Wang Lan, Tian Han, Yuan Jie, Wu Hongmei, Wu Jueheng, Zhu Xun
From the Department of Pathogen Biology and Immunology, School of Basic Courses, Guangdong Pharmaceutical University, Guangzhou, China (LW, HW); Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China (HT, XZ); and Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Ministry of Education, Guangzhou, Guangdong, China (HT, JY, JW, XZ).
Medicine (Baltimore). 2015 Dec;94(49):e2228. doi: 10.1097/MD.0000000000002228.
Breast cancer stem cells (BCSCs) are considered to be responsible for recurrence in breast cancer. The 68 kDa Src-associated protein in mitosis (Sam68) has been linked to the development and progression of breast cancer; however, the posttranscriptional regulation and role of Sam68 in BCSC self-renewal remain unclear.Sam68 was ectopically overexpressed or knocked down using a siRNA; the self-renewal potential of breast cancer cell lines was assessed using flow cytometry, in vitro mammosphere culture and a xenograft model in NOD/SCID mice. Activation of beta-catenin was assessed by immunohistochemical staining, Western blotting, and luciferase reporter gene assays. The ArrayExpress dataset GSE45666 was used to identify conserved microRNAs downregulated in breast cancer; real-time PCR, Western blotting, luciferase reporter assay, and xenografted tumor model were used to confirm miR-204 regulated Sam68.We found that endogenous Sam68 expression correlated positively with the self-renewal potential of breast cancer cell lines. Overexpression of Sam68 promoted, whereas knockdown reduced, breast cancer cell self-renewal potential in vitro and tumorigenicity in vivo. The Wnt/beta-catenin pathway was identified as a functional mediator of Sam68-induced self-renewal in SKBR-3 and MCF-7 cells. Furthermore, miR-204 was found to be frequently downregulated in human breast cancer and confirmed to directly target Sam68; miR-204 inhibited the self-renewal of breast cancer cell lines by targeting and suppressing Sam68.Our study reveals that Sam68 is regulated by miR-204 and may play an important role in the self-renewal of BCSCs via activating the Wnt/beta-catenin pathway. Sam68 may represent a novel therapeutic target for breast cancer.
乳腺癌干细胞（BCSCs）被认为是乳腺癌复发的原因。有丝分裂中68 kDa的Src相关蛋白（Sam68）与乳腺癌的发生和发展有关；然而，Sam68在BCSC自我更新中的转录后调控及作用仍不清楚。使用siRNA异位过表达或敲低Sam68；通过流式细胞术、体外乳腺球培养和NOD/SCID小鼠异种移植模型评估乳腺癌细胞系的自我更新潜能。通过免疫组织化学染色、蛋白质印迹法和荧光素酶报告基因检测评估β-连环蛋白的激活情况。使用ArrayExpress数据集GSE45666来鉴定在乳腺癌中下调的保守微小RNA；采用实时PCR、蛋白质印迹法、荧光素酶报告基因检测和异种移植肿瘤模型来证实miR-204对Sam68的调控。我们发现内源性Sam68表达与乳腺癌细胞系的自我更新潜能呈正相关。Sam68的过表达促进了乳腺癌细胞的自我更新潜能，而敲低则降低了其体外自我更新潜能和体内致瘤性。Wnt/β-连环蛋白通路被确定为Sam68诱导的SKBR-3和MCF-7细胞自我更新的功能介质。此外，发现miR-204在人类乳腺癌中经常下调，并证实其直接靶向Sam68；miR-204通过靶向和抑制Sam68来抑制乳腺癌细胞系的自我更新。我们的研究表明，Sam68受miR-204调控，并可能通过激活Wnt/β-连环蛋白通路在BCSCs的自我更新中发挥重要作用。Sam68可能是乳腺癌的一个新的治疗靶点。
