LNCaP Human Prostate Cancer Research Articles

Pyruvate-lactate exchange and glucose uptake in human prostate cancer cell models. A study in xenografts and suspensions by hyperpolarized [1-13 C]pyruvate MRS and [18 F]FDG-PET.

Reprogramming of energy metabolism in the development of prostate cancer can be exploited for a better diagnosis and treatment of the disease. The goal of this study was to determine whether differences in glucose and pyruvate metabolism of human prostate cancer cells with dissimilar aggressivenesses can be detected using hyperpolarized [1‐13C]pyruvate MRS and [18F]FDG‐PET imaging, and to evaluate whether these measures correlate. For this purpose, we compared murine xenografts of human prostate cancer LNCaP cells with those of more aggressive PC3 cells. [1‐13C]pyruvate was hyperpolarized by dissolution dynamic nuclear polarization (dDNP) and [1‐13C]pyruvate to lactate conversion was followed by 13C MRS. Subsequently [18F]FDG uptake was investigated by static and dynamic PET measurements.Standard uptake values (SUVs) for [18F]FDG were significantly higher for xenografts of PC3 compared with those of LNCaP. However, we did not observe a difference in the average apparent rate constant k pl of 13C label exchange from pyruvate to lactate between the tumor variants. A significant negative correlation was found between SUVs from [18F]FDG PET measurements and k pl values for the xenografts of both tumor types. The k pl rate constant may be influenced by various factors, and studies with a range of prostate cancer cells in suspension suggest that LDH inhibition by pyruvate may be one of these. Our results indicate that glucose and pyruvate metabolism in the prostate cancer cell models differs from that in other tumor models and that [18F]FDG‐PET can serve as a valuable complementary tool in dDNP studies of aggressive prostate cancer with [1‐13C]pyruvate.

Abstract B089: Kaiso as a novel therapeutic target in castration-resistant prostate cancer

Abstract Androgen receptor (AR) is a critical driver in the progression of prostate cancer (PCa). However, the PCa develops to castration resistant prostate cancer (CRPC) in almost 53% of patients after 18-32 month’s therapy. Metastatic CRPC has poor prognosis and mean survival time is fewer than 2 years. Kaiso belongs to a BTB/POZ zinc finger protein family and is known as a transcriptional repressor. Kaiso expression and localization have been reported to correlate with the prognosis and metastatic potential in several human malignancies. Previous works from our lab have demonstrated that transcription factor Kaiso was upregulated in the progression of PCa. Therefore, our objective is to explore the interrelationship between Kaiso and AR, further to clarify Kaiso as a potential therapeutic target in PCa progression and CRPC. To investigate molecular mechanisms underlying how aberrant expression of Kaiso contributes to CRPC, the androgen sensitive human prostate cancer LNCaP cells were treated with 10μM anti-androgen Enzalutamide (MDV3100). Kaiso expression levels were increased as detected by RT-PCR and immunofluorescence. To systematically investigate Kaiso targets in PCa, we performed Kaiso ChIP-Seq assay using prostate cancer cell lines LNCaP, C4-2B and PC3. The Kaiso ChIP-Seq peaks indicate androgen receptor motif enrichment in AR expressing LNCaP and C4-2B cells but not in AR-negative PC3 cells, indicating Kaiso could cooperate with AR as co-regulator and potentially control transcription of a subset of its target genes. The interaction of them was confirmed by co-immunoprecipitation of AR using Kaiso antibody in LNCaP cells. To identify Kaiso target genes that interacted with AR pathway, Kaiso knockdown and overexpression stable cell lines were established in LNCaP cells and C42B cells. RT2 Profiler PCR array of AR pathway were performed using LNCaP scramble (LNCaP-Scr) cells and shKaiso cells. The results showed multiple genes were upregulated and downregulated, such as FOLH1, PTEN, RAC3 and IGF1. More importantly, LNCaP Kaiso overexpression cells were more resistant to MDV3100 treatment compared to resistance observed in LNCaP-Scr cells. There were significant negative correlations between Kaiso expression levels and castration-sensitivity. Our results suggest that Kaiso is a novel AR-interacting protein. Kaiso regulates the genes in AR pathway. Targeting Kaiso presents a potential therapeutic strategy for disrupting AR signaling in CRPC and may prevent CRPC development. Citation Format: Huixian Lin, Honghe Wang, Jason White, Balasubramanyam Karanam, Anghesom Ghebremedhin, Benjaminal Adu-Addai, William Grizzle, Clayton Yates. Kaiso as a novel therapeutic target in castration-resistant prostate cancer [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B089.

