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p14 ARF，小鼠p19 ARF）的新型结合伴侣。尽管ARF具有p53非依赖性活性，但其主要的肿瘤抑制活性归因于MDM 2的抑制，从而导致p53的活化。我们发现LZAP与ARF的结合在致癌刺激后增加，并且LZAP增加了依赖和独立于ARF的p53转录活性。本实验室还研究了LZAP与RelA结合，降低RelA磷酸化，抑制NF-？B转录，并增加RelA与组蛋白脱乙酰酶的结合。LZAP没有保守的酶结构域，也没有已知的酶活性，因此LZAP调节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%的人头颈部鳞状细胞癌中丢失，并且LZAP的丢失增加了锚定非依赖性生长、侵袭和体内异种移植肿瘤生长。另一个研究组已经表明，LZAP使细胞对遗传毒性治疗剂敏感，至少部分地由LZAP通过结合和抑制Chk 1和Chk 2消除G2/M检查点介导。人类肿瘤和异种移植小鼠肿瘤数据，以及LZAP作为p53激活剂和RelA抑制剂的活性表明，LZAP可能作为肿瘤抑制剂发挥作用;然而，需要验证LZAP肿瘤抑制状态。我们现在有数据表明，在小鼠中靶向LZAP会导致肺癌形成。为了更好地理解LZAP生物活性并获得机理见解，已经寻找了另外的LZAP结合配偶体。我们最近发现并证实，LZAP结合应激活化蛋白激酶，p38 MAPK（以下简称p38），和野生型p53诱导的磷酸酶，Wip 1。肿瘤抑制和致癌活性都归因于p38，这取决于细胞环境。初步研究表明，LZAP抑制p38磷酸化和活性（见初步数据）。Wip 1（PPM 1D，PP 2C 4）是一种磷酸酶，其水平在p53激活后增加。Wip 1作为p53的反馈抑制剂具有致癌活性;然而，Wip 1也靶向并抑制RelA的发现表明其细胞活性可能更复杂。没有定义LZAP活性的统一机制。来自我们实验室的RelA和p38数据以及来自Li实验室的Chk 1/2数据表明，LZAP的共同作用是减少结合蛋白的磷酸化。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将通过调节结合伴侣和p53发挥肿瘤抑制活性，并且这种调节将至少部分地通过Wip 1磷酸酶介导。我们建议在本补助金中检验这些假设。

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.