Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress
Clinical Collections
Advances in Alzheimer’s

At a glance: The STEP trials

A round-up of the STEP phase 3 clinical trials evaluating semaglutide for weight loss in people with overweight or obesity.
ADVANCED SEARCH
Log in
Top
Cellular Oncology
Published in: Cellular Oncology 4/2018

01-08-2018 | Original Paper

Genome-wide analysis of endogenously expressed ZEB2 binding sites reveals inverse correlations between ZEB2 and GalNAc-transferase GALNT3 in human tumors

Authors: Pelin Balcik-Ercin, Metin Cetin, Irem Yalim-Camci, Gorkem Odabas, Nurettin Tokay, A. Emre Sayan, Tamer Yagci

Published in: Cellular Oncology | Issue 4/2018

Login to get access

Abstract

Background

ZEB2 is a transcriptional repressor that regulates epithelial-to-mesenchymal transition (EMT) through binding to bipartite E-box motifs in gene regulatory regions. Despite the abundant presence of E-boxes within the human genome and the multiplicity of pathophysiological processes regulated during ZEB2-induced EMT, only a small fraction of ZEB2 targets has been identified so far. Hence, we explored genome-wide ZEB2 binding by chromatin immunoprecipitation-sequencing (ChIP-seq) under endogenous ZEB2 expression conditions.

Methods

For ChIP-Seq we used an anti-ZEB2 monoclonal antibody, clone 6E5, in SNU398 hepatocellular carcinoma cells exhibiting a high endogenous ZEB2 expression. The ChIP-Seq targets were validated using ChIP-qPCR, whereas ZEB2-dependent expression of target genes was assessed by RT-qPCR and Western blotting in shRNA-mediated ZEB2 silenced SNU398 cells and doxycycline-induced ZEB2 overexpressing colorectal carcinoma DLD1 cells. Changes in target gene expression were also assessed using primary human tumor cDNA arrays in conjunction with RT-qPCR. Additional differential expression and correlation analyses were performed using expO and Human Protein Atlas datasets.

Results

Over 500 ChIP-Seq positive genes were annotated, and intervals related to these genes were found to include the ZEB2 binding motif CACCTG according to TOMTOM motif analysis in the MEME Suite database. Assessment of ZEB2-dependent expression of target genes in ZEB2-silenced SNU398 cells and ZEB2-induced DLD1 cells revealed that the GALNT3 gene serves as a ZEB2 target with the highest, but inversely correlated, expression level. Remarkably, GALNT3 also exhibited the highest enrichment in the ChIP-qPCR validation assays. Through the analyses of primary tumor cDNA arrays and expO datasets a significant differential expression and a significant inverse correlation between ZEB2 and GALNT3 expression were detected in most of the tumors. We also explored ZEB2 and GALNT3 protein expression using the Human Protein Atlas dataset and, again, observed an inverse correlation in all analyzed tumor types, except malignant melanoma. In contrast to a generally negative or weak ZEB2 expression, we found that most tumor tissues exhibited a strong or moderate GALNT3 expression.

