Combined inhibition of c-Src and epidermal growth factor receptor abrogates growth and invasion of head and neck squamous cell carcinoma

Abstract

Purpose: Increased expression and/or activation of epidermal growth factor receptor (EGFR) is associated with tumor progression and poor prognosis in many cancers, including head and neck squamous cell carcinoma (HNSCC). Src family kinases, including c-Src, mediate a variety of intracellular or extracellular signals that contribute to tumor formation and progression. This study was undertaken to elucidate the role of c-Src in the growth and invasion of HNSCC and to determine the effects of combined targeting of EGFR and Src kinases in HNSCC cell lines.

Experimental design: HNSCC cells were engineered to stably express a dominant-active form of c-Src and investigated in cell growth and invasion assays. The biochemical effects of combined treatment with the Src inhibitor AZD0530, a potent, orally active Src inhibitor with Bcr/Abl activity, and the EGFR kinase inhibitor gefitinib were examined, as well as the consequences of dual Src/EGFR targeting on the growth and invasion of a panel of HNSCC cell lines.

Results: HNSCC cells expressing dominant-active c-Src showed increased growth and invasion compared with vector-transfected controls. Combined treatment with AZD0530 and gefitinib resulted in greater inhibition of HNSCC cell growth and invasion compared with either agent alone.

Conclusions: These results suggest that increased expression and activation of c-Src promotes HNSCC progression where combined targeting of EGFR and c-Src may be an efficacious treatment approach.

Figure 1. Characterization of dominant-active (DA) c-Src clones

(A) HNSCC cells (1483) were transfected with a DA c-Src construct (Upstate Biotechnology) followed by isolation of stable clones in G418-containing media (400 µg/ml). Expression of Tyr 418 phosphorylation and total c-Src were examined by immunoblotting with pY418 Src antibody (BioSource International, Camarillo, CA) and anti-c-Src antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Actin is shown as a control for loading (Oncogene Research Products, Boston, MA). (B) Additional HA-tagged DA c-Src clones were generated in 1483 cells. A representative western blot showing increased expression of pY418 Src as well as total c-Src is shown, in conjunction with expression of the HA-tag. The experiment was repeated 3 times with similar results.

Figure 2. Invasion of dominant-active and dominant-negative c-Src HNSCC cells

Representative vector-transfected control, DN c-Src-transfected, and DA c-Src-transfected HNSCC (1483) clones (DA c-Src #6) were serum-starved for 4 days, and then plated at a density of 5× 10³ cells/well in serum-free DMEM in Matrigel-coated inserts. Lower wells contained DMEM with 10% FBS. After 48 hours at 37°C in a 5% CO₂ incubator, the cells in the insert were removed by wiping gently with a cotton swab. Cells on the reverse side of the insert were fixed and stained with Hema 3 (Fisher Scientific) according to the manufacturer’s instructions. Invading cells in 4–8 representative fields were counted using light microscopy at 200× magnification. The experiment was repeated 4 times with similar results.

Figure 3. Proliferation of dominant-active and dominant-negative c-Src HNSCC cells

For growth experiments, (A) representative vector-transfected (VC #3), DN c-Src-transfected, and DA c-Src-transfected HNSCC (1483 DA c-Src #6) clones were plated in 48-well plates at a density of 5 × 10³ cells/well in DMEM with 1% FBS. Cells were incubated at 37°C, 5% CO₂ incubator for 3 days, followed by cell counts using trypan blue exclusion. Cumulative results are shown from 6 independent experiments, each performed in triplicate (upper panel). (B) In the lower panel, the growth kinetics of a DA c-Src transfected 1483 clone, DA c-Src clone #62 was compared over the course of 8 days with the 1483 vector-transfected control clone (VC #1) and to the DN-Src clone in low serum containing media (1% FBS). Cells (3 × 10⁴) of each clone were seeded in triplicate in 12-well plates and allowed to adhere overnight. Cell counts were carried out using trypan blue dye over the next 8 days. All data values are represented as mean ± SEM. The experiment was repeated 3 times with similar results. (C) PCI-15B cells were stably transfected with DA c-Src or vector control and plated as above in DMEM with 1% of FBS. Cells were harvested on day 4 and counted with vital dye exclusion. (D) Cell counts on days 2, 4, 6 and 8 from 3 independent experiments are represented.

Figure 3. Proliferation of dominant-active and dominant-negative c-Src HNSCC cells

For growth experiments, (A) representative vector-transfected (VC #3), DN c-Src-transfected, and DA c-Src-transfected HNSCC (1483 DA c-Src #6) clones were plated in 48-well plates at a density of 5 × 10³ cells/well in DMEM with 1% FBS. Cells were incubated at 37°C, 5% CO₂ incubator for 3 days, followed by cell counts using trypan blue exclusion. Cumulative results are shown from 6 independent experiments, each performed in triplicate (upper panel). (B) In the lower panel, the growth kinetics of a DA c-Src transfected 1483 clone, DA c-Src clone #62 was compared over the course of 8 days with the 1483 vector-transfected control clone (VC #1) and to the DN-Src clone in low serum containing media (1% FBS). Cells (3 × 10⁴) of each clone were seeded in triplicate in 12-well plates and allowed to adhere overnight. Cell counts were carried out using trypan blue dye over the next 8 days. All data values are represented as mean ± SEM. The experiment was repeated 3 times with similar results. (C) PCI-15B cells were stably transfected with DA c-Src or vector control and plated as above in DMEM with 1% of FBS. Cells were harvested on day 4 and counted with vital dye exclusion. (D) Cell counts on days 2, 4, 6 and 8 from 3 independent experiments are represented.

