TRPA1 Contributes to FGFR2c Signaling and to Its Oncogenic Outcomes in Pancreatic Ductal Adenocarcinoma-Derived Cell Lines.

Fibroblast growth factor receptor (FGFR) signaling is a key modulator of cellular processes dysregulated in cancer. We recently found that the high expression of the mesenchymal FGFR2c variant in human pancreatic ductal adenocarcinoma (PDAC)-derived cells triggers the PKCε-mediated improvement of EMT and of MCL-1/SRC-dependent cell invasion. Since other membrane proteins can affect the receptor tyrosine kinase signaling, including transient receptor potential channels (TRPs), in this work, we investigated the role of TRPs in the FGFR2c/PKCε oncogenic axis. Our results highlighted that either the FGFR2c/PKCε axis shut-off obtained by shRNA or its sustained activation via ligand stimulation induces TRPA1 downregulation, suggesting a channel/receptor dependence. Indeed, biochemical molecular and immunofluorescence approaches demonstrated that the transient depletion of TRPA1 by siRNA was sufficient to attenuate FGFR2c downstream signaling pathways, as well as the consequent enhancement of EMT. Moreover, the biochemical check of MCL1/SRC signaling and the in vitro assay of cellular motility suggested that TRPA1 also contributes to the FGFR2c-induced enhancement of PDAC cell invasiveness. Finally, the use of a selective channel antagonist indicated that the contribution of TRPA1 to the FGFR2c oncogenic potential is independent of its pore function. Thus, TRPA1 could represent a putative candidate for future target therapies in PDAC.

FGFR2c Upregulation Contributes to Cancer-Associated Fibroblast Program Activation and to Enhanced Autophagy in Actinic Keratosis-Derived Dermal Fibroblasts: A Possible Role in Precancerous Cell/Stromal Cell Crosstalk.

Actinic keratosis (AK) is a preneoplastic skin disorder which can rapidly progress to cutaneous squamous cell carcinomas (SCCs). In light of our previous findings, indicating a possible oncogenic role of the mesenchymal isoform of FGFR2 (FGFR2c) aberrantly expressed in AK keratinocytes, we analyzed the possible tumor-promoting role of this receptor in the stromal AK counterpart in this work. Molecular analysis showed that, particularly in early AK lesions, FGFR2c dermal upregulation is accompanied by the downregulation of the cancer-associated fibroblasts (CAF) transcription repressor CSL, the upregulation of the CAF activator ULK3, and the consequent CAF gene induction. Immunofluorescence and molecular analysis, coupled with silencing approaches by siRNA, applied on primary cultures of KIN I-derived fibroblasts, indicated that FGFR2c upregulation contribute to CAF signature and the increased autophagy in response to FGF2. Magnetic bead-based multiplex assay, combined with FGFR2 signaling shut-off approaches, indicated that, especially in response to FGF2, IL-6 secretion could depend on FGFR2c high expression and signaling, suggesting the possible establishment of FGFR2c-dependent secretory autophagy, contributing to tumor-promoting factor release. Overall, our results identified FGFR2c as a signaling molecule involved in controlling precancerous/stromal cell oncogenic crosstalk, pointing to this receptor as a possible early molecular marker predictive for AK's rapid malignant progression.

