Hypoxic Non-small Cell Lung Cancer Research Articles

Abstract 383: Mic19 regulates mitophagy in non-small cell lung cancer under hypoxic microenvironment

Abstract Background: Mitophagy is a pivotal cellular process crucial for maintaining homeostasis in hypoxic conditions, selectively eliminating dysfunctional mitochondria through autophagy. This process is implicated in various diseases, including cancer. Non-Small Cell Lung Cancer (NSCLC), known for its prevalence and aggressiveness, often encounters hypoxic microenvironments. Mic19, a core protein of the mitochondrial cristae membrane, plays vital roles in maintaining mitochondrial architecture and function. However, its involvement in mitophagy in hypoxic NSCLC and the underlying molecular mechanisms remain inadequately explored. Methods: NSCLC cells were cultured under conditions with 1% O2 oxygen concentration to simulate a hypoxic microenvironment. Protein expression levels of Mic19 were analyzed using Western Blot and immunofluorescence (IF). Cellular proliferation and metastasis capability were assessed through CCK8 and transwell experiments. Immunoprecipitation experiments were conducted to validate the interaction between Mic19 and key mitophagy regulatory proteins. IF, confocal microscopy, ATP measurement, oxygen consumption rate (OCR), mitochondrial membrane potential, and transmission electron microscopy were employed to assess the formation of mitochondrial cristae and the level of mitophagy. Results: In hypoxic conditions, Mic19 exhibited up-regulation in NSCLC cells. Subsequent in vitro experiments confirmed Mic19's pivotal role in promoting the growth and metastasis of lung cancer cell lines. Through clinical data analysis, Mic19 emerged as an independent prognostic indicator for NSCLC. Inhibition of Mic19 expression exacerbated the disruption of mitochondrial morphology and structure in lung cancer cells, accompanied by the loss of mitochondrial cristae. Notably, Mic19 expression closely correlated with ATP and OCR production in tumor cells. Assessment of mitochondrial membrane potential and transmission electron microscopy revealed Mic19 as a critical downstream target of hypoxia, mediating mitophagy. Overall, these findings not only underscore the significance of Mic19 in regulating mitochondrial dynamics but also highlight its multifaceted role in tumor progression, positioning Mic19 as a promising therapeutic target and prognostic biomarker for NSCLC. Conclusion: This study provides novel insights into the mechanistic understanding of hypoxia-induced mitophagy in NSCLC, emphasizing the regulatory role of Mic19. Mic19 emerges as a potential therapeutic target for future interventions in NSCLC patients. Citation Format: Hua Huang, Chen Ding, Zhanrui Zhang, Di Wu, Chen Chen, Yongwen Li, Hongyu Liu, Jun Chen. Mic19 regulates mitophagy in non-small cell lung cancer under hypoxic microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 383.

Abstract 3273: Tirapazamine sensitizes homologous recombination-proficient cancers by enhancing DNA damage andferroptosis

Abstract Despite its success in HR-deficient tumors, the application of PARP inhibitors (PARPis) in HR-proficient cancers is very limited. Since the majority of cancers are HR-proficient, there is a need for developing strategies to extend the use of PARPis to HR-proficient cancers. We investigated whether the hypoxic cytotoxin, tirapazamine (TPZ) sensitizes HR-proficient cancer cells to PARPis under normoxia and hypoxia (2% O2), and found that TPZ enhanced PARPi cell killing to a greater extent under hypoxia compared to normoxia. Mechanistically, this enhanced killing was due to the induction of DNA double-strand breaks and free radicals that lead to ferroptotic cell death. To determine the role of hypoxia in the anti-tumor effect of the combination treatment, we examined the efficacy of TPZ and PARPi treatment in hypoxic spheroids and subcutaneous xenograft models derived from a panel of HR-proficient human non-small cell lung cancer (NSCLC) cells. We found that PARPis combined with TPZ significantly inhibited the growth of the hypoxic NSCLC spheroids. In addition, the combination treatment significantly inhibited the growth of A549 and CORL105 xenografts that possess significant hypoxic fractions, but did not significantly inhibit the growth of well-oxygenated Calu3 and H3122 xenografts. Taken together, this study demonstrates that TPZ can sensitize HR-proficient tumor cells to PARPi through the introduction of DNA strand breaks and free radicals, suggesting that TPZ treatment can broaden the usage of PARPi to HR-proficient cancers. Citation Format: Yanyan Jiang, Hala Estephan, Yizhou Wang, Ftoon Aljarbou, Nuria V. Lopera, Eui Jung Moon, Ester M. Hammond, Amato J. Giaccia. Tirapazamine sensitizes homologous recombination-proficient cancers by enhancing DNA damage andferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3273.

Hypoxia Modulates Radiosensitivity and Response to Different Radiation Qualities in A549 Non-Small Cell Lung Cancer (NSCLC) Cells.