Figure 4. The effect of increasing doses of AZD0530 on expression and activation of c-Src and FAK in HNSCC cell lines

(A) UM-22B, 1483 and PCI-37B cells were treated with AZD0530 (1–10⁴ nM) or DMSO for 2 hours prior to lysis. Immunoblotting was performed with pY418 c-Src antibody, anti-c-Src antibody, pFAK Tyr 861 and total FAK. (B) Cal-33 cells were treated with increasing concentrations of AZD0530 (0.06–6µM) or DMSO for 2 hrs followed by immunoblotting for total and activated c-Src, E-cadherin and pFAK Y861. The experiment was performed 3 times with similar results.

Figure 4. The effect of increasing doses of AZD0530 on expression and activation of c-Src and FAK in HNSCC cell lines

(A) UM-22B, 1483 and PCI-37B cells were treated with AZD0530 (1–10⁴ nM) or DMSO for 2 hours prior to lysis. Immunoblotting was performed with pY418 c-Src antibody, anti-c-Src antibody, pFAK Tyr 861 and total FAK. (B) Cal-33 cells were treated with increasing concentrations of AZD0530 (0.06–6µM) or DMSO for 2 hrs followed by immunoblotting for total and activated c-Src, E-cadherin and pFAK Y861. The experiment was performed 3 times with similar results.

Figure 5. Matrigel invasion assay of HNSCC cell lines treated with AZD0530 and/or gefitinib

(A) 1483 and (B) PCI-37B cells were plated in Matrigel-coated invasion chamber at a density of 1 × 10⁴ cells/well in serum-free DMEM containing DMSO, AZD0530 (1 µM), gefitinib (at IC₅₀ value), or combination of AZD0530 and gefitinib (both at IC₅₀ values). Lower wells contained DMEM with 10% FBS. After 48 hours of treatment at 37°C in a 5% CO₂ incubator, the cells in the insert were removed by wiping gently with a cotton swab. Cells on the reverse side of the insert were fixed and stained with Hema 3 (Fisher Scientific) according to the manufacturer’s instructions. Invading cells in 4 representative fields each from 3 replicates were counted using light microscopy at 200× magnification. Cumulative data from at least 5 independent experiments for both cell lines are shown.

Figure 5. Matrigel invasion assay of HNSCC cell lines treated with AZD0530 and/or gefitinib

(A) 1483 and (B) PCI-37B cells were plated in Matrigel-coated invasion chamber at a density of 1 × 10⁴ cells/well in serum-free DMEM containing DMSO, AZD0530 (1 µM), gefitinib (at IC₅₀ value), or combination of AZD0530 and gefitinib (both at IC₅₀ values). Lower wells contained DMEM with 10% FBS. After 48 hours of treatment at 37°C in a 5% CO₂ incubator, the cells in the insert were removed by wiping gently with a cotton swab. Cells on the reverse side of the insert were fixed and stained with Hema 3 (Fisher Scientific) according to the manufacturer’s instructions. Invading cells in 4 representative fields each from 3 replicates were counted using light microscopy at 200× magnification. Cumulative data from at least 5 independent experiments for both cell lines are shown.

Figure 6. Proliferation assay of HNSCC cell lines treated with AZD0530 and/or gefitinib

(A) 1483, (B) PCI-37B, and (C) Cal-33 cells were plated in 12-well plates at a density of 3 × 10⁴ cells/well in DMEM with 10% FBS. After 24 hours, growth media was replaced with media containing DMSO, AZD0530 (at IC₅₀ value), gefitinib (at IC₅₀ value), or a combination of AZD0530 and gefitinib. On days 0, 2, 4, 6 and 8, viable cells were counted using vital dye exclusion. Four independent experiments (in triplicate) were performed for each cell line. Cell numbers on day 8 after treatment are shown.

Figure 6. Proliferation assay of HNSCC cell lines treated with AZD0530 and/or gefitinib

(A) 1483, (B) PCI-37B, and (C) Cal-33 cells were plated in 12-well plates at a density of 3 × 10⁴ cells/well in DMEM with 10% FBS. After 24 hours, growth media was replaced with media containing DMSO, AZD0530 (at IC₅₀ value), gefitinib (at IC₅₀ value), or a combination of AZD0530 and gefitinib. On days 0, 2, 4, 6 and 8, viable cells were counted using vital dye exclusion. Four independent experiments (in triplicate) were performed for each cell line. Cell numbers on day 8 after treatment are shown.

Figure 6. Proliferation assay of HNSCC cell lines treated with AZD0530 and/or gefitinib

(A) 1483, (B) PCI-37B, and (C) Cal-33 cells were plated in 12-well plates at a density of 3 × 10⁴ cells/well in DMEM with 10% FBS. After 24 hours, growth media was replaced with media containing DMSO, AZD0530 (at IC₅₀ value), gefitinib (at IC₅₀ value), or a combination of AZD0530 and gefitinib. On days 0, 2, 4, 6 and 8, viable cells were counted using vital dye exclusion. Four independent experiments (in triplicate) were performed for each cell line. Cell numbers on day 8 after treatment are shown.

Figure 7. The effect of AZD0530 and/or gefitinib on expression of total and phosphorylated c-Src, MAPK, AKT, and FAK and STAT5

HNSCC cells (1483, PCI-37B and Cal-33) were treated with DMSO, AZD0530 (at IC₅₀ value), gefitinib (at IC₅₀ value) or combination of AZD0530 and gefitinib (both at IC₅₀ values) in DMEM with 10% FBS. After 2 hours, total protein was extracted prior to lysis and immunoblotting. Beta-tubulin is shown for loading control. The experiment was performed at least 2 times, with similar results.