title: Nuclear localized Akt enhances breast cancer stem-like cells through counter-regulation of p21(Waf1/Cip1) and p27(kip1).
abstract: Cancer stem-like cells (CSCs) are a rare subpopulation of cancer cells capable of propagating the disease and causing cancer recurrence. In this study, we found that the cellular localization of PKB/Akt kinase affects the maintenance of CSCs. When Akt tagged with nuclear localization signal (Akt-NLS) was overexpressed in SKBR3 and MDA-MB468 cells, these cells showed a 10-15% increase in the number of cells with CSCs enhanced ALDH activity and demonstrated a CD44(+High)/CD24(-Low) phenotype. This effect was completely reversed in the presence of Akt-specific inhibitor, triciribine. Furthermore, cells overexpressing Akt or Akt-NLS were less likely to be in G0/G1 phase of the cell cycle by inactivating p21(Waf1/Cip1) and exhibited increased clonogenicity and proliferation as assayed by colony-forming assay (mammosphere formation). Thus, our data emphasize the importance the intracellular localization of Akt has on stemness in human breast cancer cells. It also indicates a new robust way for improving the enrichment and culture of CSCs for experimental purposes. Hence, it allows for the development of simpler protocols to study stemness, clonogenic potency, and screening of new chemotherapeutic agents that preferentially target cancer stem cells.
  
  
  CONCLUSIONS
  The presented data, (i) shows new, stemness-promoting role of nuclear Akt/PKB kinase, (ii) it underlines the effects of nuclear Akt on cell cycle regulation, and finally (iii) it suggests new ways to study cancer stem-like cells.
authors.affiliation: a Department of Clinical & Experimental Medicine; Division of Cell Biology Integrative Regenerative Med. Center (IGEN); Linköping Univ. ; Linköping , Sweden.
authors.forename: Mayur Vilas
authors.initials: MV
authors.lastname: Jain
authors.forename: Jaganmohan R
authors.initials: JR
authors.lastname: Jangamreddy
authors.forename: Jerzy
authors.initials: J
authors.lastname: Grabarek
authors.forename: Frank
authors.initials: F
authors.lastname: Schweizer
authors.forename: Thomas
authors.initials: T
authors.lastname: Klonisch
authors.forename: Artur
authors.initials: A
authors.lastname: Cieślar-Pobuda
authors.forename: Marek J
authors.initials: MJ
authors.lastname: Łos
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000502:physiology*; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000502:physiology*; D005260:Female; D006801:Humans; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000032:analysis / Q000378:metabolism*
chemical_list: C493956:CDKN1A protein, human; C436291:CDKN1B protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D051057:Proto-Oncogene Proteins c-akt
keywords: 7-AAD, 7-aminoactinomycin D; ALDH, aldehyde dehydrogenase; Akt-NLS; BPE, bovine pituitary epithelial; Bcl2, B cell lymphoma 2; CDK, cyclin-dependent kinase; CSCs, cancer stem-like cells; DEAB, diethylaminobenzaldehyde; FBS, fetal bovine serum; GAPDH, glucose-6-phosphate dehydrogenase; GPCR, G-protein-coupled receptor; GSK3, glycogen synthase kinase-3; IGF1, insulin like growth factor 1; JAK, Janus kinase; NLS, nuclear localization signal; PDK, phosphoinositide dependent kinase; PH, pleckstrin-homology; PI3K; PI3K, phoshatidylinositol-3-kinase; PKB, protein kinase B; PTEN, phosphatase and tensin homolog; PVDF, polyvinylidene fluoride; RIPA, radioimmunoprecipitation; RPMI, Roswell Park Memorial Institute; RT, room temperature; RTK, receptor tyrosine kinase; STAT, signal transducer and activator of transcription; T-ALL, T-cell acute lymphoblastic leukemia; WT, wild type; cancer stem-like cells; hEGF, human epidermal growth factor; mTOR; mTOR, mammalian target of rapamycin; poly-HEMA, poly-2-hydroxyethyl methacrylate; stemness