Carcinoid tumors are rare neuroendocrine tumors (NETs) that are increasing in incidence. Mutation and altered expression of Wnt/β-catenin signaling components have been described in many tumors but have not been well-studied in NETs. Here, we observed accumulation of β-catenin in the cytoplasm and/or nucleus in 25% of clinical NET tissues. By mutational analysis, the mutations of β-catenin (I35S) and APC (E1317Q, T1493T) were identified in NET cells and the tissues. Expression of representative Wnt inhibitors was absent or markedly decreased in BON, a human pancreatic carcinoid cell line; treatment with 5-aza-2'-deoxycytidine (5-aza-CdR) increased expression levels of the Wnt inhibitors. Methylation analyses demonstrated that CpG islands of SFRP-1 and Axin-2 were methylated, whereas the promoters of DKK-1, DKK-3 and WIF-1 were unmethylated in four NET cells. Aberrant methylation of SFRP-1 was particularly observed in most of clinical NET tissues. In addition, the repression of these unmethylated genes was associated with histone H3 lysine 9 dimethylation (H3K9me2) in BON cells. Together, 5-aza-CdR treatment inhibited cell proliferation and decreased the protein levels of H3K9me2 and G9a. Moreover, a novel G9a inhibitor, UNC0638, suppressed BON cell proliferation through inhibition of Wnt/β-catenin pathway. Overexpression of the inhibitory genes, particularly SFRP-1 and WIF-1 in BON cells, resulted in suppression of anchorage-independent growth and inhibition of tumor growth in mice. Our findings suggest that aberrant Wnt/β-catenin signaling, through either mutations or epigenetic silencing of Wnt antagonists, contributes to the pathogenesis and growth of NETs and have important clinical implications for the prognosis and treatment of NETs.
Epithelial–mesenchymal transition (EMT) is a critical process for tumor invasion and metastasis. Hypoxia may induce EMT, and upregulated β-catenin expression has been found in various tumors. In this study, we investigate the role of β-catenin in hypoxia-induced EMT in hepatocellular carcinoma (HCC). Induction of EMT in HCC cell lines by hypoxia was confirmed by altered morphology, expression change of EMT-associated markers and enhanced invasion capacity. We showed that hypoxia-induced EMT could be enhanced by addition of recombinant Wnt3a while it was repressed by β-catenin small interfering RNA. An interaction between β-catenin and hypoxia-induced factor-1α (hif-1α) was found, and an underlying competition for β-catenin between hif-1α and T-cell factor-4 was implied. Notably, increased hif-1α activity was accompanied with more significant EMT features. We also showed that the pro-EMT effect of β-catenin in hypoxia was deprived in the absence of hif-1α. Moreover, β-catenin was found to be responsible for the maintenance of viability and proliferation for tumor cells undergoing hypoxia. We further showed a correlation between hif-1α and β-catenin expression, and corresponding expression of EMT-associated markers in human HCC tissues. Our results suggest that Wnt/β-catenin signaling enhances hypoxia-induced EMT in HCC by increasing the EMT-associated activity of hif-1α and preventing tumor cell death.
Leptin overexpression is closely correlated with gastric cancer (GC) invasion, but its exact effect and the underlying mechanism in tumorigenesis remain poorly understood. Membrane type 1-matrix metalloproteinase (MT1-MMP), a surface-anchored ‘master switch’ proteinase, is overexpressed and plays crucial roles in tumor invasion. Here, we characterized the influence of leptin on the generation and surface localization of MT1-MMP in GC and elucidated its molecular mechanisms. Our results revealed that leptin promoted GC cell invasion in vitro by upregulating MT1-MMP expression. Furthermore, cell surface biotinylation assay and flow cytometry demonstrated that the surface expression of MT1-MMP was also enhanced by leptin, and knockdown of kinesin family member 1B (KIF1B, a microtubule plus end-directed monomeric motor protein) by small interference RNA inhibited this process. Notably, coimmunoprecipitation analysis indicated that leptin enhanced the interaction of MT1-MMP with KIF1B in a time-dependent manner, which consequently contributed to GC cell invasion. Moreover, leptin increased MT1-MMP or KIF1B expression by the protein kinase B (AKT) pathway and extracellular signal-regulated kinase 1/2 partially participated in this process. However, only AKT was implicated in the leptin-mediated membrane localization of MT1-MMP. Immunohistochemistry analysis revealed that leptin, MT1-MMP and KIF1B are overexpressed in GC tissues, and they positively correlated with clinical stage and lymph node metastasis. These observations indicate that this regulatory network exists in vivo. Taken together, our findings suggest that leptin is an effective intracellular stimulator of MT1-MMP and that leptin-enhanced cell surface localization of MT1-MMP is dependent on KIF1B, which consequently plays a critical role in GC invasion.
