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...

BackgroundAberrant 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.MethodsThe 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.ResultsOur 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.ConclusionsTogether, 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2200-x) contains supplementary material, which is available to authorized users.

BackgroundOncolytic viruses are considered part of immunotherapy and have shown promise in preclinical experiments and clinical trials. Results from these studies have suggested that tumor microenvironment remodeling is required to achieve an effective response in solid tumors. Here, we assess the extent to which targeting specific mechanisms underlying the immunosuppressive tumor microenvironment optimizes viroimmunotherapy.MethodsWe used RNA-seq analyses to analyze the transcriptome, and validated the results using Q-PCR, flow cytometry, and immunofluorescence. Viral activity was analyzed by replication assays and viral titration. Kyn and Trp metabolite levels were quantified using liquid chromatography–mass spectrometry. Aryl hydrocarbon receptor (AhR) activation was analyzed by examination of promoter activity. Therapeutic efficacy was assessed by tumor histopathology and survival in syngeneic murine models of gliomas, including Indoleamine 2,3-dioxygenase (IDO)-/- mice. Flow cytometry was used for immunophenotyping and quantification of cell populations. Immune activation was examined in co-cultures of immune and cancer cells. T-cell depletion was used to identify the role played by specific cell populations. Rechallenge experiments were performed to identify the development of anti-tumor memory.ResultsBulk RNA-seq analyses showed the activation of the immunosuppressive IDO-kynurenine-AhR circuitry in response to Delta-24-RGDOX infection of tumors. To overcome the effect of this pivotal pathway, we combined Delta-24-RGDOX with clinically relevant IDO inhibitors. The combination therapy increased the frequency of CD8+ T cells and decreased the rate of myeloid-derived suppressor cell and immunosupressive Treg tumor populations in animal models of solid tumors. Functional studies demonstrated that IDO-blockade-dependent activation of immune cells against tumor antigens could be reversed by the oncometabolite kynurenine. The concurrent targeting of the effectors and suppressors of the tumor immune landscape significantly prolonged the survival in animal models of orthotopic gliomas.ConclusionsOur data identified for the first time the in vivo role of IDO-dependent immunosuppressive pathways in the resistance of solid tumors to oncolytic adenoviruses. Specifically, the IDO-Kyn-AhR activity was responsible for the resurface of local immunosuppression and resistance to therapy, which was ablated through IDO inhibition. Our data indicate that combined molecular and immune therapy may improve outcomes in human gliomas and other cancers treated with virotherapy.

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.