Dapper, Dishevelled-associated antagonist of β-catenin (DACT), is involved in Xenopus embryonic development. Human DACT2 is localized on chromosome 6q27, a region of frequent loss of heterozygosity (LOH) in human cancers. However, the function and regulation of DACT2 in human lung cancer remain unclear. DNA sequencing, methylation-specific PCR (MSP), semi-quantitative RT-PCR, western blotting, and xenograft models were employed in this study. Eight lung cancer cell lines, 106 cases of primary lung cancer, four specimens of normal lung from patients without cancer, and 99 blood samples from healthy individuals were examined. We found that while there was no SNP related to lung cancer, the DACT2 promoter region is frequently methylated in human lung cancer. DACT2 is silenced by promoter region hypermethylation and re-expressed by 5-aza-2′-deoxyazacytidine treatment of lung cancer cell lines. Methylation of DACT2 was associated with poor differentiation of lung cancer. Loss of DACT2 expression was associated with promoter region hypermethylation in primary lung cancer, and was associated with increased β-catenin expression. Restoration of DACT2 expression suppressed tumour proliferation both in vitro and in vivo. DACT2 expression was down-regulated by siRNA knockdown in H727 cells. DACT2 inhibited T-cell factor/lymphoid enhancer factor (TCF/LEF) and its downstream genes. In conclusion, DACT2 methylation is a potential lung cancer detection marker. DACT2 is regulated by promoter region hypermethylation. DACT2 inhibits lung cancer proliferation by suppressing the Wnt signalling pathway in lung cancer.
DACT2; chromosome 6q27; SNP; DNA methylation; epigenetics; lung cancer
Pancreatic carcinomas with acinar differentiation, including acinar cell carcinoma, pancreatoblastoma, and carcinomas with mixed differentiation, are distinct pancreatic neoplasms with poor prognosis. Although recent whole exome sequencing analyses have defined the somatic mutations that characterize the other major neoplasms of the pancreas, the molecular alterations underlying pancreatic carcinomas with acinar differentiation remain largely unknown. In the current study, we sequenced the exomes of 23 surgically resected pancreatic carcinomas with acinar differentiation. These analyses revealed a relatively large number of genetic alterations at both the individual base pair and chromosomal levels. There was an average of 119 somatic mutations per carcinoma. When three outliers were excluded, there was an average of 64 somatic mutations per tumor (range 12–189). The mean fractional allelic loss (FAL) was 0.27 (range 0–0.89) and heterogeneity at the chromosome level was confirmed in selected cases using fluorescent in situ hybridization (FISH). No gene was mutated in >30% of the cancers. Genes altered in other neoplasms of the pancreas were occasionally targeted in carcinomas with acinar differentiation; SMAD4 was mutated in six tumors (26%), TP53 in three (13%), GNAS in two (9%), RNF43 in one (4%) and MEN1 in one tumor (4%). Somatic mutations were identified in genes in which constitutional alterations are associated with familial pancreatic ductal adenocarcinoma, such as ATM, BRCA2, and PALB2 (one tumor each), as well as in genes altered in extra-pancreatic neoplasms, such as JAK1 in four tumors (17%) BRAF in three (13%), RB1 in three (13%), APC in two (9%), PTEN in two (9%), ARID1A in two (9%), MLL3 in two (9%), and BAP1 in one (4%). Perhaps most importantly, we found that more than a third of these carcinomas have potentially targetable genetic alterations including mutations in BRCA2, PALB2, ATM, BAP1, BRAF and JAK1.
