The fibroblast growth factor receptor FGFRs including four

The fibroblast growth factor receptor (FGFRs), including four highly conserved proteins (FGFR1-4), are important membrane sensors for extracellular signals [4]. By binding to their ligands, FGFRs are activated and can trigger various downstream intracellular signaling cascades, which is required for many critical processes in either embryonic development or adult tissue repair [5]. With such fundamental embryonic and homeostatic roles, FGFRs are commonly hijacked by cancer cells. It is reported that, amplification of FGFR1 is presented in nearly 10% of breast cancers and is associated with poor prognosis [6]. In addition, oncogenic nature of mutations in FGFR2 and FGFR3 are observed in lung squamous cell carcinoma. Those mutations in the extracellular part of FGFR or FGFR3 lead to constitutive activation of FGFR, and are related with an invasive phenotype of cancer PEG Virus Precipitation Kit [7]. Further, in our previous study, tumor-associated fibroblast was found to induce FGFR4 expression in colorectal cancer cells, leading to an epithelial-to-mesenchymal transition in these cancer cells [8]. Given the important roles in tumorigenesis, FGFR dependence offers the hope of developing new therapeutic approaches for those cancer patients harboring FGFR dysregulation. Small-molecule tyrosine kinase inhibitor is the major strategy for designing FGFR-targeted drugs, and several molecules with potent anti-FGFR effects are currently under clinical trial [9]. Considering the accumulating reports regarding the side effects of FGFR inhibitors, however, it is still challenging since FGF/FGFR signaling axis is so intimately implicated in diverse basic biologic processes that will also be probably disturbed by therapeutic intervention [10]. Thus a better understanding of molecular mechanisms underlying the cellular responses to FGFR antagonists will be helpful in developing FGFR-based drugs. In this study, FGFR1-amplified breast cancer cell lines were used to investigate the cellular responses to a selective FGFR1 antagonist, PD166866. We demonstrate that PD166866 induces autophagy in breast cancer cell lines, and intervention of autophagy improves PD166866-mediated anti-cancer effects.
Methods and materials
Results
Discussion Breast cancer is a leading cause of cancer-related mortality for women [1]. Enhanced FGFR1 expression was correlated with poor prognosis of this disease, and aberrant regulation of FGFR1-mediated signaling cascades was believed to be involved in the development of breast cancer [5]. A major reason for FGFR1 up-regulation lies in the amplification of FGFR1 gene in genome, which is frequently detected in those breast tumors harboring a high FGFR1 expression level [6]. Therefore, targeting FGFR1 has long been considered as a potential strategy in treating FGFR1-amplified breast cancer. It is reported that, knockdown of FGFR1 expression in FGFR1-amplified breast cancer cell line reduced cell viability [22]. Further, inhibition of FGFR1 expression also increased the tamoxifen sensitivity of breast cancer cells [23]. Recently, a phase II trial of the mixed VEGFR/FGFR inhibitor dovitinib showed well drug response in HR+ and FGFR1-amplified breast cancer population [24]. In present data, two FGFR1-amplified breast cancer cell lines were treated with a FGFR1-selective inhibitor, PD166866. We demonstrate that PD166866 reduced proliferation and induced anoikis in these cell lines. Thus our results as well as previous studies support that FGFR1 was a promising target in treating FGFR1-amplified breast cancer. Autophagy, which is a highly conserved biological process from yeast to mammalians, recycles organelles and long-lived cytosolic materials in cells. In response to stresses, such as hypoxia or nutrients deprivation, the phagosome membrane isolates a part of cytoplasm and engulfs futile cellular constituents to form an autophagosome. The autophagosome then fuses with lysosome, where cytoplasm-derived cellular contents are degraded by lysosomal hydrolases. The products, including amino acids and lipid molecules, are released to cytosol from lysosome and could be reused [14]. Although autophagy is frequently activated in cancer cells when treated with chemo-drugs, the precise role of autophagy in regulating cancer cell death or survival remains unclear [17]. In our previous reports, histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) stimulates autophagy in Jurkat T-leukemia cells by triggering production of reactive oxygen species. Inhibition of autophagy promoted SAHA-induced apoptosis [25]. Similarly, quercetin, a natural polyphenol, also induced protective autophagy in gastric cancer cells in an Akt-mTOR pathway- and HIF-1α-dependent manner [26]. In contrast, itraconazole induced autophagy in glioblastoma cells by disrupting intracellular cholesterol transport. Suppression of autophagy largely counteracted the anti-cancer property of itraconazole [11]. Here, we found that PD166866 induced autophagy in FGFR1-amplified breast cancer cells, shown by increased double-membraned vacuoles, LC3 lipidation, LC3 dots as well as autophagic flux. Notably, blocking autophagy by Atg5 knockdown markedly decreased the viability even in low-dose PD166866-treated cells, suggesting that PD166866-induced autophagy is protective.