The tumor suppressor gene rap1GAP is silenced by miR-101-mediated EZH2 overexpression in invasive squamous cell carcinoma

Rap1GAP is a critical tumor suppressor gene that is downregulated in multiple aggressive cancers, such as head and neck squamous cell carcinoma, melanoma and pancreatic cancer. However, the mechanistic basis of rap1GAP downregulation in cancers is poorly understood. By employing an integrative approach, we demonstrate polycomb-mediated repression of rap1GAP that involves Enhancer of Zeste Homolog 2 (EZH2), a histone methyltransferase in head and neck cancers. We further demonstrate that the loss of miR-101 expression correlates with EZH2 upregulation, and the concomitant downregulation of rap1GAP in head and neck cancers. EZH2 represses rap1GAP by facilitating the trimethylation of histone 3 at lysine 27, a mark of gene repression, and also hypermethylation of rap1GAP promoter. These results provide a conceptual framework involving a microRNA-oncogene-tumor suppressor axis to understand head and neck cancer progression.     

Rap1GAP1在人胰腺癌组织中的表达及意义

目的通过检测Rap1GAP1在人胰腺癌中的表达情况及rap1GAP1在三种人胰腺癌细胞系中的突变情况,探讨其在人胰腺癌发生发展过程中的作用。方法(1)采用免疫组织化学Envision两步法,检测73例人胰腺癌组织及其癌旁胰腺组织中Rap1GAP1的表达情况,并分析其与胰腺癌临床病理特征之间的关系;(2)采用RT-PCR和测序法检测三种人胰腺癌细胞系中rap1GAP1的突变情况。结果(1)Rap1GAP1在癌旁胰腺组织、胰腺上皮内肿瘤(pancreatic intraepithelialneoplasia,PanIN)1a、1b、2及3级和浸润性胰腺癌中的阳性率分别为100%、93.8%、92.3%、70.0%、57.9%和13.7%。癌旁胰腺组织、各级PanIN与浸润性癌之间的表达差异有统计学意义(P<0.01);Rap1GAP1在高、中和低分化胰腺癌中阳性率分别为26.9%、7.1%和0,显示Rap1GAP1的表达与胰腺癌的分化程度有关(P<0.05)。而Rap1GAP1的表达与患者的年龄、性别、淋巴结转移情况和临床分期无关。(2)Panc-1、MiaPaCa-2细胞系中存在rap1GAP1关键区域(催化结构域)的大片段缺失,而Aspc-1细胞系含有完整的编码rap1GAP催化结构域的外显子。结论相比于癌旁胰腺组织和各级PanIN,浸润性胰腺癌中 Rap1GAP1的表达显著降低。另外,Rap1GAP1在低分化胰腺癌中的表达显著降低。提示Rap1GAP1可能是胰腺癌发生发展过程中的一个晚期事件。三种人胰腺癌细胞系中的突变分析结果提示其表达降低可能与遗传学上的大片段缺失有关。     

Rap1GAP alters leukemia cell differentiation, apoptosis and invasion in vitro

Rap1GAP which regulates the GTP-GDP form switch of Rap1 is a member of the GTPase-activating protein (GAP) family and has recently received substantial attention. Rap1GAP is thought of as a putative tumor suppressor gene and plays an important role in human tumor progression including pancreatic cancer, thyroid cancer and melanoma. In the current study, we found that the expression of Rap1GAP was lower in acute myeloid leukemia (AML) patients compared to non-malignant blood disease patients. The expression of Rap1GAP was also low in HL-60, NB4, U937 and SHI-1 myeloid leukemia cell lines. Upregulated Rap1GAP in NB4 and HL-60 cells promoted cell differentiation induced by ATRA or TPA compared to the empty vector control cells. Furthermore, Rap1GAP-transfected cells also showed a higher rate of apoptosis in response to arsenic trioxide compared to the control counterpart cells. In addition, we found that increased expression of Rap1GAP promoted leukemia cell invasion may be due to matrix metalloproteinase 9 (MMP9). In conclusion, these results demonstrated that Rap1GAP promoted leukemia cell differentiation and apoptosis, but increased leukemia cell invasion in?vitro.     

