Microarray data analysis of antileukemic action of Cinnamoylated benzaldehyde LQB-461 in Jurkat cell line.

BACKGROUND: Leukemias stand out for being the main type of childhood cancer in the world. Current treatments have strong side effects for patients, and there is still a high rate of development of resistance to multidrug therapy. Previously, our research group developed a structure-activity study with novel synthetic molecules analogous to LQB-278, described as an essential molecule with in vitro antileukemic action. Among these analogs, LQB-461 stood out, presenting more significant antileukemic action compared to its derivative LQB-278, with cytostatic and cytotoxicity effect by apoptosis, inducing caspase-3, and increased sub-G1 phase on cell cycle analysis. METHODS AND RESULTS: Deepening the study of the mechanism of action of LQB-461 in Jurkat cells in vitro, a microarray assay was carried out, which confirmed the importance of the apoptosis pathway in the LQB-461 activity. Through real-time PCR, we validated an increased expression of CDKN1A and BAX genes, essential mediators of the apoptosis intrinsic pathway. Through the extrinsic apoptosis pathway, we found an increased expression of the Fas receptor by flow cytometry, showing the presence of a more sensitive population and another more resistant to death. Considering the importance of autophagy in cellular resistance, it was demonstrated by western blotting that LQB-461 decreased LC-3 protein expression, an autophagic marker. CONCLUSIONS: These results suggest that this synthetic molecule LQB-461 induces cell death by apoptosis in Jurkat cells through intrinsic and extrinsic pathways and inhibits autophagy, overcoming some mechanisms of cell resistance related to this process, which differentiates LQB-461 of other drugs used for the leukemia treatment.

Overweight and obesity do not determine worst prognosis in endometrioid endometrial carcinoma.

PURPOSE: The aim of this study was to investigate the impact of body mass index (BMI) on disease-free survival (DFS) and overall survival (OS) in women diagnosed with EEC and treated at the Brazilian National Cancer Institute. METHODS: The study comprised 849 women diagnosed with EEC who underwent surgical treatment between January, 2000 and December, 2011. The demographic and clinical characteristics of these patients were collected from medical records and their nutritional status was based on the BMI criteria. Univariate (OS and DFS) and multivariate analyses were performed using the Kaplan-Meier method and Cox proportional hazards models, respectively. RESULTS: About 83.2% of patients were obese or overweight at time of diagnosis, with a mean BMI of 31.83. Patients were followed for an average of 34.97 months. There were 111 recurrences (13.1%) and 140 deaths (16.5%), with mean DFS of 51.90 months and mean OS of 52.25 months. There was no significant association between BMI and DFS or OS. In multivariate analysis we did not find an increased hazard of recurrence or death among overweight or obese patients. CONCLUSION: Overweight and obesity had no impact on EEC prognosis on the assessed cohort. Further studies are warranted.

Alterations in epidermal growth factor receptors 1 and 2 in esophageal squamous cell carcinomas.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) shows a 5-year survival rate below 10%, demonstrating the urgency in improving its treatment. Alterations in epidermal growth factor receptors are closely related to malignancy transformation in a number of tumors and recent successful targeted therapies have been directed to these molecules. Therefore, in this study, we analyzed the expression of EGFR and HER2 and evaluated EGFR mutation profile as well as the presence of mutations in hotspots of KRAS and BRAF in ESCC patients. METHODS: We performed RT-qPCR, immunohistochemistry and Fluorescent in situ hybridization to determine EGFR and HER2 expression in ESCC patients, and direct sequencing and PCR-RFLP for mutations and polymorphism analysis. RESULTS: Our results showed an increased EGFR mRNA expression in tumors compared to surrounding tissue (p <0.05), with 11% of the cases presenting at least a four-fold difference between tumor and paired adjacent mucosa. EGFR protein overexpression was present only in 4% of the cases. The median expression of HER2 mRNA was not different between tumors and adjacent mucosa. Still, 7% of the tumors presented at least a 25-fold higher expression of this gene when compared to its paired counterpart. Immunohistochemical analysis revealed that 21% of the tumors were positive for HER2 (scores 2+ and 3+), although only 3+ tumors presented amplification of this gene. Mutation analysis for EGFR (exons 18-21), KRAS (codons 12 and 13) and BRAF (V600E) showed no mutations in any of the hotspots of these genes in almost 100 patients analyzed. EGFR presented synonymous polymorphisms at codon 836 (C>T) in 2.1% of the patients, and at codon 787 (G>A) in 79.2% of the cases. This last polymorphism was also evaluated in 304 healthy controls, which presented a similar frequency (73.7%) in comparison with ESCC patients. The absence of mutations of EGFR, KRAS and BRAF as well as the overexpression of EGFR and HER2 in less than 10% of the patients suggest that this signaling pathway is altered in only a small proportion of patients with ESCC. CONCLUSION: HER receptors target therapies may have the potential to be effective in only a minor fraction of patients with ESCC.

