DNA hypermethylation contributes to colorectal cancer metastasis by regulating the binding of CEBPB and TFCP2 to the CPEB1 promoter

Abstract Background Aberrant DNA methylation has been firmly established as a factor contributing to the pathogenesis of colorectal cancer (CRC) via its capacity to silence tumour suppressor genes. However, the methylation status of multiple tumour suppressor genes and their roles in promoting CRC metastasis are not well characterised. Methods We explored the methylation and expression profiles of CPEB1 (the gene encoding cytoplasmic polyadenylation element-binding protein 1), a candidate CRC tumour suppressor gene, using The Cancer Genome Atlas (TCGA) database and validated these results in both CRC cell lines and cells from Han Chinese CRC patients (n = 104). The functional role of CPEB1 in CRC was examined in experiments performed in vitro and in vivo. A candidate transcription factor capable of regulating CPEB1 expression was predicted in silico and validated by luciferase reporter, DNA pull-down, and electrophoretic mobility shift assays. Results Hypermethylation and decreased expression of CPEB1 in CRC tumour tissues were revealed by TCGA database. We also identified a significant inverse correlation (Pearson’s R = − 0.43, P < 0.001) between promoter methylation and CPEB1 expression. We validated these results in CRC samples and two CRC cell lines. We also demonstrated that up-regulation of CPEB1 resulted in significantly decreased tumour growth, migration, invasion, and tumorigenicity and promoted tumour cell apoptosis both in vitro and in vivo. We identified the transcription factors CCAAT enhancer-binding protein beta (CEBPB) and transcription factor CP2 (TFCP2) as critical regulators of CPEB1 expression. Hypermethylation of the CPEB1 promoter resulted in a simultaneous increase in the capacity for TFCP2 binding and a decreased likelihood of CEBPB binding, both of which led to diminished expression of CPEB1. Conclusions Our results identified a novel tumour-suppressive role of CPEB1 in CRC and found that hypermethylation of the CPEB1 promoter may lead to diminished expression due to decreased chromatin accessibility and transcription factor binding. Collectively, these results suggest a potential role for CPEB1 in the diagnosis and treatment of CRC.

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Progression and Differentiation of Alveolar Rhabdomyosarcoma Is Regulated by PAX7 Transcription Factor—Significance of Tumor Subclones

Cells ◽

10.3390/cells10081870 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1870

Author(s):

Klaudia Skrzypek ◽

Grażyna Adamek ◽

Marta Kot ◽

Bogna Badyra ◽

Marcin Majka

Keyword(s):

Transcription Factor ◽

Cell Lines ◽

Migration Activity ◽

Id Proteins ◽

Rh30 Cell ◽

Str Profile ◽

And Migration

Rhabdomyosarcoma (RMS), is the most frequent soft tissue tumor in children that originates from disturbances in differentiation process. Mechanisms leading to the development of RMS are still poorly understood. Therefore, by analysis of two RMS RH30 cell line subclones, one subclone PAX7 negative, while the second one PAX7 positive, and comparison with other RMS cell lines we aimed at identifying new mechanisms crucial for RMS progression. RH30 subclones were characterized by the same STR profile, but different morphology, rate of proliferation, migration activity and chemotactic abilities in vitro, as well as differences in tumor morphology and growth in vivo. Our analysis indicated a different level of expression of adhesion molecules (e.g., from VLA and ICAM families), myogenic microRNAs, such as miR-206 and transcription factors, such as MYOD, MYOG, SIX1, and ID. Silencing of PAX7 transcription factor with siRNA confirmed the crucial role of PAX7 transcription factor in proliferation, differentiation and migration of RMS cells. To conclude, our results suggest that tumor cell lines with the same STR profile can produce subclones that differ in many features and indicate crucial roles of PAX7 and ID proteins in the development of RMS.

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A Targeted Functional Clone Tracking Assay for the Identification of Tumour Suppressor Genes in BCP- ALL Implicates the Transcription Factors FOXO3 and PRDM1

Blood ◽

10.1182/blood.v126.23.2449.2449 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2449-2449

Author(s):

Paul Sinclair ◽

Joanna Cheng ◽

Prahlad Raninga ◽

Rebecca Hanna ◽

Shaun Hollern ◽

...