The FGFR2c/PKCε Axis Controls MCL-1-Mediated Invasion in Pancreatic Ductal Adenocarcinoma Cells: Perspectives for Innovative Target Therapies.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy whose main characterizations are Kirsten Rat Sarcoma-activating mutations (KRAS) and a highly aggressive phenotype. Based on our recent findings demonstrating that the highly aberrant expression of the mesenchymal isoform of Fibroblast Growth Factor Receptor 2 (FGFR2c) in PDAC cells activates Protein-Kinase C Epsilon (PKCε), which in turn controls receptor-mediated epithelial to mesenchymal transition (EMT), here we investigated the involvement of these signaling events in the establishment of additional tumorigenic features. Using PDAC cell lines expressing divergent levels of the FGFR2c and stable protein depletion approaches by short hairpin RNA (shRNA), we found that FGFR2c expression and its PKCε downstream signaling are responsible for the invasive response to Fibroblast Growth Factor 2 (FGF2) and for anchorage-independent growth. In addition, in vitro clonogenic assays, coupled with the check of the amount of cleaved Poly Adenosine Diphosphate-Ribose Polymerase 1 (PARP1) by Western blot, highlighted the involvement of both FGFR2c and PKCε in cell viability. Finally, monitoring of Myeloid Cell Leukemia 1 (MCL-1) expression and Sarcoma kinase family (SRC) phosphorylation suggested that the FGFR2c/PKCε axis could control cell migration/invasion possibly via MCL-1/SRC-mediated reorganization of the actin cytoskeleton. Being PKCs RAS-independent substrates, the identification of PKCε as a hub molecule downstream FGFR2c at the crossroad of signaling networks governing the main malignant tumor hallmarks could represent an important advance towards innovative target therapies overcoming RAS.

Role of FGFR2c and Its PKCε Downstream Signaling in the Control of EMT and Autophagy in Pancreatic Ductal Adenocarcinoma Cells.

Pancreatic ductal adenocarcinoma (PDAC) is a treatment-resistant malignancy characterized by a high malignant phenotype including acquired EMT signature and deregulated autophagy. Since we have previously described that the aberrant expression of the mesenchymal FGFR2c and the triggering of the downstream PKCε signaling are involved in epidermal carcinogenesis, the aim of this work has been to assess the contribution of these oncogenic events also in the pancreatic context. Biochemical, molecular and immunofluorescence approaches showed that FGFR2c expression impacts on PDAC cell responsiveness to FGF2 in terms of intracellular signaling activation, upregulation of EMT-related transcription factors and modulation of epithelial and mesenchymal markers compatible with the pathological EMT. Moreover, shut-off via specific protein depletion of PKCε signaling, activated by high expression of FGFR2c resulted in a reversion of EMT profile, as well as in a recovery of the autophagic process. The detailed biochemical analysis of the intracellular signaling indicated that PKCε, bypassing AKT and directly converging on ERK1/2, could be a signaling molecule downstream FGFR2c whose inhibition could be considered as possible effective therapeutic approach in counteracting aggressive phenotype in cancer.

Expression of the E5 Oncoprotein of HPV16 Impacts on the Molecular Profiles of EMT-Related and Differentiation Genes in Ectocervical Low-Grade Lesions.

Infection with human papillomavirus type 16 (HPV16) is one of the major risk factors for the development of cervical cancer. Our previous studies have demonstrated the involvement of the early oncoprotein E5 of HPV16 (16E5) in the altered isoform switch of fibroblast growth factor receptor 2 (FGFR2) and the consequent expression in human keratinocytes of the mesenchymal FGFR2c isoform, whose aberrant signaling leads to EMT, invasiveness, and dysregulated differentiation. Here, we aimed to establish the possible direct link between these pathological features or the appearance of FGFR2c and the expression of 16E5 in low-grade squamous intraepithelial lesions (LSILs). Molecular analysis showed that the FGFR2c expression displayed a statistically significant positive correlation with that of the viral oncoprotein, whereas the expression values of the epithelial FGR2b variant, as well as those of the differentiation markers keratin 10 (K10), loricrin (LOR) and involucrin (INV), were inversely linked to the 16E5 expression. In contrast, the expression of EMT-related transcription factors Snail1 and ZEB1 overlapped with that of 16E5, becoming a statistically significant positive correlation in the case of Snail2. Parallel analysis performed in human cervical LSIL-derived W12 cells, containing episomal HPV16, revealed that the depletion of 16E5 by siRNA was able to counteract these molecular events, proving to represent an effective strategy to identify the specific role of this viral oncoprotein in determining LSIL oncogenic and more aggressive profiles. Overall, coupling in vitro approaches to the molecular transcript analysis in ectocervical early lesions could significantly contribute to the characterization of specific gene expression profiles prognostic for those LSILs with a greater probability of direct neoplastic progression.