Hypoxia-induced radioresistance reduces the efficacy of radiotherapy for solid malignancies, including non-small cell lung cancer (NSCLC). Cellular hypoxia can confer radioresistance through cellular and tumor micro-environment adaptations. Until recently, studies evaluating radioresistance secondary to hypoxia were designed to maintain cellular hypoxia only before and during irradiation, while any handling of post-irradiated cells was carried out in standard oxic conditions due to the unavailability of hypoxia workstations. This limited the possibility of simulating in vivo or clinical conditions in vitro. The presence of molecular oxygen is more important for the radiotoxicity of low-linear energy transfer (LET) radiation (e.g., X-rays) than that of high-LET carbon (12C) ions. The mechanisms responsible for 12C ions' potential to overcome hypoxia-induced radioresistance are currently not fully understood. Therefore, the radioresistance of hypoxic A549 NSCLC cells following exposure to X-rays or 12C ions was investigated along with cell cycle progression and gene expression by maintaining hypoxia before, during and after irradiation. A549 cells were incubated under normoxia (20% O2) or hypoxia (1% O2) for 48 h and then irradiated with X-rays (200 kV) or 12C ions (35 MeV/n, LET ~75 keV/µm). Cell survival was evaluated using colony-forming ability (CFA) assays immediately or 24 h after irradiation (late plating). DNA double-strand breaks (DSBs) were analyzed using γH2AX immunofluorescence microscopy. Cell cycle progression was determined by flow cytometry of 4',6-diamidino-2-phenylindole-stained cells. The global transcription profile post-irradiation was evaluated by RNA sequencing. When hypoxia was maintained before, during and after irradiation, hypoxia-induced radioresistance was observed only in late plating CFA experiments. The killing efficiency of 12C ions was much higher than that of X-rays. Cell survival under hypoxia was affected more strongly by the timepoint of plating in the case of X-rays compared to 12C ions. Cell cycle arrest following irradiation under hypoxia was less pronounced but more prolonged. DSB induction and resolution following irradiation were not significantly different under normoxia and hypoxia. Gene expression response to irradiation primarily comprised cell cycle regulation for both radiation qualities and oxygen conditions. Several PI3K target genes involved in cell migration and cell motility were differentially upregulated in hypoxic cells. Hypoxia-induced radioresistance may be linked to altered cell cycle response to irradiation and PI3K-mediated changes in cell motility and migration in A549 cells rather than less DNA damage or faster repair.

Evaluation of use of hyperbaric oxygen in suppression of hyper-proliferation in hypoxic NSCLC cells.

Hyperbaric oxygen therapy (HBO) is being researched as a potential adjuvant treatment for solid malignancies, such as NSCLC. It can reduce tumour hypoxia and has been found to slow tumour growth, stop dedifferentiation, and reduce apoptosis resistance in hypoxic NSCLC cells. Though HBO has shown promise in treating various cancers, more study is required to determine its precise mechanism of action in NSCLC. Analyze the effect of hyperbaric oxygen on the growth of hypoxic non-small cell lung cancer cells. We used the NSCLC cell lines A549 and H1299 to analyze aerobic glycolysis in vitro. Warburg effect testing included glucose absorption, lactate, adenosine triphosphate (ATP), and pyruvate measurements. Using a quantitative glycolytic flow model, we also analyzed the effect of HIF-1-induced genes on the flux of glucose metabolism. Lewis lung carcinoma (LLC) animal models in C57BL/6J mice were used to examine the development of lung tumours. The effects of pcDNA and HIF1A on glucose uptake, lactate production, pyruvate, and ATP levels were studied in A549 and H1299 NSCLC cells. While A549's glucose absorption increased over time, H1299's was dramatically decreased by HBO treatment. The pyruvate levels were more significant in H1299, particularly in hypoxia, and were lowered by HBO. In A549, the lactate content was more effective. After HBO treatment, glucose absorption was reduced while intracellular ATP levels were maintained. Overexpression of HIF-1a was able to counteract the effect of HBO on glycolytic gene expression. PFKP is a possible therapeutic target because HBO reduces the Warburg effect in NSCLC cells by downregulating HIF-1.

Roles of hypoxic environment and M2 macrophage-derived extracellular vesicles on the progression of non-small cell lung cancer