pancreas; carcinoma; acinar cell carcinoma; genetics; sequencing; pancreatoblastoma
Nuclear localization of non-phosphorylated, active β-catenin is a measure of Wnt pathway activation and is associated with adverse outcome in patients with acute myeloid leukemia (AML). While genetic alterations of the Wnt pathway are infrequent in AML, inhibitors of this pathway are silenced by promoter methylation in other malignanices. Leukemia cell lines were examined for Wnt pathway inhibitor methylation and total β-catenin levels, and had frequent methylation of Wnt inhibitors and upregulated β-catenin by Western blot and immunofluorescence. One hundred sixty-nine AML samples were examined for methylation of Wnt inhibitor genes. Diagnostic samples from 72 patients with normal cytogenetics who received standard high-dose induction chemotherapy were evaluated for associations between methylation and event-free or overall survival. Extensive methylation of Wnt pathway inhibitor genes was observed in cell lines, and 89% of primary AML samples had at least one methylated gene: DKK1 (16%), DKK3 (8%), RUNX3 (27%), sFRP1 (34%), sFRP2 (66%), sFRP4 (9%), sFRP5 (54%), SOX17 (29%), and WIF1 (32%). In contrast to epithelial tumors, methylation of APC (2%) and RASSF1A (0%) was rare. In patients with AML with normal cytogenetics, sFRP2 and sFRP5 methylation at the time of diagnosis was associated with an increased risk of relapse, and sFRP2 methylation was associated with an increased risk for death. In patients with AML: (a) there is a high frequency of Wnt pathway inhibitor methylation; (b) Wnt pathway inhibitor methylation is distinct from that observed in epithelial malignancies; and (c) methylation of sFRP2 and sFRP5 may predict adverse clinical outcome in patients with normal karyotype AML.
Cytogenetics; DNA methylation; epigenetics; leukemia; Wnt pathway
Human Dachshund homologue 1 (DACH1) is a major component of the Retinal Determination Gene Network. Loss of DACH1 expression was found in breast, prostate, lung, endometrial, colorectal and hepatocellular carcinoma. To explore the expression, regulation and function of DACH1 in human esophageal cancer, 11 esophageal cancer cell lines, 10 cases of normal esophageal mucosa, 51 cases of different grades of dysplasia and 104 cases of primary esophageal squamous cancer were employed. Methylation specific PCR, immunohistochemistry, western blot, flow cytometry, small interfering RNAs, colony formation techniques and xenograft mice model were used. We found that DACH1 expression was regulated by promoter region hypermethylation in esophageal cancer cell lines. 18.8% (6 of 32) of grade 1, 42.1% (8 of 19) of grade 2 and grade 3 dysplasia (ED2,3), and 61.5% (64 of 104) of esophageal cancer were methylated, but no methylation was found in 10 cases of normal esophageal mucosa. The methylation was increased in progression tendency during esophageal carcinogenesis (P<0.01). DACH1 methylation was associated with poor differentiation (P<0.05) and late tumor stage (P<0.05). Restoration of DACH1 expression inhibited cell growth and activated TGF-β signaling in KYSE150 and KYSE510 cells. DACH1 suppressed human esophageal cancer cell tumor growth in xenograft mice. In conclusion, DACH1 is frequently methylated in human esophageal cancer and methylation of DACH1 is involved in the early stage of esophageal carcinogenesis. DACH1 expression is regulated by promoter region hypermethylation. DACH1 suppresses esophageal cancer growth by activating TGF-β signaling.
DACT2 (Dapper, Dishevelled-associated antagonist of β-catenin homolog 2) is a member of the DACT family involved in the regulation of embryonic development. Human DACT2 is localized on 6q27, a region of frequent loss of heterozygosity in human cancers. However, the regulation of DACT2 expression and function in hepatocellular carcinoma (HCC) remains unclear. In this study, genetic and epigenetic changes of DACT2 were analyzed in HCC cell lines and primary cancer. We found no single-nucleotide polymorphism (SNP) associated with HCC. Promoter region methylation was correlated with loss or reduction of DACT2 expression, and restoration of DACT2 expression was induced by 5-aza-2’-deoxycytidine (5-AZA) in HCC cell lines. Promoter region methylation was found in 54.84% of primary HCC. Reduction of DACT2 expression was associated with promoter hypermethylation, and expression of DACT2 was inversely related to β-catenin expression in primary HCC. DACT2 suppressed cell proliferation, induced G2-M arrest in cell lines and inhibited tumor growth in xenograft nude mice. The transcriptional activity of TCF-4 and the expression of Wnt signaling downstream genes were suppressed by DACT2 re-expression and reactivated by depletion of DACT2. In conclusion, DACT2 is frequently methylated in HCC and its expression is regulated by promoter hypermethylation. DACT2 suppresses HCC by inhibiting Wnt signaling in human HCC.
