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We previously reported that heat shock protein 105 (HSP105), identified by serological analysis of a recombinant cDNA expression library (SEREX) using serum from a pancreatic cancer patient, was overexpressed in various human tumors and in the testis of adult men by immunohistochemical analysis. In the present study, to elucidate the biological function of the HSP105 protein in cancer cells, we first established NIH3T3 cells overexpressing murine HSP105 (NIH3T3-HSP105). The NIH3T3-HSP105 cells acquired resistance to apoptosis induced by heat shock or doxorubicin. (1) is a stress protein belonging to the HSP105/110 family that is expressed constitutively in most tissues at low levels, whereas HSP105 mRNA is expressed at high levels in mouse and rat brain. At the protein level, high expression levels have been reported only in the brain of mice.(1-4) Like other heat shock proteins, HSP105 plays an important role as a chaperone under physiological conditions. HSP105 is induced by various stressors and plays an important role in protecting cells from the cytotoxic effects mediated by such stressors. HSP105 is composed of an ATP-binding domain, a β-sheet, a loop and α-helical domains similar to those observed in the HSP70 family of proteins, and it binds to non-native protein substrates, thereby preventing the aggregation of denatured protein through an interaction with the β-sheet domain of HSP105. (2,(5)(6)(7) The rat neuronal cell line PC12 transfected stably with murine HSP105 exhibited resistance to caspase-mediated apoptosis induced by stressors.(8) In a spinal and bulbar muscular atrophy model, the transient expression of tAR containing an expanded polyglutamine tract caused aggregates of polyglutamine in COS-7 and SK-N-SH cells and then induced the cells to undergo apoptosis. In contrast, in cells cotransfected with tAR and HSP105, both the aggregation of polyglutamine and the degree of it's cell toxicity decreased. (9) In contrast, HSP105 has demonstrated apoptosis-enhancing activity during murine embryogenesis.(10 -13) These observations suggest that HSP105 is involved in the regulation of apoptosis.We previously reported that HSP105 is overexpressed in various human tumors, including colon cancer cells but not colorectal adenomas, thus suggesting that the overexpression of HSP105 is a late event in the colorectal adenoma-carcinoma sequence. (14,15) Recent studies have also demonstrated that the expression level of HSP105 is elevated in highly metastatic colon cancer cell lines, and is correlated with advanced clinical stages and positive lymph node involvement. (16) RNA interference is used widely for manipulating biological systems, and has also been utilized successfully as a therapeutic material in experimental animals. (17)(18)(19) Synthetic siRNA strongly inhibits the expression of target proteins when they are transfected with cationic liposomes, which are thought to be safer than viral vectors for human therapy. The local injection of synthetic siRNA against VEGF (20) or sphingosine 1-phosphate rece...

We report that HSP105, identified by serological identification of antigens by recombinant expression cloning (SEREX), is overexpressed in a variety of human cancers, including colorectal, pancreatic, thyroid, esophageal, and breast carcinoma, but is not expressed in normal tissues except for the testis. The amino acid sequences and expression patterns of HSP105 are very similar in humans and mice. In this study, we set up a preclinical study to investigate the usefulness of a DNA vaccine producing mouse HSP105 whole protein for cancer immunotherapy in vivo using BALB/c and C57BL/6 mice, Colon26, a syngeneic endogenously HSP105-expressing colorectal cancer cell line, and B16.F10, a melanoma cell line. The DNA vaccine was used to stimulate HSP105-specific T-cell responses. C olorectal cancer (CRC) and melanoma are common and serious malignancies, for which surgery remains the main treatment, although the success of the treatment depends on the stage of the disease. Although adjuvant systemic chemotherapy or chemoradiation can confer a limited but significant survival advantage, novel and more effective therapies are needed. Identification of tumor associated antigens (TAA) expressed by CRC or melanomas remains one of the goals for designing novel immunological treatments for these tumors. Ideal targets for immunotherapy are gene products that are silenced in normal tissues except immune privilege tissue such as testis tissue, and that are overexpressed in cancer cells.More than 2000 candidate TAA have been identified by using the serological identification of antigens by recombinant expression cloning (SEREX) method. We have also reported TAA identified by using this method.(1-4) We earlier found that HSP105 (often called HSP110), as identified by SEREX was overexpressed specifically in a variety of human cancers, including colorectal, pancreatic, thyroid, esophageal, and breast carcinoma, but was not expressed in normal tissues except for testis tissue.(1,5) We recently found that HSP105 was also overexpressed in melanoma (unpublished data). If HSP105 can induce strong antitumor immunity, it may be a potential candidate as a target antigen for cancer immunotherapy. In the present study, we set up a preclinical study to investigate the usefulness of a HSP105-DNA vaccine, using BALB/c and C57BL/6 mice, the syngeneic endogenously HSP105-expressing CRC cell line Colon26, and the melanoma cell line B16.F10. Using these models, we analyzed both the antitumor effects and side-effects, including autoimmunity of the HSP105 DNA vaccination.The pioneering studies of Srivastava and colleagues led to the proposal that several HSP, including HSP70, HSP90 and gp96, bind antigenic peptides and deliver these peptides (through receptor-mediated endocytosis of the HSP) into the antigen-processing pathway of the antigen presenting cell (APC) for presentation on major histocompatibility complex (MHC) class I molecules. This HSP-involved pathway has been demonstrated to evoke potent antiviral and antitumor immune responses.(6) Howeve...

Ever since immune checkpoint inhibitors have been approved for anti-cancer therapy in several cancers, including triple-negative breast cancer, the significance of programmed death-1 ligand 1 (PD-L1) expression in the tumor immune microenvironment has been a topic of interest. In the present study, we investigated the detailed mechanisms of PD-L1 overexpression on tumor-associated macrophages (TAMs) in breast cancer. In in vitro culture studies using human monocyte-derived macrophages, lymphocytes, and breast cancer cell lines, PD-L1 overexpression on macrophages was induced by the conditioned medium (CM) of activated lymphocytes, but not that of cancer cells. Granulocyte–macrophage colony-stimulating factor (GM-CSF) derived from activated lymphocytes was found to be involved in PD-L1 overexpression, in addition to interferon (IFN)-γ, via STAT3 pathway activation. Macrophages suppressed lymphocyte activation, and this inhibition was impaired by PD-1 blocking. The CM of activated lymphocytes also induced the overexpression of PD-L2, but GM-CSF did not affect PD-L2 expression. In the murine E0771 breast cancer model, anti-GM-CSF therapy did not affect PD-L1 expression on TAMs, and the mechanisms of PD-L1 expression on TAMs might differ between humans and mice. However, not only PD-L1, but also PD-L2 was overexpressed on TAMs in the E0771 tumor model, and their expression levels were significantly lower in the tumors in nude mice than in wild-type mice. Anti-PD-L1 antibody and anti-PD-L2 antibody synergistically inhibited E0771 tumor development. In conclusion, PD-L1 and PD-L2 were overexpressed on TAMs, and they potentially contributed to immunosuppression. The GM-CSF-STAT3 pathway is thought to represent a new mechanism of PD-L1 overexpression on TAMs in human breast cancer microenvironment.