Heme is an essential nutritional, metabolic, and signaling molecule in living organisms. Pathogenic microbes extract heme from hosts to obtain metallonutrient, while heme fuels mitochondrial respiration and ATP generation in lung tumor cells. Here, we generated small heme-sequestering proteins (HeSPs) based on bacterial hemophores. These HeSPs contain neutral mutations in the heme-binding pocket and hybrid sequences from hemophores of different bacteria. We showed that HeSPs bind to heme and effectively extracted heme from hemoglobin. They strongly inhibited heme uptake and cell proliferation and induced apoptosis in non–small cell lung cancer (NSCLC) cells, while their effects on nontumorigenic cell lines representing normal lung cells were not significant. HeSPs strongly suppressed the growth of human NSCLC tumor xenografts in mice. HeSPs decreased oxygen consumption rates and ATP levels in tumor cells isolated from treated mice, while they did not affect liver and blood cell functions. IHC, along with data from Western blotting and functional assays, revealed that HeSPs reduced the levels of key proteins involved in heme uptake, as well as the consumption of major fuels for tumor cells, glucose, and glutamine. Further, we found that HeSPs reduced the levels of angiogenic and vascular markers, as well as vessel density in tumor tissues. Together, these results demonstrate that HeSPs act via multiple mechanisms, including the inhibition of oxidative phosphorylation, to suppress tumor growth and progression. Evidently, heme sequestration can be a powerful strategy for suppressing lung tumors and likely drug-resistant tumors that rely on oxidative phosphorylation for survival.

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