金属蛋白酶-解整合素ADAM12积极促进claudin低表达型乳腺癌中的干细胞样表型。
Metalloprotease-disintegrin ADAM12 actively promotes the stem cell-like phenotype in claudin-low breast cancer.
作者信息
Duhachek-Muggy Sara, Qi Yue, Wise Randi, Alyahya Linda, Li Hui, Hodge Jacob, Zolkiewska Anna
机构信息
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS, 66506, USA.
Current address: Department of Radiation Oncology, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA.
出版信息
Mol Cancer. 2017 Feb 1;16(1):32. doi: 10.1186/s12943-017-0599-6.
BACKGROUND
ADAM12 is upregulated in human breast cancers and is a predictor of chemoresistance in estrogen receptor-negative tumors. ADAM12 is induced during epithelial-to-mesenchymal transition, a feature associated with claudin-low breast tumors, which are enriched in cancer stem cell (CSC) markers. It is currently unknown whether ADAM12 plays an active role in promoting the CSC phenotype in breast cancer cells.
METHODS
ADAM12 expression was downregulated in representative claudin-low breast cancer cell lines, SUM159PT and Hs578T, using siRNA transfection or inducible shRNA expression. Cell characteristics commonly associated with the CSC phenotype in vitro (cell migration, invasion, anoikis resistance, mammosphere formation, ALDH activity, and expression of the CD44 and CD24 cell surface markers) and in vivo (tumor formation in mice using limiting dilution transplantation assays) were evaluated. RNA sequencing was performed to identify global gene expression changes after ADAM12 knockdown.
RESULTS
We found that sorted SUM159PT cell populations with high ADAM12 levels had elevated expression of CSC markers and an increased ability to form mammospheres. ADAM12 knockdown reduced cell migration and invasion, decreased anoikis resistance, and compromised mammosphere formation. ADAM12 knockdown also diminished ALDEFLUOR and CD44/CD24 CSC-enriched populations in vitro and reduced tumorigenesis in mice in vivo. RNA sequencing identified a significant overlap between ADAM12- and Epidermal Growth Factor Receptor (EGFR)-regulated genes. Consequently, ADAM12 knockdown lowered the basal activation level of EGFR, and this effect was abolished by batimastat, a metalloproteinase inhibitor. Furthermore, incubation of cells with exogenously added EGF prevented the downregulation of CD44/CD24 cell population by ADAM12 knockdown.
CONCLUSIONS
These results indicate that ADAM12 actively supports the CSC phenotype in claudin-low breast cancer cells via modulation of the EGFR pathway.
背景
ADAM12在人类乳腺癌中表达上调，是雌激素受体阴性肿瘤化疗耐药的一个预测指标。ADAM12在上皮-间质转化过程中被诱导，上皮-间质转化是一种与claudin低表达乳腺癌肿瘤相关的特征，这类肿瘤富含癌症干细胞（CSC）标志物。目前尚不清楚ADAM12在促进乳腺癌细胞的CSC表型方面是否发挥积极作用。
方法
使用小干扰RNA（siRNA）转染或诱导型短发夹RNA（shRNA）表达，在代表性的claudin低表达乳腺癌细胞系SUM159PT和Hs578T中下调ADAM12的表达。评估了体外（细胞迁移、侵袭、失巢凋亡抗性、乳腺球形成、醛脱氢酶（ALDH）活性以及CD44和CD24细胞表面标志物的表达）和体内（使用极限稀释移植试验在小鼠体内形成肿瘤）与CSC表型通常相关的细胞特征。进行RNA测序以鉴定ADAM12敲低后整体基因表达的变化。
结果
我们发现，ADAM12水平高的分选SUM159PT细胞群体中CSC标志物的表达升高，形成乳腺球的能力增强。ADAM12敲低减少了细胞迁移和侵袭，降低了失巢凋亡抗性，并损害了乳腺球形成。ADAM12敲低还减少了体外ALDEFLUOR和富含CD44/CD24的CSC群体，并降低了体内小鼠的肿瘤发生。RNA测序确定了ADAM12和表皮生长因子受体（EGFR）调节基因之间存在显著重叠。因此，ADAM12敲低降低了EGFR的基础激活水平，而金属蛋白酶抑制剂batimastat可消除这种作用。此外，用外源性添加的表皮生长因子（EGF）孵育细胞可防止ADAM12敲低导致的CD44/CD24细胞群体下调。
结论
这些结果表明，ADAM12通过调节EGFR途径积极支持claudin低表达乳腺癌细胞的CSC表型。
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