靶向抑制PP2A与肺鳞癌细胞的干性逆转与化疗增敏研究

Protein Phosphatase 2A (PP2A) is a major serine/threonine phosphatase in cells. It consists of a catalytic subunit, a scaffold subunit, and a regulatory subunit. Based on loss of function analysis using PP2A catalytic inhibitors or inhibition via tumor viral antigens, limited studies suggest that PP2A is a putative tumor suppressor. However, PP2A has also been shown to facilitate the activation of oncogenic signaling pathways when associated with specific regulatory subunits. Evidence supports an important role for PP2A in cellular transformation and maintaining the “stem-ness” of cancer stem cells: (i) several viral oncoproteins can function as PP2A regulatory subunits to induce transformation, (ii) PP2A regulates several oncogenic signaling cascades including AKT, ERK1/2 and Wnt/β-catenin , (iii) PP2A regulates the tumor suppressing signaling pathway ARF/MDM2/p53, which plays a key role in blocking transformation and tumorigenesis of many cancer types, inhibition of PP2A blocks the DNA damage defense mechanisms in the cell that is being targeted by the chemotherapeutic agent and induces phosphorylation of the apoptosis inducing protein Apoptin. Apoptin is a protein derived from an avian virus which, when phosphorylated, can induce apoptosis specifically in transformed human cells,(iiii) PP2A inhibition with LB100 enhances cisplatin cytotoxicity and overcomes cisplatin resistance in many cancer cells through suppression of p53 pathway mediated DNA damage response and mitotic catastrophe. So, we presume that PP2A has regulation potential on lung squamous-celled carcinoma CSCs. . In this study，we will aim to identify an important mechanism for the maintenance of stemness in multiple distinct phenotypic subpopulations of lung squamous-celled carcinoma CSCs. Using CD166, CD133, CD44 and ALDH as surface markers, we will isolate CSCs from primary tumors and cancer cell lines. Firstly, the expression and activity of PP2A and genes involved in stem cell pathways [i.e.,Sonic hedgehog (SHH,Gli-1), Notch (Notch-1, Notch-2, Hes-1), OCT4/3, and Nanog] will be invstigated by real-time PCR or FACS. Secondly, to investigate the impact of PP2A knockin/knockdown and loss of function on self-renewing, tumorigenic potential, and chemoresistant properties of CSCs. Thirdly, to gain a deeper understanding of the molecular basis for tumorigenic capacity of PP2A, we will perform genome-wide trascriptome analysis on PP2A-inactivated CSCs, the highest-ranking genes will be validated by RT-PCR or western-blot. We also examined the relationship of PP2A activity between chemo-sensitization, tumorigenicity, apoptosis, cell cycle changes and metastatic potential of lung squamous-celled carcinoma, in order to evaluate the effect of PP2A inhibitors on the self-renewing and tumorigenic potential of CSCs. Our research will suggest that PP2A inhibition may be a new therapeutic strategy aiming at NSCLC CSCs.

蛋白磷酸酶PP2A的促癌作用近来渐显，PP2A失活的肿瘤细胞易出现有丝分裂灾难、干性信号阻抑、化疗增敏与耐药逆转，展现了PP2A作为癌干细胞(CSCs)以及以化疗为基石的肺鳞癌之理想治疗靶点的特质。为此，课题组拟从肺鳞癌临床标本/细胞系中分离CSCs，①获得CD166+/133+/44+或ALDH+等不同细胞亚群并行PP2A活性与亚基特征测定，②RT-PCR或流式细胞仪检测PP2A活性改变后CSCs常见干性基因的表达变化,③观察PP2A候选调节基因敲入/敲减及其失活对CSCs自我更新、化疗抵抗和成瘤性的影响,④芯片技术筛选PP2A调控的关键信号级联,并用WB、RT-PCR和基因调控等技术进行验证,⑤借助新辅助化疗手术标本与移植瘤模型探索PP2A活性与化疗增敏、干细胞比例、有丝分裂异常、肿瘤组织药物浓度变化等的关联。旨在明确PP2A靶向抑制对鳞癌CSCs干性逆转与化疗增敏的促进作用与机制。

蛋白磷酸酶PP2A的促癌作用近来渐显，PP2A失活的肿瘤细胞易出现有丝分裂灾难、干性信号阻抑、化疗增敏与耐药逆转，展现了PP2A作为癌干细胞（CSCs）以及以化疗为基石的肺鳞癌之理想治疗靶点的特质。课题通过研究PP2A小分子抑制剂LB100对化疗效果的影响，旨在明确PP2A靶向抑制对鳞癌CSCs干性逆转与化疗增敏的促进作用与机制。本课题主要研究成果如下：（1）从肺癌细胞系中分离出CD166+CSCs，提示其为化疗耐药的基础；（2）LB100可通过抑制PP2A活性，增加肺癌细胞对顺铂的敏感性、增加顺铂对肿瘤细胞的生长抑制作用；（3）LB100可提高耐药突变的肺癌细胞对吉非替尼的敏感性；（4）LB100可能通过调控细胞周期阻滞增加肺癌细胞对化疗敏感性，而这一功能是通过对ATM及P53基因的表达调控完成的。本课题成果提示，抑制PP2A活性可能为化疗耐药的肿瘤患者提供新的治疗思路。