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Celecoxib increases EGF signaling in colon tumor associated fibroblasts, modulating EGFR expression and degradation.

Venè R, Tosetti F, Minghelli S, Poggi A, Ferrari N, Benelli R - Oncotarget (2015)

Bottom Line: We have now identified two concomitant mechanisms explaining the EGF-Celecoxib cooperation.Celecoxib modulated the levels of target proteins similarly to the inhibitors of endosome/lysosome acidification Bafilomycin-A1 and NH(4)Cl.Cytoplasmic vesicles fractionation showed a reduced maturation of Cathepsin-D in late endosomes and an increased content of EGFR and Rab7 in lysosomes of Celecoxib-treated TAFs.Our data indicate a double mechanism mediating the increased response to EGF of colon TAFs treated with Celecoxib.

View Article: PubMed Central - PubMed

Affiliation: Immunology Lab, IRCCS AOU San Martino - IST, Genoa, Italy.

ABSTRACT
We previously demonstrated that non-toxic doses of Celecoxib induced the immediate phosphorylation of Erk1-2 in colon tumor associated fibroblasts (TAFs), increasing their responsiveness to epidermal growth factor (EGF). We have now identified two concomitant mechanisms explaining the EGF-Celecoxib cooperation. We found that a 24-48h Celecoxib priming increased EGF receptor (EGFR) mRNA and protein levels in colon TAFs, promoting EGF binding and internalization. Celecoxib-primed TAFs showed a reduced EGFR degradation after EGF challenge. This delay corresponded to a deferred dissociation of EEA1 from EGFR positive endosomes and the accumulation of Rab7, pro Cathepsin-D and SQSTM1/p62, suggesting a shared bottleneck in the pathways of late-endosomes/autophagosomes maturation. Celecoxib modulated the levels of target proteins similarly to the inhibitors of endosome/lysosome acidification Bafilomycin-A1 and NH(4)Cl. Cytoplasmic vesicles fractionation showed a reduced maturation of Cathepsin-D in late endosomes and an increased content of EGFR and Rab7 in lysosomes of Celecoxib-treated TAFs.Our data indicate a double mechanism mediating the increased response to EGF of colon TAFs treated with Celecoxib. While EGFR overexpression could be targeted using anti EGFR drugs, the effects on endosome trafficking and protein turnover represents a more elusive target and should be taken into account for any long-term therapy with Celecoxib.

No MeSH data available.


Related in: MedlinePlus

Celecoxib slows down EGFR degradationa) Western blot analysis of the kinetic of EGFR degradation. Colon TAFs pretreated with Celecoxib were challenged with EGF for the indicated times. The arrow indicates the band used for EGFR degradation quantification. The test was repeated twice. b) The early endosome marker 1 (EEA1) levels were not influenced by Celecoxib pretreatment. c) A representative image (90min EGF) of double immunofluorescence analyses: EGFR (red), EEA1 (green). Celecoxib-pretreated colon TAFs were challenged with EGF for 30, 90, 180min or 16h. Fluorescent images were acquired, with fixed expositions (EEA1-488 f1/8; EGFR-594 f1/3; DAPI f1/100), by a Leica DM-LB2 microscope equipped with I3 and M2 filters and a HCX PL Fluotar 40x non immersion optic. A 20μm scale is shown. d) Analysis of Mander's overlay coefficients for EGFR and EEA1 on the double immunofluorescence. Six random 40x fields per condition -containing at least 12 TAFs- were analyzed (see methods). The test was repeated twice. e) Flow cytometric analysis for EGFR expression in TAFs pretreated or not with Celecoxib and then challenged for 90 or 180min with EGF. The peaks, representing EGFR expression under EGF, or Celecoxib plus EGF (Cel+EGF) treatments (white peaks), were compared to EGFR levels detected in untreated controls (grey peaks). The MFI ratio (white/grey) was calculated and reported on each panel. The test was repeated twice.
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Figure 3: Celecoxib slows down EGFR degradationa) Western blot analysis of the kinetic of EGFR degradation. Colon TAFs pretreated with Celecoxib were challenged with EGF for the indicated times. The arrow indicates the band used for EGFR degradation quantification. The test was repeated twice. b) The early endosome marker 1 (EEA1) levels were not influenced by Celecoxib pretreatment. c) A representative image (90min EGF) of double immunofluorescence analyses: EGFR (red), EEA1 (green). Celecoxib-pretreated colon TAFs were challenged with EGF for 30, 90, 180min or 16h. Fluorescent images were acquired, with fixed expositions (EEA1-488 f1/8; EGFR-594 f1/3; DAPI f1/100), by a Leica DM-LB2 microscope equipped with I3 and M2 filters and a HCX PL Fluotar 40x non immersion optic. A 20μm scale is shown. d) Analysis of Mander's overlay coefficients for EGFR and EEA1 on the double immunofluorescence. Six random 40x fields per condition -containing at least 12 TAFs- were analyzed (see methods). The test was repeated twice. e) Flow cytometric analysis for EGFR expression in TAFs pretreated or not with Celecoxib and then challenged for 90 or 180min with EGF. The peaks, representing EGFR expression under EGF, or Celecoxib plus EGF (Cel+EGF) treatments (white peaks), were compared to EGFR levels detected in untreated controls (grey peaks). The MFI ratio (white/grey) was calculated and reported on each panel. The test was repeated twice.

