Heme Sequestration Effectively Suppresses the Development and Progression of Both Lung Adenocarcinoma and Squamous Cell Carcinoma.

Lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are two most common subtypes of lung cancer. Here, to identify new, targetable molecular properties of both subtypes, we monitored changes in the levels of heme- and oxidative phosphorylation (OXPHOS)-related proteins during lung tumorigenesis. Heme is a central molecule for oxidative metabolism and ATP generation via OXPHOS. Notably, both lung ADC and SCC tumors can be induced in the genetically engineered KLLuc mouse model harboring the G12D Kras mutation and a conditional Lkb1 knockout. We found that the levels of the rate-limiting heme synthesis enzyme ALAS1 and uptake protein SLC48A1, along with OXPHOS complex subunits, progressively increased as lung tumorigenesis advanced. Our data demonstrated that elevated levels of heme- and OXPHOS-related proteins were associated with both ADC and SCC. Importantly, treatment of KLLuc mice with a heme-sequestering protein, HeSP2, that inhibits heme uptake in tumor cells effectively arrested lung tumor progression, and both ADC and SCC tumors were strongly suppressed. Additionally, HeSP2 effectively suppressed the growth of both SCC and ADC tumor xenografts in NOD/SCID mice. Further analyses indicated that HeSP2 effectively diminished OXPHOS in both ADC and SCC, reduced angiogenesis, alleviated tumor hypoxia, and suppressed cell proliferation. These results show that the advancing of lung tumorigenesis requires progressive increase in cellular heme synthesis and uptake, leading to intensified OXPHOS activity and ATP generation and promoting aggressive tumorigenic functions. IMPLICATIONS: Heme sequestration is an effective strategy for the suppression of both ADC and SCC tumor initiation and development.

Elevated Heme Synthesis and Uptake Underpin Intensified Oxidative Metabolism and Tumorigenic Functions in Non-Small Cell Lung Cancer Cells.

Tumors 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 rate-limiting 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. SIGNIFICANCE: These findings show that elevated heme availability due to increased heme synthesis and uptake causes intensified oxygen consumption and ATP generation, promoting tumorigenic functions and tumor growth in NSCLC. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/10/2511/F1.large.jpg.

Cyclopamine tartrate, a modulator of hedgehog signaling and mitochondrial respiration, effectively arrests lung tumor growth and progression.

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.

Essential roles of mitochondrial and heme function in lung cancer bioenergetics and tumorigenesis.

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.

The vascular disrupting agent combretastatin A-4 phosphate causes prolonged elevation of proteins involved in heme flux and function in resistant tumor cells.

Vascular disrupting agents (VDAs) represent a promising class of anti-cancer drugs for solid tumor treatment. Here, we aim to better understand the mechanisms underlying tumor reccurrence and treatment resistance following the administration of a VDA, combretastatin A-4 phosphate (CA4P). Firstly, we used photoacoustic tomography to noninvasively map the effect of CA4P on blood oxygen levels throughout subcutaneous non-small cell lung cancer (NSCLC) tumors in mice. We found that the oxygenation of peripheral tumor vessels was significantly decreased at 1 and 3 hours post-CA4P treatment. The oxygenation of the tumor core reduced significantly at 1 and 3 hours, and reached anoxia after 24 hours. Secondly, we examined the effect of CA4P on the levels of proteins involved in heme flux and function, which are elevated in lung tumors. Using immunohistochemistry, we found that CA4P substantially enhanced the levels of enzymes involved in heme biosynthesis, uptake, and degradation, as well as oxygen-utilizing hemoproteins. Furthermore, measurements of markers of mitochondrial function suggest that CA4P did not diminish mitochondrial function in resistant tumor cells. These results suggest that elevated levels of heme flux and function contribute to tumor regrowth and treatment resistance post-VDA administration.

Cyclopamine tartrate, an inhibitor of Hedgehog signaling, strongly interferes with mitochondrial function and suppresses aerobic respiration in lung cancer cells.

BACKGROUND: Aberrant Hedgehog (Hh) signaling is associated with the development of many cancers including prostate cancer, gastrointestinal cancer, lung cancer, pancreatic cancer, ovarian cancer, and basal cell carcinoma. The Hh signaling pathway has been one of the most intensely investigated targets for cancer therapy, and a number of compounds inhibiting Hh signaling are being tested clinically for treating many cancers. Lung cancer causes more deaths than the next three most common cancers (colon, breast, and prostate) combined. Cyclopamine was the first compound found to inhibit Hh signaling and has been invaluable for understanding the function of Hh signaling in development and cancer. To find novel strategies for combating lung cancer, we decided to characterize the effect of cyclopamine tartrate (CycT), an improved analogue of cyclopamine, on lung cancer cells and its mechanism of action. METHODS: The effect of CycT on oxygen consumption and proliferation of non-small-cell lung cancer (NSCLC) cell lines was quantified by using an Oxygraph system and live cell counting, respectively. Apoptosis was detected by using Annexin V and Propidium Iodide staining. CycT's impact on ROS generation, mitochondrial membrane potential, and mitochondrial morphology in NSCLC cells was monitored by using fluorometry and fluorescent microscopy. Western blotting and fluorescent microscopy were used to detect the levels and localization of Hh signaling targets, mitochondrial fission protein Drp1, and heme-related proteins in various NSCLC cells. RESULTS: Our findings identified a novel function of CycT, as well as another Hh inhibitor SANT1, to disrupt mitochondrial function and aerobic respiration. Our results showed that CycT, like glutamine depletion, caused a substantial decrease in oxygen consumption in a number of NSCLC cell lines, suppressed NSCLC cell proliferation, and induced apoptosis. Further, we found that CycT increased ROS generation, mitochondrial membrane hyperpolarization, and mitochondrial fragmentation, thereby disrupting mitochondrial function in NSCLC cells. CONCLUSIONS: Together, our work demonstrates that CycT, and likely other Hh signaling inhibitors, can interrupt NSCLC cell function by promoting mitochondrial fission and fragmentation, mitochondrial membrane hyperpolarization, and ROS generation, thereby diminishing mitochondrial respiration, suppressing cell proliferation, and causing apoptosis. Our work provides novel mechanistic insights into the action of Hh inhibitors in cancer cells.