Tumor Suppressor In Prostate Cancer Research Articles

Abstract 2470: Defining the reciprocal regulation of Abi1 and the androgen receptor in prostate cancer

Abstract Background: Prostate cancer (PCa) is a slowly progressing disease that affects nearly 50% of males over the age of 60. Current therapies for patients diagnosed with advanced PCa include Androgen Deprivation Therapy (ADT) and anti-androgen receptor (Anti-AR) treatment. PCa treatment primarily targets the androgen receptor (AR), a ligand-activated transcription factor, which is a key driver of PCa tumor growth. Our previous research has demonstrated that ABI1, acts as a tumor suppressor in PCa. ABI1 is a scaffold protein and an integral member of the WAVE Regulatory Complex (WRC), a nucleation promoting factor. UCSC database indicates there is an AR binding site within the ABI1 gene, suggesting AR plays a role in the transcriptional regulation of ABI1. In this study, we aim to characterize the ABI1-dependent AR regulation in PCa. Method: We generated an ABI1 KO cell line model in LNCaP cells using CRISPR-Cas9. The efficiency of CRISPR-Cas9 was evaluated by Western Blotting and genomic alterations confirmed by DNA sequencing. In addition, we have generated ABI1 Isoform-specific rescue cell lines in our LNCaP ABI1 KO cell line with a binding mutation in the SH3 domain (ABI1-W485N) and an SH3 domain deleted (Abi1-ΔSH3) ABI1 protein. To understand AR and ABI1 dependent pathways we performed qPCR, co-immunoprecipitation, Western Blotting, ChIP, in vitro fluorescence spectra assay, immunofluorescence, proximity ligation assays. Results: In vitro binding assays indicate the interaction of AR, NTD poly-proline region, with the ABI1 SH3 domain. Expression of ABI1- W485N in our ABI1 CRISPR KO cell line showed decrease binding, while ABI1-ΔSH3 showed no binding to AR in co-IP assays compared to our control. Consequently, we saw decreased AR nuclear localization compared to our ABI1-WT control. Furthermore, decreased nuclear localization in our ABI1-W485N mutant was associated with decreased mRNA expression of hallmark AR target genes, Prostate-Specific-Antigen (KLK3), and TMPRSS2. ABI1 is an AR responsive gene and was confirmed with ChIP assays in an AR overexpression cell line. Further, the stimulation of AR transcriptional activity increased cell-cell adhesion in an ABI1 dependent system. Conclusions: Our preliminary studies demonstrate that AR and ABI1 have a negative feedback pathway. The loss of ABI1 resulted in a decrease of nuclear AR and a subsequent decrease in mRNA expression of AR target genes. AR can also modulate ABI1 expression on a transcriptional level as needed for this pathway to function. Future studies will investigate if anti-AR treatments could lead to the dysregulation of ABI1 and promote EMT through STAT3 activation until AR transcriptional regulation on ABI1 is restored. These findings will allow for novel insights into the mechanisms underlying AR and ABI1 relationship in neoplastic progression. Citation Format: Baylee A. Porter, Alaji Bah, Alfonso Urbanucci, Fan Zhang, Sonia Kung, Ladan Fazli, Martin Gleave, Gennady Bratslavsky, Leszek Kotula. Defining the reciprocal regulation of Abi1 and the androgen receptor in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2470.

MUC15 loss facilitates epithelial-mesenchymal transition and cancer stemness for prostate cancer metastasis through GSK3β/β-catenin signaling

Patients with prostate cancer (PCa) have a high incidence of relapse and metastasis. Unfortunately, the molecular mechanisms underlying these processes have not been fully elucidated. In our study, we demonstrate that MUC15, a member of the mucin family, is a novel tumor suppressor in PCa that modulates epithelial-mesenchymal transition (EMT) and cancer stemness, contributing to PCa metastasis. First, MUC15 expression was found to be decreased in PCa tissues compared with para-carcinoma tissues. Moreover, we observed that MUC15 suppressed cell migration and invasion, both in vitro and in vivo, but had no effect on cell proliferation. Mechanistically, knockdown of MUC15 increased GSK3β phosphorylation and promoted β-catenin nuclear translocation. Therefore, the β-catenin-specific inhibitors XAV939 and PRI-724 rescued EMT in MUC15-deficient cell lines. Taken together, these results indicate that MUC15 is downregulated in PCa tissues and serves as a potential target to prevent PCa metastasis, which can inhibit EMT and cancer stemness via the GSK3β/β-catenin signaling pathway.

