vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/VJ17.313vavilov-1268Research ArticleСИСТЕМНАЯ БИОЛОГИЯ И БИОМЕДИЦИНАSYSTEMS BIOLOGY AND BIOMEDICINEПерспективные маркеры CIMP+ опухолей толстой кишки, выявленные на основе анализа данных ресурса TCGAPromising markers of CIMP+ colon tumors identified on the basis of TCGA data analysisКрасновГ. С.KrasnovG. S.

Москва.

Moscow.

БениаминовА. Д.BeniaminovA. D.

Москва.

Moscow.

ТычкоР. А.TychkoR. A.

Москва.

Moscow.

ПузановГ. А.PuzanovG. A.

Москва.

Moscow.

НоваковскийР. О.NovakovskiyR. O.

Москва.

Moscow.

КудрявцеваА. В.KudryavtsevaA. V.

Москва.

Moscow.

ДмитриевА. А.DmitrievA. A.

Москва.

Moscow.

Alex_245@mail.ruИнститут молекулярной биологии им. В.А. Энгельгардта Российской академии наук.РоссияEngelhardt Institute of Molecular Biology RAS.Russian Federation201720012018218920924Copyright © Краснов Г.С., Бениаминов А.Д., Тычко Р.А., Пузанов Г.А., Новаковский Р.О., Кудрявцева А.В., Дмитриев А.А., 20182018Краснов Г.С., Бениаминов А.Д., Тычко Р.А., Пузанов Г.А., Новаковский Р.О., Кудрявцева А.В., Дмитриев А.А.Krasnov G.S., Beniaminov A.D., Tychko R.A., Puzanov G.A., Novakovskiy R.O., Kudryavtseva A.V., Dmitriev A.A.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/1268

CIMP+ (CpG-­Island Methylator Phenotype) опухоли характеризуют ся плотным метилированием промоторных CpG­островков одновременно многих генов и представляют отдельную группу злокачественных новообразований толстой кишки. Несмотря на то что диагностика CIMP+ опухолей имеет значительную прогностическую ценность, до сих пор не разработано эффективного  набора маркеров для их выявления. Для определения CpG­сайтов, уро вень метилирования которых может быть использован для идентификации CIMP+ опухолей, с помощью созданного ранее прило жения CrossHub нами проведен анализ профилей экспрессии и метилирования 297 образцов первичных опухолей и 38 парных к ним «условных норм» толстой кишки, представленных в базе проекта TCGA (The Cancer Genome Atlas). Разработан скоринг, учи тывающий уровень метилирования CpG­сайтов, их расположение, а также уровень экспрессии соответствующих генов.  Определено, что ста тус метилирования CpG­сайтов, относящихся к генам AMOTL1, ZNF43, ZNF134 и CHFR, является перспективным маркером CIMP+ опухолей. Более того, идентифицированы конкретные районы про моторных областей этих генов, уровень метилирования которых ассоциирован с исследуемым фенотипом. Для валидации полученных дан ных на независимой выборке, сначала оценили относительный уровень мРНК генов AMOTL1, ZNF43, ZNF134 и CHFR в 30 парных (опухоль/«условная норма») образцах толстой кишки методом количественной ПЦР. Для всех генов выявлено частое (50–60 % случаев) и значительное (2–30 раз) снижение экспрессии. Затем методом бисульфитной конверсии ДНК с последующим кло нированием и секвенированием исследовали статус метилирования CpG­сайтов, отобранных в результате биоинформатического анализа, и обна ружили высокий уровень метилирования (β­value = 0.3–0.9) в об разцах с одновременно сниженным уровнем экспрессии всех четырех генов и низкий уровень метилирования (β­value = 0.0–0.2) в образцах с неизменным уровнем экспрессии четырех генов и в «условных нормах». Таким образом, статус метилирования CpG­сайтов промоторных областей генов AMOTL1, ZNF43, ZNF134 и CHFR является перспективным потенциальным маркером CIMP+ опухолей толстой кишки.