抑癌基因ARHI研究进展

ARHI（aplasia ras homologue member I）/NOEY2是1999年新发现的一个母源性抑癌印迹基因,位于人染色体1p31,编码一个相对分子量为26kD的小GTP结合蛋白。ARHI属ras/rap超家族成员,与该家族成员有50%～60%的同源性并且两者具有相似的GTP/GDP结合域,但与该家族其它成员不同,ARHI发挥抑癌基因作用,是该家族第一个被报道的肿瘤抑制基因。ARHI基因编码的蛋白在人类多种组织表达,而该基因在人卵巢癌、乳腺癌、胰腺癌、肝癌等多种肿瘤中表达下调或缺失,提示其与上述肿瘤的发生、发展密切相关。ARHI可能通过作用于cyclin D1,使其不能与CDK结合形成活性激酶,从而使细胞停止于G1期来参与细胞周期调控;可能通过依赖caspase和calpain两条途径参与信号通路传导诱发细胞凋亡;另外,该基因可通过抑制STAT3的激活而发挥抑癌基因功能,也可以调节自体吞噬和肿瘤细胞休眠。目前研究显示,ARHI基因的表达缺失主要通过遗传事件和表观遗传学机制发生,包括DNA甲基化异常、杂合性丢失,乙酰化组蛋白的低水平表达及基因突变有关,但有待进一步深入研究。可以预见,ARHI基因的深入研究必将为早期肿瘤的基因诊治提供新的思路和理论依据。      

肿瘤抑制基因ARHI的生物学功能及研究进展

肿瘤是一种基因病,抑癌基因在抵御肿瘤发生、发展中起着重要作用。ARHI(aplysia ras hom olog I)/NOEY2 是一个新发现的抑癌基因,是 ras/rap超家族成员之一,与 ras家族有50%~60%的同源性,位于人染色体1p31,属小GTP结合蛋白,是该家族第1个被报道的肿瘤抑制基因。ARHI基因编码的蛋白在人类多种组织表达,其中正常卵巢的ARHI表达最高。ARHI是一个印迹基因,印迹机制可能与其CpG岛的差异甲基化有关。ARHI 参与细胞周期调控可能作用于cyclin D1 ,使其不能与 CDK结合形成活性激酶,从而使细胞停止于 G1 期。AR HI可能通过依赖caspase和 calpain两条途径参与信号通路传导诱发细胞凋亡。该基因的异常表达跟多种肿瘤的发生、发展有关。ARHI基因参与了乳腺癌的发生和发展,该基因的表达缺失可能与乳腺癌的转移机制有关。卵巢癌、乳腺癌存在广泛的1p31缺失,其中 ARHI 基因是最常见的一个缺失区域。ARHI基因和蛋白在胰腺癌组织中有较高比例的缺失,提示该基因和蛋白在胰腺癌的发生中起一定作用。ARHI基因在膀胱癌、肝癌、前列腺癌等其他肿瘤中也有不同程度的表达异常。     

Profiling follicle stimulating hormone-induced gene expression changes in normal and malignant human ovarian surface epithelial cells

Epidemiological data have implicated the pituitary gonadotropin follicle stimulating hormone (FSH) as both a risk factor for and a protective agent against epithelial ovarian cancer. Yet, little is known about how this hormone could play such opposing roles in ovarian carcinogenesis. Complementary DNA microarrays containing 2400 named genes were used to examine FSH-induced gene expression changes in ovarian cancer (OC) and immortalized normal human ovarian surface epithelial (HOSE) cell lines. Two-way t-statistics analyses of array data identified two distinct sets of FSH-regulated genes in HOSE and in established OC cell lines established from patients (OVCA cell lines). Among the HOSE cell lines, FSH increased expression of 57% of the 312 genes and downregulated 43%. In contrast, FSH diminished expression of 92% of the 177 genes in the OVCA cell lines. All but 18 of the genes affected by FSH in HOSE cell lines were different from those altered in OVCA cell lines. Among the 18 overlapping genes, nine genes exhibited the same direction of change following FSH challenge, while the other nine showed discordance in response between HOSE and OVCA cell lines. The FSH-induced differential expression of seven out of nine genes was confirmed by real-time RT-PCR. Gene-specific antisense oligonuleotides (ODNs) were used to inhibit the expression of genes encoding GTPase activating protein (rap1GAP), neogenin, and restin in HOSE and OVCA cells. Antisense ODNs to neogenin and restin, but not an antisense ODN to rap1GAP, were effective in inhibiting OVCA cell growth, diminishing proliferating cell nuclear antigen expression, and increasing caspase 3 activities. Furthermore, the ODN to rap1GAP was further shown to be ineffective in altering migration properties of OVCA cell lines. HOSE cell proliferation was not affected by treatment with any of the antisense ODNs. In summary, gene profiling data reveal for the first time that FSH may exert different biological actions on OVCA cells than on HOSE cells, by differential regulation of a set of putative oncogenes/tumor suppressors. Specifically, neogenin and restin were found to exhibit proproliferation/survival action on OC cells.     

