Keisuke Katsushima

Chromatin Regulator PRC2 Is a Key Regulator of Epigenetic Plasticity in Glioblastoma

Tumor cell plasticity contributes to functional and morphologic heterogeneity. To uncover the underlying mechanisms of this plasticity, we examined glioma stem-like cells (GSC) where we found that the biologic interconversion between GSCs and differentiated non-GSCs is functionally plastic and accompanied by gain or loss of polycomb repressive complex 2 (PRC2), a complex that modifies chromatin structure. PRC2 mediates lysine 27 trimethylation on histone H3 and in GSC it affected pluripotency or development-associated genes (e.g., Nanog, Wnt1, and BMP5) together with alterations in the subcellular localization of EZH2, a catalytic component of PRC2. Intriguingly, exogenous expression of EZH2-dNLS, which lacks nuclear localization sequence, impaired the repression of Nanog expression under differentiation conditions. RNA interference (RNAi)-mediated attenuation or pharmacologic inhibition of EZH2 had little to no effect on apoptosis or bromodeoxyuridine incorporation in GSCs, but it disrupted morphologic interconversion and impaired GSC integration into the brain tissue, thereby improving survival of GSC-bearing mice. Pathologic analysis of human glioma specimens revealed that the number of tumor cells with nuclear EZH2 is larger around tumor vessels and the invasive front, suggesting that nuclear EZH2 may help reprogram tumor cells in close proximity to this microenvironment. Our results indicate that epigenetic regulation by PRC2 is a key mediator of tumor cell plasticity, which is required for the adaptation of glioblastoma cells to their microenvironment. Thus, PRC2-targeted therapy may reduce tumor cell plasticity and tumor heterogeneity, offering a new paradigm for glioma treatment.

Contribution of MicroRNA-1275 to Claudin11 Protein Suppression via a Polycomb-mediated Silencing Mechanism in Human Glioma Stem-like Cells

Background: Molecular mechanisms underlying heterogeneity of glioblastoma are poorly understood. Results: Newly identified microRNA-1275, which is controlled by a polycomb-mediated silencing mechanism, regulates expression of oligodendroglial lineage protein, Claudin11, in glioma stem-like cells. Conclusion: MicroRNA-1275 may contribute to the establishment of tissue heterogeneity via epigenetic mechanisms. Significance: We identified a microRNA that is associated with tumor cell differentiation in the oligodendroglial lineage. Glioblastomas show heterogeneous histological features, and tumor cells show distinct phenotypic states that confer different functional attributes and an aggressive character. However, the molecular mechanisms underlying the heterogeneity in this disease are poorly understood. Glioma stem-like cells (GSCs) are considered able to aberrantly differentiate into diverse cell types and may contribute to the establishment of tumor heterogeneity. Using a GSC model, we investigated differentially expressed microRNAs (miRNAs) and associated epigenetic mechanisms that regulate the differentiation of GSCs. miRNA profiling using microarray technology showed that 13 and 34 miRNAs were commonly up-regulated and down-regulated in two independent GSC lines during differentiation, respectively. Among this set of miRNAs, quantitative PCR analysis showed that miRNA-1275 (miR-1275) was consistently down-regulated during GSC differentiation, along with the up-regulation of its target, CLDN11, an important protein during oligodendroglial lineage differentiation. Inhibition of miR-1275 with a specific antisense oligonucleotide (anti-miR-1275) in GSCs increased the expression of CLDN11, together with significant growth suppression. Epigenetic analysis revealed that gain of histone H3 lysine 27 trimethylation (H3K27me3) in the primary microRNA-1275 promoter was closely associated with miR-1275 expression. Treatment with 3-deazaneplanocin A, an inhibitor of H3K27 methyltransferase, attenuated CLDN11 induction by serum stimulation in parallel with sustained miR-1275 expression. Our results have illuminated the epigenetic regulatory pathways of miR-1275 that are closely associated with oligodendroglial differentiation, which may contribute to the tissue heterogeneity seen in the formation of glioblastomas. Given that inhibition of miR-1275 induces expression of oligodendroglial lineage proteins and suppresses tumor cell proliferation, this may be a potential therapeutic target for glioblastomas.

