TFAP2E-DKK4 and chemoresistance in colorectal cancer.

BACKGROUND: Chemotherapy for advanced colorectal cancer leads to improved survival; however, predictors of response to systemic treatment are not available. Genomic and epigenetic alterations of the gene encoding transcription factor AP-2 epsilon (TFAP2E) are common in human cancers. The gene encoding dickkopf homolog 4 protein (DKK4) is a potential downstream target of TFAP2E and has been implicated in chemotherapy resistance. We aimed to further evaluate the role of TFAP2E and DKK4 as predictors of the response of colorectal cancer to chemotherapy. METHODS: We analyzed the expression, methylation, and function of TFAP2E in colorectal-cancer cell lines in vitro and in patients with colorectal cancer. We examined an initial cohort of 74 patients, followed by four cohorts of patients (total, 220) undergoing chemotherapy or chemoradiation. RESULTS: TFAP2E was hypermethylated in 38 of 74 patients (51%) in the initial cohort. Hypermethylation was associated with decreased expression of TFAP2E in primary and metastatic colorectal-cancer specimens and cell lines. Colorectal-cancer cell lines overexpressing DKK4 showed increased chemoresistance to fluorouracil but not irinotecan or oxaliplatin. In the four other patient cohorts, TFAP2E hypermethylation was significantly associated with nonresponse to chemotherapy (P<0.001). Conversely, the probability of response among patients with hypomethylation was approximately six times that in the entire population (overall estimated risk ratio, 5.74; 95% confidence interval, 3.36 to 9.79). Epigenetic alterations of TFAP2E were independent of mutations in key regulatory cancer genes, microsatellite instability, and other genes that affect fluorouracil metabolism. CONCLUSIONS: TFAP2E hypermethylation is associated with clinical nonresponsiveness to chemotherapy in colorectal cancer. Functional assays confirm that TFAP2E-dependent resistance is mediated through DKK4. In patients who have colorectal cancer with TFAP2E hypermethylation, targeting of DKK4 may be an option to overcome TFAP2E-mediated drug resistance. (Funded by Deutsche Forschungsgemeinschaft and others.).

DNA methylation represses IFN-gamma-induced and signal transducer and activator of transcription 1-mediated IFN regulatory factor 8 activation in colon carcinoma cells.

IFN regulatory factor 8 (IRF8) is both constitutively expressed and IFN-gamma inducible in hematopoietic and nonhematopoietic cells. We have shown that IRF8 expression is silenced by DNA methylation in human colon carcinoma cells, but the molecular mechanism underlying methylation-dependent IRF8 silencing remains elusive. In this study, we observed that IRF8 protein level is inversely correlated with the methylation status of the IRF8 promoter and the metastatic phenotype in human colorectal carcinoma specimens in vivo. Demethylation treatment or knocking down DNMT1 and DNMT3b expression rendered the tumor cells responsive to IFN-gamma to activate IRF8 transcription in vitro. Bisulfite genomic DNA sequencing revealed that the entire CpG island of the IRF8 promoter is methylated. Electrophoresis mobility shift assay revealed that DNA methylation does not directly inhibit IFN-gamma-activated phosphorylated signal transducer and activator of transcription 1 (pSTAT1) binding to the IFN-gamma activation site element in the IRF8 promoter in vitro. Chromatin immunoprecipitation assay revealed that pSTAT1 is associated with the IFN-gamma activation site element of the IRF8 promoter in vivo regardless of the methylation status of the IRF8 promoter. However, DNA methylation results in preferential association of PIAS1, a potent inhibitor of pSTAT1, with pSTAT1 in the methylated IRF8 promoter region. Silencing methyl-CpG binding domain protein 1 (MBD1) expression resulted in IRF8 activation by IFN-gamma in human colon carcinoma cells with methylated IRF8 promoter. Our data thus suggest that human colon carcinoma cells silence IFN-gamma-activated IRF8 expression through MBD1-dependent and PIAS1-mediated inhibition of pSTAT1 function at the methylated IRF8 promoter.

Epigenetic silencing of tumor suppressor candidate 3 confers adverse prognosis in early colorectal cancer.

Colorectal cancer (CRC) is a biologically and clinically heterogeneous disease. Even though many recurrent genomic alterations have been identified that may characterize distinct subgroups, their biological impact and clinical significance as prognostic indicators remain to be defined. The tumor suppressor candidate-3 (TUSC3/N33) locates to a genomic region frequently deleted or silenced in cancers. TUSC3 is a subunit of the oligosaccharyltransferase (OST) complex at the endoplasmic reticulum (ER) which catalyzes bulk N-glycosylation of membrane and secretory proteins. However, the consequences of TUSC3 loss are largely unknown. Thus, the aim of the study was to characterize the functional and clinical relevance of TUSC3 expression in CRC patients' tissues (n=306 cases) and cell lines. TUSC3 mRNA expression was silenced by promoter methylation in 85 % of benign adenomas (n=46 cases) and 35 % of CRCs (n =74 cases). Epidermal growth factor receptor (EGFR) was selected as one exemplary ER-derived target protein of TUSC3-mediated posttranslational modification. We found that TUSC3 inhibited EGFR-signaling and promoted apoptosis in human CRC cells, whereas TUSC3 siRNA knock-down increased EGFR-signaling. Accordingly, in stage I/II node negative CRC patients (n=156 cases) loss of TUSC3 protein expression was associated with poor overall survival. In sum, our data suggested that epigenetic silencing of TUSC3 may be useful as a molecular marker for progression of early CRC.

