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In Vitro Validation of the Hippo Pathway as a Pharmacological Target for Canine Mammary Gland Tumors

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Abstract

Canine mammary tumors (CMTs) are the most common neoplasms in intact female dogs. Some clinical and molecular similarities between certain CMT subtypes and breast cancer make them a potential model for the study of the human disease. As misregulated Hippo signaling is thought to play an important role in breast cancer development and also occurs in CMTs, we sought to determine if Hippo represents a valid pharmacological target for the treatment of CMTs. Six CMT cell lines were assessed for their expression of the Hippo pathway effectors YAP and TAZ and for their sensitivity to verteporfin, an inhibitor of YAP-mediated transcriptional coactivation. Four cell lines that expressed YAP (CMT-9, −12, −28, −47) were found to be very sensitive to verteporfin treatment, which killed the cells through induction of apoptosis with ED50 values of 14–79 nM. Conversely, two YAP-negative cell lines (CF-35, CMT-25) were an order of magnitude more resistant to verteporfin. Verteporfin suppressed the expression of YAP/TAZ target genes, particularly CYR61 and CTGF, which play important roles in breast cancer development. Verteporfin was also able to inhibit cell migration and anchorage-independent growth. Likewise, verteporfin efficiently suppressed tumor cell invasiveness in the CMT-28 and -47 lines, but not in CF-35 cells. Together, our findings provide proof of principle that pharmacological targeting of the Hippo pathway compromises the viability and attenuates the malignant behavior of CMT cells. These results will serve as the basis for the development of novel chemotherapeutic approaches for CMTs that could translate to human medicine.

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References

  1. Sleeckx N, de Rooster H, Veldhuis Kroeze EJ, et al. Canine mammary tumours, an overview. Reprod Domest Anim. 2011;46(6):1112–31.

    Article  CAS  PubMed  Google Scholar 

  2. Benjamin SA, Lee AC, Saunders WJ. Classification and behavior of canine mammary epithelial neoplasms based on life-span observations in beagles. Vet Pathol. 1999;36(5):423–36.

    Article  CAS  PubMed  Google Scholar 

  3. Sorenmo KU, Kristiansen VM, Cofone MA, et al. Canine mammary gland tumours; a histological continuum from benign to malignant; clinical and histopathological evidence. Vet Comp Oncol. 2009;7(3):162–72.

    Article  CAS  PubMed  Google Scholar 

  4. Withrow SJ, Vail DM. Withrow & MacEwen's small animal clinical oncology. 4th ed. St. Louis, Mo.: Saunders Elsevier; 2007. 1 texte électronique (xvii, 846 ) p.

  5. Meuten DJ, Moulton JE. Tumors in domestic animals. Fifth edition. ed. Ames, Iowa: Wiley Blackwell; 2017. viii, 989 pages p.

  6. Karayannopoulou M, Lafioniatis, S. Progrès récents chimiothérapie des tumeurs mammaires canines: examen des études de 2000 à ce jour. Revue de Médecine Vétérinaire. 2016;7–8(167):191–200.

  7. Simon D, Schoenrock D, Baumgartner W, et al. Postoperative adjuvant treatment of invasive malignant mammary gland tumors in dogs with doxorubicin and docetaxel. J Vet Intern Med. 2006;20(5):1184–90.

    PubMed  Google Scholar 

  8. Klopfleisch R, von Euler H, Sarli G, et al. Molecular carcinogenesis of canine mammary tumors: news from an old disease. Vet Pathol. 2011;48(1):98–116.

    Article  CAS  PubMed  Google Scholar 

  9. Marconato L, Lorenzo RM, Abramo F, et al. Adjuvant gemcitabine after surgical removal of aggressive malignant mammary tumours in dogs. Vet Comp Oncol. 2008;6(2):90–101.

    Article  CAS  PubMed  Google Scholar 

  10. Harvey KF, Zhang X, Thomas DM. The hippo pathway and human cancer. Nat Rev Cancer. 2013;13(4):246–57.

    Article  CAS  PubMed  Google Scholar 

  11. Johnson R, Halder G. The two faces of hippo: targeting the hippo pathway for regenerative medicine and cancer treatment. Nat Rev Drug Discov. 2014;13(1):63–79.

