RASSF1C modulates the expression of a stem cell renewal gene, PIWIL1

ABSTRACT: BACKGROUND: RASSF1A and RASSF1C are two major isoforms encoded by the Ras association domain family 1 (RASSF1) gene through alternative promoter selection and mRNA splicing. RASSF1A is a well established tumor suppressor gene. Unlike RASSF1A, RASSF1C appears to have growth promoting actions in lung cancer. In this article, we report on the identification of novel RASSF1C target genes in non small cell lung cancer (NSCLC). METHODS: Over-expression and siRNA techniques were used to alter RASSF1C expression in human lung cancer cells, and Affymetrix-microarray study was conducted using NCI-H1299 cells over-expressing RASSF1C to identify RASSF1C target genes. RESULTS: The microarray study intriguingly shows that RASSF1C modulates the expression of a number of genes that are involved in cancer development, cell growth and proliferation, cell death, and cell cycle. We have validated the expression of some target genes using qRT-PCR. We demonstrate that RASSF1C over-expression increases, and silencing of RASSF1C decreases, the expression of PIWIL1 gene in NSCLC cells using qRT-PCR, immunostaining, and Western blot analysis. We also show that RASSF1C over-expression induces phosphorylation of ERK1/2 in lung cancer cells, and inhibition of the MEK-ERK1/2 pathway suppresses the expression of PIWIL1 gene expression, suggesting that RASSF1C may exert its activities on some target genes such as PIWIL1 through the activation of the MEK-ERK1/2 pathway. Also, PIWIL1 expression is elevated in lung cancer cell lines compared to normal lung epithelial cells. CONCLUSIONS: Taken together, our findings provide significant data to propose a model for investigating the role of RASSF1C/PIWIL1 proteins in initiation and progression of lung cancer.     

Ras association domain family 1C protein stimulates human lung cancer cell proliferation

Recently, the Ras association domain family 1 gene (RASSF1) has been identified as a Ras effector encoding two major mRNA forms, RASSF1A and RASSF1C, derived by alternative promoter selection and alternative mRNA splicing. RASSF1A is a tumor suppressor gene. However, the function of RASSF1C, both in normal and cancer cells, is still unknown. To learn more about the function of RASSF1C in human cancer cells, we tested the effect of silencing RASSF1C mRNA with small interfering RNA on lung cancer cells (NCI H1299) that express RASSF1C but not RASSF1A. Small interfering RNA specific for RASSF1C reduced RASSF1C mRNA levels compared with controls. This reduction in RASSF1C expression caused a significant decrease in lung cancer cell proliferation. Furthermore, overexpression of RASSF1C increased cell proliferation in lung cancer cells. Finally, we found that RASSF1C, unlike RASSF1A, does not upregulate N-cadherin 2 and transglutaminase 2 protein expression in NCI H1299 lung cancer cells. This suggests that RASSF1C and RASSF1A have different effector targets. Together, our findings suggest that RASSF1C, unlike RASSF1A, is not a tumor suppressor but rather stimulates lung cancer cell proliferation.     

Epigenetic inactivation of the Ras-association domain family 1 (RASSF1A) gene and its function in human carcinogenesis

The Ras GTPases are a superfamily of molecular switches that regulate cellular proliferation and apoptosis in response to extra-cellular signals. The regulation of these pathways depends on the interaction of the GTPases with specific effectors. Recently, we have cloned and characterized a novel gene encoding a putative Ras effector: the Ras-association domain family 1 (RASSF1) gene. The RASSF1 gene is located in the chromosomal segment of 3p21.3. The high allelic loss in a variety of cancers suggested a crucial role of this region in tumorigenesis. At least two forms of RASSF1 are present in normal human cells. The RASSF1A isoform is highly epigenetically inactivated in lung, breast, ovarian, kidney, prostate, thyroid and several other carcinomas. Re-expression of RASSF1A reduced the growth of human cancer cells supporting a role for RASSF1 as a tumor suppressor gene. RASSF1A inactivation and K-ras activation are mutually exclusive events in the development of certain carcinomas. This observation could further pinpoint the function of RASSF1A as a negative effector of Ras in a pro-apoptotic signaling pathway. In malignant mesothelioma and gastric cancer RASSF1A methylation is associated with virus infection of SV40 and EBV, respectively, and suggests a causal relationship between viral infection and progressive RASSF1A methylation in carcinogenesis. Furthermore, a significant correlation between RASSF1A methylation and impaired lung cancer patient survival was reported, and RASSF1A silencing was correlated with several parameters of poor prognosis and advanced tumor stage (e.g. poor differentiation, aggressiveness, and invasion). Thus, RASSF1A methylation could serve as a useful marker for the prognosis of cancer patients and could become important in early detection of cancer.     