BackgroundHypoxia contributes to the development of invasive and metastatic cancer cells, and is detrimental to cancer treatment. This study aimed to explore the molecular mechanisms by which hypoxic microenvironments affect hypoxic non-small cell lung cancer (NSCLC) development and the effects of M2 macrophage-derived extracellular vesicles (EVs) on NSCLC cells.MethodsA549 cells were cultured in an anoxic incubator for 48 h to construct hypoxic A549 cells, and then normal and hypoxic A549 cells were harvested for RNA sequencing. Next, THP-1 cells were used to induce M2 macrophages, and EVs were isolated from THP-1 cells and M2 macrophages. Cell counting kit-8 and transwell assays were used to determine the viability and migration of hypoxic A549 cells, respectively.ResultsAfter sequencing, 2426 DElncRNAs and 501 DEmiRNAs were identified in normal A549 cells and hypoxic A549 cells. These DElncRNAs and DEmiRNAs were significantly enriched in “Wnt signaling pathway,” “Hippo signaling pathway,” “Rap1 signaling pathway,” “calcium signaling pathway,” “mTOR signaling pathway,” and “TNF signaling pathway.” Subsequently, ceRNA networks consisting of 4 lncRNA NDRG1 transcripts, 16 miRNAs and 221 target mRNAs were built, and the genes in the ceRNA networks were significantly associated with “Hippo signaling pathway” and “HIF-1 signaling pathway.” EVs were successfully extracted from THP-1 cells and M2 macrophages, and M2 macrophage-derived EVs significantly enhanced the viability and migration of hypoxic A549 cells. Finally, M2 macrophage-derived EVs further upregulated the expression of NDRG1-009, NDRG1-006, VEGFA, and EGLN3, while downregulating miR-34c-5p, miR-346, and miR-205-5p in hypoxic A549 cells.ConclusionsM2 macrophage-derived EVs may worsen the progression of NSCLC in a hypoxic microenvironment by regulating the NDRG1-009-miR-34c-5p-VEGFA, NDRG1-006-miR-346-EGLN3, NDRG1-009-miR-205-5p-VEGFA, and Hippo/HIF-1 signaling pathways.

Radio-resistance of hypoxic tumors: exploring the effects of oxygen and X-ray radiation on non-small lung cancer cell lines

BackgroundSolid tumors are often riddled with hypoxic areas, which develops as a result of high proliferation. Cancer cells willingly adapt and thrive in hypoxia by activating complex changes which contributes to survival and enhanced resistance to treatments, such as photon radiation. Photon radiation primarily relies on oxygen for the production of reactive oxygen species to induce DNA damage. The present in-vitro study aimed at investigating the biochemical responses of hypoxic non-small cell lung cancer (NSCLC) cells, particularly the effects on the DNA damage repair systems contributing to more radioresistant phenotypes and their pro- and anti-oxidant potential, within the first 24 h post-IR.MethodsNSCLC cell lines (H460, A549, Calu-1) were irradiated using varying X-ray doses under normoxia (21% O2) and hypoxia (0.1% O2). The overall cell survival was assessed by clonogenic assays. The extent of irradiation (IR)-induced DNA damage was evaluated by analyzing γ-H2AX foci induction and the altered expression of repair genes involved in non-homologous end joining and homologous recombination pathways. Moreover, cell-altered responses were investigated, including the nuclear and cytosolic hydrogen peroxide (H2O2) production, as well as the associated anti-oxidant potential, in particular some components related to the glutathione system.ResultsAnalysis of clonogenic survival revealed an enhanced radioresistance of the hypoxic NSCLC cells associated with reduced DNA damage and a downregulation of DNA repair genes. Moreover, nuclear H2O2 levels were IR-induced in a dose-dependent manner only under normoxia, and directly correlated with the DNA double-strand breaks. However, the observed nuclear H2O2 reduction in hypoxia appeared to be unaffected by IR, thus highlighting a possible reason for the enhanced radioresistance of the hypoxic NSCLC cells. The cellular antioxidant capacity was upregulated by IR in both oxygen conditions most likely helping to counteract the radiation effect on the cytosolic H2O2.ConclusionsIn conclusion, our data provide insight into the adaptive behavior of radiation-resistant hypoxic NSCLC cells, in particular their DNA repair and oxidative stress responses, which could contribute to lower DNA damage and higher cell survival rates following X-ray exposure. These findings may therefore help to identify potential targets for improving cancer treatment outcomes.

Exosomal protein angiopoietin-like 4 mediated radioresistance of lung cancer by inhibiting ferroptosis under hypoxic microenvironment

BackgroundHypoxia-mediated radioresistance is a major reason for the adverse radiotherapy outcome of non-small cell lung cancer (NSCLC) in clinical, but the underlying molecular mechanisms are still obscure.MethodsCellular and exosomal ANGPTL4 proteins under different oxygen status were examined. Colony survival, lipid peroxidation and hallmark proteins were employed to determine the correlation between ferroptosis and radioresistance. Gene regulations, western blot and xenograft models were used to explore the underlying mechanisms of the role of ANGPTL4 in radioresistance.ResultsANGPTL4 had a much higher level in hypoxic NSCLC cells compared to normoxic cells. Up- or down- regulation of ANGPTL4 positively interrelated to the radioresistance of NSCLC cells and xenograft tumours. GPX4-elicited ferroptosis suppression and lipid peroxidation decrease were authenticated to be involved in the hypoxia-induced radioresistance. ANGPTL4 encapsulated in the exosomes from hypoxic cells was absorbed by neighbouring normoxic cells, resulting in radioresistance of these bystander cells in a GPX4-dependent manner, which was diminished when ANGPTL4 was downregulated in the donor exosomes.ConclusionHypoxia-induced ANGPTL4 rendered radioresistance of NSCLC through at least two parallel pathways of intracellular ANGPTL4 and exosomal ANGPTL4, suggesting that ANGPTL4 might applicable as a therapeutic target to improve the therapeutic efficacy of NSCLC.

