shRNA-mediated downregulation of α-N-Acetylgalactosaminidase inhibits migration and invasion of cancer cell lines

Document Type: Original Article

Authors

1 Department of Molecular Medicine & Genetics, Zanjan University of Medical Sciences, Zanjan, Iran

2 Department of Immunogenetics, Buali Institute, Mashhad University of Medical Sciences, Mashhad, Iran

3 Department of Medical Biotechnology and Nanotechnology, Zanjan University of Medical Sciences, Zanjan, Iran

4 Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran

5 Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University Of Medical Sciences, Shahrekord, Iran

Abstract

Objective(s): Extracellular matrix (ECM) is composed of many kinds of glycoproteins containing glycosaminoglycans (GAGs) moiety. The research was conducted based on the N-Acetylgalactosamine (GalNAc) degradation of ECM components by α-N-acetylgalactosaminidase (Nagalase) which facilitates migration and invasion of cancer cells. This study aims to investigate the effects of Naga-shRNA downregulation on migration and invasion of cancer cell lines.
Materials and Methods: In this study, MCF-7 cell line (human mammary carcinoma cell line) and A2780 (human ovarian carcinoma cell line) were used. The level of normalized Naga expression and Nagalase protein were evaluated by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay/western blotting, respectively. Migration and invasion were determined using transwell assays, and statistical analysis was carried out by ANOVA test.
Results: Response to transduction by shRNA compared to the control group, migrative and invasive properties of the transfected cells were significantly inhibited.
Conclusion: These results indicate that Nagalase may have an important role in migration and invasion of cancer cells and can be considered as a candidate for further studies.

Keywords


1. Torre LA, Siegel RL, Ward EM, Jemal A. Global cancer incidence and mortality rates and trends--an update. Cancer Epidemiol Biomarkers Prev 2016; 25:16-27.

2. McDonald ES, Mankoff DA, Mach RH. Novel strategies for breast cancer imaging: new imaging agents to guide treatment. J Nucl Med 2016; 57:69S-74S.

3. Iwata H. Future treatment strategies for metastatic breast cancer: curable or incurable? Breast Cancer 2012; 19:200-205.

4. Greco M, Mitri MD, Chiriaco F, Leo G, Brienza E, Maffia M. Serum proteomic profile of cutaneous malignant melanoma and relation to cancer progression: association to tumor derived alpha-N-acetylgalactosaminidase activity. Cancer Lett 2009; 283:222-229.

5. Gulisano M, Pacini S, Thyer L, Morucci G, Branca JJ, Smith R, et al. Alpha-N-acetylgalactosaminidase levels in cancer patients are affected by Vitamin D binding protein-derived macrophage activating factor. Ital J AnatomyEmbryol 2013; 118:104.

6. Thyer L, Ward E, Smith R, Branca JJ, Morucci G, Gulisano M, et al. GC protein-derived macrophage-activating factor decreases alpha-N-acetylgalactosaminidase levels in advanced cancer patients. Oncoimmunology 2013; 2:e25769.

7. Korbelik M, Naraparaju VR, Yamamoto N. The value of serum alpha-N-acetylgalactosaminidase measurement for the assessment of tumour response to radio- and photodynamic therapy. Br J Cancer 1998; 77:1009-1014.

8. Matsuura T, Uematsu T, Yamaoka M, Furusawa K. Effect of salivary gland adenocarcinoma cell-derived alpha-N-acetylgalactosaminidase on the bioactivity of macrophage activating factor. Int J Oncol 2004; 24:521-528.

9. Choromanska B, Luto M, Szajda SD, Waszkiewicz N, Kepka A, Janica J, et al. Activity of N-acetyl-beta-hexosaminidase and its isoenzymes A and B in cancer. Postepy Hig Med Dosw 2011; 65:752-758.

10. Clark NE, Garman SC. The 1.9 a structure of human alpha-N-acetylgalactosaminidase: The molecular basis of Schindler and Kanzaki diseases. J Mol Biol 2009; 393:435-447.

11. Kodama K, Kobayashi H, Abe R, Ohkawara A, Yoshii N, Yotsumoto S, et al. A new case of alpha-N-acetylgalactosaminidase deficiency with angiokeratoma corporis diffusum, with Meniere's syndrome and without mental retardation. Br J Dermatol 2001; 144:363-368.

12. Sakuraba H, Matsuzawa F, Aikawa S, Doi H, Kotani M, Nakada H, et al. Structural and immunocytochemical studies on alpha-N-acetylgalactosaminidase deficiency (Schindler/ Kanzaki disease). J Hum Genet 2004; 49:1-8.

13. Häuselmann I, Borsig L. Altered tumor-cell glycosylation promotes metastasis. Front Ooncol 2014; 4:28.

14. Bapu D, Runions J, Kadhim M, Brooks SA. N-acetylgalactosamine glycans function in cancer cell adhesion to endothelial cells: A role for truncated O-glycans in metastatic mechanisms. Cancer Lett 2016; 375:367-374.

