Knockdown of AGR2 induces cellular senescence in prostate cancer cells

Anterior-gradient 2 (AGR2), overexpressed in many tumors including prostate cancer (PCa), is implicated in stimulation of cell proliferation, adhesion, anti-apoptosis and cell cycle regulation. Here, a potential role of AGR2 in cellular senescence was investigated. We first observed that AGR2 was overexpressed in Chinese Han PCa tissues and had a positive correlation with cyclin D1 and p-Rb but not with p16(INK4a). AGR2 expression profiles varied among cell lines, with PC3 cells being the highest level, LNCaP and DU145 relatively less. The expression of cyclin D1 showed similar pattern to the AGR2 in cell lines. Knockdown of AGR2 caused a decrease in cell viability in PC3 cells, whereas forced expression of AGR2 led to an increased cell proliferation of LNCaP and DU145 cells. Importantly, AGR2 depletion resulted in accumulation of cells at the G(0)/G(1) phase and induction of cellular senescence in all three PCa cell lines as indicated by an increase of flat, enlarged and senescence-associated β-galactosidase (SA-β-Gal) positive cells. Senescent response to AGR2 silencing was also evidenced by elevated γH2AX and fluorescent punctuate formation of tri-methyl-histone H3 in AGR2-depleted cells. Further studies indicated that LNCaP underwent a p21(CIP1)-dependent cellular senescence in response to AGR2 depletion that requires inactivation of ERK signaling, whereas PC-3 was also p21(CIP1) dependent but involved in suppression of PI3K/Akt. Unlike LNCaP and PC-3, senescent response of DU145 was found to be mainly p27(KIP1) dependent that may require upregulation of PTEN and inhibition of PI3K/Akt signaling. Thus, these findings suggest a novel role of AGR2 in regulation of cellular senescence.     

Global H3K27 trimethylation and EZH2 abundance in breast tumor subtypes

Polycomb repressive complex 2 (PRC2) and its core member enhancer of zeste homolog 2 (EZH2) mediate the epigenetic gene silencing mark: trimethylation of lysine 27 on histone 3 (H3K27me3). H3K27me3 is characteristic of the chromatin at genes involved in developmental regulation in undifferentiated cells. Overexpression of EZH2 has been found in several cancer types such as breast, prostate, melanoma and bladder cancer. Moreover, overexpression is associated with highly proliferative and aggressive types of breast and prostate tumors. We have analyzed the abundance of EZH2 and H3K27me3 using immunohistochemistry in two large and well-characterized breast tumor data sets encompassing more than 400 tumors. The results have been analyzed in relation to the molecular subtypes of breast tumors (basal-like, luminal A, luminal B, HER2-enriched and normal-like), as well as in subtypes defined by clinical markers (triple negative, ER+/HER2-/Ki67low, ER+/HER2-/Ki67high and HER2+), and were validated in representative breast cancer cell lines by western blot. We found significantly different expression of both EZH2 and H3K27me3 across all subtypes with high abundance of EZH2 in basal-like, triple negative and HER2-enriched tumors, and high H3K27me3 in luminal A, HER2-enriched and normal-like tumors. Intriguingly, the two markers show an inverse correlation, particularly for the basal-like and triple negative tumors. Consequently, high expression of EZH2 was associated with poor distant disease-free survival whereas high expression of H3K27me3 was associated with better survival. Additionally, none of 182 breast tumors was found to carry a previously described EZH2 mutation affecting Tyr641. Our observation that increased expression of EZH2 does not necessarily correlate with increased abundance of H3K27me3 supports the idea that EZH2 can have effects beyond epigenetic silencing of target genes in breast cancer.     

