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1.
iScience ; 26(7): 107006, 2023 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-37534190

RESUMO

This study evaluates the efficacy of combining targeted therapies with MET or SHP2 inhibitors to overcome MET-mediated resistance in different NSCLC subtypes. A prevalence study was conducted for MET amplification and overexpression in samples from patients with NSCLC who relapsed on ALK, ROS1, or RET tyrosine kinase inhibitors. MET-mediated resistance was detected in 37.5% of tissue biopsies, which allow the detection of MET overexpression, compared to 7.4% of liquid biopsies. The development of drug resistance by MET overexpression was confirmed in EGFRex19del-, KRASG12C-, HER2ex20ins-, and TPM3-NTRK1-mutant cell lines. The combination of targeted therapy with MET or SHP2 inhibitors was found to overcome MET-mediated resistance in both in vitro and in vivo assays. This study highlights the importance of considering MET overexpression as a resistance driver to NSCLC targeted therapies to better identify patients who could potentially benefit from combination approaches with MET or SHP2 inhibitors.

2.
Oncogene ; 39(38): 6053-6070, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32792685

RESUMO

BRAFV600E confers poor prognosis and is associated with a distinct subtype of colorectal cancer (CRC). Little is known, however, about the genetic events driving the initiation and progression of BRAFV600E mutant CRCs. Recent genetic analyses of CRCs indicate that BRAFV600E often coexists with alterations in the WNT- and p53 pathways, but their cooperation remains ill-defined. Therefore, we systematically compared small and large intestinal organoids from mice harboring conditional BraffloxV600E, Trp53LSL-R172H, and/or Apcflox/flox alleles. Using these isogenic models, we observe tissue-specific differences toward sudden BRAFV600E expression, which can be attributed to different ERK-pathway ground states in small and large intestinal crypts. BRAFV600E alone causes transient proliferation and suppresses epithelial organization, followed by organoid disintegration. Moreover, BRAFV600E induces a fetal-like dedifferentiation transcriptional program in colonic organoids, which resembles human BRAFV600E-driven CRC. Co-expression of p53R172H delays organoid disintegration, confers anchorage-independent growth, and induces invasive properties. Interestingly, p53R172H cooperates with BRAFV600E to modulate the abundance of transcripts linked to carcinogenesis, in particular within colonic organoids. Remarkably, WNT-pathway activation by Apc deletion fully protects organoids against BRAFV600E-induced disintegration and confers growth/niche factor independence. Still, Apc-deficient BRAFV600E-mutant organoids remain sensitive toward the MEK inhibitor trametinib, albeit p53R172H confers partial resistance against this clinically relevant compound. In summary, our systematic comparison of the response of small and large intestinal organoids to oncogenic alterations suggests colonic organoids to be better suited to model the human situation. In addition, our work on BRAF-, p53-, and WNT-pathway mutations provides new insights into their cooperation and for the design of targeted therapies.


Assuntos
Desdiferenciação Celular/genética , Transformação Celular Neoplásica/genética , Genes APC , Predisposição Genética para Doença , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteína Supressora de Tumor p53/genética , Animais , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Biologia Computacional/métodos , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Estudos de Associação Genética , Humanos , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Sistema de Sinalização das MAP Quinases , Camundongos , Modelos Biológicos , Invasividade Neoplásica , Oncogenes , Organoides , Técnicas de Cultura de Tecidos
3.
Blood ; 136(12): 1442-1455, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32542357

RESUMO

Acute graft-versus-host disease (GVHD) is a life-threatening complication after allogeneic hematopoietic cell transplantation (allo-HCT). Although currently used GVHD treatment regimens target the donor immune system, we explored here an approach that aims at protecting and regenerating Paneth cells (PCs) and intestinal stem cells (ISCs). Glucagon-like-peptide-2 (GLP-2) is an enteroendocrine tissue hormone produced by intestinal L cells. We observed that acute GVHD reduced intestinal GLP-2 levels in mice and patients developing GVHD. Treatment with the GLP-2 agonist, teduglutide, reduced de novo acute GVHD and steroid-refractory GVHD, without compromising graft-versus-leukemia (GVL) effects in multiple mouse models. Mechanistically GLP-2 substitution promoted regeneration of PCs and ISCs, which enhanced production of antimicrobial peptides and caused microbiome changes. GLP-2 expanded intestinal organoids and reduced expression of apoptosis-related genes. Low numbers of L cells in intestinal biopsies and high serum levels of GLP-2 were associated with a higher incidence of nonrelapse mortality in patients undergoing allo-HCT. Our findings indicate that L cells are a target of GVHD and that GLP-2-based treatment of acute GVHD restores intestinal homeostasis via an increase of ISCs and PCs without impairing GVL effects. Teduglutide could become a novel combination partner for immunosuppressive GVHD therapy to be tested in clinical trials.


