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Caveolae Microdomains Mediate STAT5 Signaling Induced by Insulin in MCF-7 Breast Cancer Cells

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Abstract

Caveolae are small plasma membrane invaginations constituted for membrane proteins namely caveolins and cytosolic proteins termed cavins, which can occupy up to 50% of the surface of mammalian cells. The caveolae have been involved with a variety of cellular processes including regulation of cellular signaling. Insulin is a hormone that mediates a variety of physiological processes through activation of insulin receptor (IR), which is a tyrosine kinase receptor expressed in all mammalian tissues. Insulin induces activation of signal transducers and activators of transcription (STAT) family members including STAT5. In this study, we demonstrate, for the first time, that insulin induces phosphorylation of STAT5 at tyrosine-694 (STAT5-Tyr(P)694), STAT5 nuclear accumulation and an increase in STAT5-DNA complex formation in MCF-7 breast cancer cells. Insulin also induces nuclear accumulation of STAT5-Tyr(P)694, caveolin-1, and IR in MCF-7 cells. STAT5 nuclear accumulation and the increase of STAT5-DNA complex formation require the integrity of caveolae and microtubule network. Moreover, insulin induces an increase and nuclear accumulation of STAT5-Tyr(P)694 in MDA-MB-231 breast cancer cells. In conclusion, results demonstrate that caveolae and microtubule network play an important role in STAT5-Tyr(P)694, STAT5 nuclear accumulation and STAT5-DNA complex formation induced by insulin in breast cancer cells.

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Data Availability

The authors declare that all data supporting the findings of this study are available within the article.

Abbreviations

Ab:

Antibody

DMEM:

Dulbecco’s modified Eagle’s media

DTT:

Dithiothreitol

EDTA:

Ethylenediamine tetraacetic acid

EGF:

Epidermal growth factor

EGTA:

Ethylene glycol-bis-(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid

EMSA:

Electrophoretic mobility shift assay

FBS:

Fetal bovine serum

HEPES:

4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid

IR:

Insulin receptor

PBS:

Phosphate buffered saline

PI3K:

Phosphatidylinositol 3-kinase

PMSF:

Phenylmethylsulfonyl fluoride

PTK:

Protein tyrosine kinase

Rb:

Retinoblastoma

RIPA:

Radioimmune precipitation assay

SDS:

Sodium dodecyl sulfate

STAT:

Signal transducer and activators of transcription

STAT5-Tyr(P)694 :

STAT5 phosphorylation at Tyr-694

Tyr:

Tyrosine

References

  • Amaya MJ, Oliveira AG, Guimaraes ES, Casteluber MC, Carvalho SM, Andrade LM, Pinto MC, Mennone A, Oliveira CA, Resende RR, Menezes GB, Nathanson MH, Leite MF (2014) The insulin receptor translocates to the nucleus to regulate cell proliferation in liver. Hepatology 59:274–283

    Article  CAS  Google Scholar 

  • Balbis A, Baquiran G, Mounier C, Posner BI (2004) Effect of insulin on caveolin-enriched membrane domains in rat liver. J Biol Chem 279:39348–39357

    Article  CAS  Google Scholar 

  • Bild AH, Turkson J, Jove R (2002) Cytoplasmic transport of Stat3 by receptor-mediated endocytosis. Embo J 21:3255–3263

    Article  CAS  Google Scholar 

  • Bradley MP, Rayns DG, Forrester IT (1980) Effects of filipin, digitonin, and polymyxin b on plasma membrane of ram spermatozoa–an EM study. Arch Androl 4:195–204

    Article  CAS  Google Scholar 

  • Chen J, Sadowski HB, Kohanski RA, Wang LH (1997) Stat5 is a physiological substrate of the insulin receptor. Proc Natl Acad Sci USA 94:2295–2300

    Article  CAS  Google Scholar 

  • Cotarla I, Ren S, Zhang Y, Gehan E, Singh B, Furth PA (2004) Stat5a is tyrosine phosphorylated and nuclear localized in a high proportion of human breast cancers. Int J Cancer 108:665–671

    Article  CAS  Google Scholar 

  • Dell’Albani P, Santangelo R, Torrisi L, Nicoletti VG, Giuffrida Stella AM (2003) Role of the JAK/STAT signal transduction pathway in the regulation of gene expression in CNS. Neurochem Res 28:53–64

