Cancer-associated oxidoreductase ERO1-α promotes immune escape through up-regulation of PD-L1 in human breast cancer.

Many human cancers have been reported to have enhanced expression of the immune checkpoint molecule programmed death-ligand 1 (PD-L1), which binds to programmed cell death-1 (PD-1) expressed on immune cells. PD-L1/PD-1 plays a role in inhibition of antitumor immunity by inducing T cell apoptosis and tolerance. Thus, it is crucial to elucidate mechanisms of PD-L1 expression on cancer cells. ERO1-α is an oxidase located in the endoplasmic reticulum. It is overexpressed in a variety of tumor types and it plays a role in disulfide bond formation in collaboration with PDI. Here, we investigated the influence of ERO1-α on expression of PD-L1 and immune escape. We demonstrated that ERO1-α augmented the expression of PD-L1 via facilitation of oxidative protein folding within PD-L1. In addition, we showed that overexpression of ERO1-α increased HIF-1α protein expression, resulting in an increase of PD-L1 mRNA as well as protein. In clinical cases, we observed that the expression of ERO1-α in triple negative breast cancer was related to the expression of PD-L1. Moreover, apoptosis of Jurkat leukemia T cells, which express PD-1, induced by tumor PD-L1 was inhibited when ERO1-α was depleted. The results suggest that targeting ERO1-α in tumor cells can be a novel approach for cancer immunotherapy. Therefore, the role of ERO1-α in tumor-mediated immunosuppression should be further explored.

Hypoxia augments MHC class I antigen presentation via facilitation of ERO1-α-mediated oxidative folding in murine tumor cells.

To establish an effective cancer immunotherapy, it is crucial that cancer cells present a cancer-specific antigen in a hypoxic area, a hallmark of the tumor microenvironment. Here, we show the impact of hypoxia on MHC class I antigen presentation in vitro and in vivo in murine tumors. Activation of antigen-specific CTLs by tumor cells that had been pre-incubated under a condition of hypoxia was enhanced compared with that by tumor cells pre-incubated under a condition of normoxia. Cell surface expression of MHC class I-peptide complex on the tumor cells was increased under a condition of hypoxia, thereby leading to higher susceptibility to specific CTLs. We show that the hypoxia-inducible ER-resident oxidase ERO1-α plays an important role in the hypoxia-induced augmentation of MHC class I-peptide complex expression. ERO1-α facilitated oxidative folding of MHC class I heavy chains, thereby resulting in the augmentation of cell surface expression of MHC class I-peptide complex under hypoxic conditions. These results suggest that since the expression of MHC class I-peptide complex is augmented in a hypoxic tumor microenvironment, strategies for inhibiting the function of regulatory T cells and myeloid-derived suppressor cells and/or immunotherapy with immune checkpoint inhibitors are promising for improving cancer immunotherapy.

Plasticity of lung cancer stem-like cells is regulated by the transcription factor HOXA5 that is induced by oxidative stress.

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are reasonable targets for cancer therapy. However, recent studies have revealed that some non-CSCs/CICs have plastic ability and can dedifferentiate into CSCs/CICs. Therefore, an understanding of the molecular mechanisms that control the plasticity is essential to achieve CSC/CIC-targeting therapy. In this study, we analyzed the plasticity of lung cancer cells and found that lung non-CSCs/CICs can dedifferentiate into CSCs/CICs in accordance with the expression of stem cell transcription factor SOX2. SOX2 expression was induced by the transcription factor HOXA5. Oxidative stress repressed the expression of HDAC8 and then induced histone 3 acetylation and increased the expression of HOXA5 and SOX2. These findings indicate that lung cancer cells have plasticity under a condition of oxidative stress and that HOAX5 has a critical role in dedifferentiation.

Cancer-associated oxidoreductase ERO1-α drives the production of VEGF via oxidative protein folding and regulating the mRNA level.

BACKGROUND: Endoplasmic reticulum disulfide oxidase 1-α (ERO1-α) is an oxidase that exists in the endoplasmic reticulum and has a role in the formation of disulfide bonds of secreted proteins and cell-surface proteins. Recently, we reported that ERO1-α is present in high levels in various types of tumours, and that ERO1-α is a novel factor of poor prognosis. However, how ERO1-α affects a tumour in vivo and why patients who have a tumour with a high expression level of ERO1-α have a poor prognosis are still unknown. Therefore, to clarify the mechanism, we investigated the effect of ERO1-α on a tumour from the point of view of angiogenesis. METHODS: The effect of ERO1-α on tumour growth and angiogenesis was analysed by using non-obese diabetic-severe combined immunodeficient mice. The production of vascular endothelial growth factor (VEGF) in MDA-MB-231 cells with ERO1-α- overexpression or with ERO1-α knockdown was measured. The role of ERO1-α on VEGF expression was investigated. In triple-negative breast cancer cases, the relationship between expression of ERO1-α and angiogenesis was analysed. RESULTS: We found that the expression of ERO1-α promoted tumour growth in a mouse study and angiogenesis. The effects of ERO1-α on angiogenesis were mediated via oxidative protein folding of VEGF and enhancement of VEGF mRNA expression by using MDA-MB-231. In triple-negative breast cancer cases, the expression of ERO1-α related to the number of the blood vessel. Furthermore, we found that ERO1-α was a poor prognosis factor in triple-negative breast cancer. CONCLUSIONS: Our study has established a novel link between expression of ERO1-α and secretion of VEGF, providing new evidence for the effectiveness of ERO1-α-targeted therapy in patients with ERO1-α-expressed cancer.

