Cancer‐associated upregulation of histone H3 lysine 9 trimethylation promotes cell motility in vitro and drives tumor formation in vivo

Global histone modification patterns correlate with tumor phenotypes and prognostic factors in multiple tumor types. Recent studies suggest that aberrant histone modifications play an important role in cancer. However, the effects of global epigenetic rearrangements on cell functions remain poorly understood. In this study, we show that the histone H3 lysine 9 (H3K9) methyltransferase SUV39H1 is clearly involved in regulating cell migration in vitro. Overexpression of wild‐type SUV39H1, but not enzymatically inactive SUV39H1, activated migration in breast and colorectal cancer cells. Inversely, migration was reduced by knockdown of SUV39H1 or chemical inhibition by chaetocin. In addition, H3K9 trimethylation (H3K9me3) was specifically increased in invasive regions of colorectal cancer tissues. Moreover, the presence of H3K9me3 positively correlated with lymph node metastasis in colorectal cancer patients. Furthermore, overexpression of SUV39H1 drove tumorigenesis in mouse, resulting in a considerable decrease in survival rate. These data indicate that H3K9 trimethylation plays an important role in human colorectal cancer progression, possibly by promoting collective cell invasion.     

SMYD3 promotes implant metastasis of ovarian cancer via H3K4 trimethylation of integrin promoters

Detachment of cancer cells from the primary tumor and formation of spheroids in ascites is required for implantation metastasis in epithelial ovarian cancer (EOC), but the underlying mechanism of this process has not been thoroughly elucidated. To mimic this process, ovarian cancer cells were grown in 3D and 2D culture. Hey and OVCA433 spheroids exhibited decreased cell proliferation and enhanced adhesion and invasion. SMYD3 expression was elevated in ovarian carcinoma spheroids in association with increased H3K4 methylation. Depletion of SMYD3 by transient siRNA, stable shRNA knockdown and the SMYD3 inhibitor BCI-121 all decreased spheroid invasion and adhesion. Gene expression arrays revealed downregulation of integrin family members. Inhibition assays confirmed that invasion and adhesion of spheroids are mediated by ITGB6 and ITGAM. SMYD3-deficient cells regained the ability to invade and adhere after forced overexpression of SMYD3, ITGB6 and ITGAM. However, this biological ability was not restored by forced overexpression of SMYD3 in ITGB6- and/or ITGAM-deficient cancer cells. SMYD3 and H3K4me3 binding at the ITGB6 and ITGAM promoters was increased in spheroids compared to that in monolayer cells, and the binding was decreased when SMYD3 expression was inhibited, consistent with the expression changes in integrins. SMYD3 expression and integrin-mediated adhesion were also activated in an intraperitoneal xenograft model and in EOC patient spheroids. In vivo, SMYD3 knockdown inhibited tumor metastasis and reduced ascites volume in both the intraperitoneal xenograft model and a PDX model. Overall, our results suggest that the SMYD3-H3K4me3-integrin pathway plays a crucial role in ovarian cancer metastasis to the peritoneal surface.     

Loss of histone H4K20 trimethylation predicts poor prognosis in breast cancer and is associated with invasive activity

Introduction

Loss of histone H4 lysine 20 trimethylation (H4K20me3) is associated with multiple cancers, but its role in breast tumors is unclear. In addition, the pathological effects of global reduction in H4K20me3 remain mostly unknown. Therefore, a major goal of this study was to elucidate the global H4K20me3 level in breast cancer tissue and investigate its pathological functions.

Methods

Levels of H4K20me3 and an associated histone modification, H3 lysine 9 trimethylation (H3K9me3), were evaluated by immunohistochemistry in a series of breast cancer tissues. Univariate and multivariate clinicopathological and survival analyses were performed. We also examined the effect of overexpression or knockdown of the histone H4K20 methyltransferases, SUV420H1 and SUV420H2, on cancer-cell invasion activity in vitro.

Results

H4K20me3, but not H3K9me3, was clearly reduced in breast cancer tissue. A reduced level of H4K20me3 was correlated with several aspects of clinicopathological status, including luminal subtypes, but not with HER2 expression. Multivariate analysis showed that reduced levels of H4K20me3 independently associated with lower disease-free survival. Moreover, ectopic expression of SUV420H1 and SUV420H2 in breast cancer cells suppressed cell invasiveness, whereas knockdown of SUV420H2 activated normal mammary epithelial-cell invasion in vitro.

Conclusions

H4K20me3 was reduced in cancerous regions of breast-tumor tissue, as in other types of tumor. Reduced H4K20me3 level can be used as an independent marker of poor prognosis in breast cancer patients. Most importantly, this study suggests that a reduced level of H4K20me3 increases the invasiveness of breast cancer cells in a HER2-independent manner.     

