title: FAK activity protects nucleostemin in facilitating breast cancer spheroid and tumor growth.
abstract: BACKGROUND
  Focal adhesion kinase (FAK) controls cell growth and survival downstream of integrin-matrix receptors. Upon adhesion loss or FAK inhibition, FAK can translocate to the nucleus. The nucleolus is a non-membrane nuclear structure that regulates ribosome biogenesis and cell proliferation. Nucleostemin (NS), a nucleolar-localized protein, modulates cell cycle progression, stemness, and three-dimensional tumor spheroid formation. The signaling pathways that regulate NS levels in tumors remain undefined.
  
  
  METHODS
  Human breast carcinoma cells were evaluated for growth in culture (adherent and anchorage-independent spheroid) and as orthotopic tumors. FAK signaling was evaluated by pharmacological FAK inhibitor addition (PF-271, IC50~0.1 μM) and by small hairpin RNA (shRNA) knockdown followed by re-expression of FAK wildtype (WT) or a kinase-dead (KD, K454R) FAK point mutant. Immunoblotting was used to evaluate FAK, NS, nucleolar phosphoprotein B23, and nucleolin levels. Total and phosphospecific antibody imunoblotting were used to detect changes in FAK, Akt kinase (Akt also known as protein kinase B), and 4E-binding protein 1 (4E-BP1) phosphorylation, a translation repressor protein and target of the mammalian target of rapamycin (mTOR) complex. Immunohistochemical, co-immunoprecipitation, and cellular fractionation analyses were used to evaluate FAK association with nucleoli.
  
  
  RESULTS
  Pharmacological (0.1 μM PF-271) or genetic inhibition of FAK activity prevents MDA-MB-231 and 4T1L breast carcinoma growth as spheroids and as orthotopic tumors. FAK inhibition triggers proteasome-mediated decreased NS levels but no changes in other nucleolar proteins such as B23 (nucleophosmin) or nucleolin. Active FAK was associated with purified nucleoli of anchorage-independent cells and present within nucleoli of human invasive ductal carcinoma tumor samples. FAK co-immunoprecipitated with B23 that binds NS and a complex between FAK, NS, Akt, and mTOR was detected. Constitutively-active Akt kinase promoted tumor spheroid growth, stabilized NS levels, and promoted pS65 4E-BP1 phosphorylation in the presence of inhibited FAK. Rapamycin lowered NS levels and inhibited pS65 4E-BP1 phosphorylation in cells with activated Akt-mTOR signaling.
  
  
  CONCLUSIONS
  FAK signaling occurs in the nucleolus, active FAK protects NS, and Akt-mTOR pathway regulates NS protein stability needed for breast carcinoma spheroid and tumor growth.
authors.affiliation: Department of Reproductive Medicine, University of California San Diego, Moores Cancer Center, 3855 Health Sciences Dr, La Jolla, CA, 92093, USA. itancioni@ucsd.edu.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Tancioni
authors.affiliation: Department of Reproductive Medicine, University of California San Diego, Moores Cancer Center, 3855 Health Sciences Dr, La Jolla, CA, 92093, USA. nichol.miller@pfizer.com.|Current address: Pfizer, La Jolla, CA, 92121, USA. nichol.miller@pfizer.com.
authors.forename: Nichol L G
authors.initials: NL
authors.lastname: Miller
authors.affiliation: Department of Reproductive Medicine, University of California San Diego, Moores Cancer Center, 3855 Health Sciences Dr, La Jolla, CA, 92093, USA. suryu@ucsd.edu.
authors.forename: Sean
authors.initials: S
authors.lastname: Uryu
authors.affiliation: Department of Reproductive Medicine, University of California San Diego, Moores Cancer Center, 3855 Health Sciences Dr, La Jolla, CA, 92093, USA. clawson@ucsd.edu.
authors.forename: Christine
authors.initials: C
authors.lastname: Lawson
authors.affiliation: Department of Reproductive Medicine, University of California San Diego, Moores Cancer Center, 3855 Health Sciences Dr, La Jolla, CA, 92093, USA. cjean@inserm.fr.|Current address: INSERM U1037 - Cancer Research Center, Toulouse, France. cjean@inserm.fr.
authors.forename: Christine
authors.initials: C
authors.lastname: Jean
authors.affiliation: Department of Reproductive Medicine, University of California San Diego, Moores Cancer Center, 3855 Health Sciences Dr, La Jolla, CA, 92093, USA. xlc001@ucsd.edu.
authors.forename: Xiao Lei
authors.initials: XL
authors.lastname: Chen
authors.affiliation: Department of Reproductive Medicine, University of California San Diego, Moores Cancer Center, 3855 Health Sciences Dr, La Jolla, CA, 92093, USA. ekleinsc@ucsd.edu.
authors.forename: Elizabeth G
authors.initials: EG
authors.lastname: Kleinschmidt
authors.affiliation: Department of Reproductive Medicine, University of California San Diego, Moores Cancer Center, 3855 Health Sciences Dr, La Jolla, CA, 92093, USA. dschlaepfer@ucsd.edu.
authors.forename: David D
authors.initials: DD
authors.lastname: Schlaepfer
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D002453:Cell Cycle / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004195:Disease Models, Animal; D004789:Enzyme Activation; D005260:Female; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D000090243:Nucleophosmin; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D020123:Sirolimus / Q000494:pharmacology; D018874:Spheroids, Cellular; D047368:Tumor Burden / Q000187:drug effects / Q000235:genetics; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay
chemical_list: C469949:GNL3 protein, human; C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000090243:Nucleophosmin; D051416:Focal Adhesion Protein-Tyrosine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019204:GTP-Binding Proteins; D020123:Sirolimus