    Article  CAS  PubMed  Google Scholar 

  12. Park HW, Guan KL. Regulation of the hippo pathway and implications for anticancer drug development. Trends Pharmacol Sci. 2013;34(10):581–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zhao B, Wei X, Li W, et al. Inactivation of YAP oncoprotein by the hippo pathway is involved in cell contact inhibition and tissue growth control. Genes Dev. 2007;21(21):2747–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Zhao B, Li L, Wang L, et al. Cell detachment activates the hippo pathway via cytoskeleton reorganization to induce anoikis. Genes Dev. 2012;26(1):54–68.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Halder G, Dupont S, Piccolo S. Transduction of mechanical and cytoskeletal cues by YAP and TAZ. Nat Rev Mol Cell Biol. 2012;13(9):591–600.

    Article  CAS  PubMed  Google Scholar 

  16. Low BC, Pan CQ, Shivashankar GV, et al. YAP/TAZ as mechanosensors and mechanotransducers in regulating organ size and tumor growth. FEBS Lett. 2014;588(16):2663–70.

    Article  CAS  PubMed  Google Scholar 

  17. Wada K, Itoga K, Okano T, et al. Hippo pathway regulation by cell morphology and stress fibers. Development. 2011;138(18):3907–14.

    Article  CAS  PubMed  Google Scholar 

  18. Zhao B, Ye X, Yu J, et al. TEAD mediates YAP-dependent gene induction and growth control. Genes Dev. 2008;22(14):1962–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Gomez M, Gomez V, Hergovich A. The hippo pathway in disease and therapy: cancer and beyond. Clin Transl Med. 2014;3:22.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Piccolo S, Dupont S, Cordenonsi M. The biology of YAP/TAZ: hippo signaling and beyond. Physiol Rev. 2014;94(4):1287–312.

    Article  CAS  PubMed  Google Scholar 

  21. Morin-Kensicki EM, Boone BN, Howell M, et al. Defects in yolk sac vasculogenesis, chorioallantoic fusion, and embryonic axis elongation in mice with targeted disruption of Yap65. Mol Cell Biol. 2006;26(1):77–87.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Hossain Z, Ali SM, Ko HL, et al. Glomerulocystic kidney disease in mice with a targeted inactivation of Wwtr1. Proc Natl Acad Sci U S A. 2007;104(5):1631–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Makita R, Uchijima Y, Nishiyama K, et al. Multiple renal cysts, urinary concentration defects, and pulmonary emphysematous changes in mice lacking TAZ. Am J Physiol Ren Physiol. 2008;294(3):F542–53.

    Article  CAS  Google Scholar 

  24. Vlug EJ, van de Ven RA, Vermeulen JF, et al. Nuclear localization of the transcriptional coactivator YAP is associated with invasive lobular breast cancer. Cell Oncol (Dordr). 2013;36(5):375–84.

    Article  CAS  Google Scholar 

  25. Chen Q, Zhang N, Gray RS, et al. A temporal requirement for hippo signaling in mammary gland differentiation, growth, and tumorigenesis. Genes Dev. 2014;28(5):432–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Barron DA, Kagey JD. The role of the hippo pathway in human disease and tumorigenesis. Clin Transl Med. 2014;3:25.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Steinhardt AA, Gayyed MF, Klein AP, et al. Expression of yes-associated protein in common solid tumors. Hum Pathol. 2008;39(11):1582–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Bartucci M, Dattilo R, Moriconi C, et al. TAZ is required for metastatic activity and chemoresistance of breast cancer stem cells. Oncogene. 2015;34(6):681–90.

    Article  CAS  PubMed  Google Scholar 

  29. Cordenonsi M, Zanconato F, Azzolin L, et al. The hippo transducer TAZ confers cancer stem cell-related traits on breast cancer cells. Cell. 2011;147(4):759–72.

