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1.
Am J Respir Crit Care Med ; 204(4): 431-444, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-33882264

RESUMO

Rationale: Lymphangioleiomyomatosis (LAM) is a multisystem disease that causes lung cysts and respiratory failure. Loss of TSC (tuberous sclerosis complex) gene function results in a clone of "LAM cells" with dysregulated mTOR (mechanistic target of rapamycin) activity. LAM cells and fibroblasts form lung nodules that also contain mast cells, although their significance is unknown. Objectives: To understand the mechanism of mast-cell accumulation and the role of mast cells in the pathogenesis of LAM. Methods: Gene expression was examined using transcriptional profiling and qRT-PCR. Mast cell/LAM nodule interactions were examined in vitro using spheroid TSC2-null cell/fibroblast cocultures and in vivo using an immunocompetent Tsc2-null murine homograft model. Measurements and Main Results: LAM-derived cell/fibroblast cocultures induced multiple CXC chemokines in fibroblasts. LAM lungs had increased tryptase-positive mast cells expressing CXCRs (CXC chemokine receptors) (P < 0.05). Mast cells located around the periphery of LAM nodules were positively associated with the rate of lung function loss (P = 0.016). LAM spheroids attracted mast cells, and this process was inhibited by pharmacologic and CRISPR/cas9 inhibition of CXCR1 and CXCR2. LAM spheroids caused mast-cell tryptase release, which induced fibroblast proliferation and increased LAM-spheroid size (1.36 ± 0.24-fold; P = 0.0019). The tryptase inhibitor APC366 and sodium cromoglycate (SCG) inhibited mast cell-induced spheroid growth. In vivo, SCG reduced mast-cell activation and Tsc2-null lung tumor burden (vehicle: 32.5.3% ± 23.6%; SCG: 5.5% ± 4.3%; P = 0.0035). Conclusions: LAM-cell/fibroblast interactions attract mast cells where tryptase release contributes to disease progression. Repurposing SCG for use in LAM should be studied as an alternative or adjunct to mTOR inhibitor therapy.


Assuntos
Biomarcadores Tumorais/metabolismo , Fibroblastos/metabolismo , Neoplasias Pulmonares/metabolismo , Linfangioleiomiomatose/metabolismo , Mastócitos/metabolismo , Triptases/metabolismo , Adulto , Animais , Biomarcadores Tumorais/genética , Quimiocinas/metabolismo , Progressão da Doença , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Linfangioleiomiomatose/genética , Linfangioleiomiomatose/patologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Esferoides Celulares , Células Tumorais Cultivadas
2.
BMC Womens Health ; 21(1): 423, 2021 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-34930226

RESUMO

BACKGROUND: Recent epidemiological studies suggest that reproductive factors are associated with breast cancer (BC) molecular subtypes. However, these associations have not been thoroughly studied in the African populations. The present study aimed to investigate the prevalence of BC molecular subtypes and assess their association with reproductive factors in Tanzanian BC patients. METHODS: This hospital-based case-only cross-sectional study consisted of 263 histologically confirmed BC patients in Tanzania. Clinico-pathological data, socio-demographic characteristics, anthropometric measurements, and reproductive risk factors were examined using the Chi-square test and one-way ANOVA. The association among reproductive factors and BC molecular subtypes was analyzed using multinomial logistic regression. The heterogeneity of the associations was assessed using the Wald test. RESULTS: We found evident subtype heterogeneity for reproductive factors. We observed that post-menopausal status was more prevalent in luminal-A subtype, while compared to luminal-A subtype, luminal-B and HER-2 enriched subtypes were less likely to be found in post-menopausal women (OR: 0.21, 95%CI 0.10-0.41, p = 0.001; OR: 0.39, 95%CI 0.17-0.89, p = 0.026, respectively). Also, the luminal-B subtype was more likely to be diagnosed in patients aged ≤ 40 years than the luminal-A subtype (OR: 2.80, 95%CI 1.46-5.32, p = 0.002). Women who had their first full-term pregnancy at < 30 years were more likely to be of luminal-B (OR: 2.71, 95%CI 1.18-4.17, p = 0.018), and triple-negative (OR: 2.28, 95%CI 1.02-4.07, p = 0.044) subtypes relative to luminal-A subtype. Furthermore, we observed that breastfeeding might have reduced odds of developing luminal-A, luminal-B and triple-negative subtypes. Women who never breastfed were more likely to be diagnosed with luminal-B and triple-negative subtypes when compared to luminal-A subtype (OR: 0.46, 95%CI 0.22-0.95, p = 0.035; OR: 0.41, 95%CI 0.20-0.85, p = 0.017, respectively). . CONCLUSION: Our results are the first data reporting reproductive factors heterogeneity among BC molecular subtypes in Tanzania. Our findings suggest that breast-feeding may reduce the likelihood of developing luminal-A, luminal-B, and triple-negative subtypes. Meanwhile, the first full-term pregnancy after 30 years of age could increase the chance of developing luminal-A subtype, a highly prevalent subtype in Tanzania. More interventions to promote modifiable risk factors across multiple levels may most successfully reduce BC incidence in Africa.


