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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

Src and phospho-SFK detection in human tumor samples and cell lines(A) Immunohistochemical staining of Src in a representative formalin fixed paraffin embedded (FFPE) human Squmaous Cell Carcinoma of the tongue. (bar=200μm) (B) Western blot analysis of Src and phospho-SFK (SFK-pY419) in membrane preparations of human head and neck tumor samples (10μg). Controls correspond to membranes (10μg) prepared from exponentially growing CAL33 cells treated for 5 minutes with 30 ng/ml EGF. (C) Quantitative Western blot data were normalized to control values within each series of 6-8 patient samples and plotted with respect to Src expression. Inter-assay variability calculated on control values from six independent runs was <27%. Bars represent single patient samples. (D) Western blot of Src and phospho-SFK (SFK-pY419) in total cell lysates of the indicated human tumor lines.
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Figure 1: Src and phospho-SFK detection in human tumor samples and cell lines(A) Immunohistochemical staining of Src in a representative formalin fixed paraffin embedded (FFPE) human Squmaous Cell Carcinoma of the tongue. (bar=200μm) (B) Western blot analysis of Src and phospho-SFK (SFK-pY419) in membrane preparations of human head and neck tumor samples (10μg). Controls correspond to membranes (10μg) prepared from exponentially growing CAL33 cells treated for 5 minutes with 30 ng/ml EGF. (C) Quantitative Western blot data were normalized to control values within each series of 6-8 patient samples and plotted with respect to Src expression. Inter-assay variability calculated on control values from six independent runs was <27%. Bars represent single patient samples. (D) Western blot of Src and phospho-SFK (SFK-pY419) in total cell lysates of the indicated human tumor lines.

Mentions: In human HNSCC, robust Src expression was observed in tumor epithelial cells by immunohistochemistry. Staining in adjacent non-tumoral epithelium was undetectable, or faint and limited to the basal cell layer (Figure 1A). We were unable to assess Src activity in paraffin-embedded human tumors using anti-phospho-Src antibodies therefore we turned to quantitative Western blot analysis in order to determine the status of Src expression and activation in HNSCC. Western blot analysis was performed on membrane preparations from 60 surgically resected human head and neck tumors prior to treatment of patients (Figure 1B). Patients (84% male, median age 58 years old) with histologically proven HNSCC and planned surgery for locally advanced disease with intermediate/poor prognosis (extracapsular spread, > 3 N+ and/or lymphatic or vascular emboli +/− perineural invasion +/− microscopically positive margins +/− pT4 on operative specimen) were enrolled in a French multicenter blinded institutional review board-approved randomized phase II trial of post-operative irradiation with cisplatin ± gefitinib vs placebo. Gefitinib (250mg twice-daily, AstraZeneca Pharmaceuticals) was administered orally for 9.5 weeks concurrently to chemoradiation. Of those enrolled patients, 60 had frozen tumor samples available for Western blot analyses. The phospho-Src antibody used to detect the active form of the kinase recognizes active Src phosphorylated on activation loop residue tyrosine 419 (tyrosine 416 in c-Src of avian origin). This antibody is referred to as SFK-pY419 since in addition to Src, it is able to recognize other Src family kinase (SFK) members. Src and active SFKs were detected in all but two (3.3%) and two (3.3%) tumor samples, respectively. Quantitative chemiluminescence imaging of Western blots revealed a wide range of both total Src and active SFK levels in tumors. The highest expression levels were comparable to or greater than that observed in membranes prepared from a human HNSCC line (CAL33), included as normalization control (Figure 1C). There was a non Gaussian distribution of Src and phospho-SFK expression levels with median levels of 0.139 and 0.269, respectively. A significant correlation was observed between Src expression and phospho-SFK levels in human tumors (Pearson coefficient r=0.494, p≤ 0.001). Neither high expression (above the median) of phospho-SFK or Src was significantly associated in this patient population with overall survival (p=0.83 and 0.33) or disease-free survival defined as time to primary, nodal or metastatic relapse (p= 0.47 and 0.29).


