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1.
Oncol Res ; 32(4): 607-614, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38560568

RESUMO

C-mannosylation is a post-translational modification that occurs intracellularly in the endoplasmic reticulum. In humans, biosynthesis of C-mannosylation in proteins containing thrombospondin type 1 repeat is catalyzed by the DPY19 family; nonetheless, biological functions of protein C-mannosylation are not yet fully understood, especially in tumor progression. Vasculogenic mimicry (VM) is the formation of fluid-conducting channels by highly invasive and genetically deregulated tumor cells, enabling the tumors to form matrix-embedded vasculogenic structures, containing plasma and blood cells to meet the metabolic demands of rapidly growing tumors. In this study, we focused on DPY19L3, a C-mannosyltransferase, and aimed to unravel its role in VM. Knockout of DPY19L3 inhibited the formation of VM in HT1080 human fibrosarcoma cells. Re-expression of wild-type DPY19L3 recovered VM formation; however, DPY19L3 isoform2, an enzymatic activity-defect mutant, did not restore it, suggesting that the C-mannosyltransferase activity of DPY19L3 is crucial to its function. Furthermore, the knockdown of DPY19L3 in MDA-MB-231 breast cancer cells hindered its network formation ability. Altogether, our findings suggest that DPY19L3 is required for VM formation and stipulate the relevance of C-mannosylation in oncogenesis.


Assuntos
Neoplasias da Mama , Manosiltransferases , Feminino , Humanos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Manosiltransferases/genética , Manosiltransferases/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo
2.
NPJ Precis Oncol ; 7(1): 107, 2023 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-37880373

RESUMO

Cancer cell resistance arises when tyrosine kinase inhibitor (TKI)-targeted therapies induce a drug-tolerant persister (DTP) state with growth via genetic aberrations, making DTP cells potential therapeutic targets. We screened an anti-cancer compound library and identified fibroblast growth factor receptor 1 (FGFR1) promoting alectinib-induced anaplastic lymphoma kinase (ALK) fusion-positive DTP cell's survival. FGFR1 signaling promoted DTP cell survival generated from basal FGFR1- and fibroblast growth factor 2 (FGF2)-high protein expressing cells, following alectinib treatment, which is blocked by FGFR inhibition. The hazard ratio for progression-free survival of ALK-TKIs increased in patients with ALK fusion-positive non-small cell lung cancer with FGFR1- and FGF2-high mRNA expression at baseline. The combination of FGFR and targeted TKIs enhanced cell growth inhibition and apoptosis induction in basal FGFR1- and FGF2-high protein expressing cells with ALK-rearranged and epidermal growth factor receptor (EGFR)-mutated NSCLC, human epidermal growth factor receptor 2 (HER2)-amplified breast cancer, or v-raf murine sarcoma viral oncogene homolog B1 (BRAF)-mutated melanoma by preventing compensatory extracellular signal-regulated kinase (ERK) reactivation. These results suggest that a targeted TKI-induced DTP state results from an oncogenic switch from activated oncogenic driver signaling to the FGFR1 pathway in basal FGFR1- and FGF2-high expressing cancers and initial dual blockade of FGFR and driver oncogenes based on FGFR1 and FGF2 expression levels at baseline is a potent treatment strategy to prevent acquired drug resistance to targeted TKIs through DTP cells regardless of types of driver oncogenes.

3.
Cancer Discov ; 13(9): 1998-2011, 2023 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-37377403

RESUMO

Several fibroblast growth factor receptor (FGFR) inhibitors are approved or in clinical development for the treatment of FGFR-driven urothelial cancer, and molecular mechanisms of resistance leading to patient relapses have not been fully explored. We identified 21 patients with FGFR-driven urothelial cancer treated with selective FGFR inhibitors and analyzed postprogression tissue and/or circulating tumor DNA (ctDNA). We detected single mutations in the FGFR tyrosine kinase domain in seven (33%) patients (FGFR3 N540K, V553L/M, V555L/M, E587Q; FGFR2 L551F) and multiple mutations in one (5%) case (FGFR3 N540K, V555L, and L608V). Using Ba/F3 cells, we defined their spectrum of resistance/sensitivity to multiple selective FGFR inhibitors. Eleven (52%) patients harbored alterations in the PI3K-mTOR pathway (n = 4 TSC1/2, n = 4 PIK3CA, n = 1 TSC1 and PIK3CA, n = 1 NF2, n = 1 PTEN). In patient-derived models, erdafitinib was synergistic with pictilisib in the presence of PIK3CA E545K, whereas erdafitinib-gefitinib combination was able to overcome bypass resistance mediated by EGFR activation. SIGNIFICANCE: In the largest study on the topic thus far, we detected a high frequency of FGFR kinase domain mutations responsible for resistance to FGFR inhibitors in urothelial cancer. Off-target resistance mechanisms involved primarily the PI3K-mTOR pathway. Our findings provide preclinical evidence sustaining combinatorial treatment strategies to overcome bypass resistance. See related commentary by Tripathi et al., p. 1964. This article is featured in Selected Articles from This Issue, p. 1949.


