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1.
Nat Immunol ; 22(11): 1440-1451, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34686860

RESUMO

Intestinal epithelial cell (IEC) damage by T cells contributes to graft-versus-host disease, inflammatory bowel disease and immune checkpoint blockade-mediated colitis. But little is known about the target cell-intrinsic features that affect disease severity. Here we identified disruption of oxidative phosphorylation and an increase in succinate levels in the IECs from several distinct in vivo models of T cell-mediated colitis. Metabolic flux studies, complemented by imaging and protein analyses, identified disruption of IEC-intrinsic succinate dehydrogenase A (SDHA), a component of mitochondrial complex II, in causing these metabolic alterations. The relevance of IEC-intrinsic SDHA in mediating disease severity was confirmed by complementary chemical and genetic experimental approaches and validated in human clinical samples. These data identify a critical role for the alteration of the IEC-specific mitochondrial complex II component SDHA in the regulation of the severity of T cell-mediated intestinal diseases.


Assuntos
Colite/enzimologia , Colo/enzimologia , Citotoxicidade Imunológica , Complexo II de Transporte de Elétrons/metabolismo , Células Epiteliais/enzimologia , Doença Enxerto-Hospedeiro/enzimologia , Mucosa Intestinal/enzimologia , Mitocôndrias/enzimologia , Linfócitos T/imunologia , Animais , Estudos de Casos e Controles , Comunicação Celular , Células Cultivadas , Colite/genética , Colite/imunologia , Colite/patologia , Colo/imunologia , Colo/ultraestrutura , Modelos Animais de Doenças , Complexo II de Transporte de Elétrons/genética , Células Epiteliais/imunologia , Células Epiteliais/ultraestrutura , Feminino , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Humanos , Imunidade nas Mucosas , Mucosa Intestinal/imunologia , Mucosa Intestinal/ultraestrutura , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/imunologia , Mitocôndrias/ultraestrutura , Fosforilação Oxidativa , Ácido Succínico/metabolismo , Linfócitos T/metabolismo
2.
Breast Cancer Res ; 16(5): 461, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25606592

RESUMO

Key mediators of signaling pathways in breast cancer involve post-translational protein modification, primarily mediated through phosphorylation and ubiquitination. While previous studies focused on phosphorylation events, more recent analysis suggests that ubiquitin plays a parallel and equally important role in several signaling and cell regulatory events in breast cancer. Availability of new tools capable of sensitive detection of gene mutations and aberrant expression of genes and proteins coupled with gene-specific knockdown and silencing protocols have provided insight into the previously unexplored ubiquitin regulatory process within these tumors. Ubiquitin-specific proteases are one class of enzymes with protein deubiquitinating activity, making up the majority of protein deubiquitinating diversity within mammalian cells. Ubiquitin-specific proteases are also emerging as potential therapeutic targets in many diseases, including cancer. In this report, we summarize the involvement of this class of enzymes in breast cancer signaling and cell regulation and illustrate the potential for additional studies to define novel targets and approaches in breast cancer therapy.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Inibidores de Proteases/farmacologia , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Neoplasias da Mama/enzimologia , Feminino , Humanos , Terapia de Alvo Molecular , Inibidores de Proteases/uso terapêutico , Transdução de Sinais , Proteases Específicas de Ubiquitina/metabolismo
3.
bioRxiv ; 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38562773

