Activin A triggers angiogenesis via regulation of VEGFA and its overexpression is associated with poor prognosis of oral squamous cell carcinoma

Ervolino De Oliveira,Carine; Dourado,Maurício  Rocha; Sawazaki‑Calone,Íris; Costa De Medeiros,Marcell; Rossa Júnior,Carlos; De Karla Cervigne,Nilva; Esquiche León,Jorge; Lambert,Daniel; Salo,Tuula; Graner,Edgard; Coletta,Ricardo  D.

doi:10.3892/ijo.2020.5058

Activin A triggers angiogenesis via regulation of VEGFA and its overexpression is associated with poor prognosis of oral squamous cell carcinoma

Authors:
- Carine Ervolino De Oliveira
- Maurício Rocha Dourado
- Íris Sawazaki‑Calone
- Marcell Costa De Medeiros
- Carlos Rossa Júnior
- Nilva De Karla Cervigne
- Jorge Esquiche León
- Daniel Lambert
- Tuula Salo
- Edgard Graner
- Ricardo D. Coletta
View Affiliations

Affiliations: Department of Oral Diagnosis, School of Dentistry, University of Campinas, Piracicaba, SP 13414‑018, Brazil, Department of Oral Pathology and Oral Medicine, Dentistry School, Western Paraná State University, Cascavel, PR 85819‑170, Brazil, Departament of Diagnosis and Surgery, School of Dentistry at Araraquara, Araraquara, SP 14801‑385, Brazil, Departament of Diagnosis and Surgery, School of Dentistry at Araraquara, Araraquara, SP 14801‑385, Brazil, Clinical Department, Faculty of Medicine of Jundiai, Jundiai, SP 13202‑550, Brazil, Departament of Stomatology, Public Oral Health and Forensic Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP 14040‑904, Brazil, Integrated Biosciences, School of Clinical Dentistry and Sheffield Cancer Centre, University of Sheffield, Sheffield S10 2TG, UK, Cancer and Translational Medicine Research Unit, Faculty of Medicine and Medical Research Center Oulu, Oulu University Hospital, University of Oulu, Oulu 90220, Finland
Published online on: May 4, 2020 https://doi.org/10.3892/ijo.2020.5058
Pages: 364-376

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Poor prognosis associated with the dysregulated expression of activin A in a number of malignancies has been related to with numerous aspects of tumorigenesis, including angiogenesis. The present study investigated the prognostic significance of activin A immunoexpression in blood vessels and cancer cells in a number of oral squamous cell carcinoma (OSCC) cases and applied in vitro strategies to determine the impact of activin A on angiogenesis. In a cohort of 95 patients with OSCC, immunoexpression of activin A in both blood vessels and tumor cells was quantified and the association with clinicopathological parameters and survival was analyzed. Effects of activin A on the tube formation, proliferation and migration of human umbilical vein endothelial cells (HUVECs) were evaluated in gain‑of‑function (treatment with recombinant activin A) or loss‑of‑function [treatment with activin A‑antagonist follistatin or by stable transfection with short hairpin RNA (shRNA) targeting activin A] conditions. Conditioned medium from an OSCC cell line with shRNA‑mediated depletion of activin A was also tested. The profile of pro‑ and anti‑angiogenic factors regulated by activin A was assessed with a human angiogenesis quantitative PCR (qPCR) array. Vascular endothelial growth factor A (VEGFA) and its major isoforms were evaluated by reverse transcription‑qPCR and ELISA. Activin A expression in blood vessels demonstrated an independent prognostic value in the multivariate analysis with a hazard ratio of 2.47 [95% confidence interval (CI), 1.30‑4.71; P=0.006) for disease‑specific survival and 2.09 (95% CI, 1.07‑4.08l: P=0.03) for disease‑free survival. Activin A significantly increased tubular formation of HUVECs concomitantly with an increase in proliferation. This effect was validated by reduced proliferation and tubular formation of HUVECs following inhibition of activin A by follistatin or shRNA, as well as by treatment of HUVECs with conditioned medium from activin A‑depleted OSCC cells. Activin A‑knockdown increased the migration of HUVECs. In addition, activin A stimulated the phosphorylation of SMAD2/3 and the expression and production of total VEGFA, significantly enhancing the expression of its pro‑angiogenic isoform 121. The present findings suggest that activin A is a predictor of the prognosis of patients with OSCC, and provide evidence that activin A, in an autocrine and paracrine manner, may contribute to OSCC angiogenesis through differential expression of the isoform 121 of VEGFA.

