Tumor Suppressor Qualities and Mechanisms of LZAP Activity

描述(申请人提供)：我们发现LZAP是交替阅读框蛋白ARF(人的p14ARF，小鼠的p19ARF)的新的结合伙伴。虽然ARF具有不依赖于P53的活性，但其主要的肿瘤抑制活性被归因于抑制MDM2从而激活P53。我们发现，在致癌刺激后，LZAP与ARF的结合增加，并且LZAP增加了P53的转录活性，既依赖于ARF，也不依赖于ARF。我们的实验室还描述了LZAP结合relA，减少relA的磷酸化，抑制核因子-β转录，并增加relA与组蛋白脱乙酰基酶的关联。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蛋白在大约30%的人头颈部鳞状细胞癌中丢失，LZAP蛋白的丢失增加了锚定非依赖性生长、侵袭性和体内异种移植瘤的生长。另一组研究表明，LZAP通过结合和抑制Chk1和Chk2，至少部分通过LZAP消除G2/M检查点，使细胞对遗传毒性治疗剂敏感。人类肿瘤和异种移植小鼠肿瘤数据，以及LZAP作为P53的激活因子和RelA的抑制因子的活性表明，LZAP可能作为肿瘤抑制因子发挥作用；然而，LZAP肿瘤抑制因子的状态尚需验证。我们现在有数据表明，在小鼠体内靶向LZAP会导致肺癌的形成。为了更好地了解LZAP的生物学活性并获得机理上的洞察力，已经寻找了更多的LZAP结合伙伴。我们最近发现并证实LZAP能结合应激激活蛋白激酶p38MAPK(以下简称p38)和野生型P53诱导的磷酸酶Wip1。根据细胞环境的不同，肿瘤抑制和致癌活性都归因于p38。初步研究表明，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，P53激活后其水平升高。Wip1作为P53的反馈抑制物具有致癌活性；然而，发现Wip1也靶向并抑制RelA，这表明它的细胞活性可能更复杂。LZAP活性的统一机制尚未定义。来自我们实验室对relA和p38的数据以及来自LI实验室关于Chk1/2的数据表明，LZAP的一个共同作用是减少结合蛋白的磷酸化。我们假设，在小鼠身上靶向LZAP将证明它是一种肿瘤抑制物。从机制上，我们假设LZAP将通过调节结合伙伴和P53来发挥肿瘤抑制活性，并且这种调节将至少部分地通过Wip1磷酸酶来介导。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.