潜在抑癌基因LAMC3和LAMA2调控细胞自噬参与卵巢癌耐药的机制研究

Although the platinum and paclitaxel-based chemotherapy, which is the current preferred treatment modality in human ovarian cancer, significantly reduces the mortality rates and prolongs the survival time of patients, the main obstacle to a successful treatment for ovarian cancer is the development of drug resistance to combination chemotherapy. Drug resistance results from a variety of factors including individual variations in patients and somatic cell genetic differences in tumors. However, regardless of mechanisms, abnormality of tumor suppressor expression and autophagy activity clearly contributes to drug resistance in ovarian cancer. The potential tumor suppressor LAMC3 and LAMA2 are the laminin genes and involved in the regulation of cancer progression and development, while their studies on ovarian cancer and drug resistance are rare. We revealed that the expression of LAMC3 and LAMA2 was significantly down-regulated in drug-resistant cells and tissues compared with controls in ovarian cancer. Low expression of LAMC3 in lab-collected 51 specimens and in TCGA cohort of 489 tissues was significantly associated with poor disease-free survival and overall survival. Over-expression of LAMC3 in paclitaxel-resistant SKOV3 and HeyA8 cells could greatly increase the sensitivity of the cells to paclitaxel. Furthermore, over-expression of LAMC3 inhibited the expression of autopahgy proteins such as ATG12, and resulted in a decreased ratio of LC3-II/LC3-I. Consistently, the mRNA expression of LAMC3 and LAMA2 was positively correlated in 489 tissues of TCGA cohort; LAMC3 and ATG12 was negatively correlated in 90 cases of drug resistant tissues, and LAMC3/LAMA2 were both negatively correlated with ATG16L2. Thus, in this application, a scientific hypothesis is proposed that LAMC3 and LAMA2 might be contributed to the modulation of drug resistance by autophagy in ovarian cancer. On the basis of studies on cell model, mouse model and clinical specimens, we aim to systematically illustrate the function and molecular mechanism of these two genes on drug resistance in ovarian cancer by autophagy. The results may pave the way for molecular-targeted therapies, reversal of drug resistance and clinical application in ovarian cancer.

对铂类/紫杉醇为主的耐药是卵巢癌化疗失败的主要原因，而抑癌基因和细胞自噬异常是耐药调控的重要机制。同属laminin家族的潜在抑癌基因LAMC3和LAMA2与肿瘤进展相关，但与卵巢癌耐药调控的研究极少见。我们研究发现LAMC3和LAMA2在卵巢癌耐药细胞和组织中低表达，且LAMC3低表达与不良预后显著相关。过表达LAMC3能降低卵巢癌耐药细胞的耐药性，并抑制ATG12等自噬蛋白的表达，降低LC3-II/I比值。基于大数据挖掘，LAMC3与LAMA2的表达正相关，LAMC3与ATG12在耐药组织中的表达负相关，且LAMC3和LAMA2都与ATG16L2负相关。由此我们提出科学假设：LAMC3和LAMA2通过负调控细胞自噬影响卵巢癌耐药。本项目将在细胞、动物、分子和临床水平上系统研究LAMC3和LAMA2调节自噬参与耐药的机制，为卵巢癌分子靶向治疗及耐药逆转提供依据，具有重要的理论和应用价值。

卵巢癌是女性生殖系统中致死率最高的恶性肿瘤，严重危害女性的生命健康。以铂类和紫杉醇为主的化疗是卵巢癌术后治疗的主要手段，但大多数患者会不同程度地对化疗药物产生耐药，最终导致复发和死亡，是卵巢癌治疗失败的主要原因。因此，发现新的耐药调控因子、揭示其分子机制、阐明其临床相关性对于卵巢癌临床治疗具有重要意义。LAMC3和LAMA2同属laminin家族，功能和结构上相关，在肿瘤进程中发挥着重要作用，但与卵巢癌相关研究较少。课题前期基于卵巢癌紫杉醇耐药细胞SKOV3-PTX，发现LAMC3影响卵巢癌耐药。基于以上背景，本课题利用卵巢癌卡铂和紫杉醇耐药细胞HeyA8-CBP（H-C）及HeyA8-R（H-R），结合裸鼠、临床、分子及大数据研究，阐述了LAMC3和LAMA2对卵巢癌耐药的影响及其临床相关性。干扰LAMC3表达能提高H-C和H-R细胞对卡铂和紫杉醇的敏感性，抑制细胞克隆形成，阻滞H-C细胞周期，促进细胞凋亡和死亡率，且显著影响周期和凋亡关键蛋白的变化。LAMC3低表达诱导LC3等自噬通路关键蛋白表达，且自噬抑制剂氯喹（CQ）能逆转LAMC3低表达细胞对卡铂的敏感性。进一步发现LAMC3与自噬调控基因SLC7A11显著负相关，且在耐药细胞H-C和H-R中，LAMC3低表达诱导SLC7A11高表达。过表达SLC7A11提高耐药细胞对卡铂和紫杉醇的敏感性，抑制克隆形成，诱导LC3等自噬蛋白表达。动物模型中，卡铂处理对LAMC3低表达细胞皮下移植瘤增长的抑制效果更明显。临床水平上，和8例正常卵巢组织及27例敏感组织相比，LAMC3在208例卵巢癌组织及24例耐药组织中低表达，且和肿瘤分级及预后显著相关。大数据研究表明LAMA2表达和卵巢癌耐药、肿瘤分级、肿瘤免疫浸润及预后相关，可能通过与Ras信号通路及LGR5互作影响卵巢癌进展，但在H-C细胞中干扰其表达对耐药无显著影响。综上，LAMC3潜在介导细胞自噬影响卵巢癌对化疗药物的耐药性，LAMC3和LAMA2低表达和卵巢癌预后相关，成果丰富了卵巢癌耐药调控理论，并有望作为治疗靶点和预后标志物应用于临床，具有重要的理论和应用价值。