CpG-A stimulates Hsp72 secretion from plasmacytoid dendritic cells, facilitating cross-presentation.

Plasmacytoid dendritic cells (pDCs) are the main producers of IFN-α in response to unmethylated DNA molecules, including cytosine guanine dinucleotide (CpG)-DNA in vivo. pDCs specifically express toll-like receptor (TLR) 9 and are therefore able to recognize the unmethylated DNAs. It has recently been shown that not only conventional DCs (cDCs) but also pDCs efficiently cross-present exogenous antigens after TLR9 activation. However, the precise molecular mechanism has remained unclear. Here, we show that pDCs secreted heat shock protein 72 (Hsp72) in response to CpG-A administration in a TLR9-dependent manner. Extracellular Hsp72 bound to an Hsp90-peptide complex and enhanced binding of Hsp90-peptide complex to pDC, resulting in efficient cross-presentation. Our experiments therefore suggest a mechanism for orchestration of immune responses by stimulation of pDCs with CpG-A.

Cancer-Associated Oxidase ERO1-α Regulates the Expression of MHC Class I Molecule via Oxidative Folding.

ERO1-α is an oxidizing enzyme that exists in the endoplasmic reticulum and is induced under hypoxia. It reoxidizes the reduced form of protein disulfide isomerase that has oxidized target proteins. We found that ERO1-α is overexpressed in a variety of tumor types. MHC class I H chain (HC) has two disulfide bonds in the α2 and α3 domains. MHC class I HC folding is linked to the assembly of MHC class I molecules because only fully disulfide-bonded class I HCs efficiently assemble with ß2-microglobulin. In this study, we show that ERO1-α associates with protein disulfide isomerase, calnexin, and immature MHC class I before being incorporated into the TAP-1-associated peptide-loading complex. Importantly, ERO1-α regulates the redox state as well as cell surface expression of MHC class I, leading to alteration of susceptibility by CD8(+) T cells. Similarly, the ERO1-α expression within cancer cells was associated with the expression level of MHC class I in colon cancer tissues. Thus, the cancer-associated ERO1-α regulates the expression of the MHC class I molecule via oxidative folding.

Cancer-associated oxidoreductase ERO1-α drives the production of tumor-promoting myeloid-derived suppressor cells via oxidative protein folding.

Endoplasmic reticulum disulfide oxidase ERO1-α plays a role in the formation of disulfide bonds in collaboration with protein disulfide isomerase. Disulfide bond formation is required for the proper conformation and function of secreted and cell surface proteins. We found that ERO1-α was overexpressed in a variety of tumor types; therefore, we examined its role in tumor growth. In BALB/c mice, knockdown of ERO1-α within 4T1 mouse mammary gland cancer (KD) cells caused retardation of in vivo tumor growth compared with tumor growth of scrambled control (SCR) cells. In contrast, when ERO1-α-overexpressed 4T1 (OE) cells were compared with mock control cells, OE cells showed augmented tumor growth. However, differences in tumor growth were not observed among four groups of nude mice, suggesting that expression of ERO1-α diminished antitumor immunity. We observed dense peritumoral granulocytic infiltrates in tumors of wild-type 4T1 and SCR cells but not KD cells, and these cells were identified as polymorphonuclear myeloid-derived suppressor cells (MDSCs). In addition, production of G-CSF and CXCL1/2, which have intramolecular disulfide bonds, from KD cells was significantly decreased compared with that from SCR cells. In contrast, OE cells produced a larger amount of these molecules than did mock cells. These changes were regulated at the posttranscriptional level. These results suggest that overexpression of ERO1-α in the tumor inhibits the T cell response by recruiting polymorphonuclear MDSCs via regulation of MDSC-prone cytokines and chemokines.

Heat shock protein 90 targets a chaperoned peptide to the static early endosome for efficient cross-presentation by human dendritic cells.

