CTCF Expression is Essential for Somatic Cell Viability and Protection Against Cancer

CCCTC-binding factor (CTCF) is a conserved transcription factor that performs diverse roles in transcriptional regulation and chromatin architecture. Cancer genome sequencing reveals diverse acquired mutations in CTCF, which we have shown functions as a tumour suppressor gene. While CTCF is essential for embryonic development, little is known of its absolute requirement in somatic cells and the consequences of CTCF haploinsufficiency. We examined the consequences of CTCF depletion in immortalised human and mouse cells using shRNA knockdown and CRISPR/Cas9 genome editing as well as examined the growth and development of heterozygous Ctcf (Ctcf+/−) mice. We also analysed the impact of CTCF haploinsufficiency by examining gene expression changes in CTCF-altered endometrial carcinoma. Knockdown and CRISPR/Cas9-mediated editing of CTCF reduced the cellular growth and colony-forming ability of K562 cells. CTCF knockdown also induced cell cycle arrest and a pro-survival response to apoptotic insult. However, in p53 shRNA-immortalised Ctcf+/− MEFs we observed the opposite: increased cellular proliferation, colony formation, cell cycle progression, and decreased survival after apoptotic insult compared to wild-type MEFs. CRISPR/Cas9-mediated targeting in Ctcf+/− MEFs revealed a predominance of in-frame microdeletions in Ctcf in surviving clones, however protein expression could not be ablated. Examination of CTCF mutations in endometrial cancers showed locus-specific alterations in gene expression due to CTCF haploinsufficiency, in concert with downregulation of tumour suppressor genes and upregulation of estrogen-responsive genes. Depletion of CTCF expression imparts a dramatic negative effect on normal cell function. However, CTCF haploinsufficiency can have growth-promoting effects consistent with known cancer hallmarks in the presence of additional genetic hits. Our results confirm the absolute requirement for CTCF expression in somatic cells and provide definitive evidence of CTCF’s role as a haploinsufficient tumour suppressor gene. CTCF genetic alterations in endometrial cancer indicate that gene dysregulation is a likely consequence of CTCF loss, contributing to, but not solely driving cancer growth.

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CTCF Expression Is Essential for Somatic Cell Viability and Protection against Cancer

10.20944/preprints201810.0451.v1 ◽

2018 ◽

Author(s):

Charles G. Bailey ◽

Cynthia Metierre ◽

Julie Feng ◽

Kinsha Baidya ◽

Galina N. Filippova ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Somatic Cells ◽

Suppressor Gene ◽

Genetic Alterations ◽

Tumour Suppressor Gene ◽

Tumour Suppressor ◽

Tumour Suppressor Genes ◽

Absolute Requirement ◽

The Impact

CCCTC-binding factor (CTCF) is a conserved transcription factor that performs diverse roles in transcriptional regulation and chromatin architecture. Cancer genome sequencing reveals diverse acquired mutations in CTCF, which we have shown, functions as a tumour suppressor gene. While CTCF is essential for embryonic development, little is known of its absolute requirement in somatic cells and the consequences of CTCF haploinsufficiency. We examined the consequences of CTCF depletion in immortalised human and mouse cells using shRNA knockdown and CRISPR/Cas9 genome editing and examined the growth and development of heterozygous Ctcf (Ctcf+/-) mice. We also analysed the impact of CTCF haploinsufficiency by examining gene expression changes in CTCF-altered endometrial carcinoma. Knockdown and CRISPR/Cas9-mediated editing of CTCF reduced the cellular growth and colony-forming ability of K562 cells. CTCF knockdown also induced cell cycle arrest and a pro-survival response to apoptotic insult. However, in p53 shRNA-immortalised Ctcf+/- MEFs we observed the opposite: increased cellular proliferation, colony formation, cell cycle progression and decreased survival after apoptotic insult compared to wild type MEFs. CRISPR/Cas9-mediated targeting in Ctcf+/- MEFs revealed a predominance of in-frame microdeletions in Ctcf in surviving clones, however protein expression could not be ablated. Examination of CTCF mutations in endometrial cancers showed locus-specific alterations in gene expression due to CTCF haploinsufficiency, in concert with downregulation of tumour suppressor genes and upregulation of estrogen-responsive genes. Depletion of CTCF expression imparts a dramatic negative effect on normal cell function. However, CTCF haploinsufficiency can have growth-promoting effects consistent with known cancer hallmarks in the presence of additional genetic hits. Our results confirm the absolute requirement for CTCF expression in somatic cells and provide definitive evidence of CTCF’s role as a haploinsufficient tumour suppressor gene. CTCF genetic alterations in endometrial cancer indicate that gene dysregulation is a likely consequence of CTCF loss, contributing to, but not solely driving cancer growth.