Role of class I histone deacetylases in the regulation of maspin expression in prostate cancer

Maspin repression is frequently observed in prostate cancer; however, the molecular mechanism(s) causing the loss is not completely understood. Here, we demonstrate that inhibition of class I histone deacetylases (HDACs) mediates re-expression of maspin which plays an essential role in suppressing proliferation and migration capability in prostate cancer cells. Human prostate cancer LNCaP and DU145 cells treated with HDAC inhibitors, sodium butyrate, and trichostatin A, resulted in maspin re-expression. Interestingly, an exploration into the molecular mechanisms demonstrates that maspin repression in prostate tumor and human prostate cancer cell lines occurs via epigenetic silencing through an increase in HDAC activity/expression, independent of promoter DNA hypermethylation. Furthermore, transcriptional activation of maspin was accompanied with the suppression of HDAC1 and HDAC8 with significant p53 enrichment at the maspin promoter associated with an increase in histone H3/H4 acetylation. Our results provide evidence of maspin induction as a critical epigenetic event altered by class I HDACs in the restoration of balance to delay proliferation and migration ability of prostate cancer cells.

Influence of human adipose stem cells on prostate cancer cell growth.

ABSTRACTIn a novel regenerative cell-based treatment developed by us for the patients with stress urinary incontinence, autologous adipose-derived stem cells (ASCs) are injected into the periurethral region and the external urethral sphincter. Since the candidates for this treatment included prostate cancer patients after radical prostatectomy, we investigated the effects of ASCs on prostate cancer cell proliferation in vitro and in vivo to confirm the feasibility of our therapeutic approach. The LNCaP (human prostate cancer cell line) cells and ASCs were co-cultured, and prostate-specific antigen (PSA) concentration in their culture medium supernatant was measured at 48 and 96 h. The PSA concentration significantly decreased in the coculture medium supernatant as compared to the culture medium with LNCaP cells alone. On the contrary, PSA concentrations in the culture medium of LNCaP cells were not affected by supplementation with ASC culture supernatant. After subcutaneous transplantation of LNCaP cells, with or without ASCs, in immunodeficient mice, tumor growth was compared. The growth of LNCaP xenograft tumor in immunodeficient mice was significantly suppressed by ASC addition. These results indicated that ASCs inhibit prostate cancer cell growth, without no proliferative effect on prostate cancer cells.

Piperlongumine inhibits cell growth and enhances TRAIL-induced apoptosis in prostate cancer cells

Objective: To investigate whether piperlongumine can sensitize prostate cancer cells to tumor necrosis factor-related apoptosis- inducing ligand (TRAIL) and trigger apoptosis in prostate cells. Methods: Human prostate cancer cell lines PC3, LNCaP, and VCaP were cultured with piperlongumine and TRAIL. Then, cell proliferation, migration, caspase activation, apoptotic protein expressions, and death receptor expressions were measured. Results: Piperlongumine inhibited cell proliferation at low doses ( Conclusions: Our findings indicate that piperlongumine can sensitize cancer cells to TRAIL through the upregulation of death receptors and can trigger apoptosis with the downregulation of anti- apoptotic proteins.

Integrated analysis of miRNA landscape and cellular networking pathways in stage-specific prostate cancer.

Dysregulation of miRNAs has been demonstrated in several human malignancies including prostate cancer. Due to tissue limitation and variable disease progression, stage-specific miRNAs changes in prostate cancer is unknown. Using chip-based microarray, we investigated global miRNA expression in human prostate cancer LNCaP, PC3, DU145 and 22Rv1 cells representing early-stage, advanced-stage and castration resistant prostate cancer in comparison with normal prostate epithelial cells. A total of 292 miRNAs were differentially expressed with 125 upregulated and 167 downregulated. These miRNAs were involved in pathways including drug resistance drug-efflux, adipogenesis, epithelial-to-mesenchymal transition, bone metamorphosis, and Th1/Th2 signaling. Regulation of miRNAs were interlinked with upstream regulators such as Argonaut 2 (AGO2), Double-Stranded RNA-Specific Endoribonuclease (DICER1), Sjogren syndrome antigen B (SSB), neurofibromatosis 2 (NF2), and peroxisome proliferator activated receptor alpha (PPARA), activated during stage-specific disease progression. Candidate target genes and pathways dysregulated in stage-specific prostate cancer were identified using CS-miRTar database and confirmed in clinical specimens. Integrative network analysis suggested some genes targeted by miRNAs include miR-17, let7g, miR-146, miR-204, miR-205, miR-221, miR-301 and miR-520 having a major effect on their dysregulation in prostate cancer. MiRNA-microarray analysis further identified miR-130a, miR-181, miR-328, miR146 and miR-200 as a panel of novel miRNAs associated with drug resistance drug-efflux and epithelial-to-mesenchymal transition in prostate cancer. Our findings provide evidence on miRNA dysregulation and its association with key functional components in stage-specific prostate cancer.

