Molecular Mechanism Of Poly-ubiquitination Regulated Tumorigenesis In Nasopharyngeal Carcinoma And Lung Cancer

Conjugation of ubiquitin to proteins (ubiquitilation) has emerged to be one of the most crucial post-translational modifications controlling virtually all cellular processes. Ubiquitination plays a critical role in numerous biological functions including cell cycle control, cell growth, apoptosis, DNA damage repair, as well as immune functions. The role of different linkages in polyubiquitins has begun to be elucidated in recent years. Linkage through Lys48(Ubk48) is mainly used for targeting to the proteasome, and Lys63(Ubk63) linkages seem to play important roles in DNA damage tolerance, inflammatory response, the endocytic pathway, and ribosomal protein synthesis. The deregulation of components of the ubiquitin system leads to human pathogenesis, including the development of many types of tumor.Nasopharyngeal carcinoma (NPC) has a remarkably distinctive ethnic and geographic distribution, more than80%of which were reported from China, Southeast Asia, and some other Asian countries. A unique feature of NPC is its strong association with Epstein-Barr Virus (EBV). Latent membrane protein1(LMP1) is one of the most important oncogenic properties which encoded by EBV. It has been well demonstrated that LMP1could regulate cell proliferation, transformation and apoptosis via activating MAPK, PI3K/Akt, JAK/STAT and NF-kappaB signaling pathways through the c terminal CTAR1and CTAR2domain.p53has been called a "cellular gatekeeper" or "the guardian of the genome" because of its central role in coordinating the cellular responses to a broad range of cellular stress factors, such as DNA damage, hypoxia and oncogene activation. Somatic mutations in the TP53gene are one of the most frequent alterations in human cancers. The stability and activation of p53tumor suppressor are regulated by posttranslational modifications. For example, phosphorylation and acetylation of p53have been shown to promote the expression of p53transcriptional targets, whereas other modifications, such as ubiquitination, sumoylation, and neddylation have been associated with the suppression of p53-mediated transcription and p53nuclear export. Oncogenic viral proteins such as large T antigen of papovaviruses, adenovirus E1B protein, hepatitis B virus X and hepatitis C virus core protein, have been shown to interact with p53and inactivate its functions through distinct mechanism to induce virus-related carcinogenesis.Unlike most human tumors, almost all of the p53protein accumulated in NPC is wild type. Our previous studies have demonstrated that LMP1up-regulated p53stability via MAPK cascade induced p53phosphorylation. In the present study, we found that LMP1promoted p53accumulation as well as its target gene MDM2. LMP1 significantly extended the half-life of p53and MDM but disrupted the interaction between MDM2and p53. We revealed that LMP1suppressed E3ligase MDM2-mediated p53UbK48-linked ubiquitination. Remarkably, LMP1induced p53UbK63-linked ubiquitination by interacted with tumor necrosis factor receptor-associated factor2(TRAF2), thus resulting in p53accumulation rather than proteasomal degradation. Furthermore, we found that LMP1rescued p53mediated apoptosis and cell cycle arrest. Collectively, these findings for the first time demonstrate that the regulation of p53accumulation/stability via different ubiquitin-modification by LMP1, contributed to EBV latency-associated tumorigenesis.Lung cancer is the leading cause of cancer-related death in the world. Although advanced molecular biology techniques have greatly accelerated the understanding of the biological mechanisms that underlie lung cancer development, the5-year survival rate is15%, which has hardly improved from the13%reported35years ago. A major challenge in treating lung cancer is to identify novel therapeutic targets that can complement current chemotherapy.Akt signaling plays a major role in tumorigenesis by regulating cell proliferation, cell cycle, cell survival and metabolism. Akt is constitutively activated in lung cancer, where it can regulate cancer cell growth and survival and confer resistance to chemotherapy or radiation treatment. It is activated by various stimuli, including insulin and EGF, and Akt phosphorylation at Thr308by PDK1and at Ser473by mTORC2is required for full activation. Studies detailing Akt activation indicate that Lys63-mediated ubiquitination is essential for Akt activity. Notably, different stimuli utilize distinct E3ligases for Akt ubiquitination. For example, TRAF6or Skp2mediates Akt activation through ubiquitination in IGF-1or ErbB receptor signaling, respectively.TRAF4is an atypical TRAF family member. Unlike other TRAFs, which are not relevant to the genetic phenotype, in vivo evidence strongly suggested that TRAF4is involved in embryogenesis and central nervous system myelin homeostasis. TRAF4is widely and highly expressed during development. Very recently studies demonstrated that TRAF4is involved in several signaling pathways transduction. TRAF4may possess potentional E3ligase activity for its amino terminus RING finger domain. Although TRAF4is overexpressed in various human malignancies, the mechanism regarding TRAF4’s role in carcinogenesis remains unclear.In the present study, we found that TRAF4plays an important role in lung tumorigenesis. TRAF4regulates EGF induced HaCaT cell transformation. Knockdown of TRAF4dramatically attenuated the malignant phenotype in lung cancer, including proliferation, anchorage-independent growth and tumor formation ability in nude mice. Furthermore, we identified that TRAF4is required for EGF-induced Akt activation through its ubiquitination in lung cancer. Our results demonstrated for the first time that TRAF4possesses E3ligase activity and UbcH5a is the conjugation enzyme which is required for TRAF4mediated Akt ubiquitination. TRAF4deficiency markedly impaired the activity of Akt signaling and Akt-mediated lung cancer glycolysis.Therefore, this study elucidate the mechanisms of EB virus LMP1induced p53accumulation via UbK63mediated ubiquitination in NPC and TRAF4overexpression regulated Akt activation in lung tumorigenesis. This study provided us with a novel view of comprehensive understanding in UbK63ubiquitination mediated carincogenesis, which will lead to the identification of novel targets for drug discovery and provide a basis for gene therapy.