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1.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205118

RESUMO

During metastasis, cancer cells that originate from the primary tumor circulate in the bloodstream, extravasate, and form micrometastases at distant locations. Several lines of evidence suggest that specific interactions between cancer cells and endothelial cells, in particular tumor cell adhesion to the endothelium and transendothelial migration, play a crucial role in extravasation. Here we have studied the role of vascular endothelial (VE)-cadherin which is expressed aberrantly by breast cancer cells and might promote such interactions. By comparing different human breast cancer cell lines, we observed that the number of cancer cells that adhered to endothelium correlated with VE-cadherin expression levels. VE-cadherin silencing experiments confirmed that VE-cadherin enhances cancer cell adhesion to endothelial cells. However, in contrast, the number of cancer cells that incorporated into the endothelium was not dependent on VE-cadherin. Thus, it appears that cancer cell adhesion and incorporation are distinct processes that are governed by different molecular mechanisms. When cancer cells incorporated into the endothelial monolayer, they formed VE-cadherin positive contacts with endothelial cells. On the other hand, we also observed tumor cells that had displaced endothelial cells, reflecting either different modes of incorporation, or a temporal sequence where cancer cells first form contact with endothelial cells and then displace them to facilitate transmigration. Taken together, these results show that VE-cadherin promotes the adhesion of breast cancer cells to the endothelium and is involved in the initial phase of incorporation, but not their transmigration. Thus, VE-cadherin might be of relevance for therapeutic strategies aiming at preventing the metastatic spread of breast cancer cells.


Assuntos
Antígenos CD/genética , Neoplasias da Mama/genética , Caderinas/genética , Adesão Celular/genética , Endotélio Vascular/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Técnicas de Cocultura , Endotélio Vascular/patologia , Endotélio Vascular/ultraestrutura , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Imagem Molecular/métodos , Metástase Neoplásica
2.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066490

RESUMO

There is an unmet need for simplified in vitro models of malignancy and metastasis that facilitate fast, affordable and scalable gene and compound analysis. "Adherent" cancer cell lines frequently release "free-floating" cells into suspension that are viable and can reattach. This, in a simplistic way, mimics the metastatic process. We compared the gene expression profiles of naturally co-existing populations of floating and adherent cells in SW620 (colon), C33a (cervix) and HeLa (cervix) cancer cells. We found that 1227, 1367 and 1333 genes were at least 2-fold differentially expressed in the respective cell lines, of which 122 were shared among the three cell lines. As proof of principle, we focused on the anti-metastatic gene NM23-H1, which was downregulated both at the RNA and protein level in the floating cell populations of all three cell lines. Knockdown of NM23-H1 significantly increased the number of floating (and viable) cells, whereas overexpression of NM23-H1 significantly reduced the proportion of floating cells. Other potential regulators of these cellular states were identified through pathway analysis, including hypoxia, mTOR (mechanistic target of rapamycin), cell adhesion and cell polarity signal transduction pathways. Hypoxia, a condition linked to malignancy and metastasis, reduced NM23-H1 expression and significantly increased the number of free-floating cells. Inhibition of mTOR or Rho-associated protein kinase (ROCK) significantly increased cell death specifically in the floating and not the adherent cell population. In conclusion, our study suggests that dynamic subpopulations of free-floating and adherent cells is a useful model to screen and identify genes, drugs and pathways that regulate the process of cancer metastasis, such as cell detachment and anoikis.


Assuntos
Modelos Biológicos , Neoplasias/patologia , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Metástase Neoplásica , Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologia , Reprodutibilidade dos Testes , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo
3.
Nat Commun ; 12(1): 3904, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162871

RESUMO

Due to its dynamic nature, the evolution of cancer cell-extracellular matrix (ECM) crosstalk, critically affecting metastasis and treatment resistance, remains elusive. Our results show that platinum-chemotherapy itself enhances resistance by progressively changing the cancer cell-intrinsic adhesion signaling and cell-surrounding ECM. Examining ovarian high-grade serous carcinoma (HGSC) transcriptome and histology, we describe the fibrotic ECM heterogeneity at primary tumors and distinct metastatic sites, prior and after chemotherapy. Using cell models from systematic ECM screen to collagen-based 2D and 3D cultures, we demonstrate that both specific ECM substrates and stiffness increase resistance to platinum-mediated, apoptosis-inducing DNA damage via FAK and ß1 integrin-pMLC-YAP signaling. Among such substrates around metastatic HGSCs, COL6 was upregulated by chemotherapy and enhanced the resistance of relapse, but not treatment-naïve, HGSC organoids. These results identify matrix adhesion as an adaptive response, driving HGSC aggressiveness via co-evolving ECM composition and sensing, suggesting stromal and tumor strategies for ECM pathway targeting.


