THE ROLE OF THE HEME-EXPORTER FLVCR1 IN HEPATOCELLULAR CARCINOMA

Tiago L DUARTE1,2, Nicole VIVEIROS1, Catarina GODINHO1,3, Sofia LAMAS1, José Manuel LOPES1,4, Janis L ABKOWITZ5, Maria José OLIVEIRA1,6, Delfim DUARTE1,7,8

1i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal
2IBMC – Instituto de Biologia Molecular e Celular da Universidade do Porto, Porto, Portugal
3Faculty of Medicine (FMUP), University of Porto, Porto, Portugal
4IPATIMUP – Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal
5Division of Hematology, Department of Medicine, University of Washington, Seattle, United States
6Departamento de Biologia Molecular, ICBAS – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal
7Departamento de Hematologia e Transplantação de Medula Óssea do Instituto Português de Oncologia (IPO)-Porto, Porto, Portugal
8Departamento de Biomedicina, Unidade de Bioquímica, Faculdade de Medicina da Universidade do Porto, Porto, Portugal

Hepatocellular carcinoma (HCC) is the most prevalent type of primary liver cancer, the third leading cause of cancer-related mortality. There is an unmet need to discover new biological mechanisms in HCC and develop new therapies. The heme exporter FLVCR1 is overexpressed in HCC and associated with poor prognosis, but its role in HCC pathophysiology remains unexplored. We aimed to investigate the role of FLVCR1 in HCC.

To assess the impact of liver-specific Flvcr1 KO in the development of HCC in vivo, we treated Flvcr1fl/fl;alb-cre and Flvcr1fl/fl male mice (N=50 each genotype) with diethylnitrosamine (DEN) and carbon tetrachloride (CCl4). At the age of 8 months, both groups developed hepatocellular adenomas. Hepatic Flvcr1 KO did not affect the overall tumor burden (relative liver weight, total tumor volume, largest tumor area) or serum markers of liver injury (AST or ALT activity, bilirubin). However, the incidence of HCC, determined by blind histologic analysis, was significantly reduced in Flvcr1fl/fl;alb-cre mice compared to Flvcr1fl/fl animals (13.73% vs 32.69%; p=0.0349).

In parallel, we generated FLVCR1 KO in human HCC cells (Huh7) by CRISPR/Cas9. Consistent with a defect in heme export, FLVCR1 KO Huh7 clones displayed increased levels of intracellular heme. The loss of FLVCR1 function decreased cell migration in vitro (time-lapse wound healing and individual live cell imaging), as well as the ability of Huh7 cells to invade the chorioallantoic membrane (CAM) model, while it potentiated new vessel formation in vivo.

Overall, our results suggest that FLVCR1 has a pro-tumorigenic role in HCC.