Targeting the Notch-regulated non-coding RNA TUG1 for glioma treatment

Targeting self-renewal is an important goal in cancer therapy and recent studies have focused on Notch signalling in the maintenance of stemness of glioma stem cells (GSCs). Understanding cancer-specific Notch regulation would improve specificity of targeting this pathway. In this study, we find that Notch1 activation in GSCs specifically induces expression of the lncRNA, TUG1. TUG1 coordinately promotes self-renewal by sponging miR-145 in the cytoplasm and recruiting polycomb to repress differentiation genes by locus-specific methylation of histone H3K27 via YY1-binding activity in the nucleus. Furthermore, intravenous treatment with antisense oligonucleotides targeting TUG1 coupled with a drug delivery system induces GSC differentiation and efficiently represses GSC growth in vivo. Our results highlight the importance of the Notch-lncRNA axis in regulating self-renewal of glioma cells and provide a strong rationale for targeting TUG1 as a specific and potent therapeutic approach to eliminate the GSC population.

Epigenetic dysregulation in glioma

Given that treatment options for patients with glioblastoma are limited, much effort has been made to clarify the underlying mechanisms of gliomagenesis. Recent genome‐wide genomic and epigenomic analyses have revealed that mutations in epigenetic modifiers occur frequently in gliomas and that dysregulation of epigenetic mechanisms is closely associated with glioma formation. Given that epigenetic changes are reversible, understanding the epigenetic abnormalities that arise in gliomagenesis might be key to developing more effective treatment strategies for glioma. In this review, we focus on the recent advancements in epigenetic research with respect to gliomas, consider how epigenetic mechanisms dynamically regulate tumor cells, including the cancer stem cell population, and discuss perspectives and challenges for glioma treatment in the near future.

Non-coding RNAs as epigenetic regulator of glioma stem-like cell differentiation

Glioblastomas show heterogeneous histological features. These distinct phenotypic states are thought to be associated with the presence of glioma stem cells (GSCs), which are highly tumorigenic and self-renewing sub-population of tumor cells that have different functional characteristics. Differentiation of GSCs may be regulated by multi-tiered epigenetic mechanisms that orchestrate the expression of thousands of genes. One such regulatory mechanism involves functional non-coding RNAs (ncRNAs), such as microRNAs (miRNAs); a large number of ncRNAs have been identified and shown to regulate the expression of genes associated with cell differentiation programs. Given the roles of miRNAs in cell differentiation, it is possible they are involved in the regulation of gene expression networks in GSCs that are important for the maintenance of the pluripotent state and for directing differentiation. Here, we review recent findings on ncRNAs associated with GSC differentiation and discuss how these ncRNAs contribute to the establishment of tissue heterogeneity during glioblastoma tumor formation.

Aberrant TET1 Methylation Closely Associated with CpG Island Methylator Phenotype in Colorectal Cancer

Inactivation of methylcytosine dioxygenase, ten-eleven translocation (TET) is known to be associated with aberrant DNA methylation in cancers. Tumors with a CpG island methylator phenotype (CIMP), a distinct subgroup with extensive DNA methylation, show characteristic features in the case of colorectal cancer. The relationship between TET inactivation and CIMP in colorectal cancers is not well understood. The expression level of TET family genes was compared between CIMP-positive (CIMP-P) and CIMP-negative (CIMP-N) colorectal cancers. Furthermore, DNA methylation profiling, including assessment of the TET1 gene, was assessed in colorectal cancers, as well as colon polyps. The TET1 was silenced by DNA methylation in a subset of colorectal cancers as well as cell lines, expression of which was reactivated by demethylating agent. TET1 methylation was more frequent in CIMP-P (23/55, 42%) than CIMP-N (2/113, 2%, P < 0.0001) colorectal cancers. This trend was also observed in colon polyps (CIMP-P, 16/40, 40%; CIMP-N, 2/24, 8%; P = 0.002), suggesting that TET1 methylation is an early event in CIMP tumorigenesis. TET1 methylation was significantly associated with BRAF mutation but not with hMLH1 methylation in the CIMP-P colorectal cancers. Colorectal cancers with TET1 methylation have a significantly greater number of DNA methylated genes and less pathological metastasis compared to those without TET1 methylation (P = 0.007 and 0.045, respectively). Our data suggest that TET1 methylation may contribute to the establishment of a unique pathway in respect to CIMP-mediated tumorigenesis, which may be incidental to hMLH1 methylation. In addition, our findings provide evidence that TET1 methylation may be a good biomarker for the prediction of metastasis in colorectal cancer. Cancer Prev Res; 8(8); 702–11. ©2015 AACR.

