Skip to main content

Advertisement

SpringerLink
Log in

Silencing of BRCA2 decreases anoikis and its heterologous expression sensitizes yeast cells to acetic acid-induced programmed cell death

  • Original Paper
  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

Adhesion of normal epithelial cells to the extracellular matrix (ECM) is essential for survival. Cell detachment from ECM induces a specific form of programmed cell death (PCD) termed anoikis. BRCA2, a tumor suppressor gene whose mutations confer predisposition to cancer, has been implicated in the regulation of DNA repair, transcription, cell proliferation, and apoptosis. However, the potential role of BRCA2 in the regulation of anoikis has not been investigated. Here, we found that suppression of BRCA2 expression by short hairpin RNA promoted resistance to anoikis in prostate, breast and thyroid normal epithelial cells, which was accompanied by reduced caspases 3/7 levels and activity. Using yeast as a model, we assessed that expression of human BRCA2 does not induce cell death by itself but it can promote acetic acid-induced PCD (AA-PCD). Induction of BRCA2 expression decreased cell survival and increased the number of cells positive to different apoptotic markers, including DNA fragmentation and phosphatidylserine externalization en route to AA-PCD. A higher increase in ROS levels occurred in the early phase of AA-PCD in BRCA2-expressing yeast cells compared with non-expressing cells. Accordingly, a delay in the initial burst of ROS levels was observed in BRCA2-knockdown anoikis-resistant human cells. Treatment with the antioxidants N-acetylcysteine or ascorbic acid reduced sensitivity to anoikis in human cells and inhibited AA-PCD in yeast cells expressing BRCA2. Taken together, these results show a new function of BRCA2 protein as modulator of anoikis sensitivity through an evolutionarily-conserved molecular mechanism involving regulation of ROS production and/or detoxification by BRCA2 during PCD processes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Chiarugi P, Giannoni E (2008) Anoikis: a necessary death program for anchorage-dependent cells. Biochem Pharmacol 76(11):1352–1364

    Article  CAS  PubMed  Google Scholar 

  2. Gilmore AP (2005) Anoikis. Cell Death Differ 12(Suppl 2):1473–1477

    Article  CAS  PubMed  Google Scholar 

  3. Guadamillas MC, Cerezo A, Del Pozo MA (2011) Overcoming anoikis—pathways to anchorage-independent growth in cancer. J Cell Sci 124(Pt 19):3189–3197

    Article  CAS  PubMed  Google Scholar 

  4. Paoli P, Giannoni E, Chiarugi P (2013) Anoikis molecular pathways and its role in cancer progression. Biochim Biophys Acta 1833(12):3481–3498

    Article  CAS  PubMed  Google Scholar 

  5. Li AE, Ito H, Rovira II, Kim KS, Takeda K, Yu ZY, Ferrans VJ, Finkel T (1999) A role for reactive oxygen species in endothelial cell anoikis. Circ Res 85(4):304–310

    Article  CAS  PubMed  Google Scholar 

  6. Kamarajugadda S, Stemboroski L, Cai Q, Simpson NE, Nayak S, Tan M, Lu J (2012) Glucose oxidation modulates anoikis and tumor metastasis. Mol Cell Biol 32(10):1893–1907

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Schafer ZT, Grassian AR, Song L, Jiang Z, Gerhart-Hines Z, Irie HY, Gao S, Puigserver P, Brugge JS (2009) Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment. Nature 461(7260):109–113

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Davison CA, Durbin SM, Thau MR, Zellmer VR, Chapman SE, Diener J, Wathen C, Leevy WM, Schafer ZT (2013) Antioxidant enzymes mediate survival of breast cancer cells deprived of extracellular matrix. Cancer Res 73(12):3704–3715

    Article  CAS  PubMed  Google Scholar 

  9. Kamarajugadda S, Cai Q, Chen H, Nayak S, Zhu J, He M, Jin Y, Zhang Y, Ai L, Martin SS, Tan M, Lu J (2013) Manganese superoxide dismutase promotes anoikis resistance and tumor metastasis. Cell Death Dis 4:e504

