HO1 plays an important role in iron metabolism alteration in breast cancer cells

Abstract

Heme Oxygenase-1 (HO1) catalyzes heme degradation, yielding biliverdin, carbon monoxide and iron. When iron is in excess produces oxidative stress through reactive oxygen species (ROS) generation. Since both HO1 and iron metabolism disruptions have been related to breast cancer progression, we sought to investigate how tumor cells regulate iron metabolism when HO-1 expression is altered. For this purpose, we first investigated the correlation of HO1 with several iron proteins by using in silico analyses and corroborated the strongest hits by using immunohistochemistry (IHC) performed on human biopsies (n= 33). In addition, a syngeneic model of LM3 and a xenograft model of MDA-MB-231 cells stably overexpressing HO1 were used to study these hits. We further performed in culture analyses using LM3 breast cancer cells treated with hemin (H), vehicle or the combination with an antioxidant, and studied iron storage (Prussian Blue), ROS levels (DFCA) and cell cycle progression (flow cytometry). In silico analyses showed that HO1 correlated with DMT1 (p= 9.8e-05), ZIP14 (p= 4.2e-06), Prohepcidin (p= 1.4e-12) and L-ferritin (p= 2.2e-16). In order to study the correlation between HO1 and DMT1 in breast cancer we analyzed by IHC their expression in biopsies. We observed an inverse correlation between DMT1 and HO1 expression (p<0.05). The IHC studies showed an increase in ZIP14 and prohepcidin expression and a slight decrease in L- ferritin and DMT1 expression in hemin-treated and HO1- overexpressing cells in both animal models. In culture studies showed that the iron storage was increased in hemin- treated LM3 cells and was associated to a decrease in cell viability (p<0.05), an increase in the apoptotic rate (p<0.05) and high ROS levels (p<0.01). NAC treatment reverted the apoptotic effect of H (p<0.05). Altogether these results indicate that HO1 induction plays a role in carcinogenesis through free iron accumulation, ROS production and oxidative stress.