Conclusions

Our observation that ZEB2 negatively regulates a GalNAc-transferase (GALNT3) that is involved in O-glycosylation adds another layer of complexity to the role of ZEB2 in cancer progression and metastasis. Proteins glycosylated by GALNT3 may be exploited as novel diagnostics and/or therapeutic targets.
Appendix
Available only for authorised users
Literature
1.
T. Nagase, K.-i. Ishikawa, N. Miyajima, A. Tanaka, H. Kotani, N. Nomura, O. Ohara, Prediction of the coding sequences of unidentified human genes. IX. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro. DNA Res 5, 31–39 (1998)CrossRefPubMed
2.
K. Verschueren, J.E. Remacle, C. Collart, H. Kraft, B.S. Baker, P. Tylzanowski, L. Nelles, G. Wuytens, M.-T. Su, R. Bodmer, SIP1, a novel zinc finger/homeodomain repressor, interacts with Smad proteins and binds to 5′-CACCT sequences in candidate target genes. J Biol Chem 274, 20489–20498 (1999)CrossRefPubMed
3.
J.E. Remacle, H. Kraft, W. Lerchner, G. Wuytens, C. Collart, K. Verschueren, J.C. Smith, D. Huylebroeck, New mode of DNA binding of multi-zinc finger transcription factors: δEF1 family members bind with two hands to two target sites. EMBO J 18, 5073–5084 (1999)CrossRefPubMedPubMedCentral
4.
J. Comijn, G. Berx, P. Vermassen, K. Verschueren, L. van Grunsven, E. Bruyneel, M. Mareel, D. Huylebroeck, F. Van Roy, The two-handed E box binding zinc finger protein SIP1 downregulates E-cadherin and induces invasion. Mol Cell 7, 1267–1278 (2001)CrossRefPubMed
5.
A.A. Postigo, J.L. Depp, J.J. Taylor, K.L. Kroll, Regulation of Smad signaling through a differential recruitment of coactivators and corepressors by ZEB proteins. EMBO J 22, 2453–2462 (2003)CrossRefPubMedPubMedCentral
6.
L.A. van Grunsven, C. Michiels, T. Van de Putte, L. Nelles, G. Wuytens, K. Verschueren, D. Huylebroeck, Interaction between Smad-interacting protein-1 and the corepressor C-terminal binding protein is dispensable for transcriptional repression of E-cadherin. J Biol Chem 278, 26135–26145 (2003)CrossRefPubMed
7.
A.E. Sayan, T.R. Griffiths, R. Pal, G.J. Browne, A. Ruddick, T. Yagci, R. Edwards, N.J. Mayer, H. Qazi, S. Goyal, SIP1 protein protects cells from DNA damage-induced apoptosis and has independent prognostic value in bladder cancer. Proc Natl Acad Sci USA 106, 14884–14889 (2009)CrossRefPubMed
8.
G. Maeda, T. Chiba, M. Okazaki, T. Satoh, Y. Taya, T. Aoba, K. Kato, S. Kawashiri, K. Imai, Expression of SIP1 in oral squamous cell carcinomas: implications for E-cadherin expression and tumor progression. Int J Oncol 27, 1535–1541 (2005)PubMed
9.
E. Rosivatz, I. Becker, K. Specht, E. Fricke, B. Luber, R. Busch, H. Höfler, K.-F. Becker, Differential expression of the epithelial-mesenchymal transition regulators snail, SIP1, and twist in gastric cancer. Am J Pathol 161, 1881–1891 (2002)CrossRefPubMedPubMedCentral
10.
J. Caramel, E. Papadogeorgakis, L. Hill, G.J. Browne, G. Richard, A. Wierinckx, G. Saldanha, J. Osborne, P. Hutchinson, G. Tse, A switch in the expression of embryonic EMT-inducers drives the development of malignant melanoma. Cancer Cell 24, 466–480 (2013)CrossRefPubMed
11.
N. Wakamatsu, Y. Yamada, K. Yamada, T. Ono, N. Nomura, H. Taniguchi, H. Kitoh, N. Mutoh, T. Yamanaka, K. Mushiake, Mutations in SIP1, encoding Smad interacting protein-1, cause a form of Hirschsprung disease. Nat Genet 27, 369–370 (2001)CrossRefPubMed
12.
G. Verstappen, L.A. van Grunsven, C. Michiels, T. Van de Putte, J. Souopgui, J. Van Damme, E. Bellefroid, J. Vandekerckhove, D. Huylebroeck, Atypical Mowat–Wilson patient confirms the importance of the novel association between ZFHX1B/SIP1 and NuRD corepressor complex. Hum Mol Genet 17, 1175–1183 (2008)CrossRefPubMed
13.
