Cellular stress induces cancer stem-like cells through expression of DNAJB8 by activation of heat shock factor 1.

In a previous study, we found that DNAJB8, a heat shock protein (HSP) 40 family member is expressed in kidney cancer stem-like cells (CSC)/cancer-initiating cells (CIC) and that it has a role in the maintenance of kidney CSC/CIC. Heat shock factor (HSF) 1 is a key transcription factor for responses to stress including heat shock, and it induces HSP family expression through activation by phosphorylation. In the present study, we therefore examined whether heat shock (HS) induces CSC/CIC. We treated the human kidney cancer cell line ACHN with HS, and found that HS increased side population (SP) cells. Western blot analysis and qRT-PCR showed that HS increased the expression of DNAJB8 and SOX2. Gene knockdown experiments using siRNAs showed that the increase in SOX2 expression and SP cell ratio depends on DNAJB8 and that the increase in DNAJB8 and SOX2 depend on HSF1. Furthermore, treatment with a mammalian target of rapamycin (mTOR) inhibitor, temsirolimus, decreased the expression of DNAJB8 and SOX2 and the ratio of SP cells. Taken together, the results indicate that heat shock induces DNAJB8 by activation of HSF1 and induces cancer stem-like cells.

The Antigen ASB4 on Cancer Stem Cells Serves as a Target for CTL Immunotherapy of Colorectal Cancer.

Colorectal cancer consists of a small number of cancer stem cells (CSC) and many non-CSCs. Although rare in number, CSCs are a target for cancer therapy, because they survive conventional chemo- and radiotherapies and perpetuate tumor formation in vivo In this study, we conducted an HLA ligandome analysis to survey HLA-A24 peptides displayed by CSCs and non-CSCs of colorectal cancer. The analysis identified an antigen, ASB4, which was processed and presented by a CSC subset but not by non-CSCs. The ASB4 gene was expressed in CSCs of colorectal cancer, but not in cells that had differentiated into non-CSCs. Because ASB4 was not expressed by normal tissues, its peptide epitope elicited CD8+ cytotoxic T-cell (CTL) responses, which lysed CSCs of colorectal cancer and left non-CSCs intact. Therefore, ASB4 is a tumor-associated antigen that can elicit CTL responses specific to CSCs and can discriminate between two cellular subsets of colorectal cancer. Adoptively transferred CTLs specific for the CSC antigen ASB4 could infiltrate implanted colorectal cancer cell tumors and effectively prevented tumor growth in a mouse model. As the cancer cells implanted in these mice contained very few CSCs, the elimination of a CSC subset could be the condition necessary and sufficient to control tumor formation in vivo These results suggest that CTL-based immunotherapies against colorectal CSCs might be useful for preventing relapses. Cancer Immunol Res; 6(3); 358-69. ©2018 AACR.

GRIK2 has a role in the maintenance of urothelial carcinoma stem-like cells, and its expression is associated with poorer prognosis.

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are small sub-population of cancer cells that are endowed with higher tumor-initiating ability, self-renewal ability and differentiation ability. CSCs/CICs could be isolated as high aldehyde dehydrogenase 1 activity cells (ALDH1high) from various cancer samples. In this study, we isolated urothelial carcinoma CSCs/CICs as ALDHhigh cells and investigated the molecular aspects. ALDH1high cells showed greater sphere-forming ability and higher tumor-initiating ability in immune-deficient mice than those of ALDH1low cells, indicating that CSCs/CICs were enriched in ALDH1high cells. cDNA microarray analysis revealed that an ionotropic glutamate receptor glutamate receptor, ionotropic, kainate 2 (GRIK2) was expressed in ALDH1high cells at a higher level than that in ALDH1low cells. GRIK2 gene knockdown by siRNAs decreased the sphere-forming ability and invasion ability, whereas GRIK2 overexpression increased the sphere-forming ability, invasion ability and tumorigenicity, indicating that GRIK2 has a role in the maintenance of CSCs/CICs. Immunohistochemical staining revealed that higher levels of GRIK2 and ALDH1 expression were related to poorer prognosis in urinary tract carcinoma cases. The findings indicate that GRIK2 has a role in the maintenance of urothelial CSCs/CICs and that GRIK2 and ALDH1 can be prognosis prediction markers for urinary tract carcinomas.

Phosphorylation of HSF1 at serine 326 residue is related to the maintenance of gynecologic cancer stem cells through expression of HSP27.

