Promotes Prostate Cancer Cell Invasion Research Articles

Integrative analysis of the ST6GALNAC family identifies GATA2-upregulated ST6GALNAC5 as an adverse prognostic biomarker promoting prostate cancer cell invasion

BackgroundST6GALNAC family members function as sialyltransferases and have been implicated in cancer progression. However, their aberrant expression levels, prognostic values and specific roles in metastatic prostate cancer (PCa) remain largely unclear.MethodsTwo independent public datasets (TCGA-PRAD and GSE21032), containing 648 PCa samples in total, were employed to comprehensively examine the mRNA expression changes of ST6GALNAC family members in PCa, as well as their associations with clinicopathological parameters and prognosis. The dysregulation of ST6GALNAC5 was further validated in a mouse PCa model and human PCa samples from our cohort (n = 64) by immunohistochemistry (IHC). Gene Set Enrichment Analysis, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes and drug sensitivity analyses were performed to enrich the biological processes most related to ST6GALNAC5. Sulforhodamine B, transwell, luciferase reporter and chromatin immunoprecipitation (ChIP) assays were used to examine the PCa cell proliferation, invasion and transcriptional regulation, respectively.ResultsSystematical investigation of six ST6GALNAC family members in public datasets revealed that ST6GALNAC5 was the only gene consistently and significantly upregulated in metastatic PCa, and ST6GALNAC5 overexpression was also positively associated with Gleason score and predicted poor prognosis in PCa patients. IHC results showed that (1) ST6GALNAC5 protein expression was increased in prostatic intraepithelial neoplasia and further elevated in PCa from a PbCre;PtenF/F mouse model; (2) overexpressed ST6GALNAC5 protein was confirmed in human PCa samples comparing with benign prostatic hyperplasia samples from our cohort (p < 0.001); (3) ST6GALNAC5 overexpression was significantly correlated with perineural invasion of PCa. Moreover, we first found transcription factor GATA2 positively and directly regulated ST6GALNAC5 expression at transcriptional level. ST6GALNAC5 overexpression could partially reverse GATA2-depletion-induced inhibition of PCa cell invasion. The GATA2-ST6GALNAC5 signature exhibited better prediction on the poor prognosis in PCa patients than GATA2 or ST6GALNAC5 alone.ConclusionsOur results indicated that GATA2-upregulated ST6GALNAC5 might serve as an adverse prognostic biomarker promoting prostate cancer cell invasion.

A prognostic signature consisting of N6-methyladenosine modified mRNAs demonstrates clinical potential in prediction of biochemical recurrence and guidance on precision therapy in prostate cancer

Novel biomarkers are urgently needed to improve the prediction of clinical outcomes and guide personalized treatment for prostate cancer (PCa) patients. However, the role of N6-methyladenosine (m6A) modifications in PCa initiation and progression remains largely elusive. In our study, we collected benign Prostate Hyperplasia (BPH), localized PCa, and metastatic PCa samples from patients and performed methylated RNA immunoprecipitation sequencing (MeRIP-Seq) to map m6A-methylated mRNAs. Furthermore, we developed a prognostic signature based on 239 differentially methylated RNAs and the TCGA-PRAD dataset, which can be used to calculate an m6A-modified mRNA (MMM) score for a PCa patient, validated by independent multi-center cohorts. Our findings revealed that differential m6A modifications were positively correlated with altered expressions of mapped m6A-modified mRNAs. Higher MMM scores were associated with shorter times to biochemical recurrence (BCR) in PCa patients, and the MMM scoring system outperformed three well-established signatures in nine independent validation cohorts, as demonstrated by Kaplan-Meier survival analysis, C-index and ROC. Patients who did not respond to androgen receptor signaling inhibitor (ARSI) therapy and immunotherapy were found to have high MMM scores. Two hub genes, TLE1 and PFKL, were confirmed to have m6A sites through MeRIP-qPCR, and their knockdown promoted PCa cell invasion. Bioinformatics analysis of single-cell databases identified cell types with high transcript abundance levels of these two genes. In summary, our study is the first to perform transcriptome-wide m6A mapping in prostate tissues. The translational potential of a prognostic signature, comprising m6A-methylated mRNAs, in predicting clinical outcomes and therapy responses for PCa patients, is demonstrated.

