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1.
Cell Mol Life Sci ; 78(23): 7519-7536, 2021 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-34657170

RESUMEN

CCCTC-binding factor (CTCF) plays fundamental roles in transcriptional regulation and chromatin architecture maintenance. CTCF is also a tumour suppressor frequently mutated in cancer, however, the structural and functional impact of mutations have not been examined. We performed molecular and structural characterisation of five cancer-specific CTCF missense zinc finger (ZF) mutations occurring within key intra- and inter-ZF residues. Functional characterisation of CTCF ZF mutations revealed a complete (L309P, R339W, R377H) or intermediate (R339Q) abrogation as well as an enhancement (G420D) of the anti-proliferative effects of CTCF. DNA binding at select sites was disrupted and transcriptional regulatory activities abrogated. Molecular docking and molecular dynamics confirmed that mutations in residues specifically contacting DNA bases or backbone exhibited loss of DNA binding. However, R339Q and G420D were stabilised by the formation of new primary DNA bonds, contributing to gain-of-function. Our data confirm that a spectrum of loss-, change- and gain-of-function impacts on CTCF zinc fingers are observed in cell growth regulation and gene regulatory activities. Hence, diverse cellular phenotypes of mutant CTCF are clearly explained by examining structure-function relationships.


Asunto(s)
Factor de Unión a CCCTC/química , Factor de Unión a CCCTC/metabolismo , Regulación Neoplásica de la Expresión Génica , Mutación , Neoplasias/patología , Fenotipo , Dedos de Zinc , Apoptosis , Factor de Unión a CCCTC/genética , Proliferación Celular , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Regiones Promotoras Genéticas , Relación Estructura-Actividad , Células Tumorales Cultivadas
2.
Biochem Biophys Rep ; 19: 100662, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31338436

RESUMEN

Beta-catenin is well-known as a key effector of Wnt signalling and aberrant expression is associated with several human cancers. Stabilisation of and atypical subcellular localisation of beta-catenin, regulated in part through specific protein-protein interactions has been linked to cancer development, however the mechanisms behind these pathologies is yet to be fully elucidated. Affinity purification and mass spectrometry were used to identify potential ß-catenin interacting proteins in SW480 colon cancer cells. Recombinant ß-catenin constructs were used to co-isolate interacting proteins from stable isotope labelled cells followed by detection using mass spectrometry. Several known and new putative interactors were observed. In particular, we identified interaction with a set of coatomer complex I subunits implicated in retrograde transport at the Golgi, and confirmed endogenous interaction of ß-catenin with coatomer subunit COPB using immunoprecipitation assays and immunofluorescence microscopy. These observations suggest a hitherto unrecognised role for ß-catenin in the secretory pathway and warrant further functional studies to unravel its activity at this cellular location.

3.
Methods Mol Biol ; 1888: 141-152, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30519945

RESUMEN

This chapter provides a detailed description for mass spectrometry-based phosphoproteomics analysis. We describe sample preparation, phosphopeptide enrichment, mass spectrometry acquisition, label-free data analysis and statistical analysis. This technique can be employed to characterize cell signaling networks, and is particularly useful to monitor cellular responses to kinase drug inhibitors.


Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Fosfoproteínas/metabolismo , Fosfotransferasas/antagonistas & inhibidores , Proteómica , Cromatografía Liquida , Descubrimiento de Drogas/métodos , Humanos , Espectrometría de Masas , Fosfopéptidos/metabolismo , Proteómica/métodos
4.
Int J Mol Sci ; 19(12)2018 Nov 30.
Artículo en Inglés | MEDLINE | ID: mdl-30513694

RESUMEN

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.


