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1.
Mol Cancer Res ; 18(6): 847-858, 2020 06.
Article in English | MEDLINE | ID: mdl-32102897

ABSTRACT

HSP47 is a collagen-specific protein chaperone expressed in fibroblasts, myofibroblasts, and stromal cells. HSP47 is also expressed in and involved in growth of cancer cells in which collagen levels are extremely low. However, its role in cancer remains largely unclear. Here, we showed that HSP47 maintains cancer cell growth via the unfolded protein response (UPR), the activation of which is well known to be induced by endoplasmic reticulum (ER) stress. We observed that HSP47 forms a complex with both the UPR transducer inositol-requiring enzyme 1α (IRE1α) and ER chaperone BiP in cancer cells. Moreover, HSP47 silencing triggered dissociation of BiP from IRE1α and IRE1α activation, followed by an increase in the intracellular level of reactive oxygen species (ROS). Increase in ROS induced accumulation of 4-hydroxy-2-nonenal-protein adducts and activated two UPR transducers, PKR-like ER kinase (PERK) and activating transcription factor 6α (ATF6α), resulting in impaired cancer cell growth. Our work indicates that HSP47 expressed in cancer cells relieves the ER stress arising from protein synthesis overload within these cells and tumor environments, such as stress induced by hypoxia, low glucose, and pH. We also propose that HSP47 has a biological role that is distinct from its normal function as a collagen-specific chaperone. IMPLICATIONS: HSP47 maintains cancer cell growth by inhibiting IRE1α.


Subject(s)
Biomarkers, Tumor/metabolism , Endoplasmic Reticulum Stress , Endoribonucleases/metabolism , Gene Expression Regulation, Neoplastic , HSP47 Heat-Shock Proteins/metabolism , Neoplasms/pathology , Protein Serine-Threonine Kinases/metabolism , Unfolded Protein Response , Activating Transcription Factor 6/genetics , Activating Transcription Factor 6/metabolism , Animals , Apoptosis , Biomarkers, Tumor/genetics , Cell Proliferation , Endoribonucleases/genetics , HSP47 Heat-Shock Proteins/antagonists & inhibitors , HSP47 Heat-Shock Proteins/genetics , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasms/genetics , Neoplasms/metabolism , Protein Serine-Threonine Kinases/genetics , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , eIF-2 Kinase/genetics , eIF-2 Kinase/metabolism
2.
BMC Cancer ; 19(1): 510, 2019 May 29.
Article in English | MEDLINE | ID: mdl-31142270

ABSTRACT

BACKGROUND: Hypoxia is an important factor that contributes to tumour aggressiveness and correlates with poor prognosis and resistance to conventional therapy. Therefore, identifying hypoxic environments within tumours is extremely useful for understanding cancer biology and developing novel therapeutic strategies. Several studies have suggested that carbonic anhydrase 9 (CA9) is a reliable biomarker of hypoxia and a potential therapeutic target, while pimonidazole has been identified as an exogenous hypoxia marker. However, other studies have suggested that CA9 expression is not directly induced by hypoxia and it is not expressed in all types of tumours. Thus, in this study, we focused on endoplasmic reticulum disulphide oxidase 1α (ERO1α), a protein that localises in the endoplasmic reticulum and is involved in the formation of disulphide bonds in proteins, to determine whether it could serve as a potential tumour-hypoxia biomarker. METHODS: Using quantitative real-time polymerase chain reaction, we analysed the mRNA expression of ERO1α and CA9 in different normal and cancer cell lines. We also determined the protein expression levels of ERO1α and CA9 in these cell lines by western blotting. We then investigated the hypoxia-inducible ERO1α and CA9 expression and localisation in HCT116 and HeLa cells, which express low (CA9-low) and high (CA9-high) levels of CA9, respectively. A comparative analysis was performed using pimonidazole, an exogenous hypoxic marker, as a positive control. The expression and localisation of ERO1α and CA9 in tumour spheres during hypoxia were analysed by a tumour sphere formation assay. Finally, we used a mouse model to investigate the localisation of ERO1α and CA9 in tumour xenografts using several cell lines. RESULTS: We found that ERO1α expression increased under chronic hypoxia. Our results show that ERO1α was hypoxia-induced in all the tested cancer cell lines. Furthermore, in the comparative analysis using CA9 and pimonidazole, ERO1α had a similar localisation to pimonidazole in both CA9-low and CA9-high cell lines. CONCLUSION: ERO1α can serve as a novel endogenous chronic hypoxia marker that is more reliable than CA9 and can be used as a diagnostic biomarker and therapeutic target for cancer.