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Lee JW, Kim JH (2013) Activation of the leukotriene B4 receptor 2-reactive oxygen species (BLT2-ROS) cascade following detachment confers anoikis resistance in prostate cancer cells. J Biol Chem 288(42):30054–30063

    Article  CAS  PubMed  Google Scholar 

  11. Giannoni E, Buricchi F, Grimaldi G, Parri M, Cialdai F, Taddei ML, Raugei G, Ramponi G, Chiarugi P (2008) Redox regulation of anoikis: reactive oxygen species as essential mediators of cell survival. Cell Death Differ 15(5):867–878

    Article  CAS  PubMed  Google Scholar 

  12. Dufour G, Demers MJ, Gagne D, Dydensborg AB, Teller IC, Bouchard V, Degongre I, Beaulieu JF, Cheng JQ, Fujita N, Tsuruo T, Vallee K, Vachon PH (2004) Human intestinal epithelial cell survival and anoikis. Differentiation state-distinct regulation and roles of protein kinase B/Akt isoforms. J Biol Chem 279(42):44113–44122

    Article  CAS  PubMed  Google Scholar 

  13. Beausejour M, Noel D, Thibodeau S, Bouchard V, Harnois C, Beaulieu JF, Demers MJ, Vachon PH (2012) Integrin/FAK/Src-mediated regulation of cell survival and anoikis in human intestinal epithelial crypt cells: selective engagement and roles of PI3-K isoform complexes. Apoptosis 17(6):566–578

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Singh AB, Sharma A, Dhawan P (2012) Claudin-1 expression confers resistance to anoikis in colon cancer cells in a Src-dependent manner. Carcinogenesis 33(12):2538–2547

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Cheng H, Liu P, Wang ZC, Zou L, Santiago S, Garbitt V, Gjoerup OV, Iglehart JD, Miron A, Richardson AL, Hahn WC, Zhao JJ (2009) SIK1 couples LKB1 to p53-dependent anoikis and suppresses metastasis. Sci Signal 2(80):ra35

    Article  PubMed Central  PubMed  Google Scholar 

  16. Thorslund T, West SC (2007) BRCA2: a universal recombinase regulator. Oncogene 26(56):7720–7730

    Article  CAS  PubMed  Google Scholar 

  17. Yoshida K, Miki Y (2004) Role of BRCA1 and BRCA2 as regulators of DNA repair, transcription, and cell cycle in response to DNA damage. Cancer Sci 95(11):866–871

    Article  CAS  PubMed  Google Scholar 

  18. Martin AM, Blackwood MA, Antin-Ozerkis D, Shih HA, Calzone K, Colligon TA, Seal S, Collins N, Stratton MR, Weber BL, Nathanson KL (2001) Germline mutations in BRCA1 and BRCA2 in breast–ovarian families from a breast cancer risk evaluation clinic. J Clin Oncol 19(8):2247–2253

    CAS  PubMed  Google Scholar 

  19. Consortium TBCL (1999) Cancer risks in BRCA2 mutation carriers. J Natl Cancer Inst 91(15):1310–1316

    Article  Google Scholar 

  20. Connor F, Bertwistle D, Mee PJ, Ross GM, Swift S, Grigorieva E, Tybulewicz VL, Ashworth A (1997) Tumorigenesis and a DNA repair defect in mice with a truncating Brca2 mutation. Nat Genet 17(4):423–430

    Article  CAS  PubMed  Google Scholar 

  21. Cheung AM, Hande MP, Jalali F, Tsao MS, Skinnider B, Hirao A, McPherson JP, Karaskova J, Suzuki A, Wakeham A, You-Ten A, Elia A, Squire J, Bristow R, Hakem R, Mak TW (2002) Loss of Brca2 and p53 synergistically promotes genomic instability and deregulation of T-cell apoptosis. Cancer Res 62(21):6194–6204