Asunto(s)
Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Supervivencia Celular/fisiología , Neoplasias Endometriales/genética , Edición Génica , Animales , Sistemas CRISPR-Cas , Proliferación Celular/genética , Proliferación Celular/fisiología , Supervivencia Celular/genética , Femenino , Haploinsuficiencia/genética , Haploinsuficiencia/fisiología , Humanos , Células K562 , Ratones , ARN Interferente Pequeño/genética
5.
Anticancer Res ; 38(11): 6195-6200, 2018 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-30396937

RESUMEN

BACKGROUND/AIM: Serine/threonine kinase B-Raf proto-oncogene (BRAF) mutant colon cancer has a poor prognosis and there is an absence of targeted treatments for this subtype. Here, we investigated the effects of inhibition of casein kinase 2 (CK2) on the inhibitory effects of BRAF and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) inhibition in BRAF-mutant colon cancer cells. MATERIALS AND METHODS: Colon cancer cell lines with mutations in components of the mitogen-activated protein kinase (MAPK) and PI3K signalling pathway were used. Cell viability was determined after exposure to single agent and combinations of erlotinib (EGFR inhibitor), dabrafenib (MEK inhibitor), GDC0941 (PI3K inhibitor) and CX4945 (CK2 inhibitor). Western blots were used to examine MAPK and AKT serine/threonine kinase (AKT) pathway activation. RESULTS: Addition of CX4945 to dabrafenib did not enhance the antiproliferative effects of single-agent dabrafenib. Use of GDC0941 alone was highly effective in controlling growth of both BRAF-mutant and wild-type cells and this effect was enhanced by CK2 inhibition. CONCLUSION: Inhibition of the PI3K/AKT pathway is central to regulating growth of colon cancer cells and this can be enhanced by CK2 inhibition.


Asunto(s)
Neoplasias del Colon/metabolismo , Clorhidrato de Erlotinib/farmacología , Imidazoles/farmacología , Indazoles/farmacología , Naftiridinas/farmacología , Oximas/farmacología , Sulfonamidas/farmacología , Quinasa de la Caseína II/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HT29 , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mutación , Fenazinas , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas B-raf/genética
6.
Exp Cell Res ; 341(2): 196-206, 2016 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-26844628

RESUMEN

Beta-catenin plays a key role in transducing Wnt signals from the plasma membrane to the nucleus. Here we characterize an unusual subcellular distribution of beta-catenin in MCF-7 breast cancer cells, wherein beta-catenin localizes to the cytoplasm and membrane but atypically did not relocate to the nucleus after Wnt treatment. The inability of Wnt or the Wnt agonist LiCl to induce nuclear localization of beta-catenin was not due to defective nuclear transport, as the transport machinery was intact and ectopic GFP-beta-catenin displayed rapid nuclear entry in living cells. The mislocalization is explained by a shift in the retention of beta-catenin from nucleus to cytoplasm. The reduced nuclear retention is caused by unusually low expression of lymphoid enhancer factor/T-cell factor (LEF/TCF) transcription factors. The reconstitution of LEF-1 or TCF4 expression rescued nuclear localization of beta-catenin in Wnt treated cells. In the cytoplasm, beta-catenin accumulated in recycling endosomes, golgi and beta-COP-positive coatomer complexes. The peripheral association with endosomes diminished after Wnt treatment, potentially releasing ß-catenin into the cytoplasm for nuclear entry. We propose that in MCF-7 and perhaps other breast cancer cells, beta-catenin may contribute to cytoplasmic functions such as ER-golgi transport, in addition to its transactivation role in the nucleus.


Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , beta Catenina/metabolismo , Neoplasias de la Mama/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Células MCF-7 , Activación Transcripcional/fisiología , Proteínas Wnt/metabolismo
7.
Proteomics Clin Appl ; 7(1-2): 42-54, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23027722

RESUMEN

This review documents the uses of quantitative MS applied to colorectal cancer (CRC) proteomics for biomarker discovery and molecular pathway profiling. Investigators are adopting various labeling and label-free MS approaches to quantitate differential protein levels in cells, tumors, and plasma/serum. We comprehensively review recent uses of this technology to examine mouse models of CRC, CRC cell lines, their secretomes and subcellular fractions, CRC tumors, CRC patient plasma/serum, and stool samples. For biomarker discovery these approaches are uncovering proteins with potential diagnostic and prognostic utility, while in vitro cell culture experiments are characterizing proteomic and phosphoproteomic responses to disrupted signaling pathways due to mutations or to inhibition of drugable enzymes.


Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/metabolismo , Proteómica , Animales , Neoplasias Colorrectales/terapia , Humanos , Espectrometría de Masas
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