Publications by authors named "Hayato Mizuta"

4 Publications

  • Page 1 of 1

Gilteritinib overcomes lorlatinib resistance in ALK-rearranged cancer.

Nat Commun 2021 02 24;12(1):1261. Epub 2021 Feb 24.

Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-021-21396-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904790PMC
February 2021

Requirement for C-mannosylation to be secreted and activated a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4).

Biochim Biophys Acta Gen Subj 2021 03 29;1865(3):129833. Epub 2020 Dec 29.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 223-8522, Japan. Electronic address:

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 Trp in the metalloprotease domain and at Trp, Trp, and Trp in the thrombospondin type 1 repeat (TSR). The replacement of Trp with Phe affected ADAMTS4 processing, without affecting secretion and intracellular localization. In contrast, the substitution of Trp, Trp, and Trp 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbagen.2020.129833DOI Listing
March 2021

The fibrinogen C-terminal domain is seldom C-mannosylated but its C-mannosylation is important for the secretion of microfibril-associated glycoprotein 4.

Biochim Biophys Acta Gen Subj 2020 09 19;1864(9):129637. Epub 2020 May 19.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 223-8522, Japan. Electronic address:

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 Trp 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbagen.2020.129637DOI Listing
September 2020

C‑mannosylation of R‑spondin2 activates Wnt/β‑catenin signaling and migration activity in human tumor cells.

Int J Oncol 2019 Jun 1;54(6):2127-2138. Epub 2019 Apr 1.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Kanagawa 223‑8522, Japan.

R‑spondin2 (Rspo2), one of the four members of the R‑spondin family of proteins, has agonistic activity in the Wnt/β‑catenin signaling pathway, and it is associated with normal development, as well as disease, such as cancer. The present study focused on the C‑mannosylation of Rspo2, which is a novel and unique type of glycosylation that occurs via a C‑C linkage between the tryptophan residue and an α‑mannose. Although Rspo2 has two putative C‑mannosylation sites at residues Trp150 and Trp153, it had not been reported to date whether these sites are C‑mannosylated. Firstly, results from mass spectrometry demonstrated that Rspo2 was C‑mannosylated at the Trp150 and Trp153 residues. Notably, while this C‑mannosylation of Rspo2 resulted in increased extracellular secretion in human fibrosarcoma HT1080 cells, in other human tumor cell lines it inhibited secretion. However, C‑mannosylation had consistent effects on the activation of Wnt/β‑catenin signaling in PANC1 and MDA‑MB‑231 cells, as well as HT1080 cells. Furthermore, overexpression of wild‑type Rspo2 significantly increased the migratory ability of A549 and HT1080 cells, whereas overexpression of a C‑mannosylation‑defective mutant enhanced migration to a lesser degree. These results suggested that C‑mannosylation of Rspo2 may promote cancer progression and that the inhibition of C‑mannosylation may serve as a potential novel therapeutic approach for cancer therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3892/ijo.2019.4767DOI Listing
June 2019