Combination Treatment of Retinoic Acid Plus Focal Adhesion Kinase Inhibitor Prevents Tumor Growth and Breast Cancer Cell Metastasis

Abstract

All-trans retinoic acid (RA), the primary metabolite of vitamin A, controls the development and homeostasis of organisms and tissues. RA and its natural and synthetic derivatives, both known as retinoids, are promising agents in treating and chemopreventing different neoplasias, including breast cancer (BC). Focal adhesion kinase (FAK) is a crucial regulator of cell migration, and its overexpression is associated with tumor metastatic behavior. Thus, pharmaceutical FAK inhibitors (FAKi) have been developed to counter its action. In this work, we hypothesize that the RA plus FAKi (RA + FAKi) approach could improve the inhibition of tumor progression. By in silico analysis and its subsequent validation by qPCR, we confirmed RARA, SRC, and PTK2 (encoding RARα, Src, and FAK, respectively) overexpression in all breast cells tested. We also showed a different pattern of genes up/down-regulated between RA-resistant and RA-sensitive BC cells. In addition, we demonstrated that both RA-resistant BC cells (MDA-MB-231 and MDA-MB-468) display the same behavior after RA treatment, modulating the expression of genes involved in Src-FAK signaling. Furthermore, we demonstrated that although RA and FAKi administered separately decrease viability, adhesion, and migration in mammary adenocarcinoma LM3 cells, their combination exerts a higher effect. Additionally, we show that both drugs individually, as well as in combination, induce the expression of apoptosis markers such as active-caspase-3 and cleaved-PARP1. We also provided evidence that RA effects are extrapolated to other cancer cells, including T-47D BC and the human cervical carcinoma HeLa cells. In an orthotopic assay of LM3 tumor growth, whereas RA and FAKi administered separately reduced tumor growth, the combined treatment induced a more potent inhibition increasing mice survival. Moreover, in an experimental metastatic assay, RA significantly reduced metastatic lung dissemination of LM3 cells. Overall, these results indicate that RA resistance could reflect deregulation of most RA-target genes, including genes encoding components of the Src-FAK pathway. Our study demonstrates that RA plays an essential role in disrupting BC tumor growth and metastatic dissemination in vitro and in vivo by controlling FAK expression and localization. RA plus FAKi exacerbate these effects, thus suggesting that the sensitivity to RA therapies could be increased with FAKi coadministration in BC tumors.

Potential Biomarkers Associated with Prognosis and Trastuzumab Response in HER2+ Breast Cancer

“…We found that Lp, T-DM1, and combined treatments induce nuclear FAK translocation in BT-474 and SKBR3 cells. FAK redistribution from the cytoplasm to the nucleus in response to stress agents has been previously reported by our group and others [36,39,67,68]. In addition, anti-HER2 treatments increased perinuclear cortactin, most highly in combined therapies.…”

“…Absorbance at 570 nm was measured using a microplate reader (MULTISKAN EX; Thermo Scientific). Cell viability was calculated as a percentage of viability in treated cells compared to untreated cells, as described by Castro-Guijarro et al (2022) [36].…”

“…Images were captured using a Nikon Eclipse E200 microscope coupled with a high-resolution CCD digital camera. The protocol was previously described in Castro-Guijarro et al (2022) [36].…”

“…The nuclei were counterstained with 4 -6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich), and coverslips were mounted with Vectashield mounting medium (Vector Laboratories). Immunofluorescence was visualized using a Nikon Eclipse E200 microscope and recorded with a high-resolution DP70 Olympus digital camera, as described by Mondaca et al (2021) [6] and Castro-Guijarro et al (2022) [36].…”