Abstract
In normal cells, p27 regulates cell cycle and functions as an atypical tumor suppressor. Unlike typical tumor suppressors such as p16 and pRb, p27 gene deletions or mutations are rarely observed in human cancers. Instead, p27 is deregulated through either excess degradation or through key C-terminal phosphorylations, in human cancers. Phosphorylation at T157 or T198 by different PI3K effector kinases allows p27 to take on new binding partners and promote oncogenic transformation. This thesis addresses the functional contribution of T157 and T198-phosphorylated, deregulated p27 to cancer progression and metastasis. By studying the effects of a transfected cell cycle defective (CK-) and double phosphomimetic p27 mutant (T157D/T198D) in transformed and non-transformed cells, we found that phosphorylation of p27 at T157 or T198 by PI3K/mTOR directly regulates tumor cell migration and invasion. Targeted inhibition of PI3K/mTOR impairs tumor cell motility and metastasis via modulation of p27. Furthermore, we uncovered a novel oncogenic function of p27 to promote tumor progression. p27CK-DD induced epithelial-mesenchymal transition (EMT) and transformation of human mammary epithelial cells and enhanced the mesenchymal characteristics and metastatic potential of breast cancer cell lines. Knockdown of p27 in highly metastatic EMT-transformed cell lines with enriched p27pT157pT198 (p27pTpT) reverted EMT and impaired metastatic potential. Mechanistically, we showed phosphorylation of p27 at T157 and T198 promotes the activation and induction of several EMT drivers and transcription factors to induce EMT. p27CK-DD activates STAT3 and facilitates its transactivation of TWIST1 to induce EMT. Pharmacological inhibition of STAT3 or dominant negative STAT3 (STAT3DN) decreased TWIST1 expression and reversed p27CK-DD-mediated EMT and tumor progression, while constitutively active STAT3 (STAT3CA) rescued the EMT phenotype and metastatic potential in p27 knockdown cells. We also identified a potential signaling feed-forward loop containing AKT activation, p27 phosphorylation, STAT3 activation and further AKT activation that might contribute to tumor progression. This thesis work also provides novel evidence for p27 as a transcriptional co- regulator of c-Jun. We prove evidence that C-terminally phophorylated p27 binds and activates c-Jun, and forms a complex with c-Jun at an enhancer region upstream of the TGF-B2 gene to upregulate TGF-B2. Not only does p27pTpT upregulate expression of the ligand, TGF-?2, p27pTpT is required for maximal TGF-B2-stimulated SMAD3 activation, SNAI1 induction, and matrigel invasion. Following addition of exogenous TGF-B2 ligand, p27 forms a tripartite complex with c-Jun and SMAD3 on the SNAI1 promoter, thus revealing an additional mechanism by which PI3K-activated, C-terminally phosphorylated p27 drives EMT and metastasis. These findings reveal a novel, oncogenic function of p27 to promote tumor progression through EMT via STAT3-mediated induction of TWIST1, c-Jun-mediated TGF-B2 induction, and c-Jun/SMAD3-mediated SNAI1 induction. Combined inhibition of AKT, STAT3, and TGF-B2 in PI3K/mTOR activated, p27pTpT enriched human cancers may ultimately have therapeutic potential to limit p27-mediated EMT and cancer metastasis.