Effects of the PI3K Pathway on p27 Function and Cancer Progression

PI3K 通路对 p27 功能和癌症进展的影响

基本信息

批准号：
8447371
负责人：
JOYCE MARIE SLINGERLAND
金额：
$ 22.34万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-03-23 至 2015-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8447371
关键词：
3-Phosphoinositide Dependent Protein Kinase-1 Actins Actomyosin Binding Binding Sites Cancer Detection Cell Cycle Cell Cycle Progression Cell Proliferation Cells Cyclin E Cyclins Cytoplasm Cytoskeleton Development Family Gene Deletion Goals Grant Growth Guanosine Triphosphate Human In Vitro Injection of therapeutic agent Knock-in Mouse Knockout Mice Knowledge Link Lung Lung Neoplasms Malignant Neoplasms Mediating Mediator of activation protein Metastatic Neoplasm to the Lung Modeling Neoplasm Metastasis Nuclear Nuclear Import Oncogene Activation Oncogenic PTEN gene Pathway interactions Patients Pharmaceutical Preparations Phosphorylation Phosphotransferases Play Post-Translational Protein Processing Precipitation Predisposition ROCK1 gene Raptors Reporting Role Signal Pathway Signal Transduction Site Stress Fibers Tail Testing Tissues Transfection Tumor Cell Invasion Tumorigenicity Variant Veins Work Xenograft procedure bone cancer cell cell growth cell motility cofilin cyclin-dependent kinase inhibitor 1B design gain of function human FRAP1 protein in vivo inhibitor/antagonist knock-down mTOR Inhibitor mouse model mutant neoplastic cell new therapeutic target outcome forecast overexpression prevent progenitor public health relevance response rho small hairpin RNA stem cell population therapeutic target tool tumor tumor progression tumorigenesis

项目摘要

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.

描述（由申请人提供）：虽然核 p27 经常减少，但 p27 基因缺失和 p27 完全丧失在人类癌症中很少见。细胞质 p27 存在于许多癌症中，与不良预后相关。细胞质 p27 获得致癌功能，通过结合 RhoA 抑制细胞骨架稳定性所需的 RhoA-ROCK 激活，从而促进细胞运动。 p27CK-敲入小鼠显示细胞质p27，并且细胞运动性、祖细胞/干细胞群和肺肿瘤形成增加。因此，p27 调节细胞增殖和迁移，并具有促癌作用，可促进细胞运动，而不依赖于其细胞周期效应，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 被 PI3K 下游的 AGC 家族激酶 T157、T198 或两者磷酸化，这会损害核 p27 输入，稳定细胞质中的 p27 并刺激细胞运动。过表达 AGC 激酶的细胞显示出 p27 依赖性细胞运动性增加，而通过 shRNAp27 敲低 p27 可逆转这种现象。 T198 磷酸化在体外增加了 p27:RhoA 结合，而 p27T157AT198A 与 RhoA 的结合较差，表明 T198 磷酸化可能通过抑制 Rho-ROCK1 来增加细胞运动性。 p27 似乎还介导 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 增加和细胞运动增加，但 p27 敲低可逆转这些现象。 p27 敲除将 MDA-MB-231-4715 的高肺转移逆转至与亲本 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，T198 位点的 p27 磷酸化促进其与 RhoA 的关联，从而增加肿瘤细胞的运动性并增强肿瘤的侵袭和转移。该资助进一步研究了 PI3K 效应激酶的 p27 磷酸化如何增加细胞运动性，并测试 p27pT157pT198 是否促进体内肿瘤发生和转移。 AIM1 将识别 p27:RhoA 结合位点并测试磷酸化对 RhoA 结合和增加细胞运动的重要性。 AIM 2将测试pT198和细胞质p27对异种移植物中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敏感性。细胞质 p27pT198 及其细胞骨架效应可能是人类癌症中 RTK/PI3K 激活的致瘤性和转移的主要驱动因素。破坏 p27:RhoA 相互作用的治疗性靶向药物可能会阻止肿瘤侵袭和转移。在癌症中，细胞质 p27 可以预测对 PI3K/mTOR 抑制剂药物的反应潜力。