MP20-10 ENZALUTAMIDE INCREASES PROSTATE CANCER (PCa) CELL INVASION YET DECREASES BLADDER CANCER (BCa) CELL INVASION VIA DIFFERENTIALLY REGULATING THE AR/circRNA-ARC1/miR-125b-2-3p OR MiR-4736/PPARγ/MMP-9 SIGNALS

You have accessJournal of UrologyCME1 Apr 2023MP20-10 ENZALUTAMIDE INCREASES PROSTATE CANCER (PCa) CELL INVASION YET DECREASES BLADDER CANCER (BCa) CELL INVASION VIA DIFFERENTIALLY REGULATING THE AR/circRNA-ARC1/miR-125b-2-3p OR MiR-4736/PPARγ/MMP-9 SIGNALS Gang Deng, Yixi Hu, Yin Sun, Jean Joseph, Shuyuan Yeh, Edward M. Messing, and Chawnshang Chang Gang DengGang Deng More articles by this author , Yixi HuYixi Hu More articles by this author , Yin SunYin Sun More articles by this author , Jean JosephJean Joseph More articles by this author , Shuyuan YehShuyuan Yeh More articles by this author , Edward M. MessingEdward M. Messing More articles by this author , and Chawnshang ChangChawnshang Chang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Androgen-deprivation therapy (ADT) is a standard treatment to prevent and shrink PCa growing. However, some patients undergoing ADT may finally develop to hormone-insensitive PCa. The underlying mechanisms haven't been fully elucidated. Here we found ADT-Enz led to increased PCa cell invasion, yet decreased BCa cell invasion. Our goal is to 1.reveal mechanisms of Enz induced hormone-insensitive PCa and find a potential combination therapy. 2. reveal mechanisms of ADT role in BCa and provide an evidence of applying ADT in BCa. METHODS: The human PCa and BCa cells: C4-2, CWR22Rv1, T24 and TCC-SUP were used. Lentiviral plasmids were used to generate gene engineering tools. qPCR and Western blot were used to detect gene expressions. Invasion assay were applied to determine cancer cell invasion capability. Protein-DNA binding complexes were detected by Chromatin immunoprecipitation (ChIP) assays. PPARGC1B/PDK1 3′UTR regions were cloned into the psiCHECKTM-2 vector to assay the miRNA functions. Preclinical mouse PCa model was used to evaluate the anti-cancer efficacy of Enz and circRNA-ARC1 blockage. RESULTS: Mechanistic dissection revealed that suppressing androgens/AR signals could result in differential alterations of the selective circular RNAs (circRNAs) as a result of differential endogenous AR transcription. A negative autoregulation in PCa, yet a positive autoregulation in BCa, as a result of differential binding of AR to different androgen-response elements (AREs) and a discriminating histone H3K4 methylation. Further mechanistic studies indicated that AR-encoded circRNA-ARC1 might sponge the availability of the miR-125b-2-3p and/or miR-4736 to impact the metastasis-related PPARγ/MMP-9 signals. The preclinical mouse model confirms in vitro cell lines data, showing that Enz treatment could increase PCa metastasis, which can be suppressed after iknockdown of circRNA-ARC1. Together, these results demonstrate that antiandrogen therapy with Enz treatment may differentially lead to promoting PCa cell invasion, yet decreasing BCa cell invasion. CONCLUSIONS: Our study is the first to reveal ADT-Enz treatment can promote PCa Cell Invasion yet decrease BCa cell Invasion via differentially regulating the AR/circRNA-ARC1/miR-125b-2-3p or MiR-4736/PPARγ/MMP-9 Signals. Furthermore, our findings may help in development of novel therapeutic approaches to increase the Enz efficacy with less adverse effects in patients with PCa, and providing an evidence of applying ADT in BCa. Source of Funding: This work was partially supported by URMC Urology Department Research fund and George H. Whipple Professorship Endowment © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e279 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Gang Deng More articles by this author Yixi Hu More articles by this author Yin Sun More articles by this author Jean Joseph More articles by this author Shuyuan Yeh More articles by this author Edward M. Messing More articles by this author Chawnshang Chang More articles by this author Expand All Advertisement PDF downloadLoading ...

Prostate cancer cell-platelet bidirectional signaling promotes calcium mobilization, invasion and apoptotic resistance via distinct receptor-ligand pairs