Subject(s)
Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Neoplasms/metabolism , Oxidoreductases/genetics , Oxidoreductases/metabolism , Animals , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Carbonic Anhydrase IX/genetics , Carbonic Anhydrase IX/metabolism , Cell Hypoxia , Cell Line, Tumor , HCT116 Cells , HT29 Cells , HeLa Cells , Humans , Mice , Neoplasm Transplantation , Neoplasms/genetics , Nitroimidazoles/metabolism
3.
J Biol Chem ; 292(38): 15649-15660, 2017 09 22.
Article in English | MEDLINE | ID: mdl-28774960

ABSTRACT

Upon liver injury, excessive deposition of collagen from activated hepatic stellate cells (HSCs) is a leading cause of liver fibrosis. An understanding of the mechanism by which collagen biosynthesis is regulated in HSCs will provide important clues for practical anti-fibrotic therapy. Endoplasmic reticulum oxidase 1α (ERO1α) functions as an oxidative enzyme of protein disulfide isomerase, which forms intramolecular disulfide bonds of membrane and secreted proteins. However, the role of ERO1α in HSCs remains unclear. Here, we show that ERO1α is expressed and mainly localized in the endoplasmic reticulum in human HSCs. When HSCs were transfected with ERO1α siRNA or an ERO1α shRNA-expressing plasmid, expression of ERO1α was completely silenced. Silencing of ERO1α expression in HSCs markedly suppressed their proliferation but did not induce apoptosis, which was accompanied by impaired secretion of collagen type 1. Silencing of ERO1α expression induced impaired disulfide bond formation and inhibited autophagy via activation of the Akt/mammalian target of rapamycin signaling pathway, resulting in intracellular accumulation of collagen type 1 in HSCs. Furthermore, silencing of ERO1α expression also promoted proteasome-dependent degradation of membrane type 1-matrix metalloproteinase (MT1-MMP), which stimulates cell proliferation through cleavage of secreted collagens. The inhibition of HSC proliferation was reversed by treatment with MT1-MMP-cleaved collagen type 1. The results suggest that ERO1α plays a crucial role in HSC proliferation via posttranslational modification of collagen and MT1-MMP and, therefore, may be a suitable therapeutic target for managing liver fibrosis.


Subject(s)
Collagen Type I/metabolism , Hepatic Stellate Cells/cytology , Hepatic Stellate Cells/metabolism , Matrix Metalloproteinase 14/metabolism , Membrane Glycoproteins/metabolism , Oxidoreductases/metabolism , Autophagy , Cell Line , Cell Proliferation , Enzyme Activation , Gene Silencing , Humans , Integrins/metabolism , Membrane Glycoproteins/deficiency , Membrane Glycoproteins/genetics , Oxidoreductases/deficiency , Oxidoreductases/genetics , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction
4.
Sci Rep ; 7(1): 9389, 2017 08 24.
Article in English | MEDLINE | ID: mdl-28839225

ABSTRACT

Endoplasmic reticulum disulphide oxidase 1α (ERO1α) is an oxidase localized in the endoplasmic reticulum that plays a role in the formation of disulphide bonds of secreted and cell-surface proteins. We previously showed that ERO1α is overexpressed in various types of cancer and we further identified ERO1α expression as a novel factor related to poor prognosis in cancer. However, the biological functions of ERO1α in cancer remain unclear. Here, we investigated the cell biological roles of ERO1α in the human colon-cancer cell line HCT116. ERO1α knockout (KO) by using CRISPR/Cas9 resulted in decreased tumourigenicity in vivo and reduced cell proliferation only under hypoxia in vitro, which suggested that ERO1α promotes cancer progression specifically in a low-oxygen environment. Thus, we evaluated the function of ERO1α in cell proliferation under hypoxia, and found that under hypoxic conditions, ERO1α KO resulted in a contact-inhibited morphology and diminished motility of cells. We further showed that ERO1α KO induced a change in integrin-ß1 glycosylation and thus an attenuation of cell-surface integrin-ß1 expression, which resulted in the aforementioned phenotype. Our study has established a previously unrecognized link between ERO1α expression and integrin activation, and thus provides new evidence for the effectiveness of ERO1α-targeted therapy for colorectal carcinoma.


