{"paper_id":"de321dfb-5c2b-4488-834d-e748d675d575","body_text":"Abstract\nProhibiting angiogenesis is an important therapeutic approach for fighting cancer and other angiogenic related diseases. Research focused on proteins that regulate abnormal angiogenesis has attracted intense interest in both academia and industry. Such proteins are able to target several angiogenic factors concurrently, thereby increasing the possibility of therapeutic success. Aquaporin-1 (AQP1) is a water channel membrane protein that promotes tumour angiogenesis by allowing faster endothelial cell migration. In this study we test the hypothesis that AQP1 inhibition impairs tumour growth in a mouse model of melanoma. After validating the inhibitor efficacy of two different AQP1 specific siRNAs in cell cultures, RNA interference experiments were performed by intratumoural injections of AQP1 siRNAs in mice. After 6 days of treatment, AQP1 siRNA treated tumours showed a 75 % reduction in volume when compared to controls. AQP1 protein level, in AQP1 knockdown tumours, was around 75 % that of the controls and was associated with a significant 40 % reduced expression of the endothelial marker, Factor VIII. Immunofluorescence analysis of AQP1 siRNA treated tumours showed a significantly lower microvessel density. Time course experiments demontrated that repeated injections of AQP1 siRNA over time are effective in sustaining the inhibition of tumour growth. Finally, we have confirmed the role of AQP1 in sustaining an active endothelium during angiogenesis and we have shown that AQP1 reduction causes an increase in VEGF levels. In conclusion, this study validates AQP1 as a pro-angiogenic protein, relevant for the therapy of cancer and other angiogenic-related diseases such as psoriasis, endometriosis, arthritis and atherosclerosis.\nSimilar content being viewed by others\nReferences\nAmiry-Moghaddam M, Otsuka T, Hurn PD, Traystman RJ, Haug FM, Froehner SC, Adams ME, Neely JD, Agre P, Ottersen OP et al (2003) An alpha-syntrophin-dependent pool of AQP4 in astroglial end-feet confers bidirectional water flow between blood and brain. Proc Natl Acad Sci USA 100:2106–2111\nAgre P, Kozono D (2003) Aquaporin water channels: molecular mechanisms for human diseases. FEBS Lett 555:72–78\nVerkman AS (2005) More than just water channels: unexpected cellular roles of aquaporins. J Cell Sci 118:3225–3232\nEndo M, Jain RK, Witwer B, Brown D (1999) Water channel (aquaporin 1) expression and distribution in mammary carcinomas and glioblastomas. Microvasc Res 58:89–98\nSaadoun S, Papadopoulos MC, Hara-Chikuma M, Verkman AS (2005) Impairment of angiogenesis and cell migration by targeted aquaporin-1 gene disruption. Nature 434:786–792\nVacca A, Frigeri A, Ribatti D, Nicchia GP, Nico B, Ria R, Svelto M, Dammacco F (2001) Microvessel overexpression of aquaporin 1 parallels bone marrow angiogenesis in patients with active multiple myeloma. Br J Haematol 113:415–421\nVacca A, Ribatti D, Roccaro AM, Frigeri A, Dammacco F (2001) Bone marrow angiogenesis in patients with active multiple myeloma. Semin Oncol 28:543–550\nVerkman AS, Hara-Chikuma M, Papadopoulos MC (2008) Aquaporins-new players in cancer biology. J Mol Med (Berl) 86:523–529\nOshio K, Binder DK, Liang Y, Bollen A, Feuerstein B, Berger MS, Manley GT (2005) Expression of the aquaporin-1 water channel in human glial tumours. Neurosurgery 56:375–381, discussion 375–381\nSaadoun S, Papadopoulos MC, Davies DC, Bell BA, Krishna S (2002) Increased aquaporin 1 water channel expression in human brain tumours. Br J Cancer 87:621–623\nWarth A, Simon P, Capper D, Goeppert B, Tabatabai G, Herzog H, Dietz K, Stubenvoll F, Ajaaj R, Becker R et al (2007) Expression pattern of the water channel aquaporin-4 in human gliomas is associated with blood–brain barrier disturbance but not with patient survival. J Neurosci Res 85:1336–1346\nWarth A, Mittelbronn M, Hulper P, Erdlenbruch B, Wolburg H (2007) Expression of the water channel protein aquaporin-9 in malignant brain tumours. Appl Immunohistochem Mol Morphol 15:193–198\nSaadoun S, Papadopoulos MC, Watanabe H, Yan D, Manley GT, Verkman AS (2005) Involvement of aquaporin-4 in astroglial cell migration and glial scar formation. J Cell Sci 118:5691–5698\nHendrix MJ, Seftor EA, Hess AR, Seftor RE (2003) Vasculogenic mimicry and tumour-cell plasticity: lessons from melanoma. Nat Rev Cancer 3:411–421\nKerbel RS (2000) Tumour angiogenesis: past, present and the near future. Carcinogenesis 21:505–515\nVacca A, Ribatti D, Roccaro AM, Ria R, Palermo L, Dammacco F (2001) Bone marrow angiogenesis and plasma cell angiogenic and invasive potential in patients with active multiple myeloma. Acta Haematol 106:162–169\nHuebert RC, Vasdev MM, Shergill U, Das A, Huang BQ, Charlton