DACT2; hepatocellular carcinoma; epigenetics; DNA methylation; Wnt signaling
Currently, there is no clinically validated test for the prediction of response to tubulin-targeting agents in non-small cell lung cancer (NSCLC). Here, we investigated the significance of nuclear expression of the mitotic checkpoint gene checkpoint with forkhead and ringfinger domains (CHFR) as predictor of response and overall survival (OS) with taxane-based first-line chemotherapy in advanced stage NSCLC.
We studied a cohort of 41 patients (median age 63 years) with advanced NSCLC treated at the Atlanta VAMC between 1999 and 2010. CHFR expression by immunohistochemistry (score 0–4) was correlated with clinical outcome using Chi-Square test and Cox proportional models. A cutoff score of ‘3’ was determined by ROC-analysis for “low” CHFR expression. Results were validated in an additional 20 patients who received taxane based chemotherapy at Emory University Hospital and the Atlanta VAMC.
High expression (score = 4) of CHFR is strongly associated with adverse outcomes: the risk for progressive disease (PD) after first-line chemotherapy with carboplatin-paclitaxel was 52% in patients with CHFR-high vs. only 19% in those with CHFR-low tumors (p=0.033). Median OS was strongly correlated with CHFR expression status (CHFR low: 9.9 months; CHFR high: 6.2 months; p =0.002). After multivariate adjustment, reduced CHFR expression remained a powerful predictor of improved OS (HR 0.24 (95% CI 0.1–0.58, p=0.002). In the validation set, low CHFR expression was associated with higher likelihood of clinical benefit (p=0.03) and improved OS (p=0.038).
CHFR expression is a novel predictive marker of response and OS in NSCLC patients treated with taxane-containing chemotherapy.
This issue of Cancer Discovery features an article that describes distinct epigenetic mechanisms that operate in TMPRSS2–ERG fusion-negative prostate cancers. This finding clarifies molecular features of these TMPRSS2–ERG fusion-negative tumors and may have implications for how to treat this prostate cancer subtype.
Novel insights in the biology of cancer have switched the paradigm of a “one-size-fits-all” cancer treatment to an individualized biology-driven treatment approach. In recent years, a diversity of biomarkers and targeted therapies has been discovered. Although these examples accentuate the promise of personalized cancer treatment, for most cancers and cancer subgroups no biomarkers and effective targeted therapy are available. The great majority of patients still receive unselected standard therapies with no use of their individual molecular characteristics. Better knowledge about the underlying tumor biology will lead the way toward personalized cancer treatment. In this review, we summarize the evidence for a promising cancer biomarker: checkpoint with forkhead and ring finger domains (CHFR). CHFR is a mitotic checkpoint and tumor suppressor gene, which is inactivated in a diverse group of solid malignancies, mostly by promoter CpG island methylation. CHFR inactivation has shown to be an indicator of poor prognosis and sensitivity to taxane-based chemotherapy. Here we summarize the current knowledge of altered CHFR expression in cancer, the impact on tumor biology and implications for personalized cancer treatment.
CHFR promoter methylation; Predictive biomarker; Taxane sensitivity
Epigenetics refers to a heritable change in the pattern of gene expression that is mediated by a mechanism specifically not due to alterations in the primary nucleotide sequence. Well known epigenetic mechanisms encompass DNA methylation, chromatin remodeling (histone modifications) and RNA interference. Functionally, epigenetics provides an extra layer of transcriptional control and plays a crucial role in normal physiological development, as well as in pathological conditions. Aberrant DNA methylation is implicated in immune dysfunction, inflammation and insulin resistance. Epigenetic changes may be responsible for “metabolic memory” and development of micro- and macrovascular complications of diabetes. MicroRNAs are critical in the maintenance of glomerular homeostasis and hence RNA interference may be important in the progression of renal disease. Recent studies have shown that epigenetic modifications orchestrate the epithelial-mesenchymal transition and eventually fibrosis of the renal tissue. Oxidative stress, inflammation, hyperhomocysteinemia and uremic toxins could induce epimutations in chronic kidney disease. Epigenetic alterations are associated with inflammation and cardiovascular disease in patients with chronic kidney disease. Reversible nature of the epigenetic changes gives an unique opportunity to halt or even reverse the disease process through targeted therapeutic strategies.
