Role and mechanism of arsenic in regulating angiogenesis

Arsenic is a wide spread carcinogen associated with several kinds of cancers including skin, lung, bladder, and liver cancers. Lung is one of the major targets of arsenic exposure. Angiogenesis is the pivotal process during carcinogenesis and chronic pulmonary diseases, but the role and mechanism of arsenic in regulating angiogenesis remain to be elucidated. In this study we show that short time exposure of arsenic induces angiogenesis in both human immortalized lung epithelial cells BEAS-2B and adenocarcinoma cells A549. To study the molecular mechanism of arsenic-inducing angiogenesis, we find that arsenic induces reactive oxygen species (ROS) generation, which activates AKT and ERK1/2 signaling pathways and increases the expression of hypoxia-inducible factor 1 (HIF-1) and vascular endothelial growth factor (VEGF). Inhibition of ROS production suppresses angiogenesis by decreasing AKT and ERK activation and HIF-1 expression. Inhibition of ROS, AKT and ERK1/2 signaling pathways is sufficient to attenuate arsenic-inducing angiogenesis. HIF-1 and VEGF are downstream effectors of AKT and ERK1/2 that are required for arsenic-inducing angiogenesis. These results shed light on the mechanism of arsenic in regulating angiogenesis, and are helpful to develop mechanism-based intervention to prevent arsenic-induced carcinogenesis and angiogenesis in the future.     

Macrophages Facilitate Coal Tar Pitch Extract-Induced Tumorigenic Transformation of Human Bronchial Epithelial Cells Mediated by NF-κB

Objective

Chronic respiratory inflammation has been associated with lung cancer. Tumor-associated macrophages (TAMs) play a critical role in the formation of inflammation microenvironment. We sought to characterize the role of TAMs in coal tar pitch extract (CTPE)-induced tumorigenic transformation of human bronchial epithelial cells and the underlying mechanisms.

Methods

The expression of TAMs-specific CD68 in lung cancer tissues and paired adjacent tissues from cancer patients was determined using immunostaining. Co-culture of human bronchial epithelial cells (BEAS-2B) and macrophage-like THP-1 cells were conducted to evaluate the promotive effect of macrophages on CTPE-induced tumorigenic transformation of BEAS-2B cells. BEAS-2B cells were first treated with 2.4 µg/mL CTPE for 72 hours. After removal of CTPE, the cells were continuously cultured either with or without THP-1 cells and passaged using trypsin-EDTA. Alterations of cell cycle, karyotype, colony formation in soft agar and tumor xenograft growth in nude mice of BEAS-2B cells at passages 10, 20 and 30, indicative of tumorigenecity, were determined, respectively. In addition, mRNA and protein levels of NF-κB in BEAS-2B cells were measured with RT-PCR and western blot, respectively. B(a)P was used as the positive control.

Results

The over-expression of TAMs-specific CD68 around lung tumor tissues was detected and associated with lung cancer progression. The tumorigenic alterations of BEAS-2B cells including increase in cell growth rate, number of cells with aneuploidy, clonogenicity in soft agar, and tumor size in nude mice in vivo occurred at passage 10, becoming significant at passages 20 and 30 of the co-culture following CTPE removal in compared to BEAS-2B cells alone. In addition, the expression levels of NF-κB in BEAS-2B cells were positively correlated to the malignancy of BEAS-2B cells under different conditions of treatment.

Conclusion

The presence of macrophages facilitated CTPE-induced tumorigenic transformation of BEAS-2B cells, which may be mediated by NF-κB.     

