Afatinib radiosensitizes head and neck squamous cell carcinoma cells by targeting cancer stem cells

Abstract

The dismal prognosis of locally advanced and metastatic squamous cell carcinoma of the head and neck (HNSCC) is primarily due to the development of resistance to chemoradiation therapy (CRT). Deregulation of Epidermal Growth Factor Receptor (EGFR) signaling is involved in HNSCC pathogenesis by regulating cell survival, cancer stem cells (CSCs), and resistance to CRT. Here we investigated the radiosensitizing activity of the pan-EGFR inhibitor afatinib in HNSCC in vitro and in vivo. Our results showed strong antiproliferative effects of afatinib in HNSCC SCC1 and SCC10B cells, compared to immortalized normal oral epithelial cells MOE1a and MOE1b. Comparative analysis revealed stronger antitumor effects with afatinib than observed with erlotinib. Furthermore, afatinib enhanced in vitro radiosensitivity of SCC1 and SCC10B cells by inducing mesenchymal to epithelial transition, G1 cell cycle arrest, and the attenuating ionizing radiation (IR)-induced activation of DNA double strand break repair (DSB) ATM/ATR/CHK2/BRCA1 pathway. Our studies also revealed the effect of afatinib on tumor sphere- and colony-forming capabilities of cancer stem cells (CSCs), and decreased IR-induced CSC population in SCC1 and SCC10B cells. Furthermore, we observed that a combination of afatinib with IR significantly reduced SCC1 xenograft tumors (median weight of 168.25 ± 20.85 mg; p = 0.05) compared to afatinib (280.07 ± 20.54 mg) or IR alone (324.91 ± 28.08 mg). Immunohistochemical analysis of SCC1 tumor xenografts demonstrated downregulation of the expression of IR-induced pEGFR1, ALDH1 and upregulation of phosphorylated γH2AX by afatinib. Overall, afatinib reduces tumorigenicity and radiosensitizes HNSCC cells. It holds promise for future clinical development as a novel radiosensitizer by improving CSC eradication.

Keywords: afatinib; head and neck squamous cell carcinoma (HNSCC); radiosensitization.

Figure 1

Afatinib and erlotinib differentially decreases the proliferation of HNSCC and normal cells (A) HNSCC cells SCC1 and SCC10B and immortalized normal oral epithelial cells MOE1a and MOE1b cells in 96 well plates were treated with different concentrations of afatinib and erlotinib for 48 h and viable cell number was analyzed by MTT assay. (B) Afatinib and erlotinib inhibits EGFR activation. SCC1 and SCC10B cells were treated with afatinib or erlotinib for 48 h and cell lysates were analyzed for pEGFR. (C) HNSCC SCC1, SCC10B, SCC11B, SCC23, SCC38, SCC47 and SCC104 cells were treated with either afatinib (2 μM) or erlotinib (10 μM) for 48 h and analyzed for pEGFR expression by Western blot analysis. β-actin was used as a loading control. (D) SCC1, SCC10B, MOE1a and MOE1b cells were treated with 2 μM of afatinib for 12–48 h and analyzed for phosphorylated and total forms of EGFR, HER2, HER3, AKT, ERK1/2, p38MAPK. Radiation (8 Gy)-treated SCC10B cells were used as positive controls for pHER2 and pHER3 expression in MOE1a and MOE1b cells. (E) Afatinib and erlotinib reduces colony formation of HNSCC cells. SCC1 and SCC10B cells were incubated with different doses of afatinib and erlotinib for 24 h and cells (1 × 10³) were seeded in triplicate in 10% DMEM in a 6-well plate. After 2 weeks, formed colonies were counted using the automatic colony counting tool by Quantity One Imaging software. The graphs represent the mean (± SE) number of colonies. The experiment was repeated twice (*p < 0.05).

Figure 2. Afatinib radio-sensitizes HNSCC cells

(A) SCC1 and SCC10B HNSCC were pretreated with afatinib (0.5 μM) for 24 h and then radiated with 2–8 Gy IR. After 24 h wells were washed and cells were allowed to grow for 2 weeks. Colonies were stained with 1% crystal violet, counted, and survival curves were plotted. (B) SCC1 and SCC10B cells were synchronized overnight in 1% serum containing medium and treated with afatinib alone for 24 h, or combined with 8 Gy IR. After 24 h, cells were fixed and stained with propidium iodide and analyzed by flow cytometry. (C) SCC1 and SCC10B HNSCC were plated on glass cover slips and pre-treated with afatinib (0.5 μM) for 24 h and then irradiated with 8 Gy IR. After 24 h wells were washed and analyzed by confocal microscopy for pγH2AX foci. DNA damage foci were counted and plotted as bar graphs.

