Enhancing radiosensitization in EphB4 receptor-expressing Head and Neck Squamous Cell Carcinomas

Abstract

Members of the Eph family of receptor tyrosine kinases have been implicated in a wide array of human cancers. The EphB4 receptor is ubiquitously expressed in head and neck squamous cell carcinoma (HNSCC) and has been shown to impart tumorigenic and invasive characteristics to these cancers. In this study, we investigated whether EphB4 receptor targeting can enhance the radiosensitization of HNSCC. Our data show that EphB4 is expressed at high to moderate levels in HNSCC cell lines and patient-derived xenograft (PDX) tumors. We observed decreased survival fractions in HNSCC cells following EphB4 knockdown in clonogenic assays. An enhanced G2 cell cycle arrest with activation of DNA damage response pathway and increased apoptosis was evident in HNSCC cells following combined EphB4 downregulation and radiation compared to EphB4 knockdown and radiation alone. Data using HNSCC PDX models showed significant reduction in tumor volume and enhanced delay in tumor regrowth following sEphB4-HSA administration with radiation compared to single agent treatment. sEphB4-HSA is a protein known to block the interaction between the EphB4 receptor and its ephrin-B2 ligand. Overall, our findings emphasize the therapeutic relevance of EphB4 targeting as a radiosensitizer that can be exploited for the treatment of human head and neck carcinomas.

Figure 1. EphB4 is expressed in human HNSCC cells and its knockdown sensitizes HNSCC cells to ionizing radiation.

(A) The EphB4 receptor is present at high to moderate levels in human HNSCC cells compared to the normal oral keratinocyte (NOK) cells as detected by Western blotting. (B) EphB4 expression is reduced upon transfection with the EphB4-targeting siRNAs 1 or 2 compared to the control non-specific siRNA (NS-siRNA). (C–E) Reduction in survival fractions in HNSCC cells is observed after transfection with the EphB4-targeting siRNA versus the control NS-siRNA (25-50 nM) in Cal27 (C), MSK-921 (D), and Fadu (E) cells as determined by clonogenic assay. Each clonogenic assay was repeated atleast three times. Representative survival plots are shown for each cell line. The survival plot for the MSK-921 cells was generated using 0-6 Gy dose of ionizing radiation because 8 Gy dose did not yield viable colonies. Data shown represent mean ± standard deviation. *p < 0.05; **p < 0.01.

Figure 2. EphB4 downregulation combined with ionizing radiation enhances the DNA damage response in HNSCC cells.

p-H2AX analysis shows a significant enhancement in the percentage of p-H2AX positive MSK-921 cells following EphB4 knockdown and ionizing radiation as demonstrated by immunofluorescence staining (A and B). In addition, an increase in Rad51-foci is also evident in MSK-921 cells (C and D). A similar trend was observed in Fadu cells in terms of p-H2AX (E and F) and Rad51 (G and H) expression. Data shown represent mean ± standard error from two to three independent experiments. *p < 0.05; **p < 0.005. Total magnification: 600–1000x. Western blot analyses demonstrate enhanced levels of p-H2AX, Ku80, and Rad51 indicative of the DNA damage response in the combination group compared to the other experimental groups in MSK-921 cells (I) and Fadu cells (J).

Figure 3. Combined EphB4 knockdown and ionizing radiation exposure induces apoptosis in HNSCC cells.

EphB4 knockdown combined with radiation enhances apoptosis in Fadu cells as shown by caspase 3/7 assay (A). Western blot analysis show modulation in the levels of pro-survival markers in Fadu (B) and MSK-921 (C) cells following EphB4 knockdown and radiation exposure (XRT). Each experiment was repeated atleast two times. Data shown represent mean ± standard error from two independent experiments. *p < 0.05.

Figure 4. EphB4 targeting enhances radiosensitivity in HNSCC PDX models.

Tumor growth analyses of CUHN013 PDX tumors (A,B), CUHN022 PDX tumors (C,D), and CUHN004 PDX tumors (E,F) show that sEphB4-HSA treatment combined with radiation significantly decreases tumor volume over time compared to single agent treatments. sEphB4-HSA was administered on days 0, 3, 5, 7, 10, 12, 14, 17, 19, 21, 24, 26, 28, 31, 33, 35, and 38. The symbol “↓” represent days when tumors were exposed to ionizing radiation (5 Gy/fraction for CUHN013 and CUHN004; 2 Gy/fraction for CUHN022). The mice in the PBS group were sacrificed on day 31 (CUHN013), or day 28 (CUHN004) because their tumors became too large. Data shown represent mean ± standard deviation. *p < 0.05,**p < 0.005, **p < 0.0005.

Figure 5. EphB4 targeting radiosensitizes HNSCC tumors and affects tumor growth by reducing proliferation, and enhancing apoptosis.

Immunofluorescence analysis of CUHN013 PDX tumors show reduction in PCNA (A) and enhancement in TUNEL-positive nuclei (B) in mice treated with sEphB4-HSA and ionizing radiation (XRT) compared to mice treated with a single agent. Quantitative analysis of TUNEL staining (C) show significant increase in apoptosis as evident by TUNEL-positive nuclei in the combination group compared to single agents. Data shown represent mean ± standard deviation. *p < 0.05, **p < 0.005. Total magnification: 600x.

Figure 6. EphB4 targeting in combination with ionizing radiation exposure alters the expression of apoptotic and tumor growth promoting molecules in CUHN013 tumors.

Western blot analysis shows decreased phosphorylation or decreased levels of proteins implicated in tumor growth and cell survival in CUHN013 tumors treated with sEphB4-HSA and radiation compared to tumors treated with a single agent. (B) A heat map generated based on the results of a human apoptosis antibody array using GeneE software (Broad institute, USA) depicts decreased expression of survival markers in CUHN013 tumors treated as indicated. (C) Validation of hits by western blotting shows decreased levels of pro-survival proteins such as Ho-2, Bcl-2, and survivin in tumors treated with sEphB4-HSA combined with radiation (XRT) compared to tumors treated with a single agent.