Molecular biomarkers in esophageal, gastric, and colorectal adenocarcinoma.

Cancers of the esophagus, stomach and colon contribute to a major health burden worldwide and over 20% of all cancer deaths. Biomarkers that should indicate pathogenic process and are measureable in an objective manner for these tumors are rare and not established in the clinical setting. In general biomarkers can be very useful for cancer management as they can improve clinical decision-making regarding diagnosis, surveillance, and therapy. Biomarkers can be different types of molecular entities (such as DNA, RNA or proteins), which can be detected, in different tissues or body fluids. However, more important is the type of biomarker itself, which allows diagnostic, prognostic or predictive analyses for different clinical problems. This review aims to systematically summarize the recent findings of genetic and epigenetic markers for gastrointestinal tumors within the last decade. While many biomarkers seem to be very promising, especially if used as panels, further development is urgently needed to address practical considerations of biomarkers in cancer treatment.

Bile acids down-regulate caveolin-1 in esophageal epithelial cells through sterol responsive element-binding protein.

Bile acids are synthesized from cholesterol and are major risk factors for Barrett adenocarcinoma (BAC) of the esophagus. Caveolin-1 (Cav1), a scaffold protein of membrane caveolae, is transcriptionally regulated by cholesterol via sterol-responsive element-binding protein-1 (SREBP1). Cav1 protects squamous epithelia by controlling cell growth and stabilizing cell junctions and matrix adhesion. Cav1 is frequently down-regulated in human cancers; however, the molecular mechanisms that lead to this event are unknown. We show that the basal layer of the nonneoplastic human esophageal squamous epithelium expressed Cav1 mainly at intercellular junctions. In contrast, Cav1 was lost in 95% of tissue specimens from BAC patients (n = 100). A strong cytoplasmic expression of Cav1 correlated with poor survival in a small subgroup (n = 5) of BAC patients, and stable expression of an oncogenic Cav1 variant (Cav1-P132L) in the human BAC cell line OE19 promoted proliferation. Cav1 was also detectable in immortalized human squamous epithelial, Barrett esophagus (CPC), and squamous cell carcinoma cells (OE21), but was low in BAC cell lines (OE19, OE33). Mechanistically, bile acids down-regulated Cav1 expression by inhibition of the proteolytic cleavage of 125-kDa pre-SREBP1 from the endoplasmic reticulum/Golgi apparatus and nuclear translocation of active 68-kDa SREBP1. This block in SREBP1's posttranslational processing impaired transcriptional activation of SREBP1 response elements in the proximal human Cav1 promoter. Cav1 was also down-regulated in esophagi from C57BL/6 mice on a diet enriched with 1% (wt/wt) chenodeoxycholic acid. Mice deficient for Cav1 or the nuclear bile acid receptor farnesoid X receptor showed hyperplasia and hyperkeratosis of the basal cell layer of esophageal epithelia, respectively. These data indicate that bile acid-mediated down-regulation of Cav1 marks early changes in the squamous epithelium, which may contribute to onset of Barrett esophagus metaplasia and progression to BAC.

Performance of epigenetic markers SEPT9 and ALX4 in plasma for detection of colorectal precancerous lesions.

BACKGROUND: Screening for colorectal cancer (CRC) has shown to reduce cancer-related mortality, however, acceptance and compliance to current programmes are poor. Developing new, more acceptable non-invasive tests for the detection of cancerous and precancerous colorectal lesions would not only allow preselection of individuals for colonoscopy, but may also prevent cancer by removal of precancerous lesions. METHODS: Plasma from 128 individuals (cohort I - exploratory study: 73 cases / 55 controls) was used to test the performance of a single marker, SEPT9, using a real-time quantitative PCR assay. To validate performance of SEPT9, plasma of 76 individuals (cohort II - validation study: 54 cases / 22 controls) was assessed. Additionally, improvement of predictive capability considering SEPT9 and additionally ALX4 methylation was investigated within these patients. RESULTS: In both cohorts combined, methylation of SEPT9 was observed in 9% of controls (3/33), 29% of patients with colorectal precancerous lesions (27/94) and 73% of colorectal cancer patients (24/33). The presence of both SEPT9 and ALX4 markers was analysed in cohort II and was observed in 5% of controls (1/22) and 37% of patients with polyps (18/49). Interestingly, also 3/5 (60%) patients with colorectal cancer were tested positive by the two marker panel in plasma. CONCLUSIONS: While these data confirm the detection rate of SEPT9 as a biomarker for colorectal cancer, they also show that methylated DNA from advanced precancerous colorectal lesions can be detected using a panel of two DNA methylation markers, ALX4 and SEPT9. If confirmed in larger studies these data indicate that screening for colorectal precancerous lesions with a blood-based test may be as feasible as screening for invasive cancer.

