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1.
Proc Natl Acad Sci U S A ; 114(3): 492-497, 2017 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-28034921

RESUMO

Tumor microvasculature tends to be malformed, more permeable, and more tortuous than vessels in healthy tissue, effects that have been largely attributed to up-regulated VEGF expression. However, tumor tissue tends to stiffen during solid tumor progression, and tissue stiffness is known to alter cell behaviors including proliferation, migration, and cell-cell adhesion, which are all requisite for angiogenesis. Using in vitro, in vivo, and ex ovo models, we investigated the effects of matrix stiffness on vessel growth and integrity during angiogenesis. Our data indicate that angiogenic outgrowth, invasion, and neovessel branching increase with matrix cross-linking. These effects are caused by increased matrix stiffness independent of matrix density, because increased matrix density results in decreased angiogenesis. Notably, matrix stiffness up-regulates matrix metalloproteinase (MMP) activity, and inhibiting MMPs significantly reduces angiogenic outgrowth in stiffer cross-linked gels. To investigate the functional significance of altered endothelial cell behavior in response to matrix stiffness, we measured endothelial cell barrier function on substrates mimicking the stiffness of healthy and tumor tissue. Our data indicate that barrier function is impaired and the localization of vascular endothelial cadherin is altered as function of matrix stiffness. These results demonstrate that matrix stiffness, separately from matrix density, can alter vascular growth and integrity, mimicking the changes that exist in tumor vasculature. These data suggest that therapeutically targeting tumor stiffness or the endothelial cell response to tumor stiffening may help restore vessel structure, minimize metastasis, and aid in drug delivery.


Assuntos
Matriz Extracelular/fisiologia , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/fisiopatologia , Microvasos/fisiopatologia , Animais , Fenômenos Biomecânicos , Bovinos , Células Cultivadas , Embrião de Galinha , Colágeno/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Neoplasias Mamárias Experimentais/patologia , Metaloproteinases da Matriz/metabolismo , Camundongos , Microvasos/patologia , Invasividade Neoplásica/patologia , Invasividade Neoplásica/fisiopatologia , Neovascularização Patológica/patologia , Neovascularização Patológica/fisiopatologia , Fenótipo , Microambiente Tumoral/fisiologia , Rigidez Vascular/fisiologia
2.
Proc Natl Acad Sci U S A ; 112(27): 8314-9, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26106154

RESUMO

Alternative splicing of proteins gives rise to different isoforms that play a crucial role in regulating several cellular processes. Notably, splicing profiles are altered in several cancer types, and these profiles are believed to be involved in driving the oncogenic process. Although the importance of alternative splicing alterations occurring during cancer is increasingly appreciated, the underlying regulatory mechanisms remain poorly understood. In this study, we use both biochemical and physical tools coupled with engineered models, patient samples, and a murine model to investigate the role of the mechanical properties of the tumor microenvironment in regulating the production of the extra domain-B (EDB) splice variant of fibronectin (FN), a hallmark of tumor angiogenesis. Specifically, we show that the amount of EDB-FN produced by endothelial cells increases with matrix stiffness both in vitro and within mouse mammary tumors. Matrix stiffness regulates splicing through the activation of serine/arginine rich (SR) proteins, the splicing factors involved in the production of FN isoforms. Activation of the SR proteins by matrix stiffness and the subsequent production of EDB-FN are dependent on intracellular contractility and PI3K-AKT signaling. Notably, matrix stiffness-mediated splicing is not limited to EDB-FN, but also affects splicing in the production of PKC ßII and the VEGF 165b splice variant. Together, these results demonstrate that the mechanical properties of the microenvironment regulate alternative splicing and establish a previously unidentified mechanism by which cells can adapt to their microenvironment.


Assuntos
Processamento Alternativo , Fibronectinas/genética , Neoplasias/genética , Microambiente Tumoral/genética , Animais , Arginina/genética , Arginina/metabolismo , Sítios de Ligação/genética , Fenômenos Biomecânicos , Western Blotting , Bovinos , Células Cultivadas , Células Endoteliais/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Humanos , Camundongos , Microscopia Confocal , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Serina/genética , Serina/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo
3.
Cell Metab ; 34(6): 874-887.e6, 2022 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-35504291

RESUMO

The tumor microenvironment (TME) contains a rich source of nutrients that sustains cell growth and facilitate tumor development. Glucose and glutamine in the TME are essential for the development and activation of effector T cells that exert antitumor function. Immunotherapy unleashes T cell antitumor function, and although many solid tumors respond well, a significant proportion of patients do not benefit. In patients with KRAS-mutant lung adenocarcinoma, KEAP1 and STK11/Lkb1 co-mutations are associated with impaired response to immunotherapy. To investigate the metabolic and immune microenvironment of KRAS-mutant lung adenocarcinoma, we generated murine models that reflect the KEAP1 and STK11/Lkb1 mutational landscape in these patients. Here, we show increased glutamate abundance in the Lkb1-deficient TME associated with CD8 T cell activation in response to anti-PD1. Combination treatment with the glutaminase inhibitor CB-839 inhibited clonal expansion and activation of CD8 T cells. Thus, glutaminase inhibition negatively impacts CD8 T cells activated by anti-PD1 immunotherapy.


