Abstract P5-08-01: Tyrosine phosphorylation of p27kip1 regulates the activity of cyclin D-cdk4 complexes in breast cancer

Abstract

Abstract The oncogenes Cyclin D and cdk4 are overexpressed in breast cancer, but the levels of these proteins are not always accurate indicators of oncogenic activity because p27Kip1 is required to assemble this otherwise unstable dimer. However, p27’s association activates or alternatively inhibits cyclin D-cdk4, serving as a bona fide ON/OFF "switch." Tyrosine (Y) phosphorylation of residues Y88/89 in p27 displaces its C-terminus from the cdk4 active site, permitting both ATP binding and CAK phosphorylation of cdk4’s T loop. This model leads to the following hypothesis: modulation of p27 pY controls cdk4 activity, which in turn regulates efficient cell cycle passsage, and in breast cancer where cdk4 activity is deregulated, p27 may be constitutively switched ON. Deregulated Src Family Kinase (SFK) signaling in cancer may increase p27 pY, constitutively activating oncogenic cdk4, causing continuous cell cycling. Using our p27 pY phosphospecific antibody, we have shown in primary tumors, that p27 pY is not detected in benign tissue regions, but is detected in grade 1 and progressively higher grade tumors, suggesting that p27 pY may be a marker for increased oncogenic cdk4 activity and cdk4 inhibitor sensitivity. We identified an SH3 recruitment domain within p27 that controls p27 pY, and in turn controls cdk4 activity. Blocking the SH3:p27 interaction with small peptides prevents p27 pY and cdk4 activity in vitro and in vivo. Using a phage-ELISA assay, we identified PTK6/Brk (Protein Tyrosine Kinase 6/Breast Tumor Kinase) that functions as a high-affinity kinase, able to phosphorylate p27 in vitro and associate with phosphorylated p27 in vivo. Overexpression of PTK6 in vivo increases p27 pY and increases resistance to specific cdk4 inhibition by the chemical inhibitor, PD0332991. An ALTernatively spliced form of PTK6 (ALT), which contains the SH3 domain, specifically associates with p27 in cells arrested by contact or serum-starvation, blocking pY and acting as an endogenous inhibitor of cdk4. As PTK6/Brk is overexpressed in more than 60% of human breast carcinomas, our data suggest that PTK6/Brk overexpression facilitates cell cycle progression by increasing cdk4 activity through direct p27 Y phosphorylation. As PD0332991 moves into the clinic, p27 pY could serve as a marker to identify tumors sensitive to cdk4 inhibition, while blocking the PTK6:p27 interaction with small molecules represents a novel therapeutic option to inhibit cdk4 activation. Citation Format: Priyank Patel, Elina Shetyn, Cindy Gomez, Susan RS Gottesman, Angela Tyner, Benedikt Asbach, Ralf Wagner, Stacy W Blain. Tyrosine phosphorylation of p27kip1 regulates the activity of cyclin D-cdk4 complexes in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-08-01.