腺样囊性癌驱动基因调控NDRG2通路介导氧化应激的化疗耐药机制研究

Resistance to chemotherapy is a critical character of adenoid cystic carcinoma (ACC) . The elevating of anti-oxidative stress ability in malignant tumors is an important factor leading to chemo-resistance. Our pre-experiment data showed that tumor-suppressor NDRG2 was inactivated in ACC. Overexpressing NDRG2 in ACC cell lines could significantly increase the chemotherapy sensitivity and the downstream classical NRF2 anti-oxidative pathway was inhibited. High-throughput sequencing and bioinformatics analysis of miRNAs suggested that NDRG2 was a potential target for multiple co-overexpressed oncomiRs (miRNAs act as oncogene) in ACC. Also the promoter regions of these miRNAs have the transcription binding sites of ACC driver gene MYB. We hypothesize that increase of multiple miRNAs modulated by MYB can cooperatively suppress NDRG2 and activate the NRF2 anti-oxidative pathway, resulting in the insensitivity to chemotherapy of ACC. We propose to conduct mass spectrometry, immune coprecipitation, dual luciferase reporter assay, etc. to further explore the cooperative effects of miRNAs upregulated by MYB, the inhibiting on NDRG2 and the downstream mechanisms of mediating oxidative stress through using of human tumor tissues, cells, patient-derived xenografts and transgenic mice. This study is the first to explore the molecular mechanism of chemo-resistance from the perspective of oxidative stress mediated by ACC driver gene, and will provide new ideas for screening the therapeutic target and improving the chemo-sensitivity of ACC.

腺样囊性癌（adenoid cystic carcinoma,ACC）普遍对化疗耐药。抗氧化应激能力增强是导致肿瘤耐药重要因素。我们前期发现N-myc下调基因（N-myc downregulated gene,NDRG2）在ACC中失活，细胞中过表达NDRG2可提高化疗敏感性，下游抗氧化应激NRF2经典通路抑制；miRNA高通量测序及分析提示NDRG2是ACC中多个上调miRNAs的潜在靶基因，且miRNAs存在ACC驱动基因MYB转录结合位点。我们提出假说：MYB通过上调miRNAs协同失活NDRG2而激活NRF2抗氧化应激经典通路，致ACC化疗不敏感。我们拟利用人体肿瘤、细胞、人源化移植瘤及转基因鼠等，通过质谱、免疫沉淀、荧光素酶等分别从NDRG2上、下游着手研究其介导的氧化应激相关机制。研究首次从ACC驱动基因介导NDRG2调控氧化应激探讨化疗耐药，对提高化疗敏感性具有重要科学意义。

唾液腺腺样囊性癌（salivary adenoid cystic carcinoma, SACC）是唾液腺最常见的恶性肿瘤之一，它的主要特点是侵袭性强，易沿神经、血管生长，具有较高的复发率及远处转移率，尤其易转移至肺、骨。目前的治疗手段为手术及放疗，无有效的化疗方案，患者远期生存率很低。因此，更多的研究应致力于挖掘提高治疗效果及改善患者预后的分子生物靶点。越来越多的证据表明，N-myc下游调控基因2 (N-myc downstream regulated gene 2, NDRG2)的表达失调参与了许多肿瘤生物学过程。然而，在唾液腺腺样囊性癌中，NDRG2对其生物学行为的影响及miRNA介导的NDRG2表达失调机制尚不清楚。在本课题中，我们明确了 SACC关键驱动基因MYB可结合miR-130a启动子区诱导miR-130a表达，并介导NDRG2下调，进一步激活下游AKT和Stat3信号通路，促进SACC发生、增殖和侵袭转移。同时，我们根据前期肿瘤及患者源性异种移植模型PDX外显子测序结果，对同时发生PIK3CA扩增/突变（突变位点R88Q）合并MYB-NFIB融合基因的PDX进行药效评估实验，PIK3CA抑制剂Alpelisib联合RA应用后，肿瘤的生长抑制作用明显增强。此外，不同组织学亚型中，实体型增殖活性及侵袭性最高，预后最差。因此，我们还重点研究了实体型腺样囊性癌的生物学及遗传学特征。实体型ACC s中，NOTCH信号通路和染色质重塑信号通路)基因富集。与筛状/管状成分相比，NOTCH1、MRELLA、MLL2、PPP2R2A 和FGFR2突变以及NOTCH2扩增仅见于实体成分，表明在同一肿瘤可进行克隆选择。NOTCH1突变的病例NICD表达强阳性，且与肿瘤较差的总体和远处无转移生存期有关。 在具有NOTCH1激活突变PDXs中我们评估了γ -分泌酶抑制剂BM S-906024的临床前效率，其显示了强大的抗肿瘤作用。综上所述，本课题利用ACC人体肿瘤、细胞及具有异质性的小队列PDX，分别从NDRG2上、下游着手研究了ACC发生、增殖及侵袭的生物学机制；同时，我们实现了ACC从组织学分型到分子分型的演变，并进行基于分子分型的靶向药物研究，为ACC的精准治疗奠定临床前研究基础。