Mentions: We tested if Celecoxib could alter the kinetic of EGFR degradation upon EGF triggering. Challenging colon TAFs with EGF (Fig. 3a), we noticed the appearance of low molecular weight bands indicating EGFR degradation at 60min and 90min, becoming more evident at 120min. Interestingly, in the cells pretreated with Celecoxib EGFR degradation was increased during the first hour of triggering, but delayed at 90 and even more at 120min. To track EGFR along its degradative route we needed an endosomal marker whose levels were not modulated by Celecoxib. We found that the early endosome marker 1 (EEA1) satisfied this condition (Fig. 3b), thus EEA1 was used for co-localization studies with EGFR. By double immunofluorescence and microscopic evaluation, we analyzed the overlap for EGFR and EEA1 signals in colon TAFs. The representative images shown in Fig. 3c (90min of incubation with EGF) indicated that most EGFR staining localized in EEA1 positive vesicles. Calculating Mander's coefficients for EEA1 and EGFR co-localization (Fig. 3d) it was evident that EGFR co-localization with EEA1 increased over time and it was unaffected by Celecoxib (left panel). This observation confirmed the unaltered internalization of the receptor upon EGF binding. On the contrary EEA1 dissociated from EGFR less efficiently in the presence of Celecoxib (right panel).


Celecoxib increases EGF signaling in colon tumor associated fibroblasts, modulating EGFR expression and degradation.

Venè R, Tosetti F, Minghelli S, Poggi A, Ferrari N, Benelli R - Oncotarget (2015)

Celecoxib slows down EGFR degradationa) Western blot analysis of the kinetic of EGFR degradation. Colon TAFs pretreated with Celecoxib were challenged with EGF for the indicated times. The arrow indicates the band used for EGFR degradation quantification. The test was repeated twice. b) The early endosome marker 1 (EEA1) levels were not influenced by Celecoxib pretreatment. c) A representative image (90min EGF) of double immunofluorescence analyses: EGFR (red), EEA1 (green). Celecoxib-pretreated colon TAFs were challenged with EGF for 30, 90, 180min or 16h. Fluorescent images were acquired, with fixed expositions (EEA1-488 f1/8; EGFR-594 f1/3; DAPI f1/100), by a Leica DM-LB2 microscope equipped with I3 and M2 filters and a HCX PL Fluotar 40x non immersion optic. A 20μm scale is shown. d) Analysis of Mander's overlay coefficients for EGFR and EEA1 on the double immunofluorescence. Six random 40x fields per condition -containing at least 12 TAFs- were analyzed (see methods). The test was repeated twice. e) Flow cytometric analysis for EGFR expression in TAFs pretreated or not with Celecoxib and then challenged for 90 or 180min with EGF. The peaks, representing EGFR expression under EGF, or Celecoxib plus EGF (Cel+EGF) treatments (white peaks), were compared to EGFR levels detected in untreated controls (grey peaks). The MFI ratio (white/grey) was calculated and reported on each panel. The test was repeated twice.
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Related In: Results  -  Collection

