Distinct Transcriptional Profiles in the Different Phenotypes of Neurofibroma from the Same Subject with Neurofibromatosis 1.

Neurofibromatosis 1 is a prevalent hereditary neurocutaneous disorder. Among the clinical phenotypes of neurofibromatosis 1, cutaneous neurofibroma (cNF) and plexiform neurofibroma (pNF) have distinct clinical manifestations, and pNF should be closely monitored owing to its malignant potential. However, the detailed distinct features of neurofibromatosis 1 phenotypes remain unknown. To determine whether the transcriptional features and microenvironment of cNF and pNF differ, single-cell RNA sequencing was performed on isolated cNF and pNF cells from the same patient. Six cNF and five pNF specimens from different subjects were also immunohistochemically analyzed. Our findings revealed that cNF and pNF had distinct transcriptional profiles even within the same subject. pNF is enriched in Schwann cells with characteristics similar to those of their malignant counterpart, fibroblasts, with a cancer-associated fibroblast-like phenotype, angiogenic endothelial cells, and M2-like macrophages, whereas cNF is enriched in CD8 T cells with tissue residency markers. The results of immunohistochemical analyses performed on different subjects agreed with those of single-cell RNA sequencing. This study found that cNF and pNF, the different neurofibromatosis phenotypes in neurofibromatosis 1, from the same subject are transcriptionally distinct in terms of the cell types involved, including T cells.

GREB1 isoform 4 is specifically transcribed by MITF and required for melanoma proliferation.

Growth regulation by estrogen in breast cancer 1 (GREB1) is involved in hormone-dependent and -independent tumor development (e.g., hepatoblastoma). In this study, we found that a GREB1 splicing variant, isoform 4 (Is4), which encodes C-terminal half of full-length GREB1, is specifically expressed via microphthalmia-associated transcription factor (MITF) in melanocytic melanoma, and that two MITF-binding E-box CANNTG motifs at the 5'-upstream region of GREB1 exon 19 are necessary for GREB1 Is4 transcription. MITF and GREB1 Is4 were strongly co-expressed in approximately 20% of the melanoma specimens evaluated (17/89 cases) and their expression was associated with tumor thickness. GREB1 Is4 silencing reduced melanoma cell proliferation in association with altered expression of cell proliferation-related genes in vitro. In addition, GREB1 Is4 targeting by antisense oligonucleotide (ASO) decreased melanoma xenograft tumor formation and GREB1 Is4 expression in a BRAFV600E; PTENflox melanoma mouse model promoted melanoma formation, demonstrating the crucial role of GREB1 Is4 for melanoma proliferation in vivo. GREB1 Is4 bound to CAD, the rate-limiting enzyme of pyrimidine metabolism, and metabolic flux analysis revealed that GREBI Is4 is necessary for pyrimidine synthesis. These results suggest that MITF-dependent GREB1 Is4 expression leads to melanoma proliferation and GREB1 Is4 represents a new molecular target in melanoma.

Design and synthesis of 4-acetoxypentanamide derivatives of spliceostatin A and their biological evaluation towards prostate cancer treatment.

We designed and synthesized novel 4-acetoxypentanamide derivatives of spliceostatin A, whose 4-acetoxypentenamide moiety is reduced (7), isomerized (8), or substituted with methyl at the α-position (9). The results of biological evaluation against AR-V7 and the docking analysis of each derivative suggest that the geometry of the 4-acetoxypentenamide moiety of spliceostatin A is important for its biological activity.

DNA barcoding and gene expression recording reveal the presence of cancer cells with unique properties during tumor progression.

