Limits...
Elevated Src family kinase activity stabilizes E-cadherin-based junctions and collective movement of head and neck squamous cell carcinomas.

Veracini L, Grall D, Schaub S, Beghelli-de la Forest Divonne S, Etienne-Grimaldi MC, Milano G, Bozec A, Babin E, Sudaka A, Thariat J, Van Obberghen-Schilling E - Oncotarget (2015)

Bottom Line: EGF receptor (EGFR) overexpression is thought to drive head and neck carcinogenesis however clinical responses to EGFR-targeting agents have been modest and alternate targets are actively sought to improve results.Src family kinases (SFKs), reported to act downstream of EGFR are among the alternative targets for which increased expression or activity in epithelial tumors is commonly associated to the dissolution of E-cadherin-based junctions and acquisition of a mesenchymal-like phenotype.Selective inhibition of SFKs with SU6656 delocalized E-cadherin and disrupted cellular junctions without affecting E-cadherin expression and this effect was phenocopied by knockdown of Src or Yes.

View Article: PubMed Central - PubMed

Affiliation: University of Nice Sophia Antipolis, UFR Sciences, Nice, France.

ABSTRACT
EGF receptor (EGFR) overexpression is thought to drive head and neck carcinogenesis however clinical responses to EGFR-targeting agents have been modest and alternate targets are actively sought to improve results. Src family kinases (SFKs), reported to act downstream of EGFR are among the alternative targets for which increased expression or activity in epithelial tumors is commonly associated to the dissolution of E-cadherin-based junctions and acquisition of a mesenchymal-like phenotype. Robust expression of total and activated Src was observed in advanced stage head and neck tumors (N=60) and in head and neck squamous cell carcinoma lines. In cultured cancer cells Src co-localized with E-cadherin in cell-cell junctions and its phosphorylation on Y419 was both constitutive and independent of EGFR activation. Selective inhibition of SFKs with SU6656 delocalized E-cadherin and disrupted cellular junctions without affecting E-cadherin expression and this effect was phenocopied by knockdown of Src or Yes. These findings reveal an EGFR-independent role for SFKs in the maintenance of intercellular junctions, which likely contributes to the cohesive invasion E-cadherin-positive cells in advanced tumors. Further, they highlight the need for a deeper comprehension of molecular pathways that drive collective cell invasion, in absence of mesenchymal transition, in order to combat tumor spread.

No MeSH data available.


Related in: MedlinePlus

Constitutive activity of SFK is independent of EGFR activation(A) Quantification of phosphorylated SFK (SFK-pY419) and EGFR (EGFR-pY1068) levels determined by Western analysis of membranes from human tumors. Results were normalized to control values within each series of 6-8 patient samples and plotted with respect to phospho-SFK expression (<27% inter-assay variability). Bars represent single patient samples. (B) Western blot of total Src and active SFK and EGFR in lysates prepared from the indicated HNSCC lines. Quantification of phosphorylated SFK (SFK-pY419) and EGFR (EGFR-pY1068) levels determined by Western analysis of membranes from human tumors. (C) Western blot analysis of EGFR and Src signaling pathways in CAL33 and CAL27 cells serum starved for 24h then stimulated for the indicated times with EGF (20ng/ml). (D) Western blot analysis of EGFR and Src signaling pathways in CAL33 and CAL27 cells serum starved for 24h then stimulated or not for 10min with EGF (20ng/ml) in presence or absence of Gefitinib (5μM).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4480700&req=5

Figure 2: Constitutive activity of SFK is independent of EGFR activation(A) Quantification of phosphorylated SFK (SFK-pY419) and EGFR (EGFR-pY1068) levels determined by Western analysis of membranes from human tumors. Results were normalized to control values within each series of 6-8 patient samples and plotted with respect to phospho-SFK expression (<27% inter-assay variability). Bars represent single patient samples. (B) Western blot of total Src and active SFK and EGFR in lysates prepared from the indicated HNSCC lines. Quantification of phosphorylated SFK (SFK-pY419) and EGFR (EGFR-pY1068) levels determined by Western analysis of membranes from human tumors. (C) Western blot analysis of EGFR and Src signaling pathways in CAL33 and CAL27 cells serum starved for 24h then stimulated for the indicated times with EGF (20ng/ml). (D) Western blot analysis of EGFR and Src signaling pathways in CAL33 and CAL27 cells serum starved for 24h then stimulated or not for 10min with EGF (20ng/ml) in presence or absence of Gefitinib (5μM).

