Katsunori Semi

Premature Termination of Reprogramming In Vivo Leads to Cancer Development through Altered Epigenetic Regulation

Cancer is believed to arise primarily through accumulation of genetic mutations. Although induced pluripotent stem cell (iPSC) generation does not require changes in genomic sequence, iPSCs acquire unlimited growth potential, a characteristic shared with cancer cells. Here, we describe a murine system in which reprogramming factor expression in vivo can be controlled temporally with doxycycline (Dox). Notably, transient expression of reprogramming factors in vivo results in tumor development in various tissues consisting of undifferentiated dysplastic cells exhibiting global changes in DNA methylation patterns. The Dox-withdrawn tumors arising in the kidney share a number of characteristics with Wilms tumor, a common pediatric kidney cancer. We also demonstrate that iPSCs derived from Dox-withdrawn kidney tumor cells give rise to nonneoplastic kidney cells in mice, proving that they have not undergone irreversible genetic transformation. These findings suggest that epigenetic regulation associated with iPSC derivation may drive development of particular types of cancer.

Derivation of ground-state female ES cells maintaining gamete-derived DNA methylation

Inhibitors of Mek1/2 and Gsk3β, known as 2i, enhance the derivation of embryonic stem (ES) cells and promote ground-state pluripotency in rodents. Here we show that the derivation of female mouse ES cells in the presence of 2i and leukaemia inhibitory factor (2i/L ES cells) results in a widespread loss of DNA methylation, including a massive erasure of genomic imprints. Despite this global loss of DNA methylation, early-passage 2i/L ES cells efficiently differentiate into somatic cells, and this process requires genome-wide de novo DNA methylation. However, the majority of imprinting control regions (ICRs) remain unmethylated in 2i/L-ES-cell-derived differentiated cells. Consistently, 2i/L ES cells exhibit impaired autonomous embryonic and placental development by tetraploid embryo complementation or nuclear transplantation. We identified the derivation conditions of female ES cells that display 2i/L-ES-cell-like transcriptional signatures while preserving gamete-derived DNA methylation and autonomous developmental potential. Upon prolonged culture, however, female ES cells exhibited ICR demethylation regardless of culture conditions. Our results provide insights into the derivation of female ES cells reminiscent of the inner cell mass of preimplantation embryos.

Cellular reprogramming and cancer development

Cancer develops through the accumulation of genetic and epigenetic abnormalities. The role of genetic alterations in cancer development has been demonstrated by reverse genetic approaches. However, evidence indicating the functional significance of epigenetic abnormalities remains limited due to the lack of means to actively modify coordinated epigenetic regulations in the genome. Application of the reprogramming technology may help researchers to overcome this limitation and shed new light on cancer research. Reprogramming is accompanied by dynamic changes of epigenetic modifications and is therefore considered to be a useful tool to induce global epigenetic changes in cancer genomes. We herein discuss the similarities between reprogramming processes and carcinogenesis and propose the potential use of reprogramming technology to help understanding of the significance of epigenetic regulations in cancer cells. We, also discuss the application of induced pluripotent stem cell technology to cancer modeling based on the similar characteristics between pluripotent stem cells and cancer cells.

Epigenetic regulation leading to induced pluripotency drives cancer development in vivo.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by the transient expression of reprogramming factors. During the reprogramming process, somatic cells acquire the ability to undergo unlimited proliferation, which is also an important characteristic of cancer cells, while their underlying DNA sequence remains unchanged. Based on the characteristics shared between pluripotent stem cells and cancer cells, the potential involvement of the factors leading to reprogramming toward pluripotency in cancer development has been discussed. Recent in vivo reprogramming studies provided some clues to understanding the role of reprogramming-related epigenetic regulation in cancer development. It was shown that premature termination of the in vivo reprogramming result in the development of tumors that resemble pediatric cancers. Given that epigenetic modifications play a central role during reprogramming, failed reprogramming-associated cancer development may have provided a proof of concept for epigenetics-driven cancer development in vivo.

