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1.
Cancers (Basel) ; 13(19)2021 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-34638453

RESUMO

Cancer is a group of heterogeneous diseases that results from the occurrence of genetic alterations combined with epigenetic changes and environmental stimuli that increase cancer cell plasticity. Indeed, multiple cancer cell populations coexist within the same tumour, favouring cancer progression and metastatic dissemination as well as drug resistance, thereby representing a major obstacle for treatment. Epigenetic changes contribute to the onset of intra-tumour heterogeneity (ITH) as they facilitate cell adaptation to perturbation of the tumour microenvironment. Despite being its central role, the intrinsic multi-layered and reversible epigenetic pattern limits the possibility to uniquely determine its contribution to ITH. In this review, we first describe the major epigenetic mechanisms involved in tumourigenesis and then discuss how single-cell-based approaches contribute to dissecting the key role of epigenetic changes in tumour heterogeneity. Furthermore, we highlight the importance of dissecting the interplay between genetics, epigenetics, and tumour microenvironments to decipher the molecular mechanisms governing tumour progression and drug resistance.

2.
Gut ; 2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436496

RESUMO

OBJECTIVE: Cancer stem cells are responsible for tumour spreading and relapse. Human epidermal growth factor receptor 2 (HER2) expression is a negative prognostic factor in colorectal cancer (CRC) and a potential target in tumours carrying the gene amplification. Our aim was to define the expression of HER2 in colorectal cancer stem cells (CR-CSCs) and its possible role as therapeutic target in CRC resistant to anti- epidermal growth factor receptor (EGFR) therapy. DESIGN: A collection of primary sphere cell cultures obtained from 60 CRC specimens was used to generate CR-CSC mouse avatars to preclinically validate therapeutic options. We also made use of the ChIP-seq analysis for transcriptional evaluation of HER2 activation and global RNA-seq to identify the mechanisms underlying therapy resistance. RESULTS: Here we show that in CD44v6-positive CR-CSCs, high HER2 expression levels are associated with an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which promotes the acetylation at the regulatory elements of the Erbb2 gene. HER2 targeting in combination with phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MEK) inhibitors induces CR-CSC death and regression of tumour xenografts, including those carrying Kras and Pik3ca mutation. Requirement for the triple targeting is due to the presence of cancer-associated fibroblasts, which release cytokines able to confer CR-CSC resistance to PI3K/AKT inhibitors. In contrast, targeting of PI3K/AKT as monotherapy is sufficient to kill liver-disseminating CR-CSCs in a model of adjuvant therapy. CONCLUSIONS: While PI3K targeting kills liver-colonising CR-CSCs, the concomitant inhibition of PI3K, HER2 and MEK is required to induce regression of tumours resistant to anti-EGFR therapies. These data may provide a rationale for designing clinical trials in the adjuvant and metastatic setting.

3.
Nat Genet ; 52(12): 1397-1411, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33169020

RESUMO

The genetic elements required to tune gene expression are partitioned in active and repressive nuclear condensates. Chromatin compartments include transcriptional clusters whose dynamic establishment and functioning depend on multivalent interactions occurring among transcription factors, cofactors and basal transcriptional machinery. However, how chromatin players contribute to the assembly of transcriptional condensates is poorly understood. By interrogating the effect of KMT2D (also known as MLL4) haploinsufficiency in Kabuki syndrome, we found that mixed lineage leukemia 4 (MLL4) contributes to the assembly of transcriptional condensates through liquid-liquid phase separation. MLL4 loss of function impaired Polycomb-dependent chromatin compartmentalization, altering the nuclear architecture. By releasing the nuclear mechanical stress through inhibition of the mechanosensor ATR, we re-established the mechanosignaling of mesenchymal stem cells and their commitment towards chondrocytes both in vitro and in vivo. This study supports the notion that, in Kabuki syndrome, the haploinsufficiency of MLL4 causes an altered functional partitioning of chromatin, which determines the architecture and mechanical properties of the nucleus.


Assuntos
Anormalidades Múltiplas/genética , Núcleo Celular/fisiologia , Cromatina/metabolismo , Face/anormalidades , Haploinsuficiência/genética , Doenças Hematológicas/genética , Histona-Lisina N-Metiltransferase/genética , Doenças Vestibulares/genética , Células 3T3 , Animais , Linhagem Celular , Linhagem da Célula/genética , Condrócitos/citologia , Condrogênese/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Mecanotransdução Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Camundongos , Osteócitos/citologia , Osteogênese/genética , Proteínas do Grupo Polycomb/genética , Estresse Mecânico
4.
Oncogene ; 38(40): 6647-6661, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31391555