LncRNA FAM83A-AS1 facilitates tumor proliferation and the migration via the HIF-1α/ glycolysis axis in lung adenocarcinoma

Background: Lung adenocarcinoma (LUAD), the major subtype of lung cancer, is among the leading cause of cancer-related death worldwide. Energy-related metabolic reprogramming metabolism is a hallmark of cancer shared by numerous cancer types, including LUAD. Nevertheless, the functional pathways and molecular mechanism by which FAM83A-AS1 acts in metabolic reprogramming in lung adenocarcinoma have not been fully elucidated.Methods: We used transwell, wound-healing scratch assay, and metabolic assays to explore the effect of FAM83A-AS1 in LUAD cell lines. Western blotting, Co-IP assays, and ubiquitination assays were used to detect the effects of FAM83A-AS1 on HIF-1α expression, degradation, and its binding to VHL. Moreover, an in vivo subcutaneous tumor formation assay was used to detect the effect of FAM83A-AS1 on LUAD.Results: Herein, we identified FAM83A-AS1 as a metabolism-related lncRNA, which was highly correlated with glycolysis, hypoxia, and OXPHOS pathways in LUAD patients using bioinformatics analysis. In addition, we uncovered that FAM83A-AS1 could promote the migration and invasion of LUAD cells, as well as influence the stemness of LUAD cells in vivo and vitro. Moreover, FAM83A-AS1 was shown to promote glycolysis in LUAD cell lines in vitro and in vivo, and was found to influence the expression of genes related to glucose metabolism. Besides, we revealed that FAM83A-AS1 could affect glycolysis by regulating HIF-1α degradation. Finally, we found that FAM83A-AS1 knockdown could inhibit tumor growth and suppress the expression of HIF-1α and glycolysis-related genes in vivo.Conclusion: Our study demonstrates that FAM83A-AS1 contributes to LUAD proliferation and stemness via the HIF-1α/glycolysis axis, making it a potential biomarker and therapeutic target in LUAD patients.     

Hypoxia-inducible factor 1 activation by aerobic glycolysis implicates the Warburg effect in carcinogenesis

Cancer cells display high rates of aerobic glycolysis, a phenomenon known historically as the Warburg effect. Lactate and pyruvate, the end products of glycolysis, are highly produced by cancer cells even in the presence of oxygen. Hypoxia-induced gene expression in cancer cells has been linked to malignant transformation. Here we provide evidence that lactate and pyruvate regulate hypoxia-inducible gene expression independently of hypoxia by stimulating the accumulation of hypoxia-inducible Factor 1alpha (HIF-1alpha). In human gliomas and other cancer cell lines, the accumulation of HIF-1alpha protein under aerobic conditions requires the metabolism of glucose to pyruvate that prevents the aerobic degradation of HIF-1alpha protein, activates HIF-1 DNA binding activity, and enhances the expression of several HIF-1-activated genes including erythropoietin, vascular endothelial growth factor, glucose transporter 3, and aldolase A. Our findings support a novel role for pyruvate in metabolic signaling and suggest a mechanism by which high rates of aerobic glycolysis can promote the malignant transformation and survival of cancer cells.     

Using the “reverse Warburg effect” to identify high-risk breast cancer patients: Stromal MCT4 predicts poor clinical outcome in triple-negative breast cancers

We have recently proposed a new model of cancer metabolism to explain the role of aerobic glycolysis and L-lactate production in fueling tumor growth and metastasis. In this model, cancer cells secrete hydrogen peroxide (H₂O₂), initiating oxidative stress and aerobic glycolysis in the tumor stroma. This, in turn, drives L-lactate secretion from cancer-associated fibroblasts. Secreted L-lactate then fuels oxidative mitochondrial metabolism (OXPHOS) in epithelial cancer cells, by acting as a paracrine onco-metabolite. We have previously termed this type of two-compartment tumor metabolism the “reverse Warburg effect,” as aerobic glycolysis takes place in stromal fibroblasts, rather than epithelial cancer cells. Here, we used MCT4 immunostaining of human breast cancer tissue microarrays (TMAs; >180 triple-negative patients) to directly assess the prognostic value of the “reverse Warburg effect.” MCT4 expression is a functional marker of hypoxia, oxidative stress, aerobic glycolysis and L-lactate efflux. Remarkably, high stromal MCT4 levels (score = 2) were specifically associated with decreased overall survival (<18% survival at 10 years post-diagnosis). In contrast, patients with absent stromal MCT4 expression (score = 0), had 10-year survival rates of ∼97% (p-value < 10⁻³²). High stromal levels of MCT4 were strictly correlated with a loss of stromal Cav-1 (p-value < 10⁻¹⁴), a known marker of early tumor recurrence and metastasis. In fact, the combined use of stromal Cav-1 and stromal MCT4 allowed us to more precisely identify high-risk triple-negative breast cancer patients, consistent with the goal of individualized risk-assessment and personalized cancer treatment. However, epithelial MCT4 staining had no prognostic value, indicating that the “conventional” Warburg effect does not predict clinical outcome. Thus, the “reverse Warburg effect” or “parasitic” energy-transfer is a key determinant of poor overall patient survival. As MCT4 is a druggable target, MCT4 inhibitors should be developed for the treatment of aggressive breast cancers, and possibly other types of human cancers. Similarly, we discuss how stromal MCT4 could be used as a biomarker for identifying high-risk cancer patients that could likely benefit from treatment with FDA-approved drugs or existing MCT-inhibitors (such as, AR-C155858, AR-C117977 and AZD-3965).Key words: caveolin-1, oxidative stress, pseudohypoxia, lactate shuttle, MCT4, metabolic coupling, tumor stroma, predictive biomarker, SLC16A3, monocarboxylic acid transporter, two-compartment tumor metabolism     

