Mechanisms by which DNA tumor virus oncoproteins target the Rb family of pocket proteins

Small DNA tumor viruses have evolved different mechanisms to abrogate the function of the retinoblastoma tumor suppressor (pRb). Studies of these viruses have been invaluable in uncovering the central role of the Rb family of pocket proteins in cell cycle control. While the molecular mechanisms by which the viral oncoproteins inactivate the Rb family are still being elucidated, it is clear that targeting of this family is required both for viral replication and for virus-induced transformation of mammalian cells. This review compares and contrasts the approaches DNA tumor viruses have evolved to antagonize Rb family members--ranging from relatively simple equilibrium dissociation of pRb from cellular pRb-binding factors to chaperone-mediated alterations in pocket protein stability and phosphorylation levels. The review will focus on the viral oncoproteins adenovirus E1A, human papillomavirus E7 and the large T antigens of several polyomaviruses. An understanding of these mechanisms may provide further insight into the regulation and functions of Rb family members as well as uncover new targets for the development of novel anti-viral agents, particularly against human papillomavirus, which is a significant cause of human cancer.     

Adeno-associated virus protects the retinoblastoma family of proteins from adenoviral-induced functional inactivation

Adeno-associated virus type 2 (AAV) is known to inhibit virally mediated oncogenic transformation. One of the early events of adenovirus (Ad) infection is the functional inactivation of cell cycle regulatory retinoblastoma (RB) family of proteins, which consists of retinoblastoma protein (pRB), p107, and p130. In an effort to understand the molecular basis of anti-oncogenic properties of AAV, we studied the effects of AAV expression on these proteins in cells infected with Ad. Western blot analysis showed that AAV interferes with the adenoviral-induced degradation and hyperphosphorylation of the pRB family of proteins in normal human fibroblasts as well as in HeLa and 293 cell lines. RNase protection assay showed enhanced expression of pocket protein gene by AAV expression. We also demonstrate that Rep proteins, the major AAV regulatory proteins, bind to E1A, the immediate early gene of Ad responsible for hyperphosphorylation and dissociation of pRB-E2F complex. This binding of AAV Rep proteins to E1A leads to decreased association between E1A and pRB leading to protection of pocket proteins from degradation, decreased expression of S phase genes and inhibition of cell cycle progression. These results suggest that the antiproliferative activity of AAV against Ad is mediated, at least in part, by effects of AAV Rep proteins on the Rb family of proteins.     

Ubiquitin-mediated control of oncogene and tumor suppressor gene products

Cellular levels of products from both oncogenes and tumor suppressor genes in normal cells need to be critically regulated to avoid malignant transformation. These products are often controlled by the ubiquitin proteasome pathway, the specific degradation mechanism in the cell. E3 ubiquitin ligases polyubiquitylate their specific substrates by collaborating with E1 and E2, and then the modified substrates are degraded in the proteasome. Mdm2 targets p53 and retinoblastoma protein, two major tumor suppressor gene products, for ubiquitin-dependent degradation. SCF^Skp2 targets other tumor suppressor gene products and CDK inhibitors such as p130, Tob1, p27 ^Kip1 , p57 ^Kip2 , and p21 ^Cip1 . Therefore, both E3 ligases act like oncogene products. In contrast, degradation of several oncogene products, such as Cyclin E, Notch, c-Myc, c-Jun, and c-Myb, are mediated by SCF^Fbw7. Fbw7 is often deleted or mutated in human cancers and acts like a tumor suppressor. As well as growth factor receptors and signal transduction regulators, DNA repair-related proteins are also regulated via the ubiquitin–proteasome pathway mediated by their specific E3 ligases. The stabilization of oncogene products and enhanced degradation of tumor suppressor gene products or DNA repair proteins might be associated with carcinogenesis and malignant progression, due to defects or the abnormal expression of their E3 ligases. ( Cancer Sci 2009)     

Effects of specific DNMT gene depletion on cancer cell transformation and breast cancer cell invasion; toward selective DNMT inhibitors