CircSLC8A1 Acts as a Tumor Suppressor in Prostate Cancer via Sponging miR-21.

Background There is more and more evidence showed that circRNAs played essentially role in the regulation of various biological processes. The role of circSLC8A1 in prostate cancer (PCa) is yet little known. Methods The CircSLC8A1 expression in human prostate cancer was measured by qRT-PCR. The interplay between the specific circRNA, miRNA, and mRNA was investigated by RT-PCR and luciferase reporter assay. Through transient transfection of siRNA, the impacts of circSLC8A1 on PCa were discussed. Cell cycle evaluation, transwell assay, and CCK-8 assay were employed to determine its biological influences. Results In this study, our data revealed that circSLC8A1 was downregulated in PCa tissues and cells. The reduction of circSLC8A1 resulted in the inhibition of cell proliferation and migration. In mechanism, circSLC8A1 exhibited a direct interaction with miR-21 and displayed as a miRNA sponge to inhibit PCa progression. The functional analysis revealed that the circSLC8A1/miR-21 axis may regulate the cell proliferation, angiogenesis, cell migration, epithelial to mesenchymal transition, MAPK signaling pathway, and chemokine signaling pathway. Conclusions CircSLC8A1 functioned as an inhibitor of neoplasm via modulating the miR-21 and might serve as a prospective target for the treatment of PCa.

The helix-loop-helix transcriptional regulator Id4 is required for terminal differentiation of luminal epithelial cells in the prostate.

Inhibitor of differentiation 4 (Id4), a member of the helix-loop-helix family of transcriptional regulators has emerged as a tumor suppressor in prostate cancer. In this study we investigated the effect of loss of Id4 (Id4-/-) on mouse prostate development. Histological analysis was performed on prostates from 25 days, 3 months and 6 months old Id4-/- mice. Expression of Amacr, Ck8, Ck18, Fkbp51, Fkbp52, androgen receptor, Pten, sca-1 and Nkx3.1 was investigated by immunohistochemistry. Results were compared to the prostates from Nkx3.1-/- mice. Id4-/- mice had smaller prostates with fewer and smaller tubules. Subtle PIN like lesions were observed at 6mo. Decreased Nkx3.1 and Pten and increased stem cell marker sca-1, PIN marker Amacr and basal cell marker p63 was observed at all ages. Persistent Ck8 and Ck18 expression suggested that loss of Id4 results in epithelial commitment but not terminal differentiation in spite of active Ar. Loss of Id4 attenuates normal prostate development and promotes hyperplasia/ dysplasia with PIN like lesions. The results suggest that loss of Id4 maintains stem cell phenotype of “luminal committed basal cells”, identifying a unique prostate developmental pathway regulated by Id4.

MiR-769-5p is associated with prostate cancer recurrence and modulates proliferation and apoptosis of cancer cells.

MicroRNAs (miRs) are relevant in biological processes, including human prostate cancer. In the present study, the role of miR-769-5p and its targets in prostate cancer were explored. Publicly available data on expression of genes, miRs and disease-free survival of patients with prostate cancer were analyzed along with RNAseq of transfected cell lines. miR-769-5p expression was inversely associated with patient survival and in vitro assays indicated that its inhibition reduced the proliferation and increased apoptosis of prostate cancer cells. miR-769-5p was revealed to target Rho GTPase activating protein 10 (ARHGAP10) and increased expression of ARHGAP10 in tumors was determined to be associated with a favorable prognosis regarding disease-free survival. Of note, ARHGAP10 is a purported tumor suppressor in ovarian cancer, where it inhibits cell division cycle 42 (CDC42) activity and increases apoptosis. Similar effects were observed in prostate cancer cells, where miR-769-5p inhibition increased ARHGAP10 and led to reduced CDC42 activity. Furthermore, miR-769-5p inhibition increased apoptosis, which was partly reversed by additional knockdown of ARHGAP10. These results suggested that miR-769-5p is an oncogene targeting ARHGAP10, which in turn is a candidate tumor suppressor in prostate cancer.