Tumors comprise diverse cancer cell populations with specific capabilities for adaptation to the tumor microenvironment, resistance to anticancer treatments, and metastatic dissemination. However, whether these populations are pre-existing in cancer cells or stochastically appear during tumor growth remains unclear. Here, we show the heterogeneous behaviors of cancer cells regarding response to anticancer drug treatments, formation of lung metastases, and expression of transcription factors related to cancer stem-like cells using a DNA barcoding and gene expression recording system. B16F10 cells maintained clonal diversity after treatment with HVJ-E, a UV-irradiated Sendai virus, and the anticancer drug dacarbazine. PBS treatment of the primary tumor and intravenous injection of B16F10 cells resulted in metastases formed from clones of multiple cell lineages. Conversely, BL6 and 4T1 cells developed spontaneous lung metastases by a small number of clones. Notably, an identical clone of 4T1 cells developed lung metastases in different mice, suggesting the existence of cells with high metastatic potential. Cas9-based transcription recording analysis in a human prostate cancer cell line revealed that specific cells express POU5F1 in response to an anticancer drug and sphere formation. Our findings provide insights into the diversity of cancer cells during tumor progression.

Identification of Malignant Cell Populations Associated with Poor Prognosis in High-Grade Serous Ovarian Cancer Using Single-Cell RNA Sequencing.

To reveal tumor heterogeneity in ovarian cancer, we performed single-cell RNA sequencing (RNA-seq). We obtained The Cancer Genome Atlas (TCGA) survival data and TCGA gene expression data for a Kaplan-Meier plot showing the association of each tumor population with poor prognosis. As a result, we found two malignant tumor cell subtypes associated with poor prognosis. Next, we performed trajectory analysis using scVelo and Monocle3 and cell-cell interaction analysis using CellphoneDB. We found that one malignant population included the earliest cancer cells and cancer stem-like cells. Furthermore, we identified SLC3A1 and PEG10 as the marker genes of cancer-initiating cells. The other malignant population expressing CA125 (MUC16) is associated with a decrease in the number of tumor-infiltrating cytotoxic T lymphocytes (CTLs). Moreover, cell-cell interaction analysis implied that interactions mediated by LGALS9 and GAS6, expressed by this malignant population, caused the CTL suppression. The results of this study suggest that two tumor cell populations, including a cancer-initiating cell population and a population expressing CA125, survive the initial treatment and suppress antitumor immunity, respectively, and are associated with poor prognosis. Our findings offer a new understanding of ovarian cancer heterogeneity and will aid in the development of diagnostic tools and treatments.

Dual function of SF3B2 on chromatin and RNA to regulate transcription in head and neck squamous cell carcinoma.

RNA is spliced concomitantly with transcription and the process is organized by RNA splicing factors, transcriptional regulators, and chromatin regulators. RNA is spliced in close proximity to transcription machinery. Hence, some RNA splicing factors may play a role in transcription. Here, we show that the splicing factor SF3B2 binds to gene regulatory elements and mRNA to modulate transcription and RNA stability in head and neck squamous cell carcinoma cells. High SF3B2 expression leads to poor prognosis in patients with head and neck squamous cell carcinoma and to progression of tumor growth in mice. SF3B2 promotes tumor growth, owing to its involvement in activation of gene expression associated with mitochondrial electron transport and transcription regulatory region DNA binding. SF3B2 is enriched around the promoter element on chromatin and the transcription termination site on RNA. SF3B2 is involved in the regulation of RNA stability. According to the SF3B2-binding profile, SF3B2 regulates RNA polymerase II activity, in addition to regulating RNA splicing. Mechanistically, SF3B2 promotes the binding of structural maintenance of chromosomes 1A and CCCTC-binding factor (CTCF) to the SF3B2-binding genomic regions. SF3B2 also modulates CTCF transcriptional activity. Our findings indicate that SF3B2 has a dual function in both transcription and RNA stability, leading to head and neck squamous cell carcinoma progression.

Regulation of RNA Splicing: Aberrant Splicing Regulation and Therapeutic Targets in Cancer.

RNA splicing is a critical step in the maturation of precursor mRNA (pre-mRNA) by removing introns and exons. The combination of inclusion and exclusion of introns and exons in pre-mRNA can generate vast diversity in mature mRNA from a limited number of genes. Cancer cells acquire cancer-specific mechanisms through aberrant splicing regulation to acquire resistance to treatment and to promote malignancy. Splicing regulation involves many factors, such as proteins, non-coding RNAs, and DNA sequences at many steps. Thus, the dysregulation of splicing is caused by many factors, including mutations in RNA splicing factors, aberrant expression levels of RNA splicing factors, small nuclear ribonucleoproteins biogenesis, mutations in snRNA, or genomic sequences that are involved in the regulation of splicing, such as 5' and 3' splice sites, branch point site, splicing enhancer/silencer, and changes in the chromatin status that affect the splicing profile. This review focuses on the dysregulation of RNA splicing related to cancer and the associated therapeutic methods.