    Article  CAS  Google Scholar 

  • Epand RM, Sayer BG, Epand RF (2005) Caveolin scaffolding region and cholesterol-rich domains in membranes. J Mol Biol 345:339–350

    Article  CAS  Google Scholar 

  • Fagerholm S, Ortegren U, Karlsson M, Ruishalme I, Stralfors P (2009) Rapid insulin-dependent endocytosis of the insulin receptor by caveolae in primary adipocytes. PLoS ONE 4:e5985

    Article  Google Scholar 

  • Frasca F, Pandini G, Sciacca L, Pezzino V, Squatrito S, Belfiore A, Vigneri R (2008) The role of insulin receptors and IGF-I receptors in cancer and other diseases. Arch Physiol Biochem 114:23–37

    Article  CAS  Google Scholar 

  • Gervasio OL, Phillips WD, Cole L, Allen DG (2011) Caveolae respond to cell stretch and contribute to stretch-induced signaling. J Cell Sci 124:3581–3590

    Article  CAS  Google Scholar 

  • Guo Q, Shen N, Yuan K, Li J, Wu H, Zeng Y, Fox J 3rd, Bansal AK, Singh BB, Gao H, Wu M (2012) Caveolin-1 plays a critical role in host immunity against Klebsiella pneumoniae by regulating STAT5 and Akt activity. Eur J Immunol 42:1500–1511

    Article  CAS  Google Scholar 

  • Gupta R, Toufaily C, Annabi B (2014) Caveolin and cavin family members: dual roles in cancer. Biochimie Pt B 107:188–202

    Article  CAS  Google Scholar 

  • Gustavsson J, Parpal S, Karlsson M, Ramsing C, Thorn H, Borg M, Lindroth M, Peterson KH, Magnusson KE, Stralfors P (1999) Localization of the insulin receptor in caveolae of adipocyte plasma membrane. Faseb J 13:1961–1971

    Article  CAS  Google Scholar 

  • Haeusler RA, McGraw TE, Accili D (2018) Biochemical and cellular properties of insulin receptor signalling. Nat Rev Mol Cell Biol 19:31–44

    Article  CAS  Google Scholar 

  • Hossain MB, Shifat R, Li J, Luo X, Hess KR, Rivera-Molina Y, Puerta Martinez F, Jiang H, Lang FF, Hung MC, Fueyo J, Gomez-Manzano C (2017) TIE2 associates with caveolae and regulates caveolin-1 to promote their nuclear translocation. Mol Cell Biol 37:e00142-17

    Article  Google Scholar 

  • Ikonen E, Parton RG (2000) Caveolins and cellular cholesterol balance. Traffic 1:212–217

    Article  CAS  Google Scholar 

  • Jia G, Sowers JR (2015) Caveolin-1 in cardiovascular disease: a double-edged sword. Diabetes 64:3645–3647

    Article  CAS  Google Scholar 

  • Ju H, Venema VJ, Liang H, Harris MB, Zou R, Venema RC (2000) Bradykinin activates the Janus-activated kinase/signal transducers and activators of transcription (JAK/STAT) pathway in vascular endothelial cells: localization of JAK/STAT signalling proteins in plasmalemmal caveolae. Biochem J 351:257–264

    Article  CAS  Google Scholar 

  • Kesten D, Horovitz-Fried M, Brutman-Barazani T, Sampson SR (2018) Insulin-induced translocation of IR to the nucleus in insulin responsive cells requires a nuclear translocation sequence. Biochim Biophys Acta Mol Cell Res 1865:551–559

    Article  CAS  Google Scholar 

  • Kido Y, Nakae J, Accili D (2001) Clinical review 125: The insulin receptor and its cellular targets. J Clin Endocrinol Metab 86:972–979

    CAS  Google Scholar 

  • Lackmann M, Harpur AG, Oates AC, Mann RJ, Gabriel A, Meutermans W, Alewood PF, Kerr IM, Stark GR, Wilks AF (1998) Biomolecular interaction analysis of IFN gamma-induced signaling events in whole-cell lysates: prevalence of latent STAT1 in high-molecular weight complexes. Growth Factors 16:39–51