    Article  CAS  PubMed  Google Scholar 

  30. Lamar JM, Stern P, Liu H, et al. The hippo pathway target, YAP, promotes metastasis through its TEAD-interaction domain. Proc Natl Acad Sci U S A. 2012;109(37):E2441–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Shi P, Feng J, Chen C. Hippo pathway in mammary gland development and breast cancer. Acta Biochim Biophys Sin Shanghai. 2015;47(1):53–9.

    Article  CAS  PubMed  Google Scholar 

  32. Chan SW, Lim CJ, Guo K, et al. A role for TAZ in migration, invasion, and tumorigenesis of breast cancer cells. Cancer Res. 2008;68(8):2592–8.

    Article  CAS  PubMed  Google Scholar 

  33. Beffagna G, Sacchetto R, Cavicchioli L, et al. A preliminary investigation of the role of the transcription co-activators YAP/TAZ of the hippo signalling pathway in canine and feline mammary tumours. Vet J. 2016;207:105–11.

    Article  CAS  PubMed  Google Scholar 

  34. Bressler NM. Verteporfin therapy of subfoveal choroidal neovascularization in age-related macular degeneration: two-year results of a randomized clinical trial including lesions with occult with no classic choroidal neovascularization-verteporfin in photodynamic therapy report 2. Am J Ophthalmol. 2002;133(1):168–9.

    Article  PubMed  Google Scholar 

  35. Blinder KJ, Blumenkranz MS, Bressler NM, et al. Verteporfin therapy of subfoveal choroidal neovascularization in pathologic myopia: 2-year results of a randomized clinical trial--VIP report no. 3. Ophthalmology. 2003;110(4):667–73.

    Article  PubMed  Google Scholar 

  36. Wang C, Zhu X, Feng W, et al. Verteporfin inhibits YAP function through up-regulating 14-3-3sigma sequestering YAP in the cytoplasm. Am J Cancer Res. 2016;6(1):27–37.

    CAS  PubMed  Google Scholar 

  37. Wei H, Wang F, Wang Y, et al. Verteporfin suppresses cell survival, angiogenesis and vasculogenic mimicry of PDAC via disrupting the YAP-TEAD complex. Cancer Sci 2016.

  38. Feng J, Gou J, Jia J, et al. Verteporfin, a suppressor of YAP-TEAD complex, presents promising antitumor properties on ovarian cancer. Onco Targets Ther. 2016;9:5371–81.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Felley-Bosco E, Stahel R. Hippo/YAP pathway for targeted therapy. Transl Lung Cancer Res. 2014;3(2):75–83.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Ma YW, Liu YZ, Pan JX. Verteporfin induces apoptosis and eliminates cancer stem-like cells in uveal melanoma in the absence of light activation. Am J Cancer Res. 2016;6(12):2816–30.

    PubMed  PubMed Central  Google Scholar 

  41. Zhang H, Ramakrishnan SK, Triner D, et al. Tumor-selective proteotoxicity of verteporfin inhibits colon cancer progression independently of YAP1. Sci Signal. 2015;8(397):ra98.

  42. Li H, Huang Z, Gao M, et al. Inhibition of YAP suppresses CML cell proliferation and enhances efficacy of imatinib in vitro and in vivo. J Exp Clin Cancer Res. 2016;35(1):134.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Donohue E, Thomas A, Maurer N, et al. The autophagy inhibitor verteporfin moderately enhances the antitumor activity of gemcitabine in a pancreatic ductal adenocarcinoma model. J Cancer. 2013;4(7):585–96.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Lutful Kabir FM, Agarwal P, Deinnocentes P, et al. Novel frameshift mutation in the p16/INK4A tumor suppressor gene in canine breast cancer alters expression from the p16/INK4A/p14ARF locus. J Cell Biochem. 2013;114(1):56–66.

    Article  CAS  PubMed  Google Scholar 

  45. DeInnocentes P, Li LX, Sanchez RL, et al. Expression and sequence of canine SIRT2 and p53 genes in canine mammary tumour cells - effects on downstream targets Wip1 and p21/Cip1. Vet Comp Oncol. 2006;4(3):161–77.