Phenotypic differences among cancers with the same origin may be associated with chemotherapy response. CHFR silencing associated with DNA methylation has been suggested to be predictive of taxane sensitivity in diverse tumor types. However, the use of microsatellite instability (MSI:unstable--MSS:stable) as a predictive marker for therapeutic effect has had conflicting results. We examined these molecular alterations as predictors of chemotherapy sensitivity in colorectal cancer (CRC). Differential sensitivity to docetaxel and gemcitabine was compared to potential predictive biomarkers CHFR methylation and MSI status. Cell lines that were MSI-H/CHFR-methylated, MSS/CHFR-methylated, and MSS/CHFR-unmethylated were assessed for in vivo sensitivity of CRC cell line xenografts to docetaxel and/or gemcitabine. We observed increased sensitivity in vitro to gemcitabine in cell lines with MSI and docetaxel in cell lines with CHFR inactivation via DNA methylation. In vivo treatment of human xenografts confirmed differential sensitivity, with the MSI-H/CHFR-methylated line RKO having tumor growth inhibition to each agent, and at least additive tumor growth inhibition with combination therapy. The MSS-CHFR-unmethylated line, CACO2, was resistant to single and combination therapy, while COLO205, the MSS/CHFR-methylated line, showed tumor growth inhibition with docetaxel, but not gemcitabine, therapy. CHFR methylation in CRC cell lines predicted for sensitivity in vitro and in vivo to docetaxel, while MSI-H cell lines were more sensitive to gemcitabine. These data suggest that a subset of CRC patients would be selectively sensitive to a novel combination of gemcitabine and docetaxel, and are the basis for an ongoing clinical trial of this combination in a biomarker-selected patient population.
MSI; CHFR; colorectal cancer; methylation; taxanes; gemcitabine
Barrett’s esophagus is a premalignant condition that is a risk factor for the development of esophageal adenocarcinoma, a disease whose incidence is rapidly increasing. Because aspirin and other nonsteroidal antiinflammatory drugs, such as celecoxib, may decrease the risk of developing esophageal cancer, we investigated the effect of long-term administration of celecoxib in patients with Barrett’s esophagus with dysplasia.
Chemoprevention for Barrett’s Esophagus Trial (CBET) is a phase IIb multicenter randomized placebo-controlled trial of celecoxib in patients with Barrett’s esophagus and low- or high-grade dysplasia. Patients were randomly assigned to treatment with 200 mg of celecoxib or placebo, both administered orally twice daily, and then stratified by grade of dysplasia. The primary outcome was the change from baseline to 48 weeks of treatment in the proportion of biopsy samples with dysplasia between the celecoxib and placebo arms. Secondary and tertiary outcomes included evaluation of changes in histology and expression levels of relevant biomarkers. All statistical tests were two-sided.
From April 1, 2000, through June 30, 2003, 222 patients were registered into CBET, and 100 of them with low- or high-grade Barrett’s dysplasia were randomly assigned to treatment (49 to celecoxib and 51 to placebo). After 48 weeks of treatment, no difference was observed in the median change in the proportion of biopsy samples with dysplasia or cancer between treatment groups in either the low-grade (median change with celecoxib = − 0.09, interquartile range [IQR] = − 0.32 to 0.14 and with placebo = − 0.07, IQR = − 0.26 to 0.12; P = .64) or high-grade (median change with celecoxib = 0.12, IQR = − 0.31 to 0.55, and with placebo = 0.02, IQR = − 0.24 to 0.28; P = .88) stratum. No statistically significant differences in total surface area of the Barrett’s esophagus; in prostaglandin levels; in cyclooxygenase-1/2 mRNA levels; or in methylation of tumor suppressor genes p16, adenomatous polyposis coli, and E-cadherin were found with celecoxib compared with placebo.
Administration of 200 mg of celecoxib twice daily for 48 weeks of treatment does not appear to prevent progression of Barrett’s dysplasia to cancer.
To explore the epigenetic changes and the function of TFPI-2 in esophageal cancer.
