Tumor Suppressor Qualities and Mechanisms of LZAP Activity

描述（由申请人提供）：我们发现LZAP是替代阅读框架蛋白ARF（人类为p14ARF，小鼠为p19ARF）的一种新的结合伙伴。尽管ARF具有不依赖于p53的活性，但其主要的肿瘤抑制活性归因于MDM2的抑制，从而激活p53。我们发现，在致癌刺激后，LZAP与ARF的结合增加，LZAP增加p53的转录活性，依赖于或独立于ARF。我们的实验室还描述了LZAP结合RelA，降低RelA磷酸化，抑制NF-？并增加RelA与组蛋白去乙酰化酶的关联。LZAP没有保守的酶结构域，也没有已知的酶活性，因此LZAP调控RelA磷酸化和活性的机制目前尚不清楚。我们还发现LZAP蛋白在约30%的人类头颈部鳞状细胞癌中缺失，LZAP蛋白的缺失增加了锚定独立生长、侵袭和体内异种移植肿瘤的生长。另一组研究表明，LZAP使细胞对基因毒性治疗剂敏感，至少部分是通过LZAP通过结合和抑制Chk1和Chk2来废除G2/M检查点。人类肿瘤和异种移植小鼠肿瘤数据，以及LZAP作为p53激活因子和RelA抑制因子的活性表明，LZAP可能具有肿瘤抑制作用；然而，LZAP肿瘤抑制状态的验证是必要的。我们现在有数据表明，LZAP靶向小鼠会导致肺癌的形成。为了更好地了解LZAP的生物活性和获得机制的洞察力，寻找更多的LZAP结合伙伴。我们最近发现并证实LZAP结合应激激活蛋白激酶p38 MAPK（以下简称p38）和野生型p53诱导的磷酸酶Wip1。肿瘤抑制和致癌活性都归因于p38，这取决于细胞环境。初步研究发现LZAP抑制p38的磷酸化和活性（见初步数据）。Wip1 （PPM1D, PP2C4）是一种磷酸酶，其水平在p53激活后升高。Wip1作为p53的反馈抑制剂具有致癌活性；然而，发现Wip1也靶向并抑制RelA，表明其细胞活性可能更为复杂。没有定义LZAP活动的统一机制。我们实验室对RelA和p38以及Li实验室对Chk1/2的数据表明，LZAP的一个共同作用是降低结合蛋白的磷酸化。我们假设在小鼠中靶向LZAP将使其成为肿瘤抑制因子。从机制上讲，我们假设LZAP通过调节结合伴侣和p53来发挥肿瘤抑制活性，并且这种调节至少部分通过Wip1磷酸酶介导。我们建议在这项拨款中检验这些假设。

DESCRIPTION (provided by applicant): We discovered LZAP as a novel binding partner of the alternate reading frame protein ARF (p14ARF in humans, p19ARF in mice). Although ARF has p53-independent activities, its major tumor suppressor activity has been attributed to inhibition of MDM2 with resultant activation of p53. We found that binding of LZAP to ARF is increased following oncogenic stimulation and that LZAP increases p53 transcriptional activity both dependent and independent of ARF. Our lab also described that LZAP binds RelA, decreases RelA phosphorylation, inhibits NF-?B transcription, and increases RelA association with histone deacetylases. LZAP has no conserved enzymatic domains and no known enzymatic activity, so mechanisms of LZAP regulation of RelA phosphorylation and activity are currently unknown. We also found that LZAP protein is lost in ~30% of human head and neck squamous cell carcinomas and that loss of LZAP increases anchorage independent growth, invasion, and in vivo xenograft tumor growth. Another group has shown that LZAP sensitizes cells to genotoxic therapeutic agents mediated, at least in part, by LZAP abrogation of the G2/M checkpoint through binding and inhibition of Chk1 and Chk2. Human tumor and xenograft mouse tumor data, as well as, LZAP activities as an activator of p53 and suppressor of RelA suggest that LZAP may function as a tumor suppressor; however, validation of LZAP tumor suppressor status is needed. We now have data that targeting of LZAP in mice results in lung cancer formation. To better understand LZAP biological activities and to gain mechanistic insight, additional LZAP binding partners have been sought. We have recently found and confirmed that LZAP binds the stress activated protein kinase, p38 MAPK (hereafter p38), and the wild-type p53 induced phosphatase, Wip1. Both tumor suppressor and oncogenic activities have been ascribed to p38 depending on cellular context. Initial exploration revealed that LZAP inhibited p38 phosphorylation and activity (see preliminary data). Wip1 (PPM1D, PP2C4) is a phosphatase whose levels are increased after activation of p53. Wip1 has oncogenic activity as a feedback inhibitor of p53; however, discovery that Wip1 also targets and inhibits RelA suggest that its cellular activity may be more complex. A unifying mechanism of LZAP activity is not defined. Data from our lab for both RelA and p38 and from the Li lab for Chk1/2 suggest that a common effect of LZAP is to decrease phosphorylation of bound proteins. We hypothesize that targeting LZAP in mice will establish it as a tumor suppressor. Mechanistically, we hypothesize that LZAP will exert tumor suppressor activity by regulation of binding partners and p53, and that this regulation will be mediated, at least partially, through the Wip1 phosphatase. We propose to test these hypotheses in this grant.