A Novel Tanshinone Analog Exerts Anti-Cancer Effects in Prostate Cancer by Inducing Cell Apoptosis, Arresting Cell Cycle at G2 Phase and Blocking Metastatic Ability.

Prostate cancer (PCa), an epithelial malignant tumor, is the second common cause of cancer death among males in western countries. Thus, the development of new strategies is urgently needed. Tanshinones isolated from Salvia miltiorrhiza and its synthetic analogs show various biological activities including anticancer effects. Among them, the tanshinone analog 2-((Glycine methyl ester)methyl)-naphtho (TC7) is the most effective, with better selectivity and lower toxicity. Therefore, in this work, the effect of TC7 against PCa was investigated through assessing the molecular mechanisms regulating the growth, metastasis, and invasion of PCa cells. Human PCa cells, PC3 and LNCAP, were used to evaluate TC7 mechanisms of action in vitro, while male BALB/c nude mice were used for in vivo experiments by subjecting each mouse to a subcutaneous injection of PC3 cells into the right flank to evaluate TC7 effects on tumor volume. Our in vitro results showed that TC7 inhibited cell proliferation by arresting the cell cycle at G2/M through the regulation of cyclin b1, p53, GADD45A, PLK1, and CDC2/cyclin b1. In addition, TC7 induced cell apoptosis by regulating apoptosis-associated genes such as p53, ERK1, BAX, p38, BCL-2, caspase-8, cleaved-caspase-8, PARP1, and the phosphorylation level of ERK1 and p38. Furthermore, it decreased DNA synthesis and inhibited the migration and invasion ability by regulating VEGF-1 and MMP-9 protein expression. Our in vivo evidence supports the conclusion that TC7 could be considered as a potential promising chemotherapeutic candidate in the treatment of PCa.

Biological roles of filamin a in prostate cancer cells.

ABSTRACTObjective This study aims to investigate the association of filamin A with the function and morphology of prostate cancer (PCa) cells, and explore the role of filamin A in the development of PCa, in order to analyze its significance in the evolvement of PCa.Materials and Methods A stably transfected cell line, in which filamin A expression was suppressed by RNA interference, was first established. Then, the effects of the suppression of filamin A gene expression on the biological characteristics of human PCa LNCaP cells were observed through cell morphology, in vitro cell growth curve, soft agar cloning assay, and scratch test.Results A cell line model with a low expression of filamin A was successfully constructed on the basis of LNCaP cells. The morphology of cells transfected with plasmid pSilencer-filamin A was the following: Cells were loosely arranged, had less connection with each other, had fewer tentacles, and presented a fibrous look. The growth rate of LNCap cells was faster than cells transfected with plasmid pSilencer-filamin A (P <0.05). The clones of LNCap cells in the soft agar cloning assay was significantly fewer than that of cells stably transfected with plasmid pSilencer-filamin A (P <0.05). Cells stably transfected with plasmid pSilencer-filamin A presented with a stronger healing and migration ability compared to LNCap cells (healing rate was 32.2% and 12.1%, respectively; P <0.05).Conclusion The expression of the filamin A gene inhibited the malignant development of LNCap cells. Therefore, the filamin A gene may be a tumor suppressor gene.

EGF-activated PI3K/Akt signalling coordinates leucine uptake by regulating LAT3 expression in prostate cancer

BackgroundGrowth factors, such as EGF, activate the PI3K/Akt/mTORC1 signalling pathway, which regulates a distinct program of protein synthesis leading to cell growth. This pathway relies on mTORC1 sensing sufficient levels of intracellular amino acids, such as leucine, which are required for mTORC1 activation. However, it is currently unknown whether there is a direct link between these external growth signals and intracellular amino acid levels. In primary prostate cancer cells, intracellular leucine levels are regulated by L-type amino acid transporter 3 (LAT3/SLC43A1), and we therefore investigated whether LAT3 is regulated by growth factor signalling.MethodsTo investigate how PI3K/Akt signalling regulates leucine transport, prostate cancer cells were treated with different PI3K/Akt inhibitors, or stable knock down of LAT3 by shRNA, followed by analysis of leucine uptake, western blotting, immunofluorescent staining and proximity ligation assay.ResultsInhibition of PI3K/Akt signalling significantly reduced leucine transport in LNCaP and PC-3 human prostate cancer cell lines, while growth factor addition significantly increased leucine uptake. These effects appeared to be mediated by LAT3 transport, as LAT3 knockdown blocked leucine uptake, and was not rescued by growth factor activation or further inhibited by signalling pathway inhibition. We further demonstrated that EGF significantly increased LAT3 protein levels when Akt was phosphorylated, and that Akt and LAT3 co-localised on the plasma membrane in EGF-activated LNCaP cells. These effects were likely due to stabilisation of LAT3 protein levels on the plasma membrane, with EGF treatment preventing ubiquitin-mediated LAT3 degradation.ConclusionGrowth factor-activated PI3K/Akt signalling pathway regulates leucine transport through LAT3 in prostate cancer cell lines. These data support a direct link between growth factor and amino acid uptake, providing a mechanism by which the cells rapidly coordinate amino acid uptake for cell growth.