Materials & methods
Nine esophageal cancer cell lines, nine normal esophageal mucosa, 60 esophageal dysplasia and 106 advanced esophageal cancer samples were included in this study. TFPI-2 methylation was examined by methylation-specific PCR. TFPI-2 expression was evaluated by immunohistochemistry in tissue samples. The effect of TFPI-2 on proliferation, apoptosis, invasion and migration was analyzed by colony formation assay, western blot assay, transwell assay and flow cytometric analysis.
TFPI-2 expression was regulated by promoter region hypermethylation in human esophageal cancer cell lines, and TFPI-2 expression is inversely correlated with methylation in primary cancer. Methylation was found in 28.2, 33.3 and 33.3% of grade 1, 2 and 3 esophageal dysplasia, and 67% of primary esophageal cancer, but no methylation was found in normal mucosa. Methylation is significantly related to tumor differentiation. Inhibition of invasion, migration, colony formation and proliferation, and induction of apoptosis occurred with the restoration of TFPI-2 expression in the KYSE70 cell line.
TFPI-2 is frequently methylated in esophageal cancer with a progression tendency. TFPI-2 is a potential tumor suppressor in esophageal cancer.
carcinogenesis; DNA methylation; dysplasia; esophageal cancer; TFPI-2
Gene expression profile (GEP) testing is a relatively new technology that offers the potential of personalized medicine to patients, yet little is known about its adoption into routine practice. One of the first commercially available GEP tests, a 21-gene profile, was developed to estimate the benefit of adjuvant chemotherapy for hormone receptor–positive breast cancer (HR-positive BC).
Patients and Methods
By using a prospective registry data set outlining the routine care provided to women diagnosed from 2006 to 2008 with HR-positive BC at 17 comprehensive and community-based cancer centers, we assessed GEP test adoption and the association between testing and chemotherapy use.
Of 7,375 women, 20.4% had GEP testing and 50.2% received chemotherapy. Over time, testing increased (14.7% in 2006 to 27.5% in 2008; P < .01) and use of chemotherapy decreased (53.9% in 2006 to 47.0% in 2008; P < .01). Characteristics independently associated with lower odds of testing included African American versus white race (odds ratio [OR], 0.70; 95% CI, 0.54 to 0.92) and high school or less versus more than high school education (OR, 0.63; 95% CI, 0.52 to 0.76). Overall, testing was associated with lower odds of chemotherapy use (OR, 0.70; 95% CI, 0.62 to 0.80). Stratified analyses demonstrated that for small, node-negative cancers, testing was associated with higher odds of chemotherapy use (OR, 11.13; 95% CI, 5.39 to 22.99), whereas for node-positive and large node-negative cancers, testing was associated with lower odds of chemotherapy use (OR, 0.11; 95% CI, 0.07 to 0.17).
There has been a progressive increase in use of this GEP test and an associated shift in the characteristics of and overall reduction in the proportion of women with HR-positive BC receiving adjuvant chemotherapy.
To evaluate the methylation state of 31 genes in sputum as biomarkers in an expanded nested, case-control study from the Colorado Cohort and to assess the replication of results from the most promising genes in an independent case-control study of asymptomatic Stage I lung cancer patients from New Mexico.
Cases and controls from Colorado and New Mexico were interrogated for methylation of up to 31 genes using nested, methylation specific PCR. Individual genes and methylation indices were used to assess the association between methylation and lung cancer with logistic regression modeling.
Seventeen genes with odds ratios of 1.4 – 3.6 were identified and selected for replication in the New Mexico study. Overall, the direction of effects seen in New Mexico was similar to Colorado with the largest increase in case discrimination (odds ratios, 3.2 – 4.2) seen for the PAX5α, GATA5, and SULF2 genes. ROC curves generated from seven gene panels from Colorado and New Mexico studies showed prediction accuracy of 71% and 77%, respectively. A 22-fold increase in lung cancer risk was seen for a subset of New Mexico cases with five or more genes methylated. Sequence variants associated with lung cancer did not improve the accuracy of this gene methylation panel.