Genetic and epigenetic loss of microRNA-31 leads to feed-forward expression of EZH2 in melanoma

MicroRNAs (miRs) play a key role in cancer etiology by coordinately repressing numerous target genes involved in cell proliferation, migration and invasion. The genomic region in chromosome 9p21 that encompasses miR-31 is frequently deleted in solid cancers including melanoma; however the expression and functional role of miR-31 has not been previously studied in melanoma. Here, we queried the expression status and performed functional characterization of miR-31 in melanoma tissues and cell lines. We found that down-regulation of miR-31 was a common event in melanoma tumors and cell lines and was associated with genomic loss in a subset of samples. Down-regulation of miR-31 gene expression was also a result of epigenetic silencing by DNA methylation, and via EZH2-mediated histone methylation. Ectopic overexpression of miR-31 in various melanoma cell lines inhibited cell migration and invasion. miR-31 targets include oncogenic kinases such as SRC, MET, NIK (MAP3K14) and the melanoma specific oncogene RAB27a. Furthermore, miR-31 overexpression resulted in down-regulation of EZH2 and a de-repression of its target gene rap1GAP; increased expression of EZH2 was associated with melanoma progression and overall patient survival. Taken together, our study supports a tumor suppressor role for miR-31 in melanoma and identifies novel therapeutic targets.     

Molecular mechanisms of pancreatic carcinogenesis

Pancreatic ductal adenocarcinoma is one of the most fatal malignancies. Intensive investigation of molecular pathogenesis might lead to identifying useful molecules for diagnosis and treatment of the disease. Pancreatic ductal adenocarcinoma harbors complicated aberrations of alleles including losses of 1p, 6q, 9p, 12q, 17p, 18q, and 21q, and gains of 8q and 20q. Pancreatic cancer is usually initiated by mutation of KRAS and aberrant expression of SHH. Overexpression of AURKA mapping on 20q13.2 may significantly enhance overt tumorigenesity. Aberrations of tumor suppressor genes synergistically accelerate progression of the carcinogenic pathway through pancreatic intraepithelial neoplasia (PanIN) to invasive ductal adenocarcinoma. Abrogation of CDKN2A occurs in low-grade/early PanIN, whereas aberrations of TP53 and SMAD4 occur in high-grade/late PanIN. SMAD4 may play suppressive roles in tumorigenesis by inhibition of angiogenesis. Loss of 18q precedes SMAD4 inactivation, and restoration of chromosome 18 in pancreatic cancer cells results in tumor suppressive phenotypes regardless of SMAD4 status, indicating the possible existence of a tumor suppressor gene(s) other than SMAD4 on 18q. DUSP6 at 12q21-q22 is frequently abrogated by loss of expression in invasive ductal adenocarcinomas despite fairly preserved expression in PanIN, which suggests that DUSP6 works as a tumor suppressor in pancreatic carcinogenesis. Restoration of chromosome 12 also suppresses growths of pancreatic cancer cells despite the recovery of expression of DUSP6; the existence of yet another tumor suppressor gene on 12q is strongly suggested. Understanding the molecular mechanisms of pancreatic carcinogenesis will likely provide novel clues for preventing, detecting, and ultimately curing this life-threatening disease.     

Expression of the tumor suppressor gene Maspin in human pancreatic cancers.

The tumor suppressor gene maspin, a unique member of the serpin superfamily, inhibits cell motility, invasion, and metastasis in breast and prostate cancers. Maspin is expressed in normal human mammary and prostate epithelial cells but down-regulated during cancer progression. In this study, we analyzed the expression of maspin in various human cancer cells by means of Northern blot and immunohistochemistry. Maspin gene expression proved to be up-regulated in pancreatic cancer. Maspin expression was not detected in any of 6 gastric cancers, 4 melanomas, or 6 of 7 breast cancer cell lines examined. In contrast, 5 of 9 pancreatic cancer cell lines showed maspin expression, although maspin expression was not detected in normal pancreatic tissue. Furthermore, maspin was expressed in 23 of 24 tumor specimens obtained from pancreatic cancer patients as well as all high-grade precancerous lesions (PanIN3 and intraductal carcinoma extension). In contrast, no expression was observed in normal and low-grade precancerous lesions. Our results show that maspin is a new factor associated with pancreatic cancer. In addition, the detection of maspin in pancreatic tumor tissues and its lack of expression in all normal pancreatic tissues suggests that maspin may be a useful marker of primary human pancreatic cancer.     