Synthesis of silver nanoparticles (AgNPs) from leaf extract of Salvia miltiorrhiza and its anticancer potential in human prostate cancer LNCaP cell lines

Biosynthesis of silver nanoparticles (AgNPs) from the medicinal plants has been considered as a remarkable approach of several therapeutic innovations and successful drug delivery. Silver nanoparticles were biosynthesized with Salvia miltiorrhiza, Chinese medicinal herb and assessed for its anticarcinogenic property. Synthesis of AgNPs was characterized by several studies such as UV-absorbance and it shows peak values in the range of 425–445 nm. The sizes of the nanoparticles are confirmed by dynamic light scattering analysis and it shows 100 nm. Furthermore, transmission electron microscopy (TEM) and energy dispersive X-ray analysis (EDX) was to confirm the shape and Ag particles are present in the synthesized materials. FTIR analysis to find out the active biomolecules located in the surface of the synthesized particles. This AgNPs from S. miltiorrhiza inhibits the growth of Bacillus subtillis, Staphylococcus aureus, Escherichia coli, and Klebsiella pneumonia. Furthermore, the anticancer potential of AgNPs is examined in prostate adenocarcinoma (LNCaP) cell lines. In this study, we found the AgNPs effectively induces cytotoxicity, ROS and apoptosis by modulation of intrinsic apoptoic Bcl2, Bclxl, Bax and Caspase 3 protein expressions in LNCap cell lines. Based on the study, synthesis of AgNPs from S. miltiorrhiza shows eco-friendly and it exhibits antimicrobial and anticarcinogenic effects.

Design, Synthesis, and Biological Evaluation of (E)-N'-((1-Chloro-3,4-Dihydronaphthalen-2-yl)Methylene)Benzohydrazide Derivatives as Anti-prostate Cancer Agents.

Prostate Cancer (PCa) is the most frequently diagnosed cancer in men in their late '50s. PCa growth is mainly due to the activation of the androgen receptor by androgens. The treatment for PCa may involve surgery, hormonal therapy, and oral chemotherapeutic drugs. A structural based molecular docking approach revealed the findings of (E)-N'-((1-chloro-3,4-dihydronaphthalen-2-yl)methylene)benzohydrazide derivatives, where the possible binding modes of the compounds with protein (PDB ID: 3V49) are shown. The compounds (6a-k) were synthesized and characterized by using conventional methods. The compounds, 6g, 6j, and 6k were reconfirmed through single crystal X-ray diffraction (XRD). Further, the compounds (6a-k) and standard drug were evaluated against human prostate cancer cell lines, LNCaP and PC-3 and the non-cancerous cell line, 3T3. Among these compounds, 6g and 6j showed higher cytotoxicity, and 6g exhibited dose-dependent activity and reduced cell viability. The mechanism of action was observed through the induced apoptosis and was further confirmed by western blot and ELISA. Molecular dynamics simulation studies were carried out to calculate the interaction and the stability of the protein-ligand complex in motion. ADME properties were predicted for all the tested compounds. These findings may give vital information for further development.

Knockdown of mediator complex subunit 19 suppresses the proliferation in prostate cancer LNCaP cells

Objective To study the role of mediator complex subunit 19 (Med 19) in prostate cancer by analyzing the effects of lentivirus-mediated suppression of Med 19 expression on LNCaP prostate cancer cells in vitro and explore the mechanism of proliferation. Methods The lentivirus vectors containing small interfering RNA (siRNA) targeting Med 19 gene were constructed and transfected to LNCaP cells. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting analysis were used to detect the Med 19 expression in the siRNA group (LNCaP-Med 19-siRNA cells)and the control group (LNCaP-Med 19-scRNA cells) at 72 h after the transfection. The influence of Med 19 on the proliferation of prostate cancer cells were assessed using methyl thiazol tetrazolium (MTT), Soft Agar Assay. Cell cycle was analyzed with flow cytometry assay. Results Med 19 relative mRNA level and Med19 protein expressing in the siRNA group were significantly inhibited (t=-69.1413, P<0.01). Compared with the control group, the siRNA group displayed a significant cell proliferation defect by MTT and the number of colonies (8.61±0.51) was significant decreased (t=-11.948, P<0.01; t=-98.775, P<0.01). The siRNA group displayed an increased proportion of cells (67.29±1.70)% in the G0/G1 phase compared with the control group [(56.23±1.46)%, t=-21.906, P<0.01]. Conclusion Knockdown of Med 19 gene increased the number of human prostate cancer LNCaP cells in G0/G1 phase, resulting in significantly inhibited proliferation and growth of prostate cancer. Key words: Mediator complex subunit 19; Prostate carcinoma; Proliferation