    Article  CAS  Google Scholar 

  • Lange CA, Richer JK, Shen T, Horwitz KB (1998) Convergence of progesterone and epidermal growth factor signaling in breast cancer. Potentiation of mitogen-activated protein kinase pathways. J Biol Chem 273:31308–31316

    Article  CAS  Google Scholar 

  • Le MN, Kohanski RA, Wang LH, Sadowski HB (2002) Dual mechanism of signal transducer and activator of transcription 5 activation by the insulin receptor. Mol Endocrinol 16:2764–2779

    Article  CAS  Google Scholar 

  • Lopez-Perez M, Salazar EP (2006) A role for the cytoskeleton in STAT5 activation in MCF7 human breast cancer cells stimulated with EGF. Int J Biochem Cell Biol 38:1716–1728

    Article  CAS  Google Scholar 

  • Martinez-Outschoorn UE, Sotgia F, Lisanti MP (2015) Caveolae and signalling in cancer. Nat Rev Cancer 15:225–237

    Article  CAS  Google Scholar 

  • Ndubuisi MI, Guo GG, Fried VA, Etlinger JD, Sehgal PB (1999) Cellular physiology of STAT3: Where’s the cytoplasmic monomer? J Biol Chem 274:25499–25509

    Article  CAS  Google Scholar 

  • Nevalainen MT, Xie J, Torhorst J, Bubendorf L, Haas P, Kononen J, Sauter G, Rui H (2004) Signal transducer and activator of transcription-5 activation and breast cancer prognosis. J Clin Oncol 22:2053–2060

    Article  CAS  Google Scholar 

  • Parpal S, Karlsson M, Thorn H, Stralfors P (2001) Cholesterol depletion disrupts caveolae and insulin receptor signaling for metabolic control via insulin receptor substrate-1, but not for mitogen-activated protein kinase control. J Biol Chem 276:9670–9678

    Article  CAS  Google Scholar 

  • Parton RG, Kozlov MM, Ariotti N (2020) Caveolae and lipid sorting: Shaping the cellular response to stress. J Cell Biol 219:e201905071

    Article  Google Scholar 

  • Parton RG, McMahon KA, Wu Y (2020b) Caveolae: formation, dynamics, and function. Curr Opin Cell Biol 65:8–16

    Article  CAS  Google Scholar 

  • Pelkmans L, Zerial M (2005) Kinase-regulated quantal assemblies and kiss-and-run recycling of caveolae. Nature 436:128–133

    Article  CAS  Google Scholar 

  • Phung-Koskas T, Pilon A, Pous C, Betzina C, Sturm M, Bourguet-Kondracki ML, Durand G, Drechou A (2005) STAT5B-mediated growth hormone signaling is organized by highly dynamic microtubules in hepatic cells. J Biol Chem 280:1123–1131

    Article  CAS  Google Scholar 

  • Pol A, Martin S, Fernandez MA, Ingelmo-Torres M, Ferguson C, Enrich C, Parton RG (2005) Cholesterol and fatty acids regulate dynamic caveolin trafficking through the Golgi complex and between the cell surface and lipid bodies. Mol Biol Cell 16:2091–2105

    Article  CAS  Google Scholar 

  • Richer JK, Lange CA, Manning NG, Owen G, Powell R, Horwitz KB (1998) Convergence of progesterone with growth factor and cytokine signaling in breast cancer. Progesterone receptors regulate signal transducers and activators of transcription expression and activity. J Biol Chem 273:31317–31326

    Article  CAS  Google Scholar 

  • Robledo T, Arriaga-Pizano L, Lopez-Perez M, Salazar EP (2005) Type IV collagen induces STAT5 activation in MCF7 human breast cancer cells. Matrix Biol 24:469–477

    Article  CAS  Google Scholar 

  • Saltiel AR, Pessin JE (2002) Insulin signaling pathways in time and space. Trends Cell Biol 12:65–71

    Article  CAS  Google Scholar 

  • Sawka-Verhelle D, Filloux C, Tartare-Deckert S, Mothe I, Van Obberghen E (1997) Identification of Stat 5B as a substrate of the insulin receptor. Eur J Biochem 250:411–417

    Article  CAS  Google Scholar 

  • Schnitzer JE, Oh P, Pinney E, Allard J (1994) Filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules. J Cell Biol 127:1217–1232

