The aim of this study was to clarify the significance of DNA methylation alterations during gastric carcinogenesis. Single-CpG resolution genome-wide DNA methylation analysis using the Infinium assay was performed on 109 samples of non-cancerous gastric mucosa (N) and 105 samples of tumorous tissue (T). DNA methylation alterations in T samples relative to N samples were evident for 3861 probes. Since N can be at the precancerous stage according to the field cancerization concept, unsupervised hierarchical clustering based on DNA methylation levels was performed on N samples (βN) using the 3861 probes. This divided the 109 patients into three clusters: A (n = 20), B1 (n = 20), and B2 (n = 69). Gastric carcinomas belonging to Cluster B1 showed tumor aggressiveness more frequently than those belonging to Clusters A and B2. The recurrence-free and overall survival rates of patients in Cluster B1 were lower than those of patients in Clusters A and B2. Sixty hallmark genes for which βN characterized the epigenetic clustering were identified. We then focused on DNA methylation levels in T samples (βT) of the 60 hallmark genes. In 48 of them, including the ADAM23, OLFM4, AMER2, GPSM1, CCL28, DTX1 and COL23A1 genes, βT was again significantly correlated with tumor aggressiveness, and the recurrence-free and/or overall survival rates. Multivariate analyses revealed that βT was a significant prognostic factor, being independent of clinicopathological parameters. These data indicate that DNA methylation profiles at the precancerous stage may be inherited by gastric carcinomas themselves, thus determining tumor aggressiveness and patient outcome.
Esophageal adenocarcinoma (EAC) is highly aggressive malignancy that frequently develops from Barrett’s esophagus (BE), a premalignant pathologic change occurring in the lower end of the esophagus. MicroRNAs (miRNAs) are small, non-coding RNAs that function as posttranscriptional regulators of gene expression and were repeatedly proved to play key roles in pathogenesis of BE as well as EAC. In our study, we used Affymetrix GeneChip miRNA arrays to obtain miRNA expression profiles in total of 119 tissue samples [24 normal esophageal mucosa (EM), 60 BE and 35 EAC]. We identified a number of miRNAs, that showed altered expression progressively in sequence EM, BE and EAC, including for instance miR-21, miR-25, miR-194 and miR-196a with increasing levels (P < 0.0015) and miR-203, miR-205, miR-210 and miR-378 with decreasing levels (P < 0.0001). The subsequent analysis revealed four diagnostic miRNA signatures enabling to distinguish EM and BE [12 miRNAs, area under curve (AUC) = 0.971], EM and EAC (13 miRNAs, AUC = 1.0), BE without and BE with dysplasia (21 miRNAs, AUC = 0.856) and BE without dysplastic changes and BE with dysplasia together with EAC (2 miRNAs, AUC = 0.886). We suggest that miRNA expression profiling expands current knowledge in molecular pathology of Barrett’s-based carcinogenesis and enables identification of molecular biomarkers for early detection of BE dysplasia and progression to EAC.
Accurate prognosis is a key factor in establishing optimal therapeutic decisions; yet in the case of bladder cancer (BlCa) current prognostic indicators cannot ensure optimal disease management. Here, we aimed to evaluate the previously unexplored clinical potential of the urological cancer-related miR-145, miR-143 and miR-224 in BlCa. A total of 279 bladder tissue specimens were included in this study (133 BlCa, 107 adjacent normal and 39 healthy samples). Total RNA was extracted from tissues, it was polyadenylated and reverse transcribed to cDNA. The expression of target molecules was measured via quantitative real-time PCR. The expression levels of both miR-143 and miR-145 were significantly decreased, whereas those of miR-224 were increased in BlCa. Receiver operating characteristic curve analysis indicated a significant discriminatory capacity for miR-143/miR-145 levels. Important associations with disease aggressiveness were observed for all three microRNAs; elevated levels were observed in tumors of higher stage and grade, as well as in ‘high-risk’ TaT1 patients. More importantly, high miR-143/145 levels could effectively prognose inferior overall survival for muscle-invasive patients and could independently predict the progression of superficial tumors. Finally, the combination of miR-143/145 overexpression with the widely used prognostic markers of European Organization for Research and Treatment of Cancer-risk groups or recurrence at the first follow-up cystoscopy resulted to a superior positive prediction of non-muscle-invasive bladder cancer short-term progression compared with the use of the abovementioned markers alone. The cancer-related miR-143, miR-145 and miR-224 were investigated for the first time in the clinical setting of BlCa, and miR-143/145 cluster constitutes a novel marker helpful for providing an enhanced prediction of oncologic outcome for BlCa patients.