Mitochondrial ROS drive resistance to chemotherapy and immune-killing in hypoxic non-small cell lung cancer

BackgroundSolid tumors subjected to intermittent hypoxia are characterized by resistance to chemotherapy and immune-killing by effector T-lymphocytes, particularly tumor-infiltrating Vγ9Vδ2 T-lymphocytes. The molecular circuitries determining this double resistance are not known.MethodsWe analyzed a panel of 28 human non-small cell lung cancer (NSCLC) lines, using an in vitro system simulating continuous and intermittent hypoxia. Chemosensitivity to cisplatin and docetaxel was evaluated by chemiluminescence, ex vivo Vγ9Vδ2 T-lymphocyte expansion and immune-killing by flow cytometry. Targeted transcriptomics identified efflux transporters and nuclear factors involved in this chemo-immuno-resistance. The molecular mechanism linking Hypoxia-inducible factor-1α (HIF-1α), CCAAT/Enhancer Binding Protein-β (C/EBP-β) isoforms LAP and LIP, ABCB1, ABCC1 and ABCA1 transporters were evaluated by immunoblotting, RT-PCR, RNA-IP, ChIP. Oxidative phosphorylation, mitochondrial ATP, ROS, depolarization, O2 consumption were monitored by spectrophotometer and electronic sensors. The role of ROS/HIF-1α/LAP axis was validated in knocked-out or overexpressing cells, and in humanized (Hu-CD34+NSG) mice bearing LAP-overexpressing tumors. The clinical meaning of LAP was assessed in 60 NSCLC patients prospectively enrolled, treated with chemotherapy.ResultsBy up-regulating ABCB1 and ABCC1, and down-regulating ABCA1, intermittent hypoxia induced a stronger chemo-immuno-resistance than continuous hypoxia in NSCLC cells. Intermittent hypoxia impaired the electron transport chain and reduced O2 consumption, increasing mitochondrial ROS that favor the stabilization of C/EBP-β mRNA mediated by HIF-1α. HIF-1α/C/EBP-β mRNA binding increases the splicing of C/EBP-β toward the production of LAP isoform that transcriptionally induces ABCB1 and ABCC1, promoting the efflux of cisplatin and docetaxel. LAP also decreases ABCA1, limiting the efflux of isopentenyl pyrophosphate, i.e. the endogenous activator of Vγ9Vδ2 T-cells, and reducing the immune-killing. In NSCLC patients subjected to cisplatin-based chemotherapy, C/EBP-β LAP was abundant in hypoxic tumors and was associated with lower response to treatment and survival. LAP-overexpressing tumors in Hu-CD34+NSG mice recapitulated the patients’ chemo-immuno-resistant phenotype. Interestingly, the ROS scavenger mitoquinol chemo-immuno-sensitized immuno-xenografts, by disrupting the ROS/HIF-1α/LAP cascade.ConclusionsThe impairment of mitochondrial metabolism induced by intermittent hypoxia increases the ROS-dependent stabilization of HIF-1α/LAP complex in NSCLC, producing chemo-immuno-resistance. Clinically used mitochondrial ROS scavengers may counteract such double resistance. Moreover, we suggest C/EBP-β LAP as a new predictive and prognostic factor in NSCLC patients.

Cyclophilin a represses reactive oxygen species generation and death of hypoxic non-small-cell lung cancer cells by degrading thioredoxin-interacting protein

ABSTRACT Cyclophilin A (cypA) is overexpressed in many types of carcinomas, including non-small-cell lung cancer (NSCLC). However, the effect of anoxia, a critical feature of the carcinoma cell microenvironment, on cypA expression in NSCLC is unknown. Here, formaldehyde-fixed and paraffin-embedded samples were collected from 60 subjects with NSCLC. The protein expression levels of cypA and hypoxia-inducible factor-1α (HIF-1α) were evaluated using immunohistochemistry. Kaplan–Meier analysis showed that subjects with high cypA expression had remarkably shorter progression-free survival than those with low cypA expression. Furthermore, cypA expression levels were significantly related to HIF-1α expression levels (Spearman’s correlation = 0.34, P < 0.0001). To further assess the effect of cypA, an anoxic carcinoma cell model was established. CypA expression was remarkably upregulated in H1299 and A549 cell lines under hypoxic conditions. Overexpression of cypA restored hypoxia-impaired cell growth and prevented reactive oxygen species (ROS) production and cell death in hypoxic A549 and H1299 cells. However, these phenotypes were not altered by the inactive R55A mutant of cypA. Mechanistic studies demonstrated that cypA can bind to and degrade the tumor suppressor protein TXNIP in H1299 and A549 cells. Restored TXNIP expression in cypA-overexpressed and hypoxic NSCLC cells led to increased ROS levels and apoptotic cell numbers and decreased cell growth compared with cypA-overexpressed and hypoxic NSCLC cells. These findings indicate that anoxia results in an increase in cypA expression in NSCLC. Additionally, cypA served as an oncogene during hypoxia by interacting with TXNIP.

