HPV16 E5 expression induces switching from FGFR2b to FGFR2c and epithelial-mesenchymal transition

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 TGFβ 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

Interplay between FGFR2b-induced autophagy and phagocytosis: role of PLCγ-mediated signalling

Signalling of the epithelial splicing variant of the fibroblast growth factor receptor 2 (FGFR2b) induces both autophagy and phagocytosis in human keratinocytes. Here, we investigated, in the cell model of HaCaT keratinocytes, whether the two processes might be related and the possible involvement of PLCγ signalling. Using fluorescence and electron microscopy, we demonstrated that the FGFR2b-induced phagocytosis and autophagy involve converging autophagosomal and phagosomal compartments. Moreover, the forced expression of FGFR2b signalling mutants and the use of specific inhibitors of FGFR2b substrates showed that the receptor-triggered autophagy requires PLCγ signalling, which in turn activates JNK1 via PKCδ. Finally, we found that in primary human keratinocytes derived from light or dark pigmented skin and expressing different levels of FGFR2b, the rate of phagocytosis and autophagy and the convergence of the two intracellular pathways are dependent on the level of receptor expression, suggesting that FGFR2b signalling would control in vivo the number of melanosomes in keratinocytes, determining skin pigmentation

Role of FGFR2b expression and signaling in keratinocyte differentiation. Sequential involvement of PKCδ and PKCα

The tumor suppressor epithelial isoform of the fibroblast growth factor receptor 2 (FGFR2b) induces human keratinocyte early differentiation. Moreover, protein kinases C (PKCs) are known to regulate the differentiation program in several cellular contexts, including keratinocytes. Therefore, in this paper we propose to clarify if FGFR2b could play a role also in the late steps of keratinocyte differentiation and to assess if this receptor-induced process would sequentially involve PKCδ and PKCα isoforms. Immunofluorescence, biochemical, and molecular approaches, performed on 2D cultures or 3D organotypic rafts of human keratinocytes overexpressing FGFR2b by stable transduction, showed that receptor signaling induced the precocious onset and an accelerated progression of keratinocyte differentiation, indicating that FGFR2b is a crucial regulator of the entire program of keratinocyte differentiation. In addition, the use of specific inhibitors and gene silencing approaches through specific siRNA demonstrated that PKCδ controls the onset of FGFR2b-triggered differentiation, while PKCα plays a role restricted to the terminal stages of the process. Molecular analysis revealed that the two PKC isoforms sequentially act via induction of KLF4 and DLX3, two transcription factors linked by negative loops to p63, suggesting that p63 would represent the hub molecule at the crossroad of an intricate signaling network downstream FGFR2b, involving multiple PKC-induced transcription factors

The aberrant expression of the mesenchymal variant of FGFR2 in the epithelial context inhibits autophagy

Signaling of the epithelial splice variant of fibroblast growth factor receptor 2 (FGFR2b) triggers both differentiation and autophagy, while the aberrant expression of the mesenchymal FGFR2c isoform in epithelial cells induces impaired differentiation, epithelial mesenchymal transition (EMT) and tumorigenic features. Here we analyzed in the human keratinocyte cell line, as well as in primary cultured cells, the possible impact of FGFR2c forced expression on the autophagic process. Biochemical and quantitative immunofluorescence analysis, coupled to the use of autophagic flux sensors, specific substrate inhibitors or silencing approaches, showed that ectopic expression and the activation of FGFR2c inhibit the autophagosome formation and that AKT/MTOR is the downstream signaling mainly involved. Interestingly, the selective inhibition of AKT or MTOR substrates caused a reversion of the effects of FGFR2c on autophagy, which could also arise from the imbalance of the interplay between AKT/MTOR pathway and JNK1 signaling in favor of JNK1 activation, BCL-2 phosphorylation and possibly phagophore nucleation. Finally, silencing experiments of depletion of ESRP1, responsible for FGFR2 splicing and consequent FGFR2b expression, indicated that the switching from FGFR2b to FGFR2c isoform could represent the key event underlying the inhibition of the autophagic process in the epithelial context. Our results provide the first evidence of a negative impact of the out-of-context expression of FGFR2c on autophagy, suggesting a possible role of this receptor in the modulation of the recently proposed negative loop between autophagy and EMT during carcinogenesis

Polarized Endocytosis of the Keratinocyte Growth Factor Receptor in Migrating Cells: Role of Src-Signaling and Cortactin

Cell migration is a physiological process that requires endocytic trafficking and polarization of adhesion molecules and receptor tyrosine kinases (RTKs) to the leading edge. Many growth factors are able to induce motility by binding to specific RTK on target cells. Among them, keratinocyte growth factor (KGF or FGF7) and fibroblast growth factor 10 (FGF10), members of the FGF family, are motogenic for keratinocytes, and exert their action by binding to the keratinocyte growth factor receptor (KGFR), a splicing variant of FGFR2, exclusively expressed on epithelial cells. Here we analyzed the possible role of cortactin, an F-actin binding protein which is tyrosine phosphorylated by Src and is involved in KGFR-mediated cell migration, in the KGFR endocytosis and polarization to the leading edge of migrating cells upon ligand-induced stimulation. Biochemical phosphorylation study revealed that both KGF and FGF10 were able to induce tyrosine phosphorylation of Src and in turn of cortactin, as demonstrated by using the specific pharmacological Src-inhibitor SU6656, although FGF10 effect was delayed with respect to that promoted by KGF. Immunofluorescence analysis demonstrated the polarized localization of KGFR upon ligand stimulation to the leading edge of migrating keratinocytes, process that was regulated by Src. Moreover, we showed that the colocalization of cortactin with KGFR at the plasma membrane protrusions and on early endosomes after KGF and FGF10 treatment was Src-dependent. Further, by using a RNA interference approach through microinjection, we showed that cortactin is required for KGFR endocytosis and that the clathrin-dependent internalization of the receptor is a critical event for its polarization. Finally, KGFR expression and polarization enhanced cell migration in a scratch assay. Our results indicate that both Src and cortactin play a key role in the KGFR endocytosis and polarization at the leading edge of migrating keratinocytes, supporting the crucial involvement of RTK trafficking in cell motility

HPV16 E5 expression induces switching from FGFR2b to FGFR2c and epithelial-mesenchymal transition.