Nicotinic acetylcholine receptors are important regulators of smoking behavior and tobacco carcinogenesis. We studied the association of the CHRNB3-A6 variant rs13280604 in relation to esophageal squamous cell carcinoma (ESCC) in Chinese populations. Two independent case–control studies were conducted. The first case–control study, consisted of 866 ESCC patients and 1621 healthy controls from Northern China, and the second case–control study consisted of 853 ESCC patients and 860 unrelated controls from Southern China. A logistic regression model was used to evaluate the associations of rs13280604 with cancer risk. We found that Rs13280604 GG/AG genotypes were significantly associated with increased risk for ESCC in both case–control studies from Northern [odds ratio (OR), 1.42, 95% confidence interval (CI), 1.19–1.70, P = 1.1x10–4], Southern China (OR, 1.56, 95% CI, 1.26–1.93, P = 5.2x10–5), and the combined population of both studies (OR, 1.44, 95% CI, 1.26–1.65, P = 8.7x10–8), respectively. Our results suggest that this CHRNB3-A6 variant confers susceptibility to ESCC risk. However, future larger studies are needed to validate our finding.
Compelling evidence has indicated a significant association between leukocyte mitochondrial DNA (mtDNA) content and incidence risks of several malignancies in a cancer-specific manner. However, to date, whether leukocyte mtDNA content can predict clinical outcome of cancer patients has never been investigated. In the present study, we measured leukocyte mtDNA content using real-time PCR-based method in a total of 598 colorectal cancer (CRC) patients and explored its prognostic values. To explore potential mechanism, we detected the immunophenotypes of peripheral blood mononuclear cells and plasma concentrations of several cytokines in CRC patients. We found that patients with high mtDNA content showed significantly worse overall survival (OS) and relapse-free survival (RFS) than those with low mtDNA content in all patient sets. Furthermore, mtDNA content and tumor node metastasis (TNM) stage exhibited a notable joint effect in prognosis prediction. Integration of TNM stage and leukocyte mtDNA content significantly improved the prognosis prediction efficacy for CRC. Importantly, patients with high mtDNA content showed OS and RFS benefits from adjuvant chemotherapy. In addition, we found that patients with high mtDNA content had a higher frequency of CD4+CD25+FOXP3+ regulatory T cells, higher plasma interleukin-2 and transforming growth factor-β1 and lower tumor necrosis factor-α concentration than those with low mtDNA content, suggesting a stronger immunosuppressive phenotype. In conclusion, our study for the first time demonstrates that leukocyte mtDNA content is an independent prognostic marker complementing TNM stage and associated with immunosuppression in CRC patients. Additionally, leukocyte mtDNA content might serve as a potential biomarker to select CRC patients who will benefit from adjuvant chemotherapy.
We know little concerning the expression of transforming growth factor-β1 (TGF-β1) and TGF-β1-induced epithelial-mesenchymal transition (EMT) markers in gastric mucosa and their changes after eradication of Helicobacter pylori infection have not yet been clarified. In the present study, we compared the time course of messenger RNA (mRNA) expression of TGF-β1 and five EMT markers (Twist, Snail, Slug, vimentin and E-cadherin) in 111 controls, 55 patients with gastric dysplasia and 71 patients with early gastric cancer, following eradication of H.pylori. mRNA levels in non-cancerous gastric mucosa were measured using quantitative real time-polymerase chain reaction and the histologic findings of gastric mucosa were compared before and after eradication. The average duration of follow-up was 46.7 months (6.0–112.4). The levels of TGF-β1, Twist, Snail, Slug and vimentin mRNA, in addition to levels of CD44 detected by immunohistochemistry, showed all up-regulation in patients with dysplasia or early gastric cancer compared with controls (P < 0.05); moreover, the mRNA levels of E-cadherin, an epithelial marker, were decreased in these patients compared with the control group (P < 0.001). Eradication of H.pylori reduced the expression of TGF-β1, Twist, Snail, Slug and vimentin mRNA (P-value for slope <0.001), as well as the immunohistochemical expression of CD44 (P = 0.014), whereas it enhanced the expression of E-cadherin (P-value for slope < 0.05). Thus, H.pylori infection may trigger the TGF-β1-induced EMT pathway and the emergence of gastric cancer stem cells (CSCs). Its eradication may prevent the carcinogenesis of gastric cancer by inhibiting these two pathways.
Granulosa cell tumor (GCT) is a rare and severe form of sex-cord stromal ovarian tumor that is characterized by its long natural history and tendency to recur years after surgical ablation. Because there is no efficient curative treatment beyond surgery, ~20% of patients die of the consequences of their tumor. However, very little is known of the molecular etiology of this pathology. About 70% of GCT patients present with elevated circulating estradiol (E2). Because this hormone is known to increase tumor growth and progression in a number of cancers, we investigated the possible role of E2 in GCTs. Cell-based studies with human GCT metastases and primary tumor-derived cells, ie KGN and COV434 cells, respectively, aimed at evaluating E2 effect on cell growth, migration and invasion. Importantly, we found that E2 did not affect GCT cell growth, but that it significantly decreased the migration and matrix invasion of metastatic GCT cells. Noteworthy, our molecular studies revealed that this effect was accompanied by the inhibition through non-genomic mechanisms of extracellular signal-regulated kinase 1/2 (ERK1/2), which is constitutively activated in GCTs. By using pharmacological and RNA silencing approaches, we found that E2 action was mediated by G protein-coupled estrogen receptor 1 (GPER1) signaling pathway. Analyses of GPER1 expression on tissue microarrays from human GCTs confirmed its expression in ~90% of GCTs. Overall, our study reveals that E2 would act via non-classical pathways to prevent metastasis spreading in GCTs and also reveals GPER1 as a possible target in this disease.