A hallmark of cancer is aberrant DNA methylation, consisting of global hypomethylation and regional hypermethylation of tumor suppressor genes. DNA methyltransferase inhibitors have been recognized as promising candidate anticancer drugs. Drug development has focused on DNA methylation inhibitors with the goal of activating tumor suppressor genes silenced by DNA methylation. 5-azacytidine (5-AC; Vidaza), a global DNA methyltransferase inhibitor, was Food and Drug Administration approved to treat myelodysplastic syndromes and is clinically tested for solid tumors. In this paper, it was demonstrated that 5'-aza-2'-deoxycytidine (5-azaCdR) activated both silenced tumor suppressor genes and pro-metastatic genes by demethylation, raising the concern that it would promote metastasis. 5-AzaCdR treatment increased the invasiveness of non-invasive breast cancer cell lines MCF-7 cells and ZR-75-1 and dramatically induced pro-metastatic genes; Urokinase plasminogen activator (uPA), matrix metalloproteinase 2 (MMP2), metastasis-associated gene (H-MTS1; S100A4) and C-X-C chemokine receptor 4 (CXCR4). The hypothesis that the blocking of cellular transformation activity of DNA methyltransferase inhibitor could be separated from the pro-metastatic activity was tested using short interfering RNA (siRNA) targeted to the different DNA methyltransferase (DNMT) genes. Although depletion of DNMT1 had the strongest effect on colony growth suppression in cellular transformation assays, it did not result in demethylation and activation of uPA, S100A4, MMP2 and CXCR4 in MCF-7 cells. Depletion of DNMT1 did not induce cellular invasion in MCF-7 and ZR-75-1 non-invasive breast cancer cell lines. These data have implications on the design of new DNA methyltransferase inhibitor and on the proper utilization of current inhibitors.     

Viral oncoproteins E1A and E7 and cellular LxCxE proteins repress SUMO modification of the retinoblastoma tumor suppressor

The retinoblastoma tumor suppressor protein (pRB) is a major regulator of cell-cycle progression and cellular differentiation. Central to pRB function is the pocket domain, which serves as the main binding region for cellular regulators. In tumors pRB is frequently inactivated by mutations in the pocket domain or by binding of viral oncoproteins to this region. A characteristic feature of these viral oncoproteins and many cellular pRB-binding partners is an LxCxE sequence motif, which interacts with pRB's pocket domain. Here, we show that the ubiquitin-like modifier SUMO is covalently attached to a distinct residue (K720) of pRB within the B-box of the pocket region that binds LxCxE-motif proteins. We provide evidence that SUMO preferentially targets the active, hypophosphorylated form of pRB and show that tumorigenic mutations of pRB in the pocket domain lead to a loss of SUMOylation. Notably, the level of pRB SUMOylation is controlled by the interaction of pRB with viral and cellular LxCxE-motif proteins. Inhibitors of pRB function, including the viral oncoproteins E1A and E7 and the cellular E1A-like inhibitor of differentiation EID-1, completely abolish SUMO modification of pRB. Conversely, pRB mutants deficient in binding of LxCxE-motif proteins exhibit a drastically enhanced modification by SUMO. Finally, we provide evidence that SUMOylation can influence pRB function, as the SUMO-deficient pRB(K720R) mutant exerts a slightly higher repressive potential on an E2F-responsive reporter gene than wild-type pRB. Taken together, these data identify SUMO modification as a novel post-translational modification of pRB that may control pRB activity by modulating LxCxE-pocket interactions.     