Platelets play a crucial role in cancer and thrombosis. However, the receptor-ligand repertoire mediating prostate cancer (PCa) cell-platelet interactions and ensuing consequences have not been fully elucidated. Microvilli emanating from the plasma membrane of PCa cell lines (RC77 T/E, MDA PCa 2b) directly contacted individual platelets and platelet aggregates. PCa cell-platelet interactions were associated with calcium mobilization in platelets, and translocation of P-selectin and integrin αIIbβ3 onto the platelet surface. PCa cell-platelet interactions reciprocally promoted PCa cell invasion and apoptotic resistance, and these events were insensitive to androgen receptor blockade by bicalutamide. PCa cells were exceedingly sensitive to activation by platelets in vitro, occurring at a PCa cell:platelet coculture ratio as low as 1:10 (whereas PCa patient blood contains 1:2,000,000 per ml). Conditioned medium from cocultures stimulated PCa cell invasion but not apoptotic resistance nor platelet aggregation. Candidate transmembrane signaling proteins responsible for PCa cell-platelet oncogenic events were identified by RNA-Seq and broadly divided into 4 major categories: (1) integrin-ligand, (2) EPH receptor-ephrin, (3) immune checkpoint receptor-ligand, and (4) miscellaneous receptor-ligand interactions. Based on antibody neutralization and small molecule inhibitor assays, PCa cell-stimulated calcium mobilization in platelets was found to be mediated by a fibronectin1 (FN1)-αIIbβ3 signaling axis. Platelet-stimulated PCa cell invasion was facilitated by a CD55-adhesion G protein coupled receptor E5 (ADGRE5) axis, with contribution from platelet cytokines CCL3L1 and IL32. Platelet-stimulated PCa cell apoptotic resistance relied on ephrin-EPH receptor and lysophosphatidic acid (LPA)-LPA receptor (LPAR) signaling. Of participating signaling partners, FN1 and LPAR3 overexpression was observed in PCa specimens compared to normal prostate, while high expression of CCR1 (CCL3L1 receptor), EPHA1 and LPAR5 in PCa was associated with poor patient survival. These findings emphasize that non-overlapping receptor-ligand pairs participate in oncogenesis and thrombosis, highlighting the complexity of any contemplated clinical intervention strategy.

GPR30 knockdown weakens the capacity of CAF in promoting prostate cancer cell invasion via reducing macrophage infiltration and M2 polarization.

Cancer-associated fibroblasts (CAFs) can promote the development and metastasis of prostate cancerpartly by mediating tumor-associated inflammation. An increasing amount of studies have focused on the functional interactions between CAFs and immune cells in the tumor microenvironment (TME). We previously reported that G protein-coupled receptor 30 (GPR30) was highly expressed in prostate CAFs and plays a crucial rolein prostate stromal cell activation. However, the effect and underlying mechanism of GPR30 expression in prostate CAFs affecting the interaction between CAFs and tumor-associated macrophages (TAMs) need further elucidation. Here, we found that, compared with CAF-shControl, CAF-shGPR30 inhibited macrophagemigration through transwell migration assays, which should be attributed to the decreased expression of C-X-C motif chemokine ligand 12 (CXCL12). In addition, macrophages treated with a culture medium of CAF-shGPR30 exhibited attenuated M2 polarization with downregulated M2-like markers expression. Moreover, macrophages stimulated with a culture medium of CAF-shGPR30 were less efficient in promoting activation of fibroblast cells and invasion of PCa cells. Finally, cocultured CAF-shGPR30 and macrophages suppressed PCa cell invasion compared to cocultured CAF-shControl and macrophages by decreasing interleukin-6 (IL-6) secretion, and this effect could be abrogated with rescue expression of IL-6. Our results pinpoint the function of GPR30 in prostate CAFs on regulating the CAF-TAM interaction in the TME and provide new insights into PCa therapies via regulating TME.

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling.

BackgroundS100A9, which is expressed in prostate cancer, has been reported in association with prostate cancer progression. However, the role of S100A9 in prostate cancer metastasis is largely unknown. The aim of this study was to investigate the effect of S100A9 on prostate cancer cell invasion and the involved mechanisms.Materials and methodsIntegrin β1 expression in PC-3 and DU-145 cells was determined by quantitative real-time polymerase chain reaction (PCR) (qRT-PCR) and Western blot. Cellular invasion was measured by transwell invasion assay. Western blot was used to determine protein expression. Concentrations of S100A9 and fibronectin were analyzed by enzyme-linked immunosorbent assay. The protein interaction was detected by immunoprecipitation. The NF-κB activity was measured by luciferase reporter assay. The DU-145 cells metastasis in vivo was determined in mice xenograft models after S100A9 overexpression.ResultsS100A9 promoted prostate cancer cells invasion, integrin β1 expression and fibronectin secretion. Further investigation evidenced that S100A9 interacted with Toll-like receptor 4 (TLR4) and activated NF-κB, which was responsible for tumor cell invasion, integrin β1 up-regulation and focal adhesion kinase (FAK) phosphorylation. Furthermore, integrin β1 inhibition led to decreased FAK phosphorylation and reduced tumor cell invasion. Overexpression of S100A9 increased xenograft tumor micro-metastases, integrin β1 expression and induced NF-κB and FAK activation in vivo.ConclusionOur study demonstrated that S100A9 promotes prostate cancer cell invasion, and one of the underlying molecular mechanisms is that S100A9 activates integrin β1/FAK through TLR4/NF-κB signaling leading to metastasis of prostate cancer cell.

Long noncoding RNA LUCAT1 promotes migration and invasion of prostate cancer cells by inhibiting KISS1 expression.