CIMP+ (CpG­Island Methylator Phenotype) tumors are characterized by dense methylation of promoter CpG islands of many genes at once and represent a separate group of malignant neoplasms of the colon. Despite the fact that the diagnostics of CIMP+ tumors has a significant prognostic value, an effective set of markers has not been developed yet. For the identification of CpG sites, the methylation level of which could be used to detect CIMP+ tumors, an analysis of expression and methylation profiles of 297 primary colon tumors and 38 histologically normal tissues paired to them, which are presented in the TCGA (The Cancer Genome Atlas) project database, was performed by us using the CrossHub tool created previously. We developed the scoring, which takes into account the methylation level of CpG sites, their location, and the expression level of the corresponding genes. It was revealed that the methylation status of CpG sites of the AMOTL1, ZNF43, ZNF134, and CHFR genes is a promising marker of CIMP+ tumors. Moreover, specific regions of promoters of these genes, the methylation level of which was associated with the examined phenotype, were identified. To verify the obtained data in independent sampling, first, the quantitative PCR was used to assess the relative mRNA level of the AMOTL1, ZNF43, ZNF134, and CHFR genes in 30 paired (tumor/histologically normal tissue) colon samples. For all the genes, a frequent (50–60 % of cases) and significant (2–30­fold) expression decrease was revealed. Then, the bisulfite conversion of DNA followed by cloning and sequencing was applied to examine the methylation status of CpG sites that were selected as the result of bioinformatics analysis. We observed a high methylation level (β­value = 0.3–0.9) of the CpG sites in the samples with simultaneous downregulation of all 4 genes and a low methylation level (β­value = 0.0–0.2) in the samples with the unchanged expression level of 4 genes and in histologically normal tissues. Thus, the methylation status of the CpG sites of promoter regions of the AMOTL1, ZNF43, ZNF134, and CHFR genes is a promising potential marker of CIMP+ colon tumors.