宫颈癌细胞系中HPV16 E6、E7及E6/E7基因对STK31基因甲基化状态及表达的影响

背景与目的：丝氨酸/苏氨酸蛋白激酶31(serine/threonine kinases 31,STK31)基因在人类多种癌症中扮演重要角色,且STK31基因的表达受其启动子及第一外显子区甲基化状态的影响;病毒感染与肿瘤组织中某些抑癌基因启动子区高甲基化有关。本研究旨在探讨宫颈癌细胞系中HPV16 E6、E7及E6/E7癌基因对STK31基因甲基化状态及表达的影响,以及不同种类甲基转移酶(DNA methyltransferases,DNMTs)基因在STK31基因甲基化中的潜在作用。方法：构建外源性HPV16 E6、E7以及E6/E7基因共表达慢病毒,分别感染人乳头瘤病毒(human papillomavirus,HPV)阴性宫颈癌细胞系HT-3及C33A,获得稳定转染的细胞系;采用亚硫酸氢盐基因组测序法(bisulfite genomic sequencing,BGS)和甲基化特异性PCR (methylation-specific PCR,MSP)检测3种HPV阳性宫颈癌细胞系HeLa、SiHa和CasKi以及HPV阴性宫颈癌细胞系HT-3和C33A转染前后STK31基因的甲基化状态;RT-PCR及蛋白［质］印迹法(Western blot)检测上述宫颈癌细胞系中STK31基因的表达以及DNMT1、DNMT2、DNMT3a、DNMT3b和DNMT3L基因在HPV16转染前后宫颈癌细胞系及HPV阳性、HPV阴性宫颈癌组织中的表达情况。结果：外源性HPV16 E6、E7以及E6/E7基因可在HPV阴性宫颈癌细胞系中稳定表达。3种HPV阳性细胞系HeLa、SiHa和CasKi中STK31基因呈低甲基化状态,STK31 mRNA及蛋白质表达阳性;2种HPV阴性细胞系HT-3、C33A中STK31基因则表现为高甲基化状态,STK31 mRNA及蛋白质表达缺失;与未感染慢病毒HT-3和C33A细胞系比较,外源性HPV16 E7以及E6/E7表达的HT-3和C33A细胞系STK31基因甲基化程度降低,其mRNA及蛋白质重新表达。DNMT1、DNMT3a和DNMT3b基因在HT-3E6/E7和C33AE6/E7细胞系中mRNA水平分别高于HT-3空载细胞系和C33A空载细胞系,差异有统计学意义(P<0.001)。DNMT1、DNMT3a和DNMT3b基因的mRNA水平在HPV16阳性宫颈癌组织中的表达高于其在HPV阴性宫颈癌组织中的表达,差异有统计学意义(t=5.997,P<0.001;t=6.743,P<0.001;t=7.926,P<0.001)。DNMT2在HT-3E6/E7和C33AE6/E7细胞系中mRNA表达水平分别低于HT-3空载细胞系和C33A空载细胞系,差异有统计学意义(t=7.451,P<0.001;t=2.451,P<0.05);DNMT2基因转录水平在HPV16阳性宫颈癌组织中低于HPV阴性宫颈癌组织(t=9.134,P<0.001)。DNMT3LmRNA表达水平在宫颈癌细胞系转染前后及HPV阴阳性宫颈癌组织中的差异无统计学意义(P>0.05)。结论：HPV感染可导致STK31基因启动子及第1外显子区甲基化状态降低,低甲基化状态促进该基因表达。STK31基因的表达受其启动子及第1外显子区甲基化状态的调控。HPV16 E7、E6/E7基因可能通过影响DNMT2的表达参与调控癌基因STK31基因启动子及第1外显子区甲基化状态。     

Che-1 affects cell growth by interfering with the recruitment of HDAC1 by Rb

DNA tumor virus oncoproteins bind and inactivate Rb by interfering with the Rb/HDAC1 interaction. Che-1 is a recently identified human Rb binding protein that inhibits the Rb growth suppressing function. Here we show that Che-1 contacts the Rb pocket region and competes with HDAC1 for Rb binding site, removing HDAC1 from the Rb/E2F complex in vitro and from the E2F target promoters in vivo. Che-1 overexpression activates DNA synthesis in quiescent NIH-3T3 cells through HDAC1 displacement. Consistently, Che-1-specific RNA interference affects E2F activity and cell proliferation in human fibroblasts but not in the pocket protein-defective 293 cells. These findings indicate the existence of a pathway of Rb regulation supporting Che-1 as the cellular counterpart of DNA tumor virus oncoproteins.     

Viruses, oncogenes, and cancer

Our current theories of virus-induced cellular transformation have changed with the emerging recognition that all normal cells contain proto-oncogenes which convert to oncogenes and induce transformation when activated and/or amplified. Cellular oncogenes have been identified by homology to the transforming genes of acute retroviruses and by the transforming activity of tumor cell DNA in transfection assays. More than two dozen cellular oncogenes identified to date constitute a heterogeneous group of genes which are remarkably conserved among highly diverse species. Expression of proto-oncogenes is linked to normal growth and development; whereas their expression as oncogenes due to gene mutation, rearrangement, amplification or other processes leading to altered or overexpression is associated with the development of tumors. Functions of oncogene proteins are being identified. These include unique protein kinase activity, growth factor/growth factor receptor properties, and the presence of DNA-binding polypeptides. It also appears that cooperation between several activated cellular oncogenes may be required in the multistep process of oncogenesis. Our recent in vitro experimental evidence supports that human cell carcinogenesis is indeed a multistep process. In addition, the involvement of the activated cellular transforming genes met and H-ras in chemically induced human cell carcinogenesis has been shown. Advancement in molecular biology of oncogenes and their products is likely to result in improvements in cancer diagnosis and cancer therapy.