Hyperbaric Oxygen Therapy Represses the Warburg Effect and Epithelial-Mesenchymal Transition in Hypoxic NSCLC Cells via the HIF-1α/PFKP Axis.

BackgroundTumor cells initiate hypoxia-induced mechanisms to fuel cell proliferation, invasion, and metastasis, largely mediated by low O2-responsive Hypoxia-Inducible Factor 1 Alpha (HIF-1α). Therefore, hyperbaric oxygen therapy (HBO) is now being studied in cancer patients, but its impact upon non-small-cell lung cancer (NSCLC) cell metabolism remains uncharacterized.MethodsWe employed the NSCLC cell lines A549 and H1299 for in vitro studies. Glucose uptake, pyruvate, lactate, and adenosine triphosphate (ATP) assays were used to assess aerobic glycolysis (Warburg effect). A quantitative glycolytic flux model was used to analyze the flux contributions of HIF-1α-induced glucose metabolism genes. We used a Lewis lung carcinoma (LLC) murine model to measure lung tumorigenesis in C57BL/6J mice.ResultsHBO suppressed hypoxia-induced HIF-1α expression and downstream HIF-1α signaling in NSCLC cells. One HIF-1α-induced glucose metabolism gene—Phosphofructokinase, Platelet (PFKP)—most profoundly enhanced glycolytic flux under both low- and high-glucose conditions. HBO suppressed hypoxia-induced PFKP transactivation and gene expression via HIF-1α downregulation. HBO’s suppression of the Warburg effect, suppression of hyperproliferation, and suppression of epithelial-to-mesenchymal transition (EMT) in hypoxic NSCLC cell lines is mediated by the HIF-1α/PFKP axis. In vivo, HBO therapy inhibited murine LLC lung tumor growth in a Pfkp-dependent manner.ConclusionsHBO’s repression of the Warburg effect, repression of hyperproliferation, and repression of EMT in hypoxic NSCLC cells is dependent upon HIF-1α downregulation. HIF-1α’s target gene PFKP functions as a central mediator of HBO’s effects in hypoxic NSCLC cells and may represent a metabolic vulnerability in NSCLC tumors.

Cisplatin-resistant NSCLC cells induced by hypoxia transmit resistance to sensitive cells through exosomal PKM2.

Hypoxia is commonly observed in solid tumors and contributes to the resistance of DNA damage drugs. However, the mechanisms behind this resistance are still unclear. In this study, we aimed to explore the effects of hypoxia-induced exosomes on non-small cell lung cancer (NSCLC).Methods: NSCLC cells were subjected to either normoxic or hypoxic conditions to assess cell survival and changes in the expression levels of key proteins. Comparative proteomics were performed to identify exosomal PKM2 in normoxic or hypoxic cisplatin-resistant NSCLC cells-derived exosomes. Functions of hypoxia induced-exosomal PKM2 in promoting cisplatin resistance to NSCLC cells were evaluated both in vitro and in vivo experiments and the molecular mechanisms of hypoxia induced-exosomal PKM2 were demonstrated using flow cytometry, immunoblotting, oxidative stress detection and histological examination. A series of in vitro experiments were performed to evaluate the function of hypoxia-induced exosomes on cancer-associated fibroblasts (CAFs).Results: Hypoxia exacerbated the cisplatin resistance in lung cancer cells due to the increased expression of PKM2 that was observed in the exosomes secreted by hypoxic cisplatin-resistance cells. We identified that hypoxia-induced exosomal PKM2 transmitted cisplatin-resistance to sensitive NSCLC cells in vitro and in vivo. Mechanistically, hypoxia-induced exosomal PKM2 promoted glycolysis in NSCLC cells to produce reductive metabolites, which may neutralize reactive oxygen species (ROS) induced by cisplatin. Additionally, hypoxia-induced exosomal PKM2 inhibited apoptosis in a PKM2-BCL2-dependent manner. Moreover, hypoxia-induced exosomal PKM2 reprogrammed CAFs to create an acidic microenvironment promoting NSCLC cells proliferation and cisplatin resistance.Conclusions: Our findings revealed that hypoxia-induced exosomes transmit cisplatin resistance to sensitive NSCLC cells by delivering PKM2. Exosomal PKM2 may serve as a promising biomarker and therapeutic target for cisplatin resistance in NSCLC.

Hypoxic Non-Small-Cell Lung Cancer Cell-Secreted Exosomal microRNA-582-3p Drives Cancer Cell Malignant Phenotypes by Targeting Secreted Frizzled-Related Protein 1.