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Figure 3: Celecoxib slows down EGFR degradationa) Western blot analysis of the kinetic of EGFR degradation. Colon TAFs pretreated with Celecoxib were challenged with EGF for the indicated times. The arrow indicates the band used for EGFR degradation quantification. The test was repeated twice. b) The early endosome marker 1 (EEA1) levels were not influenced by Celecoxib pretreatment. c) A representative image (90min EGF) of double immunofluorescence analyses: EGFR (red), EEA1 (green). Celecoxib-pretreated colon TAFs were challenged with EGF for 30, 90, 180min or 16h. Fluorescent images were acquired, with fixed expositions (EEA1-488 f1/8; EGFR-594 f1/3; DAPI f1/100), by a Leica DM-LB2 microscope equipped with I3 and M2 filters and a HCX PL Fluotar 40x non immersion optic. A 20μm scale is shown. d) Analysis of Mander's overlay coefficients for EGFR and EEA1 on the double immunofluorescence. Six random 40x fields per condition -containing at least 12 TAFs- were analyzed (see methods). The test was repeated twice. e) Flow cytometric analysis for EGFR expression in TAFs pretreated or not with Celecoxib and then challenged for 90 or 180min with EGF. The peaks, representing EGFR expression under EGF, or Celecoxib plus EGF (Cel+EGF) treatments (white peaks), were compared to EGFR levels detected in untreated controls (grey peaks). The MFI ratio (white/grey) was calculated and reported on each panel. The test was repeated twice.
Mentions: We tested if Celecoxib could alter the kinetic of EGFR degradation upon EGF triggering. Challenging colon TAFs with EGF (Fig. 3a), we noticed the appearance of low molecular weight bands indicating EGFR degradation at 60min and 90min, becoming more evident at 120min. Interestingly, in the cells pretreated with Celecoxib EGFR degradation was increased during the first hour of triggering, but delayed at 90 and even more at 120min. To track EGFR along its degradative route we needed an endosomal marker whose levels were not modulated by Celecoxib. We found that the early endosome marker 1 (EEA1) satisfied this condition (Fig. 3b), thus EEA1 was used for co-localization studies with EGFR. By double immunofluorescence and microscopic evaluation, we analyzed the overlap for EGFR and EEA1 signals in colon TAFs. The representative images shown in Fig. 3c (90min of incubation with EGF) indicated that most EGFR staining localized in EEA1 positive vesicles. Calculating Mander's coefficients for EEA1 and EGFR co-localization (Fig. 3d) it was evident that EGFR co-localization with EEA1 increased over time and it was unaffected by Celecoxib (left panel). This observation confirmed the unaltered internalization of the receptor upon EGF binding. On the contrary EEA1 dissociated from EGFR less efficiently in the presence of Celecoxib (right panel).

Bottom Line: We have now identified two concomitant mechanisms explaining the EGF-Celecoxib cooperation.Celecoxib modulated the levels of target proteins similarly to the inhibitors of endosome/lysosome acidification Bafilomycin-A1 and NH(4)Cl.Cytoplasmic vesicles fractionation showed a reduced maturation of Cathepsin-D in late endosomes and an increased content of EGFR and Rab7 in lysosomes of Celecoxib-treated TAFs.Our data indicate a double mechanism mediating the increased response to EGF of colon TAFs treated with Celecoxib.

View Article: PubMed Central - PubMed

Affiliation: Immunology Lab, IRCCS AOU San Martino - IST, Genoa, Italy.

ABSTRACT
We previously demonstrated that non-toxic doses of Celecoxib induced the immediate phosphorylation of Erk1-2 in colon tumor associated fibroblasts (TAFs), increasing their responsiveness to epidermal growth factor (EGF). We have now identified two concomitant mechanisms explaining the EGF-Celecoxib cooperation. We found that a 24-48h Celecoxib priming increased EGF receptor (EGFR) mRNA and protein levels in colon TAFs, promoting EGF binding and internalization. Celecoxib-primed TAFs showed a reduced EGFR degradation after EGF challenge. This delay corresponded to a deferred dissociation of EEA1 from EGFR positive endosomes and the accumulation of Rab7, pro Cathepsin-D and SQSTM1/p62, suggesting a shared bottleneck in the pathways of late-endosomes/autophagosomes maturation. Celecoxib modulated the levels of target proteins similarly to the inhibitors of endosome/lysosome acidification Bafilomycin-A1 and NH(4)Cl. Cytoplasmic vesicles fractionation showed a reduced maturation of Cathepsin-D in late endosomes and an increased content of EGFR and Rab7 in lysosomes of Celecoxib-treated TAFs.Our data indicate a double mechanism mediating the increased response to EGF of colon TAFs treated with Celecoxib. While EGFR overexpression could be targeted using anti EGFR drugs, the effects on endosome trafficking and protein turnover represents a more elusive target and should be taken into account for any long-term therapy with Celecoxib.

No MeSH data available.


Related in: MedlinePlus