Screen for reactivation of MeCP2 on the inactive X chromosome identifies the BMP/TGF-ß superfamily as a regulator of XIST expression.

Rett syndrome (RS) is a debilitating neurological disorder affecting mostly girls with heterozygous mutations in the gene encoding the methyl-CpG-binding protein MeCP2 on the X chromosome. Because restoration of MeCP2 expression in a mouse model reverses neurologic deficits in adult animals, reactivation of the wild-type copy of MeCP2 on the inactive X chromosome (Xi) presents a therapeutic opportunity in RS. To identify genes involved in MeCP2 silencing, we screened a library of 60,000 shRNAs using a cell line with a MeCP2 reporter on the Xi and found 30 genes clustered in seven functional groups. More than half encoded proteins with known enzymatic activity, and six were members of the bone morphogenetic protein (BMP)/TGF-ß pathway. shRNAs directed against each of these six genes down-regulated X-inactive specific transcript (XIST), a key player in X-chromosome inactivation that encodes an RNA that coats the silent X chromosome, and modulation of regulators of this pathway both in cell culture and in mice demonstrated robust regulation of XIST. Moreover, we show that Rnf12, an X-encoded ubiquitin ligase important for initiation of X-chromosome inactivation and XIST transcription in ES cells, also plays a role in maintenance of the inactive state through regulation of BMP/TGF-ß signaling. Our results identify pharmacologically suitable targets for reactivation of MeCP2 on the Xi and a genetic circuitry that maintains XIST expression and X-chromosome inactivation in differentiated cells.

Subtypes of Barrett's oesophagus and oesophageal adenocarcinoma based on genome-wide methylation analysis.

OBJECTIVE: To identify and characterise DNA methylation subtypes in oesophageal adenocarcinoma (EAC) and its precursor Barrett's oesophagus (BE). DESIGN: We performed genome-wide DNA methylation profiling on samples of non-dysplastic BE from cancer-free patients (n=59), EAC (n=23), normal squamous oesophagus (n=33) and normal fundus (n=9), and identified methylation subtypes using a recursively partitioned mixture model. We assessed genomic alterations for 9 BE and 22 EAC samples with massively parallel sequencing of 243 EAC-associated genes, and we conducted integrative analyses with transcriptome data to identify epigenetically repressed genes. We also carried out in vitro experiments treating EAC cell lines with 5-Aza-2'-Deoxycytidine (5-Aza-dC), short hairpin RNA knockdown and anticancer therapies. RESULTS: We identified and validated four methylation subtypes of EAC and BE. The high methylator subtype (HM) of EAC had the greatest number of activating events in ERBB2 (p<0.05, Student's t-test) and the highest global mutation load (p<0.05, Fisher's exact test). PTPN13 was silenced by aberrant methylation in the HM subtype preferentially and in 57% of EACs overall. In EAC cell lines, 5-Aza-dC treatment restored PTPN13 expression and significantly decreased its promoter methylation in HM cell lines (p<0.05, Welch's t-test). Inhibition of PTPN13 expression in the SK-GT-4 EAC cell line promoted proliferation, colony formation and migration, and increased phosphorylation in ERBB2/EGFR/Src kinase pathways. Finally, EAC cell lines showed subtype-specific responses to topotecan, SN-38 and palbociclib treatment. CONCLUSIONS: We identified and characterised methylator subtypes in BE and EAC. We further demonstrated the biological and clinical relevance of EAC methylator subtypes, which may ultimately help guide clinical management of patients with EAC.

Transposon mutagenesis identifies candidate genes that cooperate with loss of transforming growth factor-beta signaling in mouse intestinal neoplasms.

Colorectal cancer (CRC) results from the accumulation of gene mutations and epigenetic alterations in colon epithelial cells, which promotes CRC formation through deregulating signaling pathways. One of the most commonly deregulated signaling pathways in CRC is the transforming growth factor ß (TGF-ß) pathway. Importantly, the effects of TGF-ß signaling inactivation in CRC are modified by concurrent mutations in the tumor cell, and these concurrent mutations determine the ultimate biological effects of impaired TGF-ß signaling in the tumor. However, many of the mutations that cooperate with the deregulated TGF-ß signaling pathway in CRC remain unknown. Therefore, we sought to identify candidate driver genes that promote the formation of CRC in the setting of TGF-ß signaling inactivation. We performed a forward genetic screen in mice carrying conditionally inactivated alleles of the TGF-ß receptor, type II (Tgfbr2) using Sleeping Beauty (SB) transposon mediated mutagenesis. We used TAPDANCE and Gene-centric statistical methods to identify common insertion sites (CIS) and, thus, candidate tumor suppressor genes and oncogenes within the tumor genome. CIS analysis of multiple neoplasms from these mice identified many candidate Tgfbr2 cooperating genes and the Wnt/ß-catenin, Hippo and MAPK pathways as the most commonly affected pathways. Importantly, the majority of candidate genes were also found to be mutated in human CRC. The SB transposon system provides an unbiased method to identify Tgfbr2 cooperating genes in mouse CRC that are functionally relevant and that may provide further insight into the pathogenesis of human CRC.