Expression Profile of Fibroblast Growth Factor Receptors, Keratinocyte Differentiation Markers, and Epithelial Mesenchymal Transition-Related Genes in Actinic Keratosis: A Possible Predictive Factor for Malignant Progression?

Actinic keratosis (AK) is the ultra violet (UV)-induced preneoplastic skin lesion clinically classified in low (KIN I), intermediate (KIN II), and high (KIN III) grade lesions. In this work we analyzed the expression of Fibroblast Growth Factor Receptors (FGFRs), as well as of keratinocyte differentiation and epithelial-to-mesenchymal transition (EMT)-related markers in differentially graded AK lesions, in order to identify specific expression profiles that could be predictive for direct progression of some KIN I lesions towards squamous cell carcinoma (SCC). Our molecular analysis showed that the keratinocyte differentiation markers keratin 1 (K1), desmoglein-1 (DSG1), and filaggrin (FIL) were progressively downregulated in KIN I, II, and III lesions, while the modulation of epithelial/mesenchymal markers and the induction of the transcription factors Snail1 and Zinc finger E-box-binding homeobox 1 (ZEB1) compatible with pathological EMT, even if observable, did not appear to correlate with AK progression. Concerning FGFRs, a modulation of epithelial isoform of FGFR2 (FGFR2b) and the mesenchymal FGFR2c isoform compatible with an FGFR2 isoform switch, as well as FGFR4 upregulation were observed starting from KIN I lesions, suggesting that they could be events involved in early steps of AK pathogenesis. In contrast, the increase of FGFR3c expression, mainly appreciable in KIN II and KIN III lesions, suggested a correlation with AK late progression. Interestingly, the strong modulation of FIL, Snail1, as well as of FGFR2c, FGFR4, and of their ligand FGF2, observed in some of the KIN I samples, may indicate that they could be molecular markers predictive for those low graded lesions destined to a direct progression to SCC. In conclusion, our data point on the identification of molecular markers predictive for AK rapid progression through the "differentiated" pathway. Our results also represent an important step that, in future, will help to clarify the molecular mechanisms underlying FGFR signaling deregulation in epithelial tissues during the switch from the pre-neoplastic to the oncogenic malignant phenotype.

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.

Role of PKCε in the epithelial-mesenchymal transition induced by FGFR2 isoform switch.

BACKGROUND: The epithelial isoform of the fibroblast growth factor receptor 2 (FGFR2b) controls the entire program of keratinocyte differentiation via the sequential involvement of protein kinase C (PKC) δ and PKCα. In contrast, the FGFR2 isoform switch and the aberrant expression of the mesenchymal FGFR2c isoform leads to impairment of differentiation, epithelial-mesenchymal transition (EMT) and tumorigenic features. Aim of our present study was to contribute in clarifying the complex network of signaling pathways involved in the FGFR2c-mediated oncogenic outcomes focusing on PKCε, which appears to be involved in the induction of EMT and tumorigenesis in several epithelial contexts. METHODS: Biochemical and molecular analysis, as well as in vitro invasion assays, combined with the use of specific small interfering RNA (siRNA), were performed in human keratinocytes stably expressing FGFR2c or FGFR2b isoforms. RESULTS: Our results showed that aberrant expression and signaling of FGFR2c, but not those of FGFR2b, in human keratinocytes induced a strong phosphorylation/activation of PKCε. The use of siRNA approach showed that PKCε is the hub signaling downstream FGFR2c responsible for the modulation of EMT markers and for the induction of the EMT-related transcription factors STAT3, Snail1 and FRA1, as well as for the acquisition of the invasive behavior. Moreover, experiments of depletion of ESRP1, responsible for FGFR2 splicing in epithelial cells, indicated that the activation of PKCε is the key molecular event triggered by FGFR2 isoform switch and underlying EMT induction. CONCLUSIONS: Overall, our results point to the identification of the downstream PKC isoform responsible for the FGFR signaling deregulation occurring in epithelial tissues from the physiological oncosoppressive to the pathological oncogenic profile. Video Abstract.