Assuntos
Cistadenocarcinoma Seroso/genética , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/genética , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Colágeno/genética , Colágeno/metabolismo , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Evolução Molecular , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Humanos , Estimativa de Kaplan-Meier , Recidiva Local de Neoplasia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética
4.
Nat Commun ; 12(1): 2505, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947848

RESUMO

Autologous epidermal cultures restore a functional epidermis on burned patients. Transgenic epidermal grafts do so also in genetic skin diseases such as Junctional Epidermolysis Bullosa. Clinical success strictly requires an adequate number of epidermal stem cells, detected as holoclone-forming cells, which can be only partially distinguished from the other clonogenic keratinocytes and cannot be prospectively isolated. Here we report that single-cell transcriptome analysis of primary human epidermal cultures identifies categories of genes clearly distinguishing the different keratinocyte clonal types, which are hierarchically organized along a continuous, mainly linear trajectory showing that stem cells sequentially generate progenitors producing terminally differentiated cells. Holoclone-forming cells display stem cell hallmarks as genes regulating DNA repair, chromosome segregation, spindle organization and telomerase activity. Finally, we identify FOXM1 as a YAP-dependent key regulator of epidermal stem cells. These findings improve criteria for measuring stem cells in epidermal cultures, which is an essential feature of the graft.


Assuntos
Células Epidérmicas/citologia , Proteína Forkhead Box M1/metabolismo , Queratinócitos/citologia , Análise de Célula Única/métodos , Células-Tronco/citologia , Transcriptoma/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Adesão Celular/genética , Linhagem Celular , Autorrenovação Celular/genética , Células Cultivadas , Imunoprecipitação da Cromatina , Células Epidérmicas/metabolismo , Epidermólise Bolhosa Juncional/genética , Epidermólise Bolhosa Juncional/metabolismo , Proteína Forkhead Box M1/genética , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Queratinócitos/metabolismo , Camundongos , Análise em Microsséries , Família Multigênica , RNA-Seq , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo
5.
Med Sci Monit ; 27: e930215, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33990536

RESUMO

BACKGROUND Several risk factors contribute to the inflammation promoting hepatocellular carcinoma (HCC) development, but the underlying mechanisms are unknown. Human endogenous retrovirus H long terminal repeat-associating 2 (HHLA2), a B7 family member, is highly expressed in various malignant tumor tissues and is related to tumor progression and metastasis. MATERIAL AND METHODS Bioinformatics analysis was used to analyze the gene expression chip GSE33006 of HCC tissue in the GEO database, draw a heat map of differentially expressed genes, and analyze the GO pathway of gene function annotation. Then, we compared HCC tissues with para-carcinoma liver tissues from 55 patients for expression patterns and associations with HHLA2. Effects of HHLA2 knockdown were examined in the human HCC cell line HepG2 to explore effects of HHLA2 on HepG2 cells. RESULTS A significantly higher expression of HHLA2 at the mRNA and protein levels was detected in HCC tissues than in para-carcinoma liver tissues, which was similar to HHLA2 expression in the GSE33006 data. A higher expression of HHLA2 protein was associated with advanced cancer stage, tumor differentiation, and invasion of adjacent structures. In vitro knockdown of HHLA2 expression significantly increased HepG2 cell adhesion, promoted cell apoptosis, induced cell cycle arrest in the G1/S phase, and inhibited cell proliferation, migration, and invasion. CONCLUSIONS Our data indicated there was a higher expression of HHLA2 in HCC tissues than in para-carcinoma liver tissues, and HHLA2 plays a major role in the development and progression of HCC. Owing to its higher expression, HHLA2 is a potential prognostic biomarker for HCC.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Imunoglobulinas/genética , Neoplasias Hepáticas/genética , Apoptose/genética , Carcinoma Hepatocelular/patologia , Adesão Celular/genética , Pontos de Checagem do Ciclo Celular/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Biologia Computacional/métodos , Feminino , Fase G1/genética , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias/métodos , RNA Mensageiro/genética , Fase S/genética
6.
Hum Genet ; 140(7): 1061-1076, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33811546