    Article  CAS  PubMed  Google Scholar 

  46. Stokol T, Daddona JL, Mubayed LS, et al. Evaluation of tissue factor expression in canine tumor cells. Am J Vet Res. 2011;72(8):1097–106.

    Article  CAS  PubMed  Google Scholar 

  47. Abedini A, Zamberlam G, Lapointe E, et al. WNT5a is required for normal ovarian follicle development and antagonizes gonadotropin responsiveness in granulosa cells by suppressing canonical WNT signaling. FASEB J. 2016;30(4):1534–47.

    Article  CAS  PubMed  Google Scholar 

  48. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 2001;25(4):402–8.

    Article  CAS  PubMed  Google Scholar 

  49. Santucci M, Vignudelli T, Ferrari S, et al. The hippo pathway and YAP/TAZ-TEAD protein-protein interaction as targets for regenerative medicine and cancer treatment. J Med Chem. 2015;58(12):4857–73.

    Article  CAS  PubMed  Google Scholar 

  50. Fu D, Lv X, Hua G, et al. YAP regulates cell proliferation, migration, and steroidogenesis in adult granulosa cell tumors. Endocr Relat Cancer. 2014;21(2):297–310.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Aqeilan RI. Hippo signaling: to die or not to die. Cell Death Differ. 2013;20(10):1287–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Maugeri-Sacca M, De Maria R. Hippo pathway and breast cancer stem cells. Crit Rev Oncol Hematol. 2016;99:115–22.

    Article  PubMed  Google Scholar 

  53. Kleer CG. Dual roles of CCN proteins in breast cancer progression. J Cell Commun Signal. 2016;10(3):217–22.

    Article  PubMed  PubMed Central  Google Scholar 

  54. Tsai MS, Bogart DF, Castaneda JM, et al. Cyr61 promotes breast tumorigenesis and cancer progression. Oncogene. 2002;21(53):8178–85.

    Article  CAS  PubMed  Google Scholar 

  55. Jiang WG, Watkins G, Fodstad O, et al. Differential expression of the CCN family members Cyr61, CTGF and Nov in human breast cancer. Endocr Relat Cancer. 2004;11(4):781–91.

    Article  CAS  PubMed  Google Scholar 

  56. Sanchez-Bailon MP, Calcabrini A, Mayoral-Varo V, et al. Cyr61 as mediator of Src signaling in triple negative breast cancer cells. Oncotarget. 2015;6(15):13520–38.

    Article  PubMed  PubMed Central  Google Scholar 

  57. Espinoza I, Menendez JA, Kvp CM, et al. CCN1 promotes vascular endothelial growth factor secretion through alphavbeta 3 integrin receptors in breast cancer. J Cell Commun Signal. 2014;8(1):23–7.

    Article  PubMed  Google Scholar 

  58. Harris LG, Pannell LK, Singh S, et al. Increased vascularity and spontaneous metastasis of breast cancer by hedgehog signaling mediated upregulation of cyr61. Oncogene. 2012;31(28):3370–80.

    Article  CAS  PubMed  Google Scholar 

  59. Xie D, Nakachi K, Wang H, et al. Elevated levels of connective tissue growth factor, WISP-1, and CYR61 in primary breast cancers associated with more advanced features. Cancer Res. 2001;61(24):8917–23.

    CAS  PubMed  Google Scholar 

  60. Wang MY, Chen PS, Prakash E, et al. Connective tissue growth factor confers drug resistance in breast cancer through concomitant up-regulation of Bcl-xL and cIAP1. Cancer Res. 2009;69(8):3482–91.

    Article  CAS  PubMed  Google Scholar 

  61. Chien W, O'Kelly J, Lu D, et al. Expression of connective tissue growth factor (CTGF/CCN2) in breast cancer cells is associated with increased migration and angiogenesis. Int J Oncol. 2011;38(6):1741–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Lai D, Ho KC, Hao Y, et al. Taxol resistance in breast cancer cells is mediated by the hippo pathway component TAZ and its downstream transcriptional targets Cyr61 and CTGF. Cancer Res. 2011;71(7):2728–38.