Huaier shows anti-cancer activities by inhibition of cell growth,migration and energy metabolism in lung cancer through PI3K/AKT/HIF-1α pathway

Huaier has been verified to have anti-cancer effects on many tumours. However, little information is available about the effects of Huaier on non-small cell lung cancer (NSCLC). We sought to probe the anti-cancer effects and related mechanisms of Huaier on lung cancer. A549 cells were pre-treated with 2, 4 and 8 mg/mL Huaier at different time points. Thereafter, cell viability was analysed by CCK-8 and the migration and invasion were detected by Scratch test and Transwell chamber migration assay. Moreover, ELISA, Western blot, shRNA transfection and RT-PCR were conducted to discover the related gene and protein expressions of energy metabolism and phosphatidylinositol 3-kinase (PI3K)/AKT/hypoxia-inducible factor 1α (HIF-1α) pathway. Furthermore, tumour xenografts were accomplished to inspect the anti-cancer effects of Huaier. Our consequences suggested that Huaier considerably repressed cell viability and migration in a dose-dependent way. In addition, Huaier statistically suppressed glycolysis, glucose transport and lactic acid (LA) accumulation. Besides, we detected that Huaier could inactivate the PI3K/AKT/HIF-1α pathway. The in vivo data confirmed that Huaier obviously decreased tumour volume and tumour growth, reduced the glycolysis, glucose transport and HIF-1α expression in the tumour-bearing tissues. Our results suggested Huaier revealed anti-tumour effects in both in vivo and in vitro possibly through PI3K/AKT/HIF-1α pathway.     

CTGF-mediated autophagy-senescence transition in tumor stroma promotes anabolic tumor growth and metastasis