BackgroundHypoxic environment and exosomes (exos)-mediated intercellular communication are crucial for cancer invasion and metastasis, but the mechanisms are not yet fully understood. In this study, we investigated the regulatory effect of hypoxic tumor cell-secreted exosomal miR-582-3p on non-small-cell lung cancer (NSCLC) cell malignant phenotypes.MethodsThe concentration and diameters of exos were evaluated by nanosight particle tracking analysis. microRNA-582-3p (miR-582-3p) expression was detected by quantitative real-time PCR. The fluorescent dye PKH26 was used to label exos. The direct interaction between miR-582-3p and secreted frizzled-related protein 1 (SFRP1) was determined by dual-luciferase activity assay. NSCLC cell proliferation, migration, and invasion abilities were assessed by cell count kit-8 assay, wound healing assay, and transwell migration and invasion assay. Western blot analysis was performed to detect the protein expression level.ResultsHypoxic NSCLC cell-derived exos promoted the proliferation, migration, and invasion of normoxic NSCLC cells. miR-582-3p expression was upregulated in hypoxic NSCLC cells and hypoxic NSCLC cell-secreted exos. Hypoxic NSCLC cell-derived exos transmitted miR-582-3p to normoxic NSCLC cells. Hypoxic NSCLC cell-secreted exosomal miR-582-3p promoted the proliferation, migration, and invasion of normoxic NSCLC cells. miR-582-3p inhibited the expression of SFRP1 protein by binding to its 3ʹ-UTR. In addition, enforced expression of SFRP1 restrained malignant phenotypes of normoxic NSCLC cells, which was abrogated by hypoxic NSCLC cell-secreted exosomal miR-582-3p.ConclusionHypoxic NSCLC cell-secreted exosomal miR-582-3p drives cancer cell malignant phenotypes by targeting SFRP1, which provides a better understanding of cancer metastasis and may facilitate the development of therapeutics against human NSCLC.

Hypoxic non-small-cell lung cancer cell-derived exosomal miR-21 promotes resistance of normoxic cell to cisplatin.

PurposeTo explore the effects of hypoxic non-small-cell lung cancer (NSCLC)-derived exosomes on NSCLC resistance to cisplatin.Materials and methodsExosomes were isolated by differential centrifugation and characterized by transmission electron microscope and Western blotting. Quantitative real-time PCR was used to measure miR-21 levels. MTT assays and colony formation assays were performed to investigate the effects of hypoxia-induced exosomes on cisplatin resistance.ResultsHypoxic NSCLC cell-derived exosomes facilitate normoxic cell resistance to cisplatin. In addition, hypoxia enhanced the miR-21 expression in NSCLC cells and cell-derived exosomes. Interestingly, changes in miR-21 levels in the hypoxia-induced exosomes affected the sensitivity of recipient cells to cisplatin. Mechanically, exosomal miR-21 promoted cisplatin resistance by downregulating phosphatase and tensin homolog (PTEN). The expression of miR-21 in tumor cell lines and clinical NSCLC tumor samples was positively correlated with hypoxia-inducible factor-1α and negatively correlated with PTEN. Moreover, high miR-21 expression was associated with shorter median survival period in patients undergoing pharmacotherapy, but no association was observed in patients who were not under pharmacotherapy.ConclusionExosomal miR-21 derived from hypoxic NSCLC cells may promote cisplatin resistance, which indicates that exosomal miR-21 might be a potential biomarker and therapeutic target to address NSCLC chemoresistance.

P53 mutants cooperate with HIF-1 in transcriptional regulation of extracellular matrix components to promote tumor progression

Mutations in the TP53 gene and microenvironmentally driven activation of hypoxia-inducible factor-1 (HIF-1) typically occur in later stages of tumorigenesis. An ongoing challenge is the identification of molecular determinants of advanced cancer pathogenesis to design alternative last-line therapeutic options. Here, we report that p53 mutants influence the tumor microenvironment by cooperating with HIF-1 to promote cancer progression. We demonstrate that in non-small cell lung cancer (NSCLC), p53 mutants exert a gain-of-function (GOF) effect on HIF-1, thus regulating a selective gene expression signature involved in protumorigenic functions. Hypoxia-mediated activation of HIF-1 leads to the formation of a p53 mutant/HIF-1 complex that physically binds the SWI/SNF chromatin remodeling complex, promoting expression of a selective subset of hypoxia-responsive genes. Depletion of p53 mutants impairs the HIF-mediated up-regulation of extracellular matrix (ECM) components, including type VIIa1 collagen and laminin-γ2, thus affecting tumorigenic potential of NSCLC cells in vitro and in mouse models in vivo. Analysis of surgically resected human NSCLC revealed that expression of this ECM gene signature was highly correlated with hypoxic tumors exclusively in patients carrying p53 mutations and was associated with poor prognosis. Our data reveal a GOF effect of p53 mutants in hypoxic tumors and suggest synergistic activities of p53 and HIF-1. These findings have important implications for cancer progression and might provide innovative last-line treatment options for advanced NSCLC.

