Combined inhibition of PLC{gamma}-1 and c-Src abrogates epidermal growth factor receptor-mediated head and neck squamous cell carcinoma invasion

Abstract

Purpose: Mortality from head and neck squamous cell carcinoma (HNSCC) is usually associated with locoregional invasion of the tumor into vital organs, including the airway. Understanding the signaling mechanisms that abrogate HNSCC invasion may reveal novel therapeutic targets for intervention. The purpose of this study was to investigate the efficacy of combined inhibition of c-Src and PLCgamma-1 in the abrogation of HNSCC invasion.

Experimental design: PLCgamma-1 and c-Src inhibition was achieved by a combination of small molecule inhibitors and dominant negative approaches. The effect of inhibition of PLCgamma-1 and c-Src on invasion of HNSCC cells was assessed in an in vitro Matrigel-coated transwell invasion assay. In addition, the immunoprecipitation reactions and in silico database mining was used to examine the interactions between PLCgamma-1 and c-Src.

Results: Here, we show that inhibition of PLCgamma-1 or c-Src with the PLC inhibitor U73122 or the Src family inhibitor AZD0530 or using dominant-negative constructs attenuated epidermal growth factor (EGF)-stimulated HNSCC invasion. Furthermore, EGF stimulation increased the association between PLCgamma-1 and c-Src in HNSCC cells. Combined inhibition of PLCgamma-1 and c-Src resulted in further attenuation of HNSCC cell invasion in vitro.

Conclusions: These cumulative results suggest that PLCgamma-1 and c-Src activation contribute to HNSCC invasion downstream of EGF receptor and that targeting these pathways may be a novel strategy to prevent tumor invasion in HNSCC.

Figure 1

Levels of total and phosphorylated PLCγ-1 are higher in metastatic lymph node-derived cells compared to the primary tumor. HNSCC cell lines derived from the primary (PCI-37A, 15A, UM-22A) and paired metastatic (PCI-37B, 15B, UM-22B) tumor was analyzed for PLCγ-1 expression by immunoblotting. Densitometry analysis was performed and expression levels relative to β-Actin are shown as mean ± SE from two independent experiments. Primary tumor derived HNSCC cells have lower levels of phosphorylated PLCγ-1 and total PLCγ-1 compared to HNSCC cells derived from the paired metastatic lymph nodes (P<0.05).

Figure 2

HNSCC cells derived from metastatic lymph nodes are more invasive than cells derived from paired primary tumors. HNSCC cells derived from primary tumors and paired metastatic lymph nodes were plated in Matrigel coated transwell chambers at 2 × 10⁴ cells/well. After 48 h, the cells on the lower side of the chamber were fixed and stained with Hema 3 (Fisher Scientific) according to the manufacturer’s instruction. The number of cells that invaded the Matrigel coated transwell chamber was counted using a light field inverted microscope. An average of four fields of cells were counted under 200X magnification. The values are the mean ± SE of three independent experiments. Metastatic lymph node derived HNSCC cells were more invasive compared to HNSCC cell lines derived from paired primary tumors (* P<0.05).

Figure 3

Inhibition of PLCγ-1 results in reduced EGFR-mediated invasion in HNSCC (A) HNSCC cell line OSC-19 cells expressing dominant-negative PLCγ-1 (PLCz clones 4 and 7) and vector control transfected cells were serum starved for 24 h prior to EGF stimulation. Protein extracts were fractionated on a SDS-PAGE gel. Immunoblotting was performed with anti-phospho-PLCγ-1 followed by anti-PLCγ-1 antibody to demonstrate equal loading. EGFR failed to phosphorylate PLCγ-1 in dominant-negative PLCγ-1 cells compared to the vector control cells. (B) Dominant-negative PLCγ-1 expressing OSC-19 cells (PLCz-4) or vector transfected control cells were plated at a density of 2 × 10⁴ cells/well in Matrigel coated transwell chambers in the presence of EGF (10 ng/ml) or 10% FBS containing medium. After 48 h, cells on the reverse side of the upper chamber were fixed, stained, and counted. The values represent the mean ± SE of at least two independent experiments. EGFR-mediated HNSCC cell invasion to a lesser degree in cells expressing dominant-negative PLCγ-1 compared to vector control cells indicating that PLCγ-1 plays a role in EGFR-mediated invasion of HNSCC.