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)

Src and phospho-SFK detection in human tumor samples and cell lines(A) Immunohistochemical staining of Src in a representative formalin fixed paraffin embedded (FFPE) human Squmaous Cell Carcinoma of the tongue. (bar=200μm) (B) Western blot analysis of Src and phospho-SFK (SFK-pY419) in membrane preparations of human head and neck tumor samples (10μg). Controls correspond to membranes (10μg) prepared from exponentially growing CAL33 cells treated for 5 minutes with 30 ng/ml EGF. (C) Quantitative Western blot data were normalized to control values within each series of 6-8 patient samples and plotted with respect to Src expression. Inter-assay variability calculated on control values from six independent runs was <27%. Bars represent single patient samples. (D) Western blot of Src and phospho-SFK (SFK-pY419) in total cell lysates of the indicated human tumor lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Src and phospho-SFK detection in human tumor samples and cell lines(A) Immunohistochemical staining of Src in a representative formalin fixed paraffin embedded (FFPE) human Squmaous Cell Carcinoma of the tongue. (bar=200μm) (B) Western blot analysis of Src and phospho-SFK (SFK-pY419) in membrane preparations of human head and neck tumor samples (10μg). Controls correspond to membranes (10μg) prepared from exponentially growing CAL33 cells treated for 5 minutes with 30 ng/ml EGF. (C) Quantitative Western blot data were normalized to control values within each series of 6-8 patient samples and plotted with respect to Src expression. Inter-assay variability calculated on control values from six independent runs was <27%. Bars represent single patient samples. (D) Western blot of Src and phospho-SFK (SFK-pY419) in total cell lysates of the indicated human tumor lines.
Mentions: In human HNSCC, robust Src expression was observed in tumor epithelial cells by immunohistochemistry. Staining in adjacent non-tumoral epithelium was undetectable, or faint and limited to the basal cell layer (Figure 1A). We were unable to assess Src activity in paraffin-embedded human tumors using anti-phospho-Src antibodies therefore we turned to quantitative Western blot analysis in order to determine the status of Src expression and activation in HNSCC. Western blot analysis was performed on membrane preparations from 60 surgically resected human head and neck tumors prior to treatment of patients (Figure 1B). Patients (84% male, median age 58 years old) with histologically proven HNSCC and planned surgery for locally advanced disease with intermediate/poor prognosis (extracapsular spread, > 3 N+ and/or lymphatic or vascular emboli +/− perineural invasion +/− microscopically positive margins +/− pT4 on operative specimen) were enrolled in a French multicenter blinded institutional review board-approved randomized phase II trial of post-operative irradiation with cisplatin ± gefitinib vs placebo. Gefitinib (250mg twice-daily, AstraZeneca Pharmaceuticals) was administered orally for 9.5 weeks concurrently to chemoradiation. Of those enrolled patients, 60 had frozen tumor samples available for Western blot analyses. The phospho-Src antibody used to detect the active form of the kinase recognizes active Src phosphorylated on activation loop residue tyrosine 419 (tyrosine 416 in c-Src of avian origin). This antibody is referred to as SFK-pY419 since in addition to Src, it is able to recognize other Src family kinase (SFK) members. Src and active SFKs were detected in all but two (3.3%) and two (3.3%) tumor samples, respectively. Quantitative chemiluminescence imaging of Western blots revealed a wide range of both total Src and active SFK levels in tumors. The highest expression levels were comparable to or greater than that observed in membranes prepared from a human HNSCC line (CAL33), included as normalization control (Figure 1C). There was a non Gaussian distribution of Src and phospho-SFK expression levels with median levels of 0.139 and 0.269, respectively. A significant correlation was observed between Src expression and phospho-SFK levels in human tumors (Pearson coefficient r=0.494, p≤ 0.001). Neither high expression (above the median) of phospho-SFK or Src was significantly associated in this patient population with overall survival (p=0.83 and 0.33) or disease-free survival defined as time to primary, nodal or metastatic relapse (p= 0.47 and 0.29).

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