Assuntos
Neoplasias da Mama , Adulto , Neoplasias da Mama/diagnóstico , Estudos Transversais , Feminino , Humanos , Razão de Chances , Gravidez , Receptor ErbB-2 , Receptores de Progesterona , Fatores de Risco , Tanzânia/epidemiologia
3.
Int J Exp Pathol ; 101(3-4): 80-86, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32567731

RESUMO

ApcMin/+ mice are regarded as a standard animal model of colorectal cancer (CRC). Tensin4 (TNS4 or Cten) is a putative oncogene conferring features of stemness and promoting motility. Our objective was to assess TNS4 expression in intestinal adenomas and determine whether TNS4 is upregulated by Wnt signalling. ApcMin/+ mice (n = 11) were sacrificed at approximately 120 days old at the onset of anaemia signs. Small intestines were harvested, and Swiss roll preparations were tested for TNS4 expression by immunohistochemistry (IHC). Individual polyps were also separately collected (n = 14) and tested for TNS4 mRNA expression and Kras mutation. The relationship between Wnt signalling and TNS4 expression was tested by Western blotting in the human CRC cell line HCT116 after inhibition of ß-catenin activity with MSAB or its increase by transfection with a Flag ß-catenin expression vector. Overall, 135/148 (91.2%) of the total intestinal polyps were positive for TNS4 expression by IHC, whilst adjacent normal areas were negative. RT-qPCR analysis showed approximately 5-fold upregulation of TNS4 mRNA in the polyps compared to adjacent normal tissue and no Kras mutations were detected. In HCT116, ß-catenin inhibition resulted in reduced TNS4 expression, and conversely, ß-catenin overexpression resulted in increased TNS4 expression. In conclusion, this is the first report linking aberrant Wnt signalling to upregulation of TNS4 both during initiation of intestinal adenomas in mice and in in vitro models. The exact contribution of TNS4 to adenoma development remains to be investigated, but the ApcMin/+ mouse represents a good model to study this.


Assuntos
Pólipos Adenomatosos/metabolismo , Genes APC , Neoplasias Intestinais/metabolismo , Intestino Delgado/metabolismo , Tensinas/metabolismo , Via de Sinalização Wnt , Pólipos Adenomatosos/genética , Pólipos Adenomatosos/patologia , Animais , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Intestino Delgado/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tensinas/genética , Regulação para Cima , beta Catenina/metabolismo
4.
Int J Mol Sci ; 20(11)2019 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-31167387

RESUMO

Acute myeloid leukaemia (AML) is a heterogeneous clonal malignancy of hematopoietic progenitor cells. The Wnt pathway and its downstream targets are tightly regulated by ß-catenin. We recently discovered a new protein, FLYWCH1, which can directly bind nuclear ß-catenin. Herein, we studied the FLYWCH1/ß-catenin pathway in AML cells using qRT-PCR, Western blot, and immunofluorescence assays. In addition, the stemness activity and cell cycle were analysed by the colony-forming unit (CFU) using methylcellulose-based and Propidium iodide/flow cytometry assays. We found that FLYWCH1 mRNA and protein were differentially expressed in the AML cell lines. C-Myc, cyclin D1, and c-Jun expression decreased in the presence of higher FLYWCH1 expression, and vice versa. There appeared to be the loss of FLYWCH1 expression in dividing cells. The sub-G0 phase was prolonged and shortened in the low and high FLYWCH1 expression cell lines, respectively. The G0/G1 arrest correlated with FLYWCH1-expression, and these cell lines also formed colonies, whereas the low FLYWCH1 expression cell lines could not. Thus, FLYWCH1 functions as a negative regulator of the Wnt/ß-catenin pathway in AML.