    CAS  PubMed  Google Scholar 

  22. Singh KK, Shukla PC, Quan A, Desjardins JF, Lovren F, Pan Y, Garg V, Gosal S, Garg A, Szmitko PE, Schneider MD, Parker TG, Stanford WL, Leong-Poi H, Teoh H, Al-Omran M, Verma S (2012) BRCA2 protein deficiency exaggerates doxorubicin-induced cardiomyocyte apoptosis and cardiac failure. J Biol Chem 287(9):6604–6614

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Hay T, Patrick T, Winton D, Sansom OJ, Clarke AR (2005) Brca2 deficiency in the murine small intestine sensitizes to p53-dependent apoptosis and leads to the spontaneous deletion of stem cells. Oncogene 24(23):3842–3846

    Article  CAS  PubMed  Google Scholar 

  24. Yang G, Chang B, Yang F, Guo X, Cai KQ, Xiao XS, Wang H, Sen S, Hung MC, Mills GB, Chang S, Multani AS, Mercado-Uribe I, Liu J (2010) Aurora kinase A promotes ovarian tumorigenesis through dysregulation of the cell cycle and suppression of BRCA2. Clin Cancer Res 16(12):3171–3181

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  25. Rajagopalan S, Andreeva A, Rutherford TJ, Fersht AR (2010) Mapping the physical and functional interactions between the tumor suppressors p53 and BRCA2. Proc Natl Acad Sci USA 107(19):8587–8592

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Carmona-Gutierrez D, Eisenberg T, Buttner S, Meisinger C, Kroemer G, Madeo F (2010) Apoptosis in yeast: triggers, pathways, subroutines. Cell Death Differ 17(5):763–773

    Article  CAS  PubMed  Google Scholar 

  27. Guaragnella N, Antonacci L, Passarella S, Marra E, Giannattasio S (2011) Achievements and perspectives in yeast acetic acid-induced programmed cell death pathways. Biochem Soc Trans 39(5):1538–1543

    Article  CAS  PubMed  Google Scholar 

  28. Guaragnella N, Zdralevic M, Antonacci L, Passarella S, Marra E, Giannattasio S (2012) The role of mitochondria in yeast programmed cell death. Front Oncol 2:70

    Article  PubMed Central  PubMed  Google Scholar 

  29. Mager WH, Winderickx J (2005) Yeast as a model for medical and medicinal research. Trends Pharmacol Sci 26(5):265–273

    Article  CAS  PubMed  Google Scholar 

  30. Greenwood MT, Ludovico P (2010) Expressing and functional analysis of mammalian apoptotic regulators in yeast. Cell Death Differ 17(5):737–745

    Article  CAS  PubMed  Google Scholar 

  31. Pereira C, Coutinho I, Soares J, Bessa C, Leao M, Saraiva L (2012) New insights into cancer-related proteins provided by the yeast model. FEBS J 279(5):697–712

    Article  CAS  PubMed  Google Scholar 

  32. Guaragnella N, Palermo V, Galli A, Moro L, Mazzoni C, Giannattasio S (2014) The expanding role of yeast in cancer research and diagnosis: insights into the function of the oncosuppressors p53 and BRCA1/2. FEMS Yeast Res 14:2–16

    Article  CAS  Google Scholar 

  33. Jantti J, Lahdenranta J, Olkkonen VM, Soderlund H, Keranen S (1999) SEM1, a homologue of the split hand/split foot malformation candidate gene Dss1, regulates exocytosis and pseudohyphal differentiation in yeast. Proc Natl Acad Sci USA 96(3):909–914

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Marston NJ, Richards WJ, Hughes D, Bertwistle D, Marshall CJ, Ashworth A (1999) Interaction between the product of the breast cancer susceptibility gene BRCA2 and DSS1, a protein functionally conserved from yeast to mammals. Mol Cell Biol 19(7):4633–4642

