Linking IKKbeta activation to anti-autophagy in viral protein Tax-mediated oncoge

描述（由申请人提供）：T细胞白血病病毒1型（HTLV-1）是成人T细胞白血病和淋巴瘤（ATLL）的致病因子。HTLV-1基因组编码一种病毒转化蛋白Tax1，该蛋白在启动肿瘤发生中起重要作用。Tax1参与肿瘤发生的关键特征之一是诱导NF-(B)的持续激活。然而，这种活性如何与T细胞转化相关仍未得到解决。我们的研究表明，在htlv -1转化的T细胞中，Tax1是一种关键的脂质筏调节剂，可以劫持I(B)激酶（IKKs）到脂质筏微域。众所周知，脂质筏微域中IKKs的富集导致抗原刺激T细胞中NF-(B)的激活。Tax1能够在脂筏中持续招募IKKs，这与持续的NF- B活性有关。我们还发现，在脂筏中，Tax1以IKK依赖的方式隔离beclin 1 （BECN1）和Bif-1这两种必需的自噬介质。相反，BECN1通过抑制IKK来抑制NF-(B)的Tax1激活。由于BECN1或Bif-1的缺失与包括淋巴瘤在内的自发性肿瘤发生有关，我们的研究结果强烈表明，Tax1部分通过其抗自噬活性将IKK（激活）与肿瘤发生联系起来。这一建议的基本原理是基于这样的假设：Tax1-IKK（-BECN1/Bif-1）轴导致自噬介质的“功能丧失”。这项提议的研究既具有创新性又具有重要意义，代表了一个新的概念，填补了我们对Tax如何启动T细胞致癌转化的理解的巨大空白。我们的中心假设是“HTLV-1 Tax通过劫持IKK（脂质筏）来抑制自噬，导致微域中自噬介质的隔离，从而显著促进肿瘤的发生”。我们计划以以下具体目标来调查这一假设。目标1：定义对Tax1的脂质筏靶向至关重要的结构域。我们将确定对脂质筏靶向Tax1至关重要的基序和修饰，如泛素化，确定辅助Tax1脂质筏关联的细胞因子，并评估Tax1脂质筏关联在原代人CD4+ T细胞的永生化和NIH3T3细胞转化中的重要性。目的2：研究Tax1解除自噬调控的潜在机制。我们将探讨Tax1在脂筏中隔离Bif-1和BECN1的机制，评估BECN1对Tax1激活IKK(的抑制作用，并利用MEF （Bif1+/+）和（Bif1-/-）细胞检测Tax1的抗自噬和致瘤活性。目的3：确定脂质筏靶向IKK的致癌潜能和抗自噬功能。我们将评估IKK（与Bif-1和BECN1）的物理和功能相互作用，并评估脂质筏靶向IKK（）的抗自噬和体外转化活性。本研究将有助于揭示Tax- IKK轴（- becn1 /Bif-1轴）在HTLV-1肿瘤发生中的病理作用。

DESCRIPTION (provided by applicant): T cell leukemia virus type 1 (HTLV-1) is a causative factor for adult T cell leukemia and lymphoma (ATLL). HTLV-1 genome encodes a viral transforming protein, Tax1, which plays an essential role in initiating oncogenesis. One of the critical features that Tax1 contributes to oncogenesis is induction of persistent activation of NF-(B. Yet, how this activity correlates with T cell transformation remains unsolved. Our work demonstrates that Tax1 is a critical lipid raft modulator that can hijack I(B kinases (IKKs) to the lipid raft microdomains in HTLV-1-transformed T cells. It is well recognized that enrichment of IKKs in the lipid raft microdomains leads to activation of NF-(B in antigen-stimulated T cells. Tax1 is able to recruit IKKs persistently in lipid rafts, correlating with persistent NF-(B activity. We also find that Tax1 sequesters beclin 1 (BECN1) and Bif-1, the essential autophagy mediators, in lipid rafts in IKK(- dependent manner. Conversely, BECN1 represses Tax1 activation of NF-(B by inhibiting IKK(. Since loss of BECN1 or Bif-1 has been linked to spontaneous tumorigenesis including lymphoma, our findings strongly suggest that Tax1 links IKK( activation to oncogenesis partly through its anti-autophagy activity. The rationale for this proposal is based on the assumption that the axis of Tax1-IKK(-BECN1/Bif-1 leads to "loss of function" of the autophagy mediators. The proposed research is both innovative and significant, representing a new concept in filling a giant gap for our understanding about how Tax initiates oncogenic transformation of T cells. Our central hypothesis is that "HTLV-1 Tax suppresses autophagy by hijacking IKK( to lipid rafts leading to sequestration of the autophagy mediators in the microdomains, thereby contributing significantly to oncogenesis". We plan to investigate this hypothesis with following specific aims. Aim#1: Define the domain critical for lipid raft targeting of Tax1. We will determine the motifs and modifications such as ubiquitination crucial for lipid raft targeting of Tax1, identify a cellular factor in assisting lipid raft association of Tax1, and assess the importance of the lipid raft association of Tax1 in immortalizing primary human CD4+ T cells and in transforming NIH3T3 cells. Aim#2: Investigate the underlying mechanism of Tax1 to deregulate autophagy. We will investigate the mechanism of Tax1 in sequestrating Bif-1 and BECN1 in lipid rafts, evaluate the inhibitory effect of BECN1 on Tax1 activation of IKK(, and examine anti-autophagy and tumorigenic activity of Tax1 using MEF (Bif1+/+) and (Bif1-/-) cells. Aim#3: Determine oncogenic potential and anti-autophagy function of lipid raft-targeted IKK(. We will evaluate physical and functional interaction of IKK( with Bif-1 and BECN1 and assess anti-autophagy and in vitro transforming activities of the lipid raft-targeted IKK(. This study will help to decipher the pathological role of the axis of Tax- IKK(-BECN1/Bif-1 in HTLV-1 oncogenesis. PUBLIC HEALTH RELEVANCE: Adult T cell leukemia/lymphoma (ATLL) is caused by infection with human T cell leukemia virus type 1 (HTLV-1), infecting over 20 million patients worldwide. HTLV-1 encodes a viral oncogenic protein Tax, which plays an essential role in initiating malignancy of human T cells partly through persistent activation of NF-(B. We plan to investigate the mechanism that Tax mediates persistent activation of NF-(B and anti-autophagy function during development of T cell leukemia.