MR, LaRusso NF, Shah VH (2010) Aquaporin-1 facilitates angiogenic invasion in the pathological neovasculature that accompanies cirrhosis. Hepatology 52:238–248\nHuebert RC, Jagavelu K, Hendrickson HI, Vasdev MM, Arab JP, Splinter PL, Trussoni CE, Larusso NF, Shah VH (2011) Aquaporin-1 promotes angiogenesis, fibrosis, and portal hypertension through mechanisms dependent on osmotically sensitive microRNAs. Am J Pathol 179:1851–1860\nCamerino GM, Nicchia GP, Dinardo MM, Ribatti D, Svelto M, Frigeri A (2006) In vivo silencing of aquaporin-1 by RNA interference inhibits angiogenesis in the chick embryo chorioallantoic membrane assay. Cell Mol Biol (Noisy-le-grand) 52:51–56\nLangenkamp E, Vom Hagen FM, Zwiers PJ, Moorlag HE, Schouten JP, Hammes HP, Gouw AS, Molema G (2011) Tumour Vascular Morphology Undergoes Dramatic Changes during Outgrowth of B16 Melanoma While Proangiogenic Gene Expression Remains Unchanged. ISRN Oncol 2011:409308\nIkeda K, Nakano R, Uraoka M, Nakagawa Y, Koide M, Katsume A, Minamino K, Yamada E, Yamada H, Quertermous T et al (2009) Identification of ARIA regulating endothelial apoptosis and angiogenesis by modulating proteasomal degradation of cIAP-1 and cIAP-2. Proc Natl Acad Sci USA 106:8227–8232\nNicchia GP, Srinivas M, Li W, Brosnan CF, Frigeri A, Spray DC (2005) New possible roles for aquaporin-4 in astrocytes: cell cytoskeleton and functional relationship with connexin43. FASEB J 19\nNicchia GP, Mastrototaro M, Rossi A, Pisani F, Tortorella C, Ruggieri M, Lia A, Trojano M, Frigeri A, Svelto M (2009) Aquaporin-4 orthogonal arrays of particles are the target for neuromyelitis optica autoantibodies. Glia 57:1363–1373\nCarmeliet P, Tessier-Lavigne M (2005) Common mechanisms of nerve and blood vessel wiring. Nature 436:193–200\nAlon T, Hemo I, Itin A, Pe’er J, Stone J, Keshet E (1995) Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat Med 1:1024–1028\nGariano RF, Gardner TW (2005) Retinal angiogenesis in development and disease. Nature 438:960–966\nAgre P, Smith BL, Preston GM (1995) ABH and Colton blood group antigens on aquaporin-1, the human red cell water channel protein. Transfus Clin Biol 2:303–308\nChou CL, Knepper MA, Hoek AN, Brown D, Yang B, Ma T, Verkman AS (1999) Reduced water permeability and altered ultrastructure in thin descending limb of Henle in aquaporin-1 null mice. J Clin Invest 103:491–496\nBergers G, Hanahan D (2008) Modes of resistance to anti-angiogenic therapy. Nat Rev Cancer 8:592–603\nCarmeliet P (2005) Angiogenesis in life, disease and medicine. Nature 438:932–936\nPrager G.W. PM, Unseld M., Zielinski C.C. (2011) Angiogenesis in cancer: Anti-VEGF escape mechanisms. Transl Lung Cancer Res\nCasanovas O, Hicklin DJ, Bergers G, Hanahan D (2005) Drug resistance by evasion of antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet tumours. Cancer Cell 8:299–309\nLa Porta C (2010) AQP1 is not only a water channel: It contributes to cell migration through Lin7/beta-catenin. Cell Adh Migr 4:204–206\nNicchia GP, Rossi A, Mola MG, Procino G, Frigeri A, Svelto M (2008) Actin cytoskeleton remodeling governs aquaporin-4 localization in astrocytes. Glia 56:1755–1766\nAbreu-Rodriguez I, Sanchez Silva R, Martins AP, Soveral G, Toledo-Aral JJ, Lopez-Barneo J, Echevarria M (2011) Functional and transcriptional induction of aquaporin-1 gene by hypoxia; analysis of promoter and role of Hif-1alpha. PLoS One 6:e28385\nWang Y, Cohen J, Boron WF, Schulten K, Tajkhorshid E (2007) Exploring gas permeability of cellular membranes and membrane channels with molecular dynamics. J Struct Biol 157:534–544\nSemenza GL (2001) Regulation of hypoxia-induced angiogenesis: a chaperone escorts VEGF to the dance. J Clin Invest 108:39–40\nAcknowledgments\nThis work was supported by grants from “Progetto di Ricerca IDEA Giovani Ricercatori (GRBA085SIS)”, “Rete Nazionale di Proteomica (RBRN07BMCT_009)”, “FIRB Idee Progettuali (RBIP0695BB_004)” and by the Apulia region grant “Progetto Strategico APQ Ricerca (Neurobiotech) [PS124]”. The authors would like to thank Richard Lusardi for his assistance in revising the English of the article and Gaetano De Vito for his excellent technical assistance.\nDisclosure Statement\nThe authors declare they have no conflict of interest\nAuthor information\nAuthors and Affiliations\nCorresponding author\nRights and permissions\nAbout this article\nCite this article\nNicchia, G.P., Stigliano, C., Sparaneo, A. et al. Inhibition of aquaporin-1 dependent angiogenesis impairs tumour growth in a mouse model of melanoma. J Mol Med 91, 613–623 (2013). https://doi.org/10.1007/s00109-012-0977-x\nReceived:\nRevised:\nAccepted:\nPublished:\nIssue date:\nDOI: https://doi.org/10.1007/s00109-012-0977-x","source_license":"public-domain-us","license_restricted":false}