C. Vandewalle, J. Comijn, B. De Craene, P. Vermassen, E. Bruyneel, H. Andersen, E. Tulchinsky, F. Van Roy, G. Berx, SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell–cell junctions. Nucleic Acids Res 33, 6566–6578 (2005)CrossRefPubMedPubMedCentral
14.
S.-Y. Lin, S.J. Elledge, Multiple tumor suppressor pathways negatively regulate telomerase. Cell 113, 881–889 (2003)CrossRefPubMed
15.
N. Ozturk, E. Erdal, M. Mumcuoglu, K.C. Akcali, O. Yalcin, S. Senturk, A. Arslan-Ergul, B. Gur, I. Yulug, R. Cetin-Atalay, Reprogramming of replicative senescence in hepatocellular carcinoma-derived cells. Proc Natl Acad Sci USA 103, 2178–2183 (2006)CrossRefPubMed
16.
J. Mejlvang, M. Kriajevska, C. Vandewalle, T. Chernova, A.E. Sayan, G. Berx, J.K. Mellon, E. Tulchinsky, Direct repression of cyclin D1 by SIP1 attenuates cell cycle progression in cells undergoing an epithelial mesenchymal transition. Mol Biol Cell 18, 4615–4624 (2007)CrossRefPubMedPubMedCentral
17.
M. Kato, J. Zhang, M. Wang, L. Lanting, H. Yuan, J.J. Rossi, R. Natarajan, MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-β-induced collagen expression via inhibition of E-box repressors. Proc Natl Acad Sci USA 104, 3432–3437 (2007)CrossRefPubMed
18.
T. Shirakihara, M. Saitoh, K. Miyazono, Differential regulation of epithelial and mesenchymal markers by δEF1 proteins in epithelial–mesenchymal transition induced by TGF-β. Mol Biol Cell 18, 3533–3544 (2007)CrossRefPubMedPubMedCentral
19.
P.A. Gregory, A.G. Bert, E.L. Paterson, S.C. Barry, A. Tsykin, G. Farshid, M.A. Vadas, Y. Khew-Goodall, G.J. Goodall, The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol 10, 593–601 (2008)CrossRefPubMed
20.
S.-M. Park, A.B. Gaur, E. Lengyel, M.E. Peter, The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev 22, 894–907 (2008)CrossRefPubMedPubMedCentral
21.
22.
P.A. Gregory, C.P. Bracken, E. Smith, A.G. Bert, J.A. Wright, S. Roslan, M. Morris, L. Wyatt, G. Farshid, Y.-Y. Lim, An autocrine TGF-β/ZEB/miR-200 signaling network regulates establishment and maintenance of epithelial-mesenchymal transition. Mol Biol Cell 22, 1686–1698 (2011)CrossRefPubMedPubMedCentral
23.
24.
E. Oztas, M.E. Avci, A. Ozcan, A.E. Sayan, E. Tulchinsky, T. Yagci, Novel monoclonal antibodies detect Smad-interacting protein 1 (SIP1) in the cytoplasm of human cells from multiple tumor tissue arrays. Exp Mol Pathol 89, 182–189 (2010)CrossRefPubMed
25.
C. Grandori, J. Mac, F. Siëbelt, D.E. Ayer, R.N. Eisenman, Myc-Max heterodimers activate a DEAD box gene and interact with multiple E box-related sites in vivo. EMBO J 15, 4344–4357 (1996)PubMedPubMedCentralCrossRef
26.
A. Miquelajauregui, T. Van de Putte, A. Polyakov, A. Nityanandam, S. Boppana, E. Seuntjens, A. Karabinos, Y. Higashi, D. Huylebroeck, V. Tarabykin, Smad-interacting protein-1 (Zfhx1b) acts upstream of Wnt signaling in the mouse hippocampus and controls its formation. Proc Natl Acad Sci USA 104, 12919–12924 (2007)CrossRefPubMed
27.
A. Stryjewska, R. Dries, T. Pieters, G. Verstappen, A. Conidi, K. Coddens, A. Francis, L. Umans, W.F. van IJcken, G. Berx, Zeb2 regulates cell fate at the exit from epiblast state in mouse embryonic stem cells. Stem Cells 35, 611–625 (2017)CrossRefPubMed
28.
H. Yuzugullu, K. Benhaj, N. Ozturk, S. Senturk, E. Celik, A. Toylu, N. Tasdemir, M. Yilmaz, E. Erdal, K.C. Akcali, Canonical Wnt signaling is antagonized by noncanonical Wnt5a in hepatocellular carcinoma cells. Mol Cancer 8, 90 (2009)CrossRefPubMedPubMedCentral
29.
M.F. Carey, C.L. Peterson, S.T. Smale, Chromatin immunoprecipitation (chip). Cold Spring Harb Protoc 2009, pdb. prot5279 (2009)PubMed
30.
N. Gévry, S. Hardy, P.-É. Jacques, L. Laflamme, A. Svotelis, F. Robert, L. Gaudreau, Histone H2A. Z is essential for estrogen receptor signaling. Genes Dev 23, 1522–1533 (2009)CrossRefPubMedPubMedCentral
31.
32.