RESUMO

Teebi hypertelorism syndrome (THS; OMIM 145420) is a rare craniofacial disorder characterized by hypertelorism, prominent forehead, short nose with broad or depressed nasal root. Some cases of THS have been attributed to SPECC1L variants. Homozygous variants in CDH11 truncating the transmembrane and intracellular domains have been implicated in Elsahy-Waters syndrome (EWS; OMIM 211380) with hypertelorism. We report THS due to CDH11 heterozygous missense variants on 19 subjects from 9 families. All affected residues in the extracellular region of Cadherin-11 (CHD11) are highly conserved across vertebrate species and classical cadherins. Six of the variants that cluster around the EC2-EC3 and EC3-EC4 linker regions are predicted to affect Ca2+ binding that is required for cadherin stability. Two of the additional variants [c.164G > C, p.(Trp55Ser) and c.418G > A, p.(Glu140Lys)] are also notable as they are predicted to directly affect trans-homodimer formation. Immunohistochemical study demonstrates that CDH11 is strongly expressed in human facial mesenchyme. Using multiple functional assays, we show that five variants from the EC1, EC2-EC3 linker, and EC3 regions significantly reduced the cell-substrate trans adhesion activity and one variant from EC3-EC4 linker results in changes in cell morphology, focal adhesion, and migration, suggesting dominant negative effect. Characteristic features in this cohort included depressed nasal root, cardiac and umbilical defects. These features distinguished this phenotype from that seen in SPECC1L-related hypertelorism syndrome and CDH11-related EWS. Our results demonstrate heterozygous variants in CDH11, which decrease cell-cell adhesion and increase cell migratory behavior, cause a form of THS, as termed CDH11-related THS.


Assuntos
Anormalidades Múltiplas/genética , Caderinas/genética , Adesão Celular/genética , Anormalidades Craniofaciais/genética , Deformidades Congênitas do Pé/genética , Variação Genética/genética , Deformidades Congênitas da Mão/genética , Hipertelorismo/genética , Sequência de Aminoácidos , Movimento Celular/genética , Feminino , Heterozigoto , Homozigoto , Humanos , Masculino , Linhagem , Fenótipo
7.
PLoS Pathog ; 17(2): e1009256, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33524035

RESUMO

Lyme disease, which is caused by infection with Borrelia burgdorferi and related species, can lead to inflammatory pathologies affecting the joints, heart, and nervous systems including the central nervous system (CNS). Inbred laboratory mice have been used to define the kinetics of B. burgdorferi infection and host immune responses in joints and heart, however similar studies are lacking in the CNS of these animals. A tractable animal model for investigating host-Borrelia interactions in the CNS is key to understanding the mechanisms of CNS pathogenesis. Therefore, we characterized the kinetics of B. burgdorferi colonization and associated immune responses in the CNS of mice during early and subacute infection. Using fluorescence-immunohistochemistry, intravital microscopy, bacterial culture, and quantitative PCR, we found B. burgdorferi routinely colonized the dura mater of C3H mice, with peak spirochete burden at day 7 post-infection. Dura mater colonization was observed for several Lyme disease agents including B. burgdorferi, B. garinii, and B. mayonii. RNA-sequencing and quantitative RT-PCR showed that B. burgdorferi infection was associated with increased expression of inflammatory cytokines and a robust interferon (IFN) response in the dura mater. Histopathologic changes including leukocytic infiltrates and vascular changes were also observed in the meninges of infected animals. In contrast to the meninges, we did not detect B. burgdorferi, infiltrating leukocytes, or large-scale changes in cytokine profiles in the cerebral cortex or hippocampus during infection; however, both brain regions demonstrated similar changes in expression of IFN-stimulated genes as observed in peripheral tissues and meninges. Taken together, B. burgdorferi is capable of colonizing the meninges in laboratory mice, and induces localized inflammation similar to peripheral tissues. A sterile IFN response in the absence of B. burgdorferi or inflammatory cytokines is unique to the brain parenchyma, and provides insight into the potential mechanisms of CNS pathology associated with this important pathogen.


Assuntos
Borrelia burgdorferi/patogenicidade , Dura-Máter/patologia , Encefalomielite/microbiologia , Doença de Lyme/patologia , Animais , Linfócitos B/imunologia , Adesão Celular/genética , Modelos Animais de Doenças , Dura-Máter/imunologia , Encefalomielite/genética , Encefalomielite/imunologia , Encefalomielite/patologia , Matriz Extracelular/genética , Matriz Extracelular/imunologia , Feminino , Perfilação da Expressão Gênica , Mediadores da Inflamação/imunologia , Leucócitos/imunologia , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Camundongos , Linfócitos T/imunologia , Cicatrização/genética
8.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33483418

