Melanoma antigens recognized by T cells and their use for immunotherapy.

Melanoma has been a prototype for cancer immunology research, and the mechanisms of anti-tumor T-cell responses have been extensively investigated in patients treated with various immunotherapies. Individual differences in cancer-immune status are defined mainly by cancer cell characteristics such as DNA mutations generating immunogenic neo-antigens, and oncogene activation causing immunosuppression, but also by patients' genetic backgrounds such as HLA types and genetic polymorphisms of immune related molecules, and environmental and lifestyle factors such as UV rays, smoking, gut microbiota and concomitant medications; these factors have an influence on the efficacy of immunotherapy. Recent comparative studies on responders and non-responders in immune-checkpoint inhibitor therapy using various new technologies including multi-omics analyses on genomic DNA, mRNA, metabolites and microbiota and single cell analyses of various immune cells have led to the advance of human tumor immunology and the development of new immunotherapy. Based on the new findings from these investigations, personalized cancer immunotherapies along with appropriate biomarkers and therapeutic targets are being developed for patients with melanoma. Here, we will discuss one of the essential subjects in tumor immunology: identification of immunogenic tumor antigens and their effective use in various immunotherapies including cancer vaccines and adoptive T-cell therapy.

Long Noncoding RNA HOXA11-AS and Transcription Factor HOXB13 Modulate the Expression of Bone Metastasis-Related Genes in Prostate Cancer.

Long noncoding RNAs (lncRNAs) are emerging as critical regulators of gene expression, which play fundamental roles in cancer development. In this study, we found that homeobox A11 antisense RNA (HOXA11-AS), a highly expressed lncRNA in cell lines derived from prostate cancer bone metastases, promoted the cell invasion and proliferation of PC3 prostate cancer cells. Transcription factor homeobox B13 (HOXB13) was identified as an upstream regulator of HOXA11-AS.HOXA11-AS regulated bone metastasis-associated C-C motif chemokine ligand 2 (CCL2)/C-C chemokine receptor type 2 (CCR2) signaling in both PC3 prostate cancer cells and SaOS2 osteoblastic cells. The HOXB13/HOXA11-AS axis also regulated integrin subunits (ITGAV and ITGB1) specific to prostate cancer bone metastasis. HOXB13, in combination with HOXA11-AS, directly regulated the integrin-binding sialoprotein (IBSP) promoter. Furthermore, conditioned medium containing HOXA11-AS secreted from PC3 cells could induce the expression of CCL2 and IBSP in SaOS2 osteoblastic cells. These results suggest that prostate cancer HOXA11-AS and HOXB13 promote metastasis by regulation of CCL2/CCR2 cytokine and integrin signaling in autocrine and paracrine manners.

lncRNA HOTAIR Inhibits Mineralization in Osteoblastic Osteosarcoma Cells by Epigenetically Repressing ALPL.

HOTAIR is a lncRNA that plays critical role in gene regulation and chromatin dynamics through epigenetic mechanisms. In this work we studied the physiological role of HOTAIR during the process of mineralization using osteoblastic osteosarcoma cells focusing in ALPL (Tissue Non-Specific Alkaline Phosphatase), a pivotal gene that controls bone formation. HOTAIR knockdown resulted in upregulation of ALPL, increase of alkaline phosphatase (ALP) activity, and enhanced mineralization in osteoblastic SaOS-2 cells cultured in mineralizing medium. Luciferase assays using reporter vectors containing ALPL promoter showed that HOTAIR repression increases ALPL promoter activity. Furthermore, HOTAIR knockdown increased histone H3K4 methylation levels at ALPL promoter region, suggesting that ALPL repression by HOTAIR is regulated by epigenetic mechanisms. This work supports that physiological bone formation is epigenetically regulated by a lncRNA.

Significance of microRNAs in Androgen Signaling and Prostate Cancer Progression.