RASSF1C oncogene elicits amoeboid invasion,cancer stemness,and extracellular vesicle release via a SRC/Rho axis

Cell plasticity is a crucial hallmark leading to cancer metastasis. Upregulation of Rho/ROCK pathway drives actomyosin contractility, protrusive forces, and contributes to the occurrence of highly invasive amoeboid cells in tumors. Cancer stem cells are similarly associated with metastasis, but how these populations arise in tumors is not fully understood. Here, we show that the novel oncogene RASSF1C drives mesenchymal‐to‐amoeboid transition and stem cell attributes in breast cancer cells. Mechanistically, RASSF1C activates Rho/ROCK via SRC‐mediated RhoGDI inhibition, resulting in generation of actomyosin contractility. Moreover, we demonstrate that RASSF1C‐induced amoeboid cells display increased expression of cancer stem‐like markers such as CD133, ALDH1, and Nanog, and are accompanied by higher invasive potential in vitro and in vivo. Further, RASSF1C‐induced amoeboid cells employ extracellular vesicles to transfer the invasive phenotype to target cells and tissue. Importantly, the underlying RASSF1C‐driven biological processes concur to explain clinical data: namely, methylation of the RASSF1C promoter correlates with better survival in early‐stage breast cancer patients. Therefore, we propose the use of RASSF1 gene promoter methylation status as a biomarker for patient stratification.     

PIWIL4在人卵巢癌中的表达及其功能研究

采用半定量RT-PCR方法检测人卵巢透明细胞癌ES-2细胞中PIWIL1、PIWIL2、PIWIL3、PIWIL4 mRNA表达水平,以及PIWIL4在卵巢癌组织和癌旁正常组织中的表达情况．设计并化学合成针对PIWIL4的siRNA,用脂质体转染法将其转入ES-2细胞内,通过MTT和克隆形成实验,观察PIWIL4-siRNA对ES-2细胞生长活性和增殖能力的影响．半定量RT-PCR实验结果发现,ES-2细胞中,PIWIL4相对PIWIL1、PIWIL2、PIWIL3,其表达水平最高(P < 0.05),而且PIWIL4在卵巢癌组织中的表达明显高于癌旁正常组织(P < 0.01)．MTT实验和克隆形成实验结果显示,转染PIWIL4-siRNA的ES-2细胞生长活性和克隆形成率明显低于对照组(P < 0.05)．由此得出结论：PIWIL4在卵巢癌细胞系ES-2细胞表达较高,且它在卵巢癌组织中的表达明显上调,同时PIWIL4-siRNA可有效抑制ES-2细胞的生长活性和增殖能力,提示PIWIL4可能与卵巢癌发生、发展相关．     