Abstract 5219: Kaiso as a novel therapeutic target in castration-resistant prostate cancer (CRPC)

Abstract Androgen receptor (AR) is a critical driver in the progression of prostate cancer (PCa). Androgen deprivation therapy(ADT) has been a standard treatment of PCa. However, the PCa develops to castration resistant prostate cancer (CRPC) in almost 53% of patients after 18-32 month’s therapy. Metastatic CRPC has poor prognosis and mean survival time is fewer than 2 years. Kaiso belongs to a BTB/POZ zinc finger protein family and is known as a transcriptional repressor. Kaiso expression and localization have been reported to correlate with the prognosis and metastatic potential in several human malignancies. Previous works from our lab have demonstrated that transcription factor Kaiso was upregulated in the progression of PCa. Therefore, our objective is to explore the interrelationship between Kaiso and AR, further to clarify Kaiso as a potential therapeutic target in PCa progression and CRPC. To investigate molecular mechanisms underlying how aberrant expression of Kaiso contributes to CRPC, the androgen sensitive human prostate cancer LNCaP cells were treated with 10µM anti-androgen Enzalutamide (MDV3100). Kaiso expression levels were increased as detected by RT-PCR and immunofluorescence. To systematically investigate Kaiso targets in PCas, we performed Kaiso ChIP-Seq assay using prostate cancer cell lines LNCaP, C4-2B and PC3. The Kaiso ChIP-Seq peaks indicate androgen receptor motif enrichment in AR expressing LNCaP and C4-2B cells but not in AR-negative PC3 cells, indicating Kaiso could cooperate with AR as co-regulator and potentially control transcription of a subset of its target genes. The interaction of them was confirmed by co-immunoprecipitation of AR using Kaiso antibody in LNCaP cells. To identify Kaiso target genes that interacted with AR pathway, Kaiso knockdown and overexpression stable cell lines were established in LNCaP cells and C42B cells. RT2 Profiler PCR array of AR pathway were performed using LNCaP scramble (LNCaP-Scr) cells and shKaiso cells. The results showed multiple genes were upregulated and downregulated, such as FOLH1, PTEN, RAC3 and IGF1. More importantly, LNCaP Kaiso overexpression cells were more resistant to MDV3100 treatment compared to resistance observed in LNCaP-Scr cells. There were significant negative correlations between Kaiso expression levels and castration-sensitivity. Our results suggest that Kaiso is a novel AR-interacting protein. Kaiso regulates the genes in AR pathway. Targeting Kaiso presents a potential therapeutic strategy for disrupting AR signaling in CRPC and may prevent CRPC development. These studies were supported by grants U54MD007585, (NIH/RCMI) [CY], U54CA118623-01 (NIH/NCI) [CY] Citation Format: Hui-Xian Lin, Honghe Wang, Jason White, Balasubramanyam Karanam, Anghesom Ghebremedhin, Benjamin Adu Addai, William E. Grizzle, Clayton Yates. Kaiso as a novel therapeutic target in castration-resistant prostate cancer (CRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5219.

Abstract 2026: Loss of fibroblasts-dependent androgen receptor activation in prostate cancer cells develops castration-resistant prostate cancer

