Resistance to Chemotherapy Is Associated with Fibroblast Growth Factor Receptor 4 Up-Regulation

Abstract

Purpose: Establishment of antiapoptotic signaling pathways in tumor cells is a major cause for the failure of chemotherapy against cancer.Toinvestigate the underlying mechanisms, we developed an experimentalapproach that is basedonthe genetic plasticity of cancer cells and the selectionfor cell survival on treatment with chemotherapeutic agents. Experimental Design: Gene expression changes of surviving cell clones were analyzed by macroarrays. Involvement of fibroblast growth factor receptor 4 (FGFR4) in antiapoptotic pathways was elucidated by apoptosis assays, small interfering RNA experiments, and an antagonistic antibody. Results:We show that FGFR4 gene expression is up-regulated in doxorubicin-treated, apoptosisresistant cancer cell clones. Ectopic expression of FGFR4 in cancer cells led to reduced apoptosis sensitivity on treatment with doxorubicin or cyclophosphamide, whereas knockdown of endogenous FGFR4 expression in breast cancer cell lines had the opposite effect. FGFR4 overexpression resulted in Bcl-xl up-regulation at both mRNA and proteinlevels. Knockdown of FGFR4 expression by small interfering RNA caused a decrease in phospho-extracellular signal-regulated kinase 1/2 levels and reduced Bcl-xl expression. Moreover, an antagonistic FGFR4 antibody suppressed the resistance of cancer cells with endogenous FGFR4 expression against apoptosis-inducing chemotherapeutic agents. Conclusion: Based on these findings, we propose an antiapoptotic signaling pathway that is initiatedby FGFR4 andregulatingthe expressionof Bcl-xl throughthemitogen-activatedproteinkinase cascade. Our findings are exemplary for a novel strategy toward the elucidation of diverse signaling pathways that define antiapoptotic potential in cancer cells.These observations open new avenues toward the diagnosis of chemoresistant tumors and therapies targeting FGFR4-overexpressing cancers.Breast cancer is the most frequent malignancy among women in the western world. 3,4 Although much effort has been invested into designing new therapies, classic chemotherapeutic agents such as doxorubicin (Adriamycin) are still widely employed in the clinic (1). Chemotherapeutic drugs are used as primary or adjuvant therapy with response rates from 60% to 100% (2, 3). A major problem of most of the current therapies are the sometimes severe side effects and the intrinsic or acquired drug resistance of cancer cells to these drugs, which lead to relapse and metastatic progression of the tumor. This is reflected by the decline in the response rates with second-line chemotherapy to 20% to 30% (4).Motivated by these facts, a comprehensive characterization of genes contributing to a drug resistance phenotype has been done in this study. Based on an approach of Hudziak et al. (5) and Abraham et al. (6), where tumor necrosis factor-a-resistant and Fas ligand-resistant cell clones were established, we respectively took advantage of the genomic instability of breast cancer cell lines to generate doxorubicin-resistant cell clones. When treated with chemothera...

The FGFR4 Y367C mutant is a dominant oncogene in MDA-MB453 breast cancer cells

“…MDA-MB453 cells seem to become addicted to constitutive FGFR4 signalling, given that a complete knockdown of FGFR4 by stable selection produced no surviving clones (data not shown). However, in other knockdown experiments in various cell lines containing the WT receptor, we could efficiently knock down FGFR4 expression (Roidl et al, 2009). Moreover, the exceptional behaviour of MDA-MB453 cell line can be seen by the study of Wooster et al, in which the gene expression in 318 cell lines was deposited and analysed in the Oncomine database ( Rhodes et al, 2004).…”

“…Surprisingly, stable transfection of MDA-MB453 cells with a vector generating FGFR4-specific siRNA yielded no cells with reduced FGFR4 expression (data not shown) in contrast to other cell lines for which stable FGFR4 knockdown could be achieved (Roidl et al, 2009) Subsequently, MDA-MB453 and MDA-MB361 cells were treated with an FGFR4 antagonistic antibody to characterize the potential of FGFR4 as a therapeutic target. This 10F10 monoclonal antibody was raised against the extracellular domain of FGFR4 and was shown to inhibit FGFR4 signalling (Roidl et al, 2009).…”

“…This 10F10 monoclonal antibody was raised against the extracellular domain of FGFR4 and was shown to inhibit FGFR4 signalling (Roidl et al, 2009). Although we observed no effect in the cell line harbouring the Y367C mutation, Erk phosphorylation in MDA-MB361 cells treated with the 10F10 antibody was clearly inhibited (Figure 5d).…”

“…As reported previously, MDA-MB453 breast cancer cells overexpress FGFR4 in response to doxorubicin treatment to escape druginduced cell death. We revealed that an antagonistic FGFR4 antibody could not sensitise MDA-MB453 cells to chemotherapeutic drug treatment in contrast to other FGFR4-overexpressing cell lines (Roidl et al, 2009). As FGFR4 is involved in conferring cancer characteristic properties such as uncontrolled proliferation, invasiveness and motility, as well as anti-apoptosis and chemoresistance, we have further analysed the molecular effects of this mutation in cancer cells.…”

“…Importantly, FGFRs have been reported to contribute to oncogenic cell transformation in different human cancers (Yamaguchi et al, 1994;Giri et al, 1999). We have recently shown that FGFR4 overexpression is associated with chemoresistance in cancer cells and that inhibition of FGFR4 reverses resistance and leads to increased chemosensitivity (Roidl et al, 2009). Furthermore, we identified a single-nucleotide polymorphism in the transmembrane domain of the FGFR4 resulting in a codon conversion from glycine to arginine (G388R), which is homologous to that in FGFR3 causing achondroplasia (Bange et al, 2002).…”