Assuntos
Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Imunofluorescência , Humanos , Células-Tronco Neoplásicas/metabolismo , RNA Mensageiro/genética , Via de Sinalização Wnt
5.
Int J Exp Pathol ; 95(6): 392-400, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25348333

RESUMO

Wnt signalling and the signal transducer and activator of transcription 3 (STAT3) are oncogenic signalling pathways which are deregulated in colorectal cancer (CRC). Here we investigated the interaction of these two pathways. Firstly, we investigated biochemical interaction by inhibiting STAT3 and ß-catenin (through gene knock-down and dominant-negative TCF4 expression) in nine CRC cell lines. ß-catenin inhibition did not affect STAT3 levels, whereas STAT3 knock-down resulted in reduced ß-catenin mRNA and protein levels. The reduction in ß-catenin protein was not prevented by proteasome inhibition, and IL6-induced STAT3 activation resulted in increased ß-catenin mRNA. This suggests that STAT3 positively regulates ß-catenin (at a transcriptional level) and evaluation of 44 CRCs by immunostaining supported this by showing an association between nuclear STAT3 expression and nuclear ß-catenin (P = 0.022). We tested the functional interaction between STAT3 and Wnt signalling by knocking down STAT3 and ß-catenin individually and in combination. Knock-down of ß-catenin and STAT3 individually inhibited cell proliferation (P < 0. 001 for each) through G1 arrest. However, simultaneous knock-down of STAT3 and ß-catenin had a significantly weaker effect than knock-down of ß-catenin alone (P < 0.01). Knock-down of STAT3 and ß-catenin, individually and together, inhibited cell motility (P < 0.001) without evidence of interaction. We conclude that STAT3 regulates ß-catenin but ß-catenin does not regulate STAT3. The STAT3/ß-catenin interaction is complex but may reduce the proliferative activity of ß-catenin possibly by taking ß-catenin protein beyond the optimal level. This may indicate biological differences in tumours where both STAT3 and ß-catenin are activated compared to those where only one is activated.


Assuntos
Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição STAT3/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Apoptose/fisiologia , Proliferação de Células/fisiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Técnicas de Silenciamento de Genes , Células HT29 , Humanos , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/genética , beta Catenina/genética
6.
EMBO J ; 28(13): 1843-54, 2009 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-19521338

RESUMO

Wnt signalling is a crucial signalling pathway controlling intestinal homeostasis and cancer. We show here that the JNK MAP kinase pathway and one of its most important substrates, the AP-1 transcription factor c-Jun, modulates Wnt signalling strength in the intestine. Transgenic gut-specific augmentation of JNK signalling stimulated progenitor cell proliferation and migration, resulting in increased villus length. In the crypt, c-Jun protein was highly expressed in progenitor cells and the absence of c-Jun resulted in decreased proliferation and villus length. In addition to several known c-Jun/AP-1 target genes, expression of Wnt target genes Axin2 and Lgr5 were stimulated by JNK activation, suggesting a cross talk of JNK to Wnt signalling. Expression of the Wnt pathway component TCF4 was controlled by JNK activity, and chromatin immunoprecipitation and reporter assays identified tcf4 as a direct c-Jun target gene. Consequently, increased JNK activity accelerated tumourigenesis in a model of colorectal carcinogenesis. As c-jun is a direct target of the TCF4/beta-catenin complex, the control of tcf4 expression by JNK/c-Jun leads to a positive feedback loop that connects JNK and Wnt signalling. This mechanism regulates the physiological function of progenitor cells and oncogenic transformation.


Assuntos
Carcinoma/fisiopatologia , Neoplasias do Colo/fisiopatologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/metabolismo , Proteínas Wnt/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Intestinos/citologia , Intestinos/fisiopatologia , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas c-jun/genética , Transdução de Sinais , Células-Tronco/citologia , Fatores de Transcrição TCF/genética , Fator de Transcrição 4 , Fator de Transcrição AP-1/genética , Proteínas Wnt/genética
7.
Stem Cells ; 30(10): 2076-87, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22851508

RESUMO

Embryonic NANOG (NANOG1) is considered as an important regulator of pluripotency while NANOGP8 (NANOG-pseudogene) plays a role in tumorigenesis. Herein, we show NANOG is expressed from both NANOG1 and NANOGP8 in human colorectal cancers (CRC). Enforced NANOG1-expression increases clonogenic potential and tumor formation in xenograft models, although it is expressed only in a small subpopulation of tumor cells and is colocalized with endogenous nuclear ß-catenin(High) . Moreover, single NANOG1-CRCs form spherical aggregates, similar to the embryoid body of embryonic stem cells (ESCs), and express higher levels of stem-like Wnt-associated target genes. Furthermore, we show that NANOG1-expression is positively regulated by c-JUN and ß-catenin/TCF4. Ectopic expression of c-Jun in murine Apc(Min/+) -ESCs results in the development of larger xenograft tumors with higher cell density compared to controls. Chromatin immunoprecipitation assays demonstrate that c-JUN binds to the NANOG1-promoter via the octamer M1 DNA element. Collectively, our data suggest that ß-Catenin/TCF4 and c-JUN together drive a subpopulation of CRC tumor cells that adopt a stem-like phenotype via the NANOG1-promoter.


