Effects of the PI3K Pathway on p27 Function and Cancer Progression

描述(申请人提供)：虽然核p27经常减少，但p27基因缺失和完全p27缺失在人类癌症中很少见。细胞质p27可见于许多癌症，与预后不良有关。细胞质p27通过结合RhoA来抑制细胞骨架稳定所需的RhoA-ROCK激活，从而获得促进细胞运动的致癌功能。P27CK基因敲除小鼠表现为胞浆内p27蛋白表达，细胞活动度增加，祖细胞/干细胞数量增加，肺肿瘤形成。因此，p27调控细胞的增殖和迁移，并具有促进细胞运动的促癌作用，不依赖于其细胞周期效应，这可能解释了为什么p27在人类癌症中很少完全缺失。在这里，我们研究了p27的磷酸化如何调节其运动功能。我们发现p27在PI3K下游的T157和/或T198处被AGC家族的激酶磷酸化，这会损害核p27的输入，稳定细胞质中的p27，并刺激细胞的运动。过度表达AGC激酶的细胞表现出p27依赖的细胞运动增加，这种增加可以通过shRNAp27被p27基因敲除而逆转。T198的磷酸化在体外增加了p27：RhoA的结合，而p27T157AT198A与RhoA的结合很弱，提示T198的磷酸化可能通过抑制Rho-ROCK1而增加细胞的运动能力。P27似乎也介导了PI3K诱导的转移。高转移性的MDA-MB-231衍生物(4175和1833)表现出PI3K激活，胞浆p27增加，细胞活力增强，但p27基因敲除后这些结果被逆转。P27基因下调可逆转MDA-MB-231-4715细胞的高肺转移，其水平与其亲本细胞的水平相似。因此，PI3K可能通过胞浆p27的致癌效应刺激肿瘤的侵袭和转移。我们的假设是，p27在T198处的磷酸化促进其与RhoA的关联，从而增加肿瘤细胞的运动性，增强肿瘤的侵袭和转移。这项资助进一步研究了p27被PI3K效应激酶磷酸化如何增加细胞的运动性，并测试了p27pT157pT198是否促进了体内肿瘤的发生和转移。AIM1将确定p27：RhoA结合位点，并测试磷酸化对RhoA结合和增加细胞运动性的重要性。目的2检测pT198和胞浆p27对移植瘤局部侵袭转移的影响。AIM 3将建立p27CK-T198D和p27CK-T198A敲入小鼠，并测试它们对肿瘤发生的敏感性。胞浆p27pT198及其细胞骨架作用可能是RTK/PI3K激活的肿瘤发生和转移的主要驱动因素。干扰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可能预测对PI3K/mTOR抑制剂药物的潜在反应。 公共卫生相关性：目前的拨款研究如何通过激活癌基因信号通路来改变细胞周期抑制蛋白p27，使其不再抑制细胞生长，而是获得促进癌症进展的新作用。在这里，我们将测试PI3K通路的信号如何改变p27的功能，从而干扰细胞的细胞骨架，增加细胞的运动性，促进局部组织侵袭，最终促进肿瘤转移。PI3K通路是癌症中主要的生长促进途径。这项工作可能允许设计新的治疗靶向药物，破坏这一点，以防止癌症的侵袭和转移。在癌症中，免疫染色检测高胞浆p27可能被证明是PI3K激活的有用指标，并预测对PI3K/mTOR抑制剂药物的反应，并帮助选择最有可能对这些靶向药物有反应的患者。

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. PUBLIC HEALTH RELEVANCE: The present grant investigates how the cell cycle inhibitor protein, p27 can be changed by activation of oncogene signaling pathways such that it no longer inhibits cell growth, but rather gains a new role to promote cancer progression. Here we will test how signaling from the PI3K pathway, a major growth promoting pathway in cancers alters p27 function such that p27 interferes with the cell's cytoskeleton to increases cell motility, to facilitate local tissue invasion and ultimately to promote tumor metastasis. This work may permit the design of new therapeutic targeting agents that disrupt this to prevent cancer invasion and metastasis. In cancers, detection of high cytoplasmic p27 on immunostaining may prove to be a useful indicator of PI3K activation, and predict response to PI3K/mTOR inhibitor drugs and help select patients most likely to respond to these targeting agents.