These studies have identified and replicated a panel of methylated genes whose integration with other promising biomarkers could initially identify the highest risk smokers for computed tomography screening for early detection of lung cancer.
gene methylation; sputum; lung cancer; biomarker
The full molecular consequences of oncogene activation during tumorigenesis are not well understood, but several studies have recently linked oncogene activation to epigenetic silencing of specific genes.1,2 Transcriptional repressor Id1 is overexpressed in many malignancies including melanoma, and Id1 targets include tumor suppressor genes TSP1, CDKN2A (p16) and CDKN1A (p21), which are frequently epigenetically silenced in cancer. We confirmed that both TSP1 and CDKN2A have abnormal promoter region DNa methylation in primary melanoma, but the mechanism by which this silencing occurs remains unknown. Here we explore the effects of stable lentiviral Id1 overexpression on the expression of these Id1 target genes in human melanoma cell lines. Overexpressed Id1 was functional and bound transcriptional activator E2A, but did not sequester E2A from gene promoters and repress gene expression. Therefore, these Id1 target genes were resistant to Id1-mediated gene silencing. Our results suggest that Id1 activation may need to occur at discrete stages in cooperation with additional gene dysregulation to repress and induce epigenetic silencing of tumor suppressor genes during melanoma progression.
Id1; thrombospondin; melanoma; DNA methylation; oncogene
In this article we review many important epigenetic changes in early carcinogenesis and discuss the possibility of these alterations being targeted for therapeutic intervention in the future. Both regional DNA methylation and global chromatin packaging are interrelated partners that function in concert to control gene transcription. We first summarize briefly DNA methylation and its role in gene expression. Then, we focus on how the DNA is packaged into chromatin and the tight relationship between chromatin and DNA methylation. A more complete understanding of these key, regulatory events is vital in approaching a more rational drug therapy to various malignancies.
cancer; chromatin modification; DNA methylation; epigenetics
Insufficient dose of dietary methyl groups are associated with a host of conditions ranging from neural tube defects to cancer. On the other hand, it is not certain what effect excess dietary methyl groups could have on cancer. This is especially true for prostate cancer (PCa), a disease that is characterized by increasing DNA methylation changes with increasing grade of the cancer. In this three-part study in animals, we look at (i) the effect of excess methyl donors on the growth rate of PCa in vivo, (ii) the ability of 5-aza-2'-deoxycytidine, a demethylating agent, to demethylate in the presence of excess dietary methyl donors and (iii) the effect of in utero feeding of excess methyl donors to the later onset of PCa. The results show that when mice are fed a dietary excess of methyl donors, we do not see (i) an increase in the growth rate of DU-145 and PC-3 xenografts in vivo, or (ii) interference in the ability of 5-aza-2'-deoxycytidine to demethylate the promoters of Androgen Receptor or Reprimo of PCa xenografts but (iii) a protective effect on the development of higher grades of PCa in the “Hi-myc” mouse model of PCa which were fed the increased methyl donors in utero. We conclude that the impact of dietary methyl donors on PCa progression depends upon the timing of exposure to the dietary agents. When fed before the onset of cancer, i.e. in utero, excess methyl donors can have a protective effect on the progression of cancer.
Epigenetic inactivation of tumor-suppressor and other regulatory genes plays a critical role in carcinogenesis. Transcriptional silencing is often maintained by DNA methyl transferase (DNMT)-mediated hypermethylation of CpG islands in promoter DNA. Nucleoside analogs including azacytidine and decitabine have been used to inhibit DNMT and re-activate genes, and are clinically used. Their shortcomings include a short half-life and a slow onset of action due to required nucleotide incorporation during DNA replication, which may limit clinical utility. It might be useful to begin to identify lead compounds having novel properties, specifically distinct and fast-acting gene desilencing. We previously identified chemicals augmenting gene expression in multiple reporter systems. We now report that a subset of these compounds that includes quinacrine re-expresses epigenetically silenced genes implicated in carcinogenesis. p16, TFPI2, the cadherins E-cadherin and CDH13, and the secreted frizzle-related proteins (SFRPs) SFRP1 and SFRP5 were desilenced in cancer cell lines. These lead compounds were fast-acting: re-expression occurred by 12-24 hours. Reactivation of silenced genes was accompanied by depletion of DNMT1 at the promoters of activated genes and demethylation of DNA. A model compound, 5175328, induced changes more rapidly than decitabine. These gene desilencing agents belonged to a class of acridine compounds, intercalated into DNA, and inhibited DNMT1 activity in vitro. Although to define the mechanism would be outside the scope of this initial report, this class may re-activate silenced genes in part by intercalating into DNA and subsequently inhibiting full DNMT1 activity. Rapid mechanisms for chemical desilencing of methylated genes therefore exist.