Differences in DNA methylation signatures reveal multiple pathways of progression from adenoma to colorectal cancer.

BACKGROUND & AIMS: Genetic and epigenetic alterations contribute to the pathogenesis of colorectal cancer (CRC). There is considerable molecular heterogeneity among colorectal tumors, which appears to arise as polyps progress to cancer. This heterogeneity results in different pathways to tumorigenesis. Although epigenetic and genetic alterations have been detected in conventional tubular adenomas, little is known about how these affect progression to CRC. We compared methylomes of normal colon mucosa, tubular adenomas, and colorectal cancers to determine how epigenetic alterations might contribute to cancer formation. METHODS: We conducted genome-wide array-based studies and comprehensive data analyses of aberrantly methylated loci in 41 normal colon tissue, 42 colon adenomas, and 64 cancers using HumanMethylation450 arrays. RESULTS: We found genome-wide alterations in DNA methylation in the nontumor colon mucosa and cancers. Three classes of cancers and 2 classes of adenomas were identified based on their DNA methylation patterns. The adenomas separated into classes of high-frequency methylation and low-frequency methylation. Within the high-frequency methylation adenoma class a subset of adenomas had mutant KRAS. Additionally, the high-frequency methylation adenoma class had DNA methylation signatures similar to those of cancers with low or intermediate levels of methylation, and the low-frequency methylation adenoma class had methylation signatures similar to that of nontumor colon tissue. The CpG sites that were differentially methylated in these signatures are located in intragenic and intergenic regions. CONCLUSIONS: Genome-wide alterations in DNA methylation occur during early stages of progression of tubular adenomas to cancer. These findings reveal heterogeneity in the pathogenesis of colorectal cancer, even at the adenoma step of the process.

DNA methylation profile in CpG-depleted regions uncovers a high-risk subtype of early-stage colorectal cancer.

BACKGROUND: The current risk stratification system defined by clinicopathological features does not identify the risk of recurrence in early-stage (stage I-II) colorectal cancer (CRC) with sufficient accuracy. We aimed to investigate whether DNA methylation could serve as a novel biomarker for predicting prognosis in early-stage CRC patients. METHODS: We analyzed the genome-wide methylation status of CpG loci using Infinium MethylationEPIC array run on primary tumor tissues and normal mucosa of early-stage CRC patients to identify potential methylation markers for prognosis. The machine-learning approach was applied to construct a DNA methylation-based prognostic classifier for early-stage CRC (MePEC) using the 4 gene methylation markers FAT3, KAZN, TLE4, and DUSP3. The prognostic value of the classifier was evaluated in 2 independent cohorts (n = 438 and 359, respectively). RESULTS: The comprehensive analysis identified an epigenetic subtype with high risk of recurrence based on a group of CpG loci in the CpG-depleted region. In multivariable analysis, the MePEC classifier was independently and statistically significantly associated with time to recurrence in validation cohort 1 (hazard ratio = 2.35, 95% confidence interval = 1.47 to 3.76, P < .001) and cohort 2 (hazard ratio = 3.20, 95% confidence interval = 1.92 to 5.33, P < .001). All results were further confirmed after each cohort was stratified by clinicopathological variables and molecular subtypes. CONCLUSIONS: We demonstrated the prognostic statistical significance of a DNA methylation profile in the CpG-depleted region, which may serve as a valuable source for tumor biomarkers. MePEC could identify an epigenetic subtype with high risk of recurrence and improve the prognostic accuracy of current clinical variables in early-stage CRC.

Elevated EVL Methylation Level in the Normal Colon Mucosa Is a Potential Risk Biomarker for Developing Recurrent Adenomas.