Quantitative evaluation of SPRR3 expression in esophageal squamous cell carcinoma by qPCR and its potential use as a biomarker.

Esophageal squamous cell carcinoma (ESCC) is highly fatal due to late diagnosis and inefficient treatment. Early disease detection could improve diagnosis and patient survival. Esophageal squamous epithelial cells express SPRR3, a member of the small proline-rich protein family, which is downregulated in ESCC. Therefore, SPRR3 expression may be used as a biomarker to follow the transition from healthy mucosa to ESCC. Both SPRR3 mRNA splice variants, v1 and v2, were evaluated by real time PCR in tumor and histologically normal adjacent tissue biopsies from 84 ESCC patients and 18 healthy controls. SPRR3-v1 was most highly expressed in the esophageal mucosa of healthy subjects, with an increasingly lower expression in the adjacent mucosa of ESCC patients and in tumors, respectively. SPRR3-v2 expression was low in normal mucosa and in tumors but it was higher in the adjacent mucosa of ESCC patients. In addition, we found a significant correlation between a lower SPRR3-v1 and SPRR3-v2 expression and age and alcohol consumption, respectively. SPRR3 protein expression presented a good correlation with SPRR3 mRNA expression. Cut-off points to discriminate between healthy mucosa, tumor and adjacent mucosa were determined with receiver operating characteristic (ROC) curves. This analysis showed that SPRR3-v1 expression discriminates the esophageal mucosa of healthy subjects from the adjacent mucosa and the tumor of ESCC patients with high sensitivity and specificity. Our data shows that the quantitative analysis of SPRR3 mRNA is a robust and reliable method to monitor the malignant transformation of the healthy esophageal mucosa into ESCC.

Head and Neck Cancers Are Not Alike When Tarred with the Same Brush: An Epigenetic Perspective from the Cancerization Field to Prognosis.

Head and neck squamous cell carcinomas (HNSCC) are among the ten most frequent types of cancer worldwide and, despite all efforts, are still diagnosed at late stages and show poor overall survival. Furthermore, HNSCC patients often experience relapses and the development of second primary tumors, as a consequence of the field cancerization process. Therefore, a better comprehension of the molecular mechanisms involved in HNSCC development and progression may enable diagnosis anticipation and provide valuable tools for prediction of prognosis and response to therapy. However, the different biological behavior of these tumors depending on the affected anatomical site and risk factor exposure, as well as the high genetic heterogeneity observed in HNSCC are major obstacles in this pursue. In this context, epigenetic alterations have been shown to be common in HNSCC, to discriminate the tumor anatomical subsites, to be responsive to risk factor exposure, and show promising results in biomarker development. Based on this, this review brings together the current knowledge on alterations of DNA methylation and microRNA expression in HNSCC natural history, focusing on how they contribute to each step of the process and on their applicability as biomarkers of exposure, HNSCC development, progression, and response to therapy.

Synthesis and Biological Evaluation of Cyclic Analogues from Nitrone LQB-278: A New Potential Antileukemia Compound.