Keyword(s):

Transcription Factors ◽

B Cell ◽

Candidate Gene ◽

Copy Number ◽

B Cell Development ◽

Tumour Suppressor ◽

Tumour Suppressor Genes ◽

Suppressor Genes

Abstract B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is accompanied by genomic mutations and rearrangements that commonly affect cytokines, transcription factors or signalling molecules that drive B-cell development or contribute to the pre-B cell receptor (pre-BCR) checkpoint. Deletions of the long arm of chromosome 6 [del(6q)] occur in ~10% of BCP-ALL and are also frequent in mature B and T-cell malignancies. Loss of function of the 6q genes EPHA7 and PRDM1, have been implicated in the genesis of lymphoma and BACH2, as a mediator of pre-BCR negative selection, is functionally a candidate tumour suppressor gene. However loss of these or other 6q genes have not been demonstrated, for example through biallelic inactivation, to contribute to BCP-ALL. Analysis of our own and published SNP6.0 data from ALL patients defined 5 focal recurrent regions of deletion on 6q, 4 mapping to 6q15-6q21, coincident with previously published common regions of deletion in ALL. These 4 regions contain 22 candidate genes, including EPHA7 but not BACH2 or PRDM1, which nevertheless mapped close to focal deletions and were also classed as candidate tumour suppressors. To develop the clone tracking assay, we adapted the SIN-SIEW lentiviral construct that expresses EGFP under the control of a spleen focus forming virus (SFFV) promoter. Candidate gene consensus coding sequence (CCDS) or a control luciferase cDNA were cloned between the promoter and an internal ribosomal entry site immediately upstream of EGFP. Transduction of the control (pSLIEW) or candidate gene SIN-SIEW-CCDS constructs consistently expressed EGFP in 697, a BCP-ALL cell line with del(6)(q14.1-22.3). For clone tracking, SIN-SIEW-CCDS constructs were assigned to 4 pools that also included pSLIEW. Pools were transduced into 697 cells that were both cultured in vitro and transplanted by intra-femoral injection into NOD/LtSz-scid IL2Rƴ null (NSG) mice. DNA was isolated from transduced cells immediately before transplant and then at 3 to 5 day intervals from cultured cells or from cells recovered from mouse bone marrow, spleen or liver at end stage disease. The pSLIEW construct facilitated monitoring of disease progression by in vivo imaging and also served as a control to measure CCDS construct copy number changes against. To quantify changes in integrated SIN-SIEW-CCDS, we developed a multiplex targeted Illumina sequencing approach. In vitro, highly significant (p<0.01) reductions in copy number relative to pSLIEW over time, occurred for constructs expressing FOXO3, POU3F2, SIM1, PRDM13, C6orf168 and both α and β isoforms of PRDM1 (Fig 1a). With the exception of C6orf168, these genes also strongly suppressed leukemia development in vivo in all tissues analysed (Fig 1b). The known tumour suppressor genes, BACH2 and EPHA7, had no effect on cell growth in vitro. In vivo a moderate reduction for one of two EPHA7 CCDS was observed though curiously cells expressing BACH2 increased in relative copy number by approximately 3 fold. RNA sequencing data from 697 and published array data for normal pre-B cells and cases of BCP-ALL showed no, or extremely low, levels of expression for POU3F2, SIM1, PRDM13 and C6orf168 making it unlikely that they function as tumour suppressor genes in BCP-ALL. However significant expression of the transcription factors FOXO3 and PRDM1 were seen across data sets. Western blot confirmed expression of FOXO3 and PRDM1 in 697 and other BCP-ALL cell lines and demonstrated substantial increases in the corresponding proteins after transduction of 697 with FOXO3 and PRDM1 SIN-SIEW CCDS constructs. Over-expression of FOXO3 and both isoforms of PRDM1 decreased the proportion of cells in S and G2 phases of the cell cycle, but failed to induce apoptosis as measured by Annexin-5 staining. Comparison of total mRNA sequencing profiles of 697 cells, FACS sorted for ectopic expression of FOXO3, PRDM1 or control construct, showed distinctive patterns of up or down regulated mRNA. The roles of FOXO3 and PRDM1 in early B-cell development are currently undefined but notably our data suggests they influence expression of components of the pre-BCR and related signalling pathways and therefore may contribute to the pre-BCR checkpoint. Disclosures No relevant conflicts of interest to declare.

