Δ Np63α在头颈鳞癌中对Bortezomi耐药的作用及机制研究

Targeted drugs are widely used in individualized therapy to improve the clinical outcome and reduce the adverse effect for cancer patients. However, the intrinsic or acquired resistance to targeted drugs hinders their long-lasting use in patients. Thus, it is urgent to explore the underlying mechanism to overcome the inevitably occurred resistance. We constructed a bio-computational platform in 11 cell lines of squamous cell carcinoma of the head and neck (HNSCC), and we analyzed the correlation between the response of these cell lines to 110 antitumor drugs and the mRNA expression investigated by deep RNA sequencing. We found that these cell lines responded to bortezomib differentially, a small molecule inhibitor against proteasomes, and that the expression of ΔNp63α was significantly associated with the resistance to bortezomib. The pivotal role of ΔNp63α in bortezomib resistance was further validated in vitro by subsequent experiments: 1) endogenous expression of ΔNp63α mRNA and protein was positively correlated to the resistance in the 11 cell lines, 2) forced expression of ΔNp63α in sensitive lines dramatically increased the resistance, and 3) knockdown of ΔNp63α by shRNA in resistant lines marked decreased the resistance. A range of experiments, including 1) construction of cell lines with acquired resistance to bortezomib, 2) construction of cell lines stably overexpressed ΔNp63α or stably silenced ΔNp63α, 3) establishment of tumor models of above cell lines in nude mice to verify the correlation between ΔNp63α expression and bortezomib resistance in vivo, 4) searching for the proteins interacted with ΔNp63α following bortezomib treatment through 2D-gel for proteins pulldown by ΔNp63α antibody, and mass spectral analysis, 5) screen for kinase inhibitors approved by SFDA in resistant lines to find those synergistically killing cells when combined with bortezomib, and 6) the protein expression and activation status in tissues from clinical samples by IHC, will be conducted to elucidate the mechanism by which ΔNp63α mediates the resistance to bortezomib, and to find novel targets for combination therapy with bortezomib to overcome the resistance. Our work will help provide a theoretical basis for individualized therapies in clinic and pave the way for future studies on resistance mechanism of other targeted drugs in other cancers.

靶向药物治疗明显延长患者生命，应用前景广阔，但存在耐药现象，影响治疗效果，因机制不甚清楚，尚无逆转耐药良策。我们在 11 个头颈鳞癌细胞株中建立了深度 RNA 测序及 110 种抗癌药物敏感度数据库，通过功能性生物分析平台最终锁定小分子靶向药物 Bortezomib （万珂，蛋白酶体抑制剂）作为研究对象。发现Δ Np63α与 头颈部鳞癌万珂耐药密切相关，进一步体外实验干预Δ Np63α的表达，显著提高头颈鳞癌细胞对万珂的敏感性，初步明确Δ Np63α在万珂耐药中的关键作用。为进一步阐明其作用及机制，我们拟通过耐药株筛选，稳定株建立，裸鼠成瘤，蛋白质组学及临床标本验证等方法，明确Δ Np63α在万珂耐药中的作用及机制，寻找与万珂联合应用对头颈鳞癌耐药细胞具有协同杀伤作用的ΔNp63α上游激酶抑制剂，为逆转万珂耐药提供新的靶点，为临床个体化治疗提供理论依据，也为其他肿瘤耐药机制研究提供新思路。

本课题组通过生物学分析及临床情况以小分子靶向药物 Bortezomib （蛋白酶体抑制剂，万珂）作为研究对象，发现Δ Np63α是 Bortezomib 耐药的关键基因，并通过耐药株筛选，稳定株建立，裸鼠成瘤，蛋白质组学及临床标本验证等方法，在体内外研究Bortezomib 耐药关键基因Δ Np63α 的作用及机制。本课题验证了头颈鳞状细胞癌中ΔNp63α表达水平与Bortezomib药物敏感性相关，并通过慢病毒转染敲减和过表达ΔNp63α后检测到头颈鳞癌细胞对Bortezomib敏感性的改变，同时通过耐药株、敏感株裸鼠成瘤静脉给药检测到瘤体大小和生存时间的明显差异，明确ΔNp63α在Bortezomib头颈鳞癌细胞耐药的关键作用。深入探究机制，我们发现ΔNp63α诱发Bortezomib耐药主要通过Np63α-CYGB-ROS轴调控。Western-Blot和免疫荧光检测结果显示高表达的ΔNp63α上调CYGB，这有利于清除Bortezomib诱导的可导致细胞凋亡的过量ROS，最终促进了肿瘤细胞的存活。本课题通过双荧光素酶报告基因和Chip验证了ΔNp63α作为转录因子对CYGB启动子的调控,通过流式细胞仪检测出CYGB对过量ROS的清除作用，免疫组化进一步验证Np63α和CYGB相关性。研究结果可以为 Bortezomib 临床耐药和耐药逆转提供新的靶点，为开展个体化靶向药物治疗提供理论依据，也可为其他肿瘤的耐药机制研究提供可靠平台及新思路。