In human hepatocellular carcinoma (HCC) and many other cancers, somatic point mutations are highly prevalent, yet the mechanisms critical in their generation remain poorly understood. S-nitrosoglutathione reductase (GSNOR), a key regulator of protein S-nitrosylation, is frequently deficient in human HCC. Targeted deletion of the GSNOR gene in mice can reduce the activity of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) and promote both carcinogen-induced and spontaneous HCC. In this study, we report that following exposure to the environmental carcinogen diethylnitrosamine, the mutation frequency of a transgenic reporter in the liver of GSNOR-deficient mice (GSNOR–/–) is significantly higher than that in wild-type control. In wild-type mice, diethylnitrosamine treatment does not significantly increase the frequency of the transition from G:C to A:T, a mutation deriving from diethylnitrosamine-induced O6-ethylguanines that are normally repaired by AGT. In contrast, the frequency of this transition from diethylnitrosamine is increased ~20 times in GSNOR–/– mice. GSNOR deficiency also significantly increases the frequency of the transversion from A:T to T:A, a mutation not affected by AGT. GSNOR deficiency in our experiments does not significantly affect either the frequencies of the other diethylnitrosamine-induced point mutations or hepatocyte proliferation. Thus, GSNOR deficiency, through both AGT-dependent and AGT-independent pathways, significantly raises the rates of specific types of DNA mutations. Our results demonstrate a critical role for GSNOR in maintaining genomic integrity in mice and support the hypothesis that GSNOR deficiency is an important cause of the widespread mutations in human HCC.
The ionized cysteines present on the surfaces of many redox-sensitive proteins play functionally essential roles and are readily targeted by the reactive oxygen and reactive nitrogen species. Using disulfiram (DSF) and nitroaspirin (NCX4016) as the model compounds that mediate thiol-conjugating and nitrosylating reactions, respectively, we investigated the fate of p53, nuclear factor-kappaB (NF-B) and other redox-responsive proteins following the exposure of human cancer cell lines to the drugs. Both drugs induced glutathionylation of bulk proteins in tumor cells and cell-free extracts. A prominent finding of this study was a time- and dose-dependent degradation of the redox-regulated proteins after brief treatments of tumor cells with DSF or NCX4016. DSF and copper-chelated DSF at concentrations of 50–200 µM induced the disappearance of wild-type p53, mutant p53, NF-B subunit p50 and the ubiquitin-activating enzyme E1 (UBE1) in tumor cell lines. DSF also induced the glutathionylation of p53. The recombinant p53 protein modified by DSF was preferentially degraded by rabbit reticulocyte lysates. The proteasome inhibitor PS341 curtailed the DSF-induced degradation of p53 in HCT116 cells. Further, the NCX4016 induced a dose-dependent disappearance of the UBE1 and NF-B p50 proteins in cell lines, besides a time-dependent degradation of aldehyde dehydrogenase in mouse liver after a single injection of 150mg/kg. The loss of p53 and NF-kB proteins correlated with decreases in their specific binding to DNA. Our results demonstrate the hitherto unrecognized ability of the non-toxic thiolating and nitrosylating agents to degrade regulatory proteins and highlight the exploitable therapeutic benefits.
Germline mutations in genes that cause hereditary syndromes are highly predisposed to familial pancreatic cancer. However, genetic susceptibility to sporadic pancreatic cancer is largely uncovered. We conducted a two-stage association study on pancreatic cancer that included 981 cases and 1991 controls in the first stage followed by a second stage (2603 cases and 2877 controls). Using an approach based on candidate genes whose roles in pancreatic cancer have been well known, we identified two new susceptibility loci. rs11571836 located in the BRCA2 3'-untranslated region was significantly associated with lower expression of BRCA2 transcript and increased pancreatic cancer risk [odds ratio = 1.30, 95% confidence interval = 1.14–1.47, P = 7.64 x 10–5] in a recessive manner. rs12939944 located in the MAP2K4 intron was associated with decreased risk (odds ratio = 0.82, 95% confidence interval = 0.74–0.91, P = 0.0001) in a dominant manner. Our results demonstrate for the first time that common variants in BRCA2 and MAP2K4 are susceptibility to sporadic pancreatic cancer.
Micro-RNAs (miRNAs) are small non-coding RNA molecules, which can act as either oncogenes or tumor suppressors. Dysregulated expression of miRNA genes have been implicated in the development of many different cancers. We hypothesize that genetic variations in miRNA biogenesis genes may be associated with the prognosis of bladder cancer. We genotyped 76 single nucleotide polymorphisms (SNPs) in eight miRNA biogenesis genes in 421 patients with non-muscle-invasive bladder cancer (NMIBC). We analyzed the associations of SNPs with recurrence and progression in all patients as well as stratified by treatment: transurethral resection (TUR) alone or TUR plus intravesical bacillus Calmette–Guérin (BCG) instillation. Two SNPs were significantly associated with tumor recurrence in TUR only subgroup after adjustment for multiple comparisons (Q < 0.1). The most significant SNP was rs197412 in DDX20: the variant allele conferred a decreased risk of recurrence [hazard ratio (HR) = 0.58, 95% confidence interval (95% CI) = 0.40–0.82]. This SNP was validated in a separate group of 586 NMIBC patients and the pooled HR was 0.62 (95% CI = 0.48–0.81, P < 0.001). Two linked SNPs (rs2073778 and rs720012) in DGCR8 showed significant association with tumor progression (HR = 4.00, 95% CI = 1.53–10.46, P = 0.005). A strong gene-dosage effect was observed with higher risk for tumor recurrence and progression with increasing number of unfavorable genotypes. Haplotype and survival tree analyses further characterized the association of miRNA-related SNPs with tumor recurrence and progression. Taken together, our results indicate that genetic variants in miRNA biogenesis pathway may influence bladder cancer clinical outcome in NMIBC patients.