Cancer stem-like cells (CSCs)/ cancer-initiating cells (CICs) are defined by their higher tumor-initiating ability, self-renewal capacity and differentiation capacity. CSCs/CICs are resistant to several therapies including chemotherapy and radiotherapy. CSCs/CICs thus are thought to be responsible for recurrence and distant metastasis, and elucidation of the molecular mechanisms of CSCs/CICs are essential to design CSC/CIC-targeting therapy. In this study, we analyzed the molecular aspects of gynecological CSCs/CICs. Gynecological CSCs/CICs were isolated as ALDH1high cell by Aldefluor assay. The gene expression profile of CSCs/CICs revealed that several genes related to stress responses are preferentially expressed in gynecological CSCs/CICs. Among the stress response genes, a small heat shock protein HSP27 has a role in the maintenance of gynecological CSCs/CICs. The upstream transcription factor of HSP27, heat shock factior-1 (HSF1) was activated by phosphorylation at serine 326 residue (pSer326) in CSCs/CICs, and phosphorylation at serine 326 residue is essential for induction of HSP27. Immunohistochemical staining using clinical ovarian cancer samples revealed that higher expressions of HSF1 pSer326 was related to poorer prognosis. These findings indicate that activation of HSF1 at Ser326 residue and transcription of HSP27 is related to the maintenance of gynecological CSCs/CICs.

Brother of the regulator of the imprinted site (BORIS) variant subfamily 6 is a novel target of lung cancer stem-like cell immunotherapy.

Lung cancer is one of the most common malignancies with a high rate of mortality. Lung cancer stem-like cells (CSCs)/ cancer-initiating cells (CICs) play major role in resistance to treatments, recurrence and distant metastasis and eradication of CSCs/CICs is crucial to improve recent therapy. Cytotoxic T lymphocytes (CTLs) are major effectors of cancer immunotherapy, and CTLs recognize antigenic peptides derived from antigens that are presented by major histocompatibility complex (MHC) class I molecules. In this study, we analyzed the potency of a cancer-testis (CT) antigen, brother of the regulator of the imprinted site variant subfamily 6 (BORIS sf6), in lung CSC/CIC immunotherapy. BORIS sf6 mRNA was expressed in lung carcinoma cells (9/19), especially in sphere-cultured lung cancer stem-like cells, and in primary lung carcinoma tissues (4/9) by RT-PCR. Immunohistochemical staining using BORIS sf6-specific antibody revealed that high expression of BORIS sf6 is related to poorer prognosis. CTLs could be induced by using a human leukocyte antigen, (HLA)-A2 restricted antigenic peptide (BORIS C34_24(9)), from all of 3 HLA-A2-positive individuals, and CTL clone cells specific for BORIS C34_24(9) peptide could recognize BORIS sf6-positive, HLA-A2-positive lung carcinoma cells. These results indicate that BORIS sf6 is a novel target of lung cancer immunotherapy that might be useful for targeting treatment-resistant lung cancer stem-like cells.

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.

Establishment and Analysis of Cancer Stem-Like and Non-Cancer Stem-Like Clone Cells from the Human Colon Cancer Cell Line SW480.

Human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) can be isolated as side population (SP) cells, aldehyde dehydrogenase high (ALDHhigh) cells or cell surface marker-positive cells including CD44+ cells and CD133+ cells. CSCs/CICs and non-CSCs/CICs are unstable in in vitro culture, and CSCs/CICs can differentiate into non-CSCs/CICs and some non-CSCs/CICs can dedifferentiate into CSCs/CICs. Therefore, experiments using a large amount of CSCs/CICs are technically very difficult. In this study, we isolated single cell clones from SP cells and main population (MP) cells derived from the human colon cancer cell line SW480. SP analysis revealed that SP clone cells had relatively high percentages of SP cells, whereas MP clone cells showed very few SP cells, and the phenotypes were sustainable for more than 2 months of in vitro culture. Xenograft transplantation revealed that SP clone cells have higher tumor-initiating ability than that of MP clone cells and SP clone cell showed higher chemo-resistance compared with MP clone cells. These results indicate that SP clone cells derived from SW480 cells are enriched with CSCs/CICs, whereas MP clone cells are pure non-CSCs/CICs. SP clone cells and MP clone cells are a very stable in vitro CSC/CIC-enriched and non-CSC/CIC model for further analysis.

MAPK13 is preferentially expressed in gynecological cancer stem cells and has a role in the tumor-initiation.

