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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

Localization of SFKs in E-cadherin based cell-cell junctions(A) Phase contrast images of CAL33 and CAL27 cells (bar=150μm). (B) Western blot of SFK members in HEK293 (control), CAL33 and CAL27 cells. ERK1/2 expression is shown as loading control. (C) Co-staining of Src or Yes with E-cadherin and β-catenin in CAL33 cells (bar=15μm). (D) Western blot analysis of Src, EGFR and β-catenin in anti-E-cadherin immunoprecipitates from CAL33 cell lysates. (E) Western blot of lysates of CAL33 cells plated at increasing density for 36h. (F) Western analysis of E-cadherin, Src and active SFK levels in the indicated HNSCC lines. (G) Migration of non-dividing single cells or cell cohorts was recorded by time lapse videomicroscopy 12 hours after plating for 24 hours. (left) Tracings from origin and (right) diffusion coefficient (D). Diffusion coefficient was calculated from MSD assuming 2D brownian movement (error bars: 95% confidence bounds estimated from linear fit).
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Figure 3: Localization of SFKs in E-cadherin based cell-cell junctions(A) Phase contrast images of CAL33 and CAL27 cells (bar=150μm). (B) Western blot of SFK members in HEK293 (control), CAL33 and CAL27 cells. ERK1/2 expression is shown as loading control. (C) Co-staining of Src or Yes with E-cadherin and β-catenin in CAL33 cells (bar=15μm). (D) Western blot analysis of Src, EGFR and β-catenin in anti-E-cadherin immunoprecipitates from CAL33 cell lysates. (E) Western blot of lysates of CAL33 cells plated at increasing density for 36h. (F) Western analysis of E-cadherin, Src and active SFK levels in the indicated HNSCC lines. (G) Migration of non-dividing single cells or cell cohorts was recorded by time lapse videomicroscopy 12 hours after plating for 24 hours. (left) Tracings from origin and (right) diffusion coefficient (D). Diffusion coefficient was calculated from MSD assuming 2D brownian movement (error bars: 95% confidence bounds estimated from linear fit).

Mentions: We were intrigued by the cohesive epithelial morphology displayed by CAL33 and CAL27 cells with elevated SFK activities (Figure 3A). SFKs are known to be key regulators of E-cadherin-based cell-cell junctions and their activation is typically associated with a mesenchymal-like phenotype. HNSCC lines have been reported to express multiple SFK members, including the ubiquitously expressed Src, Yes and Fyn [18]. As shown in Figure 3B, CAL33 and CAL27 cells express Src and Yes however we were unable to detect Fyn in these cells. In both cell lines, Src and Yes were enriched in cell-cell junctions. Immunostaining revealed the co-localization of these SFK with the junctional proteins E-cadherin and β-catenin (Figure 3C). Further, co-immunoprecipitation of Src with anti E-cadherin antibodies yielded a molecular complex containing Src and E-cadherin (Figure 3D). Src has been shown to functionally cooperate with EGFR and form a heteromolecular complex when both kinases when co-expressed in fibroblasts, [19] but we failed to detect EGFR in the anti-E-cadherin immunoprecipitates, consistent with EGFR-independent SFK activation observed in our cells. Phosphorylation of SFKs increased with increasing cell density (Figure 3E and Supplemental Figure 1) suggesting that cell-cell adhesion may play an important role in this event. Indeed, E-cadherin signaling has been reported to activate c-Src at cell–cell contacts in mammary epithelial cells [20]. However, cell-cell adhesion per se was not sufficient to activate Src in CAL33 cells, as disruption and restoration of E-cadherin ligation in a Ca2+ switch assay had little effect on levels of phospho-SFK (Supplemental Figure 2). Importantly, we did not observe a decrease in E-cadherin expression coincident with SFK phosphorylation. Rather, E-cadherin levels remained elevated in dense cultures of CAL33 cells and in other HNSCC lines with elevated phospho-SFK (Figure 3E-F and not shown).