The presentation of an exogenous antigen in a major histocompatibility complex class-I- restricted fashion to CD8(+) T cells is called cross-presentation. Heat shock proteins (HSPs) such as Hsp70, gp96, and Hsp90 have been shown to elicit efficient CTL responses by cross-presentation through an as-yet entirely unknown mechanism. Hsp90 is the most abundant cytosolic HSP and is known to act as a molecular chaperone. We have shown that a tumor antigen peptide complexed with Hsp90 could be cross-presented by dendritic cells (DCs) through an endosomal pathway in a murine system. However, it has not been determined whether human DCs also cross-present an Hsp90-peptide complex and induce peptide-specific CTLs. In this study, we found that an Hsp90-cancer antigen peptide complex was efficiently cross-presented by human monocyte-derived DCs and induced peptide-specific CTLs. Furthermore, we observed that the internalized Hsp90-peptide complex was strictly sorted to the Rab5(+), EEA1(+) static early endosome and the Hsp90-chaperoned peptide was processed and bound to MHC class I molecules through an endosome-recycling pathway. Our data indicate that targeting of the antigen to a "static" early endosome by Hsp90 is essential for efficient cross-presentation.

Human endoplasmic reticulum oxidoreductin 1-α is a novel predictor for poor prognosis of breast cancer.

Human endoplasmic reticulum oxidoreductin 1-α (hERO1-α) is an oxidizing enzyme that exists in the endoplasmic reticulum and its expression is augmented under hypoxia. It regulates a redox state of various kinds of protein through reoxidation of "client" protein disulfide isomerase. Interestingly, although the expression of hERO1-α in normal tissues was comparatively limited, various types of cancer cells expressed it in large amounts. Therefore, we examined the role of ERO1-α in tumor growth using murine breast cancer line 4T1 and found that knockdown of murine ERO1-α inhibited in vivo tumor growth and decreased lung metastasis compared with wild-type 4T1. Moreover, we investigated the relationship between expression of hERO1-α and prognosis in breast cancer patients. Seventy-one patients with breast cancer who underwent surgery between 2005 and 2006 in Sapporo Medical University Hospital (Sapporo, Japan) were analyzed in this study. Significant differences were found between the hERO1-α-positive group (n = 33) and hERO1-α-negative group (n = 38) in nuclear grade (P < 0.001) and intrinsic subtype (P = 0.021) in univariate analysis. More importantly, in multivariate analysis of disease-free survival by Cox regression, expression of hERO1-α was the only independent prognosis factor (P = 0.035). Finally, in univariate survival analysis, patients positive for hERO1-α had significantly shorter disease-free survival and overall survival than those patients negative for hERO1-α. These findings indicate that the expression of hERO1-α in cancer cells is associated with poorer prognosis and thus can be a prognostic factor for patients with breast cancer.

CD4(+)CD8(+) thymocytes are induced to cell death by a small dose of puromycin via ER stress.

When the CD4(+)CD8(+) thymic lymphoma cells were treated with puromycin, we found that most of the cells died at 0.3-1 microg/ml of puromycin within 24h. However, cell death was greatly reduced when the dose of puromycin was increased. Similar dose-pattern of cell death was observed in thymocytes and the sensitivity to puromycin was greater in CD4(+)CD8(+) thymocytes than CD4(+)CD8(-) thymocytes. The induction of apoptosis was blocked by the protein synthesis inhibitor cycloheximide, and to some extent by transfection of Bcl-xL or Bcl-2 genes. Expression of GRP78 was up-regulated after treatment with a small dose of puromycin, and the cell death by puromycin was blocked in the presence of caspase 12 inhibitor. These results indicated that the induction of cell death by low-dose puromycin was due to endoplasmic reticulum stress. Furthermore, we found that dexamethasone, a synthetic glucocorticoid, and puromycin worked synergistically to induce cell death in thymocytes.

Cross-linking of SIGNR1 activates JNK and induces TNF-alpha production in RAW264.7 cells that express SIGNR1.

In this study, we evaluated the signaling ability of SIGNR1 in murine macrophage-like RAW264.7 cells that stably expressed FLAG-tagged SIGNR1 (SIGNR1-FLAG). Cross-linking of SIGNR1-FLAG expressed on the cells by an anti-FLAG antibody induced JNK phosphorylation without induction of phosphorylation of ERK1/2 and p38 MAP kinase, and led to phosphorylations of Src family kinases (SFKs) and Akt. The SIGNR1-FLAG molecules in the cells were found in lipid raft-enriched membrane fractions, and the tyrosine kinases Lyn, Hck, and Fgr co-precipitated with SIGNR1-FLAG in the lipid raft fractions. The antibody-induced JNK phosphorylation was inhibited by inhibitors of SFKs and tyrosine kinases. Furthermore, cross-linking of SIGNR1 led to production of TNF-alpha, and the JNK inhibitor inhibited the antibody-induced TNF-alpha production. These results show that cross-linking of SIGNR1 triggers phosphorylation of SFKs, which leads to activation of the JNK pathway and induction of TNF-alpha production in macrophage-like RAW264.7 cells.

