Identification of a novel AMPK-PEA15 axis in the anoikis-resistant growth of mammary cells

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.

The protein phosphatase 4 - PEA15 axis regulates the survival of breast cancer cells

“…In agreement with a previous study [36], down-regulation of PEA15 resulted in significant decreases in the number of viable cells (Fig. 7B) and cell viability (Fig.…”

“…PEA15 is a multi-functional protein that has been implicated in the regulation of major intracellular processes including proliferation and apoptosis, and its function is tightly regulated by its phosphorylation at two serine residues, Ser104 and Ser116 [27]. Both CaMKII and AKT phosphorylate PEA15 at Ser116 [47][48][49] and, more recently, AMPK was reported to act as an upstream kinase of PEA15 in both normal and cancerous breast epithelial cells [36]. Phosphatases play an equally important role as kinases in regulating the phosphorylation state of PEA15.…”

“…The PEA15 gene is amplified in breast cancer, as well as in other cancers [33], and the unphosphorylated form of PEA15 is more potent than the phosphorylated form in suppressing tumorigenicity in breast cancer [34]. While several kinases have been reported to be involved in the phosphorylation of PEA15 including Akt, Ca2+/calmodulin-dependent protein kinase (CaMKII) and AMP-activated protein kinase (AMPK) [35,36], the dephosphorylation of PEA15 is much less understood. between cell survival and cell death in breast cancer cells is mediated, at least partly, through the dephosphorylation of PEA15.…”

Marine sponge‐derived smenospongine preferentially eliminates breast cancer stem‐like cells via p38/AMPKα pathways

“…Mei et al42 further showed that p38 MAPK activation contributes to an increase in CSC population and metastasis. It has been reported that radioresistant glioblastoma stem cells acquired enhanced AMPK activity and in normal human mammary epithelial cells AMPK mediated the mammosphere formation, indicating that AMPK might involve in regulating the self‐renewal and therapeutic resistance of CSCs 38, 39. Our study showed that Sme induced apoptosis via activating the p38 MAPK and AMPKα pathways in MCF7‐Nanog cells.…”

“…There is growing evidence supporting that p38 MAPK and AMPK play crucial roles in cancer cell growth, proliferation, survival 38, 39, 40. Meng et al41 reported that p38 MAPK exhibits overexpression in highly metastatic human and mouse breast cancer cell lines and promotes basal‐like and metastatic properties in breast tumor samples.…”