Assuntos
Complexo 1 de Proteínas Adaptadoras/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Neoplasias Colorretais/metabolismo , Proteínas de Homeodomínio/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição/metabolismo , Complexo 1 de Proteínas Adaptadoras/genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Sítios de Ligação , Transformação Celular Neoplásica , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos , Proteína Homeobox Nanog , Transplante de Neoplasias , Células-Tronco Neoplásicas/patologia , Regiões Promotoras Genéticas , Ligação Proteica , Pseudogenes , Transdução de Sinais/genética , Fator de Transcrição 4 , Fatores de Transcrição/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética
8.
Genes Dis ; 9(5): 1220-1233, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35873024

RESUMO

The immune system plays an important role in protecting the body against malignancy. During cancer immunoediting, the immune system can recognize and keep checking the tumor cells by down-expression of some self-molecules or by increasing expression of some novel molecules. However, the microenvironment created in the course of cancer development hampers the immune ability to recognize and destroy the transforming cells. Human Leukocyte Antigen G (HLA-G) is emerging as immune checkpoint molecule produced more by cancer cells to weaken the immune response against them. HLA-G is a non-classical HLA class I molecule which is normally expressed in immune privileged tissues as a soluble or membrane-bound protein. HLA-G locus is highly polymorphic in the non-coding 3' untranslated region (UTR) and in the 5' upstream regulatory region (5' URR). HLA-G expression is controlled by polymorphisms located in these regions, and several association studies between these polymorphic sites and disease predisposition, response to therapy, and/or HLA-G protein expression have been reported. Various polymorphisms are demonstrated to modulate its expression and this is increasingly finding more significance in cancer biology. This review focuses on the relevance of the HLA-G gene and its polymorphisms in cancer development. We highlight population genetics of HLA-G as evidence to espouse the need and importance of exploring potential utility of HLA-G in cancer diagnosis, prognosis and immunotherapy in the currently understudied African population.

9.
Oncogenesis ; 11(1): 4, 2022 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-35046388

RESUMO

Little is known about the role of microRNAs (miRNAs) in rewiring the metabolism within tumours and adjacent non-tumour bearing normal tissue and their potential in cancer therapy. This study aimed to investigate the relationship between deregulated miRNAs and metabolic components in murine duodenal polyps and non-polyp-derived organoids (mPOs and mNPOs) from a double-mutant ApcMinFbxw7∆G mouse model of intestinal/colorectal cancer (CRC). We analysed the expression of 373 miRNAs and 12 deregulated metabolic genes in mPOs and mNPOs. Our findings revealed miR-135b might target Spock1. Upregulation of SPOCK1 correlated with advanced stages of CRCs. Knockdown of miR-135b decreased the expression level of SPOCK1, glucose consumption and lactic secretion in CRC patient-derived tumours organoids (CRC tPDOs). Increased SPOCK1 induced by miR-135b overexpression promoted the Warburg effect and consequently antitumour effect of 5-fluorouracil. Thus, combination with miR-135b antisense nucleotides may represent a novel strategy to sensitise CRC to the chemo-reagent based treatment.

10.
EMBO J ; 26(23): 4891-901, 2007 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-17972914

RESUMO

The ERK MAPK signalling pathway is a highly conserved kinase cascade linking transmembrane receptors to downstream effector mechanisms. To investigate the function of ERK in neurons, a constitutively active form of MEK1 (caMEK1) was conditionally expressed in the murine brain, which resulted in ERK activation and caused spontaneous epileptic seizures. ERK activation stimulated phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) and augmented NMDA receptor 2B (NR2B) protein levels. Pharmacological inhibition of NR2B function impaired synaptic facilitation in area cornus ammonicus region 3 (CA3) in acute hippocampal slices derived from caMEK1-expressing mice and abrogated epilepsy in vivo. In addition, expression of caMEK1 caused phosphorylation of the transcription factor, cAMP response element-binding protein (CREB) and increased transcription of ephrinB2. EphrinB2 overexpression resulted in increased NR2B tyrosine phosphorylation, which was essential for caMEK1-induced epilepsy in vivo, since conditional inactivation of ephrinB2 greatly reduced seizure frequency in caMEK1 transgenic mice. Therefore, our study identifies a mechanism of epileptogenesis that links MAP kinase to Eph/Ephrin and NMDA receptor signalling.