The androgen receptor (AR) plays important roles in prostate cancer development and prostate tumor growth. After binding to androgens, AR functions as a nuclear receptor and translocates to the nucleus to bind to specific AR-binding sites (ARBSs). AR regulates epigenetic factor recruitments to activate its downstream signaling. Although androgen deprivation therapy (ADT) is initially useful for prostate cancer patients, most patients eventually show resistance with hormone-refractory prostate cancers (HRPCs) or castration-resistant prostate cancers (CRPCs). Thus, new therapeutic strategies targeting HRPCs/CRPCs should be very important for clinical medicine as well as prostate cancer biology. Past studies have shown that mechanisms such as AR overexpression, hypersensitivity, variants and reprograming are responsible for developing HRPCs/CRPCs. These findings suggest that AR target genes will be major key factors. In this review article, we focus mainly on the androgen-regulated microRNAs (miRNAs) to summarize the contribution of miRNA-mediated pathways for prostate cancer progression.

Long non-coding RNAs and prostate cancer.

Long non-coding RNAs (lncRNAs) are RNA transcripts larger than 200 nucleotides that do not code for proteins the aberrant expression of which has been documented in various types of cancer, including prostate cancer. Lack of appropriate sensitive and specific biomarkers for prostate cancer has led to overdiagnosis and overtreatment, making lncRNAs promising novel biomarkers as well as therapeutic targets for the disease. The present review attempts to summarize the current knowledge of lncRNA expression patterns and mechanisms in prostate cancer, which contribute to carcinogenesis. In particular, we focused on lncRNAs regulated by androgen receptor and expressed in castration-resistant prostate cancer.

Androgen-induced lncRNA POTEF-AS1 regulates apoptosis-related pathway to facilitate cell survival in prostate cancer cells.

Although long non-coding RNAs (lncRNAs) have been associated with a variety of cancers, the interplay between lncRNAs and androgen receptor signaling in prostate cancer is still unclear. We identified an androgen-dependent lncRNA, POTEF-AS1, whose expression was regulated by androgen receptor in two androgen-dependent cells by using directional RNA sequencing analysis. POTEF-AS1 promoted cell growth, repressed genes related to the Toll-like receptor signaling and apoptosis pathways, and inhibited apoptosis in docetaxel-treated LNCaP cells. These findings suggest that POTEF-AS1 would play a key role in the progression of prostate cancer by repressing Toll-like receptor signaling.

Androgen-induced Long Noncoding RNA (lncRNA) SOCS2-AS1 Promotes Cell Growth and Inhibits Apoptosis in Prostate Cancer Cells.

Long noncoding RNAs (lncRNA) have been associated with the development of cancer. However, the interplay between lncRNAs and androgen receptor (AR) signaling in prostate cancer is still unclear. Here, we identified lncRNAs induced by androgen in AR-positive prostate cancer cells, where induction was abolished by AR knockdown as well as an anti-androgen, bicalutamide. By combining these data, we identified an androgen-regulated lncRNA, suppressor of cytokine signaling 2-antisense transcript 1 (SOCS2-AS1), the expression of which was higher in castration-resistant prostate cancer model cells, i.e long-term androgen-deprived (LTAD) cells, than in parental androgen-dependent LNCaP cells. SOCS2-AS1 promoted castration-resistant and androgen-dependent cell growth. We found that SOCS2-AS1 knockdown up-regulated genes related to the apoptosis pathway, including tumor necrosis factor superfamily 10 (TNFSF10), and sensitized prostate cancer cells to docetaxel treatment. Moreover, we also demonstrated that SOCS2-AS1 promotes androgen signaling by modulating the epigenetic control for AR target genes including TNFSF10 These findings suggest that SOCS2-AS1 plays an important role in the development of castration-resistant prostate cancer by repressing apoptosis.

TET2 repression by androgen hormone regulates global hydroxymethylation status and prostate cancer progression.

