Identification of a novel AMPK-PEA15 axis in the anoikis-resistant growth of mammary cells.
Identification of a novel AMPK-PEA15 axis in the anoikis-resistant growth of mammary cells.
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DOI：
10.1186/s13058-014-0420-z
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发表时间：
2014-08-06
期刊：
影响因子：
--
通讯作者：
Rangarajan A
中科院分区：
文献类型：
--
作者：
Hindupur SK;Balaji SA;Saxena M;Pandey S;Sravan GS;Heda N;Kumar MV;Mukherjee G;Dey D;Rangarajan A
在大多数正常上皮细胞中，基质脱落会引发失巢凋亡（一种细胞凋亡形式），而获得失巢凋亡抗性是实体瘤生长的首要条件。值得注意的是，最近的研究表明，一小群正常的人乳腺上皮细胞（HMEC）在悬浮液中存活，并产生称为“乳腺球”的多细胞球体。因此，了解正常的HMEC如何克服失巢凋亡可能会为乳腺癌的发生和进展提供见解。原代乳腺组织来源的正常HMEC生长为粘附单层或乳腺球。AMP活化蛋白激酶（AMPK）和PEA 15信号的状态进行了研究，通过免疫印迹。药理学试剂和RNA干扰（RNAi）方法被用来衡量它们在乳腺球形成中的作用。进行免疫沉淀和体外激酶测定以评估AMPK和PEA 15之间的相互作用。进行体外球体形成和肿瘤异种移植试验以了解它们在致瘤性中的作用。在这项研究中，我们表明，乳腺球形成的正常HMEC是伴随着AMPK活性的增加。AMPK的抑制或敲低损害乳腺球形成。伴随着AMPK激活，我们检测到PEA 15的Ser 116磷酸化增加，这促进了其抗凋亡功能。AMPK的抑制或敲低损害PEA 15 Ser 116磷酸化并增加细胞凋亡。PEA 15的敲除或PEA 15的非磷酸化S116 A突变体的过表达也废除了乳腺球的形成。我们进一步证明AMPK直接与PEA 15的Ser 116残基相互作用并磷酸化PEA 15，从而确定PEA 15为新的AMPK底物。总之，这些数据表明，AMPK活化通过抑制细胞凋亡促进乳腺球形成，至少部分是通过PEA 15的Ser 116磷酸化。由于失巢凋亡抵抗在实体瘤生长中起着关键作用，我们研究了这些发现在乳腺癌中的相关性。值得注意的是，我们表明AMPK-PEA 15轴在体外和体内乳腺癌细胞的锚定非依赖性生长中发挥着重要作用。我们的研究确定了一种新的AMPK-PEA 15信号轴在正常和癌性乳腺上皮细胞的锚定非依赖性生长，这表明乳腺癌细胞可能采用正常HMEC亚群中固有的失巢凋亡抗性机制。因此，靶向AMPK-PEA 15轴可能会阻止乳腺癌的扩散和转移。本文的在线版本（doi：10.1186/s13058-014-0420-z）包含补充材料，可供授权用户使用。
Matrix detachment triggers anoikis, a form of apoptosis, in most normal epithelial cells, while acquisition of anoikis resistance is a prime requisite for solid tumor growth. Of note, recent studies have revealed that a small population of normal human mammary epithelial cells (HMECs) survive in suspension and generate multicellular spheroids termed ‘mammospheres’. Therefore, understanding how normal HMECs overcome anoikis may provide insights into breast cancer initiation and progression. Primary breast tissue-derived normal HMECs were grown as adherent monolayers or mammospheres. The status of AMP-activated protein kinase (AMPK) and PEA15 signaling was investigated by immunoblotting. Pharmacological agents and an RNA interference (RNAi) approach were employed to gauge their roles in mammosphere formation. Immunoprecipitation and in vitro kinase assays were undertaken to evaluate interactions between AMPK and PEA15. In vitro sphere formation and tumor xenograft assays were performed to understand their roles in tumorigenicity. In this study, we show that mammosphere formation by normal HMECs is accompanied with an increase in AMPK activity. Inhibition or knockdown of AMPK impaired mammosphere formation. Concomitant with AMPK activation, we detected increased Ser116 phosphorylation of PEA15, which promotes its anti-apoptotic functions. Inhibition or knockdown of AMPK impaired PEA15 Ser116 phosphorylation and increased apoptosis. Knockdown of PEA15, or overexpression of the nonphosphorylatable S116A mutant of PEA15, also abrogated mammosphere formation. We further demonstrate that AMPK directly interacts with and phosphorylates PEA15 at Ser116 residue, thus identifying PEA15 as a novel AMPK substrate. Together, these data revealed that AMPK activation facilitates mammosphere formation by inhibition of apoptosis, at least in part, through Ser116 phosphorylation of PEA15. Since anoikis resistance plays a critical role in solid tumor growth, we investigated the relevance of these findings in the context of breast cancer. Significantly, we show that the AMPK-PEA15 axis plays an important role in the anchorage-independent growth of breast cancer cells both in vitro and in vivo. Our study identifies a novel AMPK-PEA15 signaling axis in the anchorage-independent growth of both normal and cancerous mammary epithelial cells, suggesting that breast cancer cells may employ mechanisms of anoikis resistance already inherent within a subset of normal HMECs. Thus, targeting the AMPK-PEA15 axis might prevent breast cancer dissemination and metastasis. The online version of this article (doi:10.1186/s13058-014-0420-z) contains supplementary material, which is available to authorized users.
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影响因子：
11.8
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