RESUMO

Survival rates for non-small cell lung cancer (NSCLC) remain low despite the advent of novel therapeutics. Tyrosine kinase inhibitors (TKIs) targeting mutant epidermal growth factor receptor (EGFR) in NSCLC have significantly improved mortality but are plagued with challenges--they can only be used in the small fraction of patients who have susceptible driver mutations, and resistance inevitably develops. Aberrant glycosylation on the surface of cancer cells is an attractive therapeutic target as these abnormal glycosylation patterns are typically specific to cancer cells and are not present on healthy cells. H84T BanLec (H84T), a lectin previously engineered by our group to separate its antiviral activity from its mitogenicity, exhibits precision binding of high mannose, an abnormal glycan present on the surface of many cancer cells, including NSCLC. Here, we show that H84T binds to and inhibits the growth of diverse NSCLC cell lines by inducing lysosomal degradation of EGFR and leading to cancer cell death through autophagy. This is a mechanism distinct from EGFR TKIs and is independent of EGFR mutation status; H84T inhibited proliferation of both cell lines expressing wild type EGFR and those expressing mutant EGFR that is resistant to all TKIs. Further, H84T binds strongly to multiple and diverse clinical samples of both pulmonary adenocarcinoma and squamous cell carcinoma. H84T is thus a promising potential therapeutic in NSCLC, with the ability to circumvent the challenges currently faced by EGFR TKIs.

4.
J Cell Sci ; 124(Pt 10): 1752-8, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-21525039

RESUMO

During progression of breast cancer, CCN6 protein exerts tumor inhibitory functions. CCN6 is a secreted protein that modulates the insulin-like growth factor-1 (IGF-1) signaling pathway. Knockdown of CCN6 in benign mammary epithelial cells triggers an epithelial to mesenchymal transition (EMT), with upregulation of the transcription factor ZEB1/δEF1. How CCN6 regulates ZEB1 expression is unknown. We hypothesized that CCN6 might regulate ZEB1, EMT and breast cancer invasion by modulating IGF-1 signaling. Exogenously added human recombinant CCN6 protein was sufficient to downregulate ZEB1 mRNA and protein levels in CCN6-deficient (CCN6 KD) HME cells and MDA-MB-231 breast cancer cells. Recombinant CCN6 protein decreased invasion of CCN6 KD cells compared with controls. We discovered that knockdown of CCN6 induced IGF-1 secretion in HME cells cultivated in serum-free medium to higher concentrations than found in MDA-MB-231 cells. Treatment with recombinant CCN6 protein was sufficient to decrease IGF-1 protein and mRNA to control levels, rescuing the effect of CCN6 knockdown. Specific inhibition of IGF-1 receptors using the pharmacological inhibitor NVP-AE541 or short hairpin shRNAs revealed that ZEB1 upregulation due to knockdown of CCN6 requires activation of IGF-1 receptor signaling. Recombinant CCN6 blunted IGF-1-induced ZEB1 upregulation in MDA-MB-231 cells. Our data define a pathway in which CCN6 attenuates IGF-1 signaling to decrease ZEB1 expression and invasion in breast cancer. These results suggest that CCN6 could be a target to prevent or halt breast cancer invasion.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Receptor IGF Tipo 1/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias da Mama/patologia , Proteínas de Sinalização Intercelular CCN , Desdiferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Imunofluorescência , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/deficiência , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/farmacologia , Fator de Crescimento Insulin-Like I/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Regulação para Cima , Homeobox 1 de Ligação a E-box em Dedo de Zinco
5.
Cancer Res ; 79(11): 2923-2932, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30996048

RESUMO

Patients with glioblastoma multiforme (GBM) survive on average 12 to 14 months after diagnosis despite surgical resection followed by radiotheraphy and temozolomide therapy. Intrinsic or acquired resistance to chemo- and radiotherapy is common and contributes to a high rate of recurrence. To investigate the therapeutic potential of protein disulfide isomerase (PDI) as a target to overcome resistance to chemoradiation, we developed a GBM tumor model wherein conditional genetic ablation of prolyl 4-hydroxylase subunit beta (P4HB), the gene that encodes PDI, can be accomplished. Loss of PDI expression induced the unfolded protein response (UPR) and decreased cell survival in two independent GBM models. Nascent RNA Bru-seq analysis of PDI-depleted cells revealed a decrease in transcription of genes involved in DNA repair and cell-cycle regulation. Activation of the UPR also led to a robust decrease in RAD51 protein expression as a result of its ubiquitination-mediated proteosomal degradation. Clonogenic survival assays demonstrated enhanced killing of GBM cells in response to a combination of PDI knockdown and ionizing radiation (IR) compared with either modality alone, which correlated with a decreased capacity to repair IR-induced DNA damage. Synergistic tumor control was also observed with the combination of PDI inhibition and IR in a mouse xenograft model compared with either single agent alone. These findings provide a strong rationale for the development of PDI inhibitors and their use in combination with DNA damage-inducing, standard-of-care therapies such as IR. SIGNIFICANCE: These findings identify PDIA1 as a therapeutic target in GBM by demonstrating efficacy of its inhibition in combination with radiotherapy through a novel mechanism involving downregulation of DNA repair genes.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2923/F1.large.jpg.


