Iron modulates cell survival in a Ras- and MAPK-dependent manner in ovarian cells

Abstract

Ovarian cancer is a leading cause of cancer death in women in the United States. While the majority of ovarian cancers are serous, some rarer subtypes (i.e. clear cell) are often associated with endometriosis, a benign gynecological disease. Iron is rich in the cyst fluid of endometriosis-associated ovarian cancers and induces persistent oxidative stress. The role of iron, an essential nutrient involved in multiple cellular functions, in normal ovarian cell survival and ovarian cancer remains unclear. Iron, presented as ferric ammonium citrate (FAC), dramatically inhibits cell survival in ovarian cancer cell types associated with Ras mutations, while it is without effect in immortalized normal ovarian surface epithelial (T80) and endometriotic epithelial cells (lacking Ras mutations). Interestingly, FAC induced changes in cytoplasmic vacuolation concurrently with increases in LC3-II levels (an autophagy marker); these changes occurred in an ATG5/ATG7-dependent, beclin-1/hVps34-independent, and Ras-independent manner. Knockdown of autophagy mediators in HEY ovarian cancer cells reversed FAC-induced LC3-II levels, but there was little effect on reversing the cell death response. Intriguingly, transmission electron microscopy of FAC-treated T80 cells demonstrated abundant lysosomes (confirmed using Lysotracker) rich in iron particles, which occurred in a Ras-independent manner. Although the mitogen-activated protein kinase (MAPK) inhibitor, U0126, reversed FAC-induced LC3-II/autophagic punctae and lysosomes in a Ras-independent manner, it was remarkable that U0126 reversed cell death in malignant ovarian cells associated with Ras mutations. Moreover, FAC increased heme oxygenase-1 expression in H-Ras-overexpressing T80 cells, which was associated with increased cell death when overexpressed in T80 cells. Disruption of intracellular iron levels, via chelation of intracellular iron (deferoxamine), was also detrimental to malignant ovarian cell survival; thus, homeostatic intracellular iron levels are essential for cell survival. Collectively, our results implicate iron in modulating cell death in a Ras- and MAPK-dependent manner in ovarian cancer cells.

Ferritinophagy drives uropathogenicEscherichia colipersistence in bladder epithelial cells

“…21,22 Other studies have also linked high intralysosomal iron accumulation with oxidative stress and resulting cytotoxicity 34 consistent with a recent study showing that lysosomal integrity is necessary for UPEC to take advantage of excess bioavailable iron. 59 Normal serum iron levels in healthy adults are typically under 31 mM 60 and patients with severe hemochromatosis or liver damage can exhibit levels far greater than 31.…”

“…Although this is the first report of ferritinophagy in bladder cells, this pathway has been observed in ovarian, liver, and brain cell types. [21][22][23][24][44][45][46][47][48] In addition to observing an effect of iron on levels of autophagy, we also noted that loss of autophagy gene, ATG16L1 as well as selective autophagy receptor for ferritin that traffics ferritin to autophagosomes for degradation, NCOA4, resulted in increased levels of ferritin, suggesting that reduction of autophagy can enhance the ability of bladder cells to store iron. Because ferritinophagy can be utilized by pathogens, it must be carefully regulated.…”

“…26 We selected 250 mM as a representation of iron overload that may occur due to hemochromatosis or severe liver disease in humans. 21,27,28 We observed an increased LC3-II to LC3-I ratio after iron treatment, indicating accumulation of autophagosomes (Fig. 1A).…”

“…5F). In contrast, blocking the iron regulatory process by treating cells with oxalomalic acid (OMA), a potent inhibitor of ACO1 aconitase activity, 21 led to decreased ferritin levels by itself and upon iron treatment and UPEC infection (Fig. 5G).…”

“…21,[34][35][36][37] To assess the effect of iron and UPEC infection on BEC viability, we measured activity of LDH (lactate dehydrogenase) in media and performed cell growth assays at 24 hpi. We found that neither iron supplementation alone nor UPEC infection alone induced cell death, but the combination of iron and UPEC led to a significant increase in host cell death: approximately 40% of BECs infected with UPEC in the presence of 250 mM iron died by 24 hpi ( Fig.…”