MiR-30d-5p suppresses proliferation and autophagy by targeting ATG5 in renal cell carcinoma.

Previous reports have shown that miR-30d-5p functions as a tumor suppressor in prostate cancer and gallbladder carcinoma, but its role in renal cell carcinoma (RCC) remains elusive. This study was designed to explore the functional role of miR-30d-5p in proliferation and autophagy of RCC. Our results show that miR-30d-5p is significantly down-regulated in RCC tissues compared with normal tissues. miR-30d-5p overexpression suppressed cell proliferation, cell-cycle G1/S transition and autophagy, but promoted apoptosis in RCC cell lines (786-O and ACHN). Intriguingly, autophagy-related gene 5 (ATG5) was directly targeted by miR-30d-5p, as shown using luciferase reporter assay and biotin-avidin pull-down assay. Moreover, overexpression of ATG5 attenuated the inhibitory effect of miR-30d-5p on proliferation and autophagy in 786-O cells. These results suggest that miR-30d-5p suppresses proliferation and autophagy in RCC cells by targeting ATG5, and this pathway may be a suitable basis for the design of novel cancer therapeutics.

Abstract 3430: The protein phosphatase 4 complex is a tumor suppressor in prostate cancer

Abstract Regions of recurring chromosomal deletions have been annotated in clinical prostate cancer (PCa), but for many of them there is an incomplete understanding of their contributions to disease progression. From an in vivo shRNA enrichment screen of the PCa deletome, we identified the serine threonine protein phosphatase 4 (PP4) regulatory subunit 2 (PPP4R2) as a candidate tumor suppressor, which undergoes copy number loss in 16% of primary PCa. Knockdown or CRISPR-Cas9 mediated knockout of PPP4R2 increased the growth rate of PCa xenograft tumors. Along with the catalytic subunit PP4C and an additional regulatory subunit (either PP4R3A or PP4R3B), PP4R2 forms two distinct heterotrimeric phosphatase holoenzymes. PP4R2 was critical for holoenzyme stability, as its deletion resulted in rapid, moderate downregulation of the other PP4 subunit proteins. The increased tumor growth rate and downregulation of PP4C resulting from loss of PP4R2 could be replicated by combined deletion of PPP4R3A and PPP4R3B, or by directly targeting PPP4C itself. Collectively, these findings implicated PP4C as the likely direct mediator of tumor suppression through the PP4 complex. This was confirmed in both PPP4R2 and PPP4R3A/PPP4R3B knockout cells in vivo by restoring PP4C to its normal physiological level through a dox-inducible cDNA, which reversed the accelerated tumor growth rate conferred by the deletion of the regulatory subunits. Phosphatase activity of PP4C was critical, as add-back of a catalytically dead PP4C mutant (D82A) was unable to reverse the growth rate of PPP4C knockout tumors. Interestingly, we found that the androgen receptor (AR), which is the principal therapeutic target for PCa, was co-immunoprecipitated by PP4C and its regulatory subunits. This suggests that AR could be a substrate for PP4C and is potentially present in a hyperphosphorylated and hyperactivated state in PCa that have decreased PP4 phosphatase activity. Our findings highlight a previously unappreciated tumor suppressor role for the PP4 complex in PCa, with the regulatory subunit PP4R2 providing a key function in stabilizing PP4C and thereby preserving its phosphatase activity. Citation Format: Philip A. Watson, Kayla E. Lawrence, Tejasveeta V. Nadkarni, Taslima F. Ishmael, Eugine Lee, Aura C. Agudelo Rivera, Jillian R. Love, John Wongvipat, Ling Cai, Abbas Nazir, Phillip J. Iaquinta, Kenneth Chang, Yupu Liang, Greg J. Hannon, Charles L. Sawyers. The protein phosphatase 4 complex is a tumor suppressor in prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3430.

