空泡膜蛋白1及其与肿瘤关系的研究进展

0 引言 空泡膜蛋白Ⅰ (vacuole membrane protein 1,Vmp1)[1]是近年来发现的一个跨膜蛋白.研究发现Vmp1为蛋白分泌、细胞器形成及多细胞发育过程所必需,在人体许多肿瘤组织中如乳腺癌、肾癌、肝癌等均有表达,且在原发与转移性肿瘤中表达不一致,被预测其可能是一个肿瘤相关蛋白.现概要介绍Vmp1的发现、结构特点、生物学功能,并着重综述Vmp1与肿瘤之间的关系及相关机制的研究进展. 1 Vmp1的介绍 1.1 Vmp1的发现与结构 Vmp1为Dusetti等[1]于2002年在小鼠体内首次发现的一种与急性胰腺炎有关的蛋白质,通过在GenBank数据库中的比对,发现Vmp1和果蝇、新杆状线虫、拟南芥属等的氨基酸序列具有高度同源性,在不同物种中具有序列保守性,提示Vmp1可能具有重要的功能.     

DNA双链修复相关基因NBS1的分子改变与肿瘤发生

NBS1基因是一种重要的DNA双链修复相关基因,对维持基因组稳定性及防止细胞癌变具有非常关键的作用.近年研究发现NBS1基因的突变、SNP基因型以及NBS1蛋白表达改变与肿瘤的发生风险和预后有关,本文对此作一综述.     

蛋白酪氨酸磷酸酶1B与血液系统肿瘤关系研究进展

酪氨酸磷酸化是一种重要的调节细胞进程和维持稳态的翻译后修饰,酪氨酸磷酸化的异常改变常与疾病发生如代谢障碍、肿瘤和心血管疾病等密切相关.蛋白酪氨酸磷酸酶(PTP)是在细胞增殖和信号转导过程中调节蛋白酪氨酸残基磷酸化水平的酶家族.目前研究发现人类共有1 12种PTP,其中蛋白酪氨酸磷酸酶1B(PTP1B)是该家族中研究最热、最清楚的成员之一.近年来,PTP1B作为一种积极参与肿瘤发生的酶,引起了广泛关注.现就PTP1B与血液系统肿瘤关系的研究进展进行综述.     

Vasohibin-1与肿瘤

Vasohibin-1(VASH1)是新近发现的在血管内皮细胞中由血管内皮生长因子(VEGF)、成纤维生长因子-2(FGF-2)等诱导产生的具有负反馈调节血管生成作用的蛋白因子.近年来研究表明,VASH1在多种肿瘤血管生成中扮演着重要的角色,提示其有望成为相关肿瘤诊断和治疗的分子标志物以及分子靶向治疗的有效蛋白因子.     

p27、cyclin D1与恶性肿瘤

细胞周期调控紊乱是恶性肿瘤发生、发展的主要原因之一,其中,蛋白p27与cyclin D1在细胞周期调控中发挥了重要作用.p27与cyclinD1是细胞周期正负调控因子.p27蛋白在多数肿瘤中低表达或不表达,而cyclinD1蛋白多呈高表达,它们与恶性肿瘤的发生、发展关系密切.目前细胞周期及其调控机制与肿瘤的关系已成为研究热点.     

KiSS-1基因与肿瘤关系的研究进展

KiSS-1蛋白在正常生理发育过程和肿瘤发生发展中发挥着重要作用,尤其是近年来研究发现KiSS-1基因在多种肿瘤中异常表达,如乳腺癌、黑色素瘤等,提示KiSS-1参与抑制肿瘤细胞迁移。本文就KiSS-1的结构功能及其与肿瘤的关系做一综述。     

RNA结合蛋白Musashi1在结直肠癌中的表达及临床意义

Musashi1(Msi1)是一种进化保守的RNA结合蛋白,同时它是肠道、神经等多种组织中的干细胞标志物,对于维持自我更新与分化之间的平衡具有重要作用.近年来研究发现Msi1在多种肿瘤组织尤其是结直肠癌中高表达,参与调控细胞周期、增殖、凋亡等过程,逐渐成为多种癌变的关键调节剂,有望成为肿瘤治疗的新靶点.     