Growth of human prostate cancer cells is significantly suppressed in vitro with sodium butyrate through apoptosis

Histone deacetylase inhibitors (HDACis) have shown significant antiproliferative and apoptotic properties in various types of cancer cells, including prostate cancer cells, and are therefore being evaluated as a treatment modality. However, the mechanism by which sodium butyrate (SB) induces apoptosis is not completely understood. We focused on SB which exists in the intestine and is therefore expected to have less adverse effects. In this study, three prostate cancer cell lines (LNCaP, DU145 and PC-3) were treated in vitro with different concentrations of SB. Cell proliferation was studied by the XTT assay; cell cycle analysis and induction of apoptosis were studied by laser scanning cytometry. Western blot analysis was used to study p21, p27, CDK2, CDK4, CDK6, caspase-3, caspase-7, Fas, FADD, TRADD, Bcl-2 and Bax protein expression. SB inhibited cell growth and induced apoptosis in a concentration-dependent manner in human prostate cancer cells (LNCaP, DU145 and PC-3). Western blot analysis showed dose-dependent increases of p21 levels in DU145 and PC-3 cells, and dose-dependent decreases of CDK2, CDK4, CDK6 and procaspase-3 protein levels in all three prostate cancer cell lines. Bcl-xL was significantly down-regulated in DU145 cells, and Bcl-2 was significantly down-regulated in PC-3 and LNCaP cells. No significant changes were observed in procaspase-7, TRADD and Bax expression, although slight decreases in Fas and FADD expression were seen in all three prostate cancer cell lines. Analysis of cell morphology using laser scanning microscopy detected condensed and fragmented nuclei. In conclusion, SB induces G1 and G2 arrest by increasing p21 expression resulting in CDK2, CDK4 and CDK6 down-regulation. SB potently induced apoptosis, which was accompanied by DNA fragmentation, down-regulated Bcl-2 in LNCaP and PC-3 cells, Bcl-xL in DU145 cells, and down-regulated procaspase-3, but not procaspase-7, in these human prostate cancer cell lines. These results suggest that SB may serve as a new modality for the treatment of hormone refractory prostate cancer.     

H2AK119Ub1 and H3K27Me3 in molecular staging for survival prediction of patients with pancreatic ductal adenocarcinoma

Polycomb group (PcG) proteins Ring1B and EZH2, which have been characterized as catalyzing the two epigenetic modifications H2AK119 monoubiquitination (H2AK119Ub1) and H3K27 trimethylation (H3K27Me3), are well-known epigenetic silencers implicated in embryonic development and tumorigenesis. However, the status of polycomb-associated histone modifications and their clinical implications in pancreatic cancer remain unclear. Here, we performed immunohistochemistry on tissue microarrays (TMAs) containing 80 pairs of human pancreatic cancer specimens to assess the expression levels of Ring1B, H2AK119Ub1, EZH2, and H3K27Me3 in tumors. More than 50% of the tumor cells showed a high expression of H2AK119Ub1, Ring1B, and EZH2, whereas more than 50% of the tumor cells showed a low level of H3K27Me3. Different expression patterns of H2AK119Ub1 and H3K27Me3 in tumors were negatively correlated (r = −0.247, P = 0.027). Both H2AK119Ub1 and H3K27Me3 independently predicted the clinical prognosis. In particular, a combinatorial pattern of elevated H2AK119Ub1 and decreased H3K27Me3 in tumors was significantly correlated with a poorer prognosis. Furthermore, compared to the tumor, lymph node, metastasis (TNM) staging system, histone modifications can discriminate the survival difference more accurately, especially for patients with stage I or stage II tumors. Simultaneous silencing of Ring1B and EZH2 via shRNA depleted H2AK119Ub1 and H3K27Me3 in the pancreatic cancer cells PanC1 and AsPC1, enhanced HOX gene derepression, and inhibited tumor cell growth in vitro and in tumor xenograft models. These results demonstrated that H2AK119Ub1 and H3K27Me3 cooperate in tumors and are associated with the clinical prognosis in combinatorial patterns. We have proposed that epigenetic modifications may serve as discriminatory biomarkers for molecular staging of pancreatic cancer.     