RESUMO

The biphasic adhesion-velocity relation is a universal observation in mesenchymal cell motility. It has been explained by adhesion-promoted forces pushing the front and resisting motion at the rear. Yet, there is little quantitative understanding of how these forces control cell velocity. We study motion of MDA-MB-231 cells on microlanes with fields of alternating Fibronectin densities to address this topic and derive a mathematical model from the leading-edge force balance and the force-dependent polymerization rate. It reproduces quantitatively our measured adhesion-velocity relation and results with keratocytes, PtK1 cells, and CHO cells. Our results confirm that the force pushing the leading-edge membrane drives lamellipodial retrograde flow. Forces resisting motion originate along the whole cell length. All motion-related forces are controlled by adhesion and velocity, which allows motion, even with higher Fibronectin density at the rear than at the front. We find the pathway from Fibronectin density to adhesion structures to involve strong positive feedbacks. Suppressing myosin activity reduces the positive feedback. At transitions between different Fibronectin densities, steady motion is perturbed and leads to changes of cell length and front and rear velocity. Cells exhibit an intrinsic length set by adhesion strength, which, together with the length dynamics, suggests a spring-like front-rear interaction force. We provide a quantitative mechanistic picture of the adhesion-velocity relation and cell response to adhesion changes integrating force-dependent polymerization, retrograde flow, positive feedback from integrin to adhesion structures, and spring-like front-rear interaction.


Assuntos
Adesão Celular/genética , Movimento Celular/genética , Fibronectinas/genética , Células-Tronco Mesenquimais/citologia , Actinas/genética , Animais , Células CHO , Linhagem Celular Tumoral , Membrana Celular/genética , Cricetinae , Cricetulus , Feminino , Humanos , Integrinas/genética , Células-Tronco Mesenquimais/metabolismo , Modelos Teóricos , Pseudópodes/genética
9.
Development ; 148(2)2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33298462

RESUMO

Formation of skeletal muscle is among the most striking examples of cellular plasticity in animal tissue development, and while muscle progenitor cells are reprogrammed by epithelial-mesenchymal transition (EMT) to migrate during embryonic development, the regulation of EMT in post-natal myogenesis remains poorly understood. Here, we demonstrate that the long noncoding RNA (lncRNA) Meg3 regulates EMT in myoblast differentiation and skeletal muscle regeneration. Chronic inhibition of Meg3 in C2C12 myoblasts induced EMT, and suppressed cell state transitions required for differentiation. Furthermore, adenoviral Meg3 knockdown compromised muscle regeneration, which was accompanied by abnormal mesenchymal gene expression and interstitial cell proliferation. Transcriptomic and pathway analyses of Meg3-depleted C2C12 myoblasts and injured skeletal muscle revealed a significant dysregulation of EMT-related genes, and identified TGFß as a key upstream regulator. Importantly, inhibition of TGFßR1 and its downstream effectors, and the EMT transcription factor Snai2, restored many aspects of myogenic differentiation in Meg3-depleted myoblasts in vitro We further demonstrate that reduction of Meg3-dependent Ezh2 activity results in epigenetic alterations associated with TGFß activation. Thus, Meg3 regulates myoblast identity to facilitate progression into differentiation.


Assuntos
Plasticidade Celular/genética , Transição Epitelial-Mesenquimal/genética , Mioblastos/citologia , Mioblastos/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Adesão Celular/genética , Diferenciação Celular/genética , Linhagem Celular , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Técnicas de Silenciamento de Genes , Histonas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Mesoderma/patologia , Metilação , Camundongos , Mitocôndrias/metabolismo , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Mutação/genética , RNA Longo não Codificante/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Regeneração , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
10.
Gene ; 768: 145269, 2021 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-33148459

RESUMO

Adipose stem cells (ASCs) represent a reliable source of stem cells with a widely demonstrated potential in regenerative medicine and tissue engineering applications. New recent insights suggest that three-dimensional (3D) models may closely mimic the native tissue properties; spheroids from adipose derived stem cells (SASCs) exhibit enhanced regenerative abilities compared with those of 2D models. Stem cell therapy success is determined by "cell-quality"; for this reason, the involvement of stress signals and cellular aging need to be further investigated. Here, we performed a comparative analysis of genes connected with stemness, aging, telomeric length and oxidative stress, in 3D and 2D primary cultures. The expression levels of stemness-related markers and anti-aging Sirtuin1 were significantly up-regulated (P < 0.001) in SASCs-3D while gene expression of aging-related p16INK4a was increased in ASCs-2D (P < 0.001). The 3D and 2D cultures also had a different gene expression profile for genes related to telomere maintenance (Shelterin complex, RNA Binding proteins and DNA repair genes) (P < 0.01 and P < 0.001) and oxidative stress (aldehyde dehydrogenase class1 and 3) (P < 0.05, P < 0.01 and P < 0.001) and presented a striking large variation in their cellular redox state. Based on our findings, we propose a "cell quality" model of SASCs, highlighting a precise molecular expression of several genes involved with stemness (SOX2, POU5F1 and NANOG), anti-aging (SIRT1), oxidative stress (ALDH3) and telomeres maintenance.