MiR-1908 Overexpression Inhibits Proliferation, Changing Akt Activity and p53 Expression in Hypoxic NSCLC Cells.

The ribosomal protein (RP)–p53 pathway has been shown to play a key role in apoptosis and senescence of cancer cells. miR-1908 is a newly found miRNA that was reported to have prognostic potential in melanoma. However, its role and mechanism in the progression of non-small cell lung cancer (NSCLC) are largely unknown. In this study, we found that expression of miR-1908 was significantly downregulated in human NSCLC cell lines, including SK-MES-1, A549, and NCI-H460. Then the role of miR-1908 in NSCLC cell proliferation was explored. The miR-1908 mimic was transfected into NSCLC cell lines, and their proliferation was detected. MTT and Cell Titer-Blue H analyses showed that the cell proliferation was notably reduced by the miR-1908 mimic transfection. Moreover, we found the RP–p53 pathway was activated by miR-1908 mimic. Moreover, the miR-1908 inhibitor transfection had a completely opposite effect on the NSCLC cell proliferation than that of miR-1908 mimic. To explore the underlying mechanism of that, TargetScan bioinformatics server and 3′-UTR luciferase reporter assay were applied to identify the targets of miR-1908. Our results showed that AKT1 substrate 1 (AKT1S1), a newly proven suppressor of the RP–p53 pathway, was a target of miR-1908, suggesting a probable mechanism for miR-191 suppressing NSCLC cell proliferation. Our findings provide a novel molecular target for the regulation of NSCLC cell proliferation.

Twist may be associated with invasion and metastasis of hypoxic NSCLC cells.

Hypoxia promotes tumor invasion and metastasis via multiple mechanisms, including epithelial-mesenchymal transition (EMT). Twist, an EMT regulator, has been disclosed to associate with invasion and metastasis as well as poor prognosis of many malignancies. However, it remains undefined whether Twist is involved in invasion and metastasis of hypoxic non-small cell lung cancer (NSCLC). In this study, protein levels of Twist, hypoxia-inducible factor-1α (HIF-1α), and EMT markers (E-cadherin and vimentin) were examined by immunohistochemistry in 76 lung cancer tissues from NSCLC patients. Expression of Twist and its correlation with HIF-1α, E-cadherin, and vimentin were analyzed. Small interfering RNA (siRNA) against Twist was used to knockdown Twist expression in hypoxic NSCLC cells, A549 and NCI-H460. Cellular invasion and protein levels of Twist, E-cadherin, and vimentin were evaluated by matrigel invasion assay and Western blot, respectively. Our results showed that in clinical samples, there was a significant association between Twist expression and differentiation degree, lymph node metastasis, and TNM stage. Correlation analysis demonstrated that expression of Twist was negatively correlated with E-cadherin expression, but positively associated with HIF-1α and vimentin expression. In cultured NSCLC cells, Twist messenger RNA (mRNA) and protein levels were upregulated under hypoxia, while knockdown of Twist suppressed potentiated invasion and expression of mesenchymal marker vimentin induced by hypoxia. Protein level of increased epithelial marker E-cadherin was shown along with Twist downregulation. These findings suggest that Twist promoting hypoxic invasion and metastasis of NSCLC may be associated with altered expression of EMT markers. Inhibition of Twist may be of therapeutic significance.

Amyloid precursor protein (APP) affects global protein synthesis in dividing human cells.

Hypoxic non‐small cell lung cancer (NSCLC) is dependent on Notch‐1 signaling for survival. Targeting Notch‐1 by means of γ‐secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post‐mortem analysis of GSI‐treated, NSCLC‐burdened mice suggested enhanced phosphorylation of 4E‐BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non‐canonical 4E‐BP1 phosphorylation pattern rearrangement—a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF‐4F composition indicating increased recruitment of eIF‐4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF‐4A assembly into eIF‐4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap‐ and IRES‐dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin‐1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC‐1) inhibition affected 4E‐BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC‐1. Key phenomena described in this study were reversed by overexpression of the APP C‐terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC‐1 regulation of cap‐dependent protein synthesis. J. Cell. Physiol. 230: 1064–1074, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

Abstract C40: Amyloid precursor protein (APP) synchronizes cell cycle progression and the rate of global protein synthesis in dividing cells

