Yasuaki Tamura

Extracellular heat shock protein 90 plays a role in translocating chaperoned antigen from endosome to proteasome for generating antigenic peptide to be cross-presented by dendritic cells

Extracellular heat shock protein can deliver associated antigens into the MHC class I presentation pathway of antigen-presenting cells, a process called cross-presentation, thus inducing antigen-specific CD8(+) T-cell responses; however, the precise mechanism for intracellular antigen translocation and the processing pathway has not been fully elucidated. Here we demonstrate that cross-presentation of extracellular Hsp90-ovalbumin (OVA) protein complexes to specific CD8(+) T cells involves both classical proteasome-transporter-associated antigen processing (TAP)-dependent and TAP-independent-endosomal pathways. Using confocal microscopy, we found that the internalized extracellular Hsp90 and OVA co-localized with cytosolic proteasomes. When anti-Hsp90 mAb was introduced to dendritic cells (DCs), we observed that the co-localization of internalized Hsp90-chaperoned OVA and proteasomes was abolished, resulting in the inhibition of TAP-dependent cross-presentation of OVA. Thus, extracellular Hsp90 may play a pivotal role for the translocation of chaperoned antigens for proteasomal degradation in the cytosol. In contrast, OVA chaperoned by Hsp90 was not presented by MHC class II molecules in vitro or in vivo, although the antigen was exogenously loaded onto DCs. Our data indicate that extracellular Hsp90 might be essential for the translocation of chaperoned antigens from the extracellular milieu into cytosol, resulting in proteasomal degradation for cross-presentation.

Immunotherapeutic benefit of α-interferon (IFNα) in survivin2B-derived peptide vaccination for advanced pancreatic cancer patients

Survivin, a member of the inhibitor of apoptosis protein (IAP) family containing a single baculovirus IAP repeat domain, is highly expressed in cancerous tissues but not in normal counterparts. Our group identified an HLA‐A24‐restricted antigenic peptide, survivin‐2B80–88 (AYACNTSTL), that is recognized by CD8 + CTLs and functions as an immunogenic molecule in patients with cancers of various histological origins such as colon, breast, lung, oral, and urogenital malignancies. Subsequent clinical trials with this epitope peptide alone resulted in clinical and immunological responses. However, these were not strong enough for routine clinical use as a therapeutic cancer vaccine, and our previous study of colon cancer patients indicated that treatment with a vaccination protocol of survivin‐2B80–88 plus incomplete Freunds adjuvant (IFA) and α‐interferon (IFNα) conferred overt clinical improvement and enhanced the immunological responses of patients. In the current study, we further investigated whether this vaccination protocol could efficiently provide not only improved immune responses but also better clinical outcomes for advanced pancreatic cancers. Tetramer and enzyme‐linked immunosorbent spot analysis data indicated that more than 50% of the patients had positive clinical and immunological responses. In contrast, assessment of treatment with IFNα only to another group of cancer patients resulted in no obvious increase in the frequency of survivin‐2B80‐88 peptide‐specific CTLs. Taken together, our data clearly indicate that a vaccination protocol of survivin‐2B80‐88 plus IFA and IFNα is very effective and useful in immunotherapy for this type of poor‐prognosis neoplasm. This trial was registered with the UMIN Clinical Trials Registry, no. UMIN000000905. (Cancer Sci 2013; 104: 124–129)

Immunogenic enhancement and clinical effect by type-I interferon of anti-apoptotic protein, survivin-derived peptide vaccine, in advanced colorectal cancer patients

We previously identified a human leukocyte antigen (HLA)‐A24‐restricted antigenic peptide, survivin‐2B80‐88, recognized by CD8+ cytotoxic T lymphocytes (CTL). Subsequently, we attempted clinical trials with this epitope peptide alone for some malignancies, resulting in clinical and immunological responses, although their potential was not strong enough for routine clinical use as a cancer vaccine. In the current study, to assess whether immunogenicity of the survivin‐2B80‐88 peptide could be enhanced with other vaccination protocols, we performed clinical trials in advanced colon cancer patients with two vaccination protocols: (i) survivin‐2B80‐88 plus incomplete Freund’s adjuvant (IFA); and (ii) survivin‐2B80‐88 plus IFA and a type‐I interferon (IFN), IFNα. Our data clearly indicated that, although the effect of survivin‐2B80‐88 plus IFA was not significantly different from that with survivin‐2B80‐88 alone, treatment with the vaccination protocol of survivin‐2B80‐88 plus IFA and IFNα resulted in clinical improvement and enhanced immunological responses of patients. Tetramer analysis of survivin‐2B80‐88 peptide‐specific CTL demonstrated that such CTL were increased at least twofold after vaccination with this protocol in four of eight patients. In these patients, enzyme‐linked immunosorbent spot (ELISPOT) results were also enhanced. Subsequent study of single‐cell clone separation by cell sorting of peptide‐specific CTL showed that each CTL clone was indeed not only peptide‐specific but also cytotoxic against human cancer cells in the context of the expression of both HLA‐A24 and survivin molecules. Taken together, these results indicate that vaccination of colon cancer patients with survivin‐2B80‐88 plus IFA and IFNα can be considered to be a very potent immunotherapeutic regimen, and that this protocol might work for other cancers. (Cancer Sci 2011; 102: 1181–1187)

