Autophagy fuels mitochondrial function through regulation of iron metabolism in pancreatic cancer

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) has one of the lowest 5-year survival rates of any cancer in the United States. Our previous work has shown that autophagy can promote PDAC progression. We recently established the importance of autophagy in regulating bioavailable iron to control mitochondrial metabolism in PDAC. We found that inhibition of autophagy in PDAC leads to mitochondrial dysfunction due to abrogation of succinate dehydrogenase complex iron sulfur subunit B (SDHB) expression. Additionally, we observed that cancer-associated fibroblasts (CAFs) can provide iron to autophagy-inhibited PDAC tumor cells, thereby increasing their resistance to autophagy inhibition. To impede such metabolic compensation, we used a low iron diet together with autophagy inhibition and demonstrated a significant improvement of tumor response in syngeneic PDAC models. Abbreviations: PDAC: Pancreatic ductal adenocarcinoma; CAFs: cancer-associated fibroblasts; SDHB: succinate dehydrogenase complex iron sulfur subunit B; ISCA1: iron sulfur cluster assembly protein 1; FPN: ferroportin; LIP: labile iron pool; FAC: ferric ammonium chloride; OCR: oxygen consumption rate; OXPHOS: oxidative phosphorylation, IL6: interleukin 6; Fe-S: iron sulfur; ATP: adenosine triphosphate.

Autophagy, particularly the form of selective autophagy known as ferritinophagy, has a critical role in supporting the bioavailable labile iron pool (LIP) and is important for pancreatic ductal adenocarcinoma (PDAC) growth.In our latest work [1], we observed that inhibition of autophagy led to growth impairment in PDAC which could be rescued by exogenous supplementation of iron in the form of ferric ammonium chloride (FAC).We found that iron supplementation could also restore the reduction of the oxygen consumption rate (OCR), a surrogate measure of oxidative phosphorylation and mitochondrial function, upon autophagy inhibition.Interestingly, upon assessing the proteins involved in oxidative phosphorylation (OXPHOS), we determined that there was a significant reduction of the iron sulfur (Fe-S) cluster protein succinate dehydrogenase complex iron sulfur subunit B (SDHB).Loss of SDHB (complex II of OXPHOS) resulted in mitochondrial dysfunction as illustrated by transmission electron microscopy analysis showing damaged mitochondrial cristae architecture, while the number of mitochondria was unaffected.Intriguingly, these alterations in the mitochondrial architecture were significantly abrogated when autophagy-inhibited PDAC were supplemented with iron.This was phenotypically reflected in the rescue of cell proliferation and OCR.We observed that autophagy influences SDHB expression by controlling its upstream regulator, the iron sulfur cluster assembly protein 1 (ISCA1).Overexpression of ISCA1 mitigates the decrease in SDHB and rescues the mitochondrial architecture defects upon autophagy inhibition.Enforced expression of either ISCA1 or SDHB in PDAC cells with autophagy inhibition significantly restored proliferation.
Autophagy and macropinocytosis, which converge at lysosomes, are two critical nutrient scavenging pathways in PDAC cells.Interestingly, when we compared PDAC to non-PDAC tumor cells, we observed that LIP was specifically regulated by autophagy in PDAC.Furthermore, we found that the non-PDAC cells required macropinocytosis to maintain LIP, while lysosomal inhibition influences LIP in all cell types tested regardless of their origin.Taken together, these findings imply that the route of iron utilization might differ between PDAC and non-PDAC cells, but ultimately, they use the lysosomal apparatus for LIP maintenance, which is consistent with other reports.
Clinically, monotherapy with autophagy inhibitors in PDAC has had limited clinical success.Our data suggests that this could be, in part, due to the ability of cancer associated fibroblasts (CAFs) to compensate for the drop in LIP and rescue proliferation in autophagy-inhibited tumor cells (Figure 1).We identified that autophagyinhibited PDAC cells secrete interleukin 6 (IL6), a known inducer of ferritin, to trigger ferritin buildup in CAF cells by a paracrine signaling effect.In turn, the CAFs exhibited enhanced ferroportin (FPN; encoded by SLC40A1 gene) expression.Ferroportin, a known exporter of iron, was inhibited pharmacologically and this abrogated the rescue effects of the CAF co-culture.As a proof of concept, we demonstrated that the dietary restriction of iron can improve the PDAC response to both a genetic and a pharmacological autophagy inhibition.The combination of autophagy inhibition and reduced iron diet decreased the bioavailable iron as well as the SDHB expression in these tumors.Our work is consistent with other reports that have recently highlighted the significance of ferritinophagy in supporting mitochondrial respiration and PDAC growth.
Further investigation will be required to effectively translate this type of therapeutic intervention into clinical practice.Our study focused on the feasibility of restricting dietary iron to improve the effect of autophagy inhibition.Currently, the mainstay of autophagy inhibition in patients involves the repurposing of antimalarials such as chloroquine/hydroxychloroquine which have limited potency and specificity.Thus, our study suggests that potency can be improved by developing combinatorial strategies involving autophagy inhibition and iron targeted therapy.

Disclosure statement
A.C.K. has financial interests in Vescor Therapeutics and is an inventor on patents pertaining to KRAS-regulated metabolic pathways and redox control pathways in pancreatic cancer, targeting GOT1 as a therapeutic approach, targeting alanine transport, and the autophagic control of iron metabolism.A.C.K. is on the scientific advisory board of Rafael/ Cornerstone Pharmaceuticals, is an advisor to OncoRev, and has been a consultant for Deciphera and Abbvie.The other authors declare that they have no competing interests.

Figure 1 .
Figure 1.Autophagy inhibition triggers mitochondrial dysfunction by reducing bioavailable iron.PDAC utilizes autophagy (ferritnophagy) machinery to maintain the labile iron pool.Loss of autophagy affects the Fe-S cluster proteins SDHB and ISCA1, which in turn impairs mitochondrial architecture and results in decreased ATP production.Autophagy-inhibited PDAC secrete IL6, inducing ferritin accumulation in neighboring CAFs through paracrine signaling.These CAFs trigger ferroportin expression that leads to compensation of the labile iron pool in the autophagy inhibited PDAC and results in therapeutic resistance.However, a low iron diet can impede compensatory crosstalk and improve autophagy inhibition therapy.Green or red arrow directions indicate up or down regulation respectively.Created with BioRender.com