Aging related methylation influences the gene expression of key control genes in colorectal cancer and adenoma

AIM: To analyze colorectal carcinogenesis and age-related DNA methylation alterations of gene sequences associated with epigenetic clock CpG sites. METHODS: In silico DNA methylation analysis of 353 epigenetic clock CpG sites published by Steve Horvath was performed using methylation array data for a set of 123 colonic tissue samples [64 colorectal cancer (CRC), 42 adenoma, 17 normal; GEO accession number: GSE48684]. Among the differentially methylated age-related genes, secreted frizzled related protein 1 (SFRP1) promoter methylation was further investigated in colonic tissue from 8 healthy adults, 19 normal children, 20 adenoma and 8 CRC patients using bisulfite-specific PCR followed by methylation-specific high resolution melting (MS-HRM) analysis. mRNA expression of age-related "epigenetic clock" genes was studied using Affymetrix HGU133 Plus2.0 whole transcriptome data of 153 colonic biopsy samples (49 healthy adult, 49 adenoma, 49 CRC, 6 healthy children) (GEO accession numbers: GSE37364, GSE10714, GSE4183, GSE37267). Whole promoter methylation analysis of genes showing inverse DNA methylation-gene expression data was performed on 30 colonic samples using methyl capture sequencing. RESULTS: Fifty-seven age-related CpG sites including hypermethylated PPP1R16B, SFRP1, SYNE1 and hypomethylated MGP, PIPOX were differentially methylated between CRC and normal tissues (P 0.5). The change of expression for several genes including SYNE1, CLEC3B, LTBP3 and SFRP1, followed the same pattern in aging and carcinogenesis, though not for all genes (e.g., MGP). CONCLUSION: Several age-related DNA methylation alterations can be observed during CRC development and progression affecting the mRNA expression of certain CRC- and adenoma-related key control genes

Szabad DNS-alapú vastagbéldaganat-szűrés perifériás vérből: a metilált szeptin-9 génmarker lehetőségei = Free circulating DNA based colorectal cancer screening from peripheral blood: the possibility of the methylated septin 9 gene marker

A DNS-metiláció szerepet játszik a daganatképződés korai szakaszában. Kimutatása szövetből, székletből és perifériás vérből egyaránt lehetséges. A szeptin-9 érzékeny metilációs jelző, amelyet több daganatban, mint emlő- és petefészekrákban, valamint neurológiai és hematológiai betegségekben is vizsgáltak. A szeptinproteinek fontos szerephez jutnak a sejtváz organizációjától az embrionális mintázat kialakulásáig. Napjainkban intenzív kutatások folynak a szeptin-9 fokozott metilációja és a vastagbélrák kialakulása közötti összefüggés felderítéseire. | DNA methylation acts in early tumorigenesis. Its detection is possible either from tissue, stool or peripheral blood. Septin 9 is a sensitive methylation marker, which has been studied in several cancers such as breast and ovarian tumors and in neurological or hematological diseases. Septin proteins have an important role from cytoskeleton organisation to development of embryonal pattern. Nowadays intensive researches are going on about the relation between the septin 9 gene hypermethylation and colorectal cancer development

Az öregedés mikroszkópos és molekuláris jelei a vastagbélben, valamint ezek lehetséges szerepe az időskori vastagbélrák kialakulásában = Age-related microscopic and molecular changes of the human colon, and their role in the development of colorectal cancer in elderly people