    CAS  PubMed Central  PubMed  Google Scholar 

  35. Gudmundsdottir K, Lord CJ, Witt E, Tutt AN, Ashworth A (2004) DSS1 is required for RAD51 focus formation and genomic stability in mammalian cells. EMBO Rep 5(10):989–993

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. Kojic M, Yang H, Kostrub CF, Pavletich NP, Holloman WK (2003) The BRCA2-interacting protein DSS1 is vital for DNA repair, recombination, and genome stability in Ustilago maydis. Mol Cell 12(4):1043–1049

    Article  CAS  PubMed  Google Scholar 

  37. Spugnesi L, Balia C, Collavoli A, Falaschi E, Quercioli V, Caligo MA, Galli A (2013) Effect of the expression of BRCA2 on spontaneous homologous recombination and DNA damage-induced nuclear foci in Saccharomyces cerevisiae. Mutagenesis 28(2):187–195

    Article  CAS  PubMed  Google Scholar 

  38. Moro L, Arbini AA, Yao JL, di Sant’Agnese PA, Marra E, Greco M (2009) Mitochondrial DNA depletion in prostate epithelial cells promotes anoikis resistance and invasion through activation of PI3K/Akt2. Cell Death Differ 16(4):571–583

    Article  CAS  PubMed  Google Scholar 

  39. Chen DC, Yang BC, Kuo TT (1992) One-step transformation of yeast in stationary phase. Curr Genet 21(1):83–84

    Article  CAS  PubMed  Google Scholar 

  40. Giannattasio S, Guaragnella N, Corte-Real M, Passarella S, Marra E (2005) Acid stress adaptation protects Saccharomyces cerevisiae from acetic acid-induced programmed cell death. Gene 354:93–98

    Article  CAS  PubMed  Google Scholar 

  41. Moro L, Arbini AA, Marra E, Greco M (2006) Up-regulation of Skp2 after prostate cancer cell adhesion to basement membranes results in BRCA2 degradation and cell proliferation. J Biol Chem 281(31):22100–22107

    Article  CAS  PubMed  Google Scholar 

  42. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  43. Ludovico P, Sousa MJ, Silva MT, Leao C, Corte-Real M (2001) Saccharomyces cerevisiae commits to a programmed cell death process in response to acetic acid. Microbiology 147(Pt 9):2409–2415

    CAS  PubMed  Google Scholar 

  44. Ludovico P, Rodrigues F, Almeida A, Silva MT, Barrientos A, Corte-Real M (2002) Cytochrome c release and mitochondria involvement in programmed cell death induced by acetic acid in Saccharomyces cerevisiae. Mol Biol Cell 13(8):2598–2606

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  45. Pereira C, Camougrand N, Manon S, Sousa MJ, Corte-Real M (2007) ADP/ATP carrier is required for mitochondrial outer membrane permeabilization and cytochrome c release in yeast apoptosis. Mol Microbiol 66(3):571–582

    Article  CAS  PubMed  Google Scholar 

  46. Giannattasio S, Atlante A, Antonacci L, Guaragnella N, Lattanzio P, Passarella S, Marra E (2008) Cytochrome c is released from coupled mitochondria of yeast en route to acetic acid-induced programmed cell death and can work as an electron donor and a ROS scavenger. FEBS Lett 582(10):1519–1525

    Article  CAS  PubMed  Google Scholar 

  47. Guaragnella N, Antonacci L, Giannattasio S, Marra E, Passarella S (2008) Catalase T and Cu, Zn–superoxide dismutase in the acetic acid-induced programmed cell death in Saccharomyces cerevisiae. FEBS Lett 582(2):210–214

    Article  CAS  PubMed  Google Scholar 

  48. Guaragnella N, Bobba A, Passarella S, Marra E, Giannattasio S (2010) Yeast acetic acid-induced programmed cell death can occur without cytochrome c release which requires metacaspase YCA1. FEBS Lett 584(1):224–228