APRIL (a proliferation-inducing ligand) is a cytokine of the tumor necrosis factor family associated mainly with hematologic malignancies. APRIL is also overexpressed in breast carcinoma tissue lesions, although neither its role in breast tumorigenesis nor the underlying molecular mechanism is known. Here, we show that several breast cancer cell lines express APRIL and both its receptors, B cell maturation antigen (BCMA) and transmembrane activator and CAML-interactor (TACI), independently of luminal or basal tumor cell phenotype, and that the mitogen-activated protein kinases p38, ERK1/2, and JNK1/2 are activated in response to APRIL. The inflammatory stimulus poly I:C, a toll-like receptor (TLR) 3 ligand, enhanced APRIL secretion. Silencing experiments decreased cell proliferation, demonstrating that APRIL is a critical autocrine factor for breast tumor growth. Studies of 4T1 orthotopic breast tumors in APRIL transgenic mice showed that an APRIL-enriched environment increased tumor growth and promoted lung metastasis associated with enhanced tumor cell proliferation; BCMA and TACI expression suggests that both participate in these processes. We detected APRIL, BCMA and TACI in human luminal, triple-negative breast carcinomas and HER2 breast carcinomas, with increased levels in more aggressive basal tumors. APRIL was observed near Ki67+ nuclei and was distributed heterogeneously in the cancer cells, in the leukocyte infiltrate, and in the myoepithelial layer adjacent to the tumor area; these results imply that APRIL provides proliferation signals to tumor cells through paracrine and autocrine signaling. Our study identifies participation of APRIL signaling in breast cancer promotion; we propose impairment of this pathway as a potential therapeutic strategy.
Snail2 is a zinc finger transcription factor involved in driving epithelial to mesenchymal transitions. Snail2 null mice are viable, but display defects in melanogenesis, gametogenesis and hematopoiesis, and are markedly radiosensitive. Here, using mouse genetics, we have studied the contributions of Snail2 to epidermal homeostasis and skin carcinogenesis. Snail2–/– mice presented a defective epidermal terminal differentiation and, unexpectedly, an increase in number, size and malignancy of tumor lesions when subjected to the two-stage mouse skin chemical carcinogenesis protocol, compared with controls. Additionally, tumor lesions from Snail2–/– mice presented a high inflammatory component with an elevated percentage of myeloid precursors in tumor lesions that was further increased in the presence of the anti-inflammatory agent dexamethasone. In vitro studies in Snail2 null keratinocytes showed that loss of Snail2 leads to a decrease in proliferation indicating a non-cell autonomous role for Snail2 in the skin carcinogenic response observed in vivo. Bone marrow (BM) cross-reconstitution assays between Snail2 wild-type and null mice showed that Snail2 absence in the hematopoietic system fully reproduces the tumor behavior of the Snail2 null mice and triggers the accumulation of myeloid precursors in the BM, blood and tumor lesions. These results indicate a new role for Snail2 in preventing myeloid precursors recruitment impairing skin chemical carcinogenesis progression.
Esophageal cells overexpressing epidermal growth factor receptor (EGFR) and TP53 mutation can invade into the extracellular matrix when grown in 3D-organotypic cultures (OTC) and mimic early invasion in esophageal squamous cell carcinoma (ESCC). We have performed laser capture microdissection with RNA microarray analysis on the invasive and non-invasive tumor cells of p53R175H-overexpressing OTC samples to determine candidate genes facilitating tumor invasion. WNT10A was found to be >4-fold upregulated in the invasive front. Since WNT10A is also prominently upregulated during placode promotion in hair follicle development, a process that requires epithelial cells to thicken and elongate, in order to allow downward growth, we hypothesized that WNT10A may be important in mediating a similar mechanism of tumor cell invasion in ESCC. We have found that WNT10A expression is significantly upregulated in human ESCC, when compared with normal adjacent tissue. Furthermore, high WNT10A expression levels correlate with poor survival. Interestingly, we observe that WNT10A is expressed early in embryogenesis, but is reduced dramatically postnatally. We demonstrate that overexpression of WNT10a promotes migration and invasion, and proliferation of transformed esophageal cells. Lastly, we show that WNT10A overexpression induces a greater CD44High/CD24Low population, which are putative markers of cancer stem cells, and increases self-renewal capability. Taken together, we propose that WNT10A acts as an oncofetal factor that is highly expressed and may promote proper development of the esophagus. During tumorigenesis, it is aberrantly overexpressed in order to promote ESCC migration and invasion, and may be linked to self-renewal of a subset of ESCC cells.