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.

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 neighboring 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 vaccines.

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.

Role of Fibroblast Growth Factor Receptor 2b in the Cross Talk between Autophagy and Differentiation: Involvement of Jun N-Terminal Protein Kinase Signaling.

Fibroblast growth factor receptor 2b (FGFR2b) is a receptor tyrosine kinase expressed exclusively in epithelial cells. We previously demonstrated that FGFR2b induces autophagy and that this process is required for the triggering of FGFR2b-mediated early differentiation of keratinocytes. However, the molecular mechanisms regulating this interplay remain to be elucidated. Since we have also recently shown that Jun N-terminal protein kinase 1 (JNK1) signaling is involved in FGFR2b-induced autophagy and a possible role of the JNK pathway in epidermal differentiation has been suggested (though it is still debated), we investigated here the cross talk between FGFR2b-mediated autophagy and differentiation, focusing on the downstream JNK signaling. Biochemical, molecular, and immunofluorescence approaches in 2-dimensional (2-D) keratinocyte cultures and three-dimensional (3-D) organotypic skin equivalents confirmed that FGFR2b overexpression increased both autophagy and early differentiation. The use of FGFR2b substrate inhibitors and the silencing of JNK1 highlighted that this signaling is required not only for autophagy but also for the triggering of early differentiation. In contrast, the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway did not appear to be involved in the two processes, and AKT signaling, whose activation contributes to the FGFR2b-mediated onset of keratinocyte differentiation, was not required for the triggering of autophagy. Overall, our results point to JNK1 as a signaling hub that regulates the interplay between FGFR2b-induced autophagy and differentiation.

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.

Expression of the FGFR2c mesenchymal splicing variant in human keratinocytes inhibits differentiation and promotes invasion.

The altered isoform switching of the fibroblast growth factor receptor 2 (FGFR2) and aberrant expression of the mesenchymal FGFR2c isoform in epithelial cells is involved in cancer progression. We have recently described that the ectopic expression of FGFR2c in normal human keratinocytes induces epithelial-mesenchymal transition and leads to invasiveness and anchorage-independent growth. Here, we extended our analysis to the effects of this FGFR2c forced expression on human keratinocyte differentiation and stratification. Our findings demonstrated that, differently from cells overexpressing the epithelial splicing variant FGFR2b, keratinocytes ectopically expressing FGFR2c are not able to form a monolayer and display decreased expression of early differentiation markers. This impaired ability to enter the differentiation program is related to the up-modulation of the transcription factor ΔNp63. In addition, FGFR2c-expressing keratinocytes undergo defective stratification and invasion of the collagen matrix in 3D organotypic cultures, further suggesting their tumorigenic potential. Taken together, our results support the hypothesis that the receptor switching and the consequent appearance of the mesenchymal FGFR2c variant in the epithelial context would drive early steps of carcinogenesis, unbalancing the p63/FGFR interplay, and altering the paracrine response to the microenvironment.

Tumor-Derived Microvesicles Modulate Antigen Cross-Processing via Reactive Oxygen Species-Mediated Alkalinization of Phagosomal Compartment in Dendritic Cells.