BACKGROUND: Individuals with adenomatous colorectal polyps undergo repeated colonoscopy surveillance to identify and remove metachronous adenomas. However, many patients with adenomas do not develop recurrent adenomas. Better methods to evaluate who benefits from increased surveillance are needed. We evaluated the use of altered EVL methylation as a potential biomarker for risk of recurrent adenomas. METHODS: Patients with ≥1 colonoscopy had EVL methylation (mEVL) measured with an ultra-accurate methylation-specific droplet digital PCR assay on normal colon mucosa. The association between EVL methylation levels and adenoma or colorectal cancer was evaluated using three case/control definitions in three models: unadjusted (model 1), adjusting for baseline characteristics (model 2), and an adjusted model excluding patients with colorectal cancer at baseline (model 3). RESULTS: Between 2001 and 2020, 136 patients were included; 74 healthy patients and 62 patients with a history of colorectal cancer. Older age, never smoking, and baseline colorectal cancer were associated with higher levels of mEVL (P ≤ 0.05). Each log base 10 difference in mEVL was associated with an increased risk of adenoma(s) or cancer at/after baseline for model 1 [OR, 2.64; 95% confidence interval (CI), 1.09-6.36], and adenoma(s) or cancer after baseline for models 1 (OR, 2.01; 95% CI, 1.04-3.90) and model 2 (OR, 3.17; 95% CI, 1.30-7.72). CONCLUSIONS: Our results suggest that EVL methylation level detected in the normal colon mucosa has the potential to be a biomarker for monitoring the risk for recurrent adenomas. IMPACT: These findings support the potential utility of EVL methylation for improving the accuracy for assigning risk for recurrent colorectal adenomas and cancer.

Autoimmune responses to the brain after stroke are associated with worse outcome.

BACKGROUND AND PURPOSE: Immune responses to brain antigens occur after stroke, and experimental studies show that the likelihood of developing a detrimental autoimmune response to these antigens is increased by systemic inflammation at the time of stroke. The aim of this study was to determine if patients who developed infection in the poststroke period would be similarly predisposed to develop autoimmune responses to central nervous system antigens. METHODS: We enrolled 114 patients within 72 hours of ischemic stroke. Clinical and demographic data were obtained, and cellular immune responses to a panel of central nervous system antigens were assessed during the initial week and again at Day 90. Outcome was assessed using the modified Rankin Scale. RESULTS: Patients who developed an infection, especially pneumonia, in the 15 days after stroke were more likely to evidence a Th1(+) response to myelin basic protein and glial fibrillary acidic protein (P=0.019 and P=0.039, respectively) at 90 days after stroke. Further, more robust Th1 responses to myelin basic protein at 90 days were associated with a decreased likelihood of good outcome, even after adjusting for baseline stroke severity and patient age (OR, 0.477; 95% CI, 0.244 to 0.935; P=0.031). CONCLUSIONS: This study demonstrates that immune responses to brain antigens occur after stroke. Although these responses are likely to be an epiphenomenon of ischemic brain injury, the response to myelin basic protein appears to have clinical consequences. The potential role of postischemic autoimmune-mediated brain injury deserves further investigation.

Anamnestic recall of stroke-related deficits: an animal model.

BACKGROUND AND PURPOSE: Anamnestic recall of stroke-related deficits is a common clinical observation, especially during periods of systemic infection. The pathophysiology of this transient re-emergence of neurological dysfunction is unknown. METHODS: Male Lewis rats underwent 3 hours middle cerebral artery occlusion and were treated with lipopolysaccharide or saline at the time of reperfusion. The delayed-type hypersensitivity (DTH) response to myelin basic protein was examined 28 days after middle cerebral artery occlusion. Changes in behavioral outcomes were assessed after DTH testing and repeat administration of lipopolysaccharide or saline at 34 days. At the time of euthanasia (36 days), the immunologic response of splenocytes to myelin basic protein, neuron-specific enolase, and proteolipid protein was determined by enzyme-linked immunospot assay and the number of lymphocytes in the brain determined by immunocytochemistry. RESULTS: Animals treated with lipopolysaccharide at middle cerebral artery occlusion had a greater DTH response to myelin basic protein than animals treated with saline. Among those animals that had fully recovered on a given behavioral test before DTH testing, those treated with lipopolysaccharide at middle cerebral artery occlusion displayed more neurological deterioration after DTH testing and had more CD8(+) lymphocytes within the ischemic core of the brain. Furthermore, the Th1 immune response to brain antigens in the spleen was more robust among those animals that deteriorated after DTH testing and there were more CD4(+) lymphocytes in the penumbral region of animals with a Th1 response to myelin basic protein. CONCLUSIONS: Our data suggest that an immune response to the brain contributes to the phenomenon of anamnestic recall of stroke-related deficits after an infection. The contribution of the immune response to this phenomenon deserves further investigation.

Dysfunctional epigenetic aging of the normal colon and colorectal cancer risk.

