Advertisement

Journal of Cell Communication and Signaling

, Volume 9, Issue 3, pp 255–265 | Cite as

Cellular senescence and autophagy of myoepithelial cells are involved in the progression of in situ areas of carcinoma ex-pleomorphic adenoma to invasive carcinoma. An in vitro model

  • Carolina Amália Barcellos Silva
  • Elizabeth Ferreira Martinez
  • Ana Paula Dias Demasi
  • Albina Altemani
  • Jeruza Pinheiro da Silveira Bossonaro
  • Ney Soares Araújo
  • Vera Cavalcanti de AraújoEmail author
RESEARCH ARTICLE

Abstract

During tumor invasion, benign myoepithelial cells of carcinoma ex-pleomorphic adenoma (CXPA) surround malignant epithelial cells and disappear. The mechanisms involved in the death and disappearance of these myoepithelial cells were investigated via analysis of the expression of regulatory proteins for apoptosis, autophagy and cellular senescence in an in situ in vitro model. Protein expression relating to apoptosis (Bax, Bcl-2, Survivin), autophagy (Beclin-1, LC3B) and cellular senescence (p21, p16) was evaluated using indirect immunofluorescence. β-galactosidase expression was assessed via histochemistry. Biopsies of CXPA (ex vivo) allowed immunhistochemical evaluation of p21 and p16, whilst LC3B, p21 and p16 protein expression was analyzed by western blotting. In the in vitro model, the myoepithelial cells were positive for LC3B (cytoplasm) and p21 (nucleus), whilst in vivo positivity for p21 and p16 was observed. In vitro, β-galactosidase activity increased in the myoepithelial cells over time. Western blotting analysis revealed an increased LC3B, p16 and p21 expression in the myoepithelial cells with previous contact with the malignant cells when compared with those without contact. The investigation of behavior of benign myoepithelial cells in ductal areas of CXAP revealed that the myoepithelial cells are involved in the autophagy-senescence phenotype that subsequently leads to their disappearance.

Keywords

Autophagy Cellular Senescence Myoepithelial Cells Tumor Microenvironment 

Abbreviations

AE1/AE3

Pan cytokeratin

Bad

Bcl-2 antagonist of cell death

Bak

Bcl-2 antagonistic killer

Bax

Bcl-2 associated X protein

Bcl-2

B cell lymphoma-2 protein

Bcl-xl

B cell lymphoma-extra long

BCA

Bicinchoninic acid

BSA

Bovine serum albumin

CDK

Cyclin dependent kinase

cIAP

Baculoviral IAP repeat containing

CK7

Cytokeratin 7

CXAP

Carcinoma ex-pleomorphic adenoma

DAPI

4′-6-diamidino-2phenylindole

DMEM

Dulbecco’s modified Eagle medium

DCIS

Ductal carcinoma in situ

EDTA

Ethylenediaminetetraacetic acid

IAP

Apoptosis inhibitors proteins

LC3

Microtubule-associated protein 1 light chain 3

Mcl-1

Myeloid cell leucemia 1 protein

NAIP

NLR family, apoptosis inhibitory proteins

PA

Pleomorphic adenoma

PBS

Phosphate buffer saline

RIPA

Radio immuno precipitation assay buffer

TBST

Tris-buffered saline and tween 20

XIAP

X-linked inhibitor of apoptosis protein

Notes

Acknowledgments

The authors wish to thank Pollyanna Tombini Montaldi, Vanessa Araújo and Nadir Freitas for their excellent technical expertise and assistance. This work was supported by grants from FAPESP/Brazil (2011/21157-0) and CNPq (473939/2011-8).

Conflict of Interest

The authors declare that they have no conflict of interest.