Assuntos
Adipócitos/metabolismo , Tecido Adiposo/citologia , Técnicas de Cultura de Células , Transplante de Células-Tronco , Células-Tronco/citologia , Adipócitos/citologia , Adolescente , Adulto , Idoso , Envelhecimento/genética , Adesão Celular/genética , Sobrevivência Celular/genética , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Reparo do DNA/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/genética , Proteínas de Ligação a RNA/metabolismo , Sirtuínas/metabolismo , Esferoides Celulares/citologia , Homeostase do Telômero/genética , Engenharia Tecidual/métodos , Adulto Jovem
11.
Biochimie ; 180: 1-9, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33132158

RESUMO

Neurite outgrowth involves reciprocal signaling interactions between tumor cells and nerves where invading tumor cells have acquired the ability to respond to pro-invasive signals within the nerve environment. Neurite outgrowth could serve as a mechanism leading to invasion of cancer cells into the nerve sheath and subsequent metastasis. Snail transcription factor can promote migration and invasion of prostate cancer cells. We hypothesized that prostate cancer cell interaction with nerve cells will be mediated by Snail expression within prostate cancer cells. For this study we utilized various prostate cancer cell lines: C4-2 non-silencing (NS, control); C4-2 Snail shRNA, (stable Snail knockdown); LNCaP Neo (empty vector control) and LNCaP Snail (stably over-expressing Snail). Cancer cell adhesion and migration towards nerve cells (snF96.2 or NS20Y) was examined by co-culture assays. Conditioned media (CM) collected from C4-2 cells was cultured with nerve cells (PC-12 or NS20Y) for 48 h followed by qualitative or quantitative neurite outgrowth assay. Our results showed that cancer cells expressing high levels of Snail (LNCaP Snail/C4-2 NS) displayed significantly higher migration adherence to nerve cells, compared to cells with lower levels of Snail (LNCaP Neo/C4-2 Snail shRNA). Additionally, LNCaP Snail or C4-2 NS (Snail-high) CM led to a higher neurite outgrowth compared to the LNCaP Neo or C4-2 Snail shRNA (Snail-low). In conclusion, Snail promotes migration and adhesion to nerve cells, as well as neurite outgrowth via secretion of soluble factors. Therefore, targeting cancer cell interaction with nerves may contribute to halting prostate cancer progression/metastasis.


Assuntos
Crescimento Neuronal/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Animais , Adesão Celular/genética , Comunicação Celular/genética , Linhagem Celular , Movimento Celular/genética , Proliferação de Células/genética , Inativação Gênica , Humanos , Masculino , Camundongos , Neurônios/metabolismo , Neoplasias da Próstata/patologia , Ratos
12.
FASEB J ; 35(2): e21170, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33184968

RESUMO

Secretory phospholipase A2 group IB (sPLA2-IB) and M-type phospholipase A2 receptor (PLA2R) are closely associated with proteinuria in idiopathic membranous nephropathy (IMN). Podocytes constitute an important component of glomerular filtration, and high basal autophagy is indispensable for podocyte function. The current study aimed to analyze the relationship between sPLA2-IB and podocyte autophagy in IMN and determine whether sPLA2-IB mediates abnormal autophagy regulation in podocytes. The serum sPLA2-IB level and podocyte autophagy were detected, and clinical data were collected from IMN patients with different proteinuria levels. Then, the effects of sPLA2-IB on autophagy signaling pathways were evaluated in cultured human podocytes treated with sPLA2-IB, rapamycin, p38 inhibition, and PLA2R-siRNA in vitro. We found that IMN patients with nephrotic-range proteinuria have a significantly higher level of sPLA2-IB and fewer autophagosomes than those with non-nephrotic-range proteinuria. In vitro sPLA2-IB-induced insufficient autophagy in podocytes and promoted podocyte injury via activation of the mTOR/ULK1ser757 signaling pathway. Moreover, inhibition of p38 MAPK evidently abrogated sPLA2-IB-induced autophagy and the activation of mTOR/ULK1ser757 . Additionally, PLA2R silencing demonstrated that sPLA2-IB-induced abnormal autophagy was also PLA2R-dependent. In conclusion, the results revealed that sPLA2-IB downregulated autophagy and contributed to podocyte injury via PLA2R though activation of the p38MAPK/mTOR/ULK1ser757 signaling pathway.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/genética , Glomerulonefrite Membranosa/sangue , Fosfolipases A2 do Grupo IB/sangue , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Podócitos/metabolismo , Receptores da Fosfolipase A2/sangue , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adulto , Idoso , Adesão Celular/genética , Movimento Celular/genética , Células Cultivadas , Feminino , Glomerulonefrite Membranosa/genética , Glomerulonefrite Membranosa/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteinúria/sangue , Receptores da Fosfolipase A2/genética , Transfecção
13.
Methods Mol Biol ; 2179: 7-12, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32939708

RESUMO

The epithelial-mesenchymal transition (EMT) is a key process required for building the early body plan of metazoa. It involves coordinated and precisely timed changes in multiple cell processes such as de-adhesion, motility, invasion, and cell polarity. While much has been learned about how embryos deploy epithelial-mesenchymal transitions since Betty Hay named the process decades ago, a number of things are still not well understood. Here I will discuss some of the big questions that remain, including how is all of this controlled, how does each of the cell biological events work, and how are they so nicely coordinated with one another?