恒河猴piwil4基因的鉴定与表达分析

为了研究人类近亲恒河猴中PIWI家族蛋白PIWIL4的结构和表达情况,文章首次利用同源比对和RT-PCR方法克隆了恒河猴piwil4基因,检测了其mRNA在恒河猴心脏、脑、结肠、附睾和睾丸5种组织中的表达情况,利用生物信息学的方法对恒河猴piwil4基因和人的PIWIL4(HIWI2)基因编码的蛋白产物进行了同源性分析和结构域分析,并进一步利用免疫组化的方法比较了PIWIL4蛋白在成人、成年恒河猴和性未成熟恒河猴睾丸组织中的表达分布。结果表明,恒河猴piwil4 mRNA在多组织中表达,恒河猴和人的PIWIL4蛋白的氨基酸序列同源性达97%以上,均含有PAZ和Piwi结构域,它们在两物种成年个体睾丸组织中空间分布一致,但在不同发育阶段恒河猴睾丸组织中的分布发生了改变,幼猴中PIWIL4蛋白主要表达于生精小管细胞的细胞核,在成年猴睾丸组织中则表达于各种细胞的胞浆中。上述结果提示,piwil4基因在人类和恒河猴精子发生过程中作用类似,PIWIL4蛋白在幼猴和成年猴睾丸组织中的表达差异提示它们在不同发育阶段功能的改变。     

Quantitative analysis of mRNA expression levels and DNA methylation profiles of three neighboring genes: FUS1, NPRL2/G21 and RASSF1A in non-small cell lung cancer patients

Background

Tumor suppressor gene (TSG) inactivation plays a crucial role in carcinogenesis. FUS1, NPRL2/G21 and RASSF1A are TSGs from LUCA region at 3p21.3, a critical chromosomal region in lung cancer development. The aim of the study was to analyze and compare the expression levels of these 3 TSGs in NSCLC, as well as in macroscopically unchanged lung tissue surrounding the primary lesion, and to look for the possible epigenetic mechanism of TSG inactivation via gene promoter methylation.

Methods

Expression levels of 3 TSGs and 2 DNA methyltransferases, DNMT1 and DNMT3B, were assessed using real-time PCR method (qPCR) in 59 primary non-small cell lung tumors and the matched macroscopically unchanged lung tissue samples. Promoter methylation status of TSGs was analyzed using methylation-specific PCRs (MSP method) and Methylation Index (MI) value was calculated for each gene.

Results

The expression of all three TSGs were significantly different between NSCLC subtypes: RASSF1A and FUS1 expression levels were significantly lower in squamous cell carcinoma (SCC), and NPRL2/G21 in adenocarcinoma (AC). RASSF1A showed significantly lower expression in tumors vs macroscopically unchanged lung tissues. Methylation frequency was 38–76 %, depending on the gene. The highest MI value was found for RASSF1A (52 %) and the lowest for NPRL2/G21 (5 %). The simultaneous decreased expression and methylation of at least one RASSF1A allele was observed in 71 % tumor samples. Inverse correlation between gene expression and promoter methylation was found for FUS1 (rs = −0.41) in SCC subtype. Expression levels of DNMTs were significantly increased in 75–92 % NSCLCs and were significantly higher in tumors than in normal lung tissue. However, no correlation between mRNA expression levels of DNMTs and DNA methylation status of the studied TSGs was found.

Conclusions

The results indicate the potential role of the studied TSGs in the differentiation of NSCLC histopathological subtypes. The significant differences in RASSF1A expression levels between NSCLC and macroscopically unchanged lung tissue highlight its possible diagnostic role in lung cancer in situ recognition. High percentage of lung tumor samples with simultaneous RASSF1A decreased expression and gene promoter methylation indicates its epigenetic silencing. However, DNMT overexpression doesn’t seem to be a critical determinate of its promoter hypermethylation.     

RASSF1A suppresses melanoma development by modulating apoptosis and cell-cycle progression