References

  1. Altemani A, Martins MT, Freitas L, Soares F, Araújo NS, Araújo VC (2005) Carcinoma ex pleomorphic adenoma (CXAP): immunoprofile of the cells involved in carcinomatous progression. Histophatology 46:635–641CrossRefGoogle Scholar
  2. Araújo VC, Altemani A, Furuse C, Martins MT, Araújo NS (2006) Immunoprofile of reatctive salivary myoepithelial cells in intraductal áreas of carcinoma ex-pleomorphic adenoma. Oral Oncol 42:1011–1116CrossRefPubMedGoogle Scholar
  3. Barsky SH, Karlin NJ (2005) Myoepithelial cells: autocrine and paracrine suppressors of breast cancer progression. J Mammary Gland Biol Neoplasia 10:249–260CrossRefPubMedGoogle Scholar
  4. Bartlett JM, Nofech-Moses S, Rakovitch E (2014) Ductal carcinoma in situ of the breast: can biomarkers improve current management? Clin Chem 60:60–67CrossRefPubMedGoogle Scholar
  5. Campisi J (2011) Cellular senescence: putting the paradoxes in perspective. Curr Opin Genet Dev 21:107–112PubMedCentralCrossRefPubMedGoogle Scholar
  6. Campisi J, Fagagna FDA (2007) Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol 8:729–740CrossRefPubMedGoogle Scholar
  7. Campisi J, Andersen JK, Kapahi P, Melov S (2011) Cellular senescence: a link between cancer and age-related degenerative disease? Semin Cancer Biol 21:354–359PubMedCentralPubMedGoogle Scholar
  8. Cao Y, Klionsky DJ (2007) Physiological functions of Atg6/Beclin-1: a unique autophagy-related protein. Cell Res 17:839–849CrossRefPubMedGoogle Scholar
  9. Capparelli C, Chiavarina B, Whitaker-Menezes D, Pestell TG, Pestell RG, Hulit J et al (2012a) CDK inibitors (p16/p19/p21) induce senescence and autophagy in cancer-associated fibroblasts, “fueling” tumor growth via paracrine interactions, wihout an increase in neo-angiogenesis. Cell Cycle 11:3599–3610PubMedCentralCrossRefPubMedGoogle Scholar
  10. Capparelli C, Guido C, Whitaker-Menezes D, Bonuccelli G, Balliet R, Pestell TG et al (2012b) Autophagy and senescence in cancer-associated fibroblasts metabolically supports tumor growth and metastasis via glycolysis and ketone production. Cell Cycle 11:2285–2302PubMedCentralCrossRefPubMedGoogle Scholar
  11. Capparelli C, Whitaker-Menezes D, Guido C, Balliet R, Pestell TG, Howell A et al (2012c) CTGF drives autophagy, glycolysis and senescence in cancer-associated fibroblasts via HIF1 activation, metabolically promoting tumor growth. Cell Cycle 11:2272–2284PubMedCentralCrossRefPubMedGoogle Scholar
  12. Chen J, Goligorsky MS (2006) Premature senescence of endothelial cells: methusaleh’s dilemma. Am J Physiol Heart Circ Physiol 290:729–739Google Scholar
  13. Chen N, Karantza-Wadsworth V (2009) Role and regulation of autophagy in cancer. Biochim Biophys Acta 1793:1516–1523PubMedCentralCrossRefPubMedGoogle Scholar
  14. Chen J, Xavier S, Moskowitz-Kassai E, Chen R, Lu CY, Sanduski K et al (2012) Cathepsin cleavage of sirtuin 1 in endothelial progenitor cells mediates stress-induced premature senescence. Am J Pathol 180:973–983PubMedCentralCrossRefPubMedGoogle Scholar
  15. Cheung CHA, Cheng LT, Chang KY, Chen HH, Chang JY (2011) Investigations of surviving: the past, present and future. Front Biosci 16:952–961CrossRefGoogle Scholar
  16. Cowell CF, Weigelt B, Sakr RA, Ng CK, Hicks J, King TA, Reis-Filho JS (2013) Progression from ductal carcinoma in situ to invasive breast cancer: revisited. Mol Oncol 7:859–869CrossRefPubMedGoogle Scholar
  17. Dimri GP (2005) What has senescence got to do with cancer? Cancer Cell 7:505–512PubMedCentralCrossRefPubMedGoogle Scholar
  18. Dulic V (2013) Senescence regulation by mTOR. Methods Mol Biol 965:15–35CrossRefPubMedGoogle Scholar
  19. Evan GI, Fagagna FDA (2009) Cellular senescence: hot or what? Curr Opin Genet Dev 19:25–31CrossRefPubMedGoogle Scholar
  20. Jones JL, Shaw JA, Pringle JH, Walker RA (2003) Primary breast myoepithelial cells exert an invasion-supressor effect on breast cancer cells via paracrine down-regulation of MMP expression in fibroblasts and tumour cells. J Pathol 201:562–572CrossRefPubMedGoogle Scholar
  21. Kondo Y, Kanzawa T, Sawaya R, Kondo S (2005) The role of autophagy in cancer development and response to therapy. Nat Rev Cancer 5:726–733CrossRefPubMedGoogle Scholar
  22. Larsson LG (2011) Oncogene and tumor suppressor gene-mediated suppression of cellular senescence. Semin Cancer Biol 21:367–376CrossRefPubMedGoogle Scholar
  23. Levine B (2007) Autophagy and cancer. Cell Biol 446:745–747Google Scholar
  24. Martinez EF, Demasi AP, Miguita L, Altemani A, Araújo NS, Araújo VC (2010) FGF-2 is overexpressed in myoepithelial cells of carcinoma ex-pleomorphic adenoma in situ structures. Oncol Rep 24:155–160CrossRefPubMedGoogle Scholar
