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

Ranieri, Danilo, Belleudi, Francesca, Magenta, Alessandra, Torrisi, Maria Rosaria·2015·International Journal of Cancer
DOI 10.1002/ijc.29373
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Abstract

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.

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Figure 1
Figure 1 CC-BY-NC-ND · From the PDF
Figure 2
Figure 2 CC-BY-NC-ND · From the PDF
Figure 3 16E5 promotes EMT through FGFR2 isoform switch and modifies the response to FGFs. (a) HaCaT pCI-neo and HaCaT E5 cells, grown in serum, were fixed and double immunofluorescence was performed using anti-HA monoclonal antibody, to visualize the 16E5 protein and TRITC-conjugated phalloidin, to analyze the actin cytoskeleton organization. Cell nuclei were visualized by DAPI. A portion of HaCaT E5 cells, positively stained for the viral protein (green), shows spindle mesenchymal morphology (HaCaT E5, left panel) and actin reorganization (red) with appearance of stress fibers (inset), while others (HaCaT E5, right panel) seem to maintain the polygonal epithelial shape. Bar: 10 m m. ( b ) HaCaT cells were transfected with small interfering RNA for FGFR2/Bek (FGFR2 siRNA) to obtain receptor silencing or with an unrelated siRNA used as control. Western blot analysis using anti-Bek polyclonal antibodies, recognizing both FGFR2b/2c proteins, confirms that the receptor expression, shown by the specific 140 kDa band corresponding to the molecular weight of FGFR2, is efficiently down-modulated in FGFR2 siRNA-transfected cells. The equal loading was assessed with anti-actin antibody. ( c ) HaCaT cells were coinjected with FGFR2 siRNA, E5 cDNA and mouse IgG to identify the injected cells. Coinjection of E5 cDNA and an unrelated siRNA was performed as control. After injection, cells were serum starved and then treated with the FGFR2b ligand KGF/FGF7 or with the FGFR2c ligand FGF2 before fixation. Alternatively, cells were kept in serum-free medium (-GFs) and then fixed. Immunofluorescence analysis to evaluate the EMT process was performed with anti-Ecadherin or anti-vimentin monoclonal antibodies and injected cells were visualized with anti-IgG polyclonal antibodies. Cell nuclei were visualized by DAPI. The pattern of staining observed in injected cells was analyzed in comparison with untreated control pCI-neo transfected cells (left panels) and with the uninjected cells surrounding the injected ones. In injected cells expressing E5 (asterisks in E5 cDNA/control siRNA), either unstimulated (-GFs) or treated with KGF, the signal corresponding to the plasma membrane epithelial marker E-cadherin appears unmodified respect to control pCI-neo transfected or uninjected cells and no staining for the cytoplasmic mesenchymal marker vimentin is visible. In contrast, treatment with FGF2 induces a clear decrease in E-cadherin staining and appearance of vimentin signal associated with an elongated mesenchymal morphology. Depletion of the FGFR2 (E5 cDNA/FGFR2 siRNA, right panels) is able to abolish the effects observed upon FGF2 treatment, since the injected cells mantain both the epithelial shape and the E-cadherin signal and are negative for the vimentin EMT marker. Bar: 10 m m. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Figure 3 16E5 promotes EMT through FGFR2 isoform switch and modifies the response to FGFs. (a) HaCaT pCI-neo and HaCaT E5 cells, grown in serum, were fixed and double immunofluorescence was performed using anti-HA monoclonal antibody, to visualize the 16E5 protein and TRITC-conjugated phalloidin, to analyze the actin cytoskeleton organization. Cell nuclei were visualized by DAPI. A portion of HaCaT E5 cells, positively stained for the viral protein (green), shows spindle mesenchymal morphology (HaCaT E5, left panel) and actin reorganization (red) with appearance of stress fibers (inset), while others (HaCaT E5, right panel) seem to maintain the polygonal epithelial shape. Bar: 10 m m. ( b ) HaCaT cells were transfected with small interfering RNA for FGFR2/Bek (FGFR2 siRNA) to obtain receptor silencing or with an unrelated siRNA used as control. Western blot analysis using anti-Bek polyclonal antibodies, recognizing both FGFR2b/2c proteins, confirms that the receptor expression, shown by the specific 140 kDa band corresponding to the molecular weight of FGFR2, is efficiently down-modulated in FGFR2 siRNA-transfected cells. The equal loading was assessed with anti-actin antibody. ( c ) HaCaT cells were coinjected with FGFR2 siRNA, E5 cDNA and mouse IgG to identify the injected cells. Coinjection of E5 cDNA and an unrelated siRNA was performed as control. After injection, cells were serum starved and then treated with the FGFR2b ligand KGF/FGF7 or with the FGFR2c ligand FGF2 before fixation. Alternatively, cells were kept in serum-free medium (-GFs) and then fixed. Immunofluorescence analysis to evaluate the EMT process was performed with anti-Ecadherin or anti-vimentin monoclonal antibodies and injected cells were visualized with anti-IgG