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are defined as small subpopulation of cancer cells that are endowed with higher tumor-initiating ability. CSCs/CICs are resistant to standard cancer therapies including chemotherapy and radiotherapy, and they are thus thought to be responsible for cancer recurrence and metastasis. Therefore, elucidation of molecular mechanisms of CSCs/CICs is essential to cure cancer. In this study, we analyzed the gene expression profiles of gynecological CSCs/CICs isolated as aldehyde dehydrogenase high (ALDH(high)) cells, and found that MAPK13, PTTG1IP, CAPN1 and UBQLN2 were preferentially expressed in CSCs/CICs. MAPK13 is expressed in uterine, ovary, stomach, colon, liver and kidney cancer tissues at higher levels compared with adjacent normal tissues. MAPK13 gene knockdown using siRNA reduced the ALDH(high) population and abrogated the tumor-initiating ability. These results indicate that MAPK13 is expressed in gynecological CSCs/CICs and has roles in the maintenance of CSCs/CICs and tumor-initiating ability, and MAPK13 might be a novel molecular target for treatment-resistant CSCs/CICs.

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.

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.

P4-006　　Natural peptidome presented by HLA-A24 of cancer and cancer stem cells.

  Circulating CD8+ T cells (CTL) survey the peptide/MHC class I complexes on cell surface to discriminate self and eliminate foreign/transformed cells. Thus, the MHC class I peptide repertoire directly influences cells fate, and provides information for immunotherapy strategy. Here we target HLA-A24 (A24), the most frequent serotype in Asia, and perform large-scale mass spectrometric profiling of natural peptides. Using the antibody specific to A24, we identified 264 peptides from colon (SW480, Colo320, HCT15/b2m) and 331 peptides from lung (LHK2, Sq-1) cancer cells. Although, known A24 binding anchors (Y/F at P2 and F/L/I at P9) are strongly conserved among the detected ligands, a subset of peptides with an unusual anchor (K/R at the C-terminal P9 or P10) is observed, suggesting diverse usage of anchors in certain types of cancer cells. Moreover, some peptides (and their genes) are exclusively expressed in cancer stem cells (cells able to exclude Hoechst dye). In summary, we identified approx. 500 non-overlapping natural peptides presented by A24 from colon and lung cancer cells. A combination of natural peptides specific to tumors could be an ideal way for a CTL-based immunotherapy.

Potential survival benefit of anti-apoptosis protein: survivin-derived peptide vaccine with and without interferon alpha therapy for patients with advanced or recurrent urothelial cancer--results from phase I clinical trials.

We previously identified a human leukocyte antigen (HLA)-A24-restricted antigenic peptide, survivin-2B80-88, a member of the inhibitor of apoptosis protein family, recognized by CD8+cytotoxic T lymphocytes (CTL). In a phase I clinical trial of survivin-2B80-88 vaccination for metastatic urothelial cancer (MUC), we achieved clinical and immunological responses with safety. Moreover, our previous study indicated that interferon alpha (IFN α ) enhanced the effects of the vaccine for colorectal cancer. Therefore, we started a new phase I clinical trial of survivin-2B80-88 vaccination with IFN α for MUC patients. Twenty-one patients were enrolled and no severe adverse event was observed. HLA-A24/survivin-2B80-88 tetramer analysis and ELISPOT assay revealed a significant increase in the frequency of the peptide-specific CTLs after vaccination in nine patients. Six patients had stable disease. The effects of IFN α on the vaccination were unclear for MUC. Throughout two trials, 30 MUO patients received survivin-2B80-88 vaccination. Patients receiving the vaccination had significantly better overall survival than a comparable control group of MUO patients without vaccination (P = 0.0009). Survivin-2B80-88 vaccination may be a promising therapy for selected patients with MUC refractory to standard chemotherapy. This trial was registered with UMIN00005859.

Fatal hypernatremia due to drinking a large quantity of shoyu (Japanese soy sauce).

We report a case of fatal salt poisoning in a 55-year-old woman who suffered from depression and drank a large quantity of shoyu (Japanese soy sauce). She presented with the highest ever documented serum sodium level of 187 mmol/L. This was associated with symptoms of cerebral damage which developed within hours after drinking the soy sauce. She died as a result of massive pulmonary edema, despite intensive medical treatment. Viewing the results of clinical and postmortem investigations together, her death could clearly be attributed to drinking a large quantity of soy sauce.