  25. Martinez EF, Montaldi PT, Araújo NS, Altemani A, Araújo VC (2012) A proposal of an in vitro model which mimics in situ áreas of carcinoma. J Cell Comun Signal 6:107–109CrossRefGoogle Scholar
  26. Martinez EF, Napimoga MH, Montalli VA, de Araújo NS, de Araújo VC (2013) In vitro cytokine expression in in situ-like areas of malignant neoplasia. Arch Oral Biol 58:552–557CrossRefPubMedGoogle Scholar
  27. Metwaly H, Maruyama S, Yamazaki M, Tsuneki M, Abé T, Jen KY et al (2012) Parenchymal-stromal switching for extracellular matrix production on invasion of oral squamous cell carcinoma. Hum Pathol 43:1973–1981CrossRefPubMedGoogle Scholar
  28. Miguita L, Martinez EF, Araújo NS, Araújo VC (2010) FGF-2, TGFß-1, PDGF-A and respective receptors expression in pleomorphic adenoma myoepithelial cells: an in vivo and in vitro study. J Appl Oral Sci 18:83–91CrossRefPubMedGoogle Scholar
  29. Miracco C, Meng GC, Franchi A, Luzi P, Cosci E, Mourmouras V et al (2010) Beclin-1 and LC3 autophagic gene expression. In cutaneous melanocytic lesions. Hum Pathol 41:503–512CrossRefPubMedGoogle Scholar
  30. Narita M, Young AR, Narita M (2009) Autophagy facilitates oncogene-induced senescence. Autophagy 5:1046–1047CrossRefPubMedGoogle Scholar
  31. Nguyen M, Lee MC, Wang JL, Tomlinson JS, Shao ZM, Alpaugh ML et al (2000) The human myoepithelial cells displays a multifaceted anti-angiogenic phenotype. Oncogene 19:3449–3459CrossRefPubMedGoogle Scholar
  32. Ouyang L, Shi Z, Zhao S, Wang FT, Zhou TT, Liu B et al (2012) Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell Prolif 45:487–498CrossRefPubMedGoogle Scholar
  33. Pattingre S, Espert L, Biard-Piechaczyk M, Codogno P (2008) Regulation of macroautophagy by mTOR and Beclin 1 complexes. Biochimie 90:313–323CrossRefPubMedGoogle Scholar
  34. Placzek WJ, Wei J, Kitada S, Zhai D, Reed JC, Pellecchia M (2010) A survey of the anti-apoptotic Bcl-2 subfamily expression in cancer types provides a platform to predict the efficacy of Bcl-2 antagonists in cancer therapy. Cell Death Dis 1:e40PubMedCentralCrossRefPubMedGoogle Scholar
  35. Plati J, Bucur O, Khosravi-Far R (2011) Apoptotic cell signaling in câncer progression and therapy. Integr Biol 3:279–296CrossRefGoogle Scholar
  36. Roberg K, Jonsson AC, Grénman R, Norberg-Spaak L (2007) Radiotherapy response in oral squamous carcinoma cell lines: evaluation of apoptotic proteins as prognostic factors. Head Neck 29:325–334CrossRefPubMedGoogle Scholar
  37. Roy S, Debnath J (2010) Autophagy and tumorigenesis. Semin Immunopathol 32:383–396PubMedCentralCrossRefPubMedGoogle Scholar
  38. Shao ZM, Nguyen M, Alpaugh ML, O’Connell JT, Barsky SH (1998) The human myoepithelial cell exerts antiproliferative effects on breast carcinoma cells characterized by p21 induction, G2/M arrest, and apoptosis. Exp Cell Res 241:394–403CrossRefPubMedGoogle Scholar
  39. Shay JW, Roninson IB (2004) Hallmarks of senescence in carcinogenesis and cancer therapy. Oncogene 23:2919–2933CrossRefPubMedGoogle Scholar
  40. Silva AD, Silva CAB, Montalli VA, Martinez EF, Araújo VC, Furuse C (2012) In vitro evaluation of the suppressor potential of conditioned medium from benign myoepithelial cells from pleomorphic adenoma in malignant cell invasion. J Oral Pathol Med 41:610–614CrossRefPubMedGoogle Scholar
  41. Sternlicht MD, Barsky SH (1997) The myoepithelial defense: a host defense against câncer. Med Hypotheses 48:37–46CrossRefPubMedGoogle Scholar
  42. Sternlicht MD, Kedeshian P, Shao ZM, Safarians S, Barsky SH (1997) The human myoepithelial cells is a natural tumor suppressor. Clin Cancer Res 3:1949–1958PubMedGoogle Scholar
  43. Townson JL, Naumov GN, Chambers AF (2003) The role of apoptosis in tumor progression and metastasis. Curr Mol Med 3:631–642CrossRefPubMedGoogle Scholar
  44. Ulukaya E, Acilan C, Yilmaz Y (2011) Apoptosis: why and how does it occur in biology? Cell Biochem Funct 29:468–480CrossRefPubMedGoogle Scholar
  45. Wong RSY (2011) Apoptosis in cancer: from pathogenesis to treatment. J Exp Clin Cancer Res 30:87–100PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© The International CCN Society 2015

Authors and Affiliations

  • Carolina Amália Barcellos Silva
    • 1
  • Elizabeth Ferreira Martinez
    • 1
  • Ana Paula Dias Demasi
    • 1
  • Albina Altemani
    • 2
  • Jeruza Pinheiro da Silveira Bossonaro
    • 1
  • Ney Soares Araújo
    • 1
  • Vera Cavalcanti de Araújo
    • 1
    Email author
  1. 1.Department of Oral PathologySão Leopoldo Mandic Institute and Research CenterCampinasBrazil
  2. 2.Department of PathologyState University of CampinasCampinasBrazil

Personalised recommendations