View Figures

View References

1	Matzuk MM, Kumar TR, Vassalli A, Bickenbach JR, Roop DR, Jaenisch R and Bradley A: Functional analysis of activins during mammalian development. Nature. 374:354–356. 1995. View Article : Google Scholar : PubMed/NCBI
2	Ying SY, Zhang Z, Furst B, Batres Y, Huang G and Li G: Activins and activin receptors in cell growth. Proc Soc Exp Biol Med. 214:114–122. 1997. View Article : Google Scholar : PubMed/NCBI
3	Antsiferova M and Werner S: The bright and the dark sides of activin in wound healing and cancer. J Cell Sci. 125:3929–3937. 2012. View Article : Google Scholar : PubMed/NCBI
4	Loomans HA and Andl CD: Intertwining of Activin A and TGFβ Signaling: Dual roles in cancer progression and cancer cell invasion. Cancers (Basel). 7:70–91. 2014. View Article : Google Scholar
5	McDowell N, Zorn AM, Crease DJ and Gurdon JB: Activin has direct long-range signalling activity and can form a concentration gradient by diffusion. Curr Biol. 7:671–681. 1997. View Article : Google Scholar : PubMed/NCBI
6	Wildi S, Kleeff J, Maruyama H, Maurer CA, Büchler MW and Korc M: Overexpression of activin A in stage IV colorectal cancer. Gut. 49:409–417. 2001. View Article : Google Scholar : PubMed/NCBI
7	Gold E and Risbridger G: Activins and activin antagonists in the prostate and prostate cancer. Mol Cell Endocrinol. 359:107–112. 2012. View Article : Google Scholar
8	Loumaye A, de Barsy M, Nachit M, Lause P, van Maanen A, Trefois P, Gruson D and Thissen JP: Circulating Activin A predicts survival in cancer patients. J Cachexia Sarcopenia Muscle. 8:768–777. 2017. View Article : Google Scholar : PubMed/NCBI
9	Seachrist DD and Keri RA: The Activin social network: Activin, inhibin and follistatin in breast development and cancer. Endocrinology. 160:1097–1110. 2019. View Article : Google Scholar : PubMed/NCBI
10	Sobral LM, Bufalino A, Lopes MA, Graner E, Salo T and Coletta RD: Myofbroblasts in the stroma of oral cancer promote tumorigenesis via secretion of activin A. Oral Oncol. 47:840–846. 2011. View Article : Google Scholar : PubMed/NCBI
11	Bufalino A, Cervigne NK, de Oliveira CE, Fonseca FP, Rodrigues PC, Macedo CC, Sobral LM, Miguel MC, Lopes MA, Paes Leme AF, et al: Low miR-143/miR-145 cluster levels induce activin A overexpression in oral squamous cell carcinomas, which contributes to poor prognosis. PLoS One. 10:e01365992015. View Article : Google Scholar : PubMed/NCBI
12	Kelner N, Rodrigues PC, Bufalino A, Fonseca FP, Santos-Silva AR, Miguel MC, Pinto CA, Leme AF, Graner E, Salo T, et al: Activin A immunoexpression as predictor of occult lymph node metastasis and overall survival in oral tongue squamous cell carcinoma. Head Neck. 37:479–486. 2015. View Article : Google Scholar
13	Maeshima K, Maeshima A, Hayashi Y, Kishi S and Kojima I: Crucial role of activin a in tubulogenesis of endo-thelial cells induced by vascular endothelial growth factor. Endocrinology. 145:3739–3745. 2004. View Article : Google Scholar : PubMed/NCBI