Assuntos
Reparo do DNA , Glioblastoma/radioterapia , Isomerases de Dissulfetos de Proteínas/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Camundongos , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Radiação Ionizante , Radiossensibilizantes/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Methods Mol Biol ; 1731: 247-260, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29318559

RESUMO

Proteases are "protein-cleaving" enzymes, which, in addition to their non-specific degrading function, also catalyze the highly specific and regulated process of proteolytic processing, thus regulating multiple biological functions. Alterations in proteolytic activity occur during pathological conditions such as cancer. One of the major deregulated classes of proteases in cancer is caspases, the proteolytic initiators and mediators of the apoptotic machinery. The ability to image apoptosis noninvasively in living cells and animal models of cancer can not only provide new insight into the biological basis of the disease but can also be used as a quantitative tool to screen and evaluate novel therapeutic strategies. Optical molecular imaging such as bioluminescence-based genetically engineered biosensors has been developed in our laboratory and exploited to study protease activity in animal models with a high signal to noise. Using the circularly permuted form of firefly luciferase, we have developed a reporter for Caspase 3/7, referred to as Caspase 3/7 GloSensor. Here, we discuss the use of the Caspase 3/7 GloSensor for imaging apoptotic activity in mouse xenografts and genetically engineered mouse models of cancer and present the potential of this powerful platform technology to image the proteolytic activity of numerous other proteases.


Assuntos
Substâncias Luminescentes/química , Imagem Molecular/métodos , Proteólise , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Apoptose , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Linhagem Celular Tumoral , Feminino , Genes Reporter/genética , Engenharia Genética/instrumentação , Engenharia Genética/métodos , Humanos , Luciferases de Vaga-Lume/química , Luciferases de Vaga-Lume/genética , Medições Luminescentes/instrumentação , Medições Luminescentes/métodos , Camundongos , Camundongos Nus , Camundongos Transgênicos , Imagem Molecular/instrumentação , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação
7.
Neoplasia ; 20(2): 152-164, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29248719

RESUMO

Usp9x has emerged as a potential therapeutic target in some hematologic malignancies and a broad range of solid tumors including brain, breast, and prostate. To examine Usp9x tumorigenicity and consequence of Usp9x inhibition in human pancreatic tumor models, we carried out gain- and loss-of-function studies using established human pancreatic tumor cell lines (PANC1 and MIAPACA2) and four spontaneously immortalized human pancreatic patient-derived tumor (PDX) cell lines. The effect of Usp9x activity inhibition by small molecule deubiquitinase inhibitor G9 was assessed in 2D and 3D culture, and its efficacy was tested in human tumor xenografts. Overexpression of Usp9x increased 3D growth and invasion in PANC1 cells and up-regulated the expression of known Usp9x substrates Mcl-1 and ITCH. Usp9x inhibition by shRNA-knockdown or by G9 treatment reduced 3D colony formation in PANC1 and PDX cell lines, induced rapid apoptosis in MIAPACA2 cells, and associated with reduced Mcl-1 and ITCH protein levels. Although G9 treatment reduced human MIAPACA2 tumor burden in vivo, in mouse pancreatic cancer cell lines established from constitutive (8041) and doxycycline-inducible (4668) KrasG12D/Tp53R172H mouse pancreatic tumors, Usp9x inhibition increased and sustained the 3D colony growth and showed no significant effect on tumor growth in 8041-xenografts. Thus, Usp9x inhibition may be therapeutically active in human PDAC, but this activity was not predicted from studies of genetically engineered mouse pancreatic tumor models.