    Article  CAS  PubMed  Google Scholar 

  49. Guaragnella N, Passarella S, Marra E, Giannattasio S (2010) Knock-out of metacaspase and/or cytochrome c results in the activation of a ROS-independent acetic acid-induced programmed cell death pathway in yeast. FEBS Lett 584(16):3655–3660

    Article  CAS  PubMed  Google Scholar 

  50. Guaragnella N, Antonacci L, Passarella S, Marra E, Giannattasio S (2007) Hydrogen peroxide and superoxide anion production during acetic acid-induced yeast programmed cell death. Folia Microbiol (Praha) 52(3):237–240

    Article  CAS  Google Scholar 

  51. Wang SC, Shao R, Pao AY, Zhang S, Hung MC, Su LK (2002) Inhibition of cancer cell growth by BRCA2. Cancer Res 62(5):1311–1314

    CAS  PubMed  Google Scholar 

  52. Tian XX, Rai D, Li J, Zou C, Bai Y, Wazer D, Band V, Gao Q (2005) BRCA2 suppresses cell proliferation via stabilizing MAGE-D1. Cancer Res 65(11):4747–4753

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  53. Moro L, Arbini AA, Marra E, Greco M (2005) Down-regulation of BRCA2 expression by collagen type I promotes prostate cancer cell proliferation. J Biol Chem 280(23):22482–22491

    Article  CAS  PubMed  Google Scholar 

  54. Moro L, Arbini AA, Marra E, Greco M (2007) Constitutive activation of MAPK/ERK inhibits prostate cancer cell proliferation through upregulation of BRCA2. Int J Oncol 30(1):217–224

    CAS  PubMed  Google Scholar 

  55. Moro L, Arbini AA, Yao JL, di Sant’Agnese PA, Marra E, Greco M (2008) Loss of BRCA2 promotes prostate cancer cell invasion through up-regulation of matrix metalloproteinase-9. Cancer Sci 99(3):553–563

    Article  CAS  PubMed  Google Scholar 

  56. Boulton SJ (2006) Cellular functions of the BRCA tumour-suppressor proteins. Biochem Soc Trans 34(Pt 5):633–645

    CAS  PubMed  Google Scholar 

  57. Badie S, Escandell JM, Bouwman P, Carlos AR, Thanasoula M, Gallardo MM, Suram A, Jaco I, Benitez J, Herbig U, Blasco MA, Jonkers J, Tarsounas M (2010) BRCA2 acts as a RAD51 loader to facilitate telomere replication and capping. Nat Struct Mol Biol 17(12):1461–1469

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  58. Guaragnella N, Zdralevic M, Lattanzio P, Marzulli D, Pracheil T, Liu Z, Passarella S, Marra E, Giannattasio S (2013) Yeast growth in raffinose results in resistance to acetic-acid induced programmed cell death mostly due to the activation of the mitochondrial retrograde pathway. Biochim Biophys Acta 1833(12):2765–2774

    Article  CAS  PubMed  Google Scholar 

  59. Mitra AV, Bancroft EK, Barbachano Y, Page EC, Foster CS, Jameson C, Mitchell G, Lindeman GJ, Stapleton A, Suthers G, Evans DG, Cruger D, Blanco I, Mercer C, Kirk J, Maehle L, Hodgson S, Walker L, Izatt L, Douglas F, Tucker K, Dorkins H, Clowes V, Male A, Donaldson A, Brewer C, Doherty R, Bulman B, Osther PJ, Salinas M, Eccles D, Axcrona K, Jobson I, Newcombe B, Cybulski C, Rubinstein WS, Buys S, Townshend S, Friedman E, Domchek S, Ramon YCT, Spigelman A, Teo SH, Nicolai N, Aaronson N, Ardern-Jones A, Bangma C, Dearnaley D, Eyfjord J, Falconer A, Gronberg H, Hamdy F, Johannsson O, Khoo V, Kote-Jarai Z, Lilja H, Lubinski J, Melia J, Moynihan C, Peock S, Rennert G, Schroder F, Sibley P, Suri M, Wilson P, Bignon YJ, Strom S, Tischkowitz M, Liljegren A, Ilencikova D, Abele A, Kyriacou K, van Asperen C, Kiemeney L, Easton DF, Eeles RA (2011) Targeted prostate cancer screening in men with mutations in BRCA1 and BRCA2 detects aggressive prostate cancer: preliminary analysis of the results of the IMPACT study. BJU Int 107(1):28–39

