Abstract
IntroductionMatrix 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.MethodsPrimary 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.ResultsIn 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0420-z) contains supplementary material, which is available to authorized users.
Highlights
IntroductionA form of apoptosis, in most normal epithelial cells, while acquisition of anoikis resistance is a prime requisite for solid tumor growth
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
Mammosphere formation requires AMP-activated protein kinase (AMPK) activity In order to understand the signaling mechanisms that facilitate MS formation by normal human mammary epithelial cells (HMECs), we first examined the status of the AMPK pathway, which has recently been implicated in anoikis resistance [15,16], between adherent HMECs and floating MS
Summary
A form of apoptosis, in most normal epithelial cells, while acquisition of anoikis resistance is a prime requisite for solid tumor growth. Recent studies have revealed that a small population of normal human mammary epithelial cells (HMECs) survive in suspension and generate multicellular spheroids termed ‘mammospheres’. Recent studies have demonstrated that a small population of normal human mammary epithelial cells (HMECs) survive under anchorage-independent conditions to generate floating spheroids termed ‘mammospheres’ (MS) when cultured in ultra-low (UL) attachment plates under serum-free conditions [7,8]. Understanding the molecular mechanisms that enable a subset of normal HMECs to withstand anoikis and generate MS is likely to provide important insights into normal mammary gland biology, as well as breast cancer progression
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