Az öregedés emésztőrendszeri hatásainak vizsgálata, molekuláris hátterének megismerése és bizonyos betegségek (mint például az idősek körében gyakrabban előforduló sporadikus vastagbélrák) kialakulásával való kapcsolatának feltérképezése új és ígéretes területe a molekuláris gasztroenterológiának. Ismert tény, hogy az emésztőrendszerben az öregedés során kialakuló molekuláris változások egy része (például a DNS-metiláció, a telomerrövidülés) a vastagbélrák bizonyos típusaiban is azonosítható. A sporadikus vastagbélrák epidemiológiai és molekuláris tulajdonságait ismerve továbbra is nyitott kérdés, hogy pontosan milyen molekuláris mechanizmusok, genetikai vagy génexpressziós szintű, illetve epigenetikai változások magyarázzák, hogy 45–50 éves kor felett a sporadikus vastagbélrákos megbetegedések száma ugrásszerűen megnő, és ezek vajon hogyan hozhatók összefüggésbe az öregedés mechanizmusával. Hosszú távon a megelőzés és a korszerű kezelés szempontjából is kiemelkedő jelentőségű lehet a megismert folyamatok célzott befolyásolása. Dolgozatunk az öregedés során a vastagbélben kialakuló mikroszkópos és molekuláris változásokat foglalja össze, és ezek lehetséges szerepét mutatja be az időskori sporadikus vastagbélrák kialakulásában. | The gastrointestinal effect of aging, the recognition of its molecular background and the mapping its connections with several diseases like sporadic colorectal cancer of elder people are a new and promising area of molecular gastroenterology. Nowadays, it is a well-known fact that some age-related molecular changes (e.g.: DNA methylation, telomere shortening) can be detected in several types of colorectal cancers. The known epidemiologic and molecular biologic features of sporadic colorectal cancer are not enough to explain the genetic, gene expression or epigenetic changes that may be involved in the increase of the disease over 45–50 age years. The connections of these alterations to the process of aging are also unclear. The understanding and custom-tailored modification of these mechanisms are of great clinical importance regarding of prevention and modern therapeutic strategies. In this review, we aimed to summarize the age-related microscopic and molecular changes of the human colon, as well as their role in the development of colorectal cancer of the elder people

A csontvelői eredetű őssejtek megjelenése az ép vastagbélhámban és a gyulladást követő hámregenerációban = Appearing of bone marrow derived stem cells in healthy- and regenerating colonic epithelium

A vastagbél gyulladásos folyamataiban kialakuló regenerációban csontvelői eredetű mesenchymalis őssejtek (MSC) is részt vehetnek. Ezek a sejtek a vastagbél hámrétegébe vándorolnak, ahol hámsejtté érnek, vagy megtartva őssejttulajdonságaikat, utódsejteket hoznak létre. Az MSC-sejtek feltehetően a lymphoid aggregátumok területén lépnek ki az erekből, majd innen vándorolnak a környező cryptákba. Vándorlásuk során a szöveti mikrokörnyezet hatására megváltoznak. Célkitűzés: Munkánkban a csontvelői őssejtek vastagbélhámba épülésének jellegzetességeit vizsgáltuk egészséges és gyulladásos mintákban. Megvizsgáltuk a lymphoid aggregátumok szerepét is a hámréteg regenerációjában. Anyagok és módszerek: Tanulmányunkhoz férfi donor csontvelővel átültetett nők vastagbeléből származó biopsziás mintákat használtunk fel. A mintákon először nemi kromoszómákat mutattunk ki fluoreszcens in situ hibridizációval (FISH), majd a metszeteket digitálisan archiváltuk, ez után CD45 és cytokeratin (CK) kettős fluoreszcens immunhisztokémiai (IF) jelölést végeztünk. A metszeteket ismételten digitalizáltuk, a mintákat virtuális mikroszkóp (Mirax Viewer) segítségével párhuzamosan értékeltük. Eredmények: A crypták hámrétegébe beépülő Y-FISH-pozitív, CD45-negatív potenciális MSC-sejtek száma lényegességi szintet meghaladó (szignifikáns) emelkedést (p = 0,002; p = 0,003) mutatott (1,075%) a lymphoid aggregátumok környezetében, mind a normális vastagbélszövet (0,027%), mind az enyhe gyulladásos (0,045%) mintákhoz képest. Következtetés: Adataink arra utalnak, hogy a szöveti őssejtek kapacitása elégséges a kisfokú gyulladásos folyamatok regenerációjához. Az aspecifikus gyulladás esetében azonban a hámrétegbe beépülő MSC-sejtek száma megnő, ezek funkcionáló hámsejtté érése gyakoribb, mint az utódsejtek létrehozása. Eredményeink azt mutatják, hogy a lymphoid aggregátumok részt vesznek a hám regenerációs folyamataiban. | In the process of regeneration following colon inflammation mesenchymal stem cells (MSC) of bone marrow origin may also take part besides their local counterparts. These cells migrate in the colon epithelium where they may contribute to epithelial regeneration or form progeny for keeping up local stem cell pool. MSC cells probably leave circulation around lymphoid aggregates to then migrate into nearby crypts. During migration they change their phenotype upon the influence of local microenvironment. Aims: In this study epithelial migration and transition of bone marrow stem cells were examined. Samples from normal healthy individuals and from aspecific inflammation were used. The possible role of lymphoid aggregates in the epithelial regeneration was also studied. Materials and methods: Samples of normal colon (2) and those showing mild aspecific colitis (3) from female patients who were initially transplanted with male bone marrow were studied. First we detected gender chromosomes with fluorescent in situ hybridization (FISH) and the samples were archived with digital scanning. Then CD45 and cytokeratin (CK) double immunofluorescent reactions (IF) were made followed by digitalization again. Digitalized samples were estimated simultaneously with virtual microscopy (Mirax Viewer). Results: Significant elevation of CD45 negative/Y-FISH positive potential MSCs were found in crypts locating to the neighborhood of lymphoid aggregates (1,075%) compared with both normal (0,027%, p = 0,002) and mild colitis (0,045%, p = 0,004) samples. Conclusion: Local stem cells probably have enough regeneration capacity in case of minor inflammation. However, in aspecific inflammation the number of MSCs contributing to epithelial regeneration was elevated, suggesting their facilitated contribution to the repair process with less probable forming of local stem cell progeny