Tob1与肿瘤关系研究进展

 Tob1是Tob／BTG家族成员之一，而Tob／BTG是一种抗增殖蛋白家族。研究发现，Tob1参与肿瘤发展、胚胎发育、骨质增生等过程。Tob1与肿瘤发生、发展关系密切，并在抑制肿瘤生长过程中起着重要的作用，就Tob1生物学特性及其与肿瘤关系的研究进展进行综述。。     

IQGAPl的功能及其在肿瘤发生发展中的作用

IQGAP1是一种能结合多种信号分子及结构分子的骨架蛋白.通过与目的蛋白相互作用.参与信号通路的整合和细胞功能的调节.越来越多的研究发现,IQGAP1可通过促进细胞增殖和转化,降低细胞黏附性以及刺激细胞活动性和侵袭性而在肿瘤的发生发展中扮演重要角色.     

新疆地区维、汉民族妇女散发性乳腺癌BARD1基因突变检测分析

背景与目的:乳腺癌是基因环境交互作用的复杂疾病,高共显的易感基因突变与家族性乳腺癌密切相关.本研究旨在探讨新疆地区维、汉民族女性散发性乳腺癌患者BARD1基因突变情况及突变位置,以期为后续研究奠定基础.方法:随机选取维、汉民族妇女散发性乳腺癌各30例,收集临床病理资料及癌组织与血液标本.应用启动子和外显子测序的方法检测占BARD1基因突变,并在血液样本中测序来验证上述结果.结果:(L)维族和汉族散发性乳腺癌患者在生育史上差异有统计学意义,且维族有很大比例为非雌激素依赖性乳腺癌.(2)维族散发性乳腺癌患者BARD1基因突变率为33.3%,而汉族为13.8%,但差异无统计学意义(X<'2>=3.11,P=0.07).新发现的14个低频SNP全部位于非编码区.(3)血液验证除一个位点rs28997575外,其余43个全部一致,提示突变为生殖细胞突变.结论:维、汉民族的乳腺癌类型有差异,治疗也应区别对待.BARD1基因突变在维族乳腺癌患者中作用可能大于汉族患者,机制可能是增加了乳腺癌易感性.     

HP1 regulates the localization of FANCJ at sites of DNA double‐strand breaks

The breast and ovarian cancer predisposition protein BRCA1 forms three mutually exclusive complexes with Fanconi anemia group J protein (FANCJ, also called BACH1 or BRIP1), CtIP, and Abraxas/RAP80 through its BRCA1 C terminus (BRCT) domains, while its RING domain binds to BRCA1‐associated RING domain 1 (BARD1). We recently found that the interaction between heterochromatin protein 1 (HP1) and BARD1 is required for the accumulation of BRCA1 and CtIP at sites of DNA double‐strand breaks. Here, we investigated the importance of HP1 and BARD1–HP1 interaction in the localization of FANCJ together with the other BRCA1–BRCT binding proteins to clarify the separate role of the HP1‐mediated pathway from the RNF8/RNF168‐induced ubiquitin‐mediated pathway for BRCA1 function. FANCJ interacts with HP1γ in a BARD1‐dependent manner, and this interaction was enhanced by ionizing radiation or irinotecan hydrochloride treatment. Simultaneous depletion of all three HP1 isoforms with shRNAs disrupts the accumulation of FANCJ and CtIP, but not RAP80, at double‐strand break sites. Replacement of endogenous BARD1 with a mutant BARD1 that is incapable of binding to HP1 also disrupts the accumulation of FANCJ and CtIP, but not RAP80. In contrast, RNF168 depletion disrupts the accumulation of only RAP80, but not FANCJ or CtIP. Consequently, the accumulation of conjugated ubiquitin was only inhibited by RNF168 depletion, whereas the accumulation of RAD51 and sister chromatid exchange were only inhibited by HP1 depletion or disruption of the BARD1–HP1 interaction. Taken together, the results suggest that the BRCA1–FANCJ and BRCA1–CtIP complexes are not downstream of the RNF8/RNF168/ubiquitin pathway, but are instead regulated by the HP1 pathway that precedes homologous recombination DNA repair.     

Nuclear-cytoplasmic shuttling of BARD1 contributes to its proapoptotic activity and is regulated by dimerization with BRCA1

The breast cancer-associated protein, BARD1, colocalizes with BRCA1 in nuclear foci in the S phase and after DNA damage, and the two proteins form a stable heterodimer implicated in DNA repair, protein ubiquitination, and control of mRNA processing. BARD1 has a BRCA1-independent proapoptotic activity; however, little is known about its regulation. Here, we show that BARD1 localization and apoptotic activity are regulated by nuclear-cytoplasmic shuttling. We identified a functional CRM1-dependent nuclear export sequence (NES) near the N-terminal RING domain of BARD1. The NES forms part of the BRCA1 dimerization domain, and coexpression of BRCA1 resulted in masking of the NES and nuclear retention of BARD1. In transient expression assays, BARD1 apoptotic activity was stimulated by nuclear export, and both apoptotic function and nuclear export were markedly reduced by BRCA1. Similar findings were obtained for endogenous BARD1. Silencing BRCA1 expression by siRNA, or disrupting the endogenous BARD1/BRCA1 interaction by peptide competition caused a reduction in BARD1 nuclear localization and foci formation, and increased the level of cytoplasmic BARD1 correlating with increased apoptosis. Our findings suggest that BRCA1/BARD1 heterodimer formation is important for optimal nuclear targeting of BARD1 and its role in DNA repair and cell survival.     

