HSP2HSP2 HEME HCP1 HRG1 HEME HEME HEME CYT NUC ER ALA SLung cancer cells exhibit elevated heme synthesis and uptake, which promotes tumorigenic functions. Heme-sequestering peptide 2 (HSP2) inhibits heme uptake and suppresses tumor growth. V [ ] Lung tumors Non-small cell lung cancer cell Normal lung I II III IV ATP ADPTumors of human non-small cell lung cancer (NSCLC) are heterogeneous but exhibit elevated glycolysis and glucose oxidation relative to benign lung tissues. Heme is a central molecule for oxidative metabolism and ATP generation via mitochondrial oxidative phosphorylation (OXPHOS). Here, we showed that levels of heme synthesis and uptake, mitochondrial heme, oxygen-utilizing hemoproteins, oxygen consumption, ATP generation, and key mitochondrial biogenesis regulators were enhanced in NSCLC cells relative to nontumorigenic cells. Likewise, proteins and enzymes relating to heme and mitochondrial functions were upregulated in human NSCLC tissues relative to normal tissues. Engineered heme-sequestering peptides (HSP) reduced heme uptake, intracellular heme levels, and tumorigenic functions of NSCLC cells. Addition of heme largely reversed the effect of HSPs on tumorigenic functions. Furthermore, HSP2 significantly suppressed the growth of human NSCLC xenograft tumors in mice. HSP2-treated tumors exhibited reduced oxygen consumption rates (OCR) and ATP levels. To further verify the importance of heme in promoting tumorigenicity, we generated NSCLC cell lines with increased heme synthesis or uptake by overexpressing either the ratelimiting heme synthesis enzyme ALAS1 or uptake protein SLC48A1, respectively. These cells exhibited enhanced migration and invasion and accelerated tumor growth in mice. Notably, tumors formed by cells with increased heme synthesis or uptake also displayed elevated OCRs and ATP levels. These data show that elevated heme flux and function underlie enhanced OXPHOS and tumorigenicity of NSCLC cells. Targeting heme flux and function offers a potential strategy for developing therapies for lung cancer. IntroductionLung cancer is the leading cause of cancer-related deaths in the United States (1). About 85% of cases are non-small cell lung cancer (NSCLC). Although several chemotherapeutic and targeted therapeutic agents are approved for treating lung cancer, the 5-year survival rate remains 18%. The effectiveness of targeted therapies for lung cancer is lowered by the presence of multiple driver genes and intratumoral genetic heterogeneity (2). Likewise, three PD-1/PD-L1 checkpoint inhibitors, nivolumab, pembrolizumab, and atezolizumab, generally extend median overall survival by about 3 months for second-line treatment of advanced NSCLC, compared with docetaxel alone (3-5). In the front-line setting, the median progression-free survival extends from 6.0 months with platinum-doublet chemotherapy to 10.3 months with pembrolizumab in patients with untreated NSCLC characterized by a high level of PD-L1 expression (6). Thus, alternative therapeutic strategies are still needed for lung canc...

Contrary to Warburg’s hypothesis, mitochondrial oxidative phosphorylation (OXPHOS) contributes significantly to fueling cancer cells. Several recent studies have demonstrated that radiotherapy-resistant and chemotherapy-resistant cancer cells depend on OXPHOS for survival and progression. Several cancers exhibit an increased risk in association with heme intake. Mitochondria are widely known to carry out oxidative phosphorylation. In addition, mitochondria are also involved in heme synthesis. Heme serves as a prosthetic group for several proteins that constitute the complexes of mitochondrial electron transport chain. Therefore, heme plays a pivotal role in OXPHOS and oxygen consumption. Further, lung cancer cells exhibit heme accumulation and require heme for proliferation and invasion in vitro. Abnormalities in mitochondrial biogenesis and mutations are implicated in cancer. This review delves into mitochondrial OXPHOS and lesser explored area of heme metabolism in lung cancer.