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Cell proliferation and tumour suppressor gene expression in iodine unstained area surrounding oral squamous cell carcinoma

International Journal of Oral and Maxillofacial Surgery ◽

10.1054/ijom.2002.0457 ◽

2004 ◽

Vol 33 (1) ◽

pp. 75-83 ◽

Cited By ~ 19

Author(s):

K. Yokoo ◽

H. Noma ◽

T. Inoue ◽

S. Hashimoto ◽

M. Shimono

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

Squamous Cell Carcinoma ◽

Oral Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Squamous Cell ◽

Suppressor Gene ◽

Tumour Suppressor Gene ◽

Tumour Suppressor

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Use of Monozygotic Twins in Search for Breast Cancer Susceptibility Loci

Twin Research ◽

10.1375/twin.4.4.251 ◽

2001 ◽

Vol 4 (4) ◽

pp. 251-259 ◽

Cited By ~ 4

Author(s):

Asta Försti ◽

Qianren Jin ◽

Lena Sundqvist ◽

Magnus Söderberg ◽

Kari Hemminki

Keyword(s):

Breast Cancer ◽

Cancer Susceptibility ◽

Monozygotic Twins ◽

Monozygotic Twin ◽

Suppressor Gene ◽

Tumour Suppressor Gene ◽

Tumour Suppressor ◽

Tumour Suppressor Genes ◽

Suppressor Genes ◽

Twin Model

AbstractWe have used Swedish monozygotic twins concordant for breast cancer to study genetic changes associated with the development of breast cancer. Because loss of heterozygosity (LOH) at a specific genomic region may reflect the presence of a tumour suppressor gene, loss of the same allele in both of the twins concordant for breast cancer may pinpoint a tumour suppressor gene that confers a strong predisposition to breast cancer. DNA samples extracted from the matched tumour and normal tissues of nine twin pairs were analysed for allelic imbalance using a set of microsatellite markers on chromosomes 1, 13, 16 and 17, containing loci with known tumour suppressor genes. The two main regions, where more twin pairs than expected had lost the same allele, were located at 16qtel, including markers D16S393, D16S305 and D16S413, and at 17p13, distal to the p53 locus. Our results show that the monozygotic twin model can be used to suggest candidate regions of potential tumour suppressor genes, even with a limited number of twin pairs.

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Molecular profile of nasopharyngeal carcinoma: analysing tumour suppressor gene promoter hypermethylation by multiplex ligation-dependent probe amplification

Journal of Clinical Pathology ◽

10.1136/jclinpath-2017-204661 ◽

2017 ◽

Vol 71 (4) ◽

pp. 351-359 ◽

Cited By ~ 5

Author(s):

Marc L Ooft ◽

Jolique van Ipenburg ◽

Rob van Loo ◽

Rick de Jong ◽

Cathy Moelans ◽

...

Keyword(s):

Treatment Options ◽

Disease Free Survival ◽

Promoter Hypermethylation ◽

Suppressor Gene ◽

Tumour Suppressor Gene ◽

Tumour Suppressor ◽

Molecular Profile ◽

Tumour Suppressor Genes ◽

Suppressor Genes ◽

Dependent Probe

AimsTo assess differences in methylation profiles, and thus pathogenesis, between Epstein-Barr virus (EBV)-positive and negative nasopharyngeal carcinomas (NPCs). Also, promoter hypermethylation is a common phenomenon in early carcinogenesis to inactivate tumour suppressor genes. Since epigenetic changes are reversible, the therapeutic application of methylation inhibitors could provide treatment options.MethodsWe evaluated promoter hypermethylation profiles of 22 common tumour suppressor genes in 108 NPCs using methylation-specific multiplex ligation-dependent probe amplification. Correlation between methylation, clinicopathological features (including EBV) and survival was examined. Cluster analysis was also performed.ResultsHypermethylation of RASSF1A and ESR1 was significantly more frequent in EBV-positive NPC, while hypermethylation of DAPK1 was more frequent in EBV-negative NPC. In logistic regression, age, with EBV-positive NPC occurring at earlier age, and RASSF1, with RASSF1 hypermethylation being more frequent in EBV-positive NPC, remained significant. In EBV-positive NPC, hypermethylation of RASSF1A predicted worse overall survival (OS) (HR 3.058,95% CI 1.027 to 9.107). In EBV-negative NPC, hypermethylated adenomatous polyposis coli (APC) was a predictor of poor disease-free survival (DFS) (HR 6.868, 95% CI 2.142 to 22.022).ConclusionThere are important epigenetic differences between EBV-negative and EBV-positive NPCs, with EBV-negative NPC having a more similar hypermethylation profile to other head and neck squamous cell carcinomas than EBV-positive NPC. Hypermethylation of RASSF1A might contribute to worse OS in EBV-positive NPC, and may be an important event in the pathogenesis of EBV-infected NPC. Hypermethylation of APC might contribute to worse DFS in EBV-negative NPC.

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