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Role of ESCRT component HD-PTP/PTPN23 in cancer

Biochemical Society Transactions ◽

10.1042/bst20160332 ◽

2017 ◽

Vol 45 (3) ◽

pp. 845-854 ◽

Cited By ~ 10

Author(s):

Marie-Claude Gingras ◽

Jalal M. Kazan ◽

Arnim Pause

Keyword(s):

Plasma Membrane ◽

Cancer Susceptibility ◽

Tumour Suppressor ◽

Tumour Suppressor Genes ◽

Surface Receptors ◽

Endosomal Sorting ◽

Bona Fide

Sustained cellular signalling originated from the receptors located at the plasma membrane is widely associated with cancer susceptibility. Endosomal sorting and degradation of the cell surface receptors is therefore crucial to preventing chronic downstream signalling and tumorigenesis. Since the Endosomal Sorting Complexes Required for Transport (ESCRT) controls these processes, ESCRT components were proposed to act as tumour suppressor genes. However, the bona fide role of ESCRT components in tumorigenesis has not been clearly demonstrated. The ESCRT member HD-PTP/PTPN23 was recently identified as a novel haplo-insufficient tumour suppressor in vitro and in vivo, in mice and humans. In this mini-review, we outline the role of the ESCRT components in cancer and summarize the functions of HD-PTP/PTPN23 in tumorigenesis.

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UPF1 Promotes Chemoresistance to Oxaliplatin Through Maintenance of Stemness by Increasing Phosphorylated TOP2A in Colorectal Cancer.

10.21203/rs.3.rs-130880/v1 ◽

2020 ◽

Author(s):

Congcong Zhu ◽

Long Zhang ◽

Senlin Zhao ◽

Weixing Dai ◽

Yun Xu ◽

...

Keyword(s):

Colorectal Cancer ◽

Overall Survival ◽

Cell Lines ◽

Clinical Relevance ◽

Poor Prognosis ◽

Therapy Strategy ◽

Underlying Mechanisms

Abstract Background: UPF1 is proved to dysregulate in multiple tumors and influence carcinogenesis. However, the role of UPF1 on oxaliplatin resistance in colorectal cancer (CRC) remains unknown.Methods: Firstly, we investigated the clinical relevance of UPF1 in CRC patients. Then, we explored the influence of UPF1 on chemoresistance to oxaliplatin in vitro and in vivo. Finally, we disclosed the underlying mechanisms of oxaliplatin resistance induced by UPF1.Results: UPF1 is upregulated in CRC and overexpression of UPF1 more likely results in recurrence in CRC patients and predicts a poorer overall survival (OS). UPF1 maintains stemness in CRC cell lines and promotes chemoresistance to oxaliplatin in CRC. UPF1-induced oxaliplatin resistance can be associated with interaction with TOP2A and increasing phosphorylated TOP2A.Conclusions: UPF1 was overexpressed and predicted a poor prognosis in CRC. UPF1 enhanced the stemness and chemoresistance to oxaliplatin by interaction with TOP2A and increase of phosphorylated TOP2A in CRC, which may provide a new therapy strategy for chemoresistance to oxaliplatin in CRC patients.

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Hypermethylation involved in DNA profiles of lung cancer specific tumour suppressor genes and epigenetic modification caused by an ultra-highly diluted homeopathic drug, Condurango 30C, in vitro and in vivo

International Journal of High Dilution Research - ISSN 1982-6206 ◽

10.51910/ijhdr.v13i47.721 ◽

2021 ◽

Vol 13 (47) ◽

pp. 99-99

Author(s):

Anisur Rahman Khuda-Bukhsh ◽

Sourav Sidkar

Keyword(s):