BACKGROUND/AIM: A new set of LQB-nitrones and analogues was synthesized to evaluate anticancer activity based on the substitution of the terpenyl moiety of the antileukemic compound LQB-278 by the conformationally restricted cinnamyl ether. MATERIALS AND METHODS: A structure-activity relationship study was performed in vitro on Jurkat cells to screen the antileukemic activity of LQB-nitrones and analogues and elucidate the mechanisms of action of the most active derivatives. RESULTS: The cynamyl ramification and its ortho position aldehyde substitution improved the antileukemic activity. Three compounds showed an in vitro antiproliferative action, but only 5b induced apoptosis. Analysis of the molecular mechanisms showed increased expression of the cell cycle inhibitor p21CIP1/WAF1/Sdi1, caspase 3, Fas receptor, and Bax/Bcl-2 ratio. CONCLUSION: The cinnamyl derivative 5b (LQB-461) presented higher antileukemic effects than the prototype terpenyl nitrone, inducing Jurkat cell death by activating both extrinsic and intrinsic pathways of apoptosis. Therefore, this compound is a new promising candidate drug against leukemia.

Upper Aerodigestive Tract Squamous Cell Carcinomas Show Distinct Overall DNA Methylation Profiles and Different Molecular Mechanisms behind WNT Signaling Disruption.

Upper aerodigestive tract (UADT) tumors present different biological behavior and prognosis, suggesting specific molecular mechanisms underlying their development. However, they are rarely considered as single entities (particularly head and neck subsites) and share the most common genetic alterations. Therefore, there is a need for a better understanding of the global DNA methylation differences among UADT tumors. We performed a genome-wide DNA methylation analysis of esophageal (ESCC), laryngeal (LSCC), oral (OSCC) and oropharyngeal (OPSCC) squamous cell carcinomas, and their non-tumor counterparts. The unsupervised analysis showed that non-tumor tissues present markedly distinct DNA methylation profiles, while tumors are highly heterogeneous. Hypomethylation was more frequent in LSCC and OPSCC, while ESCC and OSCC presented mostly hypermethylation, with the latter showing a CpG island overrepresentation. Differentially methylated regions affected genes in 127 signaling pathways, with only 3.1% of these being common among different tumor subsites, but with different genes affected. The WNT signaling pathway, known to be dysregulated in different epithelial tumors, is a frequent hit for DNA methylation and gene expression alterations in ESCC and OPSCC, but mostly for genetic alterations in LSCC and OSCC. UADT tumor subsites present differences in genome-wide methylation regarding their profile, intensity, genomic regions and signaling pathways affected.

TP53 mutation profile of esophageal squamous cell carcinomas of patients from Southeastern Brazil.

Esophageal cancer (EC) is among the 10 most common and fatal malignacies in the world, presenting a marked geographic variation in incidence rates between and within different countries. The TP53 tumor suppressor gene is highly mutated in esophageal tumors and its mutation pattern can offer clues to the etiopathology of the tumor. As Brazil presents one of the highest incidence areas in the West, a deeper knowledge of the molecular mechanisms related to EC development in the Brazilian population is needed. We analyzed the mutation profile of 110 esophageal squamous cell carcinomas (ESCC) of patients from Southeastern Brazil (Rio de Janeiro and São Paulo) and collected data regarding alcohol intake and tobacco smoking. We detected 41 mutations in tumor samples from 38 patients. There was no association between mutation frequency and tobacco smoking or alcohol drinking. The most frequently mutated codons were 179, 214, 220 and 248. Codons 179, 220 and 248 are hot-spots for ESCC, but codon 214 presents only 0.7% of the mutations registered in the IARC database. The mutation profile revealed a high percentage of mutations at A:T base pairs (34.1%) followed by deletions (17.1%). We concluded that the mutation profile detected in this study is different from that of patients from Southern Brazil but very similar to that previously seen in French patients, being characterized by a high frequency of mutations at A:T base pairs, which may be associated with acetaldehyde, the metabolic product of ethanol.

Multi-cancer V-ATPase molecular signatures: A distinctive balance of subunit C isoforms in esophageal carcinoma.