    Article  CAS  Google Scholar 

  • Sciacca L, Prisco M, Wu A, Belfiore A, Vigneri R, Baserga R (2003) Signaling differences from the A and B isoforms of the insulin receptor (IR) in 32D cells in the presence or absence of IR substrate-1. Endocrinology 144:2650–2658

    Article  CAS  Google Scholar 

  • Sehgal PB (2000) STAT-signalling through the cytoplasmic compartment: consideration of a new paradigm. Cell Signal 12:525–535

    Article  CAS  Google Scholar 

  • Smart EJ, Graf GA, McNiven MA, Sessa WC, Engelman JA, Scherer PE, Okamoto T, Lisanti MP (1999) Caveolins, liquid-ordered domains, and signal transduction. Mol Cell Biol 19:7289–7304

    Article  CAS  Google Scholar 

  • Stahlhut M, Sandvig K, van Deurs B (2000) Caveolae: uniform structures with multiple functions in signaling, cell growth, and cancer. Exp Cell Res 261:111–118

    Article  CAS  Google Scholar 

  • Storz P, Doppler H, Pfizenmaier K, Muller G (1999) Insulin selectively activates STAT5b, but not STAT5a, via a JAK2-independent signalling pathway in Kym-1 rhabdomyosarcoma cells. FEBS Lett 464:159–163

    Article  CAS  Google Scholar 

  • Stralfors P (2012) Caveolins and caveolae, roles in insulin signalling and diabetes. Adv Exp Med Biol 729:111–126

    Article  Google Scholar 

  • Tatulian SA (2015) Structural dynamics of insulin receptor and transmembrane signaling. Biochemistry 54:5523–5532

    Article  CAS  Google Scholar 

  • Thomas CM, Smart EJ (2008) Caveolae structure and function. J Cell Mol Med 12:796–809

    Article  CAS  Google Scholar 

  • Vasilenko KP, Burova EB, Tsupkina NV, Nikol’skii NN (1998) Intact microtubule network is necessary for the EGF-induced transport of transcription factor STAT1 in the nucleus of A-431 cells. Tsitologiia 40:1063–1069

    CAS  Google Scholar 

  • Watanabe K, Saito K, Kinjo M, Matsuda T, Tamura M, Kon S, Miyazaki T, Uede T (2004) Molecular dynamics of STAT3 on IL-6 signaling pathway in living cells. Biochem Biophys Res Commun 324:1264–1273

    Article  CAS  Google Scholar 

  • Welte T, Garimorth K, Philipp S, Doppler W (1994) Prolactin-dependent activation of a tyrosine phosphorylated DNA binding factor in mouse mammary epithelial cells. Mol Endocrinol 8:1091–1102

    CAS  Google Scholar 

  • Zhang Y, Yu S, Zhuang L, Zheng Z, Chao T, Fu Q (2012) Caveolin-1 is involved in radiation-induced ERBB2 nuclear transport in breast cancer cells. J Huazhong Univ Sci Technolog Med Sci 32:888–892

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to Biol. Ivonne-Grisel Sanchez-Cervantes for her technical assistance in the analysis of imagens and to the Unidad de microscopia (Facultad de Medicina, UNAM).

Funding

This research was funded by a grant from CONACYT (255429). R C-S is supported by a CONACYT Post-Doctoral grant.

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Author notes

  1. Rocio Castillo-Sanchez and Pedro Cortes-Reynosa have contributed equally to this work.

Authors and Affiliations

  1. Departamento de Biologia Celular, Cinvestav-IPN, Av. IPN # 2508, 07360, Mexico City, Mexico

    Rocio Castillo-Sanchez, Pedro Cortes-Reynosa, Mario Lopez-Perez, Alejandra Garcia-Hernandez & Eduardo Perez Salazar

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  1. Rocio Castillo-Sanchez
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  2. Pedro Cortes-Reynosa
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Correspondence to Eduardo Perez Salazar.

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Castillo-Sanchez, R., Cortes-Reynosa, P., Lopez-Perez, M. et al. Caveolae Microdomains Mediate STAT5 Signaling Induced by Insulin in MCF-7 Breast Cancer Cells. J Membrane Biol 256, 79–90 (2023). https://doi.org/10.1007/s00232-022-00253-x

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