Lung Cancer ◽

Dna Methylation ◽

Epigenetic Modification ◽

Tumour Suppressor ◽

Tumour Suppressor Genes ◽

Dna Hypermethylation ◽

Suppressor Genes ◽

Homeopathic Drug

Background and objectives: DNA hyper-methylation is an important aspect involved in carcinogenesis and cancer progression, which affects mainly CpG islands of DNA and causes inactivation of tumour suppressor genes. Therefore DNA hypermethylation status of the genomic DNA in both the transformed cancerous cell lines and in carcinogen-induced lung cancer was ascertained by analysis of expressions of certain major lung cancer specific tumour suppressor genes. The other objective was to examine if ultra highly diluted homeopathic drug, Condurango 30C, had ability to modulate DNA methylation. Methods: DNA methylation activity, if any, has been ascertained in H460-NSCLC cells in vitro and in BaP-induced lung cancer of rats in vivo, in respect of tumour suppressor genes like p15, p16, p18 and p53 by using PCR-SSCP analyses. The ability of modulation of DNA methylation, if any, by Condurango 30C was also verified against placebo control in a blinded manner. Results: Condurango 30C-treated DNA showed significant decrease in band-intensity of p15 and p53 genes especially in methylated condition, in vitro, at the IC50 dose (2.43Âµl/100Âµl). SSCP analysis of p15 and p53 genes in Condurango 30C-treated DNA also supported ability of Condurango 30C to modulate methylation state, in vitro. Inhibition of p15 hypermethylation was observed after post cancer treatment of rat with Condurango 30C. SSCP results gave a better indication of differences in band-position and single strand separation of p15 and p53 in Condurango 30C treated samples. Conclusion: Condurango 30C could trigger epigenetic modification in lung cancer via modulation of DNA hypermethylation but placebos could not.

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RRAD expression in gastric and colorectal cancer with peritoneal carcinomatosis

Scientific Reports ◽

10.1038/s41598-019-55767-7 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Hee Kyung Kim ◽

Inkyoung Lee ◽

Seung Tae Kim ◽

Jeeyun Lee ◽

Kyoung-Mee Kim ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Lines ◽

Therapeutic Target ◽

Combined Treatment ◽

Cancer Tissue ◽

Associated Proteins ◽

Emt Markers

AbstractThe role of Ras-related associated with diabetes (RRAD) in gastric cancer (GC) or colorectal cancer (CRC) has not been investigated. We aimed to investigate the biological and clinical roles of RRAD in GC and CRC and to assess RRAD as a therapeutic target. A total of 31 cancer cell lines (17 GC cell lines, 14 CRC cell lines), 59 patient-derived cells (PDCs from 48 GC patients and 11 CRC patients), and 84 matched pairs of primary cancer tissue and non-tumor tissue were used to evaluate the role of RRAD in vitro and in vivo. RRAD expression was frequently increased in GC and CRC cell lines, and siRNA/shRNA-mediated RRAD inhibition induced significant decline of tumor cell proliferation both in vitro and in vivo. A synergistic effect of RRAD inhibition was generated by combined treatment with chemotherapy. Notably, RRAD expression was markedly increased in PDCs, and RRAD inhibition suppressed PDC proliferation. RRAD inhibition also resulted in reduced cell invasion, decreased expression of EMT markers, and decreased angiogenesis and levels of associated proteins including VEGF and ANGP2. Our study suggests that RRAD could be a novel therapeutic target for treatment of GC and CRC, especially in patients with peritoneal seeding.

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Oncogenic lncRNA ZNF561-AS1 is essential for colorectal cancer proliferation and survival through regulation of miR-26a-3p/miR-128-5p-SRSF6 axis

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01882-1 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Zizhen Si ◽

Lei Yu ◽

Haoyu Jing ◽

Lun Wu ◽

Xidi Wang

Keyword(s):