BACKGROUND: V-ATPases are hetero-oligomeric enzymes consisting of 13 subunits and playing key roles in ion homeostasis and signaling. Differential expression of these proton pumps has been implicated in carcinogenesis and metastasis. To elucidate putative molecular signatures underlying these phenomena, we evaluated the expression of V-ATPase genes in esophageal squamous cell carcinoma (ESCC) and extended the analysis to other cancers. METHODS: Expression of all V-ATPase genes were analyzed in ESCC by a microarray data and in different types of tumors available from public databases. Expression of C isoforms was validated by qRT-PCR in paired ESCC samples. FINDINGS: A differential expression pattern of V-ATPase genes was found in different tumors, with combinations in up- and down-regulation leading to an imbalance in the expression ratios of their isoforms. Particularly, a high C1 and low C2 expression pattern accurately discriminated ESCC from normal tissues. Structural modeling of C2a isoform uncovered motifs for oncogenic kinases in an additional peptide stretch, and an actin-biding domain downstream to this sequence. INTERPRETATION: Altogether these data revealed that the expression ratios of subunits/isoforms could form a conformational code that controls the H+ pump regulation and interactions related to tumorigenesis. This study establishes a paradigm change by uncovering multi-cancer molecular signatures present in the V-ATPase structure, from which future studies must address the complexity of the onco-related V-ATPase assemblies as a whole, rather than targeting changes in specific subunit isoforms. FUNDING: This work was supported by grants from CNPq and FAPERJ-Brazil.

Transcriptome Analysis Identifies ALCAM Overexpression as a Prognosis Biomarker in Laryngeal Squamous Cell Carcinoma.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is one of the most incident tumors in the world, especially in developing countries, such as Brazil. Different from other tumors, LSCC prognosis did not improve during the past four decades. Therefore, the objective of this study was to develop biomarkers that can predict LSCC patient's prognosis. RESULTS: Transcriptome analysis pointed out 287 overexpressed genes in LSCC in comparison to adjacent mucosa. Among these, a gene-pattern signature was created with 24 genes associated with prognosis. The Bayesian clustering of both Brazil and The Cancer Genome Atlas (TCGA) data pointed out clusters of samples possessing significative differences in the prognosis, and the expression panel of three genes (ALCAM, GBP6, and ME1) was capable to distinguish patients with worse prognosis with an accuracy of 97%. Survival analyses with TCGA data highlighted ALCAM gene expression as an independent prognostic factor for LSCC. This was further confirmed through immunohistochemistry, using a validation set of Brazilian patients. ALCAM expression was not associated with prognosis for other head and neck tumor sites. CONCLUSION: ALCAM overexpression seems to be an independent prognosis biomarker for LSCC patients.

High infiltration of B cells in tertiary lymphoid structures, TCR oligoclonality, and neoantigens are part of esophageal squamous cell carcinoma microenvironment.

Esophageal squamous cell carcinoma (ESCA) exhibits high intratumoral molecular heterogeneity posing a challenge to cancer therapy. Immune checkpoint blockade therapy has been approved for this disease, but with modest results. RNA-Seq data from paired tumor and surrounding nonmalignant tissue from 14 patients diagnosed with ESCA without previous treatment and from The Cancer Genome Atlas-ESCA cohort were analyzed. Herein, we investigated ESCA immune landscape including mutation-derived neoantigens and immune cell subpopulations. Tumor-associated antigen expression was determined by in silico analyses and confirmed by immunohistochemistry showing that PRAME, CEACAM4, and MAGEA11 proteins are expressed on tumors. Immune checkpoint molecules gene expression was higher in the tumor compared with surrounding nonmalignant tissue, but its expression varies greatly among patients. TCR repertoire and BCR transcripts analysis evidenced low clonal diversity with one TCR clone predicted to be specific for a MAGEA11-derived peptide. A high number of B-cell clones infiltrating the tumors and the abundance of these cells in tertiary lymphoid structures observed in ESCA tumors support B cells as a potential immune modulator in this tumor.

Complete genome sequence of the sugarcane nitrogen-fixing endophyte Gluconacetobacter diazotrophicus Pal5.

