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1.
Int J Mol Sci ; 20(12)2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31238530

RESUMO

DUSP6 is a dual-specificity phosphatase (DUSP) involved in breast cancer progression, recurrence, and metastasis. DUSP6 is predominantly cytoplasmic in HER2+ primary breast cancer cells, but the expression and subcellular localization of DUSPs, especially DUSP6, in HER2-positive circulating tumor cells (CTCs) is unknown. Here we used the DEPArray system to identify and isolate CTCs from metastatic triple negative breast cancer (TNBC) patients and performed single-cell NanoString analysis to quantify cancer pathway gene expression in HER2-positive and HER2-negative CTC populations. All TNBC patients contained HER2-positive CTCs. HER2-positive CTCs were associated with increased ERK1/ERK2 expression, which are direct DUSP6 targets. DUSP6 protein expression was predominantly nuclear in breast CTCs and the brain metastases but not pleura or lung metastases of TNBC patients. Therefore, nuclear DUSP6 may play a role in the association with cancer spreading in TNBC patients, including brain metastasis.


Assuntos
Biomarcadores Tumorais , Neoplasias Encefálicas/secundário , Fosfatase 6 de Especificidade Dupla/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/genética , Modelos Animais de Doenças , Fosfatase 6 de Especificidade Dupla/antagonistas & inibidores , Fosfatase 6 de Especificidade Dupla/metabolismo , Feminino , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Invasividade Neoplásica , Estadiamento de Neoplasias , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Ligação Proteica , Transporte Proteico , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Análise de Célula Única , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/terapia , Ensaios Antitumorais Modelo de Xenoenxerto , Fatores de Transcrição de p300-CBP/metabolismo
2.
Proc Natl Acad Sci U S A ; 106(35): 14984-9, 2009 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-19706459

RESUMO

A remarkable feature of the adaptive immune system is the speed at which small numbers of antigen-specific lymphocytes can mediate a successful immune response. Rapid expansion of T and B lymphocyte clones that have receptors specific for a particular antigen is one of the primary means by which a swift response is generated. Although much of this clonal expansion is caused by the division of antigen-specific cells, here we demonstrate an additional mechanism by which the pool of effector T cells against a viral infection can quickly enlarge. Our data show that virus-specific CD8+ cytotoxic T lymphocytes (CTL) can transfer their T cell receptors (TCR) to recipient CTL of an unrelated specificity that, as a consequence, gain the antigen specificity of the donor T cell. This process occurs within minutes via membrane exchange and results in the recipient CTL acquiring the ability to recognize and eliminate cells targeted by the donor TCR, while still retaining the antigen specificity of its own TCR. Such receptor sharing allows rapid, proliferation-independent expansion of virus-specific T cell clones of low frequency and plays a highly significant antiviral role that can protect the host from an otherwise lethal infection.


Assuntos
Vírus da Ectromelia/imunologia , Ectromelia Infecciosa/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Membrana Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Ectromelia Infecciosa/sangue , Epitopos/imunologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Carga Viral
3.
Front Oncol ; 12: 862427, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35719960

RESUMO

Objective: Lysine-Specific Demethylase-1 (LSD1) is overexpressed in breast cancer cells and facilitate mesenchymal properties which may contribute to therapeutic resistance and cancer progression. The purpose of this study was to investigate the safety of combination, nab-paclitaxel and phenelzine, an irreversible LSD1 inhibitor in patients with metastatic breast cancer (mBC). Methods: Eligible patients with mBC were treated with nab-paclitaxel (100mg/m2) weekly for 3 weeks with one week break in a 28-day cycle. Dose escalation of phenelzine followed the Cumulative Cohort Design and phenelzine treatment commenced from day 2 of first cycle. Eleven patients were screened, and eligible patients were enrolled in cohorts with the dose of phenelzine ranging from 45mg to 90mg. Results: The Optimum Biological Dose was established at 60mg of phenelzine daily in combination with nab-paclitaxel and considered as the recommended phase 2 dose. Most (95%) of adverse events were grade 1 or 2 with two grade 3 events being diarrhea and neutropenia at 45mg and 60mg phenelzine respectively, with no unexpected toxicity/deaths. Commonly reported toxicities were fatigue (n=4,50%), dizziness (n=6,75%), neutropenia (n=3,37.5%), peripheral neuropathy (n=3,37.5%), diarrhea (n=2,25%), and hallucination (n=2,25%). After a median follow up of 113 weeks, all patients showed disease progression on trial with 4 patients being alive at the time of data cut off, including one patient with triple negative breast cancer. Median progression-free survival was 34 weeks. Significant inhibition of LSD1 and suppression of mesenchymal markers in circulating tumor cells were noted. Conclusion: Phenelzine in combination with nab-paclitaxel was well tolerated, without any unexpected toxicities in patients with mBC and demonstrated evidence of antitumor activity. For the first time, this proof-of-concept study showed in-vivo inhibition of LSD1 suppressed mesenchymal markers, which are known to facilitate generation of cancer stem cells with metastatic potential. Clinical Trial Registration: ClinicalTrials.Gov NCT03505528, UTN of U1111-1197-5518.