Differential Expression of SPARC in Intestinal-type Gastric Cancer Correlates with Tumor Progression and Nodal Spread.

AIMS: Nodal spread is the single most important prognostic factor of survival in gastric cancer patients. In this study, genes that were upregulated in the lymph node metastases of gastric cancer were identified and may serve as putative novel therapeutic target. METHODS: Complementary DNA (cDNA) microarray analysis and quantitative real-time polymerase chain reaction of primary gastric carcinomas and matched lymph node metastasis were carried out. Immunohistochemistry with anti-SPARC antibodies was performed on large tissue sections of 40 cases with primary gastric carcinoma (20 diffuse, 20 intestinal) and the corresponding lymph node metastases, as well as on tissue microarrays of 152 gastric cancer cases. RESULTS: A cDNA microarray identified SPARC as being upregulated in primary gastric carcinoma tissue and the corresponding lymph node metastasis compared with the nonneoplastic mucosa. SPARC was expressed in fibroblasts and, occasionally, in tumor cells. However, the level of immunoreactivity was particularly strong in stromal cells surrounding the tumor. The level of expression of SPARC, determined by immunohistochemistry, correlated in intestinal-type gastric cancer with the local tumor growth, nodal spread, and tumor stage according to the International Union Against Cancer. CONCLUSIONS: Our study provides transcriptional and translational evidence for the differential expression of SPARC in gastric cancer tissue. On the basis of our observations and those made by others, we hypothesize that SPARC is a promising novel target for the treatment of gastric cancer.

Proteomic and metabolic prediction of response to therapy in gastrointestinal cancers.

Despite substantial improvements in the diagnosis and treatment of many gastrointestinal cancers, particularly colorectal cancer, numerous patients are only diagnosed in advanced stages of disease, which can preclude curative treatment. Screening and early diagnosis of high-risk individuals might be the most promising approach to improve prognosis; however, molecular biomarkers for early diagnosis of most gastrointestinal cancers are not yet available. The prognosis of patients with advanced gastrointestinal cancers has improved through the development of multimodal treatments and the introduction of targeted therapies. Nonetheless, not all patients benefit equally from these treatment approaches, and toxicity can be substantial. The ability to predict whether a patient will respond to therapy early in their treatment for gastrointestinal cancer may be of particular value to stratify and individualize patient treatment strategies. Despite improvement in the understanding of cancer pathogenesis and progression at the molecular level, the molecular changes that underlie treatment response and/or drug resistance are still largely unknown. PET is the first technique to show promise in prediction of response to therapy, and has resulted in promising advancements, particularly in esophageal and gastric cancers. Tissue-based and blood-based molecular biomarkers are still subject to validation. Prediction of response to treatment could ultimately lead to an overall improvement in prognosis.

Frequent loss of TIMP-3 expression in progression of esophageal and gastric adenocarcinomas.

Tissue inhibitor of metalloproteinase 3 (TIMP-3) promoter methylation has been linked to loss of TIMP-3 expression in various cancers. In this study, we analyzed TIMP-3 gene methylation using MethyLight assay and TIMP-3 mRNA expression using reverse transcription-polymerase chain reaction analysis in 22 esophageal cancers, 27 gastric carcinomas, and 7 cancer cell lines. We also analyzed TIMP-3 protein expression by immunohistochemistry and its association with clinicopathological characteristics in two cohorts of gastric cancer comprising a total of 347 patients. The TIMP-3 gene was more commonly methylated in adenocarcinomas of the esophagus (9/13) and stomach (9/15) than in the corresponding nonneoplastic mucosa of the esophagus (1/8; P = .024) and stomach (2/14; P = .021). In gastric cancer patients, TIMP-3 was decreased in a diffuse-type gastric cancer and in cancers with poor differentiation and was associated with poor survival (P = .04). In summary, we observed frequent TIMP-3 promoter methylation in adenocarcinomas of the esophagus and stomach and the loss of TIMP-3 expression seems to be of clinical and prognostic relevance in these cancers.

Ancient DNA from the first European farmers in 7500-year-old Neolithic sites.

The ancestry of modern Europeans is a subject of debate among geneticists, archaeologists, and anthropologists. A crucial question is the extent to which Europeans are descended from the first European farmers in the Neolithic Age 7500 years ago or from Paleolithic hunter-gatherers who were present in Europe since 40,000 years ago. Here we present an analysis of ancient DNA from early European farmers. We successfully extracted and sequenced intact stretches of maternally inherited mitochondrial DNA (mtDNA) from 24 out of 57 Neolithic skeletons from various locations in Germany, Austria, and Hungary. We found that 25% of the Neolithic farmers had one characteristic mtDNA type and that this type formerly was widespread among Neolithic farmers in Central Europe. Europeans today have a 150-times lower frequency (0.2%) of this mtDNA type, revealing that these first Neolithic farmers did not have a strong genetic influence on modern European female lineages. Our finding lends weight to a proposed Paleolithic ancestry for modern Europeans.