Thioredoxin interacting protein (TXNIP) acts as a tumor suppressor in human prostate cancer.

Prostate cancer (PCa) is one of the most common malignant tumors in the world. Thioredoxin interacting protein (TXNIP) is downregulated in a variety of human tumors and plays an important role in tumor suppression. However, the expression level and biological functions of TXNIP in PCa have not been identified yet. Therefore, this study aims to investigate the expression and biological functions of TXNIP in PCa. We reported that the expression of TXNIP was significantly decreased in PCa and associated with clinicopathological features. Overexpression of TXNIP could significantly inhibited PC-3 cells proliferation, migration, invasion, and glucose uptake. Additionally, overexpression of TXNIP could remarkably block cell cycle in the G0/G1 phase and promoted cell apoptosis. Furthermore, TXNIP expression correlated inversely with GLUT1 expression in PCa. Taken together, our results for the first time revealed that TXNIP was decreased in PCa. Moreover, TXNIP might act as a tumor suppressor of PCa and correlated with tumor occurrence and development. Our findings cast a new light on better understanding the occurrence and development of PCa and indicated that TXNIP might be favorable for PCa molecular target therapy.

PTPN14 acts as a candidate tumor suppressor in prostate cancer and inhibits cell proliferation and invasion through modulating LATS1/YAP signaling

Protein tyrosine phosphatase, non-receptor type 14 (PTPN14) exerts a profound effect in the progression of multiple malignant tumors. However, whether PTPN14 plays a role in prostate cancer has not been well investigated. Herein, we evaluated the function and potential underlying mechanism of PTPN14 in prostate cancer. Decreased PTPN14 expression was detected in prostate cancer, and restoration of PTPN14 expression in prostate cancer cells inhibited the proliferative and invasive potential. Mechanistically, PTPN14 increased the phosphorylation of Yes-associated protein (YAP) by activation of large tumor suppressor 1 (LATS1), an action that resulted in a significant reduction in YAP-mediated transcriptional activity. Inactivation of YAP by its inhibitor markedly abrogated the PTPN14-knockdown-induced promotion effect on prostate cancer cell proliferation and invasion. Notably, PTPN14 up-regulation also exerted a remarkable suppressive impact on tumorigenesis of prostate cancer in vivo. Taken together, the study reveals a tumor-inhibition role of PTPN14 that represses the proliferation and invasion of prostate cancer by down-regulating YAP activation.

The diverse roles of SPOP in prostate cancer and kidney cancer.

Multiple studies have confirmed that speckle-type pox virus and zinc finger (POZ) protein (SPOP) functions as a substrate adaptor of cullin 3-based E3 ligase and has a crucial role in various cellular processes via specific targeting of proteins for ubiquitination and subsequent proteasomal degradation. Dysregulation of SPOP-mediated proteolysis might be involved in the development and progression of human prostate and kidney cancers. In prostate cancer, SPOP seems to function as a tumour suppressor by targeting several proteins, including androgen receptor (AR), steroid receptor coactivator 3 (SRC3) and BRD4, for degradation, whereas it might function as an oncoprotein in kidney cancer, for example, by targeting phosphatase and tensin homologue (PTEN) for proteasomal degradation. In addition, nuclear SPOP targets AR for degradation and has a role as a tumour suppressor in prostate cancer; however, in kidney cancer, SPOP largely accumulates in the cytoplasm and fails to promote degradation of AR located in the nucleus, resulting in activation of AR-driven pathways and cancer progression. Owing to the context-dependent function of SPOP in human malignancies, further assessment of the molecular mechanisms involving SPOP in prostate and kidney cancers is needed to improve our understanding of its role in the development of these cancer types. Treatments that target SPOP might become therapeutic strategies in these malignancies in the future.

Demethylation of miR-195 suppresses prostate cancer cell proliferation, migration and invasion.