Mentions: Activation of Src is frequently positioned downstream of EGFR activation in normal and transformed cells, including cells of HNSCC origin [17, 18]. However, the correlation between active SFK levels and active EGFR in our cohort of human tumors, as measured by Western blotting using antibodies directed against the EGFR phosphorylated on tyrosine 1068 (EGFR-pY1068) was quite weak (Figure 2A) (Pearson coefficient r=0.255, p≤ 0.046). Further, we did not observe equivalent levels of the active kinases in a set of exponentially growing HNSCC lines (Figure 2B). Therefore, we examined the effect of EGFR activation on SFK phosphorylation more closely using 2 HNSCC lines, CAL33 and CAL27. As shown in Figure 2C and D, addition of EGF to serum-starved cells stimulated the phosphorylation of EGFR and downstream signaling components including Akt, Erk, without affecting SFK phosphorylation. Conversely, inhibition of the EGFR tyrosine kinase with gefinitib had no effect on active SFK levels under conditions in which it abrogated EGFR, AKT and ERK phosphorylation (Figure 2D). SFK activation was found to be not only independent of EGFR activation in these cells, but also independent of serum growth factors. In contrast to EGFR, Akt and Erk phosphorylation, serum removal had no effect on phospho-SFK levels (Figure 2C, D). Phosphorylation of cortactin on tyrosine 421, a known Src phosphorylation site, was stimulated in response to EGF yet basal phosphorylation, likely dependent on Src, could be detected in absence of the growth factor or serum. Similar to SFKs, phosphorylation of the Src substrate p130Cas was constitutively active and insensitive to EGFR inhibition. Thus, an EGFR-independent SFK signaling axis was constitutively active in these cells.


Elevated Src family kinase activity stabilizes E-cadherin-based junctions and collective movement of head and neck squamous cell carcinomas.

Veracini L, Grall D, Schaub S, Beghelli-de la Forest Divonne S, Etienne-Grimaldi MC, Milano G, Bozec A, Babin E, Sudaka A, Thariat J, Van Obberghen-Schilling E - Oncotarget (2015)

Constitutive activity of SFK is independent of EGFR activation(A) Quantification of phosphorylated SFK (SFK-pY419) and EGFR (EGFR-pY1068) levels determined by Western analysis of membranes from human tumors. Results were normalized to control values within each series of 6-8 patient samples and plotted with respect to phospho-SFK expression (<27% inter-assay variability). Bars represent single patient samples. (B) Western blot of total Src and active SFK and EGFR in lysates prepared from the indicated HNSCC lines. Quantification of phosphorylated SFK (SFK-pY419) and EGFR (EGFR-pY1068) levels determined by Western analysis of membranes from human tumors. (C) Western blot analysis of EGFR and Src signaling pathways in CAL33 and CAL27 cells serum starved for 24h then stimulated for the indicated times with EGF (20ng/ml). (D) Western blot analysis of EGFR and Src signaling pathways in CAL33 and CAL27 cells serum starved for 24h then stimulated or not for 10min with EGF (20ng/ml) in presence or absence of Gefitinib (5μM).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480700&req=5