cancer; gene methylation; demethylation; DNA-intercalator; quinacrine; DNMT inhibitor; epigenetics; silencing and reactivation of gene expression; small molecule-DNA interactions
Epigenetic alterations are strongly associated with cancer development. We conducted a phase I/II trial of combined epigenetic therapy with azacitidine and entinostat, inhibitors of DNA methylation and histone deacetylation, respectively, in extensively pretreated patients with recurrent metastatic non-small cell lung cancer. This therapy is well tolerated, and objective responses were observed, including a complete response and a partial response in a patient who remains alive and without disease progression approximately 2 years after completing protocol therapy. Median survival in the entire cohort was 6.4 months (95% CI: 3.8–9.2), comparing favorably with existing therapeutic options. Demethylation of a set of four epigenetically silenced genes known to be associated with lung cancer was detectable in serial blood samples in these patients, and was associated with improved progression-free (p=0.034) and overall survival (p=0.035). Four of 19 patients had major objective responses to subsequent anti-cancer therapies given immediately following epigenetic therapy.
azacitidine; entinostat; demethylation; histone deacetylase inhibitor
Aberrant promoter region hypermethylation of upstream transcription factors may be responsible for silencing entire anti-neoplastic gene networks. In this study, we explored whether transcription factor coding gene, caudal-related homeobox 2 (CDX2), is silenced by promoter hypermethylation in lung cancer, and examined its potential tumor-suppressive functions. Semi-quantitative RT-PCR showed that four of six lung cancer cell lines exhibited no or weak CDX2 expression. Expression of CDX2 was correlated to CDX2 promoter region methylation status, as determined by methylation-specific PCR (MSP) and bisulfite sequencing. Restoration of CDX2 expression was induced by treatment with demethylating drug 5-aza-2'-deoxycytidine (5-AZA) in lung cancer cell lines. Methylation of CDX2 was common in human primary lung cancer (61 of 110 tumors, 55.45%), but no methylation was found in normal lung tissues. Re-expression of CDX2 suppressed lung cancer cell proliferation and blocked cells in G1 phase. β-catenin/TCF activity and downstream genes expression were inhibited by re-expression of CDX2, and increased by depletion of CDX2. In conclusion, CDX2 is frequently methylated in lung cancer, and expression of CDX2 is regulated by promoter region hypermethylation. CDX2 may serve as a tumor suppressor in lung cancer and inhibits lung cancer cell proliferation by suppressing Wnt signaling.
5-aza-2’-deoxycytidine; CDX2; DNA methylation; Wnt signaling pathway; epigenetics; lung cancer
Early detection and multi-modality curative treatment for pancreatic cancer remain unsatisfactory due to the insufficient understanding of the mechanisms underlying tumor progression. Epigenetic events, including aberrant methylation of tumor suppressor gene promoter regions, may contribute to tumorigenesis involving both the exocrine and endocrine pancreas. Methylation changes of specific gene promoter regions were examined in 48 resected neoplasms of the exocrine and endocrine pancreas, which were obtained as paraffin-embedded tissue samples. The pancreatic neoplasms included acinar cell carcinoma (n=12), adenocarcinoma (n=18), and islet cell tumors (n=18). DNA methylation was determined with a nested methylation-specific PCR (MSP) technique incorporating an initial bisulfite modification of tumor DNA for the promoter regions associated with 14 tumor suppressor genes. In decreasing order, the 6 most frequently methylated genes were: APC 50%, BRCA1 46%, p16INK4a 35%, p15INK4b 35%, RARβ 35%, and p73 33%. Overall, 94% of the tumors had methylation of at least one gene, and methylation of two or more genes was present in 69% of pancreatic tumors. Pancreatic adenocarcinomas had patterns of gene methylation that differed from pancreatic endocrine tumors. These differences were most notable for the APC and hMLH1 genes.