BACKGROUND: Gluconacetobacter diazotrophicus Pal5 is an endophytic diazotrophic bacterium that lives in association with sugarcane plants. It has important biotechnological features such as nitrogen fixation, plant growth promotion, sugar metabolism pathways, secretion of organic acids, synthesis of auxin and the occurrence of bacteriocins. RESULTS: Gluconacetobacter diazotrophicus Pal5 is the third diazotrophic endophytic bacterium to be completely sequenced. Its genome is composed of a 3.9 Mb chromosome and 2 plasmids of 16.6 and 38.8 kb, respectively. We annotated 3,938 coding sequences which reveal several characteristics related to the endophytic lifestyle such as nitrogen fixation, plant growth promotion, sugar metabolism, transport systems, synthesis of auxin and the occurrence of bacteriocins. Genomic analysis identified a core component of 894 genes shared with phylogenetically related bacteria. Gene clusters for gum-like polysaccharide biosynthesis, tad pilus, quorum sensing, for modulation of plant growth by indole acetic acid and mechanisms involved in tolerance to acidic conditions were identified and may be related to the sugarcane endophytic and plant-growth promoting traits of G. diazotrophicus. An accessory component of at least 851 genes distributed in genome islands was identified, and was most likely acquired by horizontal gene transfer. This portion of the genome has likely contributed to adaptation to the plant habitat. CONCLUSION: The genome data offer an important resource of information that can be used to manipulate plant/bacterium interactions with the aim of improving sugarcane crop production and other biotechnological applications.

Evidence for increased expression of tissue factor and protease-activated receptor-1 in human esophageal cancer.

It has been suggested that the blood clotting initiator protein, tissue factor (TF), participates in tumor growth, metastasis and angiogenesis. In addition, a family of G protein-coupled-receptors known as protease-activated receptors (PARs) has also been implicated in tumor biology. These receptors might be activated by blood coagulation proteases thus eliciting a number of pro-tumoral responses, including the expression of interleukin-8 (IL-8). Therefore, in this study we analyzed the expression of TF, PAR-1, PAR-2 and IL-8 genes in patients with esophageal cancer, one of the most aggressive neoplastic diseases. Total RNA was extracted from tissue samples (tumor and the corresponding normal mucosa) obtained from patients submitted to esophagectomy or endoscopy and further analyzed by semi-quantitative reverse transcriptase-polymerase (RT-PCR) and/or real-time quantitative PCR (qPCR). Expression of full-length transmembrane TF was significantly higher in tumor samples whereas no differences were observed in alternatively spliced TF transcripts. Tumor tissue showed increased mRNA levels for PAR-1 but not PAR-2. Remarkably, IL-8 expression was not detected in most normal tissues but showed very high expression in tumor samples. As expected, qPCR revealed greater differences in the expression pattern of all transcripts analyzed but the general profile was very similar to that observed by RT-PCR. Altogether our data suggest a possible role for blood clotting proteins in the biology of human esophageal cancer.

Analysis of mutations in TP53, APC, K-ras, and DCC genes in the non-dysplastic mucosa of patients with inflammatory bowel disease.

BACKGROUND AND AIMS: Patients with ulcerative colitis (UC) and Crohn's disease (CD) have a high risk for colorectal cancer (CRC). To understand the molecular basis of colitis-associated CRC, we analyzed alterations in TP53, APC, K-ras, and DCC genes in the non-dysplastic UC and CD colon. MATERIALS AND METHODS: Endoscopic biopsies were collected from six predefined colon sites of 35 UC and 12 CD patients for DNA extraction and genetic analysis. RESULTS: A mutation was found in codon 1141 of the APC gene of two CD patients, being somatic in one and germinative in the other. The mutation seen in both patients was a base exchange of thymine for cytosine, resulting in an exchange of leucine for serine. We did not detect any mutations in the other samples analyzed. CONCLUSIONS: Mutations in APC gene may occur in the non-dysplastic CD mucosa of patients with disease for more than 10 years. The follow-up of these patients will show the likelihood of mutant APC progressing to CRC in CD. Further analysis will be required for evaluating the impact of these findings in the context of cancer surveillance in inflammatory bowel disease.

Overexpression of HMGA1 Figures as a Potential Prognostic Factor in Endometrioid Endometrial Carcinoma (EEC).