Assuntos
Epilepsia/etiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Animais , AMP Cíclico/metabolismo , Ativação Enzimática , Efrina-B2/metabolismo , Epilepsia/enzimologia , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Fosforilação , Receptores de N-Metil-D-Aspartato/metabolismo , Transcrição Gênica
11.
Nature ; 437(7056): 281-5, 2005 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-16007074

RESUMO

The proto-oncoprotein c-Jun is a component of the AP-1 transcription factor, the activity of which is augmented in many tumour types. An important mechanism in the stimulation of AP-1 function is amino-terminal phosphorylation of c-Jun by the c-Jun N-terminal kinases (JNKs). Phosphorylated c-Jun is biologically more active, partially because it acquires the ability to interact with binding partners. Here we show that phosphorylated c-Jun interacts with the HMG-box transcription factor TCF4 to form a ternary complex containing c-Jun, TCF4 and beta-catenin. Chromatin immunoprecipitation assays revealed JNK-dependent c-Jun-TCF4 interaction on the c-jun promoter, and c-Jun and TCF4 cooperatively activated the c-jun promoter in reporter assays in a beta-catenin-dependent manner. In the Apc(Min) mouse model of intestinal cancer, genetic abrogation of c-Jun N-terminal phosphorylation or gut-specific conditional c-jun inactivation reduced tumour number and size and prolonged lifespan. Therefore, the phosphorylation-dependent interaction between c-Jun and TCF4 regulates intestinal tumorigenesis by integrating JNK and APC/beta-catenin, two distinct pathways activated by WNT signalling.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/patologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fatores de Transcrição/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Proteínas do Citoesqueleto/metabolismo , Genes APC , Genes jun/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Fosforilação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-jun/genética , Transdução de Sinais , Fatores de Transcrição TCF , Transativadores/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição , Proteínas Wnt , beta Catenina
12.
Clin Transl Med ; 11(2): e308, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33635003

RESUMO

Carcinomas are complex heterocellular systems containing epithelial cancer cells, stromal fibroblasts, and multiple immune cell-types. Cell-cell communication between these tumor microenvironments (TME) and cells drives cancer progression and influences response to existing therapies. In order to provide better treatments for patients, we must understand how various cell-types collaborate within the TME to drive cancer and consider the multiple signals present between and within different cancer types. To investigate how tissues function, we need a model to measure both how signals are transferred between cells and how that information is processed within cells. The interplay of collaboration between different cell-types requires cell-cell communication. This article aims to review the current in vitro and in vivo mono-cellular and multi-cellular cultures models of colorectal cancer (CRC), and to explore how they can be used for single-cell multi-omics approaches for isolating multiple types of molecules from a single-cell required for cell-cell communication to distinguish cancer cells from normal cells. Integrating the existing single-cell signaling measurements and models, and through understanding the cell identity and how different cell types communicate, will help predict drug sensitivities in tumor cells and between- and within-patients responses.


Assuntos
Comunicação Celular/fisiologia , Neoplasias Colorretais/patologia , Transdução de Sinais/fisiologia , Microambiente Tumoral/fisiologia , Animais , Técnicas de Cultura de Células , Drosophila , Humanos , Camundongos
13.
Cells ; 10(4)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33924684

RESUMO

Over recent years, several Cys2-His2 (C2H2) domain-containing proteins have emerged as critical players in repairing DNA-double strand breaks. Human FLYWCH1 is a newly characterised nuclear transcription factor with (C2H2)-type zinc-finger DNA-binding domains. Yet, our knowledge about FLYWCH1 is still in its infancy. This study explores the expression, role and regulation of FLYWCH1 in the context of DNA damage and repair. We provide evidence suggesting a potential contribution of FLYWCH1 in facilitating the recruitment of DNA-damage response proteins (DDRPs). We found that FLYWCH1 colocalises with γH2AX in normal fibroblasts and colorectal cancer (CRC) cell lines. Importantly, our results showed that enforced expression of FLYWCH1 induces the expression of γH2AX, ATM and P53 proteins. Using an ATM-knockout (ATMKO) model, we indicated that FLYWCH1 mediates the phosphorylation of H2AX (Ser139) independently to ATM expression. On the other hand, the induction of DNA damage using UV-light induces the endogenous expression of FLYWCH1. Conversely, cisplatin treatment reduces the endogenous level of FLYWCH1 in CRC cell lines. Together, our findings uncover a novel FLYWCH1/H2AX phosphorylation axis in steady-state conditions and during the induction of the DNA-damage response (DDR). Although the role of FLYWCH1 within the DDR machinery remains largely uncharacterised and poorly understood, we here report for the first-time findings that implicate FLYWCH1 as a potential participant in the DNA damage response signaling pathways.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , DNA/genética , Histonas/genética , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Linhagem Celular , Linhagem Celular Tumoral , Cisplatino/farmacologia , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HCT116 , Histonas/metabolismo , Humanos , Fosforilação , Ribonuclease III/genética , Ribonuclease III/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
14.
Acta Biomater ; 132: 272-287, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34023456