A simple method using CRISPR-Cas9 to knock-out genes in murine cancerous cell lines.

CRISPR-Cas9 system can be used to generate knock-out cancer cell lines. An insertion or deletion induced by a single guide RNA (gRNA) is often used to generate knock-out cells, however, some cells express the target gene by skipping the disrupted exon, or by using a splicing variant, thus losing the target exon. To overcome this unexpected expression of the target gene, almost the entire gene can be swapped with a selection marker. However, it is time-consuming to create a targeting vector which contains 5' and 3' homology arms flanked by a selection marker. Here, we developed a simple and easy method called SUCCESS (Single-strand oligodeoxynucleotides, Universal Cassette, and CRISPR/Cas9 produce Easy Simple knock-out System), to knock-out a target gene without constructing a targeting vector. Our method removed the targeted large genomic region by using two pX330 plasmids encoding Cas9 and gRNA, two 80mer single strand oligodeoxynucleotides (ssODN), and a blunt-ended universal selection maker sequence in B16F10 murine cancer cell and ID8 murine ovarian cancer cell. SUCCESS generated knock-out clones in two murine cancer cell lines by homozygous deletion of the target genomic region, and without constructing targeting vectors. Thus, our method can be widely applied to generate homozygous knock-out cell lines, as well as knock-in cell lines.

Evaluation of HPV16 E7 expression in head and neck carcinoma cell lines and clinical specimens.

Human papillomavirus (HPV) 16 infection in the oropharynx is one of the major risk factors for oropharyngeal carcinoma. Although the HPV E6 and E7 proteins are known to have a role in head and neck carcinogenesis, whether their expression is maintained once the tumour has developed still remains unclear. We evaluated the expression of these proteins in HPV16-positive cancer cell lines and clinical oropharyngeal specimens. Two out of the four commercially available antibodies directed against the E7 protein could detect the E7 protein overexpressed in the 293FT cells, human embryonic kidney cells, although none of the four commercially available anti-E6 antibodies could detect the overexpressed E6 protein. Whereas HPV16-positive head and neck or cervical carcinoma cell lines expressed the E7 mRNA, the antibodies with an ability to detect the E7 protein could not detect it in western blotting in these HPV16-positive cell lines. In clinical specimens, E7 protein was partially detected in p16-positive area in p16-positive and HPV16 DNA-positive samples, but not in p16-negative and HPV DNA-negative or p16-positive and HPV DNA-negative samples. Consistent with these findings, the E7 protein was poorly translated from the endogenous structure of the E7 mRNA, although significant E7 mRNA expression was detected in these samples. Our findings indicate that E7 protein is partially expressed in p16-positive area in p16-positive and HPV16 DNA-positive clinical specimens.

Design and Synthesis of 1,2-Deoxy-pyranose Derivatives of Spliceostatin A toward Prostate Cancer Treatment.

We designed and synthesized a novel 1,2-deoxy-pyranose and terminal epoxide methyl substituted derivatives of spliceostatin A using Julia-Kocienski olefination as a key step. With respect to the biological activity, the 1,2-deoxy-pyranose analogue of spliceostatin A suppressed AR-V7 expression at the nano level (IC50 = 3.3 nM). In addition, the in vivo toxicity test showed that the 1,2-deoxy-pyranose analogue was able to avoid severe toxicity compared to spliceostatin A.

NANOG helps cancer cells escape NK cell attack by downregulating ICAM1 during tumorigenesis.