Subject(s)
Hypoxia/metabolism , Integrin beta1/metabolism , Membrane Glycoproteins/metabolism , Oxidoreductases/metabolism , Signal Transduction , Animals , Cell Membrane/metabolism , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Disease Models, Animal , Epithelial-Mesenchymal Transition , Gene Knockout Techniques , Genetic Loci , Glycosylation , HCT116 Cells , Humans , Hypoxia/genetics , Membrane Glycoproteins/genetics , Mice , Oxidoreductases/genetics , Protein Transport , Sequence Deletion , Xenograft Model Antitumor Assays
5.
Exp Cell Res ; 341(1): 8-17, 2016 Feb 01.
Article in English | MEDLINE | ID: mdl-26812497

ABSTRACT

Transdifferentiation of vitamin A-storing hepatic stellate cells (HSCs) to vitamin A-depleted myofibroblastic cells leads to liver fibrosis. Vitamin A regulates lipid accumulation and gene transcription, suggesting that vitamin A is involved in the maintenance of HSC quiescence under a physiological condition. However, the precise mechanism remains elusive because there is no appropriate in vitro culture system for quiescent HSCs. Here, we show that treatment of quiescent HSCs with vitamin A partially maintained the accumulation of lipid droplets and expression of quiescent HSC markers (glial fibrillary acidic protein, peroxisome proliferator-activator receptor-γ and CCAAT/enhancer-binding protein-α) and also the expression of myofibroblastic markers (α-smooth muscle actin, heat shock protein 47 and collagen type I). On the other hand, combined treatment with vitamin A and insulin sustained the characteristic of HSC quiescence and completely suppressed the expression of myofibroblastic markers through activation of the JAK2/STAT5 signaling pathway and increased expression of sterol regulatory element binding protein-1. These treated HSCs transdifferentiated to myofibroblastic cells under a culture condition with fetal bovine serum. The results suggest an important role of vitamin A and insulin in the maintenance of HSC quiescence under a physiological condition.


Subject(s)
Hepatic Stellate Cells/cytology , Hepatic Stellate Cells/drug effects , Insulin/pharmacology , Vitamin A/pharmacology , Animals , Cell Proliferation/drug effects , Male , Rats , Rats, Sprague-Dawley
6.
Sci Rep ; 4: 6737, 2014 Oct 23.
Article in English | MEDLINE | ID: mdl-25338618

ABSTRACT

ATP-binding cassette transporter family A member 12 (ABCA12) is a keratinocyte transmembrane lipid transporter that plays a critical role in preserving the skin permeability barrier. Biallelic loss of function of the ABCA12 gene is causative of some forms of recessive congenital ichthyosis, an intractable disease marked by dry, thickened and scaly skin on the whole body. Genetic diagnosis is essential, although the results may occasionally be inconclusive, because some patients with low ABCA12 expression have one mutant allele and one apparently intact allele. Aside from aberrant splicing or deletion mutations, one possible explanation for such discrepancy is loss of promoter function. This study aims to elucidate the promoter region of ABCA12 and to locate the essential elements therein, thus providing the necessary information for genetic diagnostic screening of congenital ichthyosis. Close examination of the 2980-bp upstream regions of the ABCA12 gene revealed that a palindromic motif (tgagtca) at -2084 to -2078 is essential for the promoter function, and a short fragment of -2200/-1934 alone has potent promoter activity. Identification of the key promoter element of ABCA12 in this study may provide relevant information for genetic diagnosis of recessive congenital ichthyosis.


Subject(s)
ATP-Binding Cassette Transporters/genetics , Ichthyosis, Lamellar/diagnosis , Inverted Repeat Sequences/genetics , Keratinocytes/metabolism , ATP-Binding Cassette Transporters/metabolism , Alleles , Cells, Cultured , Humans , Ichthyosis, Lamellar/genetics , Ion Transport/genetics , Nucleotide Motifs/genetics , Promoter Regions, Genetic/genetics , RNA Splicing/genetics , Sequence Deletion/genetics
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