The SWI/SNF chromatin remodeling complex is an important regulator of gene expression that has been linked to cancer development. Expression of Brahma (BRM), a critical catalytic subunit of SWI/SNF, is lost in a variety of solid tumors. Two novel BRM promoter polymorphisms (BRM-741 and BRM-1321) have been correlated with BRM loss and elevated cancer risk. The aim(s) of this study were to examine BRM expression in head and neck squamous cell carcinoma (HNSCC) and to correlate BRM polymorphisms with HNSCC risk. BRM expression studies were performed on eight HNSCC cell lines and 76 surgically resected tumor samples. A case–control study was conducted on 668 HNSCC patients (oral cavity, oropharynx, larynx and hypopharynx) and 700 healthy matched controls. BRM expression was lost in 25% of cell lines and 16% of tumors. The homozygous genotype of each polymorphism was significantly associated with increased HNSCC risk [BRM-741: adjusted odds ratio (aOR) 1.75, 95% CI 1.2–2.3, P < 0.001; BRM-1321: aOR 1.65, 95% CI 1.2–2.2, P < 0.001]. Individuals that were homozygous for both BRM polymorphisms had a more than 2-fold increase in the risk of HNSCC (aOR 2.23, 95% CI 1.5–3.4, P < 0.001). A particularly elevated risk was seen within the oropharynx, human papillomavirus-positive subgroup for carriers of both homozygous variants (aOR 3.09, 95% CI 1.5–6.8, P = 0.004). BRM promoter polymorphisms appear to act as susceptibility markers of HNSCC with potential utility in screening, prevention and treatment.
To explore the association of polymorphisms in the region of three neighboring genes TRIT1, MYCL1 and MFSD2A with risk and clinicopathological features of gastric cancer, 19 tagging SNPs in this region were genotyped using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in a case–control study of 610 Chinese gastric cancer patients and 608 cancer-free controls. MFSD2A rs4233508 T>C CC genotype was associated with an increased risk of gastric cancer in younger patients and an increased risk of moderately/well-differentiated intestinal-type gastric cancer (adjusted odds ratio [OR], 1.74 and 1.50, respectively). TRIT1 rs11581557 T>G TG was associated with lymph node metastasis (TG versus TT/GG, adjusted OR, 1.64). MFSD2A rs12083239 GC genotype and TRIT1 rs2172362 or rs230310 homozygous genotype were associated with Lauren’s classification (GC versus GG, adjusted OR, 1.69; GC versus GG/CC, adjusted OR, 1.74) and tumor site (rs2172362: CC versus CT, adjusted OR, 1.71; CC/TT versus CT, adjusted OR, 1.62; rs230310: CC versus CT, adjusted OR, 1.75; CC/TT versus CT, adjusted OR, 1.67) of gastric cancer, respectively. One TRIT1 haplotype, CCGT, was associated with lymph node metastasis and tumor site of gastric cancer (CCGT versus TTTT, adjusted OR, 1.91 and 1.55). This is believed to be the first report that several tagging SNPs and haplotypes in TRIT1, MYCL1 and MFSD2A region are significantly associated with risk and clinicopathological features of gastric cancer in a Chinese population. The findings might be useful for risk assessment and prognosis prediction of gastric cancer.
Methyleugenol is a genotoxic carcinogen in mice and rats, the liver being the primary target tissue. Methyleugenol occurs in fennel and many herbs and spices. Furthermore, methyleugenol-containing plant extracts and chemically prepared methyleugenol are used as flavoring agents. We analyzed surgical human liver samples from 30 subjects for the presence of DNA adducts originating from methyleugenol using isotope-dilution ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). Twenty-nine samples unambiguously contained the N2-(trans-methylisoeugenol-3'-yl)-2'-deoxyguanosine adduct. A second adduct, N6-(trans-methylisoeugenol-3'-yl)-2'-deoxyadenosine, was also found in most samples, but at much lower levels, in agreement with the results from experimental models. The maximal and median levels of both adducts combined were 37 and 13 per 108 nucleosides (corresponding to 4700 and 1700, respectively, adducts per diploid genome). This is the first demonstration of DNA adducts formed by a xenobiotic in human liver using UPLC-MS/MS, the most reliable method available. It has been estimated for diverse rat and mouse hepatocarcinogens that 50–5500 adducts per 108 nucleosides are present after repeated treatment at the TD50 (daily dose that halves the probability to stay tumor-free in long-term studies). We conclude that the exposure to methyleugenol leads to substantial levels of hepatic DNA adducts and, therefore, may pose a significant carcinogenic risk.