Abstract Well-differentiated prostate cancer (PCa) cells are generally androgen/androgen receptor (AR)-dependent, i.e. AR signaling regulates cell cycle and differentiation. Loss of AR signaling after androgen deprivation therapy (ADT) triggers AR-independent outgrowth, generating poorly differentiated uncontrollable PCa cells. To prevent development and progression of castration-resistant prostate cancer (CRPC), we hypothesize that preservation of AR signaling after ADT is an important target. Tumor stroma surrounding PCa cells is enriched in fibroblasts secreting AR-activating factors, e.g. EGF, IGF1, and IL-6. Thus, we investigated the role of fibroblasts in AR activation of human prostate cancer LNCaP sublines differing in androgen sensitivity under androgen deprivation. Androgen-low-sensitive E9 and F10 cells were obtained from the parental androgen-sensitive LNCaP cell population through use of a limiting dilution method in regular culture condition. In contrast, androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging under hormone-depleted condition. Original fibroblasts pcPrFs (-M5, -M6, -M7) were isolated from needle biopsy samples of patients with PCa. The PCa cells alone or PCa cells plus pcPrF-M5 were grafted beneath the renal capsule of male athymic nude mice, and then mice were castrated on 14 days post transplantation. Tumor volume of E9 cells + pcPrF-M5 became diminished post castration but that of F10 and AIDL cells + pcPrF-M5 was gradually increased even post castration. In E9 cells + pcPrF-M5 tumors, serum PSA became detected on day 21 post castration. Interestingly, serum PSA was gradually increased even in F10 cells inoculated alone. In AIDL tumors with or without pcPrF-M5, serum PSA was not detected because of mutated AR in AIDL cells. Cell growth of E9 and AIDL cells was directly increased by treating with EGF, whereas that in F10 cells was not affected. Phosphorylation of AKT and ERK1/2 in E9 and AIDL cells was also significantly increased by treating with EGF, whereas that in F10 cells was not affected. In the condition of co-cultures with fibroblasts, PSA production was directly increased in E9 cells but not in F10 and AIDL cells. Protein expression of AR splice variant 7 (AR-V7) was only detected in F10 cells. The reduced AR-dependency of PCa cells is an important clinical development because of its association with the cell’ progression to CRPC. Here our results showed that fibroblasts secreting AR-activating factors preserved AR signaling in E9 cells after ADT, indicating that this type of PCa cells can be controlled by ADT. In contrast, we suggest that loss of fibroblasts-dependent AR activation in F10 and AIDL cells may be responsible for development and progression of CRPC. Citation Format: Kenichiro Ishii, Izumi Matsuoka, Takeshi Sasaki, Manabu Kato, Kohei Nishikawa, Hideki Kanda, Yoshifumi Hirokawa, Kazuhiro Iguchi, Kiminobu Arima, Masatoshi Watanabe, Yoshiki Sugimura. Loss of fibroblasts-dependent androgen receptor activation in prostate cancer cells develops castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2026.

Abstract 5084: Epigenetic modifications involving reactivation of RECK inhibiting MMP-9 and MMP-2 in prostate cancer

Abstract Metastasis is responsible for more than 90% of prostate cancer-associated mortality in the United States. One of the distinctive reason for metastasis has been the imbalance of matrix metalloproteinases (MMPs) as a result of reduced expression of RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) a novel tumor suppressor. RECK has been shown to be a potent inhibitor of MMP-2 and MMP-9. Loss of RECK has been frequently identified in clinical prostate cancer specimens and prostate cancer cell lines, therefore mechanistic understanding of its loss and approaches to restore its expression would facilitate inhibition of metastasis. Green tea polyphenols (GTP) and its major constituent, epigallocatechin-3-gallate (EGCG) has been shown to suppress prostate cancer metastasis, however the mechanism has not been fully elucidated. We determined whether treatment with GTP has ability to restore induction of RECK and play a key role in suppressing invasiveness in prostate cancer. Human prostate cancer LNCaP tumor were implanted in the ventral prostate of athymic nude mice for 2 weeks followed by per-oral intake of GTP at 7.5 and 15.0 mg/kg body weight freshly prepared in 100µl PBS. Other groups were treated with DNA methyltransferase inhibitor-5-aza-2'-deoxycytidine (AZA), histone deacetylase inhibitor-trichostatin A (TSA) and histone methyltransferase inhibitor-3-Deazaneplanocin A (DZNep) individually at 0.1 mg/kg body weight intraperitoneally at alternate days/week; and combination of AZA+TSA and DZNep+TSA at similar doses and times. Treatment of mice with GTP resulted in marked decrease in tumor growth and its local invasion in dose dependent manner, compared to treatment with epigenetic inhibitors and their combination after 8 weeks of intervention. GTP treatment significantly reduced serum levels of MMP-2, MMP-9 and VEGF, compared to treatment with epigenetic inhibitors alone. Combination of AZA+TSA exhibited similar effect which was equivalent to the lower dose of GTP treatment. Furthermore, GTP treatment significantly reduced EZH2 and H3K27me3 and class I HDAC protein levels in tumors. GTP partially reversed RECK hypermethylation and significantly enhanced its expression in the tumor tissue. Similar findings were noted in cell culture where treatment of PC-3 and LNCaP cells with 20µM EGCG and 10µg/mL GTP for 72 h significantly induced RECK expression at mRNA and protein levels along with decrease invasiveness in these cells. Our findings suggest that induction of RECK is a key epigenetic event reactivated by GTP/EGCG that results in suppression of MMP-2/MMP-9, matrix degradation and angiogenesis to delay prostate cancer invasion and its subsequent progression. Citation Format: Eswar Shankar, Omair Iqbal, Natarajan Bhaskaran, Gauri Deb, Gregory T. MacLennan, Pingfu Fu, Sanjay Gupta. Epigenetic modifications involving reactivation of RECK inhibiting MMP-9 and MMP-2 in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5084.

Low-Background Acyl-Biotinyl Exchange Largely Eliminates the Coisolation of Non-S-Acylated Proteins and Enables Deep S-Acylproteomic Analysis.