14	Wagner K, Peters M, Scholz A, Benckert C, Ruderisch HS, Wiedenmann B and Rosewicz S: Activin A stimulates vascular endothelial growth factor gene transcription in human hepato-cellular carcinoma cells. Gastroenterology. 126:1828–1843. 2004. View Article : Google Scholar : PubMed/NCBI
15	Bashir M, Damineni S, Mukherjee G and Kondaiah P: Activin-A signaling promotes epithelial-mesenchymal transition, invasion, and metastatic growth of breast cancer. NPJ Breast Cancer. 1:150072015. View Article : Google Scholar : PubMed/NCBI
16	Panopoulou E, Murphy C, Rasmussen H, Bagli E, Rofstad EK and Fotsis T: Activin A suppresses neuroblastoma xenograft tumor growth via antimitotic and antiangiogenic mechanisms. Cancer Res. 65:1877–1886. 2005. View Article : Google Scholar : PubMed/NCBI
17	Krneta J, Kroll J, Alves F, Prahst C, Sananbenesi F, Dullin C, Kimmina S, Phillips DJ and Augustin HG: Dissociation of angio-genesis and tumorigenesis in follistatin- and activin-expressing tumors. Cancer Res. 66:5686–5695. 2006. View Article : Google Scholar : PubMed/NCBI
18	Kaneda H, Arao T, Matsumoto K, De Velasco MA, Tamura D, Aomatsu K, Kudo K, Sakai K, Nagai T, Fujita Y, et al: Activin A inhibits vascular endothelial cell growth and suppresses tumour angiogenesis in gastric cancer. Br J Cancer. 105:1210–1217. 2011. View Article : Google Scholar : PubMed/NCBI
19	Sawazaki-Calone I, Rangel A, Bueno AG, Morais CF, Nagai HM, Kunz RP, Souza RL, Rutkauskis L, Salo T, Almangush A, et al: The prognostic value of histopathological grading systems in oral squamous cell carcinomas. Oral Dis. 21:755–761. 2015. View Article : Google Scholar : PubMed/NCBI
20	Agostini M, Almeida LY, Bastos DC, Ortega RM, Moreira FS, Seguin F, Zecchin KG, Raposo HF, Oliveira HC, Amoêdo ND, et al: The fatty acid synthase inhibitor orlistat reduces the growth and metastasis of orthotopic tongue oral squamous cell carcinomas. Mol Cancer Ther. 13:585–595. 2014. View Article : Google Scholar
21	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar
22	Salo T, Sutinen M, Hoque Apu E, Sundquist E, Cervigne NK, de Oliveira CE, Akram SU, Ohlmeier S, Suomi F, Eklund L, et al: A novel human leiomyoma tissue derived matrix for cell culture studies. BMC Cancer. 15:9812015. View Article : Google Scholar : PubMed/NCBI
23	De Palma M, Biziato D and Petrova TV: Microenvironmental regulation of tumour angiogenesis. Nat Rev Cancer. 17:457–474. 2017. View Article : Google Scholar : PubMed/NCBI
24	Chang KP, Kao HK, Liang Y, Cheng MH, Chang YL, Liu SC, Lin YC, Ko TY, Lee YS, Tsai CL, et al: Overexpression of activin A in oral squamous cell carcinoma: Association with poor prognosis and tumor progression. Ann Surg Oncol. 17:1945–1956. 2010. View Article : Google Scholar : PubMed/NCBI
25	Tsai CN, Tsai CL, Yi JS, Kao HK, Huang Y, Wang CI, Lee YS and Chang KP: Activin A regulates the epidermal growth factor receptor promoter by activating the PI3K/SP1 pathway in oral squamous cell carcinoma cells. Sci Rep. 9:51972019. View Article : Google Scholar : PubMed/NCBI
26	Togashi Y, Kogita A, Sakamoto H, Hayashi H, Terashima M, de Velasco MA, Sakai K, Fujita Y, Tomida S, Kitano M, et al: Activin signal promotes cancer progression and is involved in cachexia in a subset of pancreatic cancer. Cancer Lett. 356(2 Pt B): 819–827. 2015. View Article : Google Scholar