The tumor suppressor candidate gene Ras association domain family 1, isoform A (RASSF1A) encodes a microtubule-associated protein that is implicated in the regulation of cell proliferation, migration, and apoptosis. Several studies indicate that down-regulation of RASSF1A resulting from promoter hypermethylation is a frequent epigenetic abnormality in malignant melanoma. In this study, we report that compared with melanocytes in normal skins or benign skin lesions, RASSF1A is down-regulated in melanoma tissues as well as cell lines, and its expression negatively correlates with lymph node metastasis. Following ectopic expression in RASSF1A-deficient melanoma A375 cell line, RASSF1A reduces cell viability, suppresses cell-cycle progression but enhances apoptotic cell death. In vivo, RASSF1A expression inhibits the tumorigenic potential of A375 cells in nude mice, which also correlates with decreased cell proliferation and increased apoptosis. On the molecular level, ectopic RASSF1A expression leads to differential expression of 209 genes, including 26 down-regulated and 183 up-regulated ones. Among different signaling pathways, activation of the apoptosis signal-regulating kinase 1 (ASK1)/p38 MAP kinase signaling is essential for RASSF1A-induced mitochondrial apoptosis, and the inhibition of the Akt/p70S6 kinase/eIF4E signaling is also important for RASSF1A-mediated apoptosis and cell-cycle arrest. This is the first study exploring the biological functions and the underlying mechanisms of RASSF1A during melanoma development. It also identifies potential targets for further diagnosis and clinical therapy.     

HYAL1 and HYAL2 inhibit tumour growth in vivo but not in vitro

Background

We identified two 3p21.3 regions (LUCA and AP20) as most frequently affected in lung, breast and other carcinomas and reported their fine physical and gene maps. It is becoming increasingly clear that each of these two regions contains several TSGs. Until now TSGs which were isolated from AP20 and LUCA regions (e.g.G21/NPRL2, RASSF1A, RASSF1C, SEMA3B, SEMA3F, RBSP3) were shown to inhibit tumour cell growth both in vitro and in vivo.

Methodology/Principal Findings

The effect of expression HYAL1 and HYAL2 was studied by colony formation inhibition, growth curve and cell proliferation tests in vitro and tumour growth assay in vivo. Very modest growth inhibition was detected in vitro in U2020 lung and KRC/Y renal carcinoma cell lines. In the in vivo experiment stably transfected KRC/Y cells expressing HYAL1 or HYAL2 were inoculated into SCID mice (10 and 12 mice respectively). Tumours grew in eight mice inoculated with HYAL1. Ectopic HYAL1 was deleted in all of them. HYAL2 was inoculated into 12 mice and only four tumours were obtained. In 3 of them the gene was deleted. In one tumour it was present but not expressed. As expected for tumour suppressor genes HYAL1 and HYAL2 were down-expressed in 15 fresh lung squamous cell carcinomas (100%) and clear cell RCC tumours (60–67%).

Conclusions/Significance

The results suggest that the expression of either gene has led to inhibition of tumour growth in vivo without noticeable effect on growth in vitro. HYAL1 and HYAL2 thus differ in this aspect from other tumour suppressors like P53 or RASSF1A that inhibit growth both in vitro and in vivo. Targeting the microenvironment of cancer cells is one of the most promising venues of cancer therapeutics. As major hyaluronidases in human cells, HYAL1 and HYAL2 may control intercellular interactions and microenvironment of tumour cells providing excellent targets for cancer treatment.     

The scaffold protein CNK1 interacts with the tumor suppressor RASSF1A and augments RASSF1A-induced cell death

The connector enhancer of KSR (CNK) is a multidomain scaffold protein discovered in Drosophila, where it is necessary for Ras activation of the Raf kinase. Recent studies have shown that CNK1 also interacts with RalA and Rho and participates in some aspects of signaling by these GTPases. Herein we demonstrate a novel aspect of CNK1 function, i.e. reexpression of CNK1 suppresses tumor cell growth and promotes apoptosis. As shown previously for apoptosis induced by Ki-Ras(G12V), CNK1-induced apoptosis is suppressed by a dominant inhibitor of the mammalian sterile 20 kinases 1 and (MST1/MST2). Immunoprecipitates of MST1 endogenous to LoVo colon cancer cells contain endogenous CNK1; however, no association of these two polypeptides can be detected in a yeast two-hybrid assay. CNK1 does, however, bind directly to the RASSF1A and RASSF1C polypeptides, constitutive binding partners of the MST1/2 kinases. Deletion of the MST1 carboxyl-terminal segment that mediates its binding to RASSF1A/C eliminates the association of MST1 with CNK1. Coexpression of CNK1 with the tumor suppressive isoform, RASSF1A, greatly augments CNK1-induced apoptosis, whereas the nonsuppressive RASSF1C isoform is without effect on CNK1-induced apoptosis. Overexpression of CNK1-(1-282), a fragment that binds RASSF1A but is not proapoptotic, blocks the apoptosis induced by CNK1 and by Ki-Ras(G12V). Thus, in addition to its positive role in the proliferative outputs of active Ras, the CNK1 scaffold protein, through its binding of a RASSF1A.MST complex, also participates in the proapoptotic signaling initiated by active Ras.     