рак толстой кишкиCIMP+эпигенетикаметилирование ДНКTCGAколичественная ПЦРмолекулярные маркерыcolon cancerCIMP+epigeneticsDNA methylationTCGAquantitative PCRmolecular markersReferencesBarzily-Rokni M., Friedman N., Ron-Bigger S., Isaac S., Michlin D., Eden A. Synergism between DNA methylation and macroH2A1 occupancy in epigenetic silencing of the tumor suppressor gene p16(CDKN2A). Nucleic Acids Res. 2011;39(4):1326-1335. DOI 10.1093/nar/gkq994.Barzily-Rokni M., Friedman N., Ron-Bigger S., Isaac S., Michlin D., Eden A. Synergism between DNA methylation and macroH2A1 occupancy in epigenetic silencing of the tumor suppressor gene p16(CDKN2A). Nucleic Acids Res. 2011;39(4):1326-1335. DOI 10.1093/nar/gkq994.Berg M., Hagland H.R., Soreide K. Comparison of CpG island methylator phenotype (CIMP) frequency in colon cancer using different probe- and gene-specific scoring alternatives on recommended multi-gene panels. PloS ONE. 2014;9(1):e86657. DOI 10.1371/journal.pone.0086657.Berg M., Hagland H.R., Soreide K. Comparison of CpG island methylator phenotype (CIMP) frequency in colon cancer using different probe- and gene-specific scoring alternatives on recommended multi-gene panels. PloS ONE. 2014;9(1):e86657. DOI 10.1371/journal.pone.0086657.Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012;487(7407): 330-337. DOI 10.1038/nature11252.Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012;487(7407): 330-337. DOI 10.1038/nature11252.Couderc C., Boin A., Fuhrmann L., Vincent-Salomon A., Mandati V., Kieffer Y., Mechta-Grigoriou F., Del Maestro L., Chavrier P., Vallerand D., Brito I., Dubois T., De Koning L., Bouvard D., Louvard D., Gautreau A., Lallemand D. AMOTL1 promotes breast cancer progression and is antagonized by Merlin. Neoplasia. 2016;18(1):1024. DOI 10.1016/j.neo.2015.11.010.Couderc C., Boin A., Fuhrmann L., Vincent-Salomon A., Mandati V., Kieffer Y., Mechta-Grigoriou F., Del Maestro L., Chavrier P., Vallerand D., Brito I., Dubois T., De Koning L., Bouvard D., Louvard D., Gautreau A., Lallemand D. AMOTL1 promotes breast cancer progression and is antagonized by Merlin. Neoplasia. 2016;18(1):1024. DOI 10.1016/j.neo.2015.11.010.Derks S., Cleven A.H., Melotte V., Smits K.M., Brandes J.C., Azad N., van Criekinge W., de Bruine A.P., Herman J.G.,van Engeland M. Emerging evidence for CHFR as a cancer biomarker: from tumor biology to precision medicine. Cancer Metastasis Rev. 2014;33(1): 161-171. DOI 10.1007/s10555-013-9462-4.Derks S., Cleven A.H., Melotte V., Smits K.M., Brandes J.C., Azad N., van Criekinge W., de Bruine A.P., Herman J.G.,van Engeland M. Emerging evidence for CHFR as a cancer biomarker: from tumor biology to precision medicine. Cancer Metastasis Rev. 2014;33(1): 161-171. DOI 10.1007/s10555-013-9462-4.Dmitriev A.A., Kashuba V.I., Haraldson K., Senchenko V.N., Pavlova T.V., Kudryavtseva A.V., Anedchenko E.A., Krasnov G.S., Pronina I.V., Loginov V.I., Kondratieva T.T., Kazubskaya T.P., Braga E.A., Yenamandra S.P., Ignatjev I., Ernberg I., Klein G., Lerman M.I., Zabarovsky E.R. Genetic and epigenetic analysis of nonsmall cell lung cancer with NotI-microarrays. Epigenetics. 2012; 7(5):502-513. DOI 10.4161/epi.19801.Dmitriev A.A., Kashuba V.I., Haraldson K., Senchenko V.N., Pavlova T.V., Kudryavtseva A.V., Anedchenko E.A., Krasnov G.S., Pronina I.V., Loginov V.I., Kondratieva T.T., Kazubskaya T.P., Braga E.A., Yenamandra S.P., Ignatjev I., Ernberg I., Klein G., Lerman M.I., Zabarovsky E.R. Genetic and epigenetic analysis of nonsmall cell lung cancer with NotI-microarrays. Epigenetics. 