Heat-shock proteins as endogenous ligands building a bridge between innate and adaptive immunity

There has been growing evidence that heat-shock protein (HSP) functions as an endogenous immunomodulator for innate and adaptive immune responses. Since HSPs inherently act as chaperones within cells, passive release (e.g., by cell necrosis) and active release (including release by secretion in the form of an exosome) have been suggested as mechanisms of HSP release into the extracellular milieu. Such extracellular HSPs have been shown to be activators of innate immune responses through Toll-like receptors. However, it has also been suggested that HSPs augment the ability of associated innate ligands such as lipopolysaccharides to stimulate cytokine production and dendritic cell maturation. More interestingly, a recent study has demonstrated that innate immune responses elicited by danger signals were regulated spatiotemporally and that can be manipulated by HSPs, thereby controlling immune responses. We will discuss how spatiotemporal regulation of HSP-chaperoned molecules within antigen-presenting cells affects adaptive immunity via antigen cross-presentation and innate immune responses. Precise analysis of HSP biology should lead to the establishment of effective HSP-based immunotherapy.

Preferential expression of cancer/testis genes in cancer stem‐like cells: proposal of a novel sub‐category, cancer/testis/stem gene

Cancer/testis (CT) antigens encoded by CT genes are immunogenic antigens, and the expression of CT gene is strictly restricted to only the testis among mature organs. Therefore, CT antigens are promising candidates for cancer immunotherapy. In a previous study, we identified a novel CT antigen, DNAJB8. DNAJB8 was found to be preferentially expressed in cancer stem-like cells (CSCs)/cancer-initiating cells (CICs), and it is thus a novel CSC antigen. In this study, we hypothesized that CT genes are preferentially expressed in CSCs/CICs rather than in non-CSCs/-CICs and we examined the expression of CT genes in CSCs/CICs. The expression of 74 CT genes was evaluated in side population (SP) cells (=CSC) and main population (MP) cells (=non-CSC) derived from LHK2 lung adenocarcinoma cells, SW480 colon adenocarcinoma cells and MCF7 breast adenocarcinoma cells by RT-PCR and real-time PCR. Eighteen genes (MAGEA2, MAGEA3, MAGEA4, MAGEA6, MAGEA12, MAGEB2, GAGE1, GAGE8, SPANXA1, SPANXB1, SPANXC, XAGE2, SPA17, BORIS, PLU-1, SGY-1, TEX15 and CT45A1) showed higher expression levels in SP cells than in MP cells, whereas 10 genes (BAGE1, BAGE2, BAGE4, BAGE5, XAGE1, LIP1, D40, HCA661, TDRD1 and TPTE) showed similar expression levels in SP cells and MP cells. Thus, considerable numbers of CT genes showed preferential expression in CSCs/CICs. We therefore propose a novel sub-category of CT genes in this report: cancer/testis/stem (CTS) genes.

High expression of ALDH1 and SOX2 diffuse staining pattern of oral squamous cell carcinomas correlates to lymph node metastasis.