Az ösztrogének lehetséges szerepe a vastagbéldaganatok kialakulásában = Potential role of estrogens in colorectal tumour development

Absztrakt: A colorectalis carcinoma (CRC) az egyik leggyakrabban előforduló daganatos megbetegedés világszerte. A sporadikus vastagbélrák incidenciája ötvenéves kor alatt alacsonyabb, majd az életkor előrehaladtával nő, továbbá jellegzetes klinikai, lokalizáció szerinti és molekuláris eltérést mutathat a nők és a férfiak között. Epidemiológiai és molekuláris biológiai kutatások eredményei szerint az ösztradiol (E2) által szabályozott jelútrendszer meghatározó szerepet játszik a CRC kialakulásában és prognosztikájában, döntően a vastagbélhámban domináns ösztrogénreceptor-bétán (ERβ) keresztül. Az ösztradiol emésztőrendszeri hatásai igen sokrétűek, az ép és tumoros vastagbélhámsejtekre gyakorolt hatását in vitro és in vivo vizsgálatok egyaránt igazolták. Az ösztrogénreceptor-alfával (ERα) ellentétben az ERβ aktivációja a sejtosztódást gátolja és az apoptózist fokozza, a béta-receptor kifejeződése ugyanakkor mind az élettani öregedés, mind a vastagbél kórállapotaiban megváltozhat. Az ösztradiol ERβ által közvetített daganatellenes hatása a sejtproliferáció gátlása, az apoptózis serkentése, az áttétképzés gátlása és gyulladáscsökkentő hatása révén valósulhat meg. Sejtkultúra- és állatkísérletes kutatások eredményei alapján az ösztrogénreceptor-bétára szelektíven ható receptormodulátorok (szelektív ösztrogénreceptor-modulátor [SERM]) és a fitoösztrogének új, hozzáadott kezelési lehetőséget jelenthetnek az idült gyulladással és a kóros sejtproliferációval jellemezhető colorectalis megbetegedésekben. Orv Hetil. 2020; 161(14): 532–543. | Abstract: Colorectal cancer (CRC) is one of the most common types of cancers worldwide. The incidence of sporadic CRC is lower in individuals below 50 years and increases with age, furthermore, it shows typical clinical, macroscopic and molecular differences between females and males. According to the results of epidemiological and molecular biology studies, the estradiol-regulating signaling pathway plays an important role in the development and prognosis of CRC, predominantly through estrogen receptor beta (ERβ), which is dominant in the colonic epithelium. Estradiol has multiple gastrointestinal effects, which were confirmed by in vitro and in vivo studies on histologically intact and cancerous cells as well. In contrast to estrogen receptor alpha (ERα), the activation of ERβ inhibits cell proliferation and enhances apoptosis, nevertheless, the expression of estrogen receptor beta can change both during physiological ageing and in colorectal disorders. The ERβ-mediated antitumour effects of estradiol may be exerted through inhibition of cell proliferation, stimulation of apoptosis, inhibition of metastasis formation and its anti-inflammatory activity. Based on the results of cell culture and animal studies, selective modulators of estrogen receptor beta (selective estrogen receptor modulator [SERM]) and phytoestrogens can be new, additional therapeutic options in the treatment of colorectal diseases characterized by chronic inflammation and uncontrolled cell proliferation. Orv Hetil. 2020; 161(14): 532–543