EWS/ATF1 expression induces sarcomas from neural crest–derived cells in mice

Clear cell sarcoma (CCS) is an aggressive soft tissue malignant tumor characterized by a unique t(12;22) translocation that leads to the expression of a chimeric EWS/ATF1 fusion gene. However, little is known about the mechanisms underlying the involvement of EWS/ATF1 in CCS development. In addition, the cellular origins of CCS have not been determined. Here, we generated EWS/ATF1-inducible mice and examined the effects of EWS/ATF1 expression in adult somatic cells. We found that forced expression of EWS/ATF1 resulted in the development of EWS/ATF1-dependent sarcomas in mice. The histology of EWS/ATF1-induced sarcomas resembled that of CCS, and EWS/ATF1-induced tumor cells expressed CCS markers, including S100, SOX10, and MITF. Lineage-tracing experiments indicated that neural crest-derived cells were subject to EWS/ATF1-driven transformation. EWS/ATF1 directly induced Fos in an ERK-independent manner. Treatment of human and EWS/ATF1-induced CCS tumor cells with FOS-targeted siRNA attenuated proliferation. These findings demonstrated that FOS mediates the growth of EWS/ATF1-associated sarcomas and suggest that FOS is a potential therapeutic target in human CCS.

Reducing DNA methylation suppresses colon carcinogenesis by inducing tumor cell differentiation

The forced reduction of global DNA methylation suppresses tumor development in several cancer models in vivo. Nevertheless, the mechanisms underlying these suppressive effects remain unclear. In this report, we describe our findings showing that a genome-wide reduction in the DNA methylation levels induces cellular differentiation in association with decreased cell proliferation in Apc (Min/+) mouse colon tumor cells in vivo. Colon tumor-specific DNA methylation at Cdx1 is reduced in the DNA-hypomethylated tumors accompanied by Cdx1 derepression and an increased expression of intestinal differentiation-related genes. Furthermore, a histological analysis revealed that Cdx1 derepression in the DNA-hypomethylated tumors is correlated with the differentiation of colon tumor cells. Similarly, the treatment of human colon cancer cell lines with a hypomethylating agent induces differentiation-related genes, including CDX1. We herein propose that DNA demethylation exerts a tumor suppressive effect in the colon by inducing tumor cell differentiation.

Induced pluripotent stem cell technology for dissecting the cancer epigenome

Cancer arises through the accumulation of both genetic and epigenetic alterations. Although the causal role of genetic mutations on cancer development has been established in vivo, similar evidence for epigenetic alterations is limited. Moreover, mutual interactions between genetic mutations and epigenetic alterations remain unclear. Cellular reprogramming technology can be used to actively modify the epigenome without affecting the underlying genomic sequences. Here we introduce recent studies that have utilized this property for cancer research. We propose that just as it has potential for regenerative medicine and disease modeling, cell reprogramming could also be a powerful tool for dissecting the role of the cancer epigenome in the development and maintenance of cancer cells.

Cellular context-dependent consequences of Apc mutations on gene regulation and cellular behavior.

Significance Genotype-linked disease phenotypes are often observed in a cell type-specific manner, implying a cellular context-dependent effect of the genetic aberrations. However, the extent to which cellular context affects the biological consequences of oncogenic mutations is unclear. Here, we reprogrammed colon tumor cells in an ApcMin/+ (adenomatous polyposis coli) mouse model and showed the divergent in vivo consequences of Apc mutation that arise in different cellular contexts. We also showed that the reprogrammed tumor cells remain in a pretumoral microadenoma stage after differentiation into colonic epithelium, suggesting that macroscopic colon tumor cells are reprogrammable into microadenoma cells. Our results underscore the significance of epigenetic regulation on gene expression, cellular plasticity, and cellular behavior in response to cancer-causing mutations. The spectrum of genetic mutations differs among cancers in different organs, implying a cellular context-dependent effect for genetic aberrations. However, the extent to which the cellular context affects the consequences of oncogenic mutations remains to be fully elucidated. We reprogrammed colon tumor cells in an ApcMin/+ (adenomatous polyposis coli) mouse model, in which the loss of the Apc gene plays a critical role in tumor development and subsequently, established reprogrammed tumor cells (RTCs) that exhibit pluripotent stem cell (PSC)-like signatures of gene expression. We show that the majority of the genes in RTCs that were affected by Apc mutations did not overlap with the genes affected in the intestine. RTCs lacked pluripotency but exhibited an increased expression of Cdx2 and a differentiation propensity that was biased toward the trophectoderm cell lineage. Genetic rescue of the mutated Apc allele conferred pluripotency on RTCs and enabled their differentiation into various cell types in vivo. The redisruption of Apc in RTC-derived differentiated cells resulted in neoplastic growth that was exclusive to the intestine, but the majority of the intestinal lesions remained as pretumoral microadenomas. These results highlight the significant influence of cellular context on gene regulation, cellular plasticity, and cellular behavior in response to the loss of the Apc function. Our results also imply that the transition from microadenomas to macroscopic tumors is reprogrammable, which underscores the importance of epigenetic regulation on tumor promotion.

