One classical feature of cancer cells is their metabolic acquisition of a highly glycolytic phenotype. Carbon monoxide (CO), one of the products of the cytoprotective molecule heme oxygenase-1 (HO-1) in cancer cells, has been implicated in carcinogenesis and therapeutic resistance. However, the functional contributions of CO and HO-1 to these processes are poorly defined. In human prostate cancers, we found that HO-1 was nuclear localized in malignant cells, with low enzymatic activity in moderately differentiated tumors correlating with relatively worse clinical outcomes. Exposure to CO sensitized prostate cancer cells but not normal cells to chemotherapy, with growth arrest and apoptosis induced in vivo in part through mitotic catastrophe. CO targeted mitochondria activity in cancer cells as evidenced by higher oxygen consumption, free radical generation, and mitochondrial collapse. Collectively, our findings indicated that CO transiently induces an anti-Warburg effect by rapidly fueling cancer cell bioenergetics, ultimately resulting in metabolic exhaustion. Cancer Res; 73(23); 7009–21. ©2013 AACR.

Emerging evidence suggests that extracellular vesicle (EV)–containing miRNAs mediate intercellular communications in response to noxious stimuli. It remains unclear how a cell selectively sorts the cellular miRNAs into EVs. We report that caveolin-1 (cav-1) is essential for sorting of selected miRNAs into microvesicles (MVs), a main type of EVs generated by outward budding of the plasma membrane. We found that cav-1 tyrosine 14 (Y14)–phosphorylation leads to interactions between cav-1 and hnRNPA2B1, an RNA-binding protein. The cav-1/hnRNPA2B1 complex subsequently traffics together into MVs. Oxidative stress induces O-GlcNAcylation of hnRNPA2B1, resulting in a robustly altered hnRNPA2B1-bound miRNA repertoire. Notably, cav-1 pY14 also promotes hnRNPA2B1 O-GlcNAcylation. Functionally, macrophages serve as the principal recipient of epithelial MVs in the lung. MV-containing cav-1/hnRNPA2B1 complex-bound miR-17/93 activate tissue macrophages. Collectively, cav-1 is the first identified membranous protein that directly guides RNA-binding protein into EVs. Our work delineates a novel mechanism by which oxidative stress compels epithelial cells to package and secrete specific miRNAs and elicits an innate immune response.

The most salient feature of carbon monoxide (CO)-mediated cytoprotection is the suppression of inflammation and cell death. One of the important cellular targets of CO is the macrophage (mφ). Many studies have shown that exposure of mφ to CO results in the generation of an antiinflammatory phenotype; however, these reports have ignored the effect of CO alone on the cell before stimulation. Most investigations have focused on the actions of CO in modulating the response to noxious stimuli. We demonstrate here that exposure of mφ to CO results in a significant and transient burst of reactive oxygen species (ROS) arising from the mitochondria (mitochondria-deficient mφ do not respond to CO to produce ROS). The ROS promote rapid activation and stabilization of the transcription factor hypoxia-inducible factor 1α (HIF-1α), which regulates expression of genes involved in inflammation, metabolism, and cell survival. The increase in HIF-1α expression induced by CO results in regulated expression of TGF-β, a potent antiinflammatory cytokine. CO-induced HIF-1α and TGF-β expression are necessary to prevent anoxia/reoxygenation-induced apoptosis in mφ. Furthermore, blockade of HIF-1α using RNA interference and HIF-1α-cre-lox mφ resulted in a loss of TGF-β expression and CO-induced protection. A similar mechanism of CO-induced protection was operationalin vivoto protect against lung ischemia-reperfusion injury. Taken together, we conclude that CO conditions the mφ via a HIF-1α and TGF-β-dependent mechanism and we elucidate the earliest events in mφ signaling that lead to and preserve cellular homeostasis at the site of injury.