2012; 7(5):502-513. DOI 10.4161/epi.19801.Dmitriev A.A., Krasnov G.S., Rozhmina T.A., Kishlyan N.V., Zyablitsin A.V., Sadritdinova A.F., Snezhkina A.V., Fedorova M.S., Yurkevich O.Y., Muravenko O.V., Bolsheva N.L., Kudryavtseva A.V., Melnikova N.V. Glutathione S-transferases and UDP-glycosyltransferases are involved in response to aluminum stress in flax. Front. Plant Sci. 2016;7:1920. DOI 10.3389/fpls.2016.01920.Dmitriev A.A., Krasnov G.S., Rozhmina T.A., Kishlyan N.V., Zyablitsin A.V., Sadritdinova A.F., Snezhkina A.V., Fedorova M.S., Yurkevich O.Y., Muravenko O.V., Bolsheva N.L., Kudryavtseva A.V., Melnikova N.V. Glutathione S-transferases and UDP-glycosyltransferases are involved in response to aluminum stress in flax. Front. Plant Sci. 2016;7:1920. DOI 10.3389/fpls.2016.01920.Dmitriev A.A., Rudenko E.E., Kudryavtseva A.V., Krasnov G.S., Gordiyuk V.V., Melnikova N.V., Stakhovsky E.O., Kononenko O.A., Pavlova L.S., Kondratieva T.T., Alekseev B.Y., Braga E.A., Senchenko V.N., Kashuba V.I. Epigenetic alterations of chromosome 3 revealed by NotI-microarrays in clear cell renal cell carcinoma. BioMed Res. Int. 2014;2014:735292. DOI 10.1155/2014/735292.Dmitriev A.A., Rudenko E.E., Kudryavtseva A.V., Krasnov G.S., Gordiyuk V.V., Melnikova N.V., Stakhovsky E.O., Kononenko O.A., Pavlova L.S., Kondratieva T.T., Alekseev B.Y., Braga E.A., Senchenko V.N., Kashuba V.I. Epigenetic alterations of chromosome 3 revealed by NotI-microarrays in clear cell renal cell carcinoma. BioMed Res. Int. 2014;2014:735292. DOI 10.1155/2014/735292.Hughes L.A., Melotte V., de Schrijver J., de Maat M., Smit V.T., Bovee J.V., French P.J., van den Brandt P.A., Schouten L.J., de Meyer T., van Criekinge W., Ahuja N., Herman J.G., Weijenberg M.P., van Engeland M. The CpG island methylator phenotype: what’s in a name? Cancer Res. 2013;73(19):5858-5868. DOI 10.1158/00085472.CAN-12-4306.Hughes L.A., Melotte V., de Schrijver J., de Maat M., Smit V.T., Bovee J.V., French P.J., van den Brandt P.A., Schouten L.J., de Meyer T., van Criekinge W., Ahuja N., Herman J.G., Weijenberg M.P., van Engeland M. The CpG island methylator phenotype: what’s in a name? Cancer Res. 2013;73(19):5858-5868. DOI 10.1158/00085472.CAN-12-4306.Jen J., Wang Y.C. Zinc finger proteins in cancer progression. J. Biomed. Sci. 2016;23(1):53. DOI 10.1186/s12929-016-0269-9.Jen J., Wang Y.C. Zinc finger proteins in cancer progression. J. Biomed. Sci. 2016;23(1):53. DOI 10.1186/s12929-016-0269-9.Kashuba V., Dmitriev A.A., Krasnov G.S., Pavlova T., Ignatjev I., Gordiyuk V.V., Gerashchenko A.V., Braga E.A., Yenamandra S.P., Lerman M., Senchenko V.N., Zabarovsky E. NotI microarrays: novel epigenetic markers for early detection and prognosis of high grade serous ovarian cancer. Int. J. Mol. Sci. 2012;13(10):1335213377. DOI 10.3390/ijms131013352.Kashuba V., Dmitriev A.A., Krasnov G.S., Pavlova T., Ignatjev I., Gordiyuk V.V., Gerashchenko A.V., Braga E.A., Yenamandra S.P., Lerman M., Senchenko V.N., Zabarovsky E. NotI microarrays: novel epigenetic markers for early detection and prognosis of high grade serous ovarian cancer. Int. J. Mol. Sci. 2012;13(10):1335213377. DOI 10.3390/ijms131013352.Krasnov G.S., Dmitriev A.A., Melnikova N.V., Zaretsky A.R., Nasedkina T.V., Zasedatelev A.S., Senchenko V.N., Kudryavtseva A.V. CrossHub: a tool for multi-way analysis of The Cancer Genome Atlas (TCGA) in the context of gene expression regulation mechanisms. Nucleic Acids Res. 2016;44(7):e62. DOI 10.1093/nar/gkv1478.Krasnov G.S., Dmitriev A.A., Melnikova N.V., Zaretsky A.R., Nasedkina T.V., Zasedatelev A.S., Senchenko V.N., Kudryavtseva A.V. CrossHub: a tool for multi-way analysis of The Cancer Genome Atlas (TCGA) in the context of gene expression regulation mechanisms. Nucleic Acids Res. 2016;44(7):e62. DOI 10.1093/nar/gkv1478.Krasnov G.S., Oparina N.Y., Dmitriev A.A., Kudryavtseva A.V., Anedchenko E.A., Kondrat’eva T.T., Zabarovsky E.R., Senchenko V.N. RPN1, a new reference gene for quantitative data normalization in lung and kidney cancer. Mol. Biol. 2011;45(2):211-220. DOI 10.1134/S0026893311020129.Krasnov G.S., Oparina N.Y., Dmitriev A.A., Kudryavtseva A.V., Anedchenko E.A., Kondrat’eva T.T., Zabarovsky E.R., Senchenko V.N. RPN1, a new reference gene for quantitative data normalization in lung and kidney cancer. Mol. Biol. 2011;45(2):211-220. DOI 10.1134/S0026893311020129.Lee M.S., Menter D.G., Kopetz S. Right versus left colon cancer biology: integrating the consensus molecular subtypes. J. Natl. Compr. Canc. Netw. 2017;15(3):411-419.Lee M.S., Menter D.G., Kopetz S. Right versus left colon cancer biology: integrating the consensus molecular subtypes. J. Natl. Compr. Canc. Netw. 2017;15(3):411-419.Mani S., Herceg Z. DNA demethylating agents and epigenetic therapy of cancer. Adv. Genet. 