PTPRJ通过Src/PI3K/Akt信号通路促进前列腺癌细胞的黏附、迁移和侵袭

目的:探究PTPRJ基因表达对前列腺癌DU145细胞黏附、迁移和侵袭的影响以及可能的调控机制。方法:实时荧光定量PCR、Western blot检测PTPRJ在前列腺肿瘤组织和细胞系中的表达;用携带PTPRJ特异shRNA的重组慢病毒(LV-shPTPRJ)感染沉默PTPRJ表达;MTT检测细胞黏附力,Transwell检测细胞迁移和侵袭;实时荧光定量PCR、Western blot检测信号通路分子mRNA和蛋白表达。结果:与正常前列腺组织和细胞相比,PTPRJ在前列腺肿瘤组织和PC-3、DU145细胞系中表达升高（P＜0.05）;与对照组相比,沉默PTPRJ后前列腺癌DU145细胞黏附、迁移和侵袭能力显著下降（P＜0.01）、信号通路蛋白pY418Src、p-PI3K和p-Akt表达水平均显著降低（P＜0.05）;SC79激活PI3K/Akt可逆转PTPRJ下调对DU145细胞黏附和侵袭的影响;沉默PTPRJ下调裸鼠瘤体组织中pY418Src、p-PI3K和p-Akt表达（P＜0.05）。结论:PTPRJ可能通过激活Src/PI3K/Akt信号通路来促进DU145细胞的黏附、迁移和侵袭,预示PTPRJ可能成为前列腺癌治疗的潜在靶点。     

Prostate cancer mediates osteoclastogenesis through two different pathways

The present study was undertaken to test the effects of prostate cancer cell lines (LNCaP, DU145, PC3, and MDA PCa 2b) on osteoclastogenesis. Crude conditioned medium (CM) from all four prostate cancer cell lines enhanced expression of the mRNA for receptor activator of NF-kappaB ligand (RANKL) in a mouse osteoblast cell line, MC3T3-E1; however, CM had no effect on expression of osteoprotegerin (OPG) mRNA. Coculture of MC3T3-E1 with prostate cancer cells yielded similar results. The number of mature osteoclasts induced by soluble RANKL increased significantly when osteoclast precursor cells were cultured with CM from LNCaP and DU145 cells. CM from LNCaP and DU145 cells also induced maturation from precursor in the absence of soluble RANKL, and this effect was not blocked by OPG. Addition of CM from DU145 cells increased expression of MMP-9 mRNA by osteoclast precursors. Our findings indicate that prostate cancer mediates osteoclastogenesis through induction of RANKL expression by osteoblasts and through direct actions on osteoclast precursors mediated by some factors other than RANKL.     

Antiproliferative effects of AVN944, a novel inosine 5-monophosphate dehydrogenase inhibitor, in prostate cancer cells

Inosine 5-monophosphate dehydrogenase II, a key enzyme in the de novo synthesis of purine nucleotides, is expressed in prostate tumors and prostate cancer cells. AVN944 is a new, specific, noncompetitive IMPDH inhibitor. In this study, we investigated the effects of IMPDH inhibitor AVN944 on LNCaP, CWR22Rv1, DU145 and PC-3 human prostate cancer cells. AVN944 inhibited proliferation of these 4 prostate cancer cell lines and was associated with cell cycle G1 arrest of LNCaP cells and S-phase block of androgen-independent CWR22Rv1, DU145 and PC-3 cells. AVN944 induced caspase-dependentand caspase-independent cell death in LNCaP, CWR22Rv1, and DU145 cells. AVN944 induced expression of p53-target proteins Bok, Bax and Noxa in androgen-responsive cell lines and suppressed expression of survivin in prostate cancer cells regardless of their androgen sensitivity. AVN944 also induced differentiation of androgen-independent prostate cancer cells as indicated by morphological changes and increased expression of genes coding for prostasomal proteins, keratins and other proteins, including tumor suppressor genes MIG-6 and NDRG1. AVN944-differentiated androgen-independent DU145 and PC-3 cells are sensitized to TRAIL-induced apoptosis as demonstrated by induction of caspases and PARP cleavage. In summary, AVN944 inhibited the growth of human prostate cancer cells by inducing cell cycle arrest, cell death as well as differentiation. AVN944 is a novel, promising therapeutic agent that might be combined with other agents for treatment of human prostate cancer.     