Assuntos
Carcinoma Ductal Pancreático/patologia , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas/patologia , Ubiquitina Tiolesterase/metabolismo , Animais , Apoptose , Carcinoma Ductal Pancreático/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Pancreáticas/metabolismo , RNA Interferente Pequeno/genética , Células Tumorais Cultivadas , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/genética , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Int J Radiat Biol ; 83(11-12): 803-11, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18058368

RESUMO

PURPOSE: The first reports that ionizing radiation (IR) induces rapid and persistent activation of transforming growth factor beta1 (TGFbeta) were nearly two decades ago. Subsequent studies have shown that TGFbeta is a major mediator of cellular and tissue responses to IR and have revealed novel facets of its complex biology. RESULTS: We and others have recently shown that inhibition of production or signaling of TGFbeta in epithelial cells modulates radiosensitivity and impedes activation of the DNA damage response program. The primary transducer of cellular response to DNA damage caused by ionizing radiation is the nuclear protein kinase ataxia telangiectasia mutated, whose activity is severely compromised when TGFbeta is inhibited. Thus, in conjunction, with its well-recognized contribution to normal tissue fibrosis, the role of TGFbeta in the genotoxic stress program provides a previously unsuspected avenue to modulate radiotherapy. CONCLUSIONS: We hypothesize that identification of the circumstances and tumors in which TGFbeta manipulation enhances tumor cell radiosensitivity, while protecting normal tissues, could significantly increase therapeutic index.


Assuntos
Neoplasias/radioterapia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Humanos , Modelos Biológicos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/fisiologia , Radiobiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transdução de Sinais/efeitos da radiação , Fator de Crescimento Transformador beta/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo
9.
Nat Commun ; 8: 14449, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28198367

RESUMO

ETS transcription factors are commonly deregulated in cancer by chromosomal translocation, overexpression or post-translational modification to induce gene expression programs essential in tumorigenicity. Targeted destruction of these proteins may have therapeutic impact. Here we report that Ets-1 destruction is regulated by the deubiquitinating enzyme, Usp9x, and has major impact on the tumorigenic program of metastatic melanoma. Ets-1 deubiquitination blocks its proteasomal destruction and enhances tumorigenicity, which could be reversed by Usp9x knockdown or inhibition. Usp9x and Ets-1 levels are coincidently elevated in melanoma with highest levels detected in metastatic tumours versus normal skin or benign skin lesions. Notably, Ets-1 is induced by BRAF or MEK kinase inhibition, resulting in increased NRAS expression, which could be blocked by inactivation of Usp9x and therapeutic combination of Usp9x and MEK inhibitor fully suppressed melanoma growth. Thus, Usp9x modulates the Ets-1/NRAS regulatory network and may have biologic and therapeutic implications.


Assuntos
Carcinogênese/patologia , GTP Fosfo-Hidrolases/genética , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Melanoma/patologia , Proteínas de Membrana/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Humanos , Melanoma/tratamento farmacológico , Proteínas de Membrana/metabolismo , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Regiões Promotoras Genéticas/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Estabilidade Proteica , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo
10.
J Vis Exp ; (86)2014 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-24797513

RESUMO

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior(1). However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex vivo system(2,3). Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D(4). 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved(3). One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D(6,7). Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.


Assuntos
Células Acinares/citologia , Neoplasias da Mama/patologia , Mama/citologia , Técnicas de Cultura de Células/métodos , Transformação Celular Neoplásica/patologia , Mama/patologia , Feminino , Humanos
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