Comment on: Capparelli C, et al. Cell Cycle 2012; 11:2272-84 and Capparelli C, et al. Cell Cycle 2012; 11:2285-302.Otto Warburg first observed that cancer cells preferentially undergo glycolysis instead of the mitochondrial TCA cycle even under oxygen-rich conditions. This form of energy metabolism in cancer cells is called “aerobic glycolysis” or the “Warburg effect.”¹ Lisanti and colleagues have previously proposed an alternative model called the “the reverse Warburg effect,” in which aerobic glycolysis predominantly occurs in stromal fibroblasts.² During this process, cancer cells secrete oxidative stress factors, such as hydrogen peroxide, into the tumor microenvironment, which induces autophagy. This leads to degradation of mitochondria (mitophagy) and elevated glycolysis in cancer-associated fibroblasts.³ Aerobic glycolysis results in the elevated production of pyruvate, ketone bodies and L-lactate, which can be utilized by cancer cells for anabolic growth and metastasis. At the molecular level, stromal fibroblasts lose expression of caveolin-1 and activate HIF-1a (Fig. 1), TGFβ and NFκB signaling.⁴ Stromal caveolin-1 expression predicts clinical outcome in breast cancer patients.⁵Open in a separate windowFigure 1. CTGF-mediated autophagy-senescence transition in tumor stroma promotes anabolic tumor growth and metastasis. Cancer cells secrete oxidative stress factors (H₂O₂) that induce autophagy in cancer-associated fibroblasts. Additionally, caveolin-1 (cav-1) loss leads to activation of connective tissue growth factor (CTGF) and HIF-1α that mediate autophagy and senescence in these stromal cells. This is called the autophagy-senescence transition (AST). AST leads to mitophagy and elevated glycolysis in cancer-associated fibroblasts. Aerobic glycolysis results in the elevated production of several nutrients (pyruvate, ketone bodies and L-lactate), which can be utilized by cancer cells for tumor growth and metastasis.In the June 15, 2012 issue of Cell Cycle, two studies by Capparelli et al. further validate the “autophagic tumor stroma model of cancer” described above, as well as identify novel mechanisms involved in this process.^6,7 Autophagy and senescence are induced by the same stimuli and are known to occur simultaneously in cells. In the first study, the authors hypothesize that the onset of senescence in the tumor stroma in response to autophagy/mitophagy contributes to mitochondrial dysfunction and aerobic glycolysis. In order to genetically validate this process of autophagy-senescence transition (AST) (Fig. 1), Capparelli et al. overexpressed several autophagy-promoting factors (BNIP3, cathepsin B, Beclin-1 and ATG16L1) in hTERT fibroblasts to constitutively induce autophagy. Autophagic fibroblasts lost caveolin-1 expression and displayed enhanced tumor growth and metastasis when co-injected with breast cancer cells in mice, without an increase in angiogenesis. In contrast, constitutive activation of autophagy in breast cancer cells inhibited in vivo tumor growth. Autophagic fibroblasts also showed mitochondrial dysfunction, increased production of nutrients (L-lactate and ketone bodies) and features of senescence (β-galactosidase activity and p21 activation). AST was also demonstrated in human breast cancer patient samples.⁷ In the second study, using a similar experimental approach, the authors evaluated the role of the TGFβ target gene, connective tissue growth factor (CTGF), in the induction of AST and aerobic glycolysis in cancer-associated fibroblasts. CTGF would be activated in the tumor stroma upon loss of caveolin-1. CTGF overexpression in fibroblasts induced autophagy/mitophagy, glycolysis and L-lactate production in a HIF-1α-dependent manner along with features of senescence and oxidative stress. CTGF overexpression in fibroblasts also promoted tumor growth when co-injected with breast cancer cells in mice (Fig. 1), independent of angiogenesis. As expected, CTGF overexpression in breast cancer cells inhibited tumor growth. CTGF is known to be involved in extracellular matrix synthesis; however, the effects of CTGF overexpression in fibroblasts and tumor cells were found to be independent of this function.⁶Overall, the authors have identified a novel mechanism by which CTGF promotes AST and aerobic glycolysis in cancer-associated fibroblasts. In turn, the stromal cells stimulate anabolic tumor growth and metastasis. The authors also genetically validate the two-compartment model of cancer metabolism, whereby autophagy genes and CTGF have differential effects in stromal cells and tumor cells. The current studies have several implications for cancer therapy. The finding that HIF-1 activation is necessary for the induction of autophagy and senescence downstream of caveolin-1 loss and CTGF activation in stromal fibroblasts is intriguing. Activation of HIF-1 in the hypoxic tumor microenvironment is known to promote tumor cell growth, survival and therapeutic resistance.⁸ Therefore, targeting HIF-1 has the potential to block tumor progression through dual inhibitory effects on hypoxic cancer cell growth and survival as well as the induction of autophagy in stromal fibroblasts. CTGF and AST in the tumor stroma could serve as biomarkers for predicting clinical outcome, therapy response and metastasis. The two-compartment model of tumor metabolism raises further questions regarding the use of antioxidants and autophagy inhibitors/inducers for cancer therapy. The use of these agents in the clinic should be carefully evaluated considering their differential effects on stromal cells and cancer cells.     

