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. 2015 May 20;6(14):12310-25.
doi: 10.18632/oncotarget.3678.

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

Roberta Venè  1   2 , Francesca Tosetti  2 , Simona Minghelli  1   2 , Alessandro Poggi  2 , Nicoletta Ferrari  2 , Roberto Benelli  1
Affiliations

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

Roberta Venè et al. Oncotarget. .

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.

Keywords: EGFR; celecoxib; colon; fibroblast.

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Conflict of interest statement

CONFLICTS OF INTEREST

No potential conflict of interest to be disclosed.

Figures

Figure 1
Figure 1. Celecoxib increases colon TAFs responsiveness to EGF
a) TAF cell growth was evaluated on day 7 of culture in the presence of Celecoxib (Cel, 10μM), EGF (50ng/ml) or Cel+EGF. Ctrl: control TAFs in absence of stimuli. The test was run in six replicates and repeated three times. b) Cell adhesion of TAFs seeded on collagen type IV. TAFs were primed or not with Celecoxib in culture flasks, afterwards they were plated in microwells either without additional treatment or with EGF, for 30min. The test was run in quadruplicates and repeated three times. c) Western blot for p-Akt and p-Erk1-2 of TAFs primed with Celecoxib and/or incubated with EGF for the indicated period of time. Beta-actin was used as a loading control. d) Relative quantification of WB replicates run as shown in panel c.
Figure 2
Figure 2. Celecoxib increases EGFR mRNA and protein expression
a) Real time PCR for EGFR. Colon TAFs were treated with Celecoxib (Cel, 10μM) for 48 hours; EGF (50ng/ml) was added as indicated during the last 16h of incubation. EGFR mRNA levels were normalized against the RP2 housekeeping gene. The mean values of three independent tests are shown. b) A western blot for EGFR under the same condition reported for Real Time PCR. c) The relative mean intensity of bands from six independent western blots, on three different TAFs primary cell cultures, was calculated by densitometry and plotted. d) Flow cytometric analysis of surface and total EGFR. TAFs were treated as described above. The peaks, representing EGFR expression under Celecoxib (Cel), 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. e) Surface and total EGFR increase induced by Celecoxib, calculated as Cel+EGF / EGF ratio of three independent flow cytometric analyses as reported in panel d. f) Binding and internalization of biotin-EGF in colon TAFs primed or not with Celecoxib and treated with biotin-EGF (50ng/ml) for 45 or 30min respectively. The test was run in six replicates and repeated three times.
Figure 3
Figure 3. Celecoxib slows down EGFR degradation
a) 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.
Figure 4
Figure 4. Celecoxib affects protein turnover mimicking the inhibitors of endo-lysosome acidification
a) Western blot for EGFR and p62/SQSTM1 of colon TAFs pretreated with Celecoxib and triggered for 3 or 16h with EGF. b) Western blot analysis of the effects of proteasome-lysosome inhibitors. 48h treatment with MG132 (proteasome inhibitor, 2μM), Bafilomycin-A1 (Baf, V-ATPase inhibitor, 25nM), NH4Cl (lysosomal pH -buffering molecule, 10mM) were compared to Celecoxib (10μM) as modulators of EGFR, p62, HSP70 and IkBα. c) Relative quantification of EGFR and p62 levels from replicate tests as shown in panel b; p values were calculated as compared to untreated controls. d) Western blot comparison of the effects of Celecoxib and lysosome acidification inhibitors. The effects of Celecoxib (10μM), Bafilomycin-A1 (2.5nM) and NH4Cl (2.5mM) pretreatment were tested in the absence/presence of EGF (3h) on several target proteins: EGFR, p62, IkBα, EEA1, LAMP1, Rab7, pro Cathepsin-D and Cathepsin-D. Loading controls: beta-actin (1) normalizes EEA1, p62 and Cathepsin-D; beta-actin (2) normalizes EGFR, IkBα, LAMP1 and Rab7. e) Relative quantification of EGFR, p62, Rab7, pro and active Cathepsin-D levels from replicate tests as shown in panel d; p values defined in Materials and Methods were calculated as compared to untreated controls.
Figure 5
Figure 5. Celecoxib retards Cathepsin-D maturation in late endosomes, causing EGFR and Rab7 accumulation
a) Cytoplasmic vesicles from TAFs treated as shown were fractioned by centrifugation (see methods) obtaining samples enriched in lysosomes (Lys: EEA1 low; Rab7, LAMP1 high; Cathepsin-D very high), late endosomes (LE: EEA1 low; Cathepsin middle; Rab7, LAMP1 high) and early endosomes (EE: EEA1 high; Rab7, LAMP1, Cathepsin-D low). Post nuclear supernatants (Pns) from untreated controls were used as loading controls. b-e) Relative quantification of EGFR, Rab7, pro Cathepsin-D and active Cathepsin-D bands in Lys, LE and EE-enriched fractions from replicates of the test shown in panel a. f) EGFR, Rab7 and pro/active Cathepsin-D gain in the Lys, LE and EE-enriched fractions of Celecoxib-treated samples, calculated from data shown in graphs b-e as ratios against the relative controls (both untreated or EGF-treated).
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