CRISPR/Cas9文库筛选发现的TMEM106A抑制三阴性乳腺癌转移的机制研究

Breast cancer is the most common malignancies among women. The majority of cancer-associated deaths are caused by metastasis. Triple-negative breast cancer (TNBC) is a subtype of breast cancer with high malignancy, high frequency of early metastasis, and no specific treatment. Therefore, it is high time to investigate the mechanisms of metastasis in TNBC and seek for novel targets. We performed functional screening with CRISPR/Cas9 library, confirmed the functions with in vitro and in vivo experiments, and identified TMEM106A as a suppressor of metastasis in TNBC. Further，we found that TMEM106A interacted with WD repeat domain 77 (WDR77). Up-regulation of TMEM106A repressed expression of WDR77, and WDR77 cellular localization could be affected by TMEM106A. However, the underlying mechanism remains unclear. Therefore, we pose the hypothesis that TMEM106A could suppress invasion and metastasis of TNBC via downregulating WDR77 and promoting nuclear localization of WDR77. We plan to investigate function of TMEM106A and mechanisms of suppressing TNBC metastasis via multiple aspects including molecular and cellular experiments, mouse models and paired samples. We aim to provide the foundation of new strategies for treatment of metastatic TNBC.

乳腺癌是全球女性最常见的恶性肿瘤。远处转移是乳腺癌患者死亡的主要原因。三阴性乳腺癌（Triple-Negative Breast Cancer, TNBC）恶性程度高、易早期出现复发转移，且无特异的治疗方法。因而研究TNBC的转移机制、寻找新的治疗靶点是临床亟待解决的难题。本研究采用CRISPR/Cas9文库技术在小鼠体内功能性筛选并进一步验证TMEM106A可以抑制TNBC的侵袭和转移。我们进一步发现TMEM106A与WDR77相互结合，过表达TMEM106A，WDR77表达下降，且TMEM106A可以影响WDR77的细胞定位，但机制尚不明确。因此，我们提出假说：TMEM106A可通过下调WDR77的表达和促进WDR77入核而抑制TNBC的转移。为验证该假说，我们将从分子、细胞、组织及动物模型等多方面阐述TMEM106A的功能及作用机制，为TNBC临床转化性研究提供新的靶点及理论依据。

明确TMEM106A的下游或相互作用蛋白对于探索TMEM106A的抑癌作用有着至关重要的作用。在前期试验中，我们初步筛选出TMEM106A的相互作用蛋白WDR77。本次我们通过一系列实验进一步确认了二者的相互结合作用。首先，我们通过免疫荧光发现TMEM106A与WDR77在乳腺癌细胞中共定位，验证了二者的相互作用。我们已经证实敲低TMEM106A能够促进三阴性乳腺癌细胞的侵袭和转移，接下来，我们通过Werstern-blot、Transwell等试验发现敲低TMEM106A同时下调WDR77，231 sg-TM的促转移作用被逆转。敲低TMEM106A，E-cadherin表达下调，N-cadherin表达上调，同时敲低TMEM106A和WDR77，E-cadherin和N-cadherin表达与前相反。以上结果说明二者相互结合且功能相关。为了明确TMEM106A与WDR77的结合位点，我们分别设计了TMEM106A与WDR77的截短蛋白，联合免疫共沉淀（co-IP）发现WDR77与TMEM106A的C端相结合，TMEM106A可能影响WDR77的NLS2发挥作用。TMEM106A-WDR77复合体通过影响EMT通路蛋白Snail影响EMT的过程，从而发挥抑制肿瘤转移的作用。