Since this article has been suspected of research misconduct and the corresponding authors did not respond to our request to prove originality of data and figures, "Long noncoding RNA LUCAT1 promotes migration and invasion of prostate cancer cells by inhibiting KISS1 expression, by C. Liu, L. Wang, Y.-W. Li, Y.-S. Cui, Y.-Q. Wang, S. Liu, published in Eur Rev Med Pharmacol Sci 2019; 23 (8): 3277-3283-DOI:10.26355/eurrev_201904_17689-PMID: 31081080" has been withdrawn. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/17689.

Wnt Signaling Drives Prostate Cancer Bone Metastatic Tropism and Invasion

Wnt signaling has been implicated as a driver of prostate cancer-related osteoblast differentiation, and previous studies have linked modifications in Wnt function with the induction of tumor metastasis. A unique aspect of prostate cancer bone metastases in mouse models is their relative predilection to the hindlimb (femur) compared to the forelimb (humerus). Comparative gene expression profiling was performed within the humerus and femur from non–tumor-bearing mice to evaluate differences in the microenvironments of these locations. This revealed the relative overexpression of the Wnt signaling inhibitors WIF1 and SOST in the humerus compared to the femur, with increased WNT5A expression in femur bone marrow, suggesting a coordinated upregulation of Wnt signals within the femur compared to the humerus. Conditioned medium (CM) from bone marrow stromal cells (HS-5 cells) was used to mimic the bone marrow microenvironment, which strongly promoted prostate cancer cell invasion (3.3-fold increase in PC3 cells, P < .05; 7-fold increase in LNCaP cells, P < .05). WNT5A shRNA knockdown within the CM-producing HS-5 cells significantly decreased PC3 (56%, P < .05) and LNCaP (60%, P < .05) cell invasion. Similarly, preincubation of CM with WIF1 significantly blocked LNCaP cell invasion (40%, P < .05). shRNA-mediated knockdown of the Wnt receptors FZD4 and FZD8 also strongly inhibited tumor cell invasion (60% inhibition shFZD4, P < .05; 63% shFZD8, P < .05). Furthermore, small molecule inhibition of JNK, which is an important component of the noncanonical Wnt signaling pathway, significantly inhibited CM-mediated tumor invasion. Overall, this study reveals a role for Wnt signaling as a driver of prostate cancer bone metastatic tropism and invasion.

Androgen receptor reverses the oncometabolite R-2-hydroxyglutarate-induced prostate cancer cell invasion via suppressing the circRNA-51217/miRNA-646/TGFβ1/p-Smad2/3 signaling.

IDH1 (Isocitrate dehydrogenase 1) mutation occurring at codon 132 (R132) in prostate cancer (PCa) is considered as a classifier for a subgroup of PCas with accumulation of oncometabolite R-2HG (R-2-hydroxyglutarate). Here we found that adding R-2HG or the mutant IDH1 R132H could promote PCa cell invasion in androgen receptor (AR)-negative PC3 cells or suppressing the AR in AR-positive C4-2cells. Mechanism dissection revealed that R-2HG could increase circRNA-51217 expression to sponge miRNA-646, which might then lead to increase TGFβ1 expression and thus induce TGFβ1/p-Smad2/3 signaling to increase PCa cell invasion. AR can suppress this R-2HG/circRNA-51217/miRNA-646/TGFβ1/p-Smad2/3 signaling-increased PCa cell invasion via repressing TGFβ1 transcription and inhibiting circRNA-51217 expression through regulating ADAR2 expression. Preclinical studies with an in vivo xenograft mouse model also revealed that PCa cells with the IDH1 R132H mutation have more invasive metastasis. This study demonstrates that IDH1 R132H mutation with increased oncometabolite R-2HG in PCa cells may play important roles to increase PCa cell invasion.

MicroRNA‑301a‑3p overexpression promotes cell invasion and proliferation by targeting runt‑related transcription factor 3 in prostate cancer.

MicroRNAs (miRNAs) are known to serve a role in tumorigenic programs. The dysregulated expression of miR‑301a‑3p may affect the progression of various types of human cancer; however, the expression and the role of miR‑301a‑3p in prostate cancer are still unclear. The present study aimed to clarify the role and molecular mechanism of miR‑301a‑3p in prostate cancer. The results demonstrated that the expression of miR‑301a‑3p was significantly upregulated in human prostate cancer tissues and in several prostate cancer cell lines. Invitro overexpression of miR‑301a‑3p notably increased prostate cancer cell proliferation and invasion. Bioinformatics analysis revealed that runt‑related transcription factor 3 (RUNX3) may be a target of miR‑301a‑3p, which was confirmed by Dual‑luciferase reporter assay. Western blot analysis also demonstrated that miR‑301a‑3p regulated the protein expression levels of RUNX3. In addition, the results indicated that miR‑301a‑3p may regulate the Wnt signaling pathway, and rescue experiments indicated that RUNX3 contributed to the effects of miR‑301a‑3p on cell proliferation and invasion through Wnt signaling. In conclusion, these findings suggested that miR‑301a‑3p may promote prostate cancer cell invasion and proliferation by targeting RUNX3, and provided insight into understanding prostate cancer pathogenesis. miR‑301a‑3p may be a potential therapeutic candidate to treat prostate cancer.