4.
Cancers (Basel) ; 14(6)2022 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-35326747

RESUMO

Protein kinase C (PKC)-θ is a serine/threonine kinase with both cytoplasmic and nuclear functions. Nuclear chromatin-associated PKC-θ (nPKC-θ) is increasingly recognized to be pathogenic in cancer, whereas its cytoplasmic signaling is restricted to normal T-cell function. Here we show that nPKC-θ is enriched in circulating tumor cells (CTCs) in patients with triple-negative breast cancer (TNBC) brain metastases and immunotherapy-resistant metastatic melanoma and is associated with poor survival in immunotherapy-resistant disease. To target nPKC-θ, we designed a novel PKC-θ peptide inhibitor (nPKC-θi2) that selectively inhibits nPKC-θ nuclear translocation but not PKC-θ signaling in healthy T cells. Targeting nPKC-θ reduced mesenchymal cancer stem cell signatures in immunotherapy-resistant CTCs and TNBC xenografts. PKC-θ was also enriched in the nuclei of CD8+ T cells isolated from stage IV immunotherapy-resistant metastatic cancer patients. We show for the first time that nPKC-θ complexes with ZEB1, a key repressive transcription factor in epithelial-to-mesenchymal transition (EMT), in immunotherapy-resistant dysfunctional PD1+/CD8+ T cells. nPKC-θi2 inhibited the ZEB1/PKC-θ repressive complex to induce cytokine production in CD8+ T cells isolated from patients with immunotherapy-resistant disease. These data establish for the first time that nPKC-θ mediates immunotherapy resistance via its activity in CTCs and dysfunctional CD8+ T cells. Disrupting nPKC-θ but retaining its cytoplasmic function may offer a means to target metastases in combination with chemotherapy or immunotherapy.

5.
Front Immunol ; 11: 1228, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32612611

RESUMO

Lysine specific demethylase 1 (LSD1) is a key epigenetic eraser enzyme implicated in cancer metastases and recurrence. Nuclear LSD1 phosphorylated at serine 111 (nLSD1p) has been shown to be critical for the development of breast cancer stem cells. Here we show that circulating tumor cells isolated from immunotherapy-resistant metastatic melanoma patients express higher levels of nLSD1p compared to responders, which is associated with co-expression of stem-like, mesenchymal genes. Targeting nLSD1p with selective nLSD1 inhibitors better inhibits the stem-like mesenchymal signature than traditional FAD-specific LSD1 catalytic inhibitors such as GSK2879552. We also demonstrate that nLSD1p is enriched in PD-1+CD8+ T cells from resistant melanoma patients and 4T1 immunotherapy-resistant mice. Targeting the LSD1p nuclear axis induces IFN-γ/TNF-α-expressing CD8+ T cell infiltration into the tumors of 4T1 immunotherapy-resistant mice, which is further augmented by combined immunotherapy. Underpinning these observations, nLSD1p is regulated by the key T cell exhaustion transcription factor EOMES in dysfunctional CD8+ T cells. EOMES co-exists with nLSD1p in PD-1+CD8+ T cells in resistant patients, and nLSD1p regulates EOMES nuclear dynamics via demethylation/acetylation switching of critical EOMES residues. Using novel antibodies to target these post-translational modifications, we show that EOMES demethylation/acetylation is reciprocally expressed in resistant and responder patients. Overall, we show for the first time that dual inhibition of metastatic cancer cells and re-invigoration of the immune system requires LSD1 inhibitors that target the nLSD1p axis.