Cooperation of specific ICAM-3 grabbing nonintegrin-related 1 (SIGNR1) and complement receptor type 3 (CR3) in the uptake of oligomannose-coated liposomes by macrophages.

Resident peritoneal macrophages (PEMs) express SIGNR1 on the cell surface as a major mannose receptor. These cells also ingest oligomannose-coated liposomes (OMLs) in an oligomannose-dependent manner following intraperitoneal administration. Therefore, the current study was conducted to investigate the possible role of SIGNR1 in capture of OMLs. Transient expression of several SIGN-related lectins potentially expressed on PEMs in CHO cells revealed that only SIGNR1 contributed to capture of OMLs. When SIGNR1 was introduced into mouse macrophage-like RAW264.7 cells, SIGNR1-expressing RAW (RAW-SIGNR1) cells recognized OMLs under serum-free conditions. OML recognition by RAW-SIGNR1 cells as well as that by PEMs was partially inhibited by an anti-SIGNR1 antibody (ER-TR9) and by mannan, and completely inhibited by EDTA. Interestingly, OML recognition by RAW-SIGNR1 cells was accelerated in the presence of serum, partially inhibited by an anti-complement receptor 3 (CR3) antibody (M1/70), and almost completely inhibited by a combination of ER-TR9 and M1/70. Complete inhibition of OML ingestion by the combination of ER-TR9 and M1/70 was also observed under serum-free conditions, suggesting that SIGNR1 and CR3 cooperate in an additive way in capture of OMLs by macrophage-like RAW cells. Administration of ER-TR9 or M1/70 into the peritoneal cavity led to a significant decrease of OML uptake by PEMs. Therefore, SIGNR1 expressed on macrophages acts as a receptor for recognition of OMLs under physiological conditions.

Oligomannose-coated liposomes activate ERK via Src kinases and PI3K/Akt in J774A.1 cells.

We have previously shown that liposomes coated with a neoglycolipid constructed from mannotriose and dipalmitoylphosphatidylethanolamine (Man3-DPPE) activate peritoneal macrophages to induce enhanced expression of co-stimulatory molecules and MHC class II. In this study, we investigated the signaling pathways activated by the Man3-DPPE-coated liposomes (OMLs) in a murine macrophage cell line, J774A.1. In response to OML stimulation, ERK among MAPKs was clearly and transiently phosphorylated in J774 cells. ERK phosphorylation was also induced by treatment of the cells with Man3-DPPE and Man3-BSA, but not by uncoated liposomes. In addition, rapid and transient phosphorylation of Akt and Src family kinases (SFKs) was observed in response to OMLs. OML-induced ERK phosphorylation was inhibited by specific inhibitors of PI3K and SFKs, and OML-induced Akt phosphorylation was inhibited by a inhibitor of SFKs. Therefore, OMLs may activate the PI3K/Akt pathway through phosphorylation of Src family kinases to induce ERK activation.

Intracellular trafficking of beta2-glycoprotein I complexes with lipid vesicles in macrophages: implications on the development of antiphospholipid syndrome.

Beta(2)-glycoprotein I (beta(2)GPI) is known as a major autoantigen for antiphospholipid antibodies. Our recent data show that binding of beta(2)GPI to oxidized low-density lipoprotein (oxLDL) or to liposomes containing anionic phospholipid(s) may facilitate the presentation of beta(2)GPI's epitope by macrophages/dendritic cells to autoreactive T cells. In the present study, we investigated intracellular trafficking of beta(2)GPI and its complexes with oxLDL or liposomes containing phosphatidylserine (PS-liposomes) in mouse macrophage-like J774 cells. A relatively small amount of non-complexed beta(2)GPI was taken up and stagnated in the late endosome after incubating for 16h. In contrast, beta(2)GPI complexes with oxLDL or PS-liposomes were transported into the lysosome. In the presence of the IgG anti-beta(2)GPI autoantibody, WB-CAL-1, beta(2)GPI/oxLDL complexes were rapidly incorporated into intracellular space and were finally localized in the lysosome. Interestingly, in vitro pulses by beta(2)GPI/oxLDL complexes together with WB-CAL-1 led to the expression of membranous CD36 as well as Fcgamma type I receptors (FcgammaRI). These observations suggest that IgG immune complexes of beta(2)GPI/oxLDL provide not only FcgammaRI- but also scavenger receptor-mediated uptake of beta(2)GPI/oxLDL complexes by macrophages. Thus, beta(2)GPI/oxLDL complexes as a major atherogenic autoantigen and IgG anti-beta(2)GPI autoantibodies may facilitate antigen presentation and foam cell formation in antiphospholipid syndrome.