RESUMO

At the molecular level, epithelial-to-mesenchymal transition (EMT) necessitates extensive transcriptional reprogramming which is orchestrated by a small group of gene-regulatory factors that include the zinc-finger DNA-binding protein SNAIL1. Although SNAIL1 is a well-known master regulator of EMT, knowledge of its immediate target genes is incomplete. Here, we used ChIP-seq to identify genes directly regulated by SNAIL1 in colorectal adenocarcinoma cells. When comparing the genomic distribution of SNAIL1 to that of the intestinal stem cell (ISC) transcription factors ASCL2 and TCF7L2, we observed a significant overlap. Furthermore, SNAIL1 ChIP-seq peaks are associated with a substantial fraction of ISC signature genes. In two colorectal cancer cell lines, we verified that SNAIL1 decreases ISC marker expression. Likewise, SNAIL1 directly represses the proto-oncogene MYB, and the long noncoding RNA (lncRNA) WiNTRLINC1, a recently described regulator of ASCL2. SNAIL1 targets multiple regulatory elements at the MYB and WiNTRLINC1 loci, and displaces ASCL2 and TCF7L2 from their binding regions at a MYB downstream regulatory element. Correlation analyses and expression profiling showed antiparallel expression of SNAIL1 and MYB in colorectal and breast cancer cell lines and tumor transcriptomes, suggesting that SNAIL1 controls MYB expression in different tissues. MYB loss-of-function attenuated proliferation and impaired clonogenicity in two- and three-dimensional cell cultures. Therefore, SNAIL1-mediated downregulation of MYB and ISC markers like WiNTRLINC1 likely contributes to the decrease in proliferation known to be associated with EMT, while simultaneously abrogating stemness features of colorectal cancer cells. Apparently, the relationship between EMT and stemness varies in different tumor entities.


Assuntos
Adenocarcinoma/metabolismo , Mapeamento Cromossômico , Neoplasias Colorretais/metabolismo , DNA de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Sítios de Ligação , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas de Neoplasias/metabolismo
5.
PLoS Genet ; 13(11): e1007109, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29155818

RESUMO

Phenotypic conversion of tumor cells through epithelial-mesenchymal transition (EMT) requires massive gene expression changes. How these are brought about is not clear. Here we examined the impact of the EMT master regulator SNAIL1 on the FOXA family of transcription factors which are distinguished by their particular competence to induce chromatin reorganization for the activation of transcriptional enhancer elements. We show that the expression of SNAIL1 and FOXA genes is anticorrelated in transcriptomes of colorectal tumors and cell lines. In cellular EMT models, ectopically expressed Snail1 directly represses FOXA1 and triggers downregulation of all FOXA family members, suggesting that loss of FOXA expression promotes EMT. Indeed, cells with CRISPR/Cas9-induced FOXA-deficiency acquire mesenchymal characteristics. Furthermore, ChIP-seq data analysis of FOXA chromosomal distribution in relation to chromatin structural features which characterize distinct states of transcriptional activity, revealed preferential localization of FOXA factors to transcriptional enhancers at signature genes that distinguish epithelial from mesenchymal colon tumors. To validate the significance of this association, we investigated the impact of FOXA factors on structure and function of enhancers at the CDH1, CDX2 and EPHB3 genes. FOXA-deficiency and expression of dominant negative FOXA2 led to chromatin condensation at these enhancer elements. Site-directed mutagenesis of FOXA binding sites in reporter gene constructs and by genome-editing in situ impaired enhancer activity and completely abolished the active chromatin state of the EPHB3 enhancer. Conversely, expression of FOXA factors in cells with inactive CDX2 and EPHB3 enhancers led to chromatin opening and de novo deposition of the H3K4me1 and H3K27ac marks. These findings establish the pioneer function of FOXA factors at enhancer regions of epithelial genes and demonstrate their essential role in maintaining enhancer structure and function. Thus, by repressing FOXA family members, SNAIL1 targets transcription factors at strategically important positions in gene-regulatory hierarchies, which may facilitate transcriptional reprogramming during EMT.


Assuntos
Neoplasias Colorretais/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral/metabolismo , Neoplasias Colorretais/metabolismo , Elementos Facilitadores Genéticos , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Genes Reguladores , Humanos , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Elementos Reguladores de Transcrição , Fatores de Transcrição da Família Snail/genética
6.
Biochim Biophys Acta ; 1859(11): 1353-1367, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27504909

RESUMO

Transcriptional silencing is a major cause for the inactivation of tumor suppressor genes, however, the underlying mechanisms are only poorly understood. The EPHB2 gene encodes a receptor tyrosine kinase that controls epithelial cell migration and allocation in intestinal crypts. Through its ability to restrict cell spreading, EPHB2 functions as a tumor suppressor in colorectal cancer whose expression is frequently lost as tumors progress to the carcinoma stage. Previously we reported that EPHB2 expression depends on a transcriptional enhancer whose activity is diminished in EPHB2 non-expressing cells. Here we investigated the mechanisms that lead to EPHB2 enhancer inactivation. We show that expression of EPHB2 and SNAIL1 - an inducer of epithelial-mesenchymal transition (EMT) - is anti-correlated in colorectal cancer cell lines and tumors. In a cellular model of Snail1-induced EMT, we observe that features of active chromatin at the EPHB2 enhancer are diminished upon expression of murine Snail1. We identify the transcription factors FOXA1, MYB, CDX2 and TCF7L2 as EPHB2 enhancer factors and demonstrate that Snail1 indirectly inactivates the EPHB2 enhancer by downregulation of FOXA1 and MYB. In addition, Snail1 induces the expression of Lymphoid enhancer factor 1 (LEF1) which competitively displaces TCF7L2 from the EPHB2 enhancer. In contrast to TCF7L2, however, LEF1 appears to repress the EPHB2 enhancer. Our findings underscore the importance of transcriptional enhancers for gene regulation under physiological and pathological conditions and show that SNAIL1 employs a combinatorial mechanism to inactivate the EPHB2 enhancer based on activator deprivation and competitive displacement of transcription factors.