Histone Deacetylase Inhibition in Prostate Cancer Triggers miR-320–Mediated Suppression of the Androgen Receptor

Targeting androgen receptor (AR) by pharmacologic intervention is one of the effective approaches for treatment of malignant prostate cancers. Histone deacetylase (HDAC) alters the epigenetic status of tumor-associated genes, including those for miRNAs (miRNA), and affects the behavior of cancers. Here, we examined the molecular effects of a HDAC inhibitor, OBP-801, on AR expression and tumor cell growth in prostate cancers. Treatment with OBP-801 efficiently suppressed cell growth of three prostate cancer lines (22Rv1, VCaP, and LNCaP), together with AR downregulation, regardless of their hormone sensitivity. Intriguingly, this effect by OBP-801 was not due to decreased transcriptional activity of the AR gene, but due to posttranscriptional regulation, namely by miRNA-mediated suppression. Among the upregulated miRNAs after OBP-801 treatment in the three prostate cancer cell lines, miR-320a, whose expression was significantly correlated with prognosis of prostate cancers (P = 0.0185), was the most closely associated with AR expression. An miR-320a mimic suppressed AR protein expression together with growth suppression, while anti-miR-320a oligonucleotide significantly abrogated the growth suppression by OBP-801 treatment. FISH analysis revealed that miR-320a was highly expressed in human normal prostate luminal cells, but was rarely expressed in prostate cancer cells. In an AR-dependent prostate tumorigenic rat model, OBP-801 treatment profoundly increased miR-320a expression and repressed prostate tumorigenesis. Our data demonstrated that OBP-801 effectively suppressed AR activity via epigenetic upregulation of miR-320a, which resulted in tumor cell growth suppression of prostate cancers. OBP-801 may be a potent AR-targeting therapeutic reagent in AR-positive prostate cancer regardless of androgen dependency. Cancer Res; 76(14); 4192-204. ©2016 AACR.

Oncogenic effects of evolutionarily conserved noncoding RNA ECONEXIN on gliomagenesis

Accumulating studies have demonstrated the importance of long noncoding RNAs (lncRNAs) during oncogenic transformation. However, because most lncRNAs are currently uncharacterized, the identification of novel oncogenic lncRNAs is difficult. Given that intergenic lncRNA have substantially less sequence conservation patterns than protein-coding genes across species, evolutionary conserved intergenic lncRNAs are likely to be functional. The current study identified a novel intergenic lncRNA, LINC00461 (ECONEXIN) using a combined approach consisting of searching lncRNAs by evolutionary conservation and validating their expression in a glioma mouse model. ECONEXIN was the most highly conserved intergenic lncRNA containing 83.0% homology with the mouse ortholog (C130071C03Rik) for a region over 2500 bp in length within its exon 3. Expressions of ECONEXIN and C130071C03Rik were significantly upregulated in both human and mouse glioma tissues. Moreover, the expression of C130071C03Rik was upregulated even in precancerous conditions and markedly increased during glioma progression. Functional analysis of ECONEXIN in glioma cell lines, U87 and U251, showed it was dominantly located in the cytoplasm and interacted with miR-411-5p via two binding sites within ECONEXIN. Inhibition of ECONEXIN upregulated miR-411-5p together with the downregulation of its target, Topoisomerase 2 alpha (TOP2A), in glioma cell lines, resulting in decreased cell proliferation. Our data demonstrated that ECONEXIN is a potential oncogene that regulates TOP2A by sponging miR-411-5p in glioma. In addition, our investigative approaches to identify conserved lncRNA and their molecular characterization by validation in mouse tumor models may be useful to functionally annotate novel lncRNAs, especially cancer-associated lncRNAs.