BACKGROUND: Chronological age is a prominent risk factor for many types of cancers including colorectal cancer (CRC). Yet, the risk of CRC varies substantially between individuals, even within the same age group, which may reflect heterogeneity in biological tissue aging between people. Epigenetic clocks based on DNA methylation are a useful measure of the biological aging process with the potential to serve as a biomarker of an individual's susceptibility to age-related diseases such as CRC. METHODS: We conducted a genome-wide DNA methylation study on samples of normal colon mucosa (N = 334). Subjects were assigned to three cancer risk groups (low, medium, and high) based on their personal adenoma or cancer history. Using previously established epigenetic clocks (Hannum, Horvath, PhenoAge, and EpiTOC), we estimated the biological age of each sample and assessed for epigenetic age acceleration in the samples by regressing the estimated biological age on the individual's chronological age. We compared the epigenetic age acceleration between different risk groups using a multivariate linear regression model with the adjustment for gender and cell-type fractions for each epigenetic clock. An epigenome-wide association study (EWAS) was performed to identify differential methylation changes associated with CRC risk. RESULTS: Each epigenetic clock was significantly correlated with the chronological age of the subjects, and the Horvath clock exhibited the strongest correlation in all risk groups (r > 0.8, p < 1 × 10-30). The PhenoAge clock (p = 0.0012) revealed epigenetic age deceleration in the high-risk group compared to the low-risk group. CONCLUSIONS: Among the four DNA methylation-based measures of biological age, the Horvath clock is the most accurate for estimating the chronological age of individuals. Individuals with a high risk for CRC have epigenetic age deceleration in their normal colons measured by the PhenoAge clock, which may reflect a dysfunctional epigenetic aging process.

Senescence-associated tissue microenvironment promotes colon cancer formation through the secretory factor GDF15.

The risk of colorectal cancer (CRC) varies between people, and the cellular mechanisms mediating the differences in risk are largely unknown. Senescence has been implicated as a causative cellular mechanism for many diseases, including cancer, and may affect the risk for CRC. Senescent fibroblasts that accumulate in tissues secondary to aging and oxidative stress have been shown to promote cancer formation via a senescence-associated secretory phenotype (SASP). In this study, we assessed the role of senescence and the SASP in CRC formation. Using primary human colon tissue, we found an accumulation of senescent fibroblasts in normal tissues from individuals with advanced adenomas or carcinomas in comparison with individuals with no polyps or CRC. In in vitro and ex vivo model systems, we induced senescence using oxidative stress in colon fibroblasts and demonstrated that the senescent fibroblasts secrete GDF15 as an essential SASP factor that promotes cell proliferation, migration, and invasion in colon adenoma and CRC cell lines as well as primary colon organoids via the MAPK and PI3K signaling pathways. In addition, we observed increased mRNA expression of GDF15 in primary normal colon tissue from people at increased risk for CRC in comparison with average risk individuals. These findings implicate the importance of a senescence-associated tissue microenvironment and the secretory factor GDF15 in promoting CRC formation.

Implications of Epigenetic Drift in Colorectal Neoplasia.

Many normal tissues undergo age-related drift in DNA methylation, providing a quantitative measure of tissue age. Here, we identify and validate 781 CpG islands (CGI) that undergo significant methylomic drift in 232 normal colorectal tissues and show that these CGI continue to drift in neoplasia while retaining significant correlations across samples. However, compared with normal colon, this drift advanced (∼3-4-fold) faster in neoplasia, consistent with increased cell proliferation during neoplastic progression. The observed drift patterns were broadly consistent with modeled adenoma-to-carcinoma sojourn time distributions from colorectal cancer incidence data. These results support the hypothesis that, beginning with the founder premalignant cell, cancer precursors frequently sojourn for decades before turning into cancer, implying that the founder cell typically arises early in life. At least 77% to 89% of the observed drift variance in distal and rectal tumors was explained by stochastic variability associated with neoplastic progression, whereas only 55% of the variance was explained for proximal tumors. However, gene-CGI pairs in the proximal colon that underwent drift were significantly and primarily negatively correlated with cancer gene expression, suggesting that methylomic drift participates in the clonal evolution of colorectal cancer. Methylomic drift advanced in colorectal neoplasia, consistent with extended sojourn time distributions, which accounts for a significant fraction of epigenetic heterogeneity in colorectal cancer. Importantly, these estimated long-duration premalignant sojourn times suggest that early dietary and lifestyle interventions may be more effective than later changes in reducing colorectal cancer incidence. SIGNIFICANCE: These findings present age-related methylomic drift in colorectal neoplasia as evidence that premalignant cells can persist for decades before becoming cancerous.See related commentary by Sapienza, p. 437.

MethyLight droplet digital PCR for detection and absolute quantification of infrequently methylated alleles.