Evidence of MKK4 pro-oncogenic activity in breast and pancreatic tumors

MKK4, located in close proximity to p53 gene, is thought to be a tumor suppressor and a metastasis suppressor gene. A low-rate MKK4 gene alteration has been found in a few tumor types, including breast and pancreatic. A suppressor activity for prostate and ovarian tumor metastasis has also been suggested. To understand the pathobiologic roles of MKK4 in tumorigenesis, we examined the phenotypic changes in response to perturbation of the MKK4 expression in breast and pancreatic cancer cell lines. Ectopic expression of MKK4 by adenoviral delivery in MKK4-negative cancer lines stimulated the cell proliferation and invasion, whereas knockdown of MKK4 expression by small interference RNA in an MKK4-positive breast cancer cell line, MDA-MB-231, resulted in decreased anchorage-independent growth, suppressed tumor growth in mouse xenograft model, and increased cell susceptibility to apoptosis brought by stress signals such as serum deprivation. These results argue that MKK4 functions as a pro-oncogenic molecule instead of a suppressor in breast and pancreatic tumors.     

Downregulation and CpG island hypermethylation of NES1/hK10 gene in the pathogenesis of human gastric cancer

The normal epithelial cell-specific-1 (NES1)/Kallikrein 10 gene is proposed to be a novel putative tumor suppressor gene in several malignant diseases. The role of NES1 gene in gastric cancer has not been fully understood. Our study revealed that CpG island hypermethylation plays an important role in the downregulation of NES1 mRNA expression in gastric cancer. In situ hybridization showed that loss or reduction of NES1 mRNA expression is associated with differentiation level during tumor progression suggesting that NES1 inactivation might contribute to the malignant progression of human gastric cancers.     

KAI1 inhibits anchorage-dependent and -independent pancreatic cancer cell growth

Decreased expression of the tumor suppressor gene, KAI1, is associated with metastasis formation in pancreatic cancer. The aim of the present study was to investigate whether KAI1 influences pancreatic cancer cell growth and colony formation. A full-length KAI1 cDNA expression vector was stably transfected into Panc-1 and MiaPaCa-2 pancreatic cancer cell lines. Transfection was confirmed by Western blot analysis and immunohistochemistry. Tumor cell growth and cell cycle distribution were determined by MTT cell growth assays, colony formation assays, and flow cytometric analysis. KAI1-transfected, but not control-transfected pancreatic cancer cells displayed cytoplasmic KAI1 immunoreactivity. Cell proliferation decreased in the KAI1-transfected cells compared to parental and control cells together with a Go/G1-phase cell cycle arrest. Colony formation was reduced by 2.6- and 3.5-fold in the KAI1-transfected Panc-1 and MiaPaCa-2 pancreatic cancer cells, respectively, compared with parental cells. KAI1 blocks pancreatic cancer cell growth through cell cycle arrest and inhibits anchorage-independent cell growth. These findings support the premise that KAI1 functions as a tumor suppressor in this malignancy.     

Disruption of p16 and activation of Kras in pancreas increase ductal adenocarcinoma formation and metastasis in vivo

Inactivation of tumor suppressor gene p16/INK4A and oncogenic activation of KRAS occur in almost all pancreatic cancers. To better understand the roles of p16 in pancreatic tumorigenesis, we created a conditional p16 knockout mouse line (p16flox/flox), in which p16 is specifically disrupted in a tissue-specific manner without affecting p19/ARF expression. p16flox/flox; LSL-KrasG12D; Pdx1-Cre mice developed the full spectrum of pancreatic intraepithelial neoplasia (mPanIN) lesions, pancreatic ductal adenocarcinoma (PDA), and metastases were observed in all the mice. Here we report a mouse model that simulates human pancreatic tumorigenesis at both genetic and histologic levels and is ideal for studies of metastasis. During the progression from primary tumors to metastases, the wild-type allele of Kras was progressively lost (loss of heterozygosity at Kras or LOH at Kras) in p16flox/flox; LSL- KrasG12D; Pdx1-Cre mice. These observations suggest a role for Kras beyond tumor initiation. In vitro assays performed with cancer cell lines derived from primary pancreatic tumors of these mice showed that cancer cells with LOH at Kras exhibited more aggressive phenotypes than those retained the wild-type Kras allele, indicating that LOH at Kras can provide cancer cells functional growth advantages and promote metastasis. Increased LOH at KRAS was also observed in progression of human pancreatic primary tumors to metastases, again supporting a role for the KRAS gene in cancer metastasis. This finding has potential translational implications- future KRAS target therapies may need to consider targeting oncogenic KRAS specifically without inhibiting wild-type KRAS function.     