Protein S-acylation (also called palmitoylation) is a common post-translational modification whose deregulation plays a key role in the pathogenesis of many diseases. Acyl-biotinyl exchange (ABE), a widely used method for the enrichment of S-acylated proteins, has the potential of capturing the entire S-acylproteome in any type of biological sample. Here, we showed that current ABE methods suffer from a high background arising from the coisolation of non-S-acylated proteins. The background can be substantially reduced by an additional blockage of residual free cysteine residues with 2,2'-dithiodipyridine prior to the biotin-HPDP reaction. Coupling the low-background ABE (LB-ABE) method with label-free proteomics, 2 895 high-confidence candidate S-acylated proteins (including 1 591 known S-acylated proteins) were identified from human prostate cancer LNCaP cells, representing so-far the largest S-acylproteome data set identified in a single study. Immunoblotting analysis confirmed the S-acylation of five known and five novel prostate cancer-related S-acylated proteins in LNCaP cells and suggested that their S-acylation levels were about 0.6-1.8%. In summary, the LB-ABE method largely eliminates the coisolation of non-S-acylated proteins and enables deep S-acylproteomic analysis. It is expected to facilitate a much more comprehensive and accurate quantification of S-acylproteomes than previous ABE methods.

Triptonide acts as a novel antiprostate cancer agent mainly through inhibition of mTOR signaling pathway.

The increasing incidence of prostate cancer (PCa) indicates an urgent need for the development of new effective drugs in PCa therapy. Triptonide has been reported to have a strong inhibition activity in cancers through screening of Chinese herbal medicine. This study aims to investigate the effects of triptonide on anti-PCa activity and its mechanisms. Three human advanced PCa cell lines PC3, DU145, and LNCap, and a human normal prostate epithelial cell line RWPE were treated with a range (0, 1.25, 2.5, 5, 10, 20, 40, 80, 160, and 320 nM) of triptonide concentrations for 72 hours respectively. Then, cell viability was assessed by cell counting kit-8. PCa cells were treated with different doses (0-20 nM) of triptonide for 72 hours. Cell cycle and apoptosis were assessed by flow cytometry assays. Nude mice bearing human PCa xenografts were intraperitoneally injected daily with either triptonide (10 mg/kg/d) or phosphate-buffered saline as a control for 35 days. RNA-sequencing (RNA-seq) was performed by an Illumina Hiseq Sequencing platform and confirmed by a real-time polymerase chain reaction. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway analysis, and ingenuity pathway analysis were used to analyze RNA-seq results. Triptonide effectively inhibits the proliferation of human PCa cells PC3, DU145, and LNCap in vitro with their IC50 values as 11.961, 10.259, and 12.012 nM, respectively. Triptonide (10 mg/kg) potently inhibits the growth of PCa cell xenografts in vivo at an inhibition rate of over 97.95%. Treatment with triptonide (5 nM) significantly promotes cell apoptosis and retaining cell-cycle arrest in the G2/M phase. RNA-seq data revealed that total of 936 genes were upregulated or downregulated in triptonide treated. Moreover, the phosphorylation of mechanistic target of rapamycin (mTOR) and the downstream protein p70S6K were both inhibited, most obviously in PCa cells. Our findings suggest that triptonide can efficaciously suppress PCa growth in vitro and in vivo via inhibiting the phosphorylation of mTOR and the activities of related downstream signaling pathways.

Ultradeep Palmitoyl‐proteomic Analysis Uncovers Over 1,300 Novel Human Palmitoyl‐proteins

Protein palmitoylation (more accurately known as protein S‐acylation) is a reversible lipid modification that plays critical roles in protein localization, activity, stability, and complex formation. Aberrant protein palmitoylation is associated with many human diseases such as neurodegenerative disorders and cancer. The past decade has witnessed striking progress in studying the enzymes, substrates, and functions of protein palmitoylation. However, it remains unclear whether protein palmitoylation is a widespread post‐translational modification comparable to protein phosphorylation and ubiquitination. Previously, we developed a palmitoyl‐proteomics method termed Palmitoyl‐Protein Identification and Site Characterization (PalmPISC) and applied it to profile the palmitoyl‐proteomes in human cancer cells and platelets (Yang et al., Mol Cell Proteomics, 2010; Dowal et al., Blood, 2011). Nevertheless, similar to other acyl‐biotinyl exchange (ABE)‐based palmitoyl‐proteomics methods, one shortcoming of the PalmPISC method is that many non‐palmitoylated proteins are co‐isolated with palmitoyl‐proteins, resulting in a high background that masks the detection of low‐abundance palmitoyl‐proteins and interferes with accurate quantification of palmitoyl‐proteins across different conditions. In this study, we coupled stable isotope labeling by amino acid in culture (SILAC) with PalmPISC to quantify the extent of co‐isolated proteins and found that ~65% of identified proteins were co‐isolated contaminants. To address this issue, we developed a low‐background ABE enrichment method and applied it to comprehensively profile the palmitoyl‐proteome of a human prostate cancer LNCaP cell line. Using stringent cutoffs (false discovery rate &lt; 1% and fold change &gt;2), a total of 2,895 proteins, including 1,571 known and 1,324 novel human palmitoyl‐proteins, were found to be significantly enriched. Western blotting analysis confirmed the palmitoylation of six novel palmitoyl‐protein candidates that play important roles in prostate cancer. In summary, our low‐background ABE enrichment method enables proteome‐wide analysis of protein palmitoylation and the study demonstrates that protein palmitoylation is more widespread than we previously thought.Support or Funding InformationThis work was supported by the Department of Defense W81XWH‐15‐1‐0167 (to W.Y.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Green tea-induced epigenetic reactivation of tissue inhibitor of matrix metalloproteinase-3 suppresses prostate cancer progression through histone-modifying enzymes.