Prostate cancer (PCa) is the most prevalent cancer among men and the second leading cause of tumor‐associated deaths worldwide, with increasing incidence rates over the last 10 years. Recently, miR‐195 was reported to be hypermethylated at its promoter CpG island and down‐regulated in hepatocellular carcinoma. However, the function of miR‐195 and the underlying mechanisms in PCa remain unknown. Here, we report that a significant down‐regulation of microRNA‐195 (miR‐195) in PCa tissues and cell lines was associated with promoter methylation status. Overexpression of miR‐195 significantly suppressed cell proliferation, migration, invasion and epithelial–mesenchymal transition (increased E‐cadherin and decreased N‐cadherin) in PCa cells. We further demonstrated that transfection with a miR‐195 inhibitor reversed the inhibitory effect of the DNA methyltransferase inhibitor 5‐azacytidine on the proliferation, migration and invasion ability of PCa cells. In summary, our findings suggest that miR‐195 may function as a crucial tumor suppressor in PCa.

The miR-28-5p Targetome Discovery Identified SREBF2 as One of the Mediators of the miR-28-5p Tumor Suppressor Activity in Prostate Cancer Cells.

miR-28-5p is downregulated in some tumor tissues in which it has been demonstrated to have tumor suppressor (TS) activity. Here, we demonstrate that miR-28-5p acts as a TS in prostate cancer (PCa) cells affecting cell proliferation/survival, as well as migration and invasion. Using the miRNA pull out assay and next generation sequencing, we collected the complete repertoire of miR-28-5p targets, obtaining a data set (miR-28-5p targetome) of 191 mRNAs. Filtering the targetome with TargetScan 7, PITA and RNA22, we found that 61% of the transcripts had miR-28-5p binding sites. To assign a functional value to the captured transcripts, we grouped the miR-28-5p targets into gene families with annotated function and showed that six transcripts belong to the transcription factor category. Among them we selected SREBF2, a gene with an important role in PCa. We validated miR-28-5p/SREBF2 interaction, demonstrating that SREBF2 inhibition affects almost all the tumor processes altered by miR-28-5p re-expression, suggesting that SREBF2 is an important mediator of miR-28-5p TS activity. Our findings support the identification of the targetome of cancer-related miRNAs as a tool to discover genes and pathways fundamental for tumor development, and potential new targets for anti-tumor therapy.

FOXN3 Inhibits Cell Proliferation though Regulating E2F1 in Prostate Cancer

In this study, it was demonstrated that forkhead box N3 was downregulated in prostate cancer tissues compared to the matched normal adjacent tissues. Prostate cancer patients with high forkhead box N3 expression more frequently had longer survival (P<0.05) than those with low forkhead box N3 expression. Forkhead box N3 inhibited prostate cancer cell proliferation in vitro and in vivo. In addition, forkhead box N3 repressed expression of E2F1, a reported potential oncogene at the messenger ribonucleic acid and protein levels. Taken together, these data delineate an unrecognized function of forkhead box N3 as a tumor suppressor in prostate cancer via repressing the expression of E2F1.

MiR-142-3p functions as an oncogene in prostate cancer by targeting FOXO1.

Prostate cancer (PCa) is a heterogeneous malignancy, and is a primary cause of cancer-related death in males. Forkhead box transcription factor O1 (FOXO1) exerts antitumor effects in various cancers, including PCa. However, the regulatory mechanism of miR-142-3p on FOXO1 expression in human PCa has not been characterized. In this study, we showed that FOXO1 protein levels were downregulated in PCa tissues and cells. Moreover, FOXO1 expression was a predictor of disease-free survival in patients with PCa and was a predictor of prognosis. Increased expression of FOXO1 suppressed cellular proliferation and induced cell cycle arrest at G0/G1 in vitro. However, FOXO1 mRNA and protein levels were inconsistent in human PCa tissues and cell lines. We showed that miR-142-3p levels were negatively correlated with FOXO1 protein levels in PCa. We also showed that miR-142-3p suppressed FOXO1 expression by directly targeting its 3′-untranslated region. Furthermore, suppression of miR-142-3p inhibited cell proliferation and induced cell cycle arrest, and these effects were blocked by FOXO1 knockdown. In vivo experiments showed that miR-142-3p knockout impaired tumor growth. Our results validate that FOXO1 acted as a tumor suppressor in PCa and demonstrated that FOXO1 was regulated by miR-142-3p, and miR-142-3p may be a potential target for treatment of PCa.