Colorectal Cancer ◽

Rna Binding ◽

Xenograft Model ◽

Luciferase Reporter ◽

Cancer Proliferation ◽

Mouse Xenograft ◽

Mouse Xenograft Model

Abstract Background Long non-coding RNAs (lncRNA) are reported to influence colorectal cancer (CRC) progression. Currently, the functions of the lncRNA ZNF561 antisense RNA 1 (ZNF561-AS1) in CRC are unknown. Methods ZNF561-AS1 and SRSF6 expression in CRC patient samples and CRC cell lines was evaluated through TCGA database analysis, western blot along with real-time PCR. SRSF6 expression in CRC cells was also examined upon ZNF561-AS1 depletion or overexpression. Interaction between miR-26a-3p, miR-128-5p, ZNF561-AS1, and SRSF6 was examined by dual luciferase reporter assay, as well as RNA binding protein immunoprecipitation (RIP) assay. Small interfering RNA (siRNA) mediated knockdown experiments were performed to assess the role of ZNF561-AS1 and SRSF6 in the proliferative actives and apoptosis rate of CRC cells. A mouse xenograft model was employed to assess tumor growth upon ZNF561-AS1 knockdown and SRSF6 rescue. Results We find that ZNF561-AS1 and SRSF6 were upregulated in CRC patient tissues. ZNF561-AS1 expression was reduced in tissues from treated CRC patients but upregulated in CRC tissues from relapsed patients. SRSF6 expression was suppressed and enhanced by ZNF561-AS1 depletion and overexpression, respectively. Mechanistically, ZNF561-AS1 regulated SRSF6 expression by sponging miR-26a-3p and miR-128-5p. ZNF561-AS1-miR-26a-3p/miR-128-5p-SRSF6 axis was required for CRC proliferation and survival. ZNF561-AS1 knockdown suppressed CRC cell proliferation and triggered apoptosis. ZNF561-AS1 depletion suppressed the growth of tumors in a model of a nude mouse xenograft. Similar observations were made upon SRSF6 depletion. SRSF6 overexpression reversed the inhibitory activities of ZNF561-AS1 in vivo, as well as in vitro. Conclusion In summary, we find that ZNF561-AS1 promotes CRC progression via the miR-26a-3p/miR-128-5p-SRSF6 axis. This study reveals new perspectives into the role of ZNF561-AS1 in CRC.

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YAP inhibits autophagy and promotes progression of colorectal cancer via upregulating Bcl-2 expression

Cell Death and Disease ◽

10.1038/s41419-021-03722-8 ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

Lan Jin ◽

Yunhe Chen ◽

Dan Cheng ◽

Zhikai He ◽

Xinyi Shi ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Death ◽

Transcriptional Coactivator ◽

Inhibitory Protein ◽

Potential Therapeutic Target ◽

Related Cell ◽

Autophagy Inhibition

AbstractColorectal cancer (CRC) is one of the most aggressive and lethal cancers. The role of autophagy in the pathobiology of CRC is intricate, with opposing functions manifested in different cellular contexts. The Yes-associated protein (YAP), a transcriptional coactivator inactivated by the Hippo tumor-suppressor pathway, functions as an oncoprotein in a variety of cancers. In this study, we found that YAP could negatively regulate autophagy in CRC cells, and consequently, promote tumor progression of CRC in vitro and in vivo. Mechanistically, YAP interacts with TEAD forming a complex to upregulate the transcription of the apoptosis-inhibitory protein Bcl-2, which may subsequently facilitate cell survival by suppressing autophagy-related cell death; silencing Bcl-2 expression could alleviate YAP-induced autophagy inhibition without affecting YAP expression. Collectively, our data provide evidence for YAP/Bcl-2 as a potential therapeutic target for drug exploration against CRC.

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Identification of circRNA circ-CSPP1 as a potent driver of colorectal cancer by directly targeting the miR-431/LASP1 axis

Open Life Sciences ◽

10.1515/biol-2021-0053 ◽

2021 ◽

Vol 16 (1) ◽

pp. 523-536

Author(s):

Minghao Li ◽

Jianbin Zhuang ◽

Di Kang ◽

Yuzhuo Chen ◽

Weiliang Song

Keyword(s):

Colorectal Cancer ◽

Cancer Biology ◽

Spindle Pole ◽

Circular Rnas ◽

Cell Progression ◽

Crc Management ◽

Common Malignancy

Abstract Colorectal cancer (CRC) is the third most common malignancy worldwide. Circular RNAs (circRNAs) have been implicated in cancer biology. The purpose of the current work is to investigate the precise parts of circRNA centrosome and spindle pole-associated protein 1 (circ-CSPP1) in the progression of CRC. Our data showed that circ-CSPP1 was significantly overexpressed in CRC tissues and cells. The knockdown of circ-CSPP1 attenuated cell proliferation, migration, invasion and promoted apoptosis in vitro and weakened tumor growth in vivo. circ-CSPP1 directly targeted miR-431, and circ-CSPP1 knockdown modulated CRC cell progression in vitro via upregulating miR-431. Moreover, LIM and SH3 protein 1 (LASP1) was a functional target of miR-431 in modulating CRC cell malignant progression. Furthermore, circ-CSPP1 in CRC cells functioned as a posttranscriptional regulator on LASP1 expression by targeting miR-431. Our present study identified the oncogenic role of circ-CSPP1 in CRC partially by the modulation of the miR-431/LASP1 axis, providing evidence for circ-CSPP1 as a promising biomarker for CRC management.

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