Green tea polyphenols (GTPs) and their major constituent, epigallocatechin-3-gallate (EGCG), have been reported to demonstrate many interesting biological activities, including anticancer properties. Recent studies on prostate cancer provide strong evidence that epigenetic mechanisms are major players in the regulation of matrix metalloproteinases (MMPs) and their binding partner tissue inhibitor of MMPs (TIMPs) involved in prostate cancer progression. Here we demonstrate that GTP/EGCG mediate epigenetic reactivation of TIMP-3 that plays a key role in suppressing invasiveness and cancer progression. Treatment of human prostate cancer DUPRO and LNCaP cells with 10 µg/mL GTP and 20 µM EGCG induced TIMP-3 mRNA and protein expression. This transcriptional activation of TIMP-3 was associated with the decrease in the expression of both enhancers of zeste homolog 2 (EZH2) and its catalytic product trimethylation of histone H3 at lysine 27 (H3K27me3) repressive marks at the TIMP-3 promoter with an accompanying increase in histone H3K9/18 acetylation. In addition, GTP/EGCG treatment significantly reduced class I histone deacetylase (HDAC) activity/expression and EZH2 and H3K27me3 levels in prostate cancer cells. EGCG/GTP exposure also reduced MMP-2/MMP-9 gelatinolytic activity and abrogated invasion and migration capabilities in these cells. Silencing of EZH2 and class I HDACs strikingly increased the expression of TIMP-3 independent of DNA methylation. Furthermore, clinical trials performed on patients undergoing prostatectomy consuming 800 mg EGCG (Polyphenon E) up to 6 weeks and grade-matched controls demonstrate an increase in plasma TIMP-3 levels. A marked reduction in class I HDACs activity/expression and EZH2 and H3K27me3 levels were noted in GTP-supplemented prostate tissue. Our findings highlight that TIMP-3 induction, as a key epigenetic event modulated by green tea in restoring the MMP:TIMP balance suppresses prostate cancer progression.

Role of Cav3.2 T-type Ca2+ channels in prostate cancer cells

Among voltage-gated Ca2+ channels, T-type Ca2+ channels, which are activated by low voltages, regulate neuronal excitability, spontaneous neurotransmitter release, hormone secretion, etc. and also participate in proliferation of distinct cancer cells. Among three isoforms of T-type Ca2+ channels, Cav3.2 is detectable in 100% of biopsy samples from prostate cancer patients. In general, prostate cancer cells are highly sensitive to androgen deprivation therapy, but often acquire hormone-therapy resistance. The androgen deprivation may trigger neuroendocrine (NE)-like differentiation of some prostate cancer cells. We have analyzed the expression and function of Cav3.2 in human prostate cancer LNCaP cells during NE-like differentiation. NE-like LNCaP cells overexpress Cav3.2 through the CREB/Egr-1 pathway and also cystathionine-γ-lyase (CSE), which generates H2S that enhances the channel activity of Cav3.2. H2S generated by upregulated CSE appears to enhance the activity of upregulated Cav3.2 after the differentiation. The enhanced Cav3.2 activity in NE-like cells may contribute to increased secretion of mitogenic factors essential for androgen-independent proliferation of surrounding prostate cancer cells. It is known that increased extracellular glucose levels enhance Cav3.2 activity through asparagine (N)-linked glycosylation of Cav3.2, which might contribute to diabetic neuropathy. We then found that high glucose accelerates the enhanced channel function and overexpression of Cav3.2 in NE-like LNCaP cells, which might be associated with clinical evidence for diabetes-related poor prognosis of prostate cancer and development of hormone therapy resistance. Thus, Cav3.2 is considered to play a role in the pathophysiology of prostate cancer, and may serve as a therapeutic target.