The clinical and functional significance of RNA-interference machinery in lung cancer is poorly understood. Besides, microRNAs (miRNA) have the potential to serve both as biomarkers and therapeutic agents, by personalizing diagnosis and therapy. In this study, we investigated whether the expression levels of DICER1 and DROSHA, components of the RNA-interference machinery, can predict survival, and whether the miRNA expression profiles can differentiate histologic subtypes in non-small cell lung cancer (NSCLC). Levels of DICER1, DROSHA and five different miRNAs were measured in NSCLC specimens (N = 115) by qRT–PCR assay and correlated with clinical outcomes. Low expression of DROSHA was associated with an increased median survival (154.2 versus 39.8 months, P = 0.016). Also, high DROSHA expression was associated with decreased median survival in the following subgroups: adenocarcinoma (P = 0.011), grade III tumors (P = 0.038) and low-stage patients (P = 0.014). In multivariate analyses, we found two independent predictors of reduced disease-specific survival: high DROSHA expression [hazards ratio = 2.24; P = 0.04] and advanced tumor stage (hazards ratio = 1.29, P = 0.02). In general, the overall tumor miRNA expression was downregulated in our cohort compared with normal tissues. Expression levels of hsa-let-7a (P = 0.005) and miR-16 (P = 0.003) miRNA were significantly higher in squamous cell carcinoma than in adenocarcinoma samples. This study supports the value of the expression profiling of the components of the miRNA-processing machinery in the prognosis of NSCLC patients, especially DROSHA expression levels. In addition, differential expression of miRNAs, such as hsa-let-7a and miR-16 may be helpful tools in the histologic subclassification of NSCLC.
Mitochondrial DNA (mtDNA) has increased susceptibility to damage due to its close proximity to the site of reactive oxygen species production, lack of introns and protective histones, and less efficient DNA repair mechanisms than nuclear DNA. The relationship between mtDNA copy number in peripheral blood leukocytes (PBLs) and the risk of soft tissue sarcoma (STS) has not been investigated. In this study, we determined the relative mtDNA copy number in PBLs of 325 patients (cases) with histologically confirmed STS and 330 healthy controls that were frequency matched to cases according to age, sex and ethnicity. Cases had a significantly lower mtDNA copy number than controls (0.93±0.49 for cases versus 1.23±0.59 for controls; P < 0.001). In analyses stratified by sex, ethnicity and smoking status, mtDNA copy number was lower in the cases than in controls in any stratum. Using the median mtDNA copy number in controls as a cutoff, individuals with lower mtDNA copy number were associated with a significantly increased risk of STS compared with those with higher mtDNA copy number (adjusted odds ratio, 2.71; 95% confidence interval, 1.94–3.82). There was a significant dose–response relationship between reduced mtDNA copy number and increased risk of STS in tertile and quartile analyses. The present study provides the first epidemiologic evidence that reduced mtDNA copy number in PBLs is significantly associated with an increased risk of STS, thereby suggesting an important role of mtDNA in STS development.
Epidemiological studies of underground miners suggested that occupational exposure to radon causes lung cancer with squamous cell carcinoma (SCC) as the predominant histological type. However, the genetic determinants for susceptibility of radon-induced SCC in miners are unclear. Double-strand breaks induced by radioactive radon daughters are repaired primarily by non-homologous end joining (NHEJ) that is accompanied by the dynamic changes in surrounding chromatin, including nucleosome repositioning and histone modifications. Thus, a molecular epidemiological study was conducted to assess whether genetic variation in 16 genes involved in NHEJ and related histone modification affected susceptibility for SCC in radon-exposed former miners (267 SCC cases and 383 controls) from the Colorado plateau. A global association between genetic variation in the haplotype block where SIRT1 resides and the risk for SCC in miners (P = 0.003) was identified. Haplotype alleles tagged by the A allele of SIRT1 rs7097008 were associated with increased risk for SCC (odds ratio = 1.69, P = 8.2x10–5) and greater survival in SCC cases (hazard ratio = 0.79, P = 0.03) in miners. Functional validation of rs7097008 demonstrated that the A allele was associated with reduced gene expression in bronchial epithelial cells and compromised DNA repair capacity in peripheral lymphocytes. Together, these findings substantiate genetic variation in SIRT1 as a risk modifier for developing SCC in miners and suggest that SIRT1 may also play a tumor suppressor role in radon-induced cancer in miners.