RESUMO

Recent advances in intestinal organoid technologies have paved the way for in vitro recapitulation of the homeostatic renewal of adult tissues, tissue or organ morphogenesis during development, and pathogenesis of many disorders. In vitro modelling of individual patient diseases using organoid systems have been considered key in establishing rational design of personalized treatment strategies and in improving therapeutic outcomes. In addition, the transplantation of organoids into diseased tissues represents a novel approach to treat currently incurable diseases. Emerging evidence from intensive studies suggests that organoid systems' development and functional maturation depends on the presence of an extracellular matrix with suitable biophysical properties, where advanced synthetic hydrogels open new avenues for theoretical control of organoid phenotypes and potential applications of organoids in therapeutic purposes. In this review, we discuss the status, applications, challenges and perspectives of intestinal organoid systems emphasising on hydrogels and their properties suitable for intestinal organoid culture. We provide an overview of hydrogels used for intestinal organoid culture and key factors regulating their biological activity. The comparison of different hydrogels would be a theoretical basis for establishing design principles of synthetic niches directing intestinal cell fates and functions. STATEMENT OF SIGNIFICANCE: Intestinal organoid is an in vitro recapitulation of the gut, which self-organizes from intestinal stem cells and maintains many features of the native tissue. Since the development of this technology, intestinal organoid systems have made significant contribution to rapid progress in intestinal biology. Prevailing methodology for organoid culture, however, depends on animal-derived matrices and suffers from variability and potential risk for contamination of pathogens, limiting their therapeutic application. Synthetic scaffold matrices, hydrogels, might provide solutions to these issues and deepen our understanding on how intestinal cells sense and respond to key biophysical properties of the surrounding matrices. This review provides an overview of developing intestinal models and biomaterials, thereby leading to better understanding of current intestinal organoid systems for both biologists and materials scientists.


Assuntos
Materiais Biocompatíveis , Organoides , Animais , Humanos , Hidrogéis , Células-Tronco , Tecnologia
15.
J Pathol ; 218(1): 57-65, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19214987

RESUMO

The Tensin gene family encodes proteins thought to modulate integrin function. C-terminal Tensin-like (CTEN) is a member of the Tensin gene family which lacks the N-terminus actin-binding domain. Cten is reported to have both oncogenic and tumour-suppressor functions. We investigated the role that Cten may play in colorectal cancer (CRC). By quantitative RT-PCR CTEN is up-regulated (i.e. > two-fold increase) in 62% of cell lines and 69% of tumours compared with normal mucosa, consistent with CTEN being a possible oncogene. Stable transfection of HCT116 and SW480 (CRC cell lines with low endogenous Cten expression) with a Cten expression vector gave identical results in both cell lines. Forced Cten expression did not cause change in cell numbers, although it did confer resistance to staurosporine-induced apoptosis (p < 0.005). Cten also induced epithelial-mesenchymal transition (EMT) in tumour cells accompanied by a significant increase in both cell migration (transwell migration and cell wounding assays, p < 0.001 and p < 0.05, respectively) and cell invasion (invasion through Matrigel, p < 0.001). Given the observed EMT, we investigated the levels of E-cadherin. Cten induction was associated with a reduction in E-cadherin protein expression but not levels of E-cadherin mRNA. These data suggest that CTEN is an oncogene in CRC which stimulates EMT, cell migration and invasion and may therefore have a role in tumour invasion/spread. Furthermore, Cten induction is associated with post-transcriptional repression of E-cadherin.


