Effects of the PI3K Pathway on p27 Function and Cancer Progression

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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.