BACKGROUND: At the beginning of tumorigenesis, newly born cancer cells must successfully avoid attack by the immune system. Although most abnormal cells are efficiently identified and destroyed by the immune system, particularly by NK cells, the molecular mechanisms by which newly born cancer cells evade NK cell surveillance are not fully understood. METHODS: NK cell resistance of highly tumorigenic population of human prostate cancer (PCa) cells were confirmed by xenograft in SCID mice with or without NK cell neutralization. The mechanisms by which the tumorigenic PCa cells evaded NK cell attack were investigated by RNAseq, ChIPseq, generation of several transformants and xenograft in SCID mice. RESULTS: Here, we show that PCa cells have a strengthened ability to escape NK cell attack due to NANOG, a pluripotent-related transcription factor, mediating the repression of ICAM1, a cell adhesion molecule, during tumorigenesis. Mechanistically, NANOG directly binds to the region upstream of ICAM1. As the binding between NANOG and the upstream ICAM1 region increases, p300 binding to this region is diminished, resulting in decreased ICAM1 expression. High NANOG expression confers PCa cells the ability to resist NK cell attack via the repression of ICAM1. Consistent with these results, low ICAM1 expression is significantly correlated with a high recurrence rate in patients with PCa. CONCLUSIONS: Our findings indicate that repression of ICAM1 is a critical mechanism by which cancer cells evade attack from NK cells during tumorigenesis. These results suggest a pivotal role of NANOG in establishing a gene expression profile for escaping the immune system.

SF3B2-Mediated RNA Splicing Drives Human Prostate Cancer Progression.

Androgen receptor splice variant-7 (AR-V7) is a constitutively active AR variant implicated in castration-resistant prostate cancers. Here, we show that the RNA splicing factor SF3B2, identified by in silico and CRISPR/Cas9 analyses, is a critical determinant of AR-V7 expression and is correlated with aggressive cancer phenotypes. Transcriptome and PAR-CLIP analyses revealed that SF3B2 controls the splicing of target genes, including AR, to drive aggressive phenotypes. SF3B2-mediated aggressive phenotypes in vivo were reversed by AR-V7 knockout. Pladienolide B, an inhibitor of a splicing modulator of the SF3b complex, suppressed the growth of tumors addicted to high SF3B2 expression. These findings support the idea that alteration of the splicing pattern by high SF3B2 expression is one mechanism underlying prostate cancer progression and therapeutic resistance. This study also provides evidence supporting SF3B2 as a candidate therapeutic target for treating patients with cancer. SIGNIFICANCE: RNA splicing factor SF3B2 is essential for the generation of an androgen receptor (AR) variant that renders prostate cancer cells resistant to AR-targeting therapy.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5204/F1.large.jpg.

Myc Regulates Chromatin Decompaction and Nuclear Architecture during B Cell Activation.

50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.

Wolf-Hirschhorn Syndrome Candidate 1 Is Necessary for Correct Hematopoietic and B Cell Development.

Immunodeficiency is one of the most important causes of mortality associated with Wolf-Hirschhorn syndrome (WHS), a severe rare disease originated by a deletion in chromosome 4p. The WHS candidate 1 (WHSC1) gene has been proposed as one of the main genes responsible for many of the alterations in WHS, but its mechanism of action is still unknown. Here, we present in vivo genetic evidence showing that Whsc1 plays an important role at several points of hematopoietic development. Particularly, our results demonstrate that both differentiation and function of Whsc1-deficient B cells are impaired at several key developmental stages due to profound molecular defects affecting B cell lineage specification, commitment, fitness, and proliferation, demonstrating a causal role for WHSC1 in the immunodeficiency of WHS patients.

Regulation of alternative polyadenylation by Nkx2-5 and Xrn2 during mouse heart development.