Y. Zhang, T. Liu, C.A. Meyer, J. Eeckhoute, D.S. Johnson, B.E. Bernstein, C. Nusbaum, R.M. Myers, M. Brown, W. Li, Model-based analysis of ChIP-Seq (MACS). Genome Biol 9, R137 (2008)CrossRefPubMedPubMedCentral
33.
34.
M.E. Avci, O. Konu, T. Yagci, Quantification of SLIT-ROBO transcripts in hepatocellular carcinoma reveals two groups of genes with coordinate expression. BMC Cancer 8, 392 (2008)CrossRefPubMedPubMedCentral
35.
U. Burk, J. Schubert, U. Wellner, O. Schmalhofer, E. Vincan, S. Spaderna, T. Brabletz, A reciprocal repression between ZEB1 and members of the miR-200 family promotes EMT and invasion in cancer cells. EMBO Rep 9, 582–589 (2008)CrossRefPubMedPubMedCentral
36.
F. Pontén, K. Jirström, M. Uhlen, The human protein atlas—a tool for pathology. J Pathol 216, 387–393 (2008)CrossRefPubMed
37.
J.P. Thiery, Epithelial–mesenchymal transitions in tumour progression. Nat Rev Cancer 2, 442–454 (2002)CrossRefPubMed
38.
A. Sathyanarayanan, K.S. Chandrasekaran, D. Karunagaran, microRNA-145 modulates epithelial-mesenchymal transition and suppresses proliferation, migration and invasion by targeting SIP1 in human cervical cancer cells. Cell Oncol 40, 119–131 (2017)CrossRef
39.
S. Bugide, V.K. Gonugunta, V. Penugurti, V.L. Malisetty, R.K. Vadlamudi, B. Manavathi, HPIP promotes epithelial-mesenchymal transition and cisplatin resistance in ovarian cancer cells through PI3K/AKT pathway activation. Cell Oncol 40, 133–144 (2017)CrossRef
40.
A. Fortunato, The role of hERG1 ion channels in epithelial-mesenchymal transition and the capacity of riluzole to reduce cisplatin resistance in colorectal cancer cells. Cell Oncol 40, 367–378 (2017)CrossRef
41.
G. Xu, L. Zhang, A. Ma, Y. Qian, Q. Ding, Y. Liu, B. Wang, Z. Yang, Y. Liu, SIP1 is a downstream effector of GADD45G in senescence induction and growth inhibition of liver tumor cells. Oncotarget 6, 33636 (2015)PubMedPubMedCentral
42.
P. Tylzanowski, K. Verschueren, D. Huylebroeck, F.P. Luyten, Smad-interacting protein 1 is a repressor of liver/bone/kidney alkaline phosphatase transcription in bone morphogenetic protein-induced osteogenic differentiation of C2C12 cells. J Biol Chem 276, 40001–40007 (2001)CrossRefPubMed
43.
S.A. Mani, W. Guo, M.-J. Liao, E.N. Eaton, A. Ayyanan, A.Y. Zhou, M. Brooks, F. Reinhard, C.C. Zhang, M. Shipitsin, The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133, 704–715 (2008)CrossRefPubMedPubMedCentral
44.
N.E. Renthal, C.-C. Chen, C.W. Koriand’r, R.D. Gerard, J. Prange-Kiel, C.R. Mendelson, miR-200 family and targets, ZEB1 and ZEB2, modulate uterine quiescence and contractility during pregnancy and labor. Proc Natl Acad Sci USA 107, 20828–20833 (2010)CrossRefPubMed
45.
K. Yoshiura, Y. Kanai, A. Ochiai, Y. Shimoyama, T. Sugimura, S. Hirohashi, Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human carcinomas. Proc Natl Acad Sci USA 92, 7416–7419 (1995)CrossRefPubMed
46.
E.P. Bennett, U. Mandel, H. Clausen, T.A. Gerken, T.A. Fritz, L.A. Tabak, Control of mucin-type O-glycosylation: a classification of the polypeptide GalNAc-transferase gene family. Glycobiology 22, 736–756 (2011)CrossRefPubMedPubMedCentral
47.
S.R. Stowell, T. Ju, R.D. Cummings, Protein glycosylation in cancer. Annu Rev Pathol Mech 10, 473–510 (2015)CrossRef
48.
T. Liu, S. Zhang, J. Chen, K. Jiang, Q. Zhang, K. Guo, Y. Liu, The transcriptional profiling of glycogenes associated with hepatocellular carcinoma metastasis. PLoS One 9, e107941 (2014)CrossRefPubMedPubMedCentral
49.
K. Shibao, H. Izumi, Y. Nakayama, R. Ohta, N. Nagata, M. Nomoto, K. Matsuo, Y. Yamada, K. Kitazato, H. Itoh, Expression of UDP-N-acetyl-α-D-galactosamine–polypeptide galNAc N-acetylgalactosaminyl transferase-3 in relation to differentiation and prognosis in patients with colorectal carcinoma. Cancer 94, 1939–1946 (2002)CrossRefPubMed
