P34: Myoglobin and nitrite regulate cell proliferation via changes in mitochondrial dynamics

Background Rapid and uncontrolled cell proliferation is a hallmark of malignancy. In many tumor types, uncontrolled cell growth results in tumor metastasis and poor patient outcome. Many pathways involved in cell proliferation are targeted by standard chemotherapy including DNA synthesis and microtubule formation. Recent data suggests that nitrite may play a novel role in reducing mitogen-stimulated cell proliferation. While nitrite has been shown to reduce cell proliferation during both normoxia and hypoxia, the mechanism remains to be elucidated. Myoglobin is known to have several interactions with nitrite and other reactive nitrogen species, including nitric oxide (NO) scavenging during normoxia and nitrite reduction to form bioavailable NO during hypoxia. Most recently, myoglobin has been found to be abnormally expressed in several malignant epithelial tissues including breast, lung, colon, and ovarian tumors. Limited data suggests that myoglobin expression results in decreased tumor volumes, although the mechanism for this effect remains unclear. Hypothesis In the present study, we hypothesize that myoglobin expression regulates the ability of nitrite to modulate cell proliferation in malignancy. Methods We used physiological modulators, such as hypoxia and lactate, to regulate myoglobin levels in the breast cancer cell lines MCF7 and MB-MDA-231. Additionally, rat aortic smooth muscle cells (RASMC), which have abundant myoglobin expression, were used to determine the effect of myoglobin on regulating nitrite-mediated inhibition of cell proliferation. Results We demonstrate using RASMCs, containing abundant levels of myoglobin, that nitrite promotes mitochondrial fusion and the inhibition of mitogen-stimulated cell proliferation during normoxia by increasing two mitochondrial fusion signals: mitofusin 1 (Mfn-1) expression and the inhibitory phosphorylation of dynamin-related protein 1 (DRP1). Increased Mfn-1 levels are correlated with increased p21Waf1/Cip1 expression, and nitrite treatment does not increase p21Waf1/Cip1 expression in the presence of Mfn-1 siRNA, suggesting a direct effect of nitrite-mediated Mfn-1 expression on subsequent p21Waf1/Cip1 expression and cell cycle regulation. We observe in breast cancer cell lines that myoglobin expression can be induced during either prolonged hypoxia, or during normoxia through the addition of lactate to culture media. These physiological inductions of myoglobin are accompanied by inhibition of cell proliferation, an effect that is further attenuated by the addition of nitrite. Further, in breast cancer cells during hypoxia, upregulation of myoglobin and reduced cell proliferation is correlated with increased Mfn-1 and p21Waf1/Cip1 expression. Upon addition of lactate to normoxic breast cancer cells, we observed p21Waf1/Cip1 induction along with increased myoglobin expression, decreased mitochondrial respiration, and decreased cell proliferation. Conclusions Taken together, these data implicate the regulation of mitochondrial dynamics in nitrite-mediated decreases in cell proliferation. Further, these data also suggest that myoglobin expression may play a critical role in modulating this pathway. Further understanding and elucidation of this pathway is warranted, as nitrite may be a novel anti-cancer agent in tumors harboring aberrant myoglobin expression. Disclosure Nothing to disclose.