Involvement of prostate specific antigen in the stimulation of LNCaP cell growth

Since its identification in 1979, prostatic specific antigen (PSA) has been used extensively as a serum marker for diagnosis and prognosis of prostate cancer. In addition, PSA is an immunohistochemical marker for the identification of prostatic tissues and cells in histological specimens. PSA is found in normal prostate, benign prostatic hypertrophy (BPH) tissue, in cancer of the prostate, and its metastases as well as in other hormone dependent cancers, such as breast and ovarian carcinoma. However, the importance of PSA as a regulator of cell growth generally has not been appreciated. The role of PSA in the development of prostate or other hormone-dependent cancers has remained unclear. We therefore examined the role of PSA in the control of cell growth using both the PSA positive cell line, LNCaP cells and the PSA negative cell line PC-3 and DU145. LNCaP cell growth was stimulated by the conditioned medium (CM) from LNCaP cells, but not by CM from PC-3 or DU145 cells. No such stimulation was observed when PC-3 or DU145 cells were exposed to CM from LNCaP cells nor from CM produced by their own lines. The stimulation of LNCaP cell growth by its own CM could not be attributed to the high level of insulin-like growth factor binding protein-2 (IGFBP-2) present in the CM since even higher level of IGFBP-2 was also found to be present in CM from both PC-3 and DU145 CMs. High level of PSA and 66 kDa epidermal growth factor (EGF) were present in LNCaP CM as measured by Western blotting. The stimulation of LNCaP cell growth by its own CM was eliminated partially by PSA or EGF antibody. Stimulation of DNA biosynthesis in LNCaP cells by LNCaP CM or pure PSA was also observed. These data indicate that PSA and EGF are involved in the growth regulation of PSA positive LNCaP cell line.     

Epigenetic modifications of RASSF1A gene through chromatin remodeling in prostate cancer.

PURPOSE: The RAS-association domain family 1, isoform A (RASSF1A) gene is shown to be inactivated in prostate cancers. However, the molecular mechanism of silencing of the RASSFIA gene is not fully understood. The present study was designed to investigate the mechanisms of inactivation of the RASSF1A gene through the analysis of CpG methylation and histone acetylation and H3 methylation associated with the RASSF1A promoter region. EXPERIMENTAL DESIGN: Methylation status of the RASSF1A gene was analyzed in 131 samples of prostate cancer, 65 samples of benign prostate hypertrophy (BPH), and human prostate cell lines using methylation-specific PCR. Histone acetylation (acetyl-H3, acetyl-H4) and H3 methylation (dimethyl-H3-K4, dimethyl-H3-K9) status associated with the promoter region in prostate cells were analyzed by chromatin immunoprecipitation (ChIP) assay. RESULTS: Aberrant methylation was detected in 97 (74.0%) prostate cancer samples and 12 (18.5%) BPH samples. The methylation frequency of RASSF1A showed a significant increase with high Gleason sum and high stage. The ChIP assays showed enhancement of histone acetylation and dimethyl-H3-K4 methylation on the unmethylated RASSF1A promoter. TSA alone was unable to alter key components of the histone code. However, after 5-aza-2'-deoxy-cytidine treatment, there was a complete reversal of the histone components in the hypermethylated promoter. Levels of acetyl-H3, acetyl-H4, and dimethyl-H3-K4 became more enriched, whereas H3K9me2 levels were severely depleted. CONCLUSIONS: This is the first report suggesting that reduced histone acetylation or H3K4me2 methylation and increased dimethyl-H3-K9 methylation play a critical role in the maintenance of promoter DNA methylation-associated RASSF1A gene silencing in prostate cancer.     

Contributions of mitogen-activated protein kinase and nuclear factor kappa B to N-(4-hydroxyphenyl)retinamide-induced apoptosis in prostate cancer cells

The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) has been shown to induce apoptosis in various types of tumors, including prostate cancer. We sought to examine the key mechanisms affecting the resistance to 4-HPR-induced apoptosis in three human prostate cancer cell lines, PC-3, DU145, and LNCaP. Concentrations of more than 40 microM 4-HPR produced apoptosis to almost the same extent in all cell lines; however, only the LNCaP line remained highly sensitive to concentrations less than 10 microM. These differing sensitivities at low concentrations correlated well with the level of constitutive activation of nuclear factor kappa B (NFkappaB) in the individual cell lines. We found that NFkappaB activation inhibited c-jun NH(2)-terminal kinase and caspase 3 activation induced by 4-HPR and that NFkappaB inhibition by the I kappa B alpha phosphorylation inhibitor compound Bay 117082 resulted in increasing sensitization of both PC-3 and DU145 lines to apoptosis induced by 4-HPR at low concentrations. Furthermore, we found that inhibition of extracellular signal-regulated kinase (ERK) enhanced the suppression of NFkappaB by 4-HPR and also resulted in sensitization to apoptosis in the DU145 cell line, in which ERK is activated constitutively. It thus appears that mitogen-activated protein kinase associated with the activity of NFkappaB plays an important role in the degree of resistance to 4-HPR-induced apoptosis in human prostate cancer cells.     