BARD1: an independent predictor of survival in non-small cell lung cancer

BRCA1 mRNA overexpression is correlated with poor survival in NSCLC. However, BRCA1 functions depend on the interaction with BARD1 for its stability, nuclear localization and ubiquitin ligase activity. Expression of alternatively spliced BARD1 isoforms that lack the BRCA1-interaction domain was found upregulated and correlated with poor prognosis in breast and ovarian cancer. These BARD1 isoforms are essential for proliferation of cancer cells in vitro. We investigated whether BARD1 isoforms are expressed in NSCLC. While in lung tissues from healthy controls BARD1 expression was undetectable on the mRNA level and protein level, we found two novel isoforms in addition to previously identified mRNAs expressed in all NSCLC samples tested. Furthermore, the pattern of BARD1 isoform expression was similar in tumor and morphologically normal peri-tumor tissues, and only one novel isoform π was specifically upregulated in tumors. Immunohistochemistry revealed that all 100 NSCLC cases tested expressed isoform-specific BARD1 epitopes, while BARD1 expression was undetectable in biopsies from healthy controls. Statistical analysis showed that the expression of epitopes PVC and WFS, present on isoform π, or epitope WFS alone, expressed on isoforms π, κ and β, were significantly correlated with decreased patient survival. These findings were corroborated in a mouse model of chemically induced lung cancer. Immunostaining of mouse tumors showed that BARD1 epitopes PVC and WFS were specifically upregulated in invasive, but not in confined lung tumors. Thus, BARD1 isoforms might be involved in tumor initiation and invasive progression and might represent a novel prognostic marker for NSCLC.     

DNA damage-induced BARD1 phosphorylation is critical for the inhibition of messenger RNA processing by BRCA1/BARD1 complex

BRCA1-associated RING domain protein BARD1, along with its heterodimeric partner BRCA1, plays important roles in cellular response to DNA damage. Immediate cellular response to genotoxic stress is mediated by a family of phosphoinositide 3-kinase-related protein kinases, such as ataxia-telangiectasia mutated (ATM), ATM and Rad3-related, and DNA-dependent protein kinase. ATM-mediated phosphorylation of BRCA1 enhances the DNA damage checkpoint functions of BRCA1, but how BARD1 is regulated during DNA damage signaling has not been examined. Here, we report that BARD1 undergoes phosphorylation upon ionizing radiation or UV radiation and identify Thr(714) as the in vivo BARD1 phosphorylation site. Importantly, DNA damage functions of BARD1 (i.e., inhibition of pre-mRNA polyadenylation and degradation of RNA polymerase II) are abrogated in T714A and T734A mutants. Our findings suggest that phosphorylation of BARD1 is critical for the DNA damage functions of the BRCA1/BARD1 complex.     

Dysregulation of the centrosome induced by BRCA1 deficiency contributes to tissue-specific carcinogenesis

Alterations in breast cancer gene 1 (BRCA1), a tumor suppressor gene, increase the risk of breast and ovarian cancers. BRCA1 forms a heterodimer with BRCA1-associated RING domain protein 1 (BARD1) and functions in multiple cellular processes, including DNA repair and centrosome regulation. BRCA1 acts as a tumor suppressor by promoting homologous recombination (HR) repair, and alterations in BRCA1 cause HR deficiency, not only in breast and ovarian tissues but also in other tissues. The molecular mechanisms underlying BRCA1 alteration-induced carcinogenesis remain unclear. Centrosomes are the major microtubule-organizing centers and function in bipolar spindle formation. The regulation of centrosome number is critical for chromosome segregation in mitosis, which maintains genomic stability. BRCA1/BARD1 function in centrosome regulation together with Obg-like ATPase (OLA1) and receptor for activating protein C kinase 1 (RACK1). Cancer-derived variants of BRCA1, BARD1, OLA1, and RACK1 do not interact, and aberrant expression of these proteins results in abnormal centrosome duplication in mammary-derived cells, and rarely in other cell types. RACK1 is involved in centriole duplication in the S phase by promoting polo-like kinase 1 activation by Aurora A, which is critical for centrosome duplication. Centriole number is higher in cells derived from mammary tissues compared with in those derived from other tissues, suggesting that tissue-specific centrosome characterization may shed light on the tissue specificity of BRCA1-associated carcinogenesis. Here, we explored the role of the BRCA1-containing complex in centrosome regulation and the effect of its deficiency on tissue-specific carcinogenesis.     