Endometrioid endometrial carcinomas (EEC) are the most common malignant gynecologic tumors. Despite the increase in EEC molecular knowledge, the identification of new biomarkers involved in disease's development and/or progression would represent an improvement in its course. High-mobility group A protein (HMGA) family members are frequently overexpressed in a wide range of malignancies, correlating with a poor prognosis. Thus, the aim of this study was to analyze HMGA1 and HMGA2 expression pattern and their potential role as EEC biomarkers. HMGA1 and HMGA2 expression was initially evaluated in a series of 46 EEC tumors (stages IA to IV), and the findings were then validated in The Cancer Genome Atlas (TCGA) EEC cohort, comprising 381 EEC tumors (stages IA to IV). Our results reveal that HMGA1 and HMGA2 mRNA and protein are overexpressed in ECC, but only HMGA1 expression is associated with increased histological grade and tumor size. Moreover, HMGA1 but not HMGA2 overexpression was identified as a negative prognostic factor to EEC patients. Finally, a positive correlation between expression of HMGA1 pseudogenes-HMGA1-P6 and HMGA1-P7-and HMGA1 itself was detected, suggesting HMGA1 pseudogenes may play a role in HMGA1 expression regulation in EEC. Thus, these results indicate that HMGA1 overexpression possesses a potential role as a prognostic biomarker for EEC.

High-Resolution Melting (HRM) of Hypervariable Mitochondrial DNA Regions for Forensic Science.

Forensic strategies commonly are proceeding by analysis of short tandem repeats (STRs); however, new additional strategies have been proposed for forensic science. Thus, this article standardized the high-resolution melting (HRM) of DNA for forensic analyzes. For HRM, mitochondrial DNA (mtDNA) from eight individuals were extracted from mucosa swabs by DNAzol reagent, samples were amplified by PCR and submitted to HRM analysis to identify differences in hypervariable (HV) regions I and II. To confirm HRM, all PCR products were DNA sequencing. The data suggest that is possible discriminate DNA from different samples by HRM curves. Also, uncommon dual-dissociation was identified in a single PCR product, increasing HRM analyzes by evaluation of melting peaks. Thus, HRM is accurate and useful to screening small differences in HVI and HVII regions from mtDNA and increase the efficiency of laboratory routines based on forensic genetics.

UBE2C Is a Transcriptional Target of the Cell Cycle Regulator FOXM1.

FOXM1 (forkhead box protein M1) is a transcription factor that participates in all stages of tumor development, mainly through the control of cell cycle and proliferation, regulating the expression of genes involved in G1/S and G2/M transition and M phase progression. The ubiquitin conjugating enzyme E2 (UBE2C) is a member of the anaphase promoting complex/cyclosome, promoting the degradation of several target proteins along cell cycle progression, during metaphase/anaphase transition. FOXM1 and UBE2C have been found overexpressed in a wide range of different solid tumors. Therefore, the aim of this study was to investigate whether UBE2C is a transcriptional target of FOXM1, using esophageal squamous cell carcinoma (ESCC) as a model, in addition to several cancer-deposited data. Our results show that FOXM1 and UBE2C expression present a positive correlation in normal tissues and in 25 distinct tumor types, including ESCC, where these genes are overexpressed. Moreover, FOXM1 binds to UBE2C promoter region in ESCC cell line and transcriptionally activates it, leading to UBE2C upregulation. In conclusion, this study provides evidences that FOXM1 transcriptionally regulates UBE2C expression in ESCC and their deregulation may be a general phenomenon in human neoplasias.

Esophageal squamous cell carcinoma transcriptome reveals the effect of FOXM1 on patient outcome through novel PIK3R3 mediated activation of PI3K signaling pathway.

Esophageal squamous cell carcinoma (ESCC) presents poor prognosis, and patients diagnosed with this tumor currently lack target treatments. Therefore, in order to identify potential targets for ESCC treatment, we carried out a transcriptome analysis with ESCC and paired nonmalignant surrounding mucosa samples, followed by a master regulator analysis, and further explored the role of the identified central regulatory genes through in vivo and in vitro assays. Among the transcription factors deregulated/enriched in ESCC, we focused on FOXM1 because of its involvement in the regulation of critical biological processes. A new transcriptome analysis performed with ESCC cell lineage TE-1 showed that the modulation of FOXM1 expression resulted in PIK3R3 expression changes, whereas chromatin immunoprecipitation assay revealed that FOXM1 was capable of binding onto PIK3R3 promoter, thus demonstrating that PIK3R3 is a new FOXM1 target. Furthermore, FOXM1 overexpression resulted in the activation of PIK3/AKT signaling pathway through PIK3R3-mediated AKT phosphorylation. Finally, the analysis of the clinic-pathological data of ESCC patients revealed that overexpression of both FOXM1 and PIK3R3 was associated with poor prognosis, but only the latter was an independent prognosis factor for ESCC patients. In conclusion, our results show that FOXM1 seems to play a central role in ESCC carcinogenesis by upregulating many oncogenes found overexpressed in this tumor. Furthermore, PIK3R3 is a novel FOXM1 target that triggers the activation of the PI3K/AKT pathway in ESCC cells.