Assuntos
Quinases Proteína-Quinases Ativadas por AMP , Adenocarcinoma de Pulmão , Linfócitos T CD8-Positivos , Glutaminase , Neoplasias Pulmonares , Quinases Proteína-Quinases Ativadas por AMP/deficiência , Quinases Proteína-Quinases Ativadas por AMP/imunologia , Quinases Proteína-Quinases Ativadas por AMP/metabolismo , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Glutaminase/antagonistas & inibidores , Glutaminase/imunologia , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Ativação Linfocitária , Camundongos , Mutação , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Microambiente Tumoral
4.
Chirality ; 23(9): 808-19, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21919077

RESUMO

The transfer of chirality from a guest molecule to an achiral host is the subject of significant interest especially when, upon chiral induction, the chiroptical response of the host/guest complex can effectively report the absolute configuration (AC) of the guest. For more than a decade, dimeric metalloporphyrin hosts (tweezers) have been successfully applied as chirality probes for determination of the AC for a wide variety of chiral synthetic compounds and natural products. The objective of this study is to investigate the utility of a new class of melamine-bridged Zn-porphyrin tweezers as sensitive AC reporters. A combined approach based on an experimental CD analysis and a theoretical prediction of the prevailing interporphyrin helicity demonstrates that these tweezers display favorable properties for chiral recognition. Herein, we discuss the application of the melamine-bridged tweezer to the chiral recognition of a diverse set of chiral guests, such as 1,2-diamines, α-amino-esters and amides, secondary alcohols, and 1,2-amino-alcohols. The bulky periphery and the presence of a rigid porphyrin linkage lead, in some cases, to a more enhanced CD sensitivity than that reported earlier with other tweezers.


Assuntos
Dicroísmo Circular/métodos , Metaloporfirinas/química , Modelos Moleculares , Triazinas/química , Álcoois/química , Aminas/química , Amino Álcoois/química , Simulação por Computador , Diaminas/química , Espectroscopia de Ressonância Magnética/métodos , Conformação Molecular , Estrutura Molecular , Espectrofotometria Ultravioleta/métodos
5.
Methods Mol Biol ; 1983: 151-171, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31087297

RESUMO

New therapeutics directed against established and novel molecular targets are urgently needed to intervene against cancer. Recently, it was reported that several members of the sirtuin family (SIRT1-7), the mammalian orthologs of the silent information regulator 2 (Sir2) protein in Saccharomyces cerevisiae, play important roles in carcinogenesis. Although SIRT2 has been attributed both tumor-promoting and tumor-suppressing activities in different contexts, selective SIRT2 inhibition with a small molecule mechanism-based inhibitor known as Thiomyristoyl lysine (TM) repressed the growth of breast cancer cell lines. In light of the anticancer effect of SIRT2 inhibition in cell culture, it was critical to assess the efficacy of TM as a potential anticancer therapy in vivo. This was accomplished by testing the SIRT2 inhibitor in genetically engineered and xenotransplantation mouse models of breast cancer, using the procedures detailed in this chapter.


Assuntos
Antineoplásicos/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Inibidores de Histona Desacetilases/farmacologia , Neoplasias/metabolismo , Sirtuína 2/antagonistas & inibidores , Animais , Antineoplásicos/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cromatografia Líquida , Monitoramento de Medicamentos , Feminino , Inibidores de Histona Desacetilases/química , Masculino , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Cancer Cell ; 29(3): 297-310, 2016 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-26977881

RESUMO

Targeting sirtuins for cancer treatment has been a topic of debate due to conflicting reports and lack of potent and specific inhibitors. We have developed a thiomyristoyl lysine compound, TM, as a potent SIRT2-specific inhibitor with a broad anticancer effect in various human cancer cells and mouse models of breast cancer. Mechanistically, SIRT2 inhibition promotes c-Myc ubiquitination and degradation. The anticancer effect of TM correlates with its ability to decrease c-Myc level. TM had limited effects on non-cancerous cells and tumor-free mice, suggesting that cancer cells have an increased dependency on SIRT2 that can be exploited for therapeutic benefit. Our studies demonstrate that SIRT2-selective inhibitors are promising anticancer agents and may represent a general strategy to target certain c-Myc-driven cancers.


Assuntos
Antineoplásicos/farmacologia , Proteínas Oncogênicas/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sirtuína 2/antagonistas & inibidores , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Células HeLa , Humanos , Lisina/metabolismo , Células MCF-7 , Camundongos , Sirtuína 2/metabolismo , Ubiquitinação/efeitos dos fármacos
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