50.
S. Kitada, S. Yamada, A. Kuma, S. Ouchi, T. Tasaki, A. Nabeshima, H. Noguchi, K. Wang, S. Shimajiri, R. Nakano, Polypeptide N-acetylgalactosaminyl transferase 3 independently predicts high-grade tumours and poor prognosis in patients with renal cell carcinomas. Br J Cancer 109, 472–481 (2013)CrossRefPubMedPubMedCentral
51.
Y. Harada, H. Izumi, H. Noguchi, A. Kuma, Y. Kawatsu, T. Kimura, S. Kitada, H. Uramoto, K.-Y. Wang, Y. Sasaguri, Strong expression of polypeptide N. Tumour Biol 37, 1357–1368 (2016)CrossRefPubMed
52.
Z.-Q. Wang, M. Bachvarova, C. Morin, M. Plante, J. Gregoire, M.-C. Renaud, A. Sebastianelli, D. Bachvarov, Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation. Oncotarget 5, 544–560 (2014)PubMedPubMedCentral
53.
Y. Fan, R. Bi, M.J. Densmore, T. Sato, T. Kobayashi, Q. Yuan, X. Zhou, R.G. Erben, B. Lanske, Parathyroid hormone 1 receptor is essential to induce FGF23 production and maintain systemic mineral ion homeostasis. FASEB J 30, 428–440 (2016)CrossRefPubMed
54.
C.A. Yoshida, T. Kawane, T. Moriishi, A. Purushothaman, T. Miyazaki, H. Komori, M. Mori, X. Qin, A. Hashimoto, K. Sugahara, Overexpression of Galnt3 in chondrocytes resulted in dwarfism due to the increase of mucin-type O-glycans and reduction of glycosaminoglycans. J Biol Chem 289, 26584–26596 (2014)CrossRefPubMedPubMedCentral
55.
H. Izumi, O. Ryo, G. Nagatani, I. Tomoko, Y. Nakayama, M. Nomoto, K. Kohno, p300/CBP-associated factor (P/CAF) interacts with nuclear respiratory factor-1 to regulate the UDP-N-acetyl-alpha-d-galactosamine: polypeptide N-acetylgalactosaminyltransferase-3 gene. Biochem J 373, 713–722 (2003)CrossRefPubMedPubMedCentral
56.
K.A. Maupin, A. Sinha, E. Eugster, J. Miller, J. Ross, V. Paulino, V.G. Keshamouni, N. Tran, M. Berens, C. Webb, Glycogene expression alterations associated with pancreatic cancer epithelial-mesenchymal transition in complementary model systems. PLoS One 5, e13002 (2010)CrossRefPubMedPubMedCentral
Metadata
Title
Genome-wide analysis of endogenously expressed ZEB2 binding sites reveals inverse correlations between ZEB2 and GalNAc-transferase GALNT3 in human tumors
Authors
Pelin Balcik-Ercin
Metin Cetin
Irem Yalim-Camci
Gorkem Odabas
Nurettin Tokay
A. Emre Sayan
Tamer Yagci
Publication date
01-08-2018
Publisher
Springer Netherlands
Published in
Cellular Oncology / Issue 4/2018
Print ISSN: 2211-3428
Electronic ISSN: 2211-3436
DOI
https://doi.org/10.1007/s13402-018-0375-7

Other articles of this Issue 4/2018

Cellular Oncology 4/2018 Go to the issue

Review

Role of PKM2 in directing the metabolic fate of glucose in cancer: a potential therapeutic target

Review

Mutual concessions and compromises between stromal cells and cancer cells: driving tumor development and drug resistance

Original Paper

Patched-2 functions to limit Patched-1 deficient skin cancer growth

Correction

Correction to: The versatile role of exosomes in cancer progression: diagnostic and therapeutic implications

Original Paper

Identification of subsets of actionable genetic alterations in KRAS-mutant lung cancers using association rule mining

Report

AAA+ ATPases Reptin and Pontin as potential diagnostic and prognostic biomarkers in salivary gland cancer - a short report
Webinar | 19-02-2024 | 17:30 (CET)

Keynote webinar | Spotlight on antibody–drug conjugates in cancer

Watch now

Antibody–drug conjugates (ADCs) are novel agents that have shown promise across multiple tumor types. Explore the current landscape of ADCs in breast and lung cancer with our experts, and gain insights into the mechanism of action, key clinical trials data, existing challenges, and future directions.

Dr. Véronique Diéras
Prof. Fabrice Barlesi
Developed by: Springer Medicine
Image Credits