Assuntos
Peptídeo 2 Semelhante ao Glucagon/uso terapêutico , Doença Enxerto-Hospedeiro/tratamento farmacológico , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Intestinos/efeitos dos fármacos , Celulas de Paneth/efeitos dos fármacos , Peptídeos/uso terapêutico , Células-Tronco/efeitos dos fármacos , Animais , Feminino , Fármacos Gastrointestinais/uso terapêutico , Doença Enxerto-Hospedeiro/patologia , Humanos , Intestinos/citologia , Intestinos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Celulas de Paneth/patologia , Células-Tronco/patologia , Transplante Homólogo/efeitos adversos
4.
Oncogene ; 38(8): 1324-1339, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30659267

RESUMO

Copy number gains, point mutations and epigenetic silencing events are increasingly observed in genes encoding elements of the Ras/Raf/MEK/ERK signaling axis in human breast cancer. The three Raf kinases A-Raf, B-Raf, and Raf-1 have an important role as gatekeepers in ERK pathway activation and are often dysregulated by somatic alterations of their genes or by the aberrant activity of receptor tyrosine kinases (RTKs) and Ras-GTPases. B-Raf represents the most potent Raf isoform and a critical effector downstream of RTKs and RAS proteins. Aberrant RTK signaling is mimicked by the polyoma middle T antigen (PyMT), which activates various oncogenic signaling pathways, incl. the RAS/ERK axis, in a similar manner as RTKs in human breast cancer. Mammary epithelial cell directed expression of PyMT in mice by the MMTV-PyMT transgene induces mammary hyperplasia progressing over adenoma to metastatic breast cancer with an almost complete penetrance. To understand the functional role of B-Raf in this model for luminal type B breast cancer, we crossed MMTV-PyMT mice with animals that either lack B-Raf expression in the mammary gland or express the signaling impaired B-RafAVKA mutant. The AVKA mutation prevents phosphorylation of T599 and S602 in the B-Raf activation loop and thereby activation of the kinase by upstream signals. We demonstrate for the first time that B-Raf expression and activation is important for tumor initiation in vivo as well as for lung metastasis. Isogenic tumor cell lines generated from conditional Braf knock-out or knock-in mice displayed a reduction in EGF-induced ERK pathway activity as well as in proliferation and invasive growth in three-dimensional matrigel cultures. Our results suggest that B-Raf, which has been hardly studied in the context of breast cancer, represents a critical effector of the PyMT oncoprotein and invite for an assessment of its functional role in human breast cancer.


Assuntos
Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Neoplasias Mamárias Animais/genética , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Modelos Animais de Doenças , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Knockout , Mutação , Proteínas Proto-Oncogênicas A-raf/genética , Proteínas Proto-Oncogênicas B-raf/deficiência , Proteínas Proto-Oncogênicas c-raf/genética
5.
Cell Rep ; 21(1): 274-288, 2017 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-28978480

RESUMO

The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time.


Assuntos
Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia Intravital/métodos , Imagem com Lapso de Tempo/métodos , Proteínas rho de Ligação ao GTP/genética , Animais , Antineoplásicos/farmacologia , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Movimento Celular/efeitos dos fármacos , Dasatinibe/farmacologia , Cloridrato de Erlotinib/farmacologia , Feminino , Transferência Ressonante de Energia de Fluorescência/instrumentação , Regulação da Expressão Gênica , Intestino Delgado/metabolismo , Intestino Delgado/ultraestrutura , Microscopia Intravital/instrumentação , Glândulas Mamárias Animais/irrigação sanguínea , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/ultraestrutura , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/ultraestrutura , Mecanotransdução Celular , Camundongos , Camundongos Transgênicos , Neutrófilos/metabolismo , Neutrófilos/ultraestrutura , Osteócitos/metabolismo , Osteócitos/ultraestrutura , Neoplasias Pancreáticas/irrigação sanguínea , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/ultraestrutura , Imagem com Lapso de Tempo/instrumentação , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP
6.
EBioMedicine ; 20: 79-97, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28499923

