Activated endothelial cells (ECs) within the tumor microenvironment express tumor-promoting factors that are targeted by several clinically-approved drugs. In contrast, less is known about a regulatory role for ECs in maintaining vessel and tissue homeostasis. In principal, negative regulatory EC signals should act to slow tumor initiation and growth, but few models exist to study this under-explored connection. Our model centers around focal adhesion kinase (FAK), a key signaling tyrosine kinase downstream of integrin and other surface receptors. EC-specific FAK knockout (KO) is embryonic lethal with vascular defects. However, conditional FAK KO in adult mouse ECs yields surprisingly few phenotypes. Pyk2 is a kinase related to FAK. We have shown that ECs possess the adaptive capacity to switch to Pyk2-dependent survival signaling upon genetic FAK inactivation. This is a strong survival selection mechanism as FAK (or Pyk2) can form a complex with the p53 tumor suppressor in the nucleus, promoting p53 ubiquitination and degradation, lowering p21CIP1 cyclin dependent kinase inhibitor levels, and allowing for continued cell proliferation in the presence of environmental stress. Using Pyk2-/-; FAK floxed (fl/fl) mice with a tamoxifen-inducible EC-specific Cre (SCL-Cre-ERT) revealed that mice can tolerate combined Pyk2 and FAK knockout (KO) in ECs with minor changes in vessel leakage but major differences in the ability of these mice to support syngeneic melanoma, breast, or lung carcinoma tumor growth compared to Cre-negative littermate controls. Primary EC culture results support the importance of a cell intrinsic change upon FAK-loss, only in a Pyk2 KO background. In vivo, Pyk2 KO in all cells limits causality interpretations of EC specific effects. Thus, we have created new Pyk2 fl/fl mice on a C57Bl6 background and are breeding with FAK fl/fl; SCL-Cre-ERT mice to test the hypothesis that induced FAK-Pyk2 KO within blood vessel ECs alters p53 activation and gene expression and/or production-secretion of factors that create a tumor repressive microenvironment.
Aim 1 is to characterize this new Pyk2 fl/fl; FAK fl/fl; SCL-Cre-ERT model for phenotypic changes after induced Pyk2 and FAK inactivation within ECs in vivo. Assays include histological, cell biological, immunological analyses to determine tissue changes upon combined Pyk2-FAK loss over time.
Aim 2 is to determine effects of EC- specific Pyk2-FAK inactivation on the survival and growth of implanted syngeneic tumor cells.
Aim 3 is to determine primary lung EC signaling changes in vitro before and after Pyk2-FAK inactivation. Assays include protein, cytokine array, and RNA analyses to link Pyk2-FAK inactivation with p53 activation and mRNA levels changes. Characterization of this model in vivo and ECs in vitro may be exploited to guide future therapeutic strategies to recapitulate anti-tumor effects in part through EC-specific p53 activation.
Host cells within the tumor microenvironment control several aspects of tumor growth. Our studies will create a new transgenic mouse model to study novel repressive signal(s) from endothelial cells that act to limit tumor growth via cell intrinsic and extrinsic changes in the host tissue microenvironment. The identification of this pathway may uncover new regulatory factors controlling tumor growth.