Melatonin has been known to be a chemopreventive agent since its levels inversely correlate with the risk of developing cancer. We have recently shown that melatonin induces p38-dependent phosphorylation of both p53 and histone H2AX. This is associated with a p53-mediated increase in repair of both endogenous and chemotherapy-induced DNA damage. In addition, the inhibition of p38 activities impairs melatonin’s capability to induce a p53-dependent DNA damage response and thus its ability to maintain genome integrity. Since melatonin-induced p53 phosphorylation requires an intact p38 phosphorylation cascade and p38 can be activated by G proteins, we supposed that melatonin’s activities could be mediated by its G-protein-coupled membrane receptors, MT1 and MT2. Here, we show that the activation of the p53-dependent DNA damage response by melatonin is indeed mediated by MT1 and MT2. As a result, the absence of either receptor impairs melatonin’s ability to reduce both cell proliferation and clonogenic potential of cancer cells. In addition, this causes an impairment of the p53-dependent DNA damage response. By providing molecular insight, our findings might have translational impact, suggesting the involvement of melatonin receptors in tumorigenesis.
In China, esophageal cancer is the fourth leading cause of cancer death where essentially all cases are histologically esophageal squamous cell carcinoma (ESCC), in contrast to esophageal adenocarcinoma in the West. Globally, ESCC is 2.4 times more common among men than women and recently it has been suggested that sex hormones may be associated with the risk of ESCC. We examined the association between genetic variants in sex hormone metabolic genes and ESCC risk in a population from north central China with high-incidence rates. A total of 1026 ESCC cases and 1452 controls were genotyped for 797 unique tag single-nucleotide polymorphisms (SNPs) in 51 sex hormone metabolic genes. SNP-, gene- and pathway-based associations with ESCC risk were evaluated using unconditional logistic regression adjusted for age, sex and geographical location and the adaptive rank truncated product (ARTP) method. Statistical significance was determined through use of permutation for pathway- and gene-based associations. No associations were observed for the overall sex hormone metabolic pathway (P = 0.14) or subpathways (androgen synthesis: P = 0.30, estrogen synthesis: P = 0.15 and estrogen removal: P = 0.19) with risk of ESCC. However, six individual genes (including SULT2B1, CYP1B1, CYP3A7, CYP3A5, SHBG and CYP11A1) were significantly associated with ESCC risk (P < 0.05). Our examination of genetic variation in the sex hormone metabolic pathway is consistent with a potential association with risk of ESCC. These positive findings warrant further evaluation in relation to ESCC risk and replication in other populations.
HLJ1 is a novel tumour suppressor and is a potential druggable target for non-small-cell lung cancer (NSCLC). In this report, using a promoter-containing enhancer region as the HLJ1-targeting drug-screening platform, we identified several herbal compounds from a Chinese herbal bank with the capacity to enhance HLJ1 promoter activity and suppress tumour growth and invasion of NSCLC. Among the herbal drugs identified, the andrographolide (from Andrographis paniculata [Burm. f.] Nees.) most significantly induced HLJ1 expression and suppressed tumorigenesis both in vitro and in vivo. The andrographolide upregulates HLJ1 via JunB activation, which modulates AP-2α binding at the MMP-2 promoter and represses the expression of MMP-2. In addition, silencing of HLJ1 partially reverses the inhibition of cancer-cell invasion by andrographolide. Microarray transcriptomic analysis was performed to comprehensively depict the andrographolide-regulated signalling pathways. We showed that andrographolide can affect 939 genes (analysis of variance, false discovery rate < 0.05) that are dominantly involved in the cell cycle, apoptosis and adhesion-related biological signalling, including mitogen-activated protein kinase, focal adhesion and tight junction pathways, indicating the diverse effects of andrographolide on anticancer invasion and proliferation. In conclusion, the HLJ1-targeting drug-screening platform is useful for screening of novel anticancer compounds. Using this platform, we identified andrographolide is a promising new anticancer agent that could suppress tumour growth and invasion in NSCLC.
Tumor-associated inflammation is a driving force in several adult cancers and intake of low-dose aspirin has proven to reduce cancer incidence. Little is known about tumor-associated inflammation in pediatric neoplasms and no in vivo data exists on the effectiveness of low-dose aspirin on established tumors. The present study employs the transgenic TH-MYCN mouse model for neuroblastoma (NB) to evaluate inflammatory patterns paralleling tumor growth in vivo and low-dose aspirin as a therapeutic option for high-risk NB. Spontaneously arising abdominal tumors were monitored for tumor-associated inflammation ex vivo at various stages of disease and homozygous mice received daily low-dose aspirin (10mg/kg) using oral gavage or no treatment, from 4.5 to 6 weeks of age. Using flow cytometry, a transition from an adaptive immune response predominated by CD8+ T cell in early neoplastic lesions, towards enrichment in immature cells of the innate immune system, including myeloid-derived suppressor cells, dendritic cells and tumor-associated macrophages, was detected during tumor progression. An M1 to M2 transition of tumor-associated macrophages was demonstrated, paralleled by a deterioration of dendritic cell status. Treatment with low-dose aspirin to mice homozygous for the TH-MYCN transgene significantly reduced the tumor burden (P < 0.01), the presence of tumor-associated cells of the innate immune system (P < 0.01), as well as the intratumoral expression of transforming growth factor-β, thromboxane A2 (P < 0.05) and prostaglandin D2 (P < 0.01). In conclusion, tumor-associated inflammation appears as a potential therapeutic target in NB and low-dose aspirin reduces tumor burden in the TH-MYCN transgenic mouse model of NB, hence warranting further studies on aspirin in high-risk NB.