Modulation of epigenetic patterns has promising efficacy for treating cancer. 5-Hydroxymethylated cytosine (5-hmC) is an epigenetic mark potentially important in cancer. Here we report that 5-hmC is an epigenetic hallmark of prostate cancer (PCa) progression. A member of the ten-eleven translocation (TET) proteins, which catalyse the oxidation of methylated cytosine (5-mC) to 5-hmC, TET2, is repressed by androgens in PCa. Androgen receptor (AR)-mediated induction of the miR-29 family, which targets TET2, are markedly enhanced in hormone refractory PCa (HRPC) and its high expression predicts poor outcome of PCa patients. Furthermore, decreased expression of miR-29b results in reduced tumour growth and increased TET2 expression in an animal model of HRPC. Interestingly, global 5-hmC modification regulated by miR-29b represses FOXA1 activity. A reduction in 5-hmC activates PCa-related key pathways such as mTOR and AR. Thus, DNA modification directly links the TET2-dependent epigenetic pathway regulated by AR to 5-hmC-mediated tumour progression.

Estrogen-Related Receptors in Breast Cancer and Prostate Cancer.

Estrogen-signaling pathways are implicated in the development of breast cancer and prostate cancer. Various studies have focused on additional signaling pathways, mediated by estrogen-related receptors (ERRs). ERRs are constitutively active receptors that share a high degree of homology with the classical estrogen receptors (ERs). However, they do not bind to estrogen, while ERs do. ERRs are involved in the development of alternative pathways that lead to the development of cancer and are regarded as potential therapeutic targets for the treatment of breast cancer and prostate cancer that do not respond to conventional therapies. In this review, we first present general structural features of ERRs. Then, we focus on breast cancer and prostate cancer, which are primarily hormone-dependent cancers, and summarizes recent progress in elucidating the involvement of each ERR in these two types of malignancies.

Sox6 up-regulation by macrophage migration inhibitory factor promotes survival and maintenance of mouse neural stem/progenitor cells.

Macrophage migration inhibitory factor (MIF) has important roles in supporting the proliferation and/or survival of murine neural stem/progenitor cells (NSPCs), but downstream effectors remain unknown. We show here that MIF robustly increases the expression of Sox6 in NSPCs in vitro. During neural development, Sox6 is expressed in the ventricular zone of the ganglionic eminence (GE) of mouse brains at embryonic day 14.5 (E14.5), cultured NSPCs from E14.5 GE, and NSPCs in the subventricular zone (SVZ) around the lateral ventricle (LV) of the adult mouse forebrain. Retroviral overexpression of Sox6 in NSPCs increases the number of primary and secondary neurospheres and inhibits cell differentiation. This effect is accompanied with increased expression of Hes1 and Bcl-2 and Akt phosphorylation, thus suggesting a role for Sox6 in promoting cell survival and/or self-renewal ability. Constitutive activation of the transcription factor Stat3 results in up-regulation of Sox6 expression and chromatin immunoprecipitation analysis showed that MIF increases Stat3 binding to the Sox6 promoter in NSPCs, indicating that Stat3 stimulates Sox6 expression downstream of MIF. Finally, the ability of MIF to increase the number of primary and secondary neurospheres is inhibited by Sox6 gene silencing. Collectively, our data identify Sox6 as an important downstream effector of MIF signaling in stemness maintenance of NSPCs.

Macrophage migration inhibitory factor (MIF) promotes cell survival and proliferation of neural stem/progenitor cells.

In a previous study, we showed that murine dendritic cells (DCs) can increase the number of neural stem/progenitor cells (NSPCs) in vitro and in vivo. In the present study, we identified macrophage migration inhibitory factor (MIF) as a novel factor that can support the proliferation and/or survival of NSPCs in vitro. MIF is secreted by DCs and NSPCs, and its function in the normal brain remains largely unknown. It was previously shown that in macrophages, MIF binds to a CD74-CD44 complex. In the present study, we observed the expression of MIF receptors in mouse ganglionic-eminence-derived neurospheres using flow cytometry in vitro. We also found CD74 expression in the ganglionic eminence of E14 mouse brains, suggesting that MIF plays a physiological role in vivo. MIF increased the number of primary and secondary neurospheres. By contrast, retrovirally expressed MIF shRNA and MIF inhibitor (ISO-1) suppressed primary and secondary neurosphere formation, as well as cell proliferation. In the neurospheres, MIF knockdown by shRNA increased caspase 3/7 activity, and MIF increased the phosphorylation of Akt, Erk, AMPK and Stat3 (Ser727), as well as expression of Hes3 and Egfr, the products of which are known to support cell survival, proliferation and/or maintenance of NSPCs. MIF also acted as a chemoattractant for NSPCs. These results show that MIF can induce NSPC proliferation and maintenance by multiple signaling pathways acting synergistically, and it may be a potential therapeutic factor, capable of activating NSPC, for the treatment of degenerative brain disorders.