Abstract In previous studies we targeted hypoxic Non-Small Cell Lung Cancer (NSCLC) tumor tissue using gamma-secretase inhibitors (GSI) in a preclinical model of advanced NSCLC. GSI treatment of mice suggested reactivation of mTORC-1 in otherwise quiescent hypoxic NSCLC tissue. Methods included immunohistochemistry and cytochemistry, immunoblotting, immunoprecipitation, siRNA-mediated gene knockdown, nucleotide and amino acid analogs incorporation, FACS analysis, various imaging techniques, Q-PCR, gene transfection, cap binding assays, RNA and antibodies arrays. GSI treatment of mice showed a re-induced phosphorylation of 4E-BP1 at T37/46 in hypoxic tumor masses. In vitro studies indicated a role for APP in this phenomenon. APP downregulation caused a rearrangement in 4E-BP1's phosphorylation pattern at residues T37/46, S65 and T70, reversed by overexpression of the APP C-terminal domain. Increased recruitment of eIF4A on the mRNA cap was observed. These events coincided with a drastic increase in the rate of global protein synthesis, an event that appeared to be mTOR-independent. Meanwhile, NSCLC cells with depleted APP were arrested in G0/G1 through a cyclin C-mediated mechanism. Cells arrested in G0/G1 with augmented rate of global protein synthesis (both cap- and IRES-dependent) increased their size and underwent a substantial necrotic cell death due to cell membrane permeabilization. These results indicate a novel role for APP in synchronizing the rate of protein synthesis and cell cycle progression, raising questions on how these processes interact in malignant transformation. The data presented here suggest that cells of different origin, including NSCLC cells, have developed some mechanisms to couple cell growth with cell cycle progression independent of overlapping signals which derive, among others, from the PI3-K pathway. APP seems to play a central role in this synchronization, and its loss of function causes cell size abnormalities and death. Citation Format: Anna Sobol, Paola Galluzzo, Shuang Liang, Brittany Rambo, Sylvia Skucha, Megan Weber, Maurizio Bocchetta. Amyloid precursor protein (APP) synchronizes cell cycle progression and the rate of global protein synthesis in dividing cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C40.

Lysyl Oxidase May Play a Critical Role in Hypoxia-Induced NSCLC Cells Invasion and Migration

Lysyl oxidase (LOX), a copper-dependent amine oxidase known to function both intracellularly and extracellularly, is implicated in promoting tumor progression and hypoxic metastasis in certain malignancies. Nonsmall cell lung cancer (NSCLC) is a highly aggressive cancer with poor prognosis worldwide. However, the role and molecular mechanism by which LOX involving in hypoxic NSCLC invasion and migration are poorly understood. This study explores the effect of LOX on invasion and migration of NSCLC cells under hypoxic conditions. Small interfering RNA (siRNA) targeting LOX was used to silence LOX expression of hypoxic NSCLC cells, SPCA1 and A549. Cellular invasive and migratory potentials were determined by matrigel invasion and migration assays. Expression of LOX, Src, Src activation (Tyr418 phosphorylation of Src), and Snail were evaluated by real-time PCR and western blot, respectively. The results showed that LOX mRNA and protein expression were upregulated under hypoxic conditions in NSCLC cells. Knockdown of LOX led to inhibition of hypoxia-induced invasion and migration. Phosphorylated Src (Tyr418) and Snail proteins were decreased along with LOX downregulation. Our data provide molecular evidences that LOX is mechanistically linked to increased invasion and migration of hypoxic NSCLC cells, and may serve as an antimetastasis target of human NSCLC.

Multimodality Approaches to Treat Hypoxic Non-Small Cell Lung Cancer (NSCLC) Microenvironment

We found both in vitro and in vivo that survival of NSCLC cells in a hypoxic microenvironment requires Notch-1 signaling. A hypoxic tumor environment represents a problem for NSCLC treatment because it plays a critical role in cancer resistance to chemotherapy, tumor recurrence, and metastasis. Here we targeted hypoxic tumor tissue in an orthotopic NSCLC model. We inhibited the Notch-1/IGF-1R/Akt-1 axis using 3 agents: a γ-secretase inhibitor or GSI (MRK-003), a fully humanized antibody against the human IGF-1R (MK-0646), and a pan-Akt inhibitor (MK-2206), alone or in various combinations including therapeutics currently in clinical use. All treatments but Akt inhibition significantly prolonged the median survival of mice compared with controls. GSI treatment caused specific cell death of hypoxic tumors. Tumors excised from mice displayed a significant reduction of markers of hypoxia. Moreover, GSI treatment caused reduced metastasis to the liver and brain. MK-0646 was not specific to a hypoxic tumor environment but substantially increased the median survival of treated mice compared with controls. NSCLC cells evaded MK-0646 treatment by specifically overactivating EGF-R both in vivo and in 5 cell lines in vitro. This phenomenon is achieved at the level of protein stability. MK-0646 treatment caused increased erlotinib sensitivity in NSCLC cells poorly responsive to it. Sequential treatment with MK-0646 followed by erlotinib prolonged median survival of mice significantly. When the 2 drugs were administered simultaneously, no survival benefit was observed, and this combination therapy proved less effective than MK-0646 used as single agent. Our data offer novel information that may provide insights for the planning of clinical trials in humans, likely for maintenance therapy of NSCLC patients.