Figure 2: Constitutive activity of SFK is independent of EGFR activation(A) Quantification of phosphorylated SFK (SFK-pY419) and EGFR (EGFR-pY1068) levels determined by Western analysis of membranes from human tumors. Results were normalized to control values within each series of 6-8 patient samples and plotted with respect to phospho-SFK expression (<27% inter-assay variability). Bars represent single patient samples. (B) Western blot of total Src and active SFK and EGFR in lysates prepared from the indicated HNSCC lines. Quantification of phosphorylated SFK (SFK-pY419) and EGFR (EGFR-pY1068) levels determined by Western analysis of membranes from human tumors. (C) Western blot analysis of EGFR and Src signaling pathways in CAL33 and CAL27 cells serum starved for 24h then stimulated for the indicated times with EGF (20ng/ml). (D) Western blot analysis of EGFR and Src signaling pathways in CAL33 and CAL27 cells serum starved for 24h then stimulated or not for 10min with EGF (20ng/ml) in presence or absence of Gefitinib (5μM).
Mentions: Activation of Src is frequently positioned downstream of EGFR activation in normal and transformed cells, including cells of HNSCC origin [17, 18]. However, the correlation between active SFK levels and active EGFR in our cohort of human tumors, as measured by Western blotting using antibodies directed against the EGFR phosphorylated on tyrosine 1068 (EGFR-pY1068) was quite weak (Figure 2A) (Pearson coefficient r=0.255, p≤ 0.046). Further, we did not observe equivalent levels of the active kinases in a set of exponentially growing HNSCC lines (Figure 2B). Therefore, we examined the effect of EGFR activation on SFK phosphorylation more closely using 2 HNSCC lines, CAL33 and CAL27. As shown in Figure 2C and D, addition of EGF to serum-starved cells stimulated the phosphorylation of EGFR and downstream signaling components including Akt, Erk, without affecting SFK phosphorylation. Conversely, inhibition of the EGFR tyrosine kinase with gefinitib had no effect on active SFK levels under conditions in which it abrogated EGFR, AKT and ERK phosphorylation (Figure 2D). SFK activation was found to be not only independent of EGFR activation in these cells, but also independent of serum growth factors. In contrast to EGFR, Akt and Erk phosphorylation, serum removal had no effect on phospho-SFK levels (Figure 2C, D). Phosphorylation of cortactin on tyrosine 421, a known Src phosphorylation site, was stimulated in response to EGF yet basal phosphorylation, likely dependent on Src, could be detected in absence of the growth factor or serum. Similar to SFKs, phosphorylation of the Src substrate p130Cas was constitutively active and insensitive to EGFR inhibition. Thus, an EGFR-independent SFK signaling axis was constitutively active in these cells.

Bottom Line: EGF receptor (EGFR) overexpression is thought to drive head and neck carcinogenesis however clinical responses to EGFR-targeting agents have been modest and alternate targets are actively sought to improve results.Src family kinases (SFKs), reported to act downstream of EGFR are among the alternative targets for which increased expression or activity in epithelial tumors is commonly associated to the dissolution of E-cadherin-based junctions and acquisition of a mesenchymal-like phenotype.Selective inhibition of SFKs with SU6656 delocalized E-cadherin and disrupted cellular junctions without affecting E-cadherin expression and this effect was phenocopied by knockdown of Src or Yes.

View Article: PubMed Central - PubMed

Affiliation: University of Nice Sophia Antipolis, UFR Sciences, Nice, France.

ABSTRACT
EGF receptor (EGFR) overexpression is thought to drive head and neck carcinogenesis however clinical responses to EGFR-targeting agents have been modest and alternate targets are actively sought to improve results. Src family kinases (SFKs), reported to act downstream of EGFR are among the alternative targets for which increased expression or activity in epithelial tumors is commonly associated to the dissolution of E-cadherin-based junctions and acquisition of a mesenchymal-like phenotype. Robust expression of total and activated Src was observed in advanced stage head and neck tumors (N=60) and in head and neck squamous cell carcinoma lines. In cultured cancer cells Src co-localized with E-cadherin in cell-cell junctions and its phosphorylation on Y419 was both constitutive and independent of EGFR activation. Selective inhibition of SFKs with SU6656 delocalized E-cadherin and disrupted cellular junctions without affecting E-cadherin expression and this effect was phenocopied by knockdown of Src or Yes. These findings reveal an EGFR-independent role for SFKs in the maintenance of intercellular junctions, which likely contributes to the cohesive invasion E-cadherin-positive cells in advanced tumors. Further, they highlight the need for a deeper comprehension of molecular pathways that drive collective cell invasion, in absence of mesenchymal transition, in order to combat tumor spread.

No MeSH data available.


Related in: MedlinePlus