Aberrant DNA methylation is a common epigenetic alteration found in colorectal adenomas and cancers and plays a role in cancer initiation and progression. Aberrantly methylated DNA loci can also be found infrequently present in normal colon tissue, where they seem to have potential to be used as colorectal cancer (CRC) risk biomarkers. However, detection and precise quantification of the infrequent methylation events seen in normal colon is likely beyond the capability of commonly used PCR technologies. To determine the potential for methylated DNA loci as CRC risk biomarkers, we developed MethyLight droplet digital PCR (ddPCR) assays and compared their performance to the widely used conventional MethyLight PCR. Our analyses demonstrated the capacity of MethyLight ddPCR to detect a single methylated NTRK3 allele from among more than 3125 unmethylated alleles, 25-fold more sensitive than conventional MethyLight PCR. The MethyLight ddPCR assay detected as little as 19 and 38 haploid genome equivalents of methylated EVL and methylated NTRK3, respectively, which far exceeded conventional MethyLight PCR (379 haploid genome equivalents for both genes). When assessing methylated EVL levels in CRC tissue samples, MethyLight ddPCR reduced coefficients of variation (CV) to 6-65% of CVs seen with conventional MethyLight PCR. Importantly, we showed the ability of MethyLight ddPCR to detect infrequently methylated EVL alleles in normal colon mucosa samples that could not be detected by conventional MethyLight PCR. This study suggests that the sensitivity and precision of methylation detection by MethyLight ddPCR enhances the potential of methylated alleles for use as CRC risk biomarkers.

Methylated B3GAT2 and ZNF793 Are Potential Detection Biomarkers for Barrett's Esophagus.

BACKGROUND: Barrett's esophagus (BE) is a preneoplastic condition in which normal esophageal squamous epithelium (SQ) is replaced by specialized intestinal metaplasia. It is the presumed precursor for esophageal adenocarcinoma (EAC) as well as the strongest risk factor for this cancer. Unfortunately, many patients with BE go undiagnosed under the current BE screening guidelines. The development of noninvasive and accurate BE detection assays could potentially identify many of these undiagnosed BE patients. METHODS: DNA methylation is a common epigenetic alteration in BE. Therefore, we conducted a genome-wide methylation screen to identify potential BE biomarkers. Samples from SQ (N = 12), stomach (N = 28), and BE (N = 29) were analyzed and methylation levels at over 485,000 CpG sites were compared. Pyrosequencing assays were used to validate the results and MethyLight assays were developed to detect the methylated alleles in endoscopic brushings. RESULTS: We discovered two genes, B3GAT2 and ZNF793, that are aberrantly methylated in BE. Clinical validation studies confirmed B3GAT2 and ZNF793 methylation levels were significantly higher in BE samples (median = 32.5% and 33.1%, respectively) than in control tissues (median = 2.29% and 2.52%, respectively; P < 0.0001 for both genes). Furthermore, gene-specific MethyLight assays could accurately detect BE (P < 0.0001 for both) in endoscopic brushing samples. CONCLUSION: B3GAT2 and ZNF793 are hypermethylated in BE, and the methylation status of these genes can be used to detect BE in tissue samples. IMPACT: These findings support the development of methylated B3GAT2 and ZNF793 as biomarkers for noninvasive assays for the detection of BE.

Altered RECQ Helicase Expression in Sporadic Primary Colorectal Cancers.

Deregulation of DNA repair enzymes occurs in cancers and may create a susceptibility to chemotherapy. Expression levels of DNA repair enzymes have been shown to predict the responsiveness of cancers to certain chemotherapeutic agents. The RECQ helicases repair damaged DNA including damage caused by topoisomerase I inhibitors, such as irinotecan. Altered expression levels of these enzymes in colorectal cancer (CRC) may influence the response of the cancers to irinotecan. Thus, we assessed RECQ helicase (WRN, BLM, RECQL, RECQL4, and RECQL5) expression in primary CRCs, matched normal colon, and CRC cell lines. We found that BLM and RECQL4 mRNA levels are significantly increased in CRC (P = .0011 and P < .0001, respectively), whereas RECQL and RECQL5 are significantly decreased (P = .0103 and P = .0029, respectively). RECQ helicase expression patterns varied between specific molecular subtypes of CRCs. The mRNA and protein expression of the majority of the RECQ helicases was closely correlated, suggesting that altered mRNA expression is the predominant mechanism for deregulated RECQ helicase expression. Immunohistochemistry localized the RECQ helicases to the nucleus. RECQ helicase expression is altered in CRC, suggesting that RECQ helicase expression has potential to identify CRCs that are susceptible to specific chemotherapeutic agents.