15. Mittal K, Welter BH, Temesvari LA. Entamoeba histolytica: lipid rafts are involved in adhesion of trophozoites to host extracellular matrix components. Exp Parasitol 2008; 120:127-134.

16. Staquicini FI, Moreira CR, Nascimento FD, Tersariol IL, Nader HB, Dietrich CP, et al. Enzyme and integrin expression by high and low metastatic melanoma cell lines. Melanoma Res 2003; 13:11-18.

17. Vynios DH, Theocharis DA, Papageorgakopoulou N, Papadas TA, Mastronikolis NS, Goumas PD, et al. Biochemical changes of extracellular proteoglycans in squamous cell laryngeal carcinoma. Connect Tissue Res 2008;  49:239-243.

18. Yamamoto N. Pathogenic significance of alpha-N-acetylgalactosaminidase activity found in the envelope glycoprotein gp160 of human immunodeficiency virus Type 1. AIDS Res Hum Retroviruses 2006; 22:262-271.

19. Yamamoto N, Urade M. Pathogenic significance of alpha-N-acetylgalactosaminidase activity found in the hemagglutinin of influenza virus. Microbes Infect 2005; 7:674-681.

20. Mohamad SB, Nagasawa H, Uto Y, Hori H. Tumor cell alpha-N-acetylgalactosaminidase activity and its involvement in GcMAF-related macrophage activation. Comp Biochem Physiol A Mol Integr Physiol 2002; 132:1-8.

21. Ravnsborg T, Olsen DT, Thysen AH, Christiansen M, Houen G, Hojrup P. The glycosylation and characterization of the candidate Gc macrophage activating factor. Biochim Biophys Acta 2010; 1804:909-917.

22. Inui T, Kuchiike D, Kubo K, Mette M, Uto Y, Hori H, et al. Clinical experience of integrative cancer immunotherapy with GcMAF. Anticancer Res 2013; 33:2917-2919.

23. Pacini S, Punzi T, Morucci G, Gulisano M, Ruggiero M. Effects of vitamin D-binding protein-derived macrophage-activating factor on human breast cancer cells. Anticancer Res 2012; 32:45-52.

24. Inoue T, Ishikawa M, Sumiya Y, Kohda H, Inui T, Kuchiike D, et al. Establishment of a Macrophage-activating factor assay system using the human monocytic cell line THP-1. Anticancer Res 2015; 35:4441-4445.

25. Ishikawa M, Inoue T, Inui T, Kuchiike D, Kubo K, Uto Y,  et al. A novel assay system for macrophage-activating factor activity using a human U937 cell line. Anticancer Res 2014; 34:4577-45781.

26. Thyer L, Ward E, Smith R, Fiore MG, Magherini S, Branca JJ, et al. A novel role for a major component of the vitamin D axis: vitamin D binding protein-derived macrophage activating factor induces human breast cancer cell apoptosis through stimulation of macrophages. Nutrients 2013; 5:2577-2589.

27. Reddi AL, Sankaranarayanan K, Arulraj HS, Devaraj N, Devaraj H. Serum alpha-N-acetylgalactosaminidase is associated with diagnosis/prognosis of patients with squamous cell carcinoma of the uterine cervix. Cancer Lett 2000; 158:61-64.

28. Elahian F, Kalalinia F, Behravan J. Dexamethasone downregulates BCRP mRNA and protein expression in breast cancer cell lines. Oncol Res 2009; 18:9-15.

29. Wang K, Wu F, Seo BR, Fischbach C, Chen W, Hsu L, et al. Breast cancer cells alter the dynamics of stromal fibronectin-collagen interactions. Matrix Biol 2016; 60:86-95.

30. Theocharis AD, Skandalis SS, Gialeli C, Karamanos NK. Extracellular matrix structure. Adv Drug Deliv Rev 2016; 97:4-27.

31. Varki A, Freeze HH. Glycans in Acquired Human Diseases. In: Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, et al. editors. Essentials of Glycobiology. 2nd ed. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 43.

32. Sugahara K, Mikami T, Uyama T, Mizuguchi S, Nomura K, Kitagawa H. Recent advances in the structural biology of chondroitin sulfate and dermatan sulfate. Curr Opin Struct Biol 2003; 13:612-620.

33. Lo CM, Wang HB, Dembo M, Wang YL. Cell movement is guided by the rigidity of the substrate. Biophys J 2000; 79:144-152.

34. Murakami T, Murakami S, Fuyama Y, Taguchi T, Ohtsuka A. The extracellular matrix in the mature brain: its reactions to endo-alpha-N-acetylgalactosaminidase and collagenase. Ital J Anat Embryol 2001; 106:475-480.

