Papers - Kawahara, Ryota

DPY19L3 promotes vasculogenic mimicry by its C-mannosyltransferase activity

Hassan Baydoun, Yuji Kato, Hiroki Kamo, Anna Hüsch, Hayato Mizuta, Ryota Kawahara, Siro Simizu

Oncology Research (Oncology Research)  32 ( 4 ) 607 - 614 2024

Joint Work, Accepted,  ISSN  09650407

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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.

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Biogenesis of fibrils requires C-mannosylation of PMEL

Ryota Kawahara, Tomoko Usami, Satoko Arakawa, Hiroki Kamo, Takehiro Suzuki, Ryosuke Komatsu, Hiroyuki Hara, Yuki Niwa, Erina Shimizu, Naoshi Dohmae, Shigeomi Shimizu, Siro Simizu

FEBS Journal (FEBS Journal)  290 ( 22 ) 5373 - 5394 2023.11

Joint Work, Lead author, Accepted,  ISSN  1742464X

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Premelanosome protein (PMEL), a melanocyte-specific glycoprotein, has an essential role in melanosome maturation, assembling amyloid fibrils for melanin deposition. PMEL undergoes several post-translational modifications, including N- and O-glycosylations, which are associated with proper melanosome development. C-mannosylation is a rare type of protein glycosylation at a tryptophan residue that might regulate the secretion and localization of proteins. PMEL has one putative C-mannosylation site in its core amyloid fragment (CAF); however, there is no report focusing on C-mannosylation of PMEL. To investigate this, we expressed recombinant PMEL in SK-MEL-28 human melanoma cells and purified the protein. Mass spectrometry analyses demonstrated that human PMEL is C-mannosylated at multiple tryptophan residues in its CAF and N-terminal fragment (NTF). In addition to the W153 or W156 residue (CAF), which lies in the consensus sequence for C-mannosylation, the W104 residue (NTF) was C-mannosylated without the consensus sequence. To determine the effects of the modifications, we deleted the PMEL gene by using CRISPR/Cas9 technology and re-expressed wild-type or C-mannosylation-defective mutants of PMEL, in which the C-mannosylated tryptophan was replaced with a phenylalanine residue (WF mutation), in SK-MEL-28 cells. Importantly, fibril-containing melanosomes were significantly decreased in W104F mutant PMEL-re-expressing cells compared with wild-type PMEL, observed using transmission electron microscopy. Furthermore, western blot and immunofluorescence analysis suggested that the W104F mutation may cause mild endoplasmic reticulumretention, possibly associated with early misfolding, and lysosomal misaggregation, thus reducing functional fibril formation. Our results demonstrate that C-mannosylation of PMEL is required for proper melanosome development by regulating PMEL-derived fibril formation.

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Cofilin promotes vasculogenic mimicry by regulating the actin cytoskeleton in human breast cancer cells

Minami Nakajima, Ryota Kawahara, Siro Simizu

FEBS Letters (FEBS Letters)  597 ( 8 ) 1114 - 1124 2023.04

Joint Work, Accepted,  ISSN  00145793

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Vasculogenic mimicry (VM) is the formation of microvascular channels by cancer cells. VM requires cellular processes that are regulated by changes in cellular migration and morphology. Cofilin (CFL), a key regulator of actin depolymerization, has been reported to affect malignant phenotypes of cancer. We show that treatment with inhibitors of actin dynamics suppresses VM in MDA-MB-231 human breast cancer cells. We established CFL-knockout (KO) MDA-MB-231 cells and found that VM was attenuated in CFL-KO cells. Although the re-expression of wild-type CFL restored VM in CFL-KO cells, inactive phosphomimetic CFL failed to do so. Collectively, our results demonstrate that CFL is a critical regulator of VM and implicate CFL as a novel therapeutic target for breast cancer.

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Tyrosinase suppresses vasculogenic mimicry in human melanoma cells