Dendritic cells (DCs) are the only antigen-presenting cells able to prime naïve T cells and cross-prime antigen-specific CD8+ T cells. Their functionality is a requirement for the induction and maintenance of long-lasting cancer immunity. Albeit intensively investigated, the in vivo mechanisms underlying efficient antigen cross-processing and presentation are not fully understood. Several pieces of evidence indicate that antigen transfer to DCs mediated by microvesicles (MVs) enhances antigen immunogenicity. This mechanism is also relevant for cross-presentation of those tumor-associated glycoproteins such as MUC1 that are blocked in HLA class II compartment when internalized by DCs as soluble molecules. Here, we present pieces of evidence that the internalization of tumor-derived MVs modulates antigen-processing machinery of DCs. Employing MVs derived from ovarian cancer ascites fluid and established tumor cell lines, we show that MV uptake modifies DC phagosomal microenvironment, triggering reactive oxygen species (ROS) accumulation and early alkalinization. Indeed, tumor MVs carry radical species and the MV uptake by DCs counteracts the chemically mediated acidification of the phagosomal compartment. Further pieces of evidence suggest that efficacious antigen cross-priming of the MUC1 antigen carried by the tumor MVs results from the early signaling induced by MV internalization and the function of the antigen-processing machinery of DCs. These results strongly support the hypothesis that tumor-derived MVs impact antigen immunogenicity by tuning the antigen-processing machinery of DCs, besides being carrier of tumor antigens. Furthermore, these findings have important implications for the exploitation of MVs as antigenic cell-free immunogen for DC-based therapeutic strategies.

Expression of the FGFR2 mesenchymal splicing variant in epithelial cells drives epithelial-mesenchymal transition.

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.

Combination of antibodies directed against different ErbB3 surface epitopes prevents the establishment of resistance to BRAF/MEK inhibitors in melanoma.

Patients with metastatic melanoma bearing V600 mutations in BRAF oncogene clinically benefit from the treatment with BRAF inhibitors alone or in combination with MEK inhibitors. However, a limitation to such treatment is the occurrence of resistance. Tackling the adaptive changes helping cells survive from drug treatment may offer new therapeutic opportunities. Very recently the ErbB3 receptor has been shown to act as a central node promoting survival of BRAF mutated melanoma. In this paper we first demonstrate that ErbB3/AKT hyperphosphorylation occurs in BRAF mutated melanoma cell lines following exposure to BRAF and/or MEK inhibitors. This strongly correlates with increased transcriptional activation of its ligand neuregulin. Anti-ErbB3 antibodies impair the establishment of de novo cell resistance to BRAF inhibition in vitro. In order to more potently ablate ErbB3 activity we used a combination of two anti-ErbB3 antibodies directed against distinct epitopes of its extracellular domain. These two antibodies in combo with BRAF/MEK inhibitors potently inhibit in vitro cell growth and tumor regrowth after drug withdrawal in an in vivo xenograft model. Importantly, residual tumor masses from mice treated by the antibodies and BRAF/ERK inhibitors combo are characterized almost exclusively by large necrotic areas with limited residual areas of tumor growth. Taken together, our findings support the concept that triple therapy directed against BRAF/MEK/ErbB3 may be able to provide durable control of BRAF mutated metastatic melanoma.

HPV16 E5 deregulates the autophagic process in human keratinocytes.

Autophagy plays key roles during host defense against pathogens, but viruses have evolved strategies to block the process or to exploit it for replication and successful infection. The E5 oncoprotein of human papillomavirus type 16 (HPV16 E5) perturbs epithelial homeostasis down-regulating the expression of the keratinocyte growth factor receptor (KGFR/FGFR2b), whose signaling induces autophagy. Here we investigated the possible effects of 16E5 on autophagy in human keratinocytes expressing the viral protein. The 16E5 presence strongly inhibited the autophagic process, while forced expression and activation of KGFR counteracted this effect, demonstrating that the viral protein and the receptor exert opposite and interplaying roles not only on epithelial differentiation, but also in the control of autophagy. In W12 cells, silencing of the 16E5 gene in the context of the viral full length genome confirmed its role on autophagy inhibition. Finally, molecular approaches showed that the viral protein interferes with the transcriptional regulation of autophagy also through the impairment of p53 function, indicating that 16E5 uses parallel mechanisms for autophagy impairment. Overall our results further support the hypothesis that a transcriptional crosstalk among 16E5 and KGFR might be the crucial molecular driver of epithelial deregulation during early steps of HPV infection and transformation.

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 TGFß 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.