2010;70:327-340. DOI 10.1016/B978-0-12380866-0.60012-5.Mani S., Herceg Z. DNA demethylating agents and epigenetic therapy of cancer. Adv. Genet. 2010;70:327-340. DOI 10.1016/B978-0-12380866-0.60012-5.Melnikova N.V., Dmitriev A.A., Belenikin M.S., Koroban N.V., Speranskaya A.S., Krinitsina A.A., Krasnov G.S., Lakunina V.A., Snezh kina A.V., Sadritdinova A.F., Kishlyan N.V., Rozhmina T.A., Klimina K.M., Amosova A.V., Zelenin A.V., Muravenko O.V., Bolsheva N.L., Kudryavtseva A.V. Identification, expression analysis, and target prediction of flax genotroph microRNAs under normal and nutrient stress conditions. Front. Plant Sci. 2016;7:399. DOI 10.3389/fpls.2016.00399.Melnikova N.V., Dmitriev A.A., Belenikin M.S., Koroban N.V., Speranskaya A.S., Krinitsina A.A., Krasnov G.S., Lakunina V.A., Snezh kina A.V., Sadritdinova A.F., Kishlyan N.V., Rozhmina T.A., Klimina K.M., Amosova A.V., Zelenin A.V., Muravenko O.V., Bolsheva N.L., Kudryavtseva A.V. Identification, expression analysis, and target prediction of flax genotroph microRNAs under normal and nutrient stress conditions. Front. Plant Sci. 2016;7:399. DOI 10.3389/fpls.2016.00399.Rhee Y.Y., Kim K.J., Kang G.H. CpG island methylator phenotype-high colorectal cancers and their prognostic implications and relationships with the serrated neoplasia pathway. Gut Liver. 2017;11(1):3846. DOI 10.5009/gnl15535.Rhee Y.Y., Kim K.J., Kang G.H. CpG island methylator phenotype-high colorectal cancers and their prognostic implications and relationships with the serrated neoplasia pathway. Gut Liver. 2017;11(1):3846. DOI 10.5009/gnl15535.Sanbhnani S.,Yeong F.M. CHFR: a key checkpoint component implicated in a wide range of cancers. Cell. Mol. Life Sci. 2012;69(10):16691687. DOI 10.1007/s00018-011-0892-2.Sanbhnani S.,Yeong F.M. CHFR: a key checkpoint component implicated in a wide range of cancers. Cell. Mol. Life Sci. 2012;69(10):16691687. DOI 10.1007/s00018-011-0892-2.Senchenko V.N., Kisseljova N.P., Ivanova T.A., Dmitriev A.A., Krasnov G.S., Kudryavtseva A.V., Panasenko G.V., Tsitrin E.B., Lerman M.I., Kisseljov F.L., Kashuba V.I., Zabarovsky E.R. Novel tumor suppressor candidates on chromosome 3 revealed by NotImicroarrays in cervical cancer. Epigenetics. 2013;8(4):409-420. DOI 10.4161/epi.24233.Senchenko V.N., Kisseljova N.P., Ivanova T.A., Dmitriev A.A., Krasnov G.S., Kudryavtseva A.V., Panasenko G.V., Tsitrin E.B., Lerman M.I., Kisseljov F.L., Kashuba V.I., Zabarovsky E.R. Novel tumor suppressor candidates on chromosome 3 revealed by NotImicroarrays in cervical cancer. Epigenetics. 2013;8(4):409-420. DOI 10.4161/epi.24233.Suzuki H., Yamamoto E., Maruyama R., Niinuma T., Kai M. Biological significance of the CpG island methylator phenotype. Biochem. Biophys. Res. Commun. 2014;455(1-2):35-42. DOI 10.1016/j.bbrc.2014.07.007.Suzuki H., Yamamoto E., Maruyama R., Niinuma T., Kai M. Biological significance of the CpG island methylator phenotype. Biochem. Biophys. Res. Commun. 2014;455(1-2):35-42. DOI 10.1016/j.bbrc.2014.07.007.Toyota M., Ahuja N., Ohe-Toyota M., Herman J.G., Baylin S.B., Issa J.P. CpG island methylator phenotype in colorectal cancer. Proc. Natl. Acad. Sci. USA. 1999;96(15):8681-8686.Toyota M., Ahuja N., Ohe-Toyota M., Herman J.G., Baylin S.B., Issa J.P. CpG island methylator phenotype in colorectal cancer. Proc. Natl. Acad. Sci. USA. 1999;96(15):8681-8686.Zheng Y., Vertuani S., Nystrom S., Audebert S., Meijer I., Tegnebratt T., Borg J.P., Uhlen P., Majumdar A., Holmgren L. Angiomotin-like protein 1 controls endothelial polarity and junction stability during sprouting angiogenesis. Circ. Res. 2009;105(3):260-270. DOI 10.1161/CIRCRESAHA.109.195156.Zheng Y., Vertuani S., Nystrom S., Audebert S., Meijer I., Tegnebratt T., Borg J.P., Uhlen P., Majumdar A., Holmgren L. Angiomotin-like protein 1 controls endothelial polarity and junction stability during sprouting angiogenesis. Circ. Res. 2009;105(3):260-270. DOI 10.1161/CIRCRESAHA.109.195156.

The authors declare that there are no conflicts of interest present.