Hypoxia is a hallmark of solid tumors that promotes cell growth, survival, and metastasis and confers resistance to chemo and radiotherapies. Hypoxic responses are largely mediated by the transcription factors hypoxia-inducible factor 1α (HIF-1α) and HIF-2α. Our work demonstrates that HIF-2α is essential for colorectal cancer (CRC) progression. However, targeting hypoxic cells is difficult, and tumors rapidly acquire resistance to inhibitors of HIF-2α. To overcome this limitation, we performed a small molecule screen to identify HIF-2α–dependent vulnerabilities. Several known ferroptosis activators and dimethyl fumarate (DMF), a cell-permeable mitochondrial metabolite derivative, led to selective synthetic lethality in HIF-2α–expressing tumor enteroids. Our work demonstrated that HIF-2α integrated 2 independent forms of cell death via regulation of cellular iron and oxidation. First, activation of HIF-2α upregulated lipid and iron regulatory genes in CRC cells and colon tumors in mice and led to a ferroptosis-susceptible cell state. Second, via an iron-dependent, lipid peroxidation–independent pathway, HIF-2α activation potentiated ROS via irreversible cysteine oxidation and enhanced cell death. Inhibition or knockdown of HIF-2α decreased ROS and resistance to oxidative cell death in vitro and in vivo. Our results demonstrated a mechanistic vulnerability in cancer cells that were dependent on HIF-2α that can be leveraged for CRC treatment.

Cancer cells reprogram cellular metabolism to maintain adequate nutrient pools to sustain proliferation. Moreover, autophagy is a regulated mechanism to break down dysfunctional cellular components and recycle cellular nutrients. However, the requirement for autophagy and the integration in cancer cell metabolism is not clear in colon cancer. Here, we show a cell-autonomous dependency of autophagy for cell growth in colorectal cancer. Loss of epithelial autophagy inhibits tumor growth in both sporadic and colitis-associated cancer models. Genetic and pharmacological inhibition of autophagy inhibits cell growth in colon cancer–derived cell lines and patient-derived enteroid models. Importantly, normal colon epithelium and patient-derived normal enteroid growth were not decreased following autophagy inhibition. To couple the role of autophagy to cellular metabolism, a cell culture screen in conjunction with metabolomic analysis was performed. We identified a critical role of autophagy to maintain mitochondrial metabolites for growth. Loss of mitochondrial recycling through inhibition of mitophagy hinders colon cancer cell growth. These findings have revealed a cell-autonomous role of autophagy that plays a critical role in regulating nutrient pools in vivo and in cell models, and it provides therapeutic targets for colon cancer.

Mitophagy is a cargo-specific autophagic process that recycles damaged mitochondria to promote mitochondrial turnover. PTEN-induced putative kinase 1 (PINK1) mediates the canonical mitophagic pathway. However, the role of PINK1 in diseases where mitophagy has been purported to play a role, such as colorectal cancer, is unclear. Our results here demonstrate that higher PINK1 expression is positively correlated with decreased colon cancer survival, and mitophagy is required for colon cancer growth. We show that doxycycline-inducible knockdown (KD) of PINK1 in a panel of colon cancer cell lines inhibited proliferation, whereas disruption of other mitophagy receptors did not impact cell growth. We observed that PINK KD led to a decrease in mitochondrial respiration, membrane hyperpolarization, accumulation of mitochondrial DNA, and depletion of antioxidant glutathione. In addition, mitochondria are important hubs for the utilization of iron and synthesizing iron-dependent cofactors such as heme and iron sulfur clusters. We observed an increase in the iron storage protein ferritin and a decreased labile iron pool in the PINK1 KD cells, but total cellular iron or markers of iron starvation/overload were not affected. Finally, cellular iron storage and the labile iron pool are maintained via autophagic degradation of ferritin (ferritinophagy). We found overexpressing nuclear receptor coactivator 4, a key adaptor for ferritinophagy, rescued cell growth and the labile iron pool in PINK1 KD cells. These results indicate that PINK1 integrates mitophagy and ferritinophagy to regulate intracellular iron availability and is essential for maintaining intracellular iron homeostasis to support survival and growth in colorectal cancer cells.

Despite the importance of hypoxia and neutrophils in colorectal cancer (CRC), the contribution of neutrophil-specific HIFs to colon tumorigenesis is not known. We describe that neutrophil HIF-1α has no impact on colon cancer, whereas neutrophil HIF-2α loss reduces CRC growth by decreasing proinflammatory and immunosuppressive cytokines. Furthermore, neutrophil HIF-2α does not reduce preestablished tumor growth or inflammation-induced colitis. The present study offers novel potential of neutrophil HIF-2α as a therapeutic target in CRC.