LRIG1 is a pleiotropic androgen receptor-regulated feedback tumor suppressor in prostate cancer

LRIG1 has been reported to be a tumor suppressor in gastrointestinal tract and epidermis. However, little is known about the expression, regulation and biological functions of LRIG1 in prostate cancer (PCa). We find that LRIG1 is overexpressed in PCa, but its expression correlates with better patient survival. Functional studies reveal strong tumor-suppressive functions of LRIG1 in both AR+ and AR− xenograft models, and transgenic expression of LRIG1 inhibits tumor development in Hi-Myc and TRAMP models. LRIG1 also inhibits castration-resistant PCa and exhibits therapeutic efficacy in pre-established tumors. We further show that 1) AR directly transactivates LRIG1 through binding to several AR-binding sites in LRIG1 locus, and 2) LRIG1 dampens ERBB expression in a cell type-dependent manner and inhibits ERBB2-driven tumor growth. Collectively, our study indicates that LRIG1 represents a pleiotropic AR-regulated feedback tumor suppressor that functions to restrict oncogenic signaling from AR, Myc, ERBBs, and, likely, other oncogenic drivers.

Abstract B038: Dimethylaminoparthenolide-mediated inhibition of NF-κB decreases resistance to ADT by targeting AR variants in lethal prostate cancer

Abstract Background: Inflammation, mediated by NF-κB, is linked to prostate cancer (PCa) progression and is commonly observed in castrate-resistant disease. We have previously shown that low expression of tristetraprolin (TTP), which modulates the NF-κB pathway and inflammation, is prognostic for lethal PCa after surgery. It has been suggested that NF-κB-driven resistance in PCa cells may be mediated by aberrant androgen receptor (AR) activation and AR splice variant production. Reversal of resistance to androgen deprivation therapy (ADT) may therefore be achieved by utilizing NF-κB inhibitors, such as dimethylaminoparthenolide (DMAPT). Materials and Methods: To examine clinical correlations between TTP/NF-κB and AR-V7, gene-expression profiling data and clinical data was obtained from several publicly available PCa cohorts (TCGA, SU2C, Taylor et. al.). Quantitative immunofluorescence for TTP and PTEN was performed on tissue microarrays from a clinical cohort of patients treated by radical prostatectomy with known time to biochemical recurrence (BCR). In vitro, mouse (MYC-CaP) and human (VCaP-CR, LNCaP-95) PCa cell lines were treated with TNF-α (20 ng/mL) to stimulate NF-κB expression, DMAPT (5-10 μM) to inhibit NF-κB and Enzalutamide (10 μM) in various combinations. Cell lines stably expressing the IκBα super repressor (MUT IκBα) were generated for comparison to DMAPT-mediated inhibition of NF-κB. Expression of AR and AR variants was measured by western blot and qPCR. For in vivo experiments, SCID mice bearing VCaP-CR xenograft tumors were treated with surgical castration, DMAPT (100 mg/kg, oral gavage, QD), surgical castration plus DMAPT or water vehicle control (n = 7-8 per group). Xenograft tumors were measured thrice weekly until they reached an ethical endpoint or after 80 days of treatment. Time-to-progression of tumors was assessed by Kaplan-Meier analysis and molecular differences within tumors analyzed by qPCR and immunohistochemistry (IHC). Results: In multiple clinical PCa cohorts, reduced TTP expression correlated with an increased AR-V7 signature. In a cohort of patients with localized PCa, decreased TTP expression reduced time to BCR and co-loss of TTP and PTEN (a known PCa tumor suppressor) further decreased time to BCR. Activation of NF-κB in PCa cell lines resulted in a significant increase in AR variant expression and minor upregulation of AR. Chemical (DMAPT) and genetic (MUT IκBα) inhibition of NF-κB slowed tumor proliferation and decreased AR variant expression. Treatment of PCa cell lines with Enzalutamide resulted in AR variant upregulation, which was blocked by DMAPT. Castration of mice bearing VCaP-CR tumors significantly increased AR and AR-V7 tumor expression. DMAPT treatment reduced NF-κB and AR-V7 expression in xenograft tumors, but did not significantly alter tumor growth compared to vehicle treatment. The combination of castration and DMAPT slowed median time-to-progression of xenograft tumors compared to vehicle-treated tumors (79 and 36 days respectively, p &amp;lt; 0.001). Combination therapy decreased Ki-67 (IHC), AR-V7 and AR (IHC and qPCR) expression in xenograft tumors. Conclusions: Current treatments for PCa mainly target AR; however, despite initial response, these treatments commonly fail. Our data highlight the relationship between NF-κB and AR variant expression in aggressive PCa and support the rationale for clinical use of DMAPT to treat castrate-resistant tumors in combination with AR antagonists. Citation Format: Katherine L Morel, Anis A Hamid, Leigh Ellis, Christopher J Sweeney. Dimethylaminoparthenolide-mediated inhibition of NF-κB decreases resistance to ADT by targeting AR variants in lethal prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B038. doi:10.1158/1535-7163.TARG-19-B038