Functional analysis of HOXD9 in human gliomas and glioma cancer stem cells.

BACKGROUND: HOX genes encode a family of homeodomain-containing transcription factors involved in the determination of cell fate and identity during embryonic development. They also behave as oncogenes in some malignancies. RESULTS: In this study, we found high expression of the HOXD9 gene transcript in glioma cell lines and human glioma tissues by quantitative real-time PCR. Using immunohistochemistry, we observed HOXD9 protein expression in human brain tumor tissues, including astrocytomas and glioblastomas. To investigate the role of HOXD9 in gliomas, we silenced its expression in the glioma cell line U87 using HOXD9-specific siRNA, and observed decreased cell proliferation, cell cycle arrest, and induction of apoptosis. It was suggested that HOXD9 contributes to both cell proliferation and/or cell survival. The HOXD9 gene was highly expressed in a side population (SP) of SK-MG-1 cells that was previously identified as an enriched-cell fraction of glioma cancer stem-like cells. HOXD9 siRNA treatment of SK-MG-1 SP cells resulted in reduced cell proliferation. Finally, we cultured human glioma cancer stem cells (GCSCs) from patient specimens found with high expression of HOXD9 in GCSCs compared with normal astrocyte cells and neural stem/progenitor cells (NSPCs). CONCLUSIONS: Our results suggest that HOXD9 may be a novel marker of GCSCs and cell proliferation and/or survival factor in gliomas and glioma cancer stem-like cells, and a potential therapeutic target.

AP-1-Dependent miR-21 expression contributes to chemoresistance in cancer stem cell-like SP cells.

The side population (SP) of cancer cells is a minor population of cells that has been identified in a variety of cancers and harbors many cancer stem cell (CSC)-like properties, such as self-renewal potential, tumorforming capacity, and chemoresistant phenotype. CSCs are regarded as the root of cancer origin and recurrence. Thus, new therapeutic approaches targeting these malignant cells have become the topic of ongoing research. However, the chemoresistant phenotype of CSCs makes it difficult to increase their sensitivity to anticancer drugs and to decrease the rate of cancer recurrence in patients. In this study, we analyzed the chemoresistant phenotype of SP cells derived from various cancer cell lines. Microarray analysis discriminated differential gene expression profiles between SP and non-SP cells. MicroRNA-21 (miR-21) and its upstream regulator activator protein-I (AP-1), composed of c-Jun and c-Fos family transcription factors, were found to be frequently upregulated in SP cells. Downregulation of tumor suppressor programmed cell death 4, one of the miR-21 target gene products, confirmed miR-21 overexpression in SP cells. Treatment of the cells with the AP-1 inhibitor SP600125 attenuated miR-21 levels and increased topotecan sensitivity. Furthermore, specific inhibition of miR-21 by an anti-miR-21 locked nucleic acid increased drug sensitivity and decreased colony forming ability. These findings define the critical role of miR-21 in maintenance of the chemoresistant phenotype of SP cells. Targeting miR-21 may provide a new strategy for cancer therapy by impairing resistance to chemotherapy in CSCs.

Tunable chiral reaction media based on two-component liquid crystals: regio-, diastereo-, and enantiocontrolled photodimerization of anthracenecarboxylic acids.