Transcription factors organize gene expression profiles by regulating promoter activity. However, the role of transcription factors after transcription initiation is poorly understood. Here, we show that the homeoprotein Nkx2-5 and the 5'-3' exonuclease Xrn2 are involved in the regulation of alternative polyadenylation (APA) during mouse heart development. Nkx2-5 occupied not only the transcription start sites (TSSs) but also the downstream regions of genes, serving to connect these regions in primary embryonic cardiomyocytes (eCMs). Nkx2-5 deficiency affected Xrn2 binding to target loci and resulted in increases in RNA polymerase II (RNAPII) occupancy and in the expression of mRNAs with long 3'untranslated regions (3' UTRs) from genes related to heart development. siRNA-mediated suppression of Nkx2-5 and Xrn2 led to heart looping anomaly. Moreover, Nkx2-5 genetically interacts with Xrn2 because Nkx2-5(+/-)Xrn2(+/-), but neither Nkx2-5(+/-)nor Xrn2(+/-), newborns exhibited a defect in ventricular septum formation, suggesting that the association between Nkx2-5 and Xrn2 is essential for heart development. Our results indicate that Nkx2-5 regulates not only the initiation but also the usage of poly(A) sites during heart development. Our findings suggest that tissue-specific transcription factors is involved in the regulation of APA.

Elucidating the mechanisms of transcription regulation during heart development by next-generation sequencing.

Dysregulation of transcription is associated with the pathogenesis of cardiovascular diseases, including congenital heart diseases and heart failure. However, it remains unclear how transcription factors regulate transcription in the heart and which genes are associated with cardiovascular diseases in humans. Development of genome-wide analyses using next-generation sequencers provides powerful methods to determine how these transcription factors and chromatin regulators control gene expressions and to identify causative genes in cardiovascular diseases. These technologies have revealed that transcription during heart development is elaborately regulated by multiple cardiac transcription factors. In this review, we discuss the recent progress toward understanding the molecular mechanisms of how transcriptional dysregulation leads to cardiovascular diseases.

Differences in histone modifications between slow- and fast-twitch muscle of adult rats and following overload, denervation, or valproic acid administration.

Numerous studies have reported alterations in skeletal muscle properties and phenotypes in response to various stimuli such as exercise, unloading, and gene mutation. However, a shift in muscle fiber phenotype from fast twitch to slow twitch is not completely induced by stimuli. This limitation is hypothesized to result from the epigenetic differences between muscle types. The main purpose of the present study was to identify the differences in histone modification for the plantaris (fast) and soleus (slow) muscles of adult rats. Genome-wide analysis by chromatin immunoprecipitation followed by DNA sequencing revealed that trimethylation at lysine 4 and acetylation of histone 3, which occurs at transcriptionally active gene loci, was less prevalent in the genes specific to the slow-twitch soleus muscle. Conversely, gene loci specific to the fast-twitch plantaris muscle were associated with the aforementioned histone modifications. We also found that upregulation of slow genes in the plantaris muscle, which are related to enhanced muscular activity, is not associated with activating histone modifications. Furthermore, silencing of muscle activity by denervation caused the displacement of acetylated histone and RNA polymerase II (Pol II) in 5' ends of genes in plantaris, but minor effects were observed in soleus. Increased recruitment of Pol II induced by forced acetylation of histone was also suppressed in valproic acid-treated soleus. Our present data indicate that the slow-twitch soleus muscle has a unique set of histone modifications, which may relate to the preservation of the genetic backbone against physiological stimuli.

CRISPR/Cas9-mediated gene knockout of NANOG and NANOGP8 decreases the malignant potential of prostate cancer cells.

NANOG expression in prostate cancer is highly correlated with cancer stem cell characteristics and resistance to androgen deprivation. However, it is not clear whether NANOG or its pseudogenes contribute to the malignant potential of cancer. We established NANOG- and NANOGP8-knockout DU145 prostate cancer cell lines using the CRISPR/Cas9 system. Knockouts of NANOG and NANOGP8 significantly attenuated malignant potential, including sphere formation, anchorage-independent growth, migration capability, and drug resistance, compared to parental DU145 cells. NANOG and NANOGP8 knockout did not inhibit in vitro cell proliferation, but in vivo tumorigenic potential decreased significantly. These phenotypes were recovered in NANOG- and NANOGP8-rescued cell lines. These results indicate that NANOG and NANOGP8 proteins are expressed in prostate cancer cell lines, and NANOG and NANOGP8 equally contribute to the high malignant potential of prostate cancer.