To explore epigenetic regulation and the impact of chemokine CXCL14 on colorectal cancer, 7 colorectal cancer cell lines, 107 cases of primary colorectal cancer, and 10 cases of normal colorectal mucosa were evaluated in this study. Methylation specific PCR (MSP), semi-quantitative reverse-transcription PCR (RT-PCR), cell proliferation assay, colony formation, and transwell assay were performed for the evaluation. Complete methylation and loss of CXCL14 expression were found in 5 colorectal cancer cell lines. Partial methylation and weak expression were found in two cell lines. CXCL14 was methylated in 79.4% (85/107) of primary human colorectal cancer. No methylation was found in 10 cases of normal colorectal mucosa. Restoration of CXCL14 expression was induced by the 5-aza-2′-deoxycytidine (DAC) treatment. The cell viability was reduced and colony formation was inhibited by restoration of CXCL14 expression in HCT116 cells, a colorectal cancer cell line. The number of invasive and migration cells was reduced by CXCL14. The expression of MMP-2, Vimentin, and NF-κB was suppressed, and the expression of E-cadherin and IκB-α was induced by CXCL14. In conclusion, CXCL14 is frequently methylated in human colorectal cancer and promoter region hypermethylation silenced CXCL14 expression in colorectal cancer cells. Restoration of CXCL14 expression suppressed colorectal cancer proliferation. CXCL14 inhibits colorectal cancer migration, invasion, and epithelial-to-mesenchymal transition (EMT) by suppressing NF-κB signaling.
The purpose of this study was to explore epigenetic changes and functions of SOX17 in human lung cancer. Five lung cancer cell lines and 88 primary lung cancer samples were examined in this study. Methylation-specific polymerase chain reaction (MSP), semi-quantitative reverse-transcription PCR, immunohistochemistry, luciferase reporter assays, colony-formation assays, and western blotting were used to analyze methylation changes and functions of SOX17 in lung cancer. SOX17 methylation was found in 60.2% of primary human lung cancer samples, and promoter region methylation of SOX17 silenced its expression. SOX17 methylation was associated with female patients and lung cancer differentiation. Colony-formation assays revealed that SOX17 suppressed lung cancer cell proliferation. Re-expression of SOX17 inhibited Wnt signaling in H23 lung cancer cell line. SOX17 acts as a Wnt signaling inhibitor.
The diagnosis of sessile serrated adenomas (SSAs) is challenging, and there is a great deal of interobserver variability amongst pathologists in differentiating SSAs from hyperplastic polyps (HPPs). The aim of this study was (i) to assess the utility of epigenetic changes such as DNA methylation in differentiating SSAs from HPPs and (ii) to identify common methylation based molecular markers potentially useful for early detection of premalignant neoplastic lesions of gastrointestinal tract. A total of 97 primary patient adenoma samples were obtained from The Johns Hopkins Hospital pathology archive with IRB approval and HIPAA compliance. We analyzed the promoter associated CpG island methylation status of 17 genes using nested multiplex methylation specific PCR (MSP). Methylation of CDX2, hMLH1 and TLR2 was detected in SSAs and SSAs with dysplasia but not in HPPs. A subset of genes including EVL, GATAs (4 and 5), HIN-1, SFRPs (1, 2, 4 and 5), SOX17 and SYNE1 were methylated frequently in all premalignant gastrointestinal adenomas including tubular adenomas, villous adenomas, SSAs and SSAs with dysplasia but infrequently in non-premalignant polyps such as HPPs. Methylation of CDX2, hMLH1 and TLR2 may be of diagnostic utility in differentiating, histologically challenging cases of SSAs from HPPs. Genes such as EVL, GATAs, HIN-1, SFRPs, SOX17 and SYNE1, which are frequently methylated in all types of tested premalignant adenomas, may be useful as biomarkers in stool-based strategies for early detection of these adenomas and CRCs in future.
gastrointestinal adenoma; methylation; sessile serrated; classical