35. Murakami T, Ohtsuka A, Su WD, Taguchi T, Oohashi T, Abe K, et al. The extracellular matrix in the mouse brain: its reactions to endo-alpha-N-acetylgalactosaminidase and certain other enzymes. Arch Histol Cytol 1999; 62:273-281.

36. Murakami T, Murakami S, Fuyama Y, Taguchi T, Ohtsuka A. The extracellular matrix in the mature brain: its reactions to endo-alpha-N-acetylgalactosaminidase and collagenase. Ital J Anat Embryol 2001; 106:475-480.

37. Springer NL, Fischbach C. Biomaterials approaches to modeling macrophage-extracellular matrix interactions in the tumor microenvironment. Curr Opin Biotechnol 2016; 40:16-23.

38. Saburi E, Saburi A, Ghanei M. Promising role for Gc-MAF in cancer immunotherapy: from bench to bedside approach. Caspian J Intern Med 2017; 8:228-238.

39. Epp A, Sullivan KC, Herr AB, Strait RT. Immunoglobulin glycosylation effects in allergy and immunity. Curr Allergy Asthma Rep 2016; 16:79.

40. Hayes JM, Frostell A, Cosgrave EF, Struwe WB, Potter O, Davey GP, et al. Fc gamma receptor glycosylation modulates the binding of IgG glycoforms: a requirement for stable antibody interactions. J Proteome Res 2014; 13:5471-5485.

41. Liu H, Gaza-Bulseco G, Xiang T, Chumsae C. Structural effect of deglycosylation and methionine oxidation on a recombinant monoclonal antibody. Mol Immunol 2008; 45:701-708.

42. Kazuno S, Furukawa J, Shinohara Y, Murayama K, Fujime M, Ueno T, et al. Glycosylation status of serum immunoglobulin G in patients with prostate diseases. Cancer Med 2016; 5:1137-1146.

43. Soliman C, Yuriev E, Ramsland PA. Antibody recognition of aberrant glycosylation on the surface of cancer cells. Curr Opin Struct Biol 2016; 44:1-8.

44. Chen Z, Gulzar ZG, St Hill CA, Walcheck B, Brooks JD. Increased expression of GCNT1 is associated with altered O-glycosylation of PSA, PAP, and MUC1 in human prostate cancers. Prostate 2014; 74:1059-1067.

45. Yoneyama T, Ohyama C, Hatakeyama S, Narita S, Habuchi T, Koie T, et al. Measurement of aberrant glycosylation of prostate specific antigen can improve specificity in early detection of prostate cancer. BiochemBiophys Res Commun 2014; 448:390-396.

46. Brooks SA, Carter TM, Bennett EP, Clausen H, Mandel U. Immunolocalisation of members of the polypeptide N-acetylgalactosaminyl transferase (ppGalNAc-T) family is consistent with biologically relevant altered cell surface glycosylation in breast cancer. Acta Histochem 2007; 109:273-284.

47. Patani N, Jiang W, Mokabel K. Prognostic utility of glycosyltransferase expression in breast cancer. Cancer Genomics-Proteomics 2008; 5:333-340.

48. Taniuchi K, Cerny RL, Tanouchi A, Kohno K, Kotani N, Honke K, et al. Overexpression of GalNAc-transferase GalNAc-T3 promotes pancreatic cancer cell growth. Oncogene 2011; 30:4843-4854.

49. Ramessur K, Greenwell P, Nash R, Dwek M. Breast cancer invasion is mediated by β-N-acetylglucosaminidase (β-NAG) and associated with a dysregulation in the secretory pathway of cancer cells. Br J Niomed Sci 2010; 67:189-196.

50. Li D, Li Y, Wu X, Li Q, Yu J, Gen J, et al. Knockdown of Mgat5 inhibits breast cancer cell growth with activation of CD4+ T cells and macrophages. J Immunol 2008; 180:3158-3165.

51. Ren F, Tang R, Zhang X, Madushi WM, Luo D, Dang Y, et al. Overexpression of MMP Family Members Functions as Prognostic Biomarker for Breast Cancer Patients: A Systematic Review and Meta-Analysis. PLoS One 2015; 10:e0135544.

52. Singh N, Das P, Gupta SD, Sahni P, Pandey R, Gupta S, et al. Prognostic Significance of Extracellular Matrix Degrading Enzymes—Cathepsin L and Matrix Metalloproteases-2 [MMP-2] in Human Pancreatic Cancer. Cancer Invest 2013; 31:461-471.

53. McKenzie E. Heparanase: a target for drug discovery in cancer and inflammation. Brit J Pharmacol 2007; 151:1-14.

54. Glawar A, Martínez R, Ayers B, Hollas M, Ngo N, Nakagawa S, et al. Structural essentials for β-N-acetylhexosaminidase inhibition by amides of prolines, pipecolic and azetidine carboxylic acids. Org Biomol Chem 2016; 14:10371-10385.