    Article  CAS  PubMed  Google Scholar 

  63. Morishita T, Hayakawa F, Sugimoto K, et al. The photosensitizer verteporfin has light-independent anti-leukemic activity for Ph-positive acute lymphoblastic leukemia and synergistically works with dasatinib. Oncotarget. 2016;7(35):56241–52.

    Article  PubMed  PubMed Central  Google Scholar 

  64. Ciamporcero E, Shen H, Ramakrishnan S, et al. YAP activation protects urothelial cell carcinoma from treatment-induced DNA damage. Oncogene. 2016;35(12):1541–53.

    Article  CAS  PubMed  Google Scholar 

  65. Hussain RN, Jmor F, Damato B, et al. Verteporfin photodynamic therapy for the treatment of choroidal haemangioma associated with Sturge-weber syndrome. Photodiagn Photodyn Ther. 2016;15:143–6.

    Article  CAS  Google Scholar 

  66. Gupta R, Browning AC, Wu K, et al. Verteporfin photodynamic therapy for the treatment of persistent subfoveal choroidal neovascularization after external beam radiotherapy: one-year results. Am J Ophthalmol. 2005;139(3):561–2.

    Article  PubMed  Google Scholar 

  67. Mori R, Kelkar A, De Laey JJ. Photodynamic therapy with verteporfin in Belgian patients with subfoveal choroidal neovascularization secondary to age-related macular degeneration. Bull Soc Belge Ophtalmol. 2006;299:57–64.

    Google Scholar 

  68. Tayanithi P, Pisankosakul P, Laksakapuk P. Treatment of subfoveal choroidal neovascularization secondary to age related macular degeneration with single treatment of verteporfin photodynamic therapy: a safety and short-term outcome. J Med Assoc Thail. 2004;87(Suppl 2):S78–82.

    Google Scholar 

  69. Josefsen LB, Boyle RW. Photodynamic therapy and the development of metal-based photosensitisers. Metal-Based Drugs. 2008;2008:276109.

    Article  PubMed  PubMed Central  Google Scholar 

  70. Likus W, Siemianowicz K, Bienk K, et al. Could drugs inhibiting the mevalonate pathway also target cancer stem cells? Drug Resist Updat. 2016;25:13–25.

    Article  PubMed  Google Scholar 

  71. Wang Z, Wu Y, Wang H, et al. Interplay of mevalonate and hippo pathways regulates RHAMM transcription via YAP to modulate breast cancer cell motility. Proc Natl Acad Sci U S A 2014;111(1):E89–E98.

  72. Lin Q, Yang W. The hippo-YAP/TAZ pathway mediates geranylgeranylation signaling in breast cancer progression. Mol Cell Oncol. 2016;3(3):e969638.

    Article  PubMed  PubMed Central  Google Scholar 

  73. Sorrentino G, Ruggeri N, Specchia V, et al. Metabolic control of YAP and TAZ by the mevalonate pathway. Nat Cell Biol. 2014;16(4):357–66.

    Article  CAS  PubMed  Google Scholar 

  74. Undela K, Srikanth V, Bansal D. Statin use and risk of breast cancer: a meta-analysis of observational studies. Breast Cancer Res Treat. 2012;135(1):261–9.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors thank Ms. Meggie Girard for excellent technical assistance and Mr. Guy Beauchamp for statistical analyses.

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  1. Département de pathologie et de microbiologie, Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada

    Samantha Guillemette & Marilène Paquet

  2. Département de Biomédecine Vétérinaire, Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada

    Charlène Rico, Philippe Godin & Derek Boerboom

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  1. Samantha Guillemette
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Correspondence to Marilène Paquet.

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This work was supported by a First Award grant from the Morris Animal Foundation to MP. SG was supported by a bursary from the Fonds de Recherche du Québec – Nature et Technologies.

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Guillemette, S., Rico, C., Godin, P. et al. In Vitro Validation of the Hippo Pathway as a Pharmacological Target for Canine Mammary Gland Tumors. J Mammary Gland Biol Neoplasia 22, 203–214 (2017). https://doi.org/10.1007/s10911-017-9384-9

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