Effects of the PI3K Pathway on p27 Function and Cancer Progression

描述（由申请人提供）：虽然核p27经常减少，但p27基因缺失和完全p27丢失在人类癌症中是罕见的。细胞质p27，在许多癌症中可见，与预后不良有关。细胞质p27通过结合RhoA抑制RhoA- rock激活来获得促进细胞运动的致癌功能。p27CK敲入小鼠显示细胞质p27，细胞运动性、祖细胞/干细胞数量和肺肿瘤形成增加。因此，p27调节细胞增殖和迁移，并具有独立于细胞周期作用的促癌作用，促进细胞运动，这可以解释为什么p27在人类癌症中很少完全丢失。在这里，我们研究p27磷酸化如何调节其运动功能。我们发现p27在PI3K下游的T157、T198或两者都被AGC家族激酶磷酸化，这损害了核p27的输入，稳定了细胞质中的p27并刺激了细胞运动。过表达AGC激酶的细胞表现出p27依赖性的细胞运动增加，这是通过shRNAp27敲低p27而逆转的。T198磷酸化增加了p27:RhoA在体外的结合，而p27T157AT198A对RhoA的结合较差，提示T198磷酸化可能通过抑制Rho-ROCK1来增加细胞运动。p27似乎也介导PI3K驱动的转移。高转移性mda - mb -231衍生物（4175和1833）显示PI3K激活，细胞质p27增加，细胞运动增加，p27敲低逆转。p27敲除将MDA-MB-231-4715的高肺转移逆转至与亲代MDA-MB-231相似的水平。因此，PI3K可能通过细胞质p27的致癌作用刺激侵袭和转移。我们的假设是，p27在T198位点的磷酸化促进了它与RhoA的关联，从而增加了肿瘤细胞的运动性，增强了肿瘤的侵袭和转移。该基金进一步研究了PI3K效应激酶磷酸化p27如何增加细胞活力，并测试了p27pT157pT198是否促进了体内肿瘤的发生和转移。AIM1将识别p27:RhoA结合位点，并测试磷酸化对RhoA结合和细胞运动性增加的重要性。AIM 2将检测pT198和细胞质p27对异种移植物局部肿瘤侵袭和转移的影响。AIM 3将创建p27CK-T198D和p27CK-T198A敲入小鼠，并测试它们对肿瘤发生的易感性。细胞质p27pT198及其细胞骨架效应可能是RTK/PI3K激活的人类癌症致瘤性和转移的主要驱动因素。破坏p27:RhoA相互作用的治疗靶向药物可能会阻止肿瘤的侵袭和转移。在癌症中，细胞质p27可以预测对PI3K/mTOR抑制剂药物的潜在反应。

DESCRIPTION (provided by applicant): While nuclear p27 is often reduced, p27 gene deletion and complete p27 loss is rare in human cancers. Cytoplasmic p27, seen in many cancers, is associated with a poor prognosis. Cytoplasmic p27 acquires an oncogenic gain of function to promote cell motility by binding RhoA to inhibit RhoA-ROCK activation needed for cytoskeletal stability. p27CK- knock- in mice show cytoplasmic p27, and increased cell motility, progenitor/stem cell populations and lung tumor formation. Thus, p27 regulates both cell proliferation and migration, and has a pro-oncogenic action to promote cell motility independent of its cell cycle effect, which may explain why p27 is rarely entirely lost in human cancers. Here we investigate how p27 phosphorylation regulates its motility function. We showed that p27 is phosphorylated by AGC family kinases downstream of PI3K at T157, T198, or both, which impairs nuclear p27 import, stabilizes p27 in the cytoplasm and stimulates cell motility. Cells overexpressing AGC kinases show a p27-dependent increased cell motility which is reversed by p27 knock- down via shRNAp27. T198 phosphorylation increased p27:RhoA binding in vitro and p27T157AT198A bound RhoA poorly, suggesting that T198 phosphorylation may increase cell motility via Rho-ROCK1 inhibition. p27 also appears to mediate PI3K driven metastasis. High metastatic MDA-MB-231-derivatives (4175 and 1833) showed PI3K activation, increased cytoplasmic p27 and increased cell motility that were reversed by p27 knockdown. p27 knock-down reversed the high lung metastasis of MDA-MB-231-4715 to levels similar to those in parental MDA-MB-231. Thus, PI3K may stimulate invasion and metastasis via oncogenic effects of cytoplasmic p27. Our hypothesis is that p27 phosphorylation at T198 promotes its association with RhoA to increase tumor cell motility and potentiate tumor invasion and metastasis. This grant investigates further how p27 phosphorylation by PI3K effector kinases increases cell motility and tests if p27pT157pT198 promotes tumorigenesis and metastasis in vivo. AIM1 will identify the p27:RhoA binding site and test the importance of phosphorylation to RhoA binding and increased cell motility. AIM 2 will test effects of pT198 and cytoplasmic p27 on local tumor invasion and metastasis in xenografts. AIM 3 will create p27CK-T198D and p27CK-T198A knock-in mice and test their susceptibility to tumorigenesis. Cytoplasmic p27pT198 and its cytoskeleton effects may be major drivers of tumorigenicity and metastasis activated by RTK/PI3K in human cancers. Therapeutic targeting agents that disrupt p27:RhoA interaction may prevent tumor invasion and metastasis. In cancers, cytoplasmic p27 may predict potential for response to PI3K/mTOR inhibitor drugs.