University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
Radiother Oncol. 2012 Nov;105(2):241-9. doi: 10.1016/j.radonc.2012.08.010. Epub 2012 Sep 23.
Although inhibition of epidermal growth factor receptor (EGFR) signaling during radiation led to improvement of tumor control and survival, novel strategies are needed to further improve the outcome of patients with locally advanced head and neck carcinoma. Because EGFR is known to interact with c-Src kinases, the present study investigated dasatinib (BMS-354825), an inhibitor of c-Src kinases, for its efficacy in enhancing radiosensitivity of human head and neck squamous cell carcinomas (HNSCC) in vitro and examined the underlying mechanisms for this effect.
Six HNSCC lines were exposed to dasatinib, radiation, or both, and assessed for c-Src and EGFR expression, cell survival and colony forming ability. Among these cell lines, HN-5 and FaDu lines were analyzed for induction of apoptosis, cell cycle re-distribution and for nuclear localization of EGFR, γ-H2AX and 53BP1 proteins. Immuno-precipitation and Western blots were performed to analyze the levels and binding of proteins involved in cell survival, apoptosis and DNA repair pathways. Suppression of c-Src by siRNA and subsequent clonogenic assay was performed in HN-5 cells.
All six HNSCC lines that were examined expressed high levels of c-Src. Two (HN-5 and MDA-183) expressed higher levels of EGFR than other lines. Dasatinib suppressed cell survival of all cell lines tested independent of c-Src or EGFR levels but enhanced the radiosensitivity of HN-5 and MDA-183. HN-5 and FaDu were analyzed further. Dasatinib suppressed phosphorylation of c-Src in both cell lines, but decreased repair of radiation-induced DNA damage in HN-5 cells only as evidenced by suppression of c-Abl and Nbs-1 activity, inhibition of the association between c-Src and EGFR or Her-2, prolongation of nuclear γ-H2AX and 53BP1 foci and inhibition of EGFR nuclear localization and its association with DNA-PKcs. Finally, partial suppression of c-Src resulted in a small increase in HN-5 cell radiosensitivity.
Our data demonstrate that dasatinib induces apoptosis and blocks DNA repair in EGFR-expressing HNSCC cells and improves radiotherapy outcome. These findings warrant further investigation using in vivo tumor models for potential translation into clinical testing.
尽管在放疗期间抑制表皮生长因子受体（EGFR）信号通路可改善肿瘤控制和生存，但仍需要新的策略来进一步提高局部晚期头颈部癌患者的预后。因为已知 EGFR 与 c-Src 激酶相互作用，所以本研究探讨了 c-Src 激酶抑制剂 dasatinib（BMS-354825）在体外增强人头颈鳞状细胞癌（HNSCC）放射敏感性的效果，并研究了这种作用的潜在机制。
将六种 HNSCC 细胞系暴露于 dasatinib、放疗或两者中，并评估 c-Src 和 EGFR 的表达、细胞存活和集落形成能力。在这些细胞系中，对 HN-5 和 FaDu 细胞系进行了诱导细胞凋亡、细胞周期再分布以及 EGFR、γ-H2AX 和 53BP1 蛋白核定位的分析。进行免疫沉淀和 Western blot 分析以研究参与细胞存活、凋亡和 DNA 修复途径的蛋白的水平和结合。在 HN-5 细胞中通过 siRNA 抑制 c-Src 并随后进行集落形成测定。
所检查的六种 HNSCC 细胞系均表达高水平的 c-Src。两种（HN-5 和 MDA-183）表达的 EGFR 水平高于其他细胞系。Dasatinib 抑制了所有测试细胞系的细胞存活，而与 c-Src 或 EGFR 水平无关，但增强了 HN-5 和 MDA-183 的放射敏感性。进一步分析了 HN-5 和 FaDu。Dasatinib 抑制了这两种细胞系中 c-Src 的磷酸化，但仅在 HN-5 细胞中降低了辐射诱导的 DNA 损伤的修复，这表现在 c-Abl 和 Nbs-1 活性的抑制、c-Src 与 EGFR 或 Her-2 的结合减少、核 γ-H2AX 和 53BP1 焦点的延长以及 EGFR 核定位及其与 DNA-PKcs 的结合的抑制。最后，c-Src 的部分抑制导致 HN-5 细胞放射敏感性略有增加。
我们的数据表明，dasatinib 诱导 EGFR 表达的 HNSCC 细胞凋亡并阻断 DNA 修复，并改善放射治疗效果。这些发现为进一步使用体内肿瘤模型进行研究并将其转化为临床测试提供了依据。