Sporadic Colorectal Cancer Development Shows Rejuvenescence Regarding Epithelial Proliferation and Apoptosis

Background and Aims: Sporadic colorectal cancer (CRC) development is a sequential process showing age-dependency, uncontrolled epithelial proliferation and decreased apoptosis. During juvenile growth cellular proliferation and apoptosis are well balanced, which may be perturbed upon aging. Our aim was to correlate proliferative and apoptotic activities in aging human colonic epithelium and colorectal cancer. We also tested the underlying molecular biology concerning the proliferation- and apoptosis-regulating gene expression alterations. Materials and Methods: Colorectal biopsies from healthy children (n1 = 14), healthy adults (n2 = 10), adult adenomas (n3 = 10) and CRCs (n4 = 10) in adults were tested for Ki-67 immunohistochemistry and TUNEL apoptosis assay. Mitosis- and apoptosis-related gene expression was also studied in healthy children (n1 = 6), adult (n2 = 41) samples and in CRC (n3 = 34) in HGU133plus2.0 microarray platform. Measured alterations were confirmed with RT-PCR both on dependent and independent sample sets (n1=6, n2=6, n3 = 6). Results: Mitotic index (MI) was significantly higher (p,0.05) in intact juvenile (MI = 0.3360.06) and CRC samples (MI = 0.4260.10) compared to healthy adult samples (MI = 0.1560.06). In contrast, apoptotic index (AI) was decreased in children (0.1360.06) and significantly lower in cancer (0.0660.03) compared to healthy adult samples (0.1760.05). Eight proliferation- (e.g. MKI67, CCNE1) and 11 apoptosis-associated genes (e.g. TNFSF10, IFI6) had altered mRNA expression both in the course of normal aging and carcinogenesis, mainly inducing proliferation and reducing apoptosis compared to healthy adults. Eight proliferation-associated genes including CCND1, CDK1, CDK6 and 26 apoptosis-regulating genes (e.g. SOCS3) were differently expressed between juvenile and cancer groups mostly supporting the pronounced cell growth in CRC. Conclusion: Colorectal samples from children and CRC patients can be characterized by similarly increased proliferative and decreased apoptotic activities compared to healthy colonic samples from adults. Therefore, cell kinetic alterations during colorectal cancer development show uncontrolled rejuvenescence as opposed to the controlled cell growth in juvenile colonic epithelium

Promoter Hypermethylation-Related Reduced Somatostatin Production Promotes Uncontrolled Cell Proliferation in Colorectal Cancer.

BACKGROUND: Somatostatin (SST) has anti-proliferative and pro-apoptotic effects. Our aims were to analyze and compare the SST expression during normal aging and colorectal carcinogenesis at mRNA and protein levels. Furthermore, we tested the methylation status of SST in biopsy samples, and the cell growth inhibitory effect of the SST analogue octreotide in human colorectal adenocarcinoma cell line. METHODS: Colonic samples were collected from healthy children (n1 = 6), healthy adults (n2 = 41) and colorectal cancer patients (CRCs) (n3 = 34) for SST mRNA expression analysis, using HGU133 Plus2.0 microarrays. Results were validated both on original (n1 = 6; n2 = 6; n3 = 6) and independent samples ((n1 = 6; n2 = 6; n3 = 6) by real-time PCR. SST expressing cells were detected by immunohistochemistry on colonic biopsy samples (n1 = 14; n2 = 20; n3 = 23). The effect of octreotide on cell growth was tested on Caco-2 cell line. SST methylation percentage in biopsy samples (n1 = 5; n2 = 5; n3 = 9) was defined using methylation-sensitive restriction enzyme digestion. RESULTS: In case of normal aging SST mRNA expression did not alter, but decreased in cancer (p<0.05). The ratio of SST immunoreactive cells was significantly higher in children (0.70%+/-0.79%) compared to CRC (0%+/-0%) (p<0.05). Octreotide significantly increased the proportion of apoptotic Caco-2 cells. SST showed significantly higher methylation level in tumor samples (30.2%+/-11.6%) compared to healthy young individuals (3.5%+/-1.9%) (p<0.05). CONCLUSIONS: In cancerous colonic mucosa the reduced SST production may contribute to the uncontrolled cell proliferation. Our observation that in colon cancer cells octreotide significantly enhanced cell death and attenuated cell proliferation suggests that SST may act as a regulator of epithelial cell kinetics. The inhibition of SST expression in CRC can be epigenetically regulated by promoter hypermethylation

