Limits...
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 increases colon TAFs responsiveness to EGFa) 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4494940&req=5

Figure 1: Celecoxib increases colon TAFs responsiveness to EGFa) 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.

Mentions: We previously showed that colon TAFs exhibit a great tolerance to Celecoxib treatment. Moreover, Celecoxib at nontoxic concentration activated colon TAFs signaling, causing a rapid and transient phosphorylation of Erk1-2 [32]. This activation was able to synergize with low dose EGF (1ng/ml). In this study we first assessed whether Celecoxib could affect TAFs growth also in the presence of the high dose EGF, used here (Fig. 1a). Celecoxib induced a negligible effect per se, but it consistently increased the EGF-mediated triggering of TAFs growth. To analyze whether Celecoxib could affect TAFs adhesive properties, a feature of TAFs activation, we assessed binding of TAFs to collagen (Fig. 1b). Of note, Celecoxib alone increased TAFs adhesion to collagen as compared to untreated cells. More importantly, Celecoxib potentiated EGF-mediated adhesion to collagen. This effect was associated with a more intense activation of Erk1-2 phosphorylation induced by Celecoxib and EGF together, than that induced by EGF alone. On the other hand, Akt phosphorylation consequent to EGF signaling was poorly affected by Celecoxib (Fig. 1c and 1d).


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 increases colon TAFs responsiveness to EGFa) 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494940&req=5

Figure 1: Celecoxib increases colon TAFs responsiveness to EGFa) 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.
Mentions: We previously showed that colon TAFs exhibit a great tolerance to Celecoxib treatment. Moreover, Celecoxib at nontoxic concentration activated colon TAFs signaling, causing a rapid and transient phosphorylation of Erk1-2 [32]. This activation was able to synergize with low dose EGF (1ng/ml). In this study we first assessed whether Celecoxib could affect TAFs growth also in the presence of the high dose EGF, used here (Fig. 1a). Celecoxib induced a negligible effect per se, but it consistently increased the EGF-mediated triggering of TAFs growth. To analyze whether Celecoxib could affect TAFs adhesive properties, a feature of TAFs activation, we assessed binding of TAFs to collagen (Fig. 1b). Of note, Celecoxib alone increased TAFs adhesion to collagen as compared to untreated cells. More importantly, Celecoxib potentiated EGF-mediated adhesion to collagen. This effect was associated with a more intense activation of Erk1-2 phosphorylation induced by Celecoxib and EGF together, than that induced by EGF alone. On the other hand, Akt phosphorylation consequent to EGF signaling was poorly affected by Celecoxib (Fig. 1c and 1d).

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