Benzene metabolism in human lung cell lines BEAS-2B and A549 and cells overexpressing CYP2F1

Benzene is an occupational and environmental toxicant. The main human health concern associated with benzene exposure is leukemia. The toxic effects of benzene are dependent on its metabolism by the cytochrome p450 enzyme system. The cytochrome p450 enzymes CYP2E1 and CYP2F2 are the major contributors to the bioactivation of benzene in rats and mice. Although benzene metabolism has been shown to occur with mouse and human lung microsomal preparations, little is known about the ability of human CYP2F to metabolize benzene or the lung cell types that might activate this toxicant. Our studies compared bronchiolar derived (BEAS-2B) and alveolar derived (A549) human cell lines for benzene metabolizing ability by evaluating the roles of CYP2E1 and CYP2F1. BEAS-2B cells that overexpressed CYP2F1 and recombinant CYP2F1 were also evaluated. BEAS-2B cells overexpressing the enzyme CYP2F1 produced 47.4 +/- 14.7 pmols hydroxylated metabolite/10(6) cells/45 min. The use of the CYP2E1-selective inhibitor diethyldithiocarbamate and the CYP2F2-selective inhibitor 5-phenyl-1-pentyne demonstrated that both CYP2E1 and CYP2F1 are important in benzene metabolism in the BEAS-2B and A549 human lung cell lines. The recombinant expressed human CYP2F1 enzyme had a K(m) value of 3.83 microM and a V(max) value of 0.01 pmol/pmol p450 enzyme/min demonstrating a reasonably efficient catalysis of benzene metabolism (V(max)/K(m) = 2.6). Thus, these studies have demonstrated in human lung cell lines that benzene is bioactivated by two lung-expressed p450 enzymes.     

Evidence for a stromal-epithelial “lactate shuttle” in human tumors: MCT4 is a marker of oxidative stress in cancer-associated fibroblasts

Recently, we proposed a new mechanism for understanding the Warburg effect in cancer metabolism. In this new paradigm, cancer-associated fibroblasts undergo aerobic glycolysis, and extrude lactate to “feed” adjacent cancer cells, which then drives mitochondrial biogenesis and oxidative mitochondrial metabolism in cancer cells. Thus, there is vectorial transport of energy-rich substrates from the fibroblastic tumor stroma to anabolic cancer cells. A prediction of this hypothesis is that cancer-associated fibroblasts should express MCT4, a mono-carboxylate transporter that has been implicated in lactate efflux from glycolytic muscle fibers and astrocytes in the brain. To address this issue, we co-cultured MCF7 breast cancer cells with normal fibroblasts. Interestingly, our results directly show that breast cancer cells specifically induce the expression of MCT4 in cancer-associated fibroblasts; MCF7 cells alone and fibroblasts alone, both failed to express MCT4. We also show that the expression of MCT4 in cancer-associated fibroblasts is due to oxidative stress, and can be prevented by pre-treatment with the anti-oxidant N-acetyl-cysteine. In contrast to our results with MCT4, we see that MCT1, a transporter involved in lactate uptake, is specifically upregulated in MCF7 breast cancer cells when co-cultured with fibroblasts. Virtually identical results were also obtained with primary human breast cancer samples. In human breast cancers, MCT4 selectively labels the tumor stroma, e.g., the cancer-associated fibroblast compartment. Conversely, MCT1 was selectively expressed in the epithelial cancer cells within the same tumors. Functionally, we show that overexpression of MCT4 in fibroblasts protects both MCF7 cancer cells and fibroblasts against cell death, under co-culture conditions. Thus, we provide the first evidence for the existence of a stromal-epithelial lactate shuttle in human tumors, analogous to the lactate shuttles that are essential for the normal physiological function of muscle tissue and brain. These data are consistent with the “reverse Warburg effect,” which states that cancer-associated fibroblasts undergo aerobic glycolysis, thereby producing lactate, which is utilized as a metabolic substrate by adjacent cancer cells. In this model, “energy transfer” or “metabolic-coupling” between the tumor stroma and epithelial cancer cells “fuels” tumor growth and metastasis, via oxidative mitochondrial metabolism in anabolic cancer cells. Most importantly, our current findings provide a new rationale and novel strategy for anti-cancer therapies, by employing MCT inhibitors.Key words: caveolin-1, oxidative stress, pseudohypoxia, lactate shuttle, MCT1, MCT4, metabolic coupling, tumor stroma, predictive biomarker, SLC16A1, SLC16A3, monocarboxylic acid transporter     