Hypermethylation in human cancers of the RIZ1 tumor suppressor gene, a member of a histone/protein methyltransferase superfamily.

The retinoblastoma protein-interacting zinc finger gene RIZ1 is a tumor suppressor gene and a member of a nuclear histone/protein methyltransferase superfamily. RIZ1 inactivation is commonly found in many types of human cancers and occurs through loss of mRNA expression, frameshift mutation, chromosomal deletion, and missense mutation. RIZ1 is also a tumor susceptibility gene in mice. We now show that loss of RIZ1 mRNA in human cancers is associated with DNA methylation of its promoter CpG island. Methylation of the RIZ1 promoter strongly correlated with lost or decreased RIZ1 mRNA expression in breast, liver, colon, and lung cancer cell lines as well as in liver cancer tissues. Treatment with the methylation inhibitor 5-aza-2'-deoxycytidine activated RIZ1 mRNA expression in cancer cells. Furthermore, methylation was found in 11 of 25 (44%) breast cancer specimens and 20 of 32 (62%) liver cancer specimens. Our results suggest that DNA methylation is a common mechanism in inactivating the RIZ1 tumor suppressor gene in human liver and breast cancers.     

MicroRNA-218 inhibits cell invasion and migration of pancreatic cancer via regulating ROBO1

miRNA-218 is a highlighted tumor suppressor and its underlying role in tumor progression is still unknown. Here, we restored the expression of miRNA-218 in pancreatic cancer to clarify the function and potent downstream pathway of miRNA-218. The expressions of both miRNA-218 and its potent target gene ROBO1 were revealed by RT-PCR and western blotting analysis. Transfection of miRNA-218 precursor mimics and luciferase assay were performed to elucidate the regulation mechanism between miRNA-218 and ROBO1. Cells, stably expressing miRNA-218 followed by forced expression of mutant ROBO1, were established through co-transfections of both lentivirus vector and plasmid vector. The cell migration and invasion abilities were evaluated by migration assay and invasion assay respectively. An increased expression of ROBO1 was revealed in cell BxPC-3-LN compared with cell BxPC-3. Elevated expression of miRNA-218 would suppress the expression of ROBO1 via complementary binding to a specific region within 3′UTR of ROBO1 mRNA (sites 971–978) in pancreatic cancer cells. Stably restoring the expression of miRNA-218 in pancreatic cancer significantly downregulated the expression of ROBO1 and effectively inhibited cell migration and invasion. Forced expression of mutant ROBO1 could reverse the repression effects of miRNA-218 on cell migration and invasion. Consequently, miRNA-218 acted as a tumor suppressor in pancreatic cancer by inhibiting cell invasion and migration. ROBO1 was a functional target of miRNA-218’s downstream pathway involving in cell invasion and migration of pancreatic cancer.     

Epigenetic silencing of the candidate tumor suppressor gene Per1 in non-small cell lung cancer.

PURPOSE: Epigenetic events are a critical factor contributing to cancer development. The purpose of this study was to identify tumor suppressor genes silenced by DNA methylation and histone deacetylation in non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: We used microarray analysis to screen for tumor suppressor genes. RESULTS: We identified Per1, a core circadian gene, as a candidate tumor suppressor in lung cancer. Although Per1 levels were high in normal lung, its expression was low in a large panel of NSCLC patient samples and cell lines. Forced expression of Per1 in NSCLC cell lines led to significant growth reduction and loss of clonogenic survival. Recent studies showed that epigenetic regulation, particularly histone H3 acetylation, is essential for circadian function. Using bisulfite sequencing and chromatin immunoprecipitation, we found that DNA hypermethylation and histone H3 acetylation are potential mechanisms for silencing Per1 expression NSCLC. CONCLUSIONS: These results support the hypothesis that disruption of circadian rhythms plays an important role in lung tumorigenesis. Moreover, our findings suggest a novel link between circadian epigenetic regulation and cancer development.