Role of fibroblast growth factor receptor signaling in prostate cancer cell survival.

BACKGROUND: Expression of fibroblast growth factors (FGFs) is increased in a substantial fraction of human prostate cancers in vivo and in prostate cancer cell lines. Altered FGF signaling can potentially have a variety of effects, including stimulating cell proliferation and inhibiting cell death. To determine the biologic significance of altered FGF signaling in human prostate cancer, we disrupted signaling by expression of a dominant-negative (DN) FGF receptor in prostate cancer cell lines. METHODS: PC-3, LNCaP, and DU145 prostate cancer cells were stably transfected with DN FGFR constructs, and LNCaP and DU145 cells were infected with a recombinant adenovirus expressing DN FGFR-1. The effect of DN FGFR-1 expression was assessed by colony-formation assays, cell proliferation assays, flow cytometry, and cytogenetic analysis. Key regulators involved in the G(2)-to-M cell cycle transition were assessed by western blotting to examine cyclin B1 expression and by in vitro kinase assay to assess cdc2 kinase activity. RESULTS: Stable transfection of the DN FGFR-1 construct inhibited colony formation by more than 99% in all three cell lines. Infection of LNCaP and DU145 prostate cancer cells with adenovirus expressing DN FGFR-1 led to extensive cell death within 48 hours. Flow cytometry and cytogenetic analysis revealed that the DN FGFR-1 receptor led to arrest in the G(2) phase of the cell cycle before cell death. Cyclin B1 accumulated in DN FGFR-1-infected LNCaP cells, but cdc2 kinase activity was decreased. CONCLUSIONS: These findings reveal an unexpected dependence of prostate cancer cells on FGF receptor signal transduction to traverse the G(2)/M checkpoint. The mechanism for the G(2) arrest is not clear. Our results raise the possibility that FGF-signaling antagonists might enhance the cell death induced by other prostate cancer therapies.     

EZH2和H3K27me3的表达对食管癌细胞迁移和侵袭能力的影响

目的:通过转染Zeste同源物增强子2(enhancer of zeste homolog 2,EZH2)过表达或者敲低载体,探讨EZH2和Lys27位点三甲基化组蛋白H3(histone H3 methylated Lys27,H3K27me3)对食管麟状细胞癌(esophageal squamous cell cancer,ESCC)细胞迁移和侵袭能力的影响.方法:应用实时荧光定量PCR、Western blotting法检测ESCC细胞株KYSE30、KYSE170、TE1、Eca109中EZH2 mRNA水平,以及ESCC细胞过表达或者敲低EZH2对H3 K27me3表达水平的影响.用划痕实验及Transwell侵袭实验分析过表达或者敲低EZH2后ESCC细胞的迁移侵袭能力.用实时荧光定量PCR法分析ESCC细胞过表达及敲低EZH2对MMPs mRNA水平的影响.结果:食管癌Eea109及TE1细胞中EZH2和H3K27me3 mRNA和蛋白水平明显高于KYSE30及KYSE170细胞(P＜0.05).过表达EZH2的食管癌KYSE30及KYSE170细胞H3K27me3蛋白的表达水平显著升高(P＜0.05),敲低EZH2后Eca109及TE1细胞H3 K27 me3蛋白的表达水平明显降低(P＜0.05).过表达EZH2后,KYSE30及KYSE170细胞的穿膜数目明显增多[(281.33±4.10)、(241.67 ±4.04) vs(132.00 ±4.00)、(105.33 ±3.51)个,均P＜0.05]、迁移距离明显增大[(63.6±1.2)、(62.5±2.5)vs (23.0±2.3)、(21.2±1.0) μm,P＜0.05].敲低EZH2后Eca109及TE1细胞的穿膜数目显著减少(均P＜0.05),转染shEZH2后Eca109及TE1细胞迁移的距离明显减小(均P＜0.05).结论:EZH2可增加靶基因启动子上组蛋白H3第27位赖氨酸的三甲基化,并增强ESCC细胞的迁移和侵袭能力.