Assuntos
Caderinas/genética , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Proteínas dos Microfilamentos/genética , Oncogenes , Linhagem Celular , Movimento Celular , Proliferação de Células , Colo/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Imuno-Histoquímica , Microscopia Confocal , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estaurosporina/uso terapêutico , Tensinas , Transfecção
16.
Methods Mol Biol ; 2171: 257-269, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32705648

RESUMO

Organoid culture faithfully reproduces the in vivo characteristics of the intestinal/colon epithelium and elucidates molecular mechanisms underlying the regulation of stem cell compartment that, if altered, may lead tumorigenesis. CRISPR-Cas9 based editing technology has provided promising opportunities for targeted loss-of-function mutations at chosen sites in the genome of eukaryotes. Herein, we demonstrate a CRISPR/Cas9-mediated mutagenesis-based screening method using murine intestinal organoids by investigating the phenotypical morphology of Cas9-expressing murine intestinal organoids. Murine intestinal crypts can be isolated and seeded into Matrigel and grown into stable organoid lines. Organoids subsequently transduced and selected to generate Cas9 expressing organoids. These organoids can be further transduced with the second lentiviruses expressing guide RNA (gRNA) (s) and screened for 8-10 days using bright-field and fluorescent microscopy to determine possible morphological or phenotypical abnormalities. Via phenotypical screening analysis, the candidate knockouts can be selected based on differential abnormal growth pattern vs their untransduced or lenti-GFP transduced controls. Further assessment of these knockout organoids can be done via phalloidin and propidium iodide (PI) staining, proliferation assay and qRT-PCR and also biochemical analysis. This CRISPR/Cas9 organoid mutagenesis-based screening method provides a reliable and rapid approach for investigating large numbers of genes with unknown/poorly identified biological functions. Knockout intestinal organoids can be associated with the key biological function of the gene(s) in development, homeostasis, disease progression, tumorigenesis, and drug screening, thereby reducing and potentially replacing animal models.


Assuntos
Organoides/citologia , Organoides/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Edição de Genes , Lentivirus/genética , Camundongos , Camundongos Transgênicos , RNA Guia de Cinetoplastídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
Cancers (Basel) ; 12(1)2019 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-31906201

RESUMO

Tumour-promoting inflammation is involved in colorectal cancer (CRC) development and therapeutic resistance. However, the antibiotics and antibacterial drugs and signalling that regulate the potency of anticancer treatment upon forced differentiation of cancer stem-like cell (CSC) are not fully defined yet. We screened an NIH-clinical collection of the small-molecule compound library of antibacterial/anti-inflammatory agents that identified potential candidate drugs targeting CRC-SC for differentiation. Selected compounds were validated in both in vitro organoids and ex vivo colon explant models for their differentiation induction, impediment on neoplastic cell growth, and to elucidate the mechanism of their anticancer activity. We initially focused on AM404, an anandamide uptake inhibitor. AM404 is a metabolite of acetaminophen with antibacterial activity, which showed high potential in preventing CRC-SC features, such as stemness/de-differentiation, migration and drug-resistance. Furthermore, AM404 suppressed the expression of FBXL5 E3-ligase, where AM404 sensitivity was mimicked by FBXL5-knockout. This study uncovers a new molecular mechanism for AM404-altering FBXL5 oncogene which mediates chemo-resistance and CRC invasion, thereby proposes to repurpose antibacterial AM404 as an anticancer agent.

18.
Cells ; 8(10)2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31635307

RESUMO

Ataxia-telegiectasia mutated (ATM), phosphatase and tensin homolog (PTEN), and p85α are key tumour suppressors. Whether ATM regulates PTEN expression and influence platinum sensitivity is unknown. We generated ATM knockdowns (KD) and CRISPR knock outs (KO) in glioblastoma (LN18, LN229) and ovarian cancer cells (OVCAR3, OVCAR4). Doxycycline inducible PTEN expression was generated in LN18 and LN229 cells. Transient KD of p85α, CK2, and XIAP was accomplished using siRNAs. Stable p85α knock-in was isolated in LN18 cells. Molecular biology assays included proteasome activity assays, PCR, flow cytometry analysis (cell cycle, double strand break accumulation, apoptosis), immunofluorescence, co-immunoprecipitation, clonogenic, invasion, migration, and 3D neurosphere assays. The clinicopathological significance of ATM, PTEN, p85α, and XIAP (X-linked inhibitor of apoptosis protein) was evaluated in 525 human ovarian cancers using immunohistochemistry. ATM regulated PTEN is p85α dependant. ATM also controls CK2α level which in turn phosphorylates and stabilizes PTEN. In addition, p85α physically interacts with CK2α and protects CK2α from ATM regulated degradation. ATM deficiency resulted in accumulation of XIAP/p-XIAP levels which ubiquitinated PTEN and CK2α thereby directing them to degradation. ATM depletion in the context of p85α deficiency impaired cancer cell migration and invasion reduced 3D-neurosphere formation and increased toxicity to cisplatin chemotherapy. Increased sensitivity to platinum was associated with DNA double strand breaks accumulation, cell cycle arrest, and induction of autophagy. In ovarian cancer patients, ATM, PTEN, p85α, and XIAP protein levels predicted better progression free survival after platinum therapy. We unravel a previously unknown function of ATM in the regulation of PTEN throµgh XIAP mediated proteasome degradation.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Platina/uso terapêutico , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Western Blotting , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cisplatino/uso terapêutico , Doxiciclina/uso terapêutico , Feminino , Citometria de Fluxo , Células HeLa , Humanos , Neoplasias Ovarianas/genética , PTEN Fosfo-Hidrolase/genética , Plasmídeos/genética , Estabilidade Proteica/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Análise Serial de Tecidos , Ubiquitina-Proteína Ligases/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética
19.
Oncogenesis ; 8(3): 13, 2019 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-30783098