An EWS-FLI1-Induced Osteosarcoma Model Unveiled a Crucial Role of Impaired Osteogenic Differentiation on Osteosarcoma Development

Summary EWS-FLI1, a multi-functional fusion oncogene, is exclusively detected in Ewing sarcomas. However, previous studies reported that rare varieties of osteosarcomas also harbor EWS-ETS family fusion. Here, using the doxycycline-inducible EWS-FLI1 system, we established an EWS-FLI1-dependent osteosarcoma model from murine bone marrow stromal cells. We revealed that the withdrawal of EWS-FLI1 expression enhances the osteogenic differentiation of sarcoma cells, leading to mature bone formation. Taking advantage of induced pluripotent stem cell (iPSC) technology, we also show that sarcoma-derived iPSCs with cancer-related genetic abnormalities exhibited an impaired differentiation program of osteogenic lineage irrespective of the EWS-FLI1 expression. Finally, we demonstrate that EWS-FLI1 contributed to secondary sarcoma development from the sarcoma iPSCs after osteogenic differentiation. These findings demonstrate that modulating cellular differentiation is a fundamental principle of EWS-FLI1-induced osteosarcoma development. This in vitro cancer model using sarcoma iPSCs should provide a unique platform for dissecting relationships between the cancer genome and cellular differentiation.

Pluripotent stem cell models of Blau syndrome reveal an IFN-γ–dependent inflammatory response in macrophages

&NA; Figure. No caption available. Background: Blau syndrome, or early‐onset sarcoidosis, is a juvenile‐onset systemic granulomatosis associated with a mutation in nucleotide‐binding oligomerization domain 2 (NOD2). The underlying mechanisms of Blau syndrome leading to autoinflammation are still unclear, and there is currently no effective specific treatment for Blau syndrome. Objectives: To elucidate the mechanisms of autoinflammation in patients with Blau syndrome, we sought to clarify the relation between disease‐associated mutant NOD2 and the inflammatory response in human samples. Methods: Blau syndrome–specific induced pluripotent stem cell (iPSC) lines were established. The disease‐associated NOD2 mutation of iPSCs was corrected by using a CRISPR‐Cas9 system to precisely evaluate the in vitro phenotype of iPSC‐derived cells. We also introduced the same NOD2 mutation into a control iPSC line. These isogenic iPSCs were then differentiated into monocytic cell lineages, and the statuses of nuclear factor &kgr;B pathway and proinflammatory cytokine secretion were investigated. Results: IFN‐&ggr; acted as a priming signal through upregulation of NOD2. In iPSC‐derived macrophages with mutant NOD2, IFN‐&ggr; treatment induced ligand‐independent nuclear factor &kgr;B activation and proinflammatory cytokine production. RNA sequencing analysis revealed distinct transcriptional profiles of mutant macrophages both before and after IFN‐&ggr; treatment. Patient‐derived macrophages demonstrated a similar IFN‐&ggr;–dependent inflammatory response. Conclusions: Our data support the significance of ligand‐independent autoinflammation in the pathophysiology of Blau syndrome. Our comprehensive isogenic disease‐specific iPSC panel provides a useful platform for probing therapeutic and diagnostic clues for the treatment of patients with Blau syndrome.