Detection of methylated septin 9 in tissue and plasma of colorectal patients with neoplasia and the relationship to the amount of circulating cell-free DNA

BACKGROUND: Determination of methylated Septin 9 (mSEPT9) in plasma has been shown to be a sensitive and specific biomarker for colorectal cancer (CRC). However, the relationship between methylated DNA in plasma and colon tissue of the same subjects has not been reported. METHODS: Plasma and matching biopsy samples were collected from 24 patients with no evidence of disease (NED), 26 patients with adenoma and 34 patients with CRC. Following bisulfite conversion of DNA a commercial RT-PCR assay was used to determine the total amount of DNA in each sample and the fraction of mSEPT9 DNA. The Septin-9 protein was assessed using immunohistochemistry. RESULTS: The percent of methylated reference (PMR) values for SEPT9 above a PMR threshold of 1% were detected in 4.2% (1/24) of NED, 100% (26/26) of adenoma and 97.1% (33/34) of CRC tissues. PMR differences between NED vs. adenoma and NED vs. CRC comparisons were significant (p<0.001). In matching plasma samples using a PMR cut-off level of 0.01%, SEPT9 methylation was 8.3% (2/24) of NED, 30.8% (8/26) of adenoma and 88.2% (30/34) of CRC. Significant PMR differences were observed between NED vs. CRC (p<0.01) and adenoma vs. CRC (p<0.01). Significant differences (p<0.01) were found in the amount of cfDNA (circulating cell-free DNA) between NED and CRC, and a modest correlation was observed between mSEPT9 concentration and cfDNA of cancer (R2 = 0.48). The level of Septin-9 protein in tissues was inversely correlated to mSEPT9 levels with abundant expression in normals, and diminished expression in adenomas and tumors. CONCLUSIONS: Methylated SEPT9 was detected in all tissue samples. In plasma samples, elevated mSEPT9 values were detected in CRC, but not in adenomas. Tissue levels of mSEPT9 alone are not sufficient to predict mSEPT9 levels in plasma. Additional parameters including the amount of cfDNA in plasma appear to also play a role

Comprehensive DNA Methylation Analysis Reveals a Common Ten-Gene Methylation Signature in Colorectal Adenomas and Carcinomas

Microarray analysis of promoter hypermethylation provides insight into the role and extent of DNA methylation in the development of colorectal cancer (CRC) and may be co-monitored with the appearance of driver mutations. Colonic biopsy samples were obtained endoscopically from 10 normal, 23 adenoma (17 low-grade (LGD) and 6 high-grade dysplasia (HGD)), and 8 ulcerative colitis (UC) patients (4 active and 4 inactive). CRC samples were obtained from 24 patients (17 primary, 7 metastatic (MCRC)), 7 of them with synchronous LGD. Field effects were analyzed in tissues 1 cm (n = 5) and 10 cm (n = 5) from the margin of CRC. Tissue materials were studied for DNA methylation status using a 96 gene panel and for KRAS and BRAF mutations. Expression levels were assayed using whole genomic mRNA arrays. SFRP1 was further examined by immunohistochemistry. HT29 cells were treated with 5-aza-2' deoxycytidine to analyze the reversal possibility of DNA methylation. More than 85% of tumor samples showed hypermethylation in 10 genes (SFRP1, SST, BNC1, MAL, SLIT2, SFRP2, SLIT3, ALDH1A3, TMEFF2, WIF1), whereas the frequency of examined mutations were below 25%. These genes distinguished precancerous and cancerous lesions from inflamed and healthy tissue. The mRNA alterations that might be caused by systematic methylation could be partly reversed by demethylation treatment. Systematic changes in methylation patterns were observed early in CRC carcinogenesis, occuring in precursor lesions and CRC. Thus we conclude that DNA hypermethylation is an early and systematic event in colorectal carcinogenesis, and it could be potentially reversed by systematic demethylation therapy, but it would need more in vitro and in vivo experiments to support this theory