Hiroki Kamo, Ryota Kawahara, Siro Simizu

Oncology Letters (Oncology Letters)  23 ( 5 )  2022.05

Joint Work, Accepted,  ISSN  17921074

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Melanoma is a type of skin cancer that derives from melanocytes; this tumor is highly metastatic and causes poor clinical outcomes in patients. Vasculogenic mimicry (VM), a vascular-like network that is formed by tumor cells instead of endothelial cells, promotes the growth and metastasis of tumors by providing tumors with oxygen- and nutrient-containing blood. VM correlates with a poor prognosis in patients with melanoma, but the melanoma-specific mechanisms of VM are unknown. The present study revealed that treatment with the melanogenesis stimulators 3-isobutyl 1-methylxanthine (IBMX) and α-melanocyte-stimulating hormone (α-MSH) significantly inhibited VM in MNT-1 human pigmented melanoma cells. Tyrosinase (TYR), an essential enzyme in melanin production, was upregulated on treatment with α-MSH and IBMX, prompting an examination of the association between TYR and VM. A TYR inhibitor, arbutin, promoted VM in melanoma cells. Furthermore, CRISPR/Cas9-mediated knockout (KO) of TYR increased VM by melanoma cells. Notably, even in non-pigmented melanoma cells, TYR attenuated VM. Although re-expression of wild-type TYR suppressed VM in TYR-KO cells, T373K TYR, a frequently detected mutation in individuals with albinism, failed to inhibit VM. Overall, these results demonstrated that TYR negatively regulates VM, providing novel insights into the antioncogenic function of TYR in melanomas.

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ErbB4-mediated regulation of vasculogenic mimicry capability in breast cancer cells

Ryota Kawahara, Siro Simizu

Cancer Science (Cancer Science)  113 ( 3 ) 950 - 959 2022.03

Joint Work, Lead author, Accepted,  ISSN  13479032

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ErbB4 is a member of the ErbB receptor tyrosine kinase family. It has both pro- and anti-oncogenic activities in tumors. Vasculogenic mimicry (VM), a phenomenon in which cancer cells form capillary-like structures without endothelial cells, has been recognized to be a cause of malignant phenotypes in some solid tumors. Here, we used an in vitro VM formation assay, and demonstrated that ErbB4 negatively regulated VM formation in human breast cancer cells. By using CRISPR/Cas9-mediated gene knockout, we verified that the depletion of endogenous ErbB4 improved the VM formation capability. Although treatment with neuregulin 1 (NRG1), a ligand of ErbB4, induced the phosphorylation of ErbB4 and promoted VM formation in a dose-dependent manner, it did not induce such activities in kinase-dead K751M ErbB4-overexpressing cells. Moreover, we examined the effect of the missense mutation E872K of ErbB4, which has been reported in multiple tumors, on VM formation, and found that the mutation enhanced the basal phosphorylation level and ErbB4-mediated VM formation in the absence of NRG1 stimulation. Whereas NRG1 stimulated VM formation, excessive activation of ErbB4 induced a negative effect. In E872K ErbB4-overexpressing cells, but not in wild-type ErbB4-overexpressing cells, the number of VM tubes was significantly decreased by low-dose treatment with the ErbB inhibitor afatinib. Taken together, our findings demonstrated the significance of ErbB4-mediated VM formation, and suggested the possibility of ErbB4 mutations as effective targets in breast cancer.

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Methionine aminopeptidase-2 is a pivotal regulator of vasculogenic mimicry

Shota Shimizu, Ryota Kawahara, Siro Simizu

Oncology Reports (Oncology Reports)  47 ( 2 )  2022.02

Joint Work, Accepted,  ISSN  1021335X

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Vasculogenic mimicry (VM) is the formation of a blood supply system that confers aggressive and metastatic properties to tumors and correlates with a poor prognosis in cancer patients. Thus, the inhibition of VM is considered an effective approach for cancer treatment, although such a mechanism remains poorly described. In the present study, we examined methionine aminopeptidase-2 (MetAP2), a key factor of angiogenesis, and demonstrated that it is pivotal for VM, using pharmacological and genetic approaches. Fumagillin and TNP-470, angiogenesis inhibitors that target MetAP2, significantly suppressed VM in various human cancer cell lines. We established MetAP2-knockout (KO) human fibrosarcoma HT1080 cells using the CRISPR/Cas9 system and found that VM was attenuated in these cells. Furthermore, re-expression of wild-type MetAP2 restored VM in the MetAP2-KO HT1080 cells, but the substitution of D251, a conserved amino acid in MetAP2, failed to rescue the VM. Collectively, our results demonstrate that MetAP2 is critical for VM in human cancer cells and suggest fumagillin and TNP-470 as potent VM-suppressing agents.

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Identification and characterization of collagen-like glycosylation and hydroxylation of CCN1

Yudai Ishizawa, Yuki Niwa, Takehiro Suzuki, Ryota Kawahara, Naoshi Dohmae, Siro Simizu

Glycobiology 29   696 - 704 2019.09

Research paper (scientific journal), Joint Work, Accepted

Integrin β1 is an essential factor in vasculogenic mimicry of human cancer cells

Ryota Kawahara, Yuki Niwa, Siro Simizu

Cancer Science 109   2490 - 2496 2018.08

Research paper (scientific journal), Joint Work, Lead author, Accepted