27	Hoda MA, Rozsas A, Lang E, Klikovits T, Lohinai Z, Torok S, Berta J, Bendek M, Berger W, Hegedus B, et al: High circulating activin A level is associated with tumor progression and predicts poor prognosis in lung adenocarcinoma. Oncotarget. 7:13388–13399. 2016. View Article : Google Scholar : PubMed/NCBI
28	Jueckstock J, Burkhardt N, Kuhn C, Blankenstein T, Mahner S, Schindlbeck C, Janni W, Rack B and Mylonas I: Expression of activin during and after chemotherapy in peripheral blood of patients with primary breast cancer. Anticancer Res. 36:2153–2159. 2016.PubMed/NCBI
29	Staudacher JJ, Bauer J, Jana A, Tian J, Carroll T, Mancinelli G, Özden Ö, Krett N, Guzman G, Kerr D, et al: Activin signaling is an essential component of the TGF-β induced pro-metastatic phenotype in colorectal cancer. Sci Rep. 7:55692017. View Article : Google Scholar
30	Hayashi Y, Maeshima K, Goto F and Kojima I: Activin A as a critical mediator of capillary formation: Interaction with the fbroblast growth factor action. Endocr J. 54:311–318. 2007. View Article : Google Scholar : PubMed/NCBI
31	Kalli M, Mpekris F, Wong CK, Panagi M, Ozturk S, Thiagalingam S, Stylianopoulos T and Papageorgis P: Activin A signaling regulates IL13Rα2 expression to promote breast cancer metastasis. Front Oncol. 9:322019. View Article : Google Scholar
32	Donovan P, Dubey OA, Kallioinen S, Rogers KW, Muehlethaler K, Müller P, Rimoldi D and Constam DB: Paracrine activin-A signaling promotes melanoma growth and metastasis through immune evasion. J Invest Dermatol. 137:2578–2587. 2017. View Article : Google Scholar : PubMed/NCBI
33	McCarthy SA and Bicknell R: Inhibition of vascular endothelial cell growth by activin-A. J Biol Chem. 268:23066–23071. 1993.PubMed/NCBI
34	Schramm A, von Schuetz V, Christiansen H, Havers W, Papoutsi M, Wilting J and Schweigerer L: High activin A-expression in human neuroblastoma: Suppression of malignant potential and correlation with favourable clinical outcome. Oncogene. 24:680–687. 2005. View Article : Google Scholar
35	Merfeld-Clauss S, Lu H, Wu X, March KL and Traktuev DO: Hypoxia-induced activin A diminishes endothelial cell vascu-logenic activity. J Cell Mol Med. 22:173–184. 2018. View Article : Google Scholar
36	Fahmy-Garcia S, Farrell E, Witte-Bouma J, Robbesom-van den Berge I, Suarez M, Mumcuoglu D, Walles H, Kluijtmans SGJM, van der Eerden BCJ, van Osch GJVM, et al: Follistatin effects in migration, vascularization, and osteogenesis in vitro and bone repair in vivo. Front Bioeng Biotechnol. 7:382019. View Article : Google Scholar :
37	Kang HY, Huang HY, Hsieh CY, Li CF, Shyr CR, Tsai MY, Chang C, Chuang YC and Huang KE: Activin A enhances prostate cancer cell migration through activation of androgen receptor and is overexpressed in metastatic prostate cancer. J Bone Miner Res. 24:1180–1193. 2009. View Article : Google Scholar : PubMed/NCBI
38	Tahergorabi Z and Khazaei M: A review on angiogenesis and its assays. Iran J Basic Med Sci. 15:1110–1126. 2012.