Figure 4

c-Src plays a role in EGFR-mediated HNSCC invasion. (A) Abrogation of EGFR-mediated c-Src phosphorylation with the Src inhibitor AZD0530. HNSCC cell lines PCI-37A and PCI-37B were serum starved for 72 h to minimize the effects of autocrine ligands. Cells were pretreated for 4 h with the Src inhibitor AZD0530 followed by stimulation with either recombinant EGF (10 ng/ml) or 10% FBS containing medium for 5 min. EGFR failed to activate c-Src in HNSCC cells treated with AZD0530 compared to cells treated with the vehicle control. (B) Inhibition of c-Src abrogates downstream signaling through FAK. HNSCC cell line 37A was serum starved for 72 h. Cells were either treated with Src inhibitor AZD0530 or medium followed by stimulation with either recombinant EGF (10 ng/ml) or 10% FBS containing medium for 5 min. Cell lysates were analyzed by immunoblotting for phosphorylated FAK levels. Abrogation of c-Src reduced EGF-mediated phosphorylation of FAK indicating that c-Src inhibition disrupted downstream signaling that contributes to increased invasion of HNSCC cells. β-Tubulin levels demonstrate equal loading of protein in all wells. The experiment was repeated twice with similar results. (C) Abrogation of EGFR-mediated c-Src phosphorylation in cells expressing dominant-negative c-Src. PCI-37A cells expressing dominant-negative c-Src and vector control transfected cells were serum starved for 72 h prior to EGF stimulation. Protein extracts were fractionated on a SDS-PAGE gel. Immunoblotting was performed with ant-phospho-c-Src (PY418) antibody followed by anti-c-Src antibody to demonstrate equal loading. EGFR failed to phosphorylate c-Src in HNSCC cells expressing dominant-negative c-Src. (D) HNSCC cells expressing dominant-negative c-Src have reduced invasive potential on EGFR stimulation. PCI-37A expressing a c-Src dominant-negative construct and vector transfected control cells were plated in Matrigel coated transwell chambers. Cells were allowed to invade for 48 h with or without EGF or 10% FBS containing medium. Cells that invaded were stained and counted at 200 × magnification. The values are the mean ± SE of at least two independent experiments. HNSCC cells expressing dominant-negative c-Src had fewer invading cells compared with vehicle control (* P<0.05).

Figure 4

c-Src plays a role in EGFR-mediated HNSCC invasion. (A) Abrogation of EGFR-mediated c-Src phosphorylation with the Src inhibitor AZD0530. HNSCC cell lines PCI-37A and PCI-37B were serum starved for 72 h to minimize the effects of autocrine ligands. Cells were pretreated for 4 h with the Src inhibitor AZD0530 followed by stimulation with either recombinant EGF (10 ng/ml) or 10% FBS containing medium for 5 min. EGFR failed to activate c-Src in HNSCC cells treated with AZD0530 compared to cells treated with the vehicle control. (B) Inhibition of c-Src abrogates downstream signaling through FAK. HNSCC cell line 37A was serum starved for 72 h. Cells were either treated with Src inhibitor AZD0530 or medium followed by stimulation with either recombinant EGF (10 ng/ml) or 10% FBS containing medium for 5 min. Cell lysates were analyzed by immunoblotting for phosphorylated FAK levels. Abrogation of c-Src reduced EGF-mediated phosphorylation of FAK indicating that c-Src inhibition disrupted downstream signaling that contributes to increased invasion of HNSCC cells. β-Tubulin levels demonstrate equal loading of protein in all wells. The experiment was repeated twice with similar results. (C) Abrogation of EGFR-mediated c-Src phosphorylation in cells expressing dominant-negative c-Src. PCI-37A cells expressing dominant-negative c-Src and vector control transfected cells were serum starved for 72 h prior to EGF stimulation. Protein extracts were fractionated on a SDS-PAGE gel. Immunoblotting was performed with ant-phospho-c-Src (PY418) antibody followed by anti-c-Src antibody to demonstrate equal loading. EGFR failed to phosphorylate c-Src in HNSCC cells expressing dominant-negative c-Src. (D) HNSCC cells expressing dominant-negative c-Src have reduced invasive potential on EGFR stimulation. PCI-37A expressing a c-Src dominant-negative construct and vector transfected control cells were plated in Matrigel coated transwell chambers. Cells were allowed to invade for 48 h with or without EGF or 10% FBS containing medium. Cells that invaded were stained and counted at 200 × magnification. The values are the mean ± SE of at least two independent experiments. HNSCC cells expressing dominant-negative c-Src had fewer invading cells compared with vehicle control (* P<0.05).