H3K4me3高表达与肝癌患者较差生存预后相关

目的:探讨检测肝癌组织中组蛋白第三亚基四号赖氨酸的三甲基化（H3K4me3）蛋白的表达与肿瘤病理特点和肝癌患者生存预后的相关性。方法:免疫组化和Western-blot检测H3K4me3和组蛋白甲基转移酶(SET and MYND domain-containing protein 3,SMYD3)在肝癌组织（n=168）和细胞株中的表达。此外,实验结果还在另外一个肝癌组织芯片（n=147）中进行验证。H3K4me3表达的最佳分界点（optimal cut-point）由X-tile程序确定,患者的预后由Kaplan-meier生存曲线描述。结果:H3K4me3高表达于肝癌细胞系和肝癌组织,其高表达与肝癌尤其是早期TNM1/2期患者的较差总体生存显著相关。单因素和多因素分析均提示H3K4me3表达水平是患者预后的独立危险因素。此外,H3K4me3和SMYD3在两组肝癌组织中均存在正相关表达。结论:H3K4me3表达水平能成为肝癌患者术后生存的预测因子,其高表达可能与SMYD3有关。     

Targeting SMYD2 inhibits prostate cancer cell growth by regulating c-Myc signaling

SMYD2 is a lysine histone methyl transferase involved in various cancers epigenetically via methylating histone H3K4, and H3K36. c-Myc is one of the major drivers of prostate cancer (PCa) initiation and progression. The roles of SMYD2 in PCa and the regulators of c-Myc activity in PCa are still under-researched. SMYD2 expression and survival outcomes in PCa cohorts were analyzed by bioinformatics analysis. SMYD2 protein levels were detected in PCa tissues by immunohistochemistry. SMYD2 knockdown cells were established to identify the effects of SMYD2 on cell growth in vitro and in vivo. GSEA and RNA sequencing were adopted to reconnoiter the signaling regulated by SMYD2 in PCa. The relationship between SMYD2 and c-Myc was examined by western blot analysis, qPCR, and immunohistochemistry. SMYD2 specific inhibitor-AZ505 was used to pharmacologically inhibit SMYD2 function in vitro and in vivo. SMYD2 expression increased in PCa tissues compared with benign prostate tissues and higher SMYD2 expression was associated with a higher risk of biochemical relapse after radical prostatectomy. SMYD2 knockdown inhibited the growth of PCa cells both in vitro and in vivo. Furthermore, high SMYD2 levels conduced to activated c-Myc signaling in PCa cells. Importantly, the pharmacological intervention of SMYD2 by AZ505 significantly repressed PCa cell growth both in vitro and in vivo. Our findings indicate that SMYD2 inhibition restrains PCa cell proliferation by regulating c-Myc signaling and provide evidence for the potential practice of SMYD2 targeting in the treatment of PCa.     

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.     

GSK-J4: An H3K27 histone demethylase inhibitor,as a potential anti-cancer agent

Aberrant epigenetic modifications are emerging as potent drivers of tumor initiation and progression. The deregulation of H3K27me3 marks has shown to play an important role in cancer progression in several cancers. The H3K27me3 mark is associated with gene silencing. The reversible nature of these epigenetic aberrations makes them an important target for treating cancer. GSK-J4 is a histone demethylase inhibitor that inhibits the JMJD3/UTX enzyme, which results in the upregulation of H3K27me3 levels. In this review, the anti-cancer properties of GSK-J4 have been summarized, the various molecular pathways targeted, in-vivo studies, and drug combination studies in different cancer models. GSK-J4 targeted pathways like apoptosis, cell cycle, invasion, migration, DNA damage repair, metabolism, oxidative stress, stemness, etc. GSK-J4 is a promising candidate alone and in combination with other conventional anti-cancer drugs against different cancer types.     

Oct-3/4 expression reflects tumor progression and regulates motility of bladder cancer cells

Cancer and embryonic stem cells exhibit similar behavior, including immortal, undifferentiated, and invasive activities. Here, we show that in clinical samples bladder tumors with intense expression of stem cell marker Oct-3/4 (also known as POU5F1) are associated with further disease progression, greater metastasis, and shorter cancer-related survival compared with those with moderate and low expressions. Expression of Oct-3/4 is detected in human bladder transitional cell carcinoma samples and cell lines. Overexpression of Oct-3/4 enhances, whereas knockdown of Oct-3/4 expression by RNA interference reduces, migration and invasion of bladder cancer cells. Oct-3/4 can up-regulate fibroblast growth factor-4 and matrix metalloproteinase-2 (MMP-2), MMP-9, and MMP-13 production, which may contribute to tumor metastasis. Finally, we show that Ad5WS4, an E1B-55 kD-deleted adenovirus driven by the Oct-3/4 promoter, exerts potent antitumor activity against bladder cancer in a syngeneic murine tumor model. Therefore, our results implicate that Oct-3/4 may be useful as a novel tumor biological and prognostic marker and probably as a potential therapeutic target for bladder cancer.     