polyclonal antibodies. Cell nuclei were visualized by DAPI. The pattern of staining observed in injected cells was analyzed in comparison with untreated control pCI-neo transfected cells (left panels) and with the uninjected cells surrounding the injected ones. In injected cells expressing E5 (asterisks in E5 cDNA/control siRNA), either unstimulated (-GFs) or treated with KGF, the signal corresponding to the plasma membrane epithelial marker E-cadherin appears unmodified respect to control pCI-neo transfected or uninjected cells and no staining for the cytoplasmic mesenchymal marker vimentin is visible. In contrast, treatment with FGF2 induces a clear decrease in E-cadherin staining and appearance of vimentin signal associated with an elongated mesenchymal morphology. Depletion of the FGFR2 (E5 cDNA/FGFR2 siRNA, right panels) is able to abolish the effects observed upon FGF2 treatment, since the injected cells mantain both the epithelial shape and the E-cadherin signal and are negative for the vimentin EMT marker. Bar: 10 m m. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]CC-BY-NC-ND · From the PDF
Figure 4 FGF2 triggers EMT in cervical epithelial cells expressing HPV16 E5. W12p6 cells, containing episomal HPV16 and expressing 16E5 as shown in Figure 1 b , were treated with TGF b 1, as positive control of EMT induction, or with KGF/FGF7 or FGF2 ligands to activate FGFR2b and FGFR2c, respectively. Quantitative immunofluorescence analysis, using antibodies directed against the epithelial markers E-cadherin and b 4integrin or recognizing the mesenchymal marker vimentin, was performed as described in Material and Methods to evaluate the percentages of cells positive for the EMT markers. Similarly, to TGF b 1 stimulation, FGF2 treatment induces a drastic decrease in the percentage of cells showing the plasma membrane staining for the epithelial markers as well as the appearance in a significant percentage of cells of the cytosolic and filamentous signal of the mesenchymal marker vimentin. In contrast, KGF/FGF7 treatment does not modify the pattern of staining observed in unstimulated (-GFs) cells. The FGF2-induced effects are abolished in the presence of the specific FGFR tyrosine kinase inhibitor SU5402. The quantitative analysis was performed and the cut-off of the signal intensity was selected as described in Material and Methods. Results are expressed as mean values 6 SD. Student's t test was performed as reported in Material and Methods and significance level has been defined as follows: *, ***, ^^ p < 0.001 vs the corresponding untreated cells, **, ^, ^^^ p < 0.001 vs the corresponding FGF2-treated cells. Bar: 10 m m [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Figure 4 FGF2 triggers EMT in cervical epithelial cells expressing HPV16 E5. W12p6 cells, containing episomal HPV16 and expressing 16E5 as shown in Figure 1 b , were treated with TGF b 1, as positive control of EMT induction, or with KGF/FGF7 or FGF2 ligands to activate FGFR2b and FGFR2c, respectively. Quantitative immunofluorescence analysis, using antibodies directed against the epithelial markers E-cadherin and b 4integrin or recognizing the mesenchymal marker vimentin, was performed as described in Material and Methods to evaluate the percentages of cells positive for the EMT markers. Similarly, to TGF b 1 stimulation, FGF2 treatment induces a drastic decrease in the percentage of cells showing the plasma membrane staining for the epithelial markers as well as the appearance in a significant percentage of cells of the cytosolic and filamentous signal of the mesenchymal marker vimentin. In contrast, KGF/FGF7 treatment does not modify the pattern of staining observed in unstimulated (-GFs) cells. The FGF2-induced effects are abolished in the presence of the specific FGFR tyrosine kinase inhibitor SU5402. The quantitative analysis was performed and the cut-off of the signal intensity was selected as described in Material and Methods. Results are expressed as mean values 6 SD. Student's t test was performed as reported in Material and Methods and significance level has been defined as follows: *, ***, ^^ p < 0.001 vs the corresponding untreated cells, **, ^, ^^^ p < 0.001 vs the corresponding FGF2-treated cells. Bar: 10 m m [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]CC-BY-NC-ND · From the PDF
Figure 5
Figure 5 CC-BY-NC-ND · From the PDF

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Cited by

Metadata

Authors
Ranieri, D., Belleudi, F., Magenta, A., & Torrisi, M. R.
Affiliations
Istituto Pasteur‐Fondazione Cenci Bolognetti Dipartimento di Medicina Clinica e Molecolare Sapienza Università di Roma Italy
Venue
International Journal of Cancer
Publisher
Wiley
Published
2014-12-13
Volume
137
Issue
1
Pages
61-72
ISSN
  • 0020-7136
  • 1097-0215
DOI
10.1002/ijc.29373
Cited by
54
References
1
Language
en
Licence
creativecommons.org/licenses/by-nc-nd/3.0/ (vor)
Funders
  • MIUR
  • Associazione Italiana per la Ricerca sul Cancro (AIRC), Italy (IG 15858)

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