Assuntos
Adesão Celular/genética , Transição Epitelial-Mesenquimal/genética , Gastrulação/genética , Mesoderma/crescimento & desenvolvimento , Animais , Movimento Celular/genética , Polaridade Celular/genética , Embrião não Mamífero , Epitélio/metabolismo , Epitélio/patologia , Humanos , Mesoderma/metabolismo
14.
FASEB J ; 35(1): e21194, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33337553

RESUMO

Synapses are the fundamental structural unit by which neurons communicate. An orchestra of proteins regulates diverse synaptic functions, including synapse formation, maintenance, and elimination-synapse homeostasis. Some proteins of the larger C1q super-family are synaptic organizers involved in crucial neuronal processes in various brain regions. C1Q-like (C1QL) proteins bind to the adhesion G protein-coupled receptor B3 (ADGRB3) and act at synapses in a subset of circuits. To investigate the hypothesis that the secreted C1QL proteins mediate tripartite trans-synaptic adhesion complexes, we conducted an in vivo interactome study and identified new binding candidates. We demonstrate that C1QL3 mediates a novel cell-cell adhesion complex involving ADGRB3 and two neuronal pentraxins, NPTX1 and NPTXR. Analysis of single-cell RNA-Seq data from the cerebral cortex shows that C1ql3, Nptx1, and Nptxr are highly co-expressed in the same excitatory neurons. Thus, our results suggest the possibility that in vivo the three co-expressed proteins are presynaptically secreted and form a complex capable of binding to postsynaptically localized ADGRB3, thereby creating a novel trans-synaptic adhesion complex. Identifying new binding partners for C1QL proteins and deciphering their underlying molecular principles will accelerate our understanding of their role in synapse organization.


Assuntos
Proteína C-Reativa/metabolismo , Complemento C1q/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Animais , Proteína C-Reativa/genética , Adesão Celular/genética , Complemento C1q/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Complexos Multiproteicos/genética , Proteínas do Tecido Nervoso/genética , Sinapses/genética
15.
PLoS One ; 15(12): e0243272, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33270750

RESUMO

Cluster of differentiation (CD) 166 or activated leukocyte cell adhesion molecule (ALCAM) is a transmembrane molecule known to be an intercellular adhesion factor. The expression and function of ALCAM in medulloblastoma (MB), a pediatric brain tumor with highly advanced molecular genetics, remains unclear. Therefore, this study aimed to clarify the significance and functional role of ALCAM expression in MB. ALCAM expression in 45 patients with MB was evaluated by immunohistochemical analysis of formalin-fixed paraffin-embedded clinical specimens and the relationship between ALCAM expression and pathological type/molecular subgroup, such as WNT, SHH, Group 3, and Group 4, was examined. Eight ALCAM positive (18%), seven partially positive (16%), and 30 negative (67%) cases were detected. All seven cases of the WNT molecular subgroup were ALCAM positive and ALCAM expression strongly correlated with this subgroup (P < 0.0001). In addition, functional studies using MB cell lines revealed ALCAM expression affected proliferation and migration as a positive regulator in vitro. However, ALCAM silencing did not affect survival or the formation of leptomeningeal dissemination in an orthotopic mouse model, but did induce a malignant phenotype with increased tumor cell invasion at the dissemination sites (P = 0.0029). In conclusion, our results revealed that ALCAM exhibited highly specific expression in the WNT subgroup of MB. Furthermore, we demonstrated that the cell kinetics of MB cell lines can be altered by the expression of ALCAM.


Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas Fetais/metabolismo , Meduloblastoma/metabolismo , Proteínas Wnt/metabolismo , Molécula de Adesão de Leucócito Ativado/genética , Adolescente , Animais , Antígenos CD/fisiologia , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/genética , Adesão Celular/genética , Moléculas de Adesão Celular Neuronais/fisiologia , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Cerebelares/genética , Criança , Pré-Escolar , Feminino , Proteínas Fetais/fisiologia , Expressão Gênica/genética , Perfilação da Expressão Gênica , Humanos , Lactente , Japão/epidemiologia , Masculino , Meduloblastoma/fisiopatologia , Camundongos , Invasividade Neoplásica , RNA Mensageiro/genética , Proteínas Wnt/genética , Adulto Jovem
16.
PLoS One ; 15(12): e0243497, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33275637

