No abstract

The FGFRs are receptor tyrosine kinases expressed by tissue-specific alternative splicing in epithelial IIIb or mesenchymal IIIc isoforms. Deregulation of FGF/FGFR signaling unbalances the epithelial-stromal homeostasis and may lead to cancer development. In the epithelial-context, while FGFR2b/KGFR acts as tumor suppressor, FGFR2c appears to play an oncogenic role. Based on our recent observation that the switching of FGFR2b versus FGFR2c induces EMT, here we investigated the biological outcome of the ectopic expression of FGFR2c in normal human keratinocytes. Morphological analysis showed that, differently from FGFR2b overexpression, the forced expression and activation of FGFR2c drive the epithelial cells to acquire a mesenchymal-like shape and actin reorganization. Moreover, the appearance of invasiveness and anchorage-independent growth ability in FGFR2c transfected keratinocytes was consistent with the potential tumorigenic role proposed for this receptor variant. Biochemical and molecular approaches revealed that the observed phenotypic changes were accompanied by modulation of EMT biomarkers and indicated the involvement of EMT transcription factors and miRs. Finally, the analysis of the expression pattern of discriminating markers strongly suggested that activation of FGFR2c triggers a process corresponding to the initiation of the pathological type III EMT, but not to the more physiological type II EMT occurring during FGFR2b-mediated wound healing.

The E5 oncoprotein of the human papillomavirus type 16 (HPV16 E5) deregulates epithelial homeostasis through the modulation of receptor tyrosine kinases and their signaling. Accordingly, the fibroblast growth factor receptor 2b (FGFR2b/KGFR), epithelial splicing transcript variant of the FGFR2, is down-modulated by the viral protein expression, leading to impairment of keratinocyte differentiation. Here, we report that, in cell models of transfected human keratinocytes as well as in cervical epithelial cells containing episomal HPV16, the down-regulation of FGFR2b induced by 16E5 is associated with the aberrant expression of the mesenchymal FGFR2c isoform as a consequence of splicing switch: in fact, quantitative RT-PCR analysis showed that this molecular event is transcriptionally regulated by the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) and is able to produce effects synergistic with those caused by TGFb treatment. Immunofluorescence analysis revealed that this altered FGFR2 splicing leads to changes in the specificity for the ligands FGFs and in the cellular response, triggering epithelial-mesenchymal transition (EMT). Through 16E5 or FGFR2 silencing as well as inhibition of FGFR2 activity we demonstrated the direct role of the viral protein in the receptor isoform switching and EMT, suggesting that these early molecular events during HPV infection might represent additional mechanisms driving cervical transformation and tumor progression.The fibroblast growth factor receptors (FGFRs) are receptor tyrosine kinases (RTKs) expressed on different tissues and involved in the control of cellular key processes such as growth, differentiation, migration and survival.1,2 The receptor extracellular domain is characterized by three immunoglobulin (Ig) loops (IgI-III) and the Ig like-domains II and III form the high affinity binding site for their ligands. Tissue specific alternative splicing of the IgIII domain in FGFR1-3 generates IIIb and IIIc isoforms and is responsible for ligand selection. While the FGFRIIIb isoforms are mostly expressed in epithelial tissues, the FGFRIIIc isoforms are generally present on mesenchymal cells: since also the ligand expression patterns are characterized by tissue specificity, the FGF signaling is tightly controlled in a paracrine and contextdependent manner and is crucial for the epithelial-stromal interaction and homeostasis.FGF/FGFR signaling may play oncogenic roles in tumorigenesis; however, through the triggering of cell differentiation, receptor activation might lead to opposite tumor suppressive outcome. 3,4 In fact, deregulated and out-of-context oncogenic FGFR signaling is frequently associated with carcinogenesis and altered paracrine or autocrine loops of receptor activation are often established in tumor progression. Interestingly, the switching from the FGFRIIIb to the FGFRIIIc isoforms is involved in epithelial-mesenchymal transition (EMT) and cancer progression. 5,6 However, reduced FGFR expression and loss-of function mutations are also freque...