Mutations, Differential Gene Expression, and Chimeric Transcripts in Esophageal Squamous Cell Carcinoma Show High Heterogeneity.

Esophageal squamous cell carcinoma (ESCC) is a frequent and lethal neoplasia. As recent advances in targeted therapy have not improved ESCC prognosis, characterization of molecular alterations associated to this tumor is of foremost relevance. In this study, we analyze, for the first time, the complete genomic profile of ESCC by RNA-seq. TP53 was the most frequently mutated gene in the investigation and validation sets (78.6% and 67.4%, respectively). Differential expression analysis between tumor and nontumor adjacent mucosa showed 6698 differentially expressed genes, most of which were overexpressed (74%). Enrichment analysis identified overrepresentation of Wnt pathway, with overexpressed activators and underexpressed inactivators, suggesting activation of canonical and noncanonical Wnt signaling pathways. Higher WNT7B expression was associated with poor prognosis. Twenty-one gene fusions were identified in 50% of tumors, none of which involving the same genes in different patients; 71% of fusions involved syntenic genes. Comparisons with TCGA data showed co-amplification of seven gene pairs involved in fusions in the present study (~33%), suggesting that these rearrangements might have been driven by chromoanagenesis. In conclusion, genomic alterations in ESCC are highly heterogeneous, impacting negatively in target therapy development.

TFF1 hypermethylation and decreased expression in esophageal squamous cell carcinoma and histologically normal tumor surrounding esophageal cells.

Background: Esophageal squamous cell carcinoma (ESCC) is one of the 10 most incident cancer types in the world, and it is mainly associated with tobacco and alcohol consumption. ESCC mortality rates stand very close to its incidence, which is a direct consequence of a late diagnosis and an inefficient treatment. Although this scenery is quite alarming, the major molecular alterations that drive this carcinogenesis process remain unclear. We have previously shown through the first ESCC methylome analysis that TFF1 promoter is frequently hypermethylated in ESCC. Here, to evaluate TFF1 methylation as a potential biomarker of early ESCC diagnosis, we investigated the status of TFF1 promoter methylation and its expression in ESSC and histologically normal tumor surrounding tissue of ESCC patients in comparison to healthy esophagus of non-cancer individuals. Results: Analysis of TFF1 promoter methylation, and gene and protein expression in 65 ESCC patients and 88 controls revealed that TFF1 methylation levels were already increased in histologically normal tumor surrounding tissue of ESCC patients when compared to healthy esophagus of non-cancer individuals. This increase in DNA methylation was followed by the reduction of TFF1 mRNA expression. Interestingly, TFF1 expression was capable of distinguishing tumor surrounding normal tissue from normal mucosa of healthy individuals with 92% accuracy. In addition, TFF1 protein was undetectable both in tumor and surrounding mucosa by immunohistochemistry, while submucosa glands of the healthy esophagus showed positive staining. Furthermore, treatment of TE-1 and TE-13 ESCC cell lines with decitabine led to a reduction of promoter methylation and consequent upregulation of TFF1 gene and protein expression. Finally, using TCGA data we showed that TFF1 loss is observed in ESCC, but not in esophageal adenocarcinoma, highlighting the different molecular mechanisms involved in the development of each histological subtype of esophageal cancer. Conclusions: This study shows that TFF1 expression is silenced in early phases of ESCC development, which seems to be mediated at least in part by promoter hypermethylation, and provides the basis for the use of TFF1 expression as a potential biomarker for early ESCC detection.