Assuntos
Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/genética , Histona Desmetilases/genética , Neoplasias/etiologia , Proteínas com Domínio T/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Animais , Biomarcadores , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Feminino , Citometria de Fluxo , Imunofluorescência , Expressão Gênica , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Histona Desmetilases/metabolismo , Humanos , Imunoterapia , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Proteínas com Domínio T/genética , Linfócitos T/imunologia , Resultado do Tratamento
6.
Front Immunol ; 10: 1351, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31249575

RESUMO

Macrophages play an important role in regulating the tumor microenvironment (TME). Here we show that classical (M1) macrophage polarization reduced expression of LSD1, nuclear REST corepressor 1 (CoREST), and the zinc finger protein SNAIL. The LSD1 inhibitor phenelzine targeted both the flavin adenine dinucleotide (FAD) and CoREST binding domains of LSD1, unlike the LSD1 inhibitor GSK2879552, which only targeted the FAD domain. Phenelzine treatment reduced nuclear demethylase activity and increased transcription and expression of M1-like signatures both in vitro and in a murine triple-negative breast cancer model. Overall, the LSD1 inhibitors phenelzine and GSK2879552 are useful tools for dissecting the contribution of LSD1 demethylase activity and the nuclear LSD1-CoREST complex to switching macrophage polarization programs. These findings suggest that inhibitors must have dual FAD and CoREST targeting abilities to successfully initiate or prime macrophages toward an anti-tumor M1-like phenotype in triple-negative breast cancer.


Assuntos
Histona Desmetilases/metabolismo , Macrófagos/imunologia , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Diferenciação Celular , Proteínas Correpressoras/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Flavina-Adenina Dinucleotídeo/metabolismo , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/genética , Humanos , Ativação de Macrófagos , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Fenelzina/farmacologia , Células RAW 264.7 , RNA Interferente Pequeno/genética , Fatores de Transcrição da Família Snail/metabolismo , Células Th1/imunologia , Neoplasias de Mama Triplo Negativas/imunologia , Microambiente Tumoral
7.
Cell Mol Neurobiol ; 28(8): 1129-38, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18683040

RESUMO

Endothelins regulate cellular functions in the mammalian brain through the endothelin receptors A and B (EDNRA and EDNRB). In this study, we investigated the role of EDNRB on cell proliferation in the cerebellum by using the spotting lethal (sl) rat, which carries a naturally occurring deletion in the EDNRB gene. Proliferating cells in the three genotypes, wild-type (+/+), heterozygous (+/sl) and homozygous mutant (sl/sl) rats were labelled by intraperitoneal injection of 5-bromo-2'-deoxyuridine (BrdU) at postnatal day 2. The density of BrdU-positive cells (per mm(2)) in the external germinal layer of sl/sl rats (Mean +/- SEM, 977 +/- 388) was significantly reduced compared to +/+ (4915 +/- 631) and +/sl (2304 +/- 557) rats. Subsequently, we examined the effects of EDNRB mutation on neural apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labelling assay. This showed that the density of apoptotic cells in the cerebella of sl/sl rats (9.3 +/- 0.5/mm(2)) was significantly more increased than +/+ rats (4 +/- 0.7). The expression of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) were measured with standard ELISA, but were unchanged in all genotypes. These results suggest that ENDRB mediates neural proliferation and have anti-apoptotic effects in the cerebellum of the postnatal rat, and that these effects are independent of changes in the expression of BDNF and GDNF. Our findings will lead to better understanding of the morphological changes in the cerebellum of Hirschsprung's disease patients with congenital EDNRB mutation.


Assuntos
Apoptose , Cerebelo/citologia , Cerebelo/metabolismo , Neurônios/citologia , Receptor de Endotelina B/deficiência , Células-Tronco/citologia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Bromodesoxiuridina/metabolismo , Contagem de Células , Núcleo Celular/metabolismo , Proliferação de Células , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Marcação In Situ das Extremidades Cortadas , Mutação/genética , Neurônios/metabolismo , Ratos , Ratos Wistar , Receptor de Endotelina B/metabolismo , Células-Tronco/metabolismo
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