Abstract B096: CDCP1, a potential noninvasive biomarker in circulating tumor cells for treatment of prostate cancer with FAK inhibitors
Authors: Sara Pollan, Jamie Sperger, Joshua Lang, Fangjin Huang, Jie Zheng, Colm Morrissey, Anne E. Cress, Danislav Spassov, Mark Moasser, William Carter, Beatrice Knudsen
Journal: Cancer Research (2018)

Abstract Background: Tumor metastasis requires dynamic changes in cell adhesion mediated through beta 1-integrin and focal adhesion kinase (FAK). In particular, FAK is responsible for counteracting anoikis and promoting survival of CTCs. The Cub-Domain Containing Protein–1 (CDCP1) is a transmembrane glycoprotein expressed in epithelial cells that regulates cell adhesion. CDCP1 loss leads to activation of FAK in nonadherent cancer cells (na-PCC) (Spassov, 2011). As we demonstrated, another consequence of loss of CDCP1 is the inhibition of inside-out activation of beta 1-integrin in na-PCC, suggesting that FAK activation can occur in the absence of active beta 1-integrin. The main goal of this project is to determine how FAK becomes activated in na-PCC upon CDCP1 silencing and to establish that CDCP1 loss sensitizes to treatment with FAK inhibitors. Methods: CTCs were collected from nine patients with castrate-resistant prostate cancer (CRPC) (Schehr, 2016). CTC mRNA was eluted and used for CDCP1 qPCR. CDCP1 was stably silenced in DU145 with short hairpins (Spassov, 2013). In the anoikis assay, 5 x 105 DU145 shControl and shCDCP1 cells were suspended on HEMA coated plates for up to 6 hrs. Western blot membranes were probed for pMAPK1/2, MAPK1/2, FAKpY397, FAKpY925, and FAK. For MTT assays, 5,000 DU145 shControl and shCDCP1 were incubated for 24 hrs on HEMA-coated 96-well plates in regular culture medium +/- Saracatinib, FAKi-PF573228 (kinase domain inhibitor) and FAKi-LGA (FAT domain inhibitor) and/or MEKi-PD98059. A kinase dead mutant of PIP5K-D316A from the Addgene PIP5K plasmid was generated by site directed mutagenesis. Results: We find reduced CDCP1 expression in CTCs from patients with CRPC. Six patients did not express detectable CDCP1 mRNA in CTCs, 1 patient demonstrated CDCP1 expression at the limit of detection, and in 2 patients CDCP1 mRNA levels were above the limit of detection. Silencing CDCP1 in DU145 and PC3 cells results in 3.4-fold higher proliferation rates and 4.4-fold greater anchorage-independent growth rates. CDCP1-negative tumors grew in 100% of mice, versus 30% growth of CDCP1 positive control tumors. In na-PCC, CDCP1 silencing triggered FAK activation, as demonstrated by de novo pY397 and pY925. In addition, 73% of CDCP1-negative cells expressed FAK-stimulated pMAPK. FAK activation required a factor in human plasma and involved a trypsin-sensitive receptor in na-PCC. CDCP1 loss also conferred increased sensitivity to treatment with the PF573228 FAK-kinase inhibitor. At the 4uM concentration 98.1% of CDCP1-positive cells survived compared to 32.9% of CDCP1-silenced cells. An inhibitor treatment matrix showed a synergistic treatment effect of dual targeting of SRC, FAK, and MEK with up to 3-fold increased cytotoxicity of FAKi+SRCi or FAKi+MEKi. The largest treatment effect was observed when all 3 inhibitors were combined. To better understand the role of the FAK-MEK pathway in anoikis, a novel C-terminal FAT domain inhibitor was investigated and compared to PF573228. To determine the mechanism of FAK activation in na-PCC, we expressed a dominant negative PIP5K-D316A mutant. Expression of this mutant is expected to reduce CDCP1-induced FAK activation and also diminish SRC activity. Conclusion: We discovered a synergistic treatment effect between FAK and SRC and/or MEK inhibition in CDCP1-negative nonadherent cells. Expression of pFAK and pMAPK downstream of CDCP1 loss overcomes anoikis and depends on a factor in human plasma. By silencing beta 1-integrin and activating FAK, CDCP1-negative cancer cells may gain easy access to the circulation and survive during the CTC phase of cancer dissemination. CDCP1 loss may serve as a biomarker to identify patients who benefit from treatment with FAK inhibitors. Funding: This work was supported by Department of Defense Synergistic Idea Development Award W81XWH-08-1-0268. Citation Format: Sara Pollan, Jamie Sperger, Joshua Lang, Fangjin Huang, Jie Zheng, Colm Morrissey, Anne E. Cress, Danislav Spassov, Mark Moasser, William Carter, Beatrice Knudsen. CDCP1, a potential noninvasive biomarker in circulating tumor cells for treatment of prostate cancer with FAK inhibitors [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B096.