RNA干扰敲减PIWIL1表达对人结肠癌细胞SW620增殖，粘附及侵袭能力影响

PIWIL1为AGO蛋白(Argonaute proteins )PIWI亚家族的成员之一,在睾丸中特异表达. 其在干细胞自我更新、RNA干扰和翻译调节中起着重要的作用.本文采用实时PCR方法检测PIWI基因家族中PIWIL1、PIWIL2、PIWIL3、PIWIL4在人结肠癌SW620细胞中mRNA表达水平,首次证实了在SW620细胞中,PIWIL1相对PIWIL2、PIWIL3、PIWIL4,其表达水平最高（P﹤0.05）.构建表达针对PIWIL1的小发卡结构干扰RNA的重组质粒,并用Western 印迹验证其达到较高的干扰效率.用脂质体将重组质粒转染入SW620细胞中,通过MTT实验、集落形成实验、细胞聚集实验及细胞侵袭转移实验,分别观察到其可抑制SW620细胞的生长、增殖、侵袭转移能力,增强了SW620细胞的粘附能力.提示PIWIL1可能在结肠癌发生、发展过程中发挥作用.     

WT1 Promotes Cell Proliferation in Non-Small Cell Lung Cancer Cell Lines through Up-Regulating Cyclin D1 and p-pRb In Vitro and In Vivo

The Wilms’ tumor suppressor gene (WT1) has been identified as an oncogene in many malignant diseases such as leukaemia, breast cancer, mesothelioma and lung cancer. However, the role of WT1 in non-small-cell lung cancer (NSCLC) carcinogenesis remains unclear. In this study, we compared WT1 mRNA levels in NSCLC tissues with paired corresponding adjacent tissues and identified significantly higher expression in NSCLC specimens. Cell proliferation of three NSCLC cell lines positively correlated with WT1 expression; moreover, these associations were identified in both cell lines and a xenograft mouse model. Furthermore, we demonstrated that up-regulation of Cyclin D1 and the phosphorylated retinoblastoma protein (p-pRb) was mechanistically related to WT1 accelerating cells to S-phase. In conclusion, our findings demonstrated that WT1 is an oncogene and promotes NSCLC cell proliferation by up-regulating Cyclin D1 and p-pRb expression.     

IGFBP-5 regulates muscle cell differentiation by binding to IGF-II and switching on the IGF-II auto-regulation loop

IGF-II stimulates both mitogenesis and myogenesis through its binding and activation of the IGF-I receptor (IGF-IR). How this growth factor pathway promotes these two opposite cellular responses is not well understood. We investigate whether local IGF binding protein-5 (IGFBP-5) promotes the myogenic action of IGF-II. IGFBP-5 is induced before the elevation of IGF-II expression during myogenesis. Knockdown of IGFBP-5 impairs myogenesis and suppresses IGF-II gene expression. IGF-II up-regulates its own gene expression via the PI3K-Akt signaling pathway. Adding IGF-II or constitutively activating Akt rescues the IGFBP-5 knockdown-caused defects. However, an IGF analogue that binds to the IGF-IR but not IGFBP has only a limited effect. When added with low concentrations of IGF-II, IGFBP-5 restores IGF-II expression and myogenic differentiation, whereas an IGF binding–deficient IGFBP-5 mutant has no effect. These findings suggest that IGFBP-5 promotes muscle cell differentiation by binding to and switching on the IGF-II auto-regulation loop.