RETRACTED: CircLMTK2 acts as a tumor suppressor in prostate cancer via regulating the expression of microRNA-183

Prostate cancer (PCa) is one of the commonest male urinary and reproductive system malignancies with high morbidity and mortality. circLMTK2 was reported as a tumor suppressor, therefore, we attempted to investigate the potential mechanism of circLMTK2 in PCa. qRT-PCR was employed to examine the expressions of circLMTK2 and miR-183. Afterwards, cell transfection was conducted for overexpressing circLMTK2 and miR-183 in LNCaP and PC3 cells, and silencing circLMTK2 in RWPE1 cells. Then, CCK-8 assay, BrdU, transwell assay, flow cytometry and western blot were respectively conducted to examine the variations of cell growth and metastasis, as well as apoptosis. The expressions of key proteins involved in Wnt/β-catenin and PI3K/AKT pathways were further investigated utilizing western blot. circLMTK2 was lowly expressed in tumor tissues. circLMTK2 overexpression suppressed cell proliferation and metastasis, however promoted cell apoptosis in LNCaP and PC3 cells. circLMTK2 knockdown enhanced cell viability, proliferation, migration and invasion, while had no significant influences on apoptosis of RWPE1 cells. Further experiments verified that miR-183 up-regulation counteracted the influences triggered by circLMTK2 overexpression in LNCaP and PC3 cells. Besides, it markedly promoted the viability, proliferation, migration and invasion of LNCaP cells, however had no significant influence on cell apoptosis. Moreover, the inhibitory effects on Wnt/β-catenin and PI3K/AKT pathways evoked by circLMTK2 overexpression were diminished by miR-183 up-regulation in LNCaP and PC3 cells. These outcomes illustrated that circLMTK2 overexpression exerts an anti-tumor effects through down-regulating the expression of miR-183.

Abi1 loss drives prostate tumorigenesis through activation of EMT and non-canonical WNT signaling