RESUMO

Non-alcoholic steatohepatitis (NASH) is a severe, advanced form of non-alcoholic fatty liver disease (NAFLD) that is associated with features of metabolic syndrome and characterized by hepatic steatosis, inflammation, and fibrosis. In addition, NASH is associated with endothelial dysfunction within the hepatic vasculature. Treatment with CU06-1004 (previously called Sac-1004) ameliorates endothelial dysfunction by inhibiting hyperpermeability and inflammation. In this study, we investigated the protective effects of CU06-1004 in a choline-deficient L-amino acid (CDAA)-induced mouse model of NASH for 3 or 6 weeks. Specifically, we evaluated the effects of CU06-1004 on lipid accumulation, inflammation, hepatic fibrosis, and liver sinusoidal endothelial cell (LSEC) capillarization through biochemical analysis, immunohistochemistry, and real-time PCR. We found that the administration of CU06-1004 to mice improved liver triglyceride (TG) and serum alanine aminotransferase (ALT) in this CDAA-induced model of NASH for 6 weeks. In groups of NASH induced mice for both 3 and 6 weeks, CU06-1004 significantly reduced the hepatic expression of genes related to lipogenesis, inflammation, and cell adhesion. However, expression of genes related to hepatic fibrosis and vascular endothelial changes were only decreased in animals with mild NASH. These results suggest that the administration of CU06-1004 suppresses hepatic steatosis, inflammation, fibrosis, and LSEC capillarization in a CDAA-induced mouse model of NASH. This suggests that CU06-1004 has therapeutic potential for the treatment of mild NASH.


Assuntos
Dieta/veterinária , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Substâncias Protetoras/uso terapêutico , Alanina Transaminase/sangue , Aminoácidos/deficiência , Aminoácidos/metabolismo , Animais , Adesão Celular/genética , Colina/metabolismo , Modelos Animais de Doenças , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Inflamação/genética , Lipogênese/genética , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia , Substâncias Protetoras/química , Substâncias Protetoras/farmacologia , Triglicerídeos/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
17.
Aging (Albany NY) ; 13(2): 2073-2088, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33323549

RESUMO

Janus kinase 1 (JAK1) is a member of the JAK family, which plays an essential and non-redundant role in tumorigenesis. However, the potential role of JAK1 in immune infiltration and prognosis of lung adenocarcinoma (LUAD) remains unclear. The mRNA expression and methylation level of JAK1 in LUAD were examined using the Oncomine and The Cancer Genome Atlas (TCGA) databases, respectively. The correlations between JAK1 expression and its methylation level and clinicopathological parameters were analyzed. The Kaplan-Meier plotter database was used to evaluate the prognostic value of JAK1 in LUAD. The signaling pathways associated with JAK1 expression were identified by performing a GSEA. The CIBERSORT and TIMER databases were used to analyze the correlations between JAK1 and tumor-infiltrating immune cells. In addition, the JAK1 expression and proportion of immune cells in LUAD cell lines were analyzed. The JAK1 expression was remarkably decreased in patients with LUAD and significantly correlated with the clinical features of patients with LUAD. The JAK1 methylation level was increased and negatively correlated with its mRNA expression. A decrease in JAK1 expression was correlated with poor prognosis. The results of GSEA showed that cell adhesion, tumorigenesis, and immune-related signaling pathways were mainly enriched. JAK1 was positively associated with tumor-infiltrating immune cells, and the results of CIBERSORT analysis suggested that JAK1 was correlated with monotypes and M1 macrophages. The results of the TIMER database analysis confirmed that JAK1 was closely associated with the gene markers of M1 macrophages. Thus, JAK1 may serve as a potential prognostic biomarker in LUAD and is associated with immune infiltration.


Assuntos
Adenocarcinoma de Pulmão/genética , Janus Quinase 1/genética , Neoplasias Pulmonares/genética , Linfócitos do Interstício Tumoral/imunologia , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/imunologia , Adenocarcinoma de Pulmão/imunologia , Carcinogênese/genética , Adesão Celular/genética , Bases de Dados Genéticas , Humanos , Janus Quinase 1/imunologia , Neoplasias Pulmonares/imunologia , Monócitos , Prognóstico , RNA Mensageiro/metabolismo
18.
Development ; 147(23)2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33310787

RESUMO

Planar cell polarity (PCP) is essential for tissue morphogenesis and homeostasis; however, the mechanisms that orchestrate the cell shape and packing dynamics required to establish PCP are poorly understood. Here, we identified a major role for the globular (G)-actin-binding protein thymosin-ß4 (TMSB4X) in PCP establishment and cell adhesion in the developing epidermis. Depletion of Tmsb4x in mouse embryos hindered eyelid closure and hair-follicle angling owing to PCP defects. Tmsb4x depletion did not preclude epidermal cell adhesion in vivo or in vitro; however, it resulted in abnormal structural organization and stability of adherens junction (AJ) due to defects in filamentous (F)-actin and G-actin distribution. In cultured keratinocytes, TMSB4X depletion increased the perijunctional G/F-actin ratio and decreased G-actin incorporation into junctional actin networks, but it did not change the overall actin expression level or cellular F-actin content. A pharmacological treatment that increased the G/F-actin ratio and decreased actin polymerization mimicked the effects of Tmsb4x depletion on both AJs and PCP. Our results provide insights into the regulation of the actin pool and its involvement in AJ function and PCP establishment.