RESUMO

Colorectal cancer (CRC) patients develop recurrence after chemotherapy owing to the survival of stem cell-like cells referred to as cancer stem-like cells (CSCs). The origin of CSCs is linked to the epithelial-mesenchymal transition (EMT) process. Currently, it remains poorly understood how EMT programmes enable CSCs residing in the tumour microenvironment to escape the effects of chemotherapy. This study identifies a key molecular pathway that is responsible for the formation of drug-resistant CSC populations. Using a modified yeast-2-hybrid system and 2D gel-based proteomics methods, we show that the E3-ubiquitin ligase FBXW7 directly binds and degrades the EMT-inducing transcription factor ZEB2 in a phosphorylation-dependent manner. Loss of FBXW7 induces an EMT that can be effectively reversed by knockdown of ZEB2. The FBXW7-ZEB2 axis regulates such important cancer cell features, as stemness/dedifferentiation, chemoresistance and cell migration in vitro, ex vivo and in animal models of metastasis. High expression of ZEB2 in cancer tissues defines the reduced ZEB2 expression in the cancer-associated stroma in patients and in murine intestinal organoids, demonstrating a tumour-stromal crosstalk that modulates a niche and EMT activation. Our study thus uncovers a new molecular mechanism, by which the CRC cells display differences in resistance to chemotherapy and metastatic potential.

20.
Neuron ; 43(1): 57-67, 2004 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-15233917

RESUMO

Nerve injury triggers numerous changes in the injured neurons and surrounding nonneuronal cells that ultimately result in successful target reinnervation or cell death. c-Jun is a component of the heterodimeric AP-1 transcription factor, and c-Jun is highly expressed in response to neuronal trauma. Here we have investigated the role of c-jun during axonal regeneration using mice lacking c-jun in the central nervous system. After transection of the facial nerve, the absence of c-Jun caused severe defects in several aspects of the axonal response, including perineuronal sprouting, lymphocyte recruitment, and microglial activation. c-Jun-deficient motorneurons were atrophic, resistant to axotomy-induced cell death, and showed reduced target muscle reinnervation. Expression of CD44, galanin, and alpha7beta1 integrin, molecules known to be involved in regeneration, was greatly impaired, suggesting a mechanism for c-Jun-mediated axonal growth. Taken together, our results identify c-Jun as an important regulator of axonal regeneration in the injured central nervous system.


Assuntos
Traumatismos do Nervo Facial/metabolismo , Cones de Crescimento/metabolismo , Regeneração Nervosa/genética , Proteínas Proto-Oncogênicas c-jun/genética , Fator de Transcrição AP-1/metabolismo , Animais , Atrofia/genética , Atrofia/metabolismo , Axotomia , Morte Celular/genética , Regulação para Baixo/genética , Nervo Facial/citologia , Nervo Facial/crescimento & desenvolvimento , Nervo Facial/metabolismo , Traumatismos do Nervo Facial/genética , Galanina/metabolismo , Gliose/genética , Cones de Crescimento/ultraestrutura , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Integrinas/metabolismo , Ativação Linfocitária/genética , Camundongos , Camundongos Transgênicos , Microglia/citologia , Microglia/metabolismo , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Músculo Esquelético/inervação , Plasticidade Neuronal/genética , Proteínas Proto-Oncogênicas c-jun/deficiência , Recuperação de Função Fisiológica/genética
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