Fenretinide Perturbs Focal Adhesion Kinase in Premalignant and Malignant Human Oral Keratinocytes. Fenretinide's Chemopreventive Mechanisms Include ECM Interactions

Abstract

The membrane-associated protein, focal adhesion kinase (FAK), modulates cell-extracellular matrix interactions and also conveys pro-survival and proliferative signals. Notably, increased intraepithelial FAK levels accompany transformation of premalignant oral intraepithelial neoplasia (OIN) to oral squamous cell carcinoma (OSCC). OIN chemoprevention is a patient-centric, optimal strategy to prevent OSCC’s co-morbidities and mortality. The cancer chemopreventive and synthetic vitamin A derivative, fenretinide, has demonstrated protein-binding capacities e.g. mTOR and retinol binding protein interactions. These studies employed a continuum of human oral keratinocytes (normal-HPV E6/E7-transduced-OSCC) to assess potential fenretinide-FAK drug protein interactions and functional consequences on cellular growth regulation and motility. Molecular modeling studies demonstrated fenretinide has ~200-fold greater binding affinity relative to the natural ligand (ATP) at FAK’s kinase domain. Fenretinide also shows intermediate binding at FAK’s FERM domain and interacts at the ATP-binding site of the closest FAK analogue, Pyk2. Fenretinide significantly suppressed proliferation via induction of apoptosis and G2/M cell cycle blockade. Fenretinide-treated cells also demonstrated F-actin disruption, significant inhibition of both directed migration and invasion of a synthetic basement membrane, and decreased phosphorylation of growth-promoting kinases. A commercially available FAK inhibitor did not suppress cell invasion. Notably, while FAK’s FERM domain directs cell invasion, FAK inhibitors target the kinase domain. In addition, FAK-specific siRNA treated cells showed an intermediate cell migration capacity; data which suggest co-contribution of the established migrating-enhancing Pyk2. Our data imply that fenretinide is uniquely capable of disrupting FAK’s and Pyk2’s pro-survival and mobility-enhancing effects and further extend fenretinide’s chemopreventive contributions beyond induction of apoptosis and differentiation.

Benefits of Multifaceted Chemopreventives in the Suppression of the Oral Squamous Cell Carcinoma (OSCC) Tumorigenic Phenotype

“…As the Abl ATP binding site remains constant in the Bcr-Abl fusion protein [31], 4-HPR's ATP-blocking effects should extend to this oncoprotein. This current and a previous study from our lab [21] depict 4-HPR's capacity to perturb tyrosine kinases via binding at higher affinities than the natural ligand at functional sites. Although chemically derived from retinol i.e.…”

“…gratuitous signaling, constitutive transcription factor activation and DNA binding, anchorage independent growth and tumorigenesis [21-25]. Collectively, our results show combinations of agents with complementary mechanisms of action provided enhanced efficacy at both the in vitro and in vivo levels.…”

“…Structure function analyses to assess 4-HPR-retinol binding protein interactions suggested bound 4-HPR induced both steric hindrance and target protein conformational changes [39]. Our current and previous modeling-functional analyses [21] suggest 4-HPR's high affinity protein interactions extend to tyrosine kinases integral for carcinogenesis.…”

“…Our lab recently showed fenretinide binds to and perturbs two proteins i.e. FAK and PYK2 essential for signaling and OSCC-ECM interactions including invasion [21]. This current study evaluated the abilities of three chemopreventives i.e.…”

“…the vitamin A derivative fenretinide (4-HPR), the estrogen metabolite 2-methoxyestradiol (2-ME) and the humanized monoclonal antibody to the IL-6R receptor tocilizumab (TOC) to modulate OSCC cell gratuitous signaling and tumorigenesis. These agents possess complementary mechanisms of action that include induction of apoptosis (4-HPR, 2-ME) and differentiation (4-HPR), capacity to inhibit intracellular signaling proteins and invasion (4-HPR) and reduce IL-6-mediated signaling (TOC) [15, 21-25]. Corresponding studies on OSCC tumors (from which cell lines were isolated) provided corresponding in situ data while molecular modeling studies depicted 4-HPR-cell target interactions.…”