39	Levchenko T, Veitonmäki N, Lundkvist A, Gerhardt H, Ming Y, Berggren K, Kvanta A, Carlsson R and Holmgren L: Therapeutic antibodies targeting angiomotin inhibit angiogenesis in vivo. FASEB J. 22:880–889. 2008. View Article : Google Scholar
40	Douglass S, Goyal A and Iozzo RV: The role of perlecan and endorepellin in the control of tumor angiogenesis and endothelial cell autophagy. Connect Tissue Res. 56:381–391. 2015. View Article : Google Scholar : PubMed/NCBI
41	Li Y, Zhu H, Klausen C, Peng B and Leung PC: Vascular endothelial growth factor-A (VEGF-A) mediates activin A-induced human trophoblast endothelial-like tube formation. Endocrinology. 156:4257–4268. 2015. View Article : Google Scholar : PubMed/NCBI
42	Schuster C, Akslen LA, Stokowy T and Straume O: Predictive value of angiogenic proteins in patients with metastatic melanoma treated with bevacizumab monotherapy. J Pathol Clin Res. 5:53–62. 2019. View Article : Google Scholar :
43	Caporarello N, Lupo G, Olivieri M, Cristaldi M, Cambria MT, Salmeri M and Anfuso CD: Classical VEGF, Notch and Ang signalling in cancer angiogenesis, alternative approaches and future directions (Review). Mol Med Rep. 16:4393–4402. 2017. View Article : Google Scholar : PubMed/NCBI
44	Vempati P, Popel AS and Mac Gabhann F: Extracellular regulation of VEGF: Isoforms, proteolysis, and vascular patterning. Cytokine Growth Factor Rev. 25:1–19. 2014. View Article : Google Scholar :
45	Zhao SF, Yang XD, Lu MX, Sun GW, Wang YX, Zhang YK, Pu YM and Tang EY: Prognostic significance of VEGF immuno-histochemical expression in oral cancer: A meta-analysis of the literature. Tumour Biol. 34:3165–3171. 2013. View Article : Google Scholar : PubMed/NCBI
46	Cai C, Böttcher MC, Werner JA and Mandic R: Differential expression of VEGF121, VEGF165 and VEGF189 in angiomas and squamous cell carcinoma cell lines of the head and neck. Anticancer Res. 30:805–810. 2010.PubMed/NCBI
47	Ehrbar M, Djonov VG, Schnell C, Tschanz SA, Martiny-Baron G, Schenk U, Wood J, Burri PH, Hubbell JA and Zisch AH: Cell-demanded liberation of VEGF121 from fibrin implants induces local and controlled blood vessel growth. Circ Res. 94:1124–1132. 2004. View Article : Google Scholar : PubMed/NCBI
48	Nakatsu MN, Sainson RC, Pérez-del-Pulgar S, Aoto JN, Aitkenhead M, Taylor KL, Carpenter PM and Hughes CC: VEGF(121) and VEGF(165) regulate blood vessel diameter through vascular endothelial growth factor receptor 2 in an in vitro angiogenesis model. Lab Invest. 83:1873–1885. 2003. View Article : Google Scholar : PubMed/NCBI
49	Tozer GM, Kanthou C and Baguley BC: Disrupting tumour blood vessels. Nat Rev Cancer. 5:423–435. 2005. View Article : Google Scholar : PubMed/NCBI
50	Shang B, Cao Z and Zhou Q: Progress in tumor vascular normalization for anticancer therapy: Challenges and perspectives. Front Med. 6:67–78. 2012. View Article : Google Scholar : PubMed/NCBI
51	Goel S, Duda DG, Xu L, Munn LL, Boucher Y, Fukumura D and Jain RK: Normalization of the vasculature for treatment of cancer and other diseases. Physiol Rev. 91:1071–1121. 2011. View Article : Google Scholar : PubMed/NCBI