RESUMO

Despite being overexpressed in different tumor entities, RIO kinases are hardly characterized in mammalian cells. We investigated the role of these atypical kinases in different cancer cells. Using isogenic colon-, breast- and lung cancer cell lines, we demonstrate that knockdown of RIOK1, but not of RIOK2 or RIOK3, strongly impairs proliferation and invasiveness in conventional and 3D culture systems. Interestingly, these effects were mainly observed in RAS mutant cancer cells. In contrast, growth of RAS wildtype Caco-2 and Bcr-Abl-driven K562 cells is not affected by RIOK1 knockdown, suggesting a specific requirement for RIOK1 in the context of oncogenic RAS signaling. Furthermore, we show that RIOK1 activates NF-κB signaling and promotes cell cycle progression. Using proteomics, we identified the pro-invasive proteins Metadherin and Stathmin1 to be regulated by RIOK1. Additionally, we demonstrate that RIOK1 promotes lung colonization in vivo and that RIOK1 is overexpressed in different subtypes of human lung- and breast cancer. Altogether, our data suggest RIOK1 as a potential therapeutic target, especially in RAS-driven cancers.


Assuntos
Antígenos de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Animais , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Modelos Animais de Doenças , Expressão Gênica , Técnicas de Inativação de Genes , Xenoenxertos , Humanos , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Metástase Neoplásica , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas p21(ras)/genética , Células Tumorais Cultivadas
7.
Cell Rep ; 14(1): 152-167, 2016 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-26725115

RESUMO

E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments.


Assuntos
Caderinas/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Imagem Óptica/métodos , Microambiente Tumoral , Animais , Caderinas/genética , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/genética , Especificidade de Órgãos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
8.
Cell Cycle ; 12(19): 3203-18, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-24013422

RESUMO

Fifty years of genetic and molecular experiments have revealed a wealth of molecular interactions involved in the control of cell division. In light of the complexity of this control system, mathematical modeling has proved useful in analyzing biochemical hypotheses that can be tested experimentally. Stochastic modeling has been especially useful in understanding the intrinsic variability of cell cycle events, but stochastic modeling has been hampered by a lack of reliable data on the absolute numbers of mRNA molecules per cell for cell cycle control genes. To fill this void, we used fluorescence in situ hybridization (FISH) to collect single molecule mRNA data for 16 cell cycle regulators in budding yeast, Saccharomyces cerevisiae. From statistical distributions of single-cell mRNA counts, we are able to extract the periodicity, timing, and magnitude of transcript abundance during the cell cycle. We used these parameters to improve a stochastic model of the cell cycle to better reflect the variability of molecular and phenotypic data on cell cycle progression in budding yeast.


Assuntos
Modelos Biológicos , Pontos de Checagem do Ciclo Celular , Ciclinas/genética , Ciclinas/metabolismo , Redes Reguladoras de Genes , Hibridização in Situ Fluorescente , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica
9.
Perspect Biol Med ; 55(4): 490-502, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23502560

RESUMO

The emerging field of synthetic biology is a novel biological discipline at the interface between traditional biology, chemistry, and engineering sciences. Synthetic biology aims at the rational design of complex synthetic biological devices and systems with desired properties by combining compatible, modular biological parts in a systematic manner. While the first engineered systems were mainly proof-of-principle studies to demonstrate the power of the modular engineering approach of synthetic biology, subsequent systems focus on applications in the health, environmental, and energy sectors. This review describes recent approaches for biomedical applications that were developed along the synthetic biology design hierarchy, at the level of individual parts, of devices, and of complex multicellular systems. It describes how synthetic biological parts can be used for the synthesis of drug-delivery tools, how synthetic biological devices can facilitate the discovery of novel drugs, and how multicellular synthetic ecosystems can give insight into population dynamics of parasites and hosts. These examples demonstrate how this new discipline could contribute to novel solutions in the biopharmaceutical industry.


Assuntos
Pesquisa Biomédica , Biologia Sintética , Animais , Descoberta de Drogas , Regulação da Expressão Gênica , Genótipo , Interações Hospedeiro-Parasita , Humanos , Biologia Molecular , Fenótipo , Biologia de Sistemas , Tecnologia Farmacêutica
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