Selenium (Se) has long been known for its cancer prevention properties, but the molecular basis remains unclear. The principal questions in assessing the effect of dietary Se in cancer are whether selenoproteins, small molecule selenocompounds, or both, are involved, and under which conditions and genotypes Se may be protective. In this study, we examined diethylnitrosamine-induced hepatocarcinogenesis in mice lacking a subset of selenoproteins due to expression of a mutant selenocysteine tRNA gene (TrspA37G mice). To uncouple the effects of selenocompounds and selenoproteins, these animals were examined at several levels of dietary Se. Our analysis revealed that tumorigenesis in TrspA37G mice maintained on the adequate Se diet was increased. However, in the control, wild-type mice, both Se deficiency and high Se levels protected against tumorigenesis. We further found that the Se-deficient diet induced severe neurological phenotypes in TrspA37G mice. Surprisingly, a similar phenotype could be induced in these mice at high dietary Se intake. Overall, our results show a complex role of Se in chemically induced hepatocarcinogenesis, which involves interaction among selenoproteins, selenocompounds and toxins, and depends on genotype and background of the animals.
Betel quid (BQ) chewing is an oral habit that increases the risk of oral cancer and oral submucous fibrosis (OSF), a precancerous condition showing epithelial atrophy and tissue fibrosis. Persistent fibroblast contraction may induce the fibrotic contracture of tissue. In this study, we found that areca nut extract (ANE) (200–1200 µg/ml) stimulated buccal mucosa fibroblast (OMF)-populated collagen gel contraction. Arecoline but not arecaidine—two areca alkaloids, slightly induced the OMF contraction. Exogenous addition of carboxylesterase (2U/ml) prevented the arecoline- but not ANE-induced OMF contraction. OMF expressed inositol triphosphate (IP3) receptors. ANE-induced OMF (800 µg/ml) contraction was inhibited by U73122 [phospholipase C (PLC) inhibitor] and 2-aminoethoxydiphenyl borate (IP3 receptor antagonist), respectively. Ethylene glycol tetraacetic acid and verapamil, two calcium mobilization modulators, also suppressed the ANE-induced OMF contraction. ANE induced calcium/calmodulin kinase II and myosin light chain (MLC) phosphorylation in OMF. Moreover, W7 (a Ca2+/calmodulin inhibitor), HA1077 (Rho kinase inhibitor), ML-7 (MLC kinase inhibitor) and cytochalasin B (actin filament polymerization inhibitor) inhibited the ANE-induced OMF contraction. Although ANE elevated reactive oxygen species (ROS) level in OMF, catalase, superoxide dismutase and N-acetyl-l-cysteine showed no obvious effect on ANE-elicited OMF contraction. These results indicate that BQ chewing may affect the wound healing and fibrotic processes in OSF via inducing OMF contraction by ANE and areca alkaloids. AN components-induced OMF contraction was related to PLC/IP3/Ca2+/calmodulin and Rho signaling pathway as well as actin filament polymerization, but not solely due to ROS production.
In this study, primary murine prostate cancer (PCa) cells were derived using the well-established TRAMP model. These PCa cells were treated with the histone deacetylase inhibitor, valproic acid (VPA), and we demonstrated that VPA treatment has an antimigrative, antiinvasive and antiproliferative effect on PCa cells. Using microarray analyses, we discovered several candidate genes that could contribute to the cellular effects we observed. In this study, we could demonstrate that VPA treatment of PCa cells causes the re-expression of cyclin D2, a known regulator that is frequently lost in PCa as we could show using immunohistochemical analyses on PCa specimens. We demonstrate that VPA specifically induces the re-expression of cyclin D2, one of the highly conserved D-type cyclin family members, in several cancer cell lines with weak or no cyclin D2 expression. Interestingly, VPA treatment had no effect in fibroblasts, which typically have high basal levels of cyclin D2 expression. The re-expression of cyclin D2 observed in PCa cells is activated by increased histone acetylation in the promoter region of the Ccnd2 gene and represents one underlying molecular mechanism of VPA treatment that inhibits the proliferation of cancer cells. Altogether, our results confirm that VPA is an anticancer therapeutic drug for the treatment of tumors with epigenetically repressed cyclin D2 expression.
