be a major player due to its ability to impair prostate cancer cell motility. The present work elucidates the molecular mechanism through which TRPM8 exerts its inhibitory effect on cell migration. We found that this effect is mediated by a direct interaction between residues E207 and Y240 in the N-terminal TRPM8 tail and residue Y32 in the Switch I region of the Rap1-GDP bound inactive form. Since these results have also been validated in breast and cervical cancer cells, the TRPM8-Rap1 interaction could constitute a therapeutic target against metastatic progression in several types of tumors.Emerging evidence indicates that the TRPM8 channel plays an important role in prostate cancer (PCa) progression, by impairing the motility of these cancer cells. Here, we reveal a novel facet of PCa motility control via direct protein-protein interaction (PPI) of the channel with the small GTPase Rap1A. The functional interaction of the two proteins was assessed by active Rap1 pull-down assays and live-cell imaging experiments. Molecular modeling analysis allowed the identification of four putative residues involved in TRPM8-Rap1A interaction. Point mutations of these sites impaired PPI as shown by GST-pull-down, co-immunoprecipitation, and PLA experiments and revealed their key functional role in the adhesion and migration of PC3 prostate cancer cells. More precisely, TRPM8 inhibits cell migration and adhesion by trapping Rap1A in its GDP-bound inactive form, thus preventing its activation at the plasma membrane. In particular, residues E207 and Y240 in the sequence of TRPM8 and Y32 in that of Rap1A are critical for the interaction between the two proteins not only in PC3 cells but also in cervical (HeLa) and breast (MCF-7) cancer cells. This study deepens our knowledge of the mechanism through which TRPM8 would exert a protective role in cancer progression and provides new insights into the possible use of TRPM8 as a new therapeutic target in cancer treatment.

6-Gingerol suppresses cell viability, migration and invasion via inhibiting EMT, and inducing autophagy and ferroptosis in LPS-stimulated and LPS-unstimulated prostate cancer cells.

6-Gingerol is a bioactive compound isolated from Zingiber officinale. 6-Gingerol has been shown to have anticancer effects in numerous types of cancer cell. The mechanisms underlying the anticancer effect of 6-Gingerol in prostate cancer requires investigation. In the present study, the effect on cell viability of 6-Gingerol on LNCaP, PC3 and DU145 prostate cancer cells were determined using the MTT and colony formation assays. 6-Gingerol significantly inhibited cell migration, adhesion and invasion in LPS-stimulated and LPS-unstimulated prostate cancer cells. Furthermore, these changes were accompanied by alterations in the protein expression levels of epithelial-mesenchymal transition biomarkers, including E-cadherin, N-cadherin, Vimentin and zonula occludens-1. 6-Gingerol also induced autophagy by significantly increasing LC3B-II and Beclin-1 protein expression levels in prostate cancer cells. Combining 6-Gingerol with LY294002, an autophagy inhibitor, significantly increased cell survival in DU145 cells. Furthermore, 6-Gingerol significantly decreased the protein expression levels of glutathione (GSH) peroxidase 4 and nuclear factor erythroid 2-related factor 2 in prostate cancer cells. Reactive oxygen species (ROS) levels were significantly increased but GSH levels were decreased following 6-Gingerol treatment in prostate cancer cells. Co-treatment with the ferroptosis inhibitor, ferrostatin-1, significantly increased cell viability and significantly decreased ROS levels in 6-Gingerol-treated cells. These results suggested that 6-Gingerol may have inhibited prostate cell cancer viability via the regulation of autophagy and ferroptosis. In addition, 6-Gingerol inhibited cell migration, adhesion and invasion via the regulation of EMT-related protein expression levels in LPS-stimulated and LPS-unstimulated prostate cancer cells. In conclusion, 6-Gingerol may induce protective autophagy, autophagic cell death and ferroptosis-mediated cell death in prostate cancer cells. These findings may provide a strategy for the treatment and prevention of prostate cancer.

OCT1-target neural gene PFN2 promotes tumor growth in androgen receptor-negative prostate cancer

Androgen and androgen receptor (AR) targeted therapies are the main treatment for most prostate cancer (PC) patients. Although AR signaling inhibitors are effective, tumors can evade this treatment by transforming to an AR-negative PC via lineage plasticity. OCT1 is a transcription factor interacting with the AR to enhance signaling pathways involved in PC progression, but its role in the emergence of the AR-negative PC is unknown. We performed chromatin immunoprecipitation sequencing (ChIP-seq) in patient-derived castration-resistant AR-negative PC cells to identify genes that are regulated by OCT1. Interestingly, a group of genes associated with neural precursor cell proliferation was significantly enriched. Then, we focused on neural genes STNB1 and PFN2 as OCT1-targets among them. Immunohistochemistry revealed that both STNB1 and PFN2 are highly expressed in human AR-negative PC tissues. Knockdown of SNTB1 and PFN2 by siRNAs significantly inhibited migration of AR-negative PC cells. Notably, knockdown of PFN2 showed a marked inhibitory effect on tumor growth in vivo. Thus, we identified OCT1-target genes in AR-negative PC using a patient-derived model, clinicopathologial analysis and an animal model.

Overexpression of CDCA8 Predicts Poor Prognosis and Promotes Tumor Cell Growth in Prostate Cancer.

Human cell division cycle-related protein 8 (CDCA8) is an essential component of the vertebrate chromosomal passenger complex (CPC). CDCA8 was confirmed to play a role in promoting malignant tumor progression. However, the exact function of CDCA8 in the development and progression of prostate cancer (PCa) remains unclear. In this study, the database GSE69223 was downloaded by the gene expression omnibus (GEO) database, as well as CDCA8 expression differences in multiple tumor tissues and normal tissues were detected by The Cancer Genome Atlas (TCGA), TIMER, Oncomine, and Ualcan databases. Kaplan-Meier and Cox regression methods were used to analyze the correlation between CDCA8 expression and prognosis in PCa. We confirmed the expression of CDCA8 in PCa tissues by HPA. We also analyzed the association of CDCA8 expression with PCa clinical characteristics in the TCGA database. To further understand the role of CDCA8 in PCa, we assessed the effects of CDCA8 on PCa cell growth, proliferation, and migration in vitro studies. As a result, CDCA8 was significantly overexpressed in PCa cells compared with normal prostate cells. High CDCA8 expression predicts poor prognosis in PCa patients, and CDCA8 expression was higher in high-grade PCa. In addition, silencing of CDCA8 significantly inhibited PCa cell proliferation and migration. In summary, CDCA8 promoted the proliferation and migration of PCa cells.