Nuclear-cytoplasmic translocation of BARD1 is linked to its apoptotic activity

The tumor suppressor protein BARD1 plays a dual role in response to genotoxic stress: DNA repair as a BARD1-BRCA1 heterodimer and induction of apoptosis in a BRCA1-independent manner. We have constructed a series of BARD1 deletion mutants and analysed their cellular distribution and capacity to induce apoptosis. As opposed to previous studies suggesting an exclusively nuclear localization of BARD1, we found, both in tissues and cell cultures, nuclear and cytoplasmic localization of BARD1. Enhanced cytoplasmic localization of BARD1, as well as appearance of a 67 kDa C-terminal proteolytic cleavage product, coincided with apoptosis. BARD1 translocates to the nucleus independently of BRCA1. For recruitment to nuclear dots, however, the BRCA1-interacting RING finger domain is required but not sufficient. Protein levels of N-terminal RING finger deletion mutants were much higher than those of full-length BARD1, despite comparable mRNA levels, suggesting that the N-terminal region comprising the RING finger is important for BARD1 degradation. Sequences required for apoptosis induction were mapped between the ankyrin repeats and the BRCT domains coinciding with two known cancer-associated missense mutations. We suggest that nuclear and cytoplasmic localization of BARD1 reflect its dual function and that the increased cytoplasmic localization of BARD1 is associated with apoptosis.     

Identification of domains of BRCA1 critical for the ubiquitin-dependent inhibition of centrosome function

The breast and ovarian cancer specific tumor suppressor BRCA1, bound to BARD1, has multiple functions aimed at maintaining genomic stability in the cell. We have shown earlier that the BRCA1/BARD1 E3 ubiquitin ligase activity regulates centrosome-dependent microtubule nucleation. In this study, we tested which domains of BRCA1 and BARD1 were required to control the centrosome function. In the present study, (a) we confirmed that the ubiquitination activity of BRCA1 regulates centrosome number and function in Hs578T breast cancer cells; (b) we observed that both the amino and carboxyl termini of BRCA1 are required for regulation of centrosome function in vitro; (c) an internal domain (770-1,290) is dispensable for centrosome regulation; (d) BARD1 is required for regulation of centrosome function and protein sequences within the terminal 485 amino acids are necessary for activity; and (e) BARD1 is localized at the centrosome throughout the cell cycle. We conclude that the BRCA1-dependent E3 ubiquitin ligase functions to restrain centrosomes in mammary cells, and loss of BRCA1 in the precancerous breast cell leads to centrosomal hypertrophy, a phenotype commonly observed in incipient breast cancer.     

Expression of oncogenic BARD1 isoforms affects colon cancer progression and correlates with clinical outcome

Background:

Colon cancer predisposition is associated with mutations in BRCA1. BRCA1 protein stability depends on binding to BARD1. In different cancers, expression of differentially spliced BARD1 isoforms is correlated with poor prognosis and decreased patient survival. We therefore suspected a role of BARD1 isoforms in colon cancer.

Methods:

We performed immunohistochemistry in 168 colorectal cancers, using four antibodies directed against differentially expressed regions of BARD1. We determined structure and relative expression of BARD1 mRNA isoforms in 40 tumour and paired normal peri-tumour tissues. BARD1 expression was correlated with clinical outcome.

Results:

BARD1 isoforms were expressed in 98% of cases and not correlated with BRCA1. BARD1 mRNA isoforms were upregulated in all tumours as compared with paired normal peri-tumour tissues. Non-correlated expression and localisation of different epitopes suggested insignificant expression of full-length (FL) BARD1. Expression of N- and C-terminal epitopes correlated with increased survival, but expression of epitopes mapping to the middle of BARD1 correlated with decreased survival. Middle epitopes are present in oncogenic BARD1 isoforms, which have pro-proliferative functions. Correlated upregulation of only N- and C-terminal epitopes reflects the expression of isoforms BARD1δ and BARD1ϕ.

Conclusion:

Our results suggest that BARD1 isoforms, but not FL BARD1, are expressed in colon cancer and affect its progression and clinical outcome.