Activation of sphingosine kinase by lipopolysaccharide promotes prostate cancer cell invasion and metastasis via SphK1/S1PR4/matriptase.

Gram-negative bacteria have been found to be a major population in prostatitis and prostate cancer (PCa) tissues. Lipopolysaccharide (LPS), a major compound of Gram-negative bacteria, with stimulatory activities in some cancer types, but has not been fully studied in PCa. In this study, we examined the effect of LPS on the invasion of PCa cells. Interestingly, LPS can enhance the invasiveness of PCa, but had no significant effect on PCa cell viability. Using protease inhibitor screening and biochemical analyses, matriptase, a member of the membrane-anchored serine protease family, is found to play a key role in LPS-induced PCa cell invasion. Mechanistically, Toll-like receptor 4 (TLR4, LPS receptor)-sphingosine kinase 1 (SphK1) signaling underlies LPS-induced matriptase activation and PCa cell invasion. Specifically, LPS induced the S225 phosphorylation of SphK1 and the translocation of SphK1 to plasma membrane, leading to the production of sphingosine 1-phosphate (S1P), ERK1/2 and matriptase activation via S1P receptor 4 (S1PR4). This phenomenon is further validated using the patient-derived explant (PDE) model. Indeed, there is a significant correlation among the elevated SphK1 levels, the Gleason grades of PCa specimens, and the poor survival of PCa patients. Taken together, this study demonstrates a potential impact of LPS on PCa progression. Our results provide not only a new finding of the role of bacterial infection in PCa progression but also potential therapeutic target(s) associated with PCa metastasis.

Abstract A081: Activation of sphingosine kinase 1 by lipopolysaccharide promotes prostate cancer cell invasion via matriptase

Abstract A 15-year cohort study indicates that bacterial-induced prostatitis is correlated to prostate cancer (PCa) incidence. Several gram-negative bacteria have been identified in PCa patient specimens and inflammatory cytokines derived from bacterial infection have been involved in PCa tumorigenesis. However, the direct effect of lipopolysaccharide (LPS), a potent inflammatory substance from gram-negative bacteria, on PCa development is not well studied. In this study, we examine the effect of LPS on the growth (MTT assay) and invasion (Transwell assay) of several PCa cells lines including LNCaP, PC3, and DU145. Although LPS does not change the cell growth, our results reveal that LPS can enhance the PCa cell invasiveness and increase pericellular proteolytic activities. Using protease inhibitor screening and proteolytic analyses, we have identified that matriptase (MTX), a member of the membrane-anchored serine protease family, plays a key role in LPS-induced PCa cell invasiveness. The activated levels of MTX can be detected in the cells and the conditioned medium (CM) from LPS-treated cells. Indeed, the elevation of MTX expression is associated with high-grade PCa in archival specimens. Knowing LPS as a ligand for toll-like receptor 4 (TLR4), specific antagonists and shRNA knockdown methodology were used to delineate the mechanism by which TLR4/sphingosine kinase 1 (SphK1) signaling cascade plays a role in LPS-induced PCa cell invasion. The results show that SphK1 was important for MTX activation and PCa cell invasion. Moreover, we found that LPS could induce the activation of SphK1 based on an elevated phosphorylation level and SphK1 translocation to plasma membrane for the production of sphingosine 1-phosphate (S1P). S1P can subsequently induce MTX activation and PCa cell invasion through S1P receptor 4 (S1PR4) in a paracrine or autocrine manner. S1PR4 is further identified as an important receptor for S1P to mediate MAPK signaling. Furthermore, there is a significant correlation between the elevated expression of SphK1 and the Gleason scores of PCa specimens. The phenomenon was further corroborated by the fact that LPS could induce SphK1 activation in patient-derived explant model (PDeX). Taken together, we have unveiled a novel LPS-elicited pathway leading to PCa cell invasion. Our results provide not only the potential impact of bacterial infection on PCa progression but also several therapeutic target(s) against PCa metastasis. Citation Format: Cheng-Fan Lee, Benjamin Chen, Jer-Tsong Hsieh, Ming-Shyue Lee. Activation of sphingosine kinase 1 by lipopolysaccharide promotes prostate cancer cell invasion via matriptase [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A081.