Three kinds of enantiopure amphiphilic amino alcohols (1a-c) were newly synthesized, of which the stereochemistry of the stereogenic carbons adjacent to the amino (C2) and hydroxy (C1) groups was systematically varied. By using these amino alcohols and four photoreactive carboxylic acids, 12 kinds of salts were prepared. The structure and thermal behavior of the salts were thoroughly investigated by various techniques, which revealed that the stereochemistry of the amino alcohol unit has significant effects on the properties of the salts; the salts of 1a with (1R,2S)-configuration did not exhibit any liquid crystal (LC) phase but showed high crystallinity, whereas 1b and 1c with (1S,2S)- and (1S)-configurations, respectively, generally afforded stable LC salts with smectic structure(s). Within the matrix of these amphiphilic salts, the in situ photodimerizations of 2-anthracenecarboxylic acid (2c) and 1-anthracenecarboxylic acid (2d) were conducted by the irradiation with UV/vis light (500 W, a high-pressure mercury arc lamp, >380 nm). Concerning reactivity and regio-/diastereo-/enantioselectivities, the LC phases were found to be superior to isotropic and crystalline phases. For the two substrates 2c and 2d, every LC phase promoted the photodimerization with unprecedentedly high head-to-head selectivity. Particularly in the case of 2c, diastereoselecitivity (syn(HH) vs anti(HH)) could be rationally controlled by the choice of the amino alcohol unit and mesophase (syn(HH):anti(HH) = 61:37 to 26:72). Moreover, one of the LC phases exhibited by 1b·2c afforded the anti(HH)-dimer of 2c with excellent enantioselectivity (up to 86% ee). On the basis of the hypothesis that the present photochemical outcome arises from the preorientation of the substrates, a preliminary structural model of these LCs was proposed.

SUV39H1 interacts with HTLV-1 Tax and abrogates Tax transactivation of HTLV-1 LTR.

BACKGROUND: Tax is the oncoprotein of HTLV-1 which deregulates signal transduction pathways, transcription of genes and cell cycle regulation of host cells. Transacting function of Tax is mainly mediated by its protein-protein interactions with host cellular factors. As to Tax-mediated regulation of gene expression of HTLV-1 and cellular genes, Tax was shown to regulate histone acetylation through its physical interaction with histone acetylases and deacetylases. However, functional interaction of Tax with histone methyltransferases (HMTase) has not been studied. Here we examined the ability of Tax to interact with a histone methyltransferase SUV39H1 that methylates histone H3 lysine 9 (H3K9) and represses transcription of genes, and studied the functional effects of the interaction on HTLV-1 gene expression. RESULTS: Tax was shown to interact with SUV39H1 in vitro, and the interaction is largely dependent on the C-terminal half of SUV39H1 containing the SET domain. Tax does not affect the methyltransferase activity of SUV39H1 but tethers SUV39H1 to a Tax containing complex in the nuclei. In reporter gene assays, co-expression of SUV39H1 represses Tax transactivation of HTLV-1 LTR promoter activity, which was dependent on the methyltransferase activity of SUV39H1. Furthermore, SUV39H1 expression is induced along with Tax in JPX9 cells. Chromatin immunoprecipitation (ChIP) analysis shows localization of SUV39H1 on the LTR after Tax induction, but not in the absence of Tax induction, in JPX9 transformants retaining HTLV-1-Luc plasmid. Immunoblotting shows higher levels of SUV39H1 expression in HTLV-1 transformed and latently infected cell lines. CONCLUSION: Our study revealed for the first time the interaction between Tax and SUV39H1 and apparent tethering of SUV39H1 by Tax to the HTLV-1 LTR. It is speculated that Tax-mediated tethering of SUV39H1 to the LTR and induction of the repressive histone modification on the chromatin through H3 K9 methylation may be the basis for the dose-dependent repression of Tax transactivation of LTR by SUV39H1. Tax-induced SUV39H1 expression, Tax-SUV39H1 interaction and tethering to the LTR may provide a support for an idea that the above sequence of events may form a negative feedback loop that self-limits HTLV-1 viral gene expression in infected cells.