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)

Localization of SFKs in E-cadherin based cell-cell junctions(A) Phase contrast images of CAL33 and CAL27 cells (bar=150μm). (B) Western blot of SFK members in HEK293 (control), CAL33 and CAL27 cells. ERK1/2 expression is shown as loading control. (C) Co-staining of Src or Yes with E-cadherin and β-catenin in CAL33 cells (bar=15μm). (D) Western blot analysis of Src, EGFR and β-catenin in anti-E-cadherin immunoprecipitates from CAL33 cell lysates. (E) Western blot of lysates of CAL33 cells plated at increasing density for 36h. (F) Western analysis of E-cadherin, Src and active SFK levels in the indicated HNSCC lines. (G) Migration of non-dividing single cells or cell cohorts was recorded by time lapse videomicroscopy 12 hours after plating for 24 hours. (left) Tracings from origin and (right) diffusion coefficient (D). Diffusion coefficient was calculated from MSD assuming 2D brownian movement (error bars: 95% confidence bounds estimated from linear fit).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Localization of SFKs in E-cadherin based cell-cell junctions(A) Phase contrast images of CAL33 and CAL27 cells (bar=150μm). (B) Western blot of SFK members in HEK293 (control), CAL33 and CAL27 cells. ERK1/2 expression is shown as loading control. (C) Co-staining of Src or Yes with E-cadherin and β-catenin in CAL33 cells (bar=15μm). (D) Western blot analysis of Src, EGFR and β-catenin in anti-E-cadherin immunoprecipitates from CAL33 cell lysates. (E) Western blot of lysates of CAL33 cells plated at increasing density for 36h. (F) Western analysis of E-cadherin, Src and active SFK levels in the indicated HNSCC lines. (G) Migration of non-dividing single cells or cell cohorts was recorded by time lapse videomicroscopy 12 hours after plating for 24 hours. (left) Tracings from origin and (right) diffusion coefficient (D). Diffusion coefficient was calculated from MSD assuming 2D brownian movement (error bars: 95% confidence bounds estimated from linear fit).
Mentions: We were intrigued by the cohesive epithelial morphology displayed by CAL33 and CAL27 cells with elevated SFK activities (Figure 3A). SFKs are known to be key regulators of E-cadherin-based cell-cell junctions and their activation is typically associated with a mesenchymal-like phenotype. HNSCC lines have been reported to express multiple SFK members, including the ubiquitously expressed Src, Yes and Fyn [18]. As shown in Figure 3B, CAL33 and CAL27 cells express Src and Yes however we were unable to detect Fyn in these cells. In both cell lines, Src and Yes were enriched in cell-cell junctions. Immunostaining revealed the co-localization of these SFK with the junctional proteins E-cadherin and β-catenin (Figure 3C). Further, co-immunoprecipitation of Src with anti E-cadherin antibodies yielded a molecular complex containing Src and E-cadherin (Figure 3D). Src has been shown to functionally cooperate with EGFR and form a heteromolecular complex when both kinases when co-expressed in fibroblasts, [19] but we failed to detect EGFR in the anti-E-cadherin immunoprecipitates, consistent with EGFR-independent SFK activation observed in our cells. Phosphorylation of SFKs increased with increasing cell density (Figure 3E and Supplemental Figure 1) suggesting that cell-cell adhesion may play an important role in this event. Indeed, E-cadherin signaling has been reported to activate c-Src at cell–cell contacts in mammary epithelial cells [20]. However, cell-cell adhesion per se was not sufficient to activate Src in CAL33 cells, as disruption and restoration of E-cadherin ligation in a Ca2+ switch assay had little effect on levels of phospho-SFK (Supplemental Figure 2). Importantly, we did not observe a decrease in E-cadherin expression coincident with SFK phosphorylation. Rather, E-cadherin levels remained elevated in dense cultures of CAL33 cells and in other HNSCC lines with elevated phospho-SFK (Figure 3E-F and not shown).

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