    Article  PubMed  Google Scholar 

  60. Castro E, Goh C, Olmos D, Saunders E, Leongamornlert D, Tymrakiewicz M, Mahmud N, Dadaev T, Govindasami K, Guy M, Sawyer E, Wilkinson R, Ardern-Jones A, Ellis S, Frost D, Peock S, Evans DG, Tischkowitz M, Cole T, Davidson R, Eccles D, Brewer C, Douglas F, Porteous ME, Donaldson A, Dorkins H, Izatt L, Cook J, Hodgson S, Kennedy MJ, Side LE, Eason J, Murray A, Antoniou AC, Easton DF, Kote-Jarai Z, Eeles R (2013) Germline BRCA mutations are associated with higher risk of nodal involvement, distant metastasis, and poor survival outcomes in prostate cancer. J Clin Oncol 31(14):1748–1757

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  61. Rodriguez-Berriguete G, Galvis L, Fraile B, de Bethencourt FR, Martinez-Onsurbe P, Olmedilla G, Paniagua R, Royuela M (2012) Immunoreactivity to caspase-3, caspase-7, caspase-8, and caspase-9 forms is frequently lost in human prostate tumors. Hum Pathol 43(2):229–237

    Article  CAS  PubMed  Google Scholar 

  62. Winter RN, Kramer A, Borkowski A, Kyprianou N (2001) Loss of caspase-1 and caspase-3 protein expression in human prostate cancer. Cancer Res 61(3):1227–1232

    CAS  PubMed  Google Scholar 

  63. Chen T, Yang I, Irby R, Shain KH, Wang HG, Quackenbush J, Coppola D, Cheng JQ, Yeatman TJ (2003) Regulation of caspase expression and apoptosis by adenomatous polyposis coli. Cancer Res 63(15):4368–4374

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank Professor Ileana Ferrero for critical discussions and Ms. Annarita Armenise for skilful assistance. This work has been funded by Grants from Project FIRB-MERIT RBNE08HWLZ to E.M., FIRB-Merit RBNE08YFN3_005 and PRIN 2009R8LJPS_002 to L.M., and the Italian Ministry of Economy and Finance to the CNR for the Project “FaReBio di Qualità” to L.M., S.G.

Author information

Authors and Affiliations

  1. Institute of Biomembranes and Bioenergetics, National Research Council (CNR), Via Amendola 165/A, 70126, Bari, Italy

    Nicoletta Guaragnella, Ersilia Marra, Loredana Moro & Sergio Giannattasio

  2. Institute of Clinical Physiology, National Research Council (CNR), Via Moruzzi 1, 56124, Pisa, Italy

    Alvaro Galli

Authors
  1. Nicoletta Guaragnella
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ersilia Marra
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alvaro Galli
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Loredana Moro
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sergio Giannattasio
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Loredana Moro or Sergio Giannattasio.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Guaragnella, N., Marra, E., Galli, A. et al. Silencing of BRCA2 decreases anoikis and its heterologous expression sensitizes yeast cells to acetic acid-induced programmed cell death. Apoptosis 19, 1330–1341 (2014). https://doi.org/10.1007/s10495-014-1006-z

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-014-1006-z

Keywords

Search

Navigation