title: Identification of a novel AMPK-PEA15 axis in the anoikis-resistant growth of mammary cells.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.forename: Sravanth K
authors.initials: SK
authors.lastname: Hindupur
authors.forename: Sai A
authors.initials: SA
authors.lastname: Balaji
authors.forename: Meera
authors.initials: M
authors.lastname: Saxena
authors.forename: Shubham
authors.initials: S
authors.lastname: Pandey
authors.forename: Gopalkrishnashetty Sreenivasmurthy
authors.initials: GS
authors.lastname: Sravan
authors.forename: Namrata
authors.initials: N
authors.lastname: Heda
authors.forename: M Vijaya
authors.initials: MV
authors.lastname: Kumar
authors.forename: Geetashree
authors.initials: G
authors.lastname: Mukherjee
authors.forename: Devaveena
authors.initials: D
authors.lastname: Dey
authors.forename: Annapoorni
authors.initials: A
authors.lastname: Rangarajan
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D023102:Anoikis; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000378:metabolism; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000378:metabolism*; D005260:Female; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D042361:Mammary Glands, Human / Q000166:cytology* / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D018874:Spheroids, Cellular
chemical_list: D051017:Apoptosis Regulatory Proteins; D047908:Intracellular Signaling Peptides and Proteins; C092039:PEA15 protein, human; D010750:Phosphoproteins; D055372:AMP-Activated Protein Kinases
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