Endothelial Akt1 loss promotes prostate cancer metastasis via \u03b2-catenin-regulated tight-junction protein turnover

BackgroundCancer research, in general, is focused on targeting tumour cells to limit tumour growth. These studies, however, do not account for the specific effects of chemotherapy on tumour endothelium, in turn, affecting metastasis.MethodsWe determined how endothelial deletion of Akt1 promotes prostate cancer cell invasion in vitro and metastasis to the lungs in vivo in endothelial-specific Akt1 knockdown mice.ResultsHere we show that metastatic human PC3 and DU145 prostate cancer cells invade through Akt1-deficient human lung endothelial cell (HLEC) monolayer with higher efficiency compared to control HLEC. Although the endothelial Akt1 loss in mice had no significant effect on RM1 tumour xenograft growth in vivo, it promoted metastasis to the lungs compared to the wild-type mice. Mechanistically, Akt1-deficient endothelial cells exhibited increased phosphorylation and nuclear translocation of phosphorylated β-catenin, and reduced expression of tight-junction proteins claudin-5, ZO-1 and ZO-2. Pharmacological inhibition of β-catenin nuclear translocation using compounds ICG001 and IWR-1 restored HLEC tight-junction integrity and inhibited prostate cancer cell transendothelial migration in vitro and lung metastasis in vivo.ConclusionsHere we show for the first time that endothelial-specific loss of Akt1 promotes cancer metastasis in vivo involving β-catenin pathway.

Differential Expression of Cannabinoid Receptors, CB1R and CB2R, in Prostate Tumor Samples with Perineural Invasion

Perineural invasion (PNI) affects over 20% of patients with prostate cancer who often recur with metastasis primarily to the bones. Despite its association with the patient's poor prognosis and dissemination of various cancers, knowledge of the regulatory mechanisms that drive invasion of prostate cancer cells through and around nerves is limited. Cannabinoid receptors (CBRs) are highly enriched in prostate cancer cells and exogenous application of CBR ligands attenuates the cell's survival, migration and invasion ability. Our objective is to define whether activation of the endocannabinoid system (ECS) associates with PNI and to identify the endogenous regulatory mechanisms through which the ECS promotes prostate cancer cell invasion through nerves. We used immunohistochemistry technique to determine the expression levels of the CB1R and the CB2R in formalin‐fixed paraffin embedded (FFPE) prostate tumor samples with PNI. Preliminary data from our laboratory show that CB1R is highly enriched in FFPE prostate tumor samples with PNI when compared to CB2R. Given the increase of the CB1R in PNI FFPE samples, we used total internal fluorescence reflection microscopy (TIRFm) to elucidate in vivo the trafficking kinetics of CB1Rs in highly invasive versus less invasive prostate cancer cells, PC3 and 22RV1, respectively. We found that PC3 cells display constitutive CB1R trafficking. However, this was not the case for the less invasive prostate cancer cells, 22RV1. Our results suggest that the elevated CB1R levels detected in the FFPE PNI positive samples is not coincidental, but rather an indication of an association of the ECS and PNI. Ongoing studies aim to determine how nerve‐PC tumor interactions encourage PNI by using an in vitro model in which we can modulate the ECS.Support or Funding InformationGrants: RCMI‐NIMHD 8G12MD007600 to JFO.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 2122: Pharmacologic interconversion of EMT to MET for prostate cancer

