Goro Kutomi

Efficient Cross-Presentation by Heat Shock Protein 90-Peptide Complex-Loaded Dendritic Cells via an Endosomal Pathway

It is well-established that heat shock proteins (HSPs)-peptides complexes elicit antitumor responses in prophylactic and therapeutic immunization protocols. HSPs such as gp96 and Hsp70 have been demonstrated to undergo receptor-mediated uptake by APCs with subsequent representation of the HSP-associated peptides to MHC class I molecules on APCs, facilitating efficient cross-presentation. On the contrary, despite its abundant expression among HSPs in the cytosol, the role of Hsp90 for the cross-presentation remains unknown. We show here that exogenous Hsp90-peptide complexes can gain access to the MHC class I presentation pathway and cause cross-presentation by bone marrow-derived dendritic cells. Interestingly, this presentation is TAP independent, and followed chloroquine, leupeptin-sensitive, as well as cathepsin S-dependent endosomal pathways. In addition, we show that Hsp90-chaperoned precursor peptides are processed and transferred onto MHC class I molecules in the endosomal compartment. Furthermore, we demonstrate that immunization with Hsp90-peptide complexes induce Ag-specific CD8+ T cell responses and strong antitumor immunity in vivo. These findings have significant implications for the design of T cell-based cancer immunotherapy.

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)

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.

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.

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.

Targeting to Static Endosome Is Required for Efficient Cross-Presentation of Endoplasmic Reticulum-Resident Oxygen-Regulated Protein 150-Peptide Complexes

Heat shock proteins (HSPs) such as Hsp70, gp96, and Hsp90 have been shown to elicit intriguing, efficient CTL responses by cross-presentation via an as yet entirely unknown mechanism. Oxygen-regulated protein 150 (ORP150), also known as grp170, is an endoplasmic reticulum-resident HSP and is up-regulated by hypoxia. It has been demonstrated that ORP150 binds tumor-associated Ag peptides within cancer cells. Immunization with an ORP150-tumor Ag complex has been shown to generate tumor-specific CTLs. Most recently, it has been shown that exogenous ORP150 induces cross-presentation of a chaperoned Ag, thereby stimulating Ag-specific CTLs. However, the mechanism underlying this efficient cross-presentation is still unsolved. In this study, we show that the ORP150-precursor peptide complex can elicit CTL response through cross-presentation as well as the CD4+ T cell response by dendritic cells. Furthermore, we observed that the internalized ORP150-peptide complex, but not OVA protein, which was not cross-presented, was sorted to the Rab5+, EEA1+ static early endosome, followed by translocation to a recycling endosome, where the ORP150-chaperoned peptide was processed and bound to MHC class I molecules. Moreover, we observed that immunization of mice with ORP150-peptide complexes elicited strong peptide-specific CTLs and antitumor effects in vivo. Our data indicate that targeting of the Ag to a “static” early endosme by ORP150 is required for the efficient cross-presentation.

Olfactory Receptor Family 7 Subfamily C Member 1 Is a Novel Marker of Colon Cancer-Initiating Cells and Is a Potent Target of Immunotherapy.

Purpose: Cancer-initiating cells (CICs) are thought to be essential for tumor maintenance, recurrence, and distant metastasis, and they are therefore reasonable targets for cancer therapy. Cancer immunotherapy is a novel approach to target cancer. In this study, we aimed to establish novel CIC-targeting immunotherapy. Experimental Design: Colorectal cancer (CRC) CICs were isolated as side population (SP) cells. The gene expression profile of CRC CICs was analyzed by cDNA microarray and RT-PCR. Protein expression of olfactory receptor family 7 subfamily C member 1 (OR7C1) were analyzed by Western blot and immunohistochemical staining. The functions of OR7C1 were analyzed by gene overexpression and gene knockdown using siRNAs. OR7C1-positive cells were isolated by a flow cytometer and analyzed. CTLs specific for OR7C1 peptide were generated, and the antitumor effect was addressed by mice adoptive transfer model. Results: OR7C1 has essential roles in the maintenance of colon CICs, and the OR7C1-positive population showed higher tumorigenicity than that of the OR7C1-negative population, indicating that OR7C1 is a novel functional marker for colon CIC. Immunohistochemical staining revealed that OR7C1 high expression was correlated with poorer prognosis in CRC patients. OR7C1-derived antigenic peptide-specific CTLs showed specific cytotoxicity for CICs, and an OR7C1-specific CTL clone showed a greater antitumor effect than did a CTL clone targeting all cancer cells in a CTL adoptive transfer mouse model. Conclusions: OR7C1 is a novel marker for colon CICs and can be a target of potent CIC-targeting immunotherapy. Clin Cancer Res; 22(13); 3298–309. ©2016 AACR.

Equol, Adiponectin, Insulin Levels and Risk of Breast Cancer

Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer death among women. Soy isoflavones have been widely studied and among all isoflavones equol has been gaining interest with regard to its relationship with breast cancer risk. Obesity has been revealed as one of the breast cancer risk factors, known to be associated with high levels of circulating insulin and decreased levels of adiponectin. Hence there have been many studies investigating relationships between insulin and adiponectin levels and breast cancer risk. Additionally recent findings have suggested that insulin and adiponectin themselves may have influence on breast cancer development, independent of obesity. In the present review, we discuss the relationships between breast cancer risk and equol, insulin and adiponectin levels, which are three important factors in our ongoing hospital-based case-control study. Herein these factors are reviewed not only from the clinical viewpoint but also from possible chemical and biological points of view which may explain clinical observations.

The potential for oral combination chemotherapy of 5′-deoxy-5-fluorouridine, a 5-FU prodrug, and cyclophosphamide for metastatic breast cancer

Preclinical studies have demonstrated the synergistic anti-tumour activity of combination therapy with the oral cytostatics, 5′-deoxy-5-fluorouridine (5′-DFUR) and cyclophosphamide (CPA), in human breast cancer xenograft models. This study was performed to evaluate the efficacy and safety of this oral combination chemotherapy in the treatment of metastatic breast cancer. In all, 101 patients with metastatic breast cancer were enrolled in the study, and the data for 94 eligible patients of these were evaluated. The patients received twice daily oral combinations of 5′-DFUR (1200 mg/body/day) and CPA (100 mg/body/day) for 2 weeks, followed by a 1-week rest period. After a median of 19 treatment cycles (range 1–66 cycles), 16 patients (17.0%) had a complete response, and 40 patients (42.6%) had partial responses. The response rate was 59.6% (95% CI, 49.0–69.6%). The median time to progression and overall survival times were 11.7 and 40.3 months, respectively. The toxicity was mild and tolerable, and the related grade 3/4 clinical adverse effects consisted of haematological toxicity in 21 patients (22%) and nonhaematological toxicity in five patients (5%). These results suggest that the oral combination chemotherapy of 5′-DFUR and CPA has low toxicity and is a novel, very convenient and effective treatment for metastatic breast cancer.