Abstract 3135: Epigenetic regulation of miR-1275 through histone H3 lysine 27 trimethylation during human glioma stem-like cell differentiation

Functional and morphological heterogeneity characterizes aggressive glioblastoma and contributes to invasion, metastasis and drug resistance. The molecular mechanisms underlying this heterogeneity are poorly understood. Recent studies have revealed that tumors contain a minor population of tumor-initiating cells, called cancer stem cells (CSCs). The CSCs are considered able to aberrantly differentiate into diverse cell types. Such stem-like cancer cells have been well characterized in glioblastoma multiforme, and are referred to as glioma stem-like cells (GSCs). Using GSCs as a model, we investigated the roles of micro-RNA (miR) in GSCs differentiation. miR-microarray showed that 19 and 48 miRs were commonly upregulated and downregulated in two GSC lines during the differentiation, respectively. Among those miRs, quantitative-PCR analysis showed that miR-1275 was consistently downregulated during the GSC differentiation, while the expression of its target, oligodendrocyte specific protein (OSP/CLDN11), a marker of oligodendrocyte, was upregulated. Compellingly, inhibition of miR-1275 using specific antisense oligonucleotide increased the expression of OSP in GSC. Epigenetic analysis revealed that level of histone H3 lysine 27 trimethylation(H3K27me3) was enriched in the promoter of pri-miR-1275 during the GSC differentiation. Treatment of 3-Dezaneplanocin-A, an inhibitor of H3K27me3, impaired the GSC differentiation in parallel with sustained miR-1275 expression. In general, astrocytic component is the most prevalent in glioblastomas, while a subset of cases also contain an oligodendroglial component in the tumors. Our results illuminated the epigenetic regulatory pathways of miR-1275 closely associated with oligodendroglial differentiation, which may contribute to the tissue heterogeneity formation of glioblastomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3135. doi:1538-7445.AM2012-3135

Abstract 1110: Inhibition of histone deacetylase induces miR-320-mediated androgen receptor suppression in prostate cancer

Targeting androgen receptor (AR) by pharmacological intervention is one of the effective approaches for treatment of malignant prostate cancers. Histone deacetylase (HDAC) alters epigenetic status of tumor-associated genes, including microRNAs (miRNAs), and affects the behavior of cancers. Here we examined molecular effects of a HDAC inhibitor, OBP-801, on AR expression and tumor cell growth in prostate cancers. Treatment of OBP-801 efficiently suppressed cell growth of three prostate cancer lines (22Rv1, VCaP, and LNCaP), together with AR downregulation, regardless of their hormone sensitivity. Intriguingly, this effect by OBP-801 is not due to downregulation of transcriptional activity of the AR gene, but due to post-transcriptional regulation, namely miRNA-mediated suppression. Among the upregulated miRNAs after OBP-801 treatment in three prostate cancer cell lines, miR-320a, of which expression was significantly correlated with prognosis of prostate cancers (P = 0.0185), was the most closely associated with AR expression. miR-320a mimic suppressed AR protein expression together with growth suppression, while anti-miR320a significantly abrogated the growth suppression by OBP-801 treatment. Fluorescent in situ hybridization analysis revealed that miR-320a was highly expressed in human normal prostate luminal cells but rarely expressed in prostate cancer cells. In AR-dependent prostate tumorigenic rat model, OBP-801 treatment profoundly increased the miR-320 expression and repressed prostate tumorigenesis. Our data demonstrated that OBP-801 effectively suppressed AR activity via epigenetic upregulation of miR-320a, which resulted in tumor cell growth suppression of prostate cancers. OBP-801 may be a potent drug as an AR targeting therapy in AR positive prostate cancer regardless of androgen dependency. Citation Format: Shinya Sato, Keisuke Katsushima, Keiko Shinjo, Satoru Takahashi, Yutaka Kondo. Inhibition of histone deacetylase induces miR-320-mediated androgen receptor suppression in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1110.