Abstract Introduction and objection: Dysregulation of transforming growth factor-β (TGF-β) and insulin-like growth factor (IGF) axis has been linked to reactive stroma dynamics in the tumor microenvironment during prostate cancer progression. IGFBP3 induction is initiated by stroma remodeling and represents a potential therapeutic target for advanced prostate cancer. A lead quinazoline-based Doxazosin® derivative, DZ-50, generated in our laboratory (US Patent # 8377948), inhibits prostate tumor growth via inducing anoikis and disrupting focal adhesions. Molecular profiling revealed that the process of epithelial-mesenchymal-transition (EMT) is targeted by DZ-50. In this study, we investigated the effect of DZ-50 on EMT landscape, EMT to mesenchymal-epithelial-transition (MET) conversion, and invasive properties of prostate cancer cells. Methods: Human prostate cancer cells LNCaP, LNCaP overexpressing TGF-β type II receptor (TβRII), and cancer associated fibroblasts (CAFs) derived from human prostate cancer specimens, were used. The antitumor effect of DZ-50 against prostate cancer epithelial cells and CAFs was evaluated using cell viability assays. Effect of the drug on EMT key regulators (including IGFBP3) was determined using RT-PCR and Western blot analysis. Drug-induced phenotypic conversions of EMT were evaluated by confocal microscopy. Impact of TGF-β from the stroma microenvironment or exogenous cytokine, on prostate tumor cell migration and invasion, was assessed in co-cultures with CAFs. The functional contribution of IGFBP3 to EMT-MET interconversion in response to DZ-50 was assessed using siRNA approaches. Results: DZ-50 induced cell death in prostate cancer epithelial cells and CAFs, in a concentration-dependent manner. DZ-50 downregulated IGFBP3 mRNA and protein expression and promoted EMT-MET conversion in both LNCaP and LNCaPTβRII cells. IGFBP3 knockdown in LNCaPTβRII cells led to E-cadherin upregulation and MET induction, implicating IGFBP3 as a potential target of DZ-50 to reverse EMT to MET. Moreover exposure to TGF-β reversed DZ-50-induced MET by upregulating IGFBP3 in LNCaPTβRII cells. Co-cultures of LNCaPTβRII with CAFs promoted prostate cancer cell invasion via TGF-β and IGFBP3, an effect that was inhibited by the drug. Conclusions: Treatment of prostate cancer cells with the novel agent DZ-50 inhibits cell migration and invasion and causes reversal of EMT to MET by regulating IGFBP3. This study integrates IGFBP3 as new signaling effector driving the antitumor action of DZ-50 via targeting the EMT-MET phenotypic landscape in the prostate tumor microenvironment. Ongoing work in pre-clinical models will establish the therapeutic value of this novel compound in advanced metastatic prostate cancer. Citation Format: Zheng Cao, Shahriar Koochekpour, Stephen E. Strup, Natasha Kyprianou. Pharmacologic interconversion of EMT to MET for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2122. doi:10.1158/1538-7445.AM2017-2122

Inhibition of cyclooxygenase-2-mediated matriptase activation contributes to the suppression of prostate cancer cell motility and metastasis.

Chronic inflammation plays an important role in cancer development and progression. Cyclooxygenases-2 (COX-2) is a key enzyme in generating prostaglandins causing inflammation, is often found to be overexpressed in prostate cancer (PCa) and is correlated with PCa cell invasion and metastasis. We aim to investigate the molecular mechanism of how COX-2 promotes PCa cell invasion and metastasis and to evaluate the effect of COX-2 inhibitors in a selected model of PCa progression. Our results showed that the expression of COX-2 and Interleukin 1β (IL-1β) was upregulated in highly invasive PCa cells and was correlated with the activated levels of membrane-anchored serine protease matriptase. The expression levels of COX-2 were increased and were correlated with matriptase levels in PCa specimens. Moreover, results showed that COX-2 overexpression or a COX-2 product Prostaglandin E2 (PGE2) caused an increase in matriptase activation and PCa cell invasion, whereas COX-2 silencing antagonized matriptase activation and cell invasion. In addition, the inhibition of COX-2-mediated matriptase activation by Celebrex and sulindac sulfide suppressed the androgen-independent and COX2-overexpressing PCa PC-3 cell invasion, tumor growth and lung metastasis in an orthotopic xenograft model. Our results indicate that COX-2/matriptase signaling contributes to the invasion, tumor growth and metastasis of COX-2-overexpressing and androgen-independent PCa cells.

MiR-150 promotes the cell invasion of prostate cancer cells by directly regulating the expression of p53.

Therapeutic targeting of p53 has been implicated as a promising strategy for cancer treatment. MiRNAs are emerging as important modulators of invasion, regulation of p53. Resent reports have shown that miR-150 is involved in the growth, invasion and metastasis in numerous tumor types. However, the role of miR-150 in prostate cancer pathology is unclear. In this study, we firstly determined the miR-150 expression levels of prostate cancer cell lines by quantitative real-time PCR (qRT-PCR). The effects of miR-150 on prostate cancer cell proliferation and invasion were evaluated using MTT, colony and transwell assays. The target of miR-150 was identified and confirmed using a luciferase activity assay. The results revealed that miR-150 was significantly upregulated in prostate cancer cells compared with RWPE-1 normal prostate epithelial cells. The ectopic expression of miR-150 significantly promoted prostate cancer cell proliferation, colony formation and invasion. In addition, p53 was confirmed as a downstream target of miR-150 in the prostate cancer cells by western blot and qRT-PCR analysis as well as luciferase activity assays. Together, these findings show that miR-150 promotes prostate cancer cell proliferation and invasion by targeting p53, suggesting that targeting miR-150 may be a potential therapeutic strategy in prostate cancer patients.

BET bromodomain-mediated interaction between ERG and BRD4 promotes prostate cancer cell invasion.