Ovarian cancer is the deadliest gynecological malignancy due to detection of cancer at a late stage when the disease has metastasized. One likely progenitor cell type of ovarian cancer is the ovarian surface epithelium (OSE), which proliferates rapidly in the presence of inflammatory cytokines and oxidative stress following ovulation. To determine whether oxidative stress induces DNA damage leading to spontaneous transformative changes in normal OSE, an immortalized mouse OSE cell line (MOSE cells) or normal mouse ovarian organoids were treated with hydrogen peroxide (H2O2) and loss of contact inhibition was assessed by soft agar assay. In response to H2O2, OSE cells grown in 3D exhibited growth in soft agar but MOSE cells grown on 2D plastic did not, indicating a critical role for epithelial–stromal interactions in neoplastic initiation. Loss of contact inhibition in response to H2O2 correlated with an increase in proliferation, DNA damage and upregulation of the oncogene Akt1. Use of a reactive oxygen species scavenger or Akt inhibitor blocked H2O2-induced proliferation and growth in soft agar. Although parental MOSE cells did not undergo transformation by H2O2, MOSE cells stably overexpressing constitutively active myristoylated Akt or knockdown of phosphatase and tensin homolog (PTEN) exhibited loss of contact inhibition and increased proliferation. This study indicates that normal OSE undergo transformative changes induced by oxidative stress and that this process requires Akt upregulation and activation. A 3D model that retains tissue architecture is critical for studying this process and may lead to development of new intervention strategies directed at early stages of ovarian cancer.
The mitogen-activated protein kinase kinase 1 and 2 signaling pathway is a major component of the RAS (Rat sarcoma)/RAF (Radpidly accelerated fibrosarcoma)/MEK (mitogen-activated protein kinase kinase)/ERKs (Extracellular signal-regulated kinases) signaling axis that regulates tumorigenesis and cancer cell growth. MEK is frequently activated in various cancers that have mutations in the KRAS and BRAF oncogenes. Therefore, MEK has been suggested as a therapeutic target for inhibitor development against tumors that are dependent on the activating mutations in mitogen-activated protein kinase signaling. Herein, we report the discovery of three novel MEK inhibitors, herein referred to as CInQ-01, CInQ-03 and CInQ-06. All three inhibitors were highly effective in suppressing MEK1 and MEK2 in vitro kinase activity as well as anchorage-dependent and anchorage-independent cell growth. The inhibitory activity was associated with markedly reduced phosphorylation of ERKs and ribosomal S6 kinases. Furthermore, administration of CInQ-03 inhibited colon cancer cell growth in an in vivo xenograft mouse model and showed no skin toxicity. Overall, these results suggest that these novel MEK inhibitors might be used for chemotherapy or prevention.
Nicotinamide (vitamin B3) protects from ultraviolet (UV) radiation-induced carcinogenesis in mice and from UV-induced immunosuppression in mice and humans. Recent double-blinded randomized controlled Phase 2 studies in heavily sun-damaged individuals have shown that oral nicotinamide significantly reduces premalignant actinic keratoses, and may reduce new non-melanoma skin cancers. Nicotinamide is a precursor of nicotinamide adenine dinucleotide (NAD+), an essential coenzyme in adenosine triphosphate (ATP) production. Previously, we showed that nicotinamide prevents UV-induced ATP decline in HaCaT keratinocytes. Energy-dependent DNA repair is a key determinant of cellular survival after exposure to DNA-damaging agents such as UV radiation. Hence, in this study we investigated whether nicotinamide protection from cellular energy loss influences DNA repair. We treated HaCaT keratinocytes with nicotinamide and exposed them to low-dose solar-simulated UV (ssUV). Excision repair was quantified using an assay of unscheduled DNA synthesis. Nicotinamide increased both the proportion of cells undergoing excision repair and the repair rate in each cell. We then investigated ssUV-induced cyclobutane pyrimidine dimers (CPDs) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxoG) formation and repair by comet assay in keratinocytes and with immunohistochemistry in human skin. Nicotinamide reduced CPDs and 8oxoG in both models and the reduction appeared to be due to enhancement of DNA repair. These results show that nicotinamide enhances two different pathways for repair of UV-induced photolesions, supporting nicotinamide’s potential as an inexpensive, convenient and non-toxic agent for skin cancer chemoprevention.
