Tumor Suppressor Qualities and Mechanisms of LZAP Activity

描述(申请人提供)：我们发现LZAP结合交替阅读框架蛋白ARF(在人类中为p14ARF，在小鼠中为p19ARF)，并将LZAP描述为P53的激活剂，既依赖于也不依赖于ARF。我们还描述了LZAP对RelA(核因子-B)的抑制以及LZAP的缺失加速了肿瘤细胞的侵袭，以及异种移植瘤的生长和血管生成。从机制上讲，我们发现LZAP降低了RELA的磷酸化，抑制了核因子？B的转录。其他研究小组已经证明，LZAP可加速化疗药物对细胞的死亡，并通过抑制Chk1和Chk2改变G2/M细胞周期检查点。随着我们的描述，LZAP在30%的人HNSCC中表达缺失，LZAP潜在的肿瘤抑制活性继续显现，并且LZAP的缺失与选择的NF-B靶点的表达增加有关。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肿瘤抑制因子状态的验证一直缺乏。我们现在有初步数据表明，与野生型小鼠相比，LZAP杂合子丢失的小鼠患肺癌的多发性和发病率更高。LZAP调控的具有看似无关活动的癌症中心蛋白的数量是惊人的(例如，p53、MDM2、relA、Chk1、Chk2、p38MAPK)。我们注意到，无一例外，所有这些LZAP靶标也都是野生型P53诱导的磷酸酶1(Wip1)的靶标。Wip1最初被描述为p53的转录靶点，但它作为p53和p38MAPK(P38)的有效抑制物的致癌活性很快被认识到。最近，Wip1对RelA的抑制表明Wip1也可能具有肿瘤抑制活性。Wip1的活性被报道控制在表达水平，但我们最近发表的数据表明，LZAP调节Wip1结合和抑制一个靶点的能力(P38)。由于P53和RelA都受LZAP调控，并在肺癌发生中发挥关键作用，我们将利用我们的LZAP小鼠肺癌模型来确定P53和RELA是否对这些小鼠的肿瘤发展起关键作用。鉴于LZAP调节Wip1活性朝向p38，我们将确定LZAP是否也调节Wip1对其他Wip1底物(例如P53、MDM2、RelA、Chk1、Chk2)的活性，并通过这样做将对LZAP的活性有一个更全面的了解。在相关但不依赖的研究中，我们将确定LZAP的肿瘤抑制活性是否全部或部分依赖于Wip1，以及Wip1是否调节LZAP的磷酸化、泛素化和表达。这些创新性和机械性的研究将极大地推动LZAP领域的发展，通过确立LZAP作为肿瘤抑制物，通过确定LZAP作为致癌磷酸酶Wip1的第一调节因子，通过确定P53和RelA是否是LZAP在小鼠肿瘤发生中的关键靶点，以及通过确定Wip1作为主要效应器和潜在的LZAP活性调节器的作用。 公共卫生相关性：LZAP诱导细胞周期停滞，抑制细胞凋亡和侵袭，在30%的人头颈部鳞状细胞癌和小鼠肺肿瘤模型中LZAP的表达缺失。LZAP调节许多肿瘤中心蛋白(P53、MDM2、RELA、Chk1、Chk2、p38MAPK)，现在，我们有数据表明LZAP的活性至少部分依赖于致癌磷酸酶Wip1。基于我们的初步数据，利用小鼠模型和LZAP特异性试剂，我们将探索LZAP的作用机制，这可能导致依赖LZAP表达缺失的新的靶向肿瘤的策略。

DESCRIPTION (provided by applicant): We discovered that LZAP binds the alternate reading frame protein ARF (p14ARF in humans, p19ARF in mice) and described LZAP as an activator of p53, both dependent and independent of ARF. We also described LZAP inhibition of RelA (NF-?B) and that loss of LZAP accelerates tumor cell invasion, as well as xenograft tumor growth and angiogenesis. Mechanistically, we found that LZAP decreases RelA phosphorylation and inhibits NF-?B transcription. Other groups have shown that LZAP accelerates cell death in response to chemotherapeutic agents and alters the G2/M cell cycle checkpoint through inhibition of Chk1 and Chk2. The potential tumor suppressor activities of LZAP have continued to emerge with our description that LZAP expression is lost in 30% of human HNSCC and loss of LZAP is associated with increased expression of select NF-?B targets. 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 has been lacking. We now have preliminary data that mice with heterozygous loss of LZAP develop lung tumors at higher multiplicity and incidence compared to wild-type littermates. The number of cancer-centric proteins with seemingly unrelated activities that LZAP regulates is remarkable (e.g. p53, MDM2, RelA, Chk1, Chk2, p38MAPK). We noted that without exception, all of these LZAP targets are also targets of the wild-type p53-induced phosphatase 1 (Wip1). Wip1 was originally described as a p53- transcriptional target, but its oncogenic activity as a potent inhibitor of p53 and p38MAPK (p38) was soon recognized. More recently, Wip1 inhibition of RelA suggests that Wip1 may also have tumor suppressive activities. Wip1 activity has been reported to be controlled at the level of expression, but our recently published data show that LZAP regulates Wip1 ability to bind and inhibit one target (p38). Because both p53 and RelA are regulated by LZAP and serve critical roles in lung tumorigenesis, we will leverage our LZAP murine model of lung cancer to determine if p53 and RelA are critical for tumor development in these mice. Given that LZAP regulates Wip1 activity toward p38, we will determine if LZAP also regulates Wip1 activity toward other Wip1 substrates (e.g. p53, MDM2, RelA, Chk1, Chk2) and in doing so will develop a more comprehensive understanding of LZAP activity. In related, but not dependent, studies we will determine if all or a portion of LZAP tumor suppressive activities depend on Wip1 and if Wip1 regulates LZAP phosphorylation, ubiquitination and expression. These innovative and mechanistic studies will significantly advance the LZAP field by establishing LZAP as a tumor suppressor, by identification of LZAP as the first regulator of the oncogenic phosphatase Wip1, by determining if p53 and RelA are critical targets of LZAP for murine tumorigenesis, and by determining the role of Wip1 as a major effector and potentially as a regulator of LZAP activities. PUBLIC HEALTH RELEVANCE: LZAP induces cell cycle arrest, inhibits apoptosis and invasion, and expression of LZAP is lost in 30% of human head & neck squamous cell carcinomas and in a murine lung tumor model. LZAP regulates many tumor-centric proteins (p53, MDM2, RelA, Chk1, Chk2, p38MAPK), and now, we have data suggesting that LZAP activity is at least partially dependent the oncogenic phosphatase Wip1. Based on our preliminary data and using murine models and LZAP-specific reagents, we will explore mechanisms of LZAP action which may lead to new strategies to target tumors dependent on loss of LZAP expression.