Assuntos
Regulação para Baixo , Elementos Facilitadores Genéticos , Transição Epitelial-Mesenquimal/genética , Inativação Gênica , Receptor EphB2/genética , Fatores de Transcrição da Família Snail/fisiologia , Transativadores/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Linhagem Celular , Cromatina/metabolismo , Humanos
7.
Oncotarget ; 7(33): 53526-53539, 2016 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-27447549

RESUMO

Here we study the effects of inducible oncogenic K-Ras (G12V) expression on the polarized morphogenesis of colonic epithelial cells. We provide evidence that the autocrine production of heregulins, ligands for the ErbB3 receptor tyrosine kinase, is responsible for the hyperproliferation and aberrant 3D morphogenesis upon oncogenic K-Ras expression. This is in line with results obtained in primary intestinal organoid cultures, in which exogenous heregulin is shown to interfere with normal tissue architecture. Importantly, ErbB3 inhibition and heregulin gene silencing rescued K-RasG12V-induced features of cell transformation. Together with the increased ErbB3 positivity detected in human high-grade primary colorectal cancers, our findings provide support for an autocrine signaling loop engaged by oncogenic K-Ras involving ErbB3 that contributes to the dedifferentiation of the intestinal epithelium during tumor initiation and progression.


Assuntos
Comunicação Autócrina/fisiologia , Transformação Celular Neoplásica/metabolismo , Mucosa Intestinal/patologia , Receptor ErbB-3/metabolismo , Proteínas ras/metabolismo , Desdiferenciação Celular/fisiologia , Linhagem Celular Tumoral , Polaridade Celular/fisiologia , Proliferação de Células , Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Mucosa Intestinal/metabolismo , Morfogênese/fisiologia
8.
PLoS One ; 9(9): e107165, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25198428

RESUMO

TRAIL is a death receptor ligand that induces cell death preferentially in tumor cells. Recombinant soluble TRAIL, however, performs poorly as an anti-cancer therapeutic because oligomerization is required for potent biological activity. We previously generated a diabody format of tumor-targeted TRAIL termed Db(αEGFR-sc)TRAIL, comprising single-stranded TRAIL molecules (scTRAIL) and the variable domains of a humanized variant of the EGFR blocking antibody Cetuximab. Here we define the bioactivity of Db(αEGFR)-scTRAIL with regard to both EGFR inhibition and TRAIL receptor activation in 3D cultures of Caco-2 colorectal cancer cells, which express wild-type K-Ras. Compared with conventional 2D cultures, Caco-2 cells displayed strongly enhanced sensitivity toward Db(αEGFR)-scTRAIL in these 3D cultures. We show that the antibody moiety of Db(αEGFR-sc)TRAIL not only efficiently competed with ligand-induced EGFR function, but also determined the apoptotic response by specifically directing Db(αEGFR)-scTRAIL to EGFR-positive cells. To address how aberrantly activated K-Ras, which leads to Cetuximab resistance, affects Db(αEGFR-sc)TRAIL sensitivity, we generated stable Caco-2tet cells inducibly expressing oncogenic K-Ras(G12V). In the presence of doxycycline, these cells showed increased resistance to Db(αEGFR-sc)TRAIL, associated with the elevated expression of the anti-apoptotic proteins cIAP2, Bcl-xL and FlipS. Co-treatment of cells with the Smac mimetic SM83 restored the Db(αEGFR)-scTRAIL-induced apoptotic response. Importantly, this synergy between Db(αEGFR)-scTRAIL and SM83 also translated to 3D cultures of oncogenic K-Ras expressing HCT-116 and LoVo colorectal cancer cells. Our findings thus support the notion that Db(αEGFR)-scTRAIL therapy in combination with apoptosis-sensitizing agents may be promising for the treatment of EGFR-positive colorectal cancers, independently of their KRAS status.


Assuntos
Apoptose/efeitos dos fármacos , Materiais Biomiméticos/farmacologia , Neoplasias Colorretais/patologia , Receptores ErbB/metabolismo , Mutação , Proteínas Proto-Oncogênicas/genética , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteínas ras/genética , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose , Linhagem Celular Tumoral , Cetuximab/farmacologia , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas p21(ras)
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