Figure 3. Afatinib inhibits Epithelial to Mesenchymal transition in HNSCC cells

(A) SCC1 and SCC10B cells were treated with afatinib for 24 h and protein lysates were checked for pEGFR, pFAK, E-Cadherin, Snail, and Slug expression. β-actin was used as an internal loading control (B) SCC1 and SCC10B cells were allowed to form confluent layer in a 6 well plate and a scratch were made using a 200 μl sterile pipette tip. Unattached cells were washed with PBS and images were taken (t = 0 h). Cells were then treated with afatinib (2 μM) for 24 h and again photographed. The width of the wound was calculated with or without afatinib and bar graph plotted. (C) Afatinib treated and untreated SCC1 and SCC10B HNSCC cells (250 × 10³ cells) were seeded into the upper chamber of Matrigel gel coated Boyden chamber in serum-free αMEM media. Invading cells after 24h were stained using Diff Kit and quantified in 10 random fields under a light microscope (magnification, 100). Histograms represent mean of invasive cell number from three independent experiments; bars, SD.

Figure 4. Afatinib attenuates IR-induced DNA repair machinery and induced mitotic catastrophe in HNSCC cells

(A) HNSCC cells SCC1 and SCC10B were treated with afatinib alone for 48 h, or combined with 8 Gy IR. Cell lysates were analyzed by Western blot analysis for DNA repair pathway proteins including pEGFR (Tyr-1068), pAkt, pERK1/2 (Thr202/Tyr204), pCHK2 (Thr-68), pBRCA1 (Ser-1524), pATM (Ser-1981), and pATR (Ser-428). β-actin was used as an internal loading control. (B) SCC1 and SCC10B were treated with afatinib alone for 48 h or combined with 8 Gy IR. Cells were washed and observed under microscope for DAPI staining. The number of cells containing fragmented nuclei (catastrophic nuclei) were photographed.

Figure 5. Afatinib affects cancer stem cells in HNSCC cells

(A) SCC1 and SCC10B cells were treated with 2 μM of afatinib for 12–48 h and analyzed for expression of cancer stem cell markers including CD44, ESA, SHH and Oct3/4 by Western blot analysis. (B) SCC1 and SCC10B cells were treated with 0.5–2μM of afatinib alone for 48 h and SP and NSP cells were isolated by FACS analysis using Hoechst 33342 (5 mg/ml) staining. 1 × 10³ SP and NSP cells were plated in 24-well low attachment plates and analyzed for sphere formation on 14th day and photographed using light microscope. (C) SCC1 and SCC10B cells were treated with either afatinib (48 h) alone or combined with IR. After 48 h, cells were trypsinized and analyzed for SP and NSP cells by FACS analysis using Hoechst 33342 (5 mg/ml) staining. (D) Isolated SP cells (250) were plated on 6 well plate and analyzed for colony formation assay after 2 weeks. The graphs represent the mean (± SE) number of colonies. The experiment was repeated twice (*p < 0.05).

Figure 6. Afatinib radiosensitizes HNSCC tumors in vivo

(A) SCC1 cells were subcutaneously implanted on contralateral flanks in athymic nude mice and randomized into group 1 (8 animals each) with afatinib treatment and radiation on the right tumors, and afatinib only on the left side tumors; group 2 (8 animals each) with vehicle gavages and radiation on the right tumors. Tumor volume and animal weights were measured every 3 days starting from the day of drug administration. All the mice were sacrificed on the 15th day after afatinib treatment and body weight and tumor weight measured. The graphs show a significant decrease in tumor volume (A) and tumor weights (B) in afatinib + IR treated animals compared to control, afatinib only, or IR-treated mice. (C) Excised tumors were analyzed for pEGFR (Tyr-1068), pγH2AX (Ser-139), CD24, and ALDH1 expression using immunohistochemical analysis (×20 magnification). (D) Schematic diagram illustrating the potential molecular mechanism of afatinib mediated radio-sensitization of HNSCC. Treatment of ionizing radiation (IR) kills the bulk of tumor cells but enriches cancer stem cells shown as red (left side) that leads to tumor recurrence. However, pre-treatment of afatinib radio-sensitizes tumors and inhibits both CSCs and the bulk of tumor cells, and results in significant tumor shrinkage. The molecular mechanism revealed that afatinib significantly inhibited the phosphorylation of EGFR, HER2, and HER3 coupled with inhibition of downstream signaling molecules, including pAkt (Ser-473) and pERK1/2 (Thr202/Tyr204). Afatinib pre-treatment abrogated IR-induced activation of DNA DSB repair by inhibiting pAkt (Ser-473) and pERK1/2 (Thr202/Tyr204), pATM (Ser-1981), pChk2 (Thr-68), pATR (Ser-428) and pBRCA1 (Ser-1524). In addition, afatinib inhibited IR-induced SP and NSP population and downregulated expression of cancer stem cell markers CD44 and Oct3/4.