基于LAMB1调控细胞外基质重塑诠释唾液腺腺样囊性癌转移分子机制研究

High metastasis potential is a main cause for the unfavorable prognosis in patients with salivary adenoid cystic carcinoma (ACC). Extracellular matrix (ECM) remodeling plays a key role in tumor metastasis. However, whether ECM remodeling correlates with metastasis in ACC and the molecular mechanism remains unknown. Our preliminary results showed that ECM related signaling pathways was enriched in ACC samples with metastasis compared with non-metastatic sample using RNA sequencing. Among those signaling pathways, we found that LAMB1 gene had showed a significant higher expression level in ACC samples with distant metastasis. We also detected LAMB1 gene expression in 18 ACC samples with distant metastasis and 15 non-metastatic samples via Q-PCR, the results also showed that LAMB1 gene had a significant higher expression level in samples with distant metastasis. As LAMB1 encode a subunit of laminin, a major component of basement membrane, we want to know whether LAMB1 can regulate ECM remodeling and tumor metastasis of ACC and clarify the underlying molecular mechanism. We will determine the relationship between LAMB1 expression and ECM remodeling, ACC metastasis, clinicopathological parameters and patient's prognosis in clinical samples. Then we will detect whether LAMB1 can regulate ACC metastasis through generating ECM component, changing ECM structure with or without interfering in vitro and in vivo. At last, we will investigate whether LAMB1 regulated ECM modeling and ACC metastasis via binding to ITGA9 to modulate downstream signaling pathways, including FAK, PI3K and also TGF-β. The study will focus on how LAMB1 modulates extracellular matrix remodeling to regulate ACC metastasis. The results will provide a theoretical basis for identifying biomarkers for ACC metastasis, inhibiting metastasis and screening new target for ACC treatment.

唾液腺腺样囊性癌（ACC）高转移性是导致患者预后不佳的重要原因。细胞外基质（ECM）重塑在肿瘤转移过程中发挥了关键作用，但其与ACC转移的关系及分子机制尚不明确。我们前期通过RNA测序发现发生转移和未转移ACC样本中与ECM相关信号通路富集，差异表达最显著的基因是LAMB1，在临床样本中通过Q-PCR验证了这一结果。为了明确LAMB1调控ECM重塑促进ACC转移的分子机制，本课题首先从临床样本分析LAMB1表达与ECM重塑、肿瘤转移、患者预后的关系，然后进行体外、体内实验阐明LAMB1是否通过改变ECM成分及结构从而调控ACC转移，最后探讨LAMB1是否与ITGA9结合并激活下游信号通路而导致ACC中ECM重塑和肿瘤转移。本研究将从LAMB1调控ECM重塑的角度诠释ACC肿瘤转移的分子机制，为寻找预测ACC转移的分子标志物、抑制ACC转移并筛选ACC治疗新靶点提供理论基础。

唾液腺腺样囊性癌（adenoid cystic carcinoma，ACC）是最常见的唾液腺恶性肿瘤之一，其特征是易发生血行转移。本项目首先在208例ACC样本中检测了LAMB1蛋白表达情况，发现LAMB1高表达组以老年、组织学分级III级以及晚期患者居多（p值分别为0.001、0.006和<0.001），LAMB1高表达组存在总生存率较差的趋势。进一步通过体外、体内实验分别验证ACC细胞系中沉默LAMB1后对肿瘤细胞生物学行为的影响。发现与对照组相比，LAMB1敲低组裸鼠皮下成瘤肿瘤体积小（p<0.01）、重量轻（p<0.05），提示LAMB1敲低可能影响ACC增殖与生长。在裸鼠尾静脉注射两组细胞后，LAMB1敲低组肺转移结节比对照组少。LAMB1与其受体ITGA9表达存在相关性，这二者在ACC发生和发展中的作用还需进一步深入研究。分析了少见的儿童和青少年ACC的临床病理特征，组织学分型上这部分病例以筛状型（66.7%）为主，大部分病例至少表达一个与细胞外基质相关的蛋白（LAMB1、type IV Collagen和Laminin），且83.3%的病例存在丰富的细胞外基质成分。37.5%的儿童和青少年ACC存在MYB基因重排。两例患者分别在225个月和261个月后出现肿瘤复发，提示针对这部分患者应长期随访。本项目探讨了β-catenin和Ki-67在鉴别ACC和基底细胞腺癌（basal cell adenocarcinoma，BCAC）中的作用。ACC患者β-catenin均表达于细胞膜和细胞质，而90.6%的BCAC患者可见肿瘤细胞核β-catenin表达（p<0.01）。ACC中Ki-67指数高于BCAC（p<0.01）。β-catenin在细胞内不同的表达定位和Ki-67指数可以作为鉴别唾液腺ACC和BCAC的指标。本项目采用构建人源肿瘤细胞系（Patient Derived Cell line，PDC）的方法对具有MYB基因重排的ACC细胞进行原代培养，细胞可稳定传代。通过本研究，我们初步探讨了LAMB1在ACC发生和发展中的作用，分析了一类少见的儿童和青少年ACC的临床病理特征和细胞外基质蛋白表达情况，提出了可用于鉴别ACC和BCAC的指标，并构建了ACC原代细胞系，为ACC发生、发展、鉴别诊断及后续深入地进行体外研究提供了实验理论依据。