Assuntos
Polaridade Celular/genética , Desenvolvimento Embrionário/genética , Morfogênese/genética , Timosina/genética , Citoesqueleto de Actina/genética , Actinas/genética , Junções Aderentes/genética , Animais , Adesão Celular/genética , Forma Celular/genética , Células Epidérmicas/metabolismo , Epiderme/crescimento & desenvolvimento , Homeostase/genética , Queratinócitos/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética
19.
Aging (Albany NY) ; 12(24): 24734-24777, 2020 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-33349623

RESUMO

Patients with acute myeloid leukemia (AML) have a median age of 65-70 years at diagnosis. Elderly patients have more chemoresistant disease, and this is partly due to decreased frequencies of favorable and increased frequencies of adverse genetic abnormalities. However, aging-dependent differences may also contribute. We therefore compared AML cell proteomic and phosphoproteomic profiles for (i) elderly low-risk and younger low-risk patients with favorable genetic abnormalities; and (ii) high-risk patients with adverse genetic abnormalities and a higher median age against all low-risk patients with lower median age. Elderly low-risk and younger low-risk patients showed mainly phosphoproteomic differences especially involving transcriptional regulators and cytoskeleton. When comparing high-risk and low-risk patients both proteomic and phosphoproteomic studies showed differences involving cytoskeleton and immunoregulation but also transcriptional regulation and cell division. The age-associated prognostic impact of cyclin-dependent kinases was dependent on the cellular context. The protein level of the adverse prognostic biomarker mitochondrial aldehyde dehydrogenase (ALDH2) showed a similar significant upregulation both in elderly low-risk and elderly high-risk patients. Our results suggest that molecular mechanisms associated with cellular aging influence chemoresistance of AML cells, and especially the cytoskeleton function may then influence cellular hallmarks of aging, e.g. mitosis, polarity, intracellular transport and adhesion.


Assuntos
Envelhecimento/genética , Aldeído-Desidrogenase Mitocondrial/genética , Citoesqueleto/metabolismo , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/metabolismo , Aldeído-Desidrogenase Mitocondrial/metabolismo , Adesão Celular/genética , Polaridade Celular , Senescência Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Masculino , Pessoa de Meia-Idade , Mitose/genética , Fosfoproteínas , Prognóstico , Proteômica , Fatores de Risco , Regulação para Cima
20.
Int J Mol Sci ; 21(21)2020 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-33158289

RESUMO

Fibronectin (FN) expressed by tumor cells has been known to be tumor suppressive but the pericellular FN (periFN) assembled on circulating tumor cells appears to evidently promote distant metastasis. Whereas the regulation of periFN assembly in suspended cells has currently been under investigation, how it is regulated in adherent tumor cells and the role of periFN in primary tumor growth remain elusive. Techniques of RNAi, plasmid transfections, immunoblotting, fluorescence/immunohistochemistry staining, cell proliferation assays, and primary tumor growth in C57BL6 mice and Fischer 344 rats were employed in this study. We found that endogenously synthesized FN in adherent tumor cells was required for periFN assembly which was aligned by RhoA-organized actin stress fiber (SF). Depleting periFN on adherent tumor cells congruently promoted in vivo tumor growth but surprisingly did not autonomously impact on in vitro tumor cell proliferation and apoptosis, suggestive of a non-autonomous role of periFN in in vivo tumor growth. We showed that the proliferative ability of shFN-expressing tumor cells was higher than shScramble cells did in the presence of fibroblasts. Altogether, these results suggested that depriving RhoA/SF-regulated periFN matrices non-autonomously promotes fibroblast-mediated tumor cell growth.


Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/fisiologia , Fibronectinas/metabolismo , Neoplasias/patologia , Fibras de Estresse/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Adesão Celular/genética , Proliferação de Células/genética , Matriz Extracelular/patologia , Fibroblastos/patologia , Fibronectinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Neoplasias/metabolismo , Ratos , Ratos Endogâmicos F344 , Fibras de Estresse/patologia , Carga Tumoral/fisiologia , Células Tumorais Cultivadas , Proteína rhoA de Ligação ao GTP/genética
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