◥Multispectral optoacoustic tomography (MSOT) is an emerging noninvasive imaging modality that can detect real-time dynamic information about the tumor microenvironment in humans and animals. Oxygen enhanced (OE)-MSOT can monitor tumor vasculature and oxygenation during disease development or therapy. Here, we used MSOT and OE-MSOT to examine in mice the response of human non-small cell lung cancer (NSCLC) xenografts to a new class of antitumor drugs, heme-targeting agents heme-sequestering peptide 2 (HSP2) and cyclopamine tartrate (CycT). HSP2 inhibits heme uptake, while CycT inhibits heme synthesis in NSCLC cells, where heme is essential for ATP generation via oxidative phosphorylation. HSP2 and CycT can inhibit ATP generation and thereby suppress NSCLC cell tumorigenic functions. MSOT showed that treatment of NSCLC tumors with HSP2 or CycT reduced total hemoglobin, increased oxygen saturation, and enhanced the amplitude of response to oxygen gas breathing challenge. HSP2 and CycT normalized tumor vasculature and improved tumor oxygenation, where levels of several hypoxia markers in NSCLC tumors were reduced by treatment with HSP2 or CycT. Furthermore, treatment with HSP2 or CycT reduced levels of angiogenic factor VEGFA, its receptor VEGFR1, and vascular marker CD34. Together, our data show that heme-targeting drugs HSP2 and CycT elicit multiple tumor-suppressing functions, such as inhibiting angiogenic function, normalizing tumor vasculature, alleviating tumor hypoxia, and inhibiting oxygen consumption and ATP generation.Significance: Heme-targeting agents HSP2 and CycT effectively normalize tumor vasculature and alleviate tumor hypoxia, raising the possibility of their combination with chemo-, radio-, and immunotherapies to improve antitumor efficacy.

Lung cancer remains the leading cause of cancer-related death, despite the advent of targeted therapies and immunotherapies. Therefore, it is crucial to identify novel molecular features unique to lung tumors. Here, we show that cyclopamine tartrate (CycT) strongly suppresses the growth of subcutaneously implanted non-small cell lung cancer (NSCLC) xenografts and nearly eradicated orthotopically implanted NSCLC xenografts. CycT reduces heme synthesis and degradation in NSCLC cells and suppresses oxygen consumption in purified mitochondria. In orthotopic tumors, CycT decreases the levels of proteins and enzymes crucial for heme synthesis, uptake, and oxidative phosphorylation (OXPHOS). CycT also decreases the levels of two regulators promoting OXPHOS, MYC and MCL1, and effectively alleviates tumor hypoxia. Evidently, CycT acts via multiple modes to suppress OXPHOS. One mode is to directly inhibit mitochondrial respiration/OXPHOS. Another mode is to inhibit heme synthesis and degradation. Both modes appear to be independent of hedgehog signaling. Addition of heme to NSCLC cells partially reverses the effect of CycT on oxygen consumption, proliferation, and tumorigenic functions. Together, our results strongly suggest that CycT suppress tumor growth in the lung by inhibiting heme metabolism and OXPHOS. Targeting heme metabolism and OXPHOS may be an effective strategy to combat lung cancer.

The yeast Gis1 protein is a transcriptional regulator belonging to the JMJD2/KDM4 subfamily of demethylases that contain a JmjC domain, which are highly conserved from yeast to humans. They have important functions in histone methylation, cellular signaling and tumorigenesis. Besides serving as a cofactor in many proteins, heme is known to directly regulate the activities of proteins ranging from transcriptional regulators to potassium channels. Here, we report a novel mechanism governing heme regulation of Gis1 transcriptional and histone demethylase activities. We found that two Gis1 modules, the JmjN + JmjC domain and the zinc finger (ZnF), can bind to heme specifically in vitro. In vivo functional analysis showed that the ZnF, not the JmjN + JmjC domain, promotes heme activation of transcriptional activity. Likewise, measurements of the demethylase activity of purified Gis1 proteins showed that full-length Gis1 and the JmjN + JmjC domain both possess demethylase activity. However, heme potentiates the demethylase activity of full-length Gis1, but not that of the JmjN + JmjC domain, which can confer heme activation of transcriptional activity in an unrelated protein. These results demonstrate that Gis1 represents a novel class of multi-functional heme sensing and signaling proteins, and that heme binding to the ZnF stimulates Gis1 demethylase and transcriptional activities.