Assuntos
Carcinoma de Células de Transição , Neoplasias da Bexiga Urinária , Humanos , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Carcinoma de Células de Transição/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Serina-Treonina Quinases TOR , Classe I de Fosfatidilinositol 3-Quinases , Fosfatidilinositol 3-Quinases
4.
FEBS J ; 290(1): 196-208, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-35942636

RESUMO

C-mannosylation is a unique type of protein glycosylation via C-C linkage between an α-mannose and a tryptophan residue. This modification has been identified in about 30 proteins and regulates several functions, such as protein secretion and intracellular localization, as well as protein stability. About half of C-mannosylated proteins are categorized as proteins containing thrombospondin type 1 repeat domain or type I cytokine receptors. To evaluate whether C-mannosylation broadly affects protein functions regardless of protein domain or family, we have sought to identify other types of C-mannosylated protein and analyse their functions. In this study, we focused on receptor activity modifying protein 1, which neither contains thrombospondin type 1 repeat domain nor belongs to the type I cytokine receptors. Our mass spectrometry analysis demonstrated that RAMP1 is C-mannosylated at Trp56 . It has been shown that RAMP1 transports to the plasma membrane after dimerization with calcitonin receptor-like receptor and is important for ligand-dependent downstream signalling activation. Our results showed that C-mannosylation has no effect on this transport activity. On the other hand, C-mannosylation did enhance protein stability and cell migration activity. Our data may provide new insight into both C-mannosylation research and novel RAMP1 analysis.


Assuntos
Receptores de Citocinas , Trombospondinas , Glicosilação , Membrana Celular , Movimento Celular
5.
Nat Commun ; 12(1): 1261, 2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33627640

RESUMO

ALK gene rearrangement was observed in 3%-5% of non-small cell lung cancer patients, and multiple ALK-tyrosine kinase inhibitors (TKIs) have been sequentially used. Multiple ALK-TKI resistance mutations have been identified from the patients, and several compound mutations, such as I1171N + F1174I or I1171N + L1198H are resistant to all the approved ALK-TKIs. In this study, we found that gilteritinib has an inhibitory effect on ALK-TKI-resistant single mutants and I1171N compound mutants in vitro and in vivo. Surprisingly, EML4-ALK I1171N + F1174I compound mutant-expressing tumors were not completely shrunk but regrew within a short period of time after alectinib or lorlatinib treatment. However, the relapsed tumor was markedly shrunk after switching to the gilteritinib in vivo model. In addition, gilteritinib was effective against NTRK-rearranged cancers including entrectinib-resistant NTRK1 G667C-mutant and ROS1 fusion-positive cancer.


Assuntos
Compostos de Anilina/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Lactamas Macrocíclicas/uso terapêutico , Pirazinas/uso terapêutico , Aminopiridinas , Animais , Apoptose/fisiologia , Benzamidas/uso terapêutico , Carbazóis/uso terapêutico , Linhagem Celular , Sobrevivência Celular/fisiologia , Crizotinibe/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Immunoblotting , Indazóis/uso terapêutico , Lactamas , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Dinâmica Molecular , Recidiva Local de Neoplasia , Piperidinas/uso terapêutico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Pirazóis , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo
6.
Biochim Biophys Acta Gen Subj ; 1865(3): 129833, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33358865