Prostate cancer (PCa) that becomes resistant to hormone castration and next-generation androgen receptor (AR)-targeted therapies, called castration-resistant prostate cancer (CRPC), poses a significant clinical challenge. A better understanding of PCa progression and key molecular mechanisms could bring novel therapies to light. One potential therapeutic target is ERG, a transcription factor aberrantly up-regulated in PCa due to chromosomal rearrangements between androgen-regulated gene TMPRSS2 and ERG. Here we show that the most common PCa-associated truncated ERG T1–E4 (ERGΔ39), encoded by fusion between TMPRSS2 exon 1 and ERG exon 4, binds to bromodomain-1 (BD1) of bromodomain containing protein 4 (BRD4), a member of the bromodomain and extraterminal domain (BET) family. This interaction is partially abrogated by BET inhibitors JQ1 and iBET762. Meta-analysis of published ERG (T1–E4) and BRD4 chromatin immunoprecipitation-sequencing (ChIP-seq) data demonstrates overlap in a substantial portion of their binding sites. Gene expression profile analysis shows some ERG-BRD4 co-target genes are upregulated in CRPC compared to hormone-naïve counterparts. We provide further evidence that ERG-mediated invasion of PCa cells was significantly enhanced by an acetylation-mimicking mutation in ERG that augments the ERG-BRD4 interaction. Our findings reveal that PCa-associated ERG can interact and co-occupy with BRD4 in the genome, and suggest this druggable interaction is critical for ERG-mediated cell invasion and PCa progression.

Abstract B67: Matriptase is involved in COX-2 signaling-induced prostate cancer cell invasion

Abstract Chronic inflammation plays an important role in the early development and progression of cancer. Cyclooxygenases-2 (COX-2) is a key rate-limiting enzyme which regulates of inflammation-producing prostaglandins. Several studies have shown that COX-2 is found with overexpression in human cancers (colon, breast, lung, prostate cancers, etc), and implicated in cancer cell invasion. Moreover, high COX-2 expression has been correlated with prostate cancer metastasis. However, the molecular mechanism that COX-2 promotes prostate cancer cell invasion is still unclear. In this study, we established a PC-3 cell invasion progression model (parental and M2I2 PC-3 cells) and found out that several inflammation-associated proteins, COX-2, p-JNK and IL-1β were up-regulated in M2I2 PC-3 cells. Therefore, we purposed that if COX-2 signaling plays a role in prostate cancer cell invasion and the involvement of matriptase in COX-2 signaling-induced cancer cell invasion. The results showed that COX-2 inhibitors (celebrex and sulindac sulfide) could suppress prostate cancer PC3 cell invasion and inhibited matriptase expression and activation. A COX-2 product PGE2 could induce matriptase activation and prostate cancer cell invasion. COX-2 overexpression also could up-regulate matriptase and enhance the cancer cell invasion, while COX-2 silencing antagonized both matriptase activation and cell invasion. Thus, our results indicate that COX-2 signaling can promote prostate cancer cell invasion, at least in part via up-regulating matriptase. Citation Format: Chun-Jung Ko, Ying-Chieh Lu, Pee-Fang Lai, Pei-Wen Hsiao, Ming-Shyue Lee. Matriptase is involved in COX-2 signaling-induced prostate cancer cell invasion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B67.

AB173. TR4 nuclear receptor increases prostate cancer invasion via decreasing the miR-373-3p expression to alter TGFβR2/p-Smad3 signals

ObjectiveTesticular nuclear receptor 4 (TR4) is a member of the nuclear receptor superfamily, which may play key roles to influence the metabolic diseases and prostate tumorigenesis. The purpose of our study is to elucidate the mechanisms how TR4 influences the prostate cancer (PCa) metastasis.MethodsWe constructed three different PCa cell lines including C4-2, PC3 and CWR22Rv1 with differential stable expression of TR4. RT-PCR and Western blot analysis were used to validate identified downstream genes. To explore the function of genes, we manipulated cells 2D and 3D invasion assays and mice experiment.Resultswe found TR4 could promote PCa cell invasion using two different cell invasion assays. Mechanism dissection revealed that TR4 might enhance PCa cell invasion via modulation of the microRNA-373-3p (miR-373-3p) expression. An interruption approach using miR-373-3p partially reversed TR4-enhanced PCa cell invasion. Furthermore, we found TR4-miR-373-3p might function through modulation of the TGFβR2/p-Smad3 signals to enhance the PCa cell invasion. The in vivo mouse model using orthotopic xenografted CWR22Rv1 cell line transfected with luciferase-reporter also confirmed in vitro cell line studies showing TR4 enhanced PCa metastasis via modulation of miR-373-3p.ConclusionsOur data suggest that TR4 may represent a key player to influence the PCa metastasis and targeting TR4 miR-373-3p→ TGFβR2/p-Smad3 axis using TR4 antagonist or TR4-siRNA or miR-373-3p may become a new potential therapeutic approach to better suppress PCa metastasis.