Comparison of gene expression profiles in chromate transformed BEAS-2B cells

Background

Hexavalent chromium [Cr(VI)] is a potent human carcinogen. Occupational exposure has been associated with increased risk of respiratory cancer. Multiple mechanisms have been shown to contribute to Cr(VI) induced carcinogenesis, including DNA damage, genomic instability, and epigenetic modulation, however, the molecular mechanism and downstream genes mediating chromium''s carcinogenicity remain to be elucidated.

Methods/Results

We established chromate transformed cell lines by chronic exposure of normal human bronchial epithelial BEAS-2B cells to low doses of Cr(VI) followed by anchorage-independent growth. These transformed cell lines not only exhibited consistent morphological changes but also acquired altered and distinct gene expression patterns compared with normal BEAS-2B cells and control cell lines (untreated) that arose spontaneously in soft agar. Interestingly, the gene expression profiles of six Cr(VI) transformed cell lines were remarkably similar to each other yet differed significantly from that of either control cell lines or normal BEAS-2B cells. A total of 409 differentially expressed genes were identified in Cr(VI) transformed cells compared to control cells. Genes related to cell-to-cell junction were upregulated in all Cr(VI) transformed cells, while genes associated with the interaction between cells and their extracellular matrices were down-regulated. Additionally, expression of genes involved in cell proliferation and apoptosis were also changed.

Conclusion

This study is the first to report gene expression profiling of Cr(VI) transformed cells. The gene expression changes across individual chromate exposed clones were remarkably similar to each other but differed significantly from the gene expression found in anchorage-independent clones that arose spontaneously. Our analysis identified many novel gene expression changes that may contribute to chromate induced cell transformation, and collectively this type of information will provide a better understanding of the mechanism underlying chromate carcinogenicity.     

Oxidative stress-mediated epidermal growth factor receptor activation regulates PM2.5-induced over-secretion of pro-inflammatory mediators from human bronchial epithelial cells

BackgroundExposure to PM_2.5 has been associated with increased morbidity and mortality of lung diseases although the underlying mechanisms have not been fully uncovered. Airway inflammation is a critical event in the pathogenesis of lung diseases. This study aimed to examine the role of oxidative stress and epidermal growth factor receptor (EGFR) in PM_2.5-induced pro-inflammatory response in a human bronchial epithelial cell line, BEAS-2B.MethodsBEAS-2B cells were exposed to 0, 20, 50, 100 and 150 μg/ml of PM_2.5. Secretion of pro-inflammatory mediators including interleukin-6 (IL-6), IL-8 and IL-1β was determined using enzyme linked immunosorbent assay. Levels of intracellular reactive oxygen species (ROS) were determined using flow cytometry. Phosphorylation of the EGFR was examined with immunoblotting.ResultsPM_2.5 exposure increased the secretion of IL-6, IL-8, and IL-1β in a concentration-dependent fashion. Moreover, exposure to PM_2.5 elevated intracellular levels of ROS, and phosphorylation of the EGFR (Y1068). Pretreatment of BEAS-2B cells with either an antioxidant or a specific EGFR inhibitor significantly reduced PM_2.5-induced IL-6, IL-8 and IL-1β secretion, implying that both oxidative stress and EGFR activation were involved in PM_2.5-induced pro-inflammatory response. Furthermore, pre-treatment of BEAS-2B cells with an antioxidant significantly blunted PM_2.5-induced EGFR activation, suggesting that oxidative stress was required for PM_2.5-induced EGFR activation.ConclusionPM_2.5 exposure induces pro-inflammatory response in human bronchial epithelial cells through oxidative stress-mediated EGFR activation.