RESUMO

BACKGROUND: C-mannosylation is a unique type of glycosylation. A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) is a multidomain extracellular metalloproteinase that contains several potential C-mannosylation sites. Although some ADAMTS family proteins have been reported to be C-mannosylated proteins, whether C-mannosylation affects the activation and protease activity of these proteins is unclear. METHODS: We established wild-type and mutant ADAMTS4-overexpressing HT1080 cell lines. Recombinant ADAMTS4 was purified from the conditioned medium of the wild-type ADAMTS4-overexpressing cells, and the C-mannosylation sites of ADAMTS4 were identified by LC-MS/MS. The processing, secretion, and intracellular localization of ADAMTS4 were examined by immunoblot and immunofluorescence analyses. ADAMTS4 enzymatic activity was evaluated by assessing the cleavage of recombinant aggrecan. RESULTS: We identified that ADAMTS4 is C-mannosylated at Trp404 in the metalloprotease domain and at Trp523, Trp526, and Trp529 in the thrombospondin type 1 repeat (TSR). The replacement of Trp404 with Phe affected ADAMTS4 processing, without affecting secretion and intracellular localization. In contrast, the substitution of Trp523, Trp526, and Trp529 with Phe residues suppressed ADAMTS4 secretion, processing, intracellular trafficking, and enzymatic activity. CONCLUSIONS: Our results demonstrated that the C-mannosylation of ADAMTS4 plays important roles in protein processing, intracellular trafficking, secretion, and enzymatic activity. GENERAL SIGNIFICANCE: Because C-mannosylation appears to regulate many ADAMTS4 functions, C-mannosylation may also affect other members of the ADAMTS superfamily.


Assuntos
Proteína ADAMTS4/metabolismo , Agrecanas/metabolismo , Manose/metabolismo , Processamento de Proteína Pós-Traducional , Proteína ADAMTS4/genética , Motivos de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica
7.
Biochim Biophys Acta Gen Subj ; 1864(9): 129637, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32442478

RESUMO

BACKGROUND: C-mannosylation is the one of glycosylations. Microfibril-associated glycoprotein 4 (MFAP4), an important protein for tissue homeostasis and cell adhesion, contains a consensus sequence of C-mannosylation in its fibrinogen C-terminal domain. In this study, we sought to demonstrate that fibrinogen C-terminal domain is a new substrate domain for C-mannosylation. METHODS: We established an MFAP4-overexpresssing HT1080 cell line and purified recombinant MFAP4 protein from the conditioned medium for LC-MS/MS analysis. Subcellular localization of MFAP4 was observed under confocal fluorescence microscope. RESULTS: We found that MFAP4 is C-mannosylated at Trp235 in the fibrinogen C-terminal domain by LC-MS/MS. To determine the functions of the C-mannosylation of MFAP4, we established a C-mannosylation-defective mutant MFAP4-overexpresssing HT1080 cell line and measured its secretion of MFAP4. The secretion of MFAP4 decreased significantly in the C-mannosylation-defective mutant MFAP4-overexpresssing cell line versus wild-type cells. Moreover, co-transfection experiments indicated that C-mannosylated MFAP4 accelerated its secretion. CONCLUSIONS: Our results demonstrate that the fibrinogen C-terminal domain is a novel C-mannosylation domain and that the C-mannosylation of MFAP4 is important for its secretion. GENERAL SIGNIFICANCE: These results suggest that C-mannosylation has a role for dominant effect for MFAP4 secretion.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fibrinogênio/química , Fibrinogênio/metabolismo , Glicoproteínas/metabolismo , Manose/metabolismo , Linhagem Celular Tumoral , Humanos , Domínios Proteicos , Transporte Proteico
8.
Int J Oncol ; 54(6): 2127-2138, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30942431

RESUMO

R­spondin2 (Rspo2), one of the four members of the R­spondin family of proteins, has agonistic activity in the Wnt/ß­catenin signaling pathway, and it is associated with normal development, as well as disease, such as cancer. The present study focused on the C­mannosylation of Rspo2, which is a novel and unique type of glycosylation that occurs via a C­C linkage between the tryptophan residue and an α­mannose. Although Rspo2 has two putative C­mannosylation sites at residues Trp150 and Trp153, it had not been reported to date whether these sites are C­mannosylated. Firstly, results from mass spectrometry demonstrated that Rspo2 was C­mannosylated at the Trp150 and Trp153 residues. Notably, while this C­mannosylation of Rspo2 resulted in increased extracellular secretion in human fibrosarcoma HT1080 cells, in other human tumor cell lines it inhibited secretion. However, C­mannosylation had consistent effects on the activation of Wnt/ß­catenin signaling in PANC1 and MDA­MB­231 cells, as well as HT1080 cells. Furthermore, overexpression of wild­type Rspo2 significantly increased the migratory ability of A549 and HT1080 cells, whereas overexpression of a C­mannosylation­defective mutant enhanced migration to a lesser degree. These results suggested that C­mannosylation of Rspo2 may promote cancer progression and that the inhibition of C­mannosylation may serve as a potential novel therapeutic approach for cancer therapy.


Assuntos
Movimento Celular , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias/patologia , Via de Sinalização Wnt , Linhagem Celular Tumoral , Humanos , Manose/metabolismo , Manosiltransferases/metabolismo , Triptofano/metabolismo , beta Catenina/metabolismo
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