International audienceThe 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 TGFβ 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 aberrant expression in epithelial cells of the mesenchymal isoform of FGFR2 controls the negative crosstalk between EMT and autophagy

Signalling of the epithelial splicing variant of fibroblast growth factor receptor 2 (FGFR2b) triggers both differentiation and autophagy, while the aberrant expression of the mesenchymal FGFR2c isoform in epithelial cells induces impaired differentiation, inhibition of autophagy as well as the induction of the epithelial-mesenchymal transition (EMT). In light of the widely proposed negative loop linking autophagy and EMT in the early steps of carcinogenesis, here we investigated the possible involvement of FGFR2c aberrant expression and signalling in orchestrating this crosstalk in human keratinocytes. Biochemical, molecular, quantitative immunofluorescence analysis and in vitro invasion assays, coupled to the use of specific substrate inhibitors and transient or stable silencing approaches, showed that AKT/MTOR and PKCε are the two hub signalling pathways, downstream FGFR2c, intersecting with each other in the control of both the inhibition of autophagy and the induction of EMT and invasive behaviour. These results indicate that the expression of FGFR2c, possibly resulting from FGFR2 isoform switch, could represent a key upstream event responsible for the establishment of a negative interplay between autophagy and EMT, which contributes to the assessment of a pathological oncogenic profile in epithelial cells

Tumor derived Microvesicles enhance cross-processing ability of clinical grade Dendritic Cells

Tumor cells release extracellular microvesicles (MVs) in the microenvironment to deliver biological signals to neighbouring cells as well as to cells in distant tissues. Tumor-derived MVs appear to play contradictory role promoting both immunosuppression and tumor growth and both evoking tumor specific immune response. Recent evidences indicate that tumor-derived MVs can positively impact Dendritic Cells (DCs) immunogenicity by reprogramming DC antigen processing machinery and intracellular signaling pathways, thus promoting anti-tumor response. DCs are considered pivot cells of the immune system due to their exclusive ability to coordinate the innate and acquired immune responses, cross-present exogenous antigens and prime naïve T cells. DCs are required for the induction and maintenance of long-lasting anti-tumor immunity and their exploitation has been extensively investigated for the design of anti-tumor vaccines. However, the clinical grade culture conditions that are required to generate DCs for therapeutic use can strongly affect their functions. Here, we investigated the immunomodulatory impact of MVs carrying the MUC1 tumor glycoantigen (MVsMUC1) as immunogen formulation on clinical grade DCs grown in X-VIVO 15 (X-DCs). Results indicated that X-DCs displayed reduced performance of the antigen processing machinery in term of diminished phagocytosis and acidification of the phagosomal compartment suggesting an altered immunogenicity of clinical grade DCs. Pulsing DCs with MVsMUC1 restored phagosomal alkalinization, triggering ROS increase. This was not observed when a soluble MUC1 protein was employed (rMUC1). Concurrently, MVsMUC1 internalization by X-DCs allowed MUC1 cross-processing. Most importantly, MVsMUC1 pulsed DCs activated IFNγ response mediated by MUC1 specific CD8+ T cells. These results strongly support the employment of tumor-derived MVs as immunogen platforms for the implementation of DC-based vaccine

Temporal and geographic heterogeneity of the association between socioeconomic position and hospitalisation in Italy: an income based indicator

<p>Abstract</p> <p>Background</p> <p>The inverse association between socioeconomic position (SEP) and health has been extensively explored in Italy; however few studies have been carried out on the relationship between income inequalities and health status or health services utilisation, particularly at a local level.</p> <p>The objective of this study is to test the association between the demand for hospital care and a small area indicator based on income in four Italian cities, over a four-year period (1997-2000), in the adult population.</p> <p>Methods</p> <p>Census Block (median 260 residents) Median per capita Income (CBMI) was computed through record linkage between 1998 national tax and local population registries in the cities of Rome, Turin, Milan and Bologna (total population approximately 5.5 million). CBMI was linked to acute hospital discharges among residents, based on patient's residence.</p> <p>Age-standardized gender-specific hospitalisation rates were computed by CBMI quintiles (first quintile indicating lowest income), overall, and by city and year. Heterogeneity of the association between income level and hospitalisation was analysed through a Poisson model.</p> <p>Results</p> <p>We found an inverse association between small area income level and hospitalisation rates, which decreased continuously from 153 per 1000 inhabitants in the first quintile to 107 per 1000 inhabitants in the fifth quintile. Income differences in hospitalisation were confirmed in each city and year. However, the magnitude of the association and the absolute level of hospitalisation rates were quite different in each city and tended to slightly decrease over time in all cities considered, except Bologna.</p> <p>Conclusion</p> <p>Our study confirms an inverse association between income level and the use of hospitalization in four Italian cities, using a small area economic indicator, based on population tax data. Further analysis of the association between income and cause-specific hospitalization rates will allow to better understand the capability of the Italian National Health System to compel with socio-economic inequalities in health needs.</p> <p>Furthermore the SEP indicator we propose can represent a contribution to the improvement of tools for monitoring inequalities in health and in health services utilization.</p