BackgroundProstate cancer development involves various mechanisms, which are poorly understood but pointing to epithelial mesenchymal transition (EMT) as the key mechanism in progression to metastatic disease. ABI1, a member of WAVE complex and actin cytoskeleton regulator and adaptor protein, acts as tumor suppressor in prostate cancer but the role of ABI1 in EMT is not clear.MethodsTo investigate the molecular mechanism by which loss of ABI1 contributes to tumor progression, we disrupted the ABI1 gene in the benign prostate epithelial RWPE-1 cell line and determined its phenotype. Levels of ABI1 expression in prostate organoid tumor cell lines was evaluated by Western blotting and RNA sequencing. ABI1 expression and its association with prostate tumor grade was evaluated in a TMA cohort of 505 patients and metastatic cell lines.ResultsLow ABI1 expression is associated with biochemical recurrence, metastasis and death (p = 0.038). Moreover, ABI1 expression was significantly decreased in Gleason pattern 5 vs. pattern 4 (p = 0.0025) and 3 (p = 0.0012), indicating an association between low ABI1 expression and highly invasive prostate tumors. Disruption of ABI1 gene in RWPE-1 cell line resulted in gain of an invasive phenotype, which was characterized by a loss of cell-cell adhesion markers and increased migratory ability of RWPE-1 spheroids. Through RNA sequencing and protein expression analysis, we discovered that ABI1 loss leads to activation of non-canonical WNT signaling and EMT pathways, which are rescued by re-expression of ABI1. Furthermore, an increase in STAT3 phosphorylation upon ABI1 inactivation and the evidence of a high-affinity interaction between the FYN SH2 domain and ABI1 pY421 support a model in which ABI1 acts as a gatekeeper of non-canonical WNT-EMT pathway activation downstream of the FZD2 receptor.ConclusionsABI1 controls prostate tumor progression and epithelial plasticity through regulation of EMT-WNT pathway. Here we discovered that ABI1 inhibits EMT through suppressing FYN-STAT3 activation downstream from non-canonical WNT signaling thus providing a novel mechanism of prostate tumor suppression.

ING5 inhibits cancer aggressiveness by inhibiting Akt and activating p53 in prostate cancer.

Prostate cancer (PCa) is one of the most common types of cancer in men. In several recent studies, chromosomal deletions in the q arm of chromosome 2, where ING5 resides within, have been identified in various cancer types including PCa. In this study, we investigate the role of ING5 as a tumor suppressor in PCa. We examined the expression level of ING5 in tissue samples and cell lines using quantitative real-time polymerase chain reaction and western blot analysis. We tested the in vitro tumor suppressor potential of ING5 in PC3 and LNCaP cells stably overexpressing it using cell viability, colony formation, migration, invasion, and apoptosis assays. We then investigated the effects of ING5 on the Akt and p53 signaling using western blot analysis. We show that ING5 is significantly downregulated in PCa tumor tissue samples and cell lines compared with the corresponding controls. In vitro assays demonstrate that ING5 effectively suppresses proliferative, clonogenic, migratory, and invasive potential and induce apoptosis in PCa cells. ING5 may potentially exert its anti-tumor potential by inhibiting AKT and inducing p53 signaling pathways. Our findings demonstrate that ING5 possesses tumor suppressor roles in vitro, pointing its importance during the prostatic carcinogenesis processes.

Lactoferrin CpG Island Hypermethylation and Decoupling of mRNA and Protein Expression in the Early Stages of Prostate Carcinogenesis

Lactoferrin (LTF) is an iron-binding protein canonically known for its innate and adaptive immune functions. LTF may also act as a tumor suppressor with antiproliferative action. LTF is inactivated genetically or epigenetically in various cancers, and a CpG island spanning the transcriptional start site of LTF is hypermethylated in prostate cancer cell lines. We, therefore, hypothesized that LTF expression is silenced via CpG island hypermethylation in the early stages of prostate tumorigenesis carcinogenesis. Targeted methylation analysis was performed using a combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes, and laser-capture microdissection followed by bisulfite sequencing on DNA isolated from prostate tissue samples, including both primary and metastatic disease. LTF mRNA in situ hybridization and LTF protein immunohistochemistry were also performed. We report that the LTF CpG island is frequently and densely methylated in high-grade prostatic intraepithelial neoplasia, primary prostate carcinoma, and metastases. We further report a decoupling of lactoferrin mRNA and protein expression, including in lesions where LTF mRNA has presumably been silenced via CpG island methylation. We conclude that LTF mRNA expression is silenced in prostate tumorigenesis via hypermethylation, supporting a role for LTF as a prostate cancer tumor suppressor gene. Likewise, the frequency at which the LTF CpG island is methylated across samples suggests it is an important and conserved step in prostate cancer initiation.