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Triple-negative breast cancer (TNBC) is an aggressive breast tumor subtype that currently lacks targeted treatment options. The role played by the insulin-like growth factor-1 (IGF-1) and its cognate receptor IGF-1R in TNBC has been reported. Nevertheless, the molecular mechanisms by which the IGF-1/IGF-1R system may contribute to TNBC progression still remains to be fully understood. By computational analysis of the vast cancer genomics information in public databases (TCGA and METABRIC), we obtained evidence that high IGF-1 or IGF-1R levels correlate with a worse clinical outcome in TNBC patients. Further bioinformatics analysis revealed that both the focal adhesion and the Hippo pathways are enriched in TNBC harboring an elevated expression of IGF-1 or IGF-1R. Mechanistically, we found that in TNBC cells, the IGF-1/IGF-1R system promotes the activation of the FAK signal transduction pathway, which in turn regulates the nuclear accumulation of YAP (yes-associated protein/yes-related protein) and the expression of its target genes. At the biological level, we found that the IGF-1/IGF-1R-FAK-YAP network cascade triggers the growth potential of TNBC cells, as evaluated in different experimental systems. Overall, our results suggest that the IGF-1/IGF-1R/FAK/YAP axis may contribute to the progression of the aggressive TNBC subtype.

Background: Focal adhesion kinase (FAK) is a cytoplasmatic protein tyrosine kinase that associates with both integrins and growth factor receptors toward the adhesion, migration and invasion of cancer cells. The G-protein coupled estrogen receptor (GPER) has been involved in the stimulatory action of estrogens in breast tumor. In this study, we have investigated the engagement of FAK by GPER signaling in triple negative breast cancer (TNBC) cells., Methods: Publicly available large-scale database and patient data sets derived from "The Cancer Genome Atlas" (TCGA; www.cbioportal.org ) were used to assess FAK expression in TNBC, non-TNBC tumors and normal breast tissues. MDA-MB 231 and SUM159 TNBC cells were used as model system. The levels of phosphorylated FAK, other transduction mediators and target genes were detected by western blotting analysis. Focal adhesion assay was carried out in order to determine the focal adhesion points and the formation of focal adhesions (FAs). Luciferase assays were performed to evaluate the promoters activity of c-FOS, EGR1 and CTGF upon GPER activation. The mRNA expression of the aforementioned genes was measured by real time-PCR. Boyden chamber and wound healing assays were used in order to evaluate cell migration. The statistical analysis was performed by ANOVA., Results: We first determined by bioinformatic analysis that the mRNA expression levels of the gene encoding FAK, namely PTK2, is higher in TNBC respect to non-TNBC and normal breast tissues. Next, we found that estrogenic GPER signaling triggers Y397 FAK phosphorylation as well as the increase of focal adhesion points (FAs) in TNBC cells. Besides, we ascertained that GPER and FAK activation are involved in the STAT3 nuclear accumulation and gene expression changes. As biological counterpart, we show that FAK inhibition prevents the migration of TNBC cells upon GPER activation., Conclusions: The present data provide novel insights regarding the action of FAK in TNBC. Moreover, on the basis of our findings estrogenic GPER signaling may be considered among the transduction mechanisms engaging FAK toward breast cancer progression.

Overexpression of ATP-binding cassette (ABC) transporters is one of the most important mechanisms responsible for multi-drug resistance (MDR). VS-4718, a tyrosine kinase inhibitor targeting focal adhesion kinase (FAK) with a potential anticancer effect, is currently evaluated in clinical trials. In this study, we investigated whether VS-4718 could reverse MDR mediated by ABC transporters, including ABCB1, ABCG2, and ABCC1. The results showed that VS-4718 significantly reversed ABCB1- and ABCG2-mediated MDR, but not MDR mediated by ABCC1. Treatment of VS-4718 did not alter the protein level and subcellular localization of ABCB1 or ABCG2. Mechanism studies indicated that the reversal effects of VS-4718 were related to attenuation of the efflux activity of ABCB1 and ABCG2 transporters. ATPase analysis indicated that VS-4718 stimulated the ATPase activity of ABCB1 and ABCG2. Docking study showed that VS-4718 interacted with the substrate-binding sites of both ABCB1 and ABCG2, suggesting that VS-4718 may affect the activity of ABCB1 and ABCG2 competitively. This study provided a novel insight for MDR cancer treatment. It indicated that combination of VS-4718 with antineoplastic drugs could attenuate MDR mediated by ABCB1 or ABCG2 in ABCB1- or ABCG2-overexpressing cancer cells.

Because cancer stem cells (CSCs) have been implicated in chemo-resistance, metastasis and tumor recurrence, therapeutic targeting of CSCs holds promise to address these clinical challenges to cancer treatment. VS-4718 and VS-6063 are potent inhibitors of focal adhesion kinase (FAK), a non-receptor tyrosine kinase that mediates cell signals transmitted by integrins and growth factor receptors. We report here that inhibition of FAK kinase activity by VS-4718 or VS-6063 preferentially targets CSCs, as demonstrated by a panel of orthogonal CSC assays in cell line models and surgically resected primary breast tumor specimens cultured ex vivo . Oral administration of VS-4718 or VS-6063 to mice bearing xenograft models of triple-negative breast cancer (TNBC) significantly reduced the proportion of CSCs in the tumors, as evidenced by a reduced tumor-initiating capability upon re-implantation in limiting dilutions of cells prepared from these tumors. In contrast, the cytotoxic chemotherapeutic agents, paclitaxel and carboplatin, enriched for CSCs, consistent with previous reports that these cytotoxic agents preferentially target non-CSCs. Importantly, VS-4718 and VS-6063 attenuated the chemotherapy-induced enrichment of CSCs in vitro and delayed tumor regrowth following cessation of chemotherapy. An intriguing crosstalk between FAK and the Wnt/β-catenin pathway was revealed wherein FAK inhibition blocks β-catenin activation by reducing tyrosine 654 phosphorylation of β-catenin. Furthermore, a constitutively active mutant form of β-catenin reversed the preferential targeting of CSCs by FAK inhibition, suggesting that this targeting is mediated, at least in part, through attenuating β-catenin activation. The preferential targeting of cancer stem cells by FAK inhibitors provides a rationale for the clinical development of FAK inhibitors aimed to increase durable responses for cancer patients., Competing Interests: CONFLICTS OF INTEREST Authors Vihren N. Kolev, Winnie F. Tam, Quentin Wright, Christian M. Vidal, Irina S. Shapiro, and Qunli Xu are former Verastem employees. Authors Jonathan A. Pachter and David T. Weaver are current Verastem employees.

VS-4718, a novel inhibitor of focal adhesion kinase (FAK), was tested against the Pediatric Preclinical Testing Program's (PPTP's) in vitro cell line panel and showed a median relative IC 50 of 1.22 μM. VS-4718 was tested in vivo against the PPTP xenograft models using a dose of 50 mg/kg administered by the oral route twice daily for 21 days. VS-4718 induced significant differences in an event-free survival distribution compared with control in 18 of 36 of the evaluable solid tumor xenografts and in 0 of 8 acute lymphoblastic leukemia (ALL) xenografts, but no xenograft lines showed tumor regression. Future plans include further evaluation of the role of FAK inhibition in combination with ABL kinase inhibitors for Ph + ALL., (© 2016 Wiley Periodicals, Inc.)