HNF4A充当肿瘤抑制基因抑制结直肠肿瘤进展的实验

目的 探究HNF4A在结直肠肿瘤进展中的作用。方法 构建HNF4A过表达或敲除的结直肠癌细胞系,通过实时定量PCR和Western blot检测构建的细胞系HNF4A的表达水平;通过软琼脂及平板克隆形成实验检测细胞系的克隆形成能力,CCK-8法检测细胞的增殖能力;Western blot检测干细胞标志物的表达水平;最后通过Transwell迁移与侵袭实验检测细胞的迁移侵袭能力,R2基因分析平台分析结直肠癌中HNF4A与基质金属蛋白酶MMP2及MMP9表达的相关性并通过Western blot验证。结果 过表达HNF4A抑制了结直肠肿瘤细胞的增殖与克隆形成能力及迁移侵袭能力,敲低HNF4A促进了结直肠癌细胞的增殖与克隆能力及迁移侵袭能力,Western blot显示HNF4A抑制了结直肠癌干细胞的标志物CD133、CD44及EpCAM的表达和基质金属蛋白酶MMP2及MMP9的表达,在HNF4A敲低的细胞系中得出了同样的结论。结论 HNF4A通过抑制结直肠癌干细胞特征及金属蛋白酶的表达来抑制结直肠肿瘤的进展。     

BMAL1基因抑制耐放射鼻咽癌细胞株增殖、迁移和侵袭能力机制研究

目的探究BMAL1基因对耐放射鼻咽癌细胞株（5‐8FR）增殖、迁移和侵袭能力的影响及分子机制。方法 小剂量分次照射构建鼻咽癌耐放射细胞5‐8FR,运用克隆形成实验结果拟合多靶单击模型并计算放疗增敏比。通过蛋白质印迹实验检测5‐8FR和对照组5‐8F细胞株中PI3K/Akt/MMP‐2/9信号通路相关蛋白的表达。构建BMAL1基因的高表达及敲除载体,分别转染鼻咽癌细胞株5‐8F和耐放射细胞株5‐8FR,获得BMAL1基因过表达（pcDNA‐BMAL1）及其对照组（pcDNA）和干扰（BMAL1‐shRNA）及对照组（con‐shRNA）的稳转细胞株。蛋白质印迹法验证感染效率,检测两组细胞过表达或干扰BMAL1基因后PI3K/Akt/MMP‐2/9信号通路相关蛋白的改变情况。采用CCK‐8法、划痕实验、Transwell法检测过表达及干扰BMAL1基因后耐放疗细胞株5‐8FR增殖、迁移和侵袭能力的变化。结果 鼻咽癌耐放射细胞株中BMAL1基因表达下调,PI3K/Akt通路蛋白及下游相关分子MMP‐2、MMP‐9表达增加,TIMP‐2、TIMP‐1表达降低。过表达BMAL1基因可抑制PI3K/Akt通路蛋白及下游相关分子MMP‐2、MMP‐9表达,促进TIMP‐2、TIMP‐1表达,抑制耐放射鼻咽癌细胞的增殖、迁移和侵袭能力,干扰BMAL1基因则结果相反。结论 BMAL1基因可以逆转耐放射鼻咽癌细胞株PI3K/Akt/MMP‐2/9信号通路相关蛋白的表达,并抑制耐放射鼻咽癌细胞株的增殖、迁移和侵袭能力。     

Overexpression of Fn14 promotes androgen-independent prostate cancer progression through MMP-9 and correlates with poor treatment outcome

Fibroblast growth factor-inducible 14 (Fn14), a transmembrane receptor binding to the multifunctional cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK), is known to modulate many cellular activities including cancer progression. Here, we demonstrated the significant role of Fn14 in invasion, migration and proliferation of androgen-independent prostate cancer (AIPC) cells. Fn14 and its ligand TWEAK were highly expressed in two AIPC cell lines, DU 145 and PC-3, whereas expression was weak in androgen-sensitive LNCaP cells. Fn14 knockdown using small-interfering RNAs attenuated migration, invasion and proliferation and enhanced apoptosis in the AIPC cell lines. Both forced overexpression of Fn14 by stable Fn14 complementary DNA transfection to PC-3 cells (PC-3/Fn14) and ligand activation by recombinant TWEAK in PC-3 cells enhanced invasion. Fn14 was shown to modulate expression of matrix metalloproteinase (MMP)-9, and MMP-9 mediated the invasive potential influenced by Fn14 in PC-3 cells. In vivo, subcutaneous xenografts of PC-3/Fn14 grew significantly faster than xenograft of PC-3/Mock, and the invasive capacity in PC-3/Fn14 was found to be higher than that of PC-3/Mock as evaluated in an invasion model of the diaphragm. Furthermore, the messenger RNA expressions of MMP-9 in PC-3/Fn14 xenografts were significantly higher than those in PC-3/Mock xenografts. Clinically, high expression of Fn14 was significantly associated with higher prostate-specific antigen recurrence rate in patients who underwent radical prostatectomy. In conclusion, the overexpression of Fn14 may contribute to multiple malignant cellular phenotypes associated with prostate cancer (PCa) progression, in part via MMP-9. TWEAK-Fn14 signaling may be a novel therapeutic target of PCa.