Figure 5

Combined inhibition of PLCγ-1 and c-Src results in attenuation of EGFR-mediated invasion in HNSCC cells. (A) Pharmacologic inhibitors of PLCγ-1 and c-Src are active against EGFR-mediated invasion in HNSCC. HNSCC cells PCI-37B were plated at a density of 2.5 × 10⁴ cells/well in Matrigel coated transwell chambers in the presence of EGF (10 ng/ml) or 10% FBS containing medium, and treated with AZD0530 (1 μM), PLC inhibitor (U73122, 3 μM), control inactive compound (U73343, 3 μM) and AZD0530 (1 μM) plus U73122 or U73343 (3 μM) for 48h. Combined inhibition of PLC and c-Src in HNSCC cells results in abrogation of EGFR-mediated invasion compared to single agent treated cells (P<0.05). (B) PLC inhibition in HNSCC cells expressing dominant-negative c-Src results in minimal EGFR-mediated invasion. Dominant-negative c-Src expressing HNSCC cells or vector transfected control cells were plated at a density of 2.5 × 10⁴ cells/well in the presence of EGF (10 ng/ml) or 10% FBS containing medium and treated with U73122 (3 μM). Treatment of dominant-negative c-Src expressing cells with PLC inhibitor U73122 attenuated EGFR-mediated invasion in HNSCC cells compared to vector control cells (* P<0.05). (C) c-Src inhibition in HNSCC cells expressing dominant-negative PLCγ-1 results in minimal EGFR-mediated invasion. Dominant-negative PLCγ-1 expressing HNSCC cells were plated and treated with AZD0530 (1 μM). After 48 h of treatment, the cells on the lower side of the chamber were fixed, stained and counted at 200X magnification. The values are the mean ± SE of at least two independent experiments. HNSCC cells expressing dominant-negative PLCγ-1 when treated with the Src inhibitor AZD0530 significantly fewer invading cells of EGFR stimulation compared with vector transfected control under the same conditions (* P<0.05).

Figure 5

Combined inhibition of PLCγ-1 and c-Src results in attenuation of EGFR-mediated invasion in HNSCC cells. (A) Pharmacologic inhibitors of PLCγ-1 and c-Src are active against EGFR-mediated invasion in HNSCC. HNSCC cells PCI-37B were plated at a density of 2.5 × 10⁴ cells/well in Matrigel coated transwell chambers in the presence of EGF (10 ng/ml) or 10% FBS containing medium, and treated with AZD0530 (1 μM), PLC inhibitor (U73122, 3 μM), control inactive compound (U73343, 3 μM) and AZD0530 (1 μM) plus U73122 or U73343 (3 μM) for 48h. Combined inhibition of PLC and c-Src in HNSCC cells results in abrogation of EGFR-mediated invasion compared to single agent treated cells (P<0.05). (B) PLC inhibition in HNSCC cells expressing dominant-negative c-Src results in minimal EGFR-mediated invasion. Dominant-negative c-Src expressing HNSCC cells or vector transfected control cells were plated at a density of 2.5 × 10⁴ cells/well in the presence of EGF (10 ng/ml) or 10% FBS containing medium and treated with U73122 (3 μM). Treatment of dominant-negative c-Src expressing cells with PLC inhibitor U73122 attenuated EGFR-mediated invasion in HNSCC cells compared to vector control cells (* P<0.05). (C) c-Src inhibition in HNSCC cells expressing dominant-negative PLCγ-1 results in minimal EGFR-mediated invasion. Dominant-negative PLCγ-1 expressing HNSCC cells were plated and treated with AZD0530 (1 μM). After 48 h of treatment, the cells on the lower side of the chamber were fixed, stained and counted at 200X magnification. The values are the mean ± SE of at least two independent experiments. HNSCC cells expressing dominant-negative PLCγ-1 when treated with the Src inhibitor AZD0530 significantly fewer invading cells of EGFR stimulation compared with vector transfected control under the same conditions (* P<0.05).

Figure 6

PLCγ-1 and c-Src interact with each other upon EGFR stimulation in HNSCC cells. HNSCC cells expressing dominant-negative c-Src (A) or PLCγ-1 (B) and vector transfected control cells were stimulated with EGF (10 ng/ml) or 10% FBS containing medium after serum starvation for 72 h. PLCγ-1 was immunoprecipitated from cell lysates followed by immunoblotting for c-Src and PLCγ-1. Densitometric analysis was performed and expression levels relative to PLCγ-1 are shown as mean ± SE from at least two independent experiments. A significant decrease in EGFR stimulated PLCγ-1 and c-Src interaction was observed in dominant-negative cell lines compared with vector transfected control under similar conditions (* P<0.05). IgG control lysates demonstrate that there is no non-specific binding of anti-mouse antibody to PLCγ-1 or c-Src.