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.

Zinc Supplementation Enhances the Pro-Death Function of UPR in Lymphoma Cells Exposed to Radiation.

We have previously shown that Zinc supplementation triggered ER stress/UPR in cancer cells undergoing treatment by genotoxic agents, reactivated wtp53 in cancer cells harboring mutant p53 (mutp53) and potentiated the activity of wtp53 in those carrying wtp53. In this study, we used Zinc chloride alone or in combination with 2 Gy radiation to treat Primary Effusion Lymphoma (PEL) cells, an aggressive B-cell lymphoma associated with KSHV that harbors wt or partially functioning p53. We found that Zinc triggered a mild ER stress/UPR in these lymphoma cells and activated ERK1/2, molecule known to sustain cell survival in the course of UPR activation. In combination with radiations, Zinc triggered a stronger p53 activation that counteracted its mediated ERK1/2 phosphorylation, further upregulating the UPR molecule CHOP and promoting cell death. These data suggest that Zinc supplementation could be a promising strategy to reduce the doses of radiation and possibly of other DNA-damaging agents to obtain an efficient capacity to induce lymphoma cell death.

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.

IRE1 Alpha/XBP1 Axis Sustains Primary Effusion Lymphoma Cell Survival by Promoting Cytokine Release and STAT3 Activation.

Primary Effusion Lymphoma (PEL) is a highly aggressive B cell lymphoma associated with Kaposi's Sarcoma-associated Herpesvirus (KSHV). It is characterized by a high level of basal Endoplasmic Reticulum (ER) stress, Unfolded Protein Response (UPR) activation and constitutive phosphorylation of oncogenic pathways such as the Signal Transducer and activator of Transcription (STAT3). In this study, we found that the inositol requiring kinase (IRE) 1alpha/X-box binding protein (XBP1) axis of UPR plays a key role in the survival of PEL cells, while double stranded RNA-activated protein kinase-like ER kinase (PERK) and activating transcription factor (ATF) 6 slightly influence it, in correlation with the capacity of the IRE1alpha/XBP1 axis to induce the release of interleukin (IL)-6, IL-10 and Vascular-Endothelial Growth Factor (VEGF). Moreover, we found that IRE1alpha/XBP1 inhibition reduced STAT3 Tyr705 phosphorylation and induced a pro-survival autophagy in PEL cells. In conclusion, this study suggests that targeting the IRE1alpha/XBP1 axis represents a promising strategy against PEL cells and that the cytotoxic effect of this treatment may be potentiated by autophagy inhibition.