Cyclooxygenase-2 is overexpressed in the majority of colorectal tumours leading to elevated levels of prostaglandin E2 (PGE2), promoting many hallmarks of cancer. Importantly, PGE2 is reported to enhance Wnt/β-catenin signalling in colorectal carcinoma cells and in normal haematopoietic stem cells where it promotes stem cell function. Although Wnt signalling plays a crucial role in intestinal stem cells, the relationship between PGE2 and intestinal stem cells is unclear. Given that the key intestinal cancer stem cell marker LGR5 (leucine-rich G-protein coupled receptor 5) is a Wnt target and PGE2 enhances Wnt signalling, the focus of this study was to investigate whether PGE2 regulated LGR5 expression in colorectal adenoma cells and whether LGR5 was important for tumour cell survival. PGE2 upregulated LGR5 protein in adenoma (RG/C2) and carcinoma (DLD-1) cell lines. LGR5 knockdown induced cell death in RG/C2 and AA/C1 adenoma cells, suggesting that LGR5 has an important survival-promoting role in adenoma cells. Indeed, we detected LGR5 protein expression in 4 of 4 human adenoma cell lines. Furthermore, LGR5 small interfering RNA inhibited the survival-promoting effects of PGE2 in RG/C2, suggesting that PGE2 promotes adenoma cell survival, at least in part, by increasing LGR5 expression. These studies, therefore, show the first link between PGE2 and LGR5 in human colorectal adenoma and carcinoma cells and demonstrate a survival-promoting role of LGR5. As non-steroidal anti-inflammatory drugs (NSAIDs) cause adenomas to regress in FAP patients, these studies could have important implications for the mechanism by which NSAIDs are chemopreventive, as lowering PGE2 levels could reduce LGR5 expression and survival of LGR5+ adenoma stem cells.
The cells of origin of oral cavity squamous cell carcinoma (OCSCC) are unknown. We used a cell lineage tracing approach (adult K14-CreERTAM; ROSA26 mice transiently treated with tamoxifen) to identify and track normal epithelial stem cells (SCs) in mouse tongues by X-gal staining and to determine if these cells become neoplastically transformed by treatment with a carcinogen, 4-nitroquinoline 1-oxide (4-NQO). Here, we show that in normal tongue epithelia, X-gal(+) cells formed thin columns throughout the entire epithelium 12 weeks after tamoxifen treatment, indicating that the basal layer contains long-lived SCs that produce progeny by asymmetric division to maintain homeostasis. Carcinogen treatment results in a ~10-fold reduction in the total number of X-gal(+) clonal cell populations and horizontal expansion of X-gal(+) clonal cell columns, a pattern consistent with symmetric division of some SCs. Finally, X-gal(+) SCs are present in papillomas and invasive OCSCCs, and these long-lived X-gal(+) SCs are the cells of origin of these tumors. Moreover, the resulting 4-NQO-induced tumors are multiclonal. These findings provide insights into the identity of the initiating cells of oral cancer.
MicroRNAs (miRNA) are small non-coding RNAs that regulate the expression of approximately 60% of all human genes and play important roles in disease processes. Recent studies have demonstrated a link between dysregulated expression of miRNAs and breast carcinogenesis. Long-term estrogen exposure is implicated in development of human breast cancers, yet underlying mechanisms remain elusive. We have recently demonstrated that antioxidant vitamin C (vit C) prevents estrogen-induced breast tumor development. In this study, we investigated the role of vit C in the regulation of microRNA-93 (miR-93) and its target gene(s) in a rat model of mammary carcinogenesis. Female August Copenhagen Irish (ACI) rats were treated with vit C in the presence or absence of 17β-estradiol (E2) for 8 months. We demonstrate an increased expression of the miR-93 in E2-treated mammary tissues and in human breast cell lines and vit C treatment reverted E2-mediated increase in miR-93 levels. MiRNA target prediction programs suggest one of the target genes of miR-93 to be nuclear factor erythroid 2-related factor 2 (NRF2). In contrast with miR-93 expression, NRF2 protein expression was significantly decreased in E2-treated mammary tissues, mammary tumors, and in breast cancer cell lines, and its expression was significantly increased after vit C treatment. Ectopic expression of miR-93 decreased protein expression of NRF2 and NRF2-regulated genes. Furthermore, miR-93 decreased apoptosis, increased colony formation, mammosphere formation, cell migration and DNA damage in breast epithelial cells, whereas silencing of miR-93 in these cells inhibited these carcinogenic processes. Taken together, our findings suggest an oncogenic potential of miR-93 during E2-induced breast carcinogenesis.
Telomere shortening is a major source of chromosome instability (CIN) at early stages during carcinogenesis. However, the mechanisms through which telomere-driven CIN (T-CIN) contributes to the acquisition of tumor phenotypes remain uncharacterized. We discovered that human epithelial kidney cells undergoing T-CIN display massive microRNA (miR) expression changes that are not related to local losses or gains. This widespread miR deregulation encompasses a miR-200-dependent epithelial-to-mesenchymal transition (EMT) that confers to immortalized pre-tumoral cells phenotypic traits of metastatic potential. Remarkably, a miR signature of these cells, comprising a downregulation of miRs with conserved expression in kidney, was retrieved in poorly differentiated aggressive renal cell carcinomas. Our results reveal an unanticipated connection between telomere crisis and the activation of the EMT program that occurs at pre-invasive stages of epithelial cancers, through mechanisms that involve miR deregulation. Thus, this study provides a new rational into how telomere instability contributes to the acquisition of the malignant phenotype.