Stefania Cannito1, Beatrice Foglia1, Gianmarco Villano2, Cristian Turato3, Teresa C Delgado4, Elisabetta Morello1, Fabrizio Pin1, Erica Novo1, Lucia Napione5,6, Santina Quarta7, Mariagrazia Ruvoletto2, Silvano Fasolato2, Giacomo Zanus8, Sebastiano Colombatto9, Fernando Lopitz-Otsoa4, David Fernández-Ramos4, Federico Bussolino6,9, Salvatore Sutti10, Emanuele Albano10, Maria Luz Martínez-Chantar4, Patrizia Pontisso7, Maurizio Parola1. 1. Department of Clinical and Biological Sciences, Unit of Experimental Medicine & Clinical Pathology, University of Torino, 10125 Torino, Italy. 2. Department of Surgery, Oncology and Gastroenterology, University of Padova, 35128 Padova, Italy. 3. Veneto Institute of Oncology IOV-IRCCS, 35128 Padova, Italy. 4. Liver Disease and Metabolism Laboratory, CIC bioGUNE, Centro de Investigacion Biomedica en Red de Enfermedades Hepaticas y Digestivas (Ciberehd), Technology Park of Bizkaia, 48160 Derio, Bizkaia, Spain. 5. Department of Applied Science and Technology, Politecnico di Torino, 10129 Torino, Italy. 6. Laboratory of Vascular Oncology, Candiolo Cancer Institute-IRCCS, 10060 Candiolo, Italy. 7. Department of Medicine, University of Padova, 35128 Padova, Italy. 8. Hepatobiliary Surgery, University of Padova, 35128 Padova, Italy. 9. Department of Oncology, University of Torino, 10125 Torino, Italy. 10. Department of Health Sciences and Interdisciplinary Research Center for Autoimmune Diseases, University Amedeo Avogadro of East Piedmont, 28100 Novara, Italy.
Abstract
BACKGROUND: SerpinB3 (SB3) is a hypoxia and hypoxia-inducible factor (HIF)-2α-dependent cysteine-protease inhibitor up-regulated in hepatocellular carcinoma (HCC), released by cancer cells and able to stimulate proliferation and epithelial-to-mesenchymal-transition. Methods: In the study we employed transgenic and knock out SerpinB3 mice, liver cancer cell line, human HCC specimens, and mice receiving diethyl-nitrosamine (DEN) administration plus choline-deficient L-amino acid refined (CDAA) diet (DEN/CDAA protocol). Results: We provide detailed and mechanistic evidence that SB3 can act as a paracrine mediator able to affect the behavior of surrounding cells by differentially up-regulating, in normoxic conditions, HIF-1α and HIF-2α. SB3 acts by (i) up-regulating HIF-1α transcription, facilitating cell survival in a harsh microenvironment and promoting angiogenesis, (ii) increasing HIF-2α stabilization via direct/selective NEDDylation, promoting proliferation of liver cancer cells, and favoring HCC progression. Moreover (iii) the highest levels of NEDD8-E1 activating enzyme (NAE1) mRNA were detected in a subclass of HCC patients expressing the highest levels of HIF-2α transcripts; (iv) mice undergoing DEN/CDAA carcinogenic protocol showed a positive correlation between SB3 and HIF-2α transcripts with the highest levels of NAE1 mRNA detected in nodules expressing the highest levels of HIF-2α transcripts. Conclusions: These data outline either HIF-2α and NEDDylation as two novel putative therapeutic targets to interfere with the procarcinogenic role of SerpinB3 in the development of HCC.
BACKGROUND:SerpinB3 (SB3) is a hypoxia and hypoxia-inducible factor (HIF)-2α-dependent cysteine-protease inhibitor up-regulated in hepatocellular carcinoma (HCC), released by cancer cells and able to stimulate proliferation and epithelial-to-mesenchymal-transition. Methods: In the study we employed transgenic and knock out SerpinB3mice, liver cancer cell line, humanHCC specimens, and mice receiving diethyl-nitrosamine (DEN) administration plus choline-deficient L-amino acid refined (CDAA) diet (DEN/CDAA protocol). Results: We provide detailed and mechanistic evidence that SB3 can act as a paracrine mediator able to affect the behavior of surrounding cells by differentially up-regulating, in normoxic conditions, HIF-1α and HIF-2α. SB3 acts by (i) up-regulating HIF-1α transcription, facilitating cell survival in a harsh microenvironment and promoting angiogenesis, (ii) increasing HIF-2α stabilization via direct/selective NEDDylation, promoting proliferation of liver cancer cells, and favoring HCC progression. Moreover (iii) the highest levels of NEDD8-E1 activating enzyme (NAE1) mRNA were detected in a subclass of HCCpatients expressing the highest levels of HIF-2α transcripts; (iv) mice undergoing DEN/CDAA carcinogenic protocol showed a positive correlation between SB3 and HIF-2α transcripts with the highest levels of NAE1 mRNA detected in nodules expressing the highest levels of HIF-2α transcripts. Conclusions: These data outline either HIF-2α and NEDDylation as two novel putative therapeutic targets to interfere with the procarcinogenic role of SerpinB3 in the development of HCC.