One of the major factors involved in the prognosis of oral squamous cell carcinoma (OSCC) patients is metastasis. Recent progress in cancer stem‐like cell/cancer‐initiating cell (CSC/CIC) research indicates that CSCs are related to metastasis. Aldehyde dehydrogenase 1 – (ALDH1) and SRY‐related HMG‐box gene 2 (SOX2) have recently been shown to be putative CSC markers for several human malignancies. The aim of this study was to determine the association of ALDH1 and SOX2 expression in oral squamous cell carcinoma (OSCC) with lymph node metastasis. Immunohistochemical staining of ALDH1, SOX2 and Ki67 was performed in 80 OSCC tissues. High expression rates of ALDH1 (2%–40%) were found to be related to lymph node metastasis (Pu2003=u20030.0017). Interestingly, we found that SOX2 staining could be classified into two patterns: (i) peripheral staining pattern; and (ii) diffuse staining pattern. The diffuse staining pattern showed a significant correlation with lymph node metastasis (Pu2003<u20030.001). No correlation was found between Ki67 staining and lymph node metastasis (Pu2003=u20030.4724). The ALDH1 positive staining rates in metastatic lymph nodes were higher than that in corresponding primary OSCC tissues. These results indicate that high expression rates of ALDH1 and SOX2 diffuse staining patterns might be novel prediction markers for OSCC lymph node metastasis.

New paradigm for intrinsic function of heat shock proteins as endogenous ligands in inflammation and innate immunity.

Recently, growing evidences that extracellular heat shock protein (HSP) functions as endogenous immunomodulator for innate and adaptive immune responses have been demonstrated. Because HSPs inherently act as chaperones within the cells, passive release such as cell necrosis and active release including secretion in the form of exosome have been suggested for HSP release into extracellular milieu. Such extracellular HSPs have been shown to be activators for innate immune responses through Toll-like receptors (TLRs). However, it has also been suggested that HSPs augmented the ability of associated innate ligands such as LPS to stimulate cytokine production and dendritic cell (DC) maturation. More interestingly, recent study demonstrated that innate immune responses elicited by both endogenous and exogenous danger signals were spatially and temporally regulated and this can be manipulated using Hsp90 or oxygen-regulated protein 150 (ORP150), thereby controlling the immune responses. We will discuss how spatiotemporal regulation of HSP-chaperoned molecules within antigen-presenting cells affects the antigen cross-presentation and innate immune responses. Precise analysis of HSP biology can lead us to establish outstanding HSPbased immunotherapy.

Establishment of a monoclonal anti-pan HLA class I antibody suitable for immunostaining of formalin-fixed tissue : Unusually high frequency of down-regulation in breast cancer tissues

A novel monoclonal anti‐pan human leukocyte antigen (HLA) class I heavy chain antibody, EMR8‐5, was established. It could detect HLA‐A, ‐B, and ‐C antigens in formalin‐fixed paraffin embedded tissues. By immunohistochemical staining using the EMR8‐5 antibody, various cancer tissues from 246 cases were examined for HLA class I expression. It was found that HLA class I expression was decreased in 20% to 42% of the cases of lung cancer, hepatocellular carcinoma, colon cancer, renal cell carcinoma, and urothelial carcinoma. In contrast, 85% of breast cancer cases had loss of or decreased HLA class I expression. Of the 35 breast cancer cases that had decreased HLA class I heavy chain expression, 33 (94%) also had decreased beta2‐microglobulin expression detected by immunohistochemical staining. It was suggested that HLA class I down‐regulation might be a common characteristic of breast cancer mostly caused by the down‐regulation of beta2‐microglobulin expression.

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.

DNA methyltransferase 1 is essential for initiation of the colon cancers.

DNA methyltransferase 1 (Dnmt1) is essential for the maintenance of hematopoietic and somatic stem cells in mice; however, its roles in human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are still elusive. In the present study, we investigated DNMT1 functions in the maintenance of human colon CSCs/CICs using the human colon cancer cell line HCT116 (HCT116 w/t) and its DNMT1 knockout cell line (DNMT1(-/-)). The rates of CSCs/CICs were evaluated by side population (SP) analysis, ALDEFLUOR assay and expression of CD44 and CD24. SP, ALDEFLUOR-positive (ALDEFLUOR(+)) and CD44-positive and CD24-positive (CD44(+)CD24(+)) cell rates were lower in DNMT1(-/-) cells than in HCT116 w/t cells. Since CSCs/CICs have higher tumor-initiating ability than that of non-CSCs/CICs, the tumor-initiating abilities were addressed by injecting immune deficient (NOD/SCID) mice. DNMT1(-/-) cells showed less tumor-initiating ability than did HCT116 w/t cells, whereas the growing rate of DNMT1(-/-) cells showed no significant difference from that of HCT116 cells both in vitro and in vivo. Similar results were obtained for cells in which DNMT1 had been transiently knocked-down using gene-specific siRNAs. Taken together, these results indicate that DNMT1 is essential for maintenance of colon CSCs/CICs and that short-term suppression of DNMT1 might be sufficient to disrupt CSCs/CICs.