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FAK activity protects nucleostemin in facilitating breast cancer spheroid and tumor growth

INTRODUCTION: 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....

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Autores principales: Tancioni, Isabelle, Miller, Nichol LG, Uryu, Sean, Lawson, Christine, Jean, Christine, Chen, Xiao Lei, Kleinschmidt, Elizabeth G, Schlaepfer, David D
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4407832/
https://www.ncbi.nlm.nih.gov/pubmed/25880415
http://dx.doi.org/10.1186/s13058-015-0551-x
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author Tancioni, Isabelle
Miller, Nichol LG
Uryu, Sean
Lawson, Christine
Jean, Christine
Chen, Xiao Lei
Kleinschmidt, Elizabeth G
Schlaepfer, David D
author_facet Tancioni, Isabelle
Miller, Nichol LG
Uryu, Sean
Lawson, Christine
Jean, Christine
Chen, Xiao Lei
Kleinschmidt, Elizabeth G
Schlaepfer, David D
author_sort Tancioni, Isabelle
collection PubMed
description INTRODUCTION: 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13058-015-0551-x) contains supplementary material, which is available to authorized users.
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spelling pubmed-44078322015-04-24 FAK activity protects nucleostemin in facilitating breast cancer spheroid and tumor growth Tancioni, Isabelle Miller, Nichol LG Uryu, Sean Lawson, Christine Jean, Christine Chen, Xiao Lei Kleinschmidt, Elizabeth G Schlaepfer, David D Breast Cancer Res Research Article INTRODUCTION: 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13058-015-0551-x) contains supplementary material, which is available to authorized users. BioMed Central 2015-03-28 2015 /pmc/articles/PMC4407832/ /pubmed/25880415 http://dx.doi.org/10.1186/s13058-015-0551-x Text en © Tancioni et al.; licensee BioMed Central. 2015 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Tancioni, Isabelle
Miller, Nichol LG
Uryu, Sean
Lawson, Christine
Jean, Christine
Chen, Xiao Lei
Kleinschmidt, Elizabeth G
Schlaepfer, David D
FAK activity protects nucleostemin in facilitating breast cancer spheroid and tumor growth
title FAK activity protects nucleostemin in facilitating breast cancer spheroid and tumor growth
title_full FAK activity protects nucleostemin in facilitating breast cancer spheroid and tumor growth
title_fullStr FAK activity protects nucleostemin in facilitating breast cancer spheroid and tumor growth
title_full_unstemmed FAK activity protects nucleostemin in facilitating breast cancer spheroid and tumor growth
title_short FAK activity protects nucleostemin in facilitating breast cancer spheroid and tumor growth
title_sort fak activity protects nucleostemin in facilitating breast cancer spheroid and tumor growth
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4407832/
https://www.ncbi.nlm.nih.gov/pubmed/25880415
http://dx.doi.org/10.1186/s13058-015-0551-x
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