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1.
EMBO J ; 42(22): e114032, 2023 Nov 15.
Article in English | MEDLINE | ID: mdl-37781951

ABSTRACT

Bone marrow-derived cells (BMDCs) infiltrate hypoxic tumors at a pre-angiogenic state and differentiate into mature macrophages, thereby inducing pro-tumorigenic immunity. A critical factor regulating this differentiation is activation of SREBP2-a well-known transcription factor participating in tumorigenesis progression-through unknown cellular mechanisms. Here, we show that hypoxia-induced Golgi disassembly and Golgi-ER fusion in monocytic myeloid cells result in nuclear translocation and activation of SREBP2 in a SCAP-independent manner. Notably, hypoxia-induced SREBP2 activation was only observed in an immature lineage of bone marrow-derived cells. Single-cell RNA-seq analysis revealed that SREBP2-mediated cholesterol biosynthesis was upregulated in HSCs and monocytes but not in macrophages in the hypoxic bone marrow niche. Moreover, inhibition of cholesterol biosynthesis impaired tumor growth through suppression of pro-tumorigenic immunity and angiogenesis. Thus, our findings indicate that Golgi-ER fusion regulates SREBP2-mediated metabolic alteration in lineage-specific BMDCs under hypoxia for tumor progression.


Subject(s)
Monocytes , Neoplasms , Humans , Monocytes/metabolism , Bone Marrow , Cholesterol/metabolism , Sterol Regulatory Element Binding Protein 2/genetics , Sterol Regulatory Element Binding Protein 2/metabolism , Hypoxia
2.
Breast Cancer Res ; 25(1): 21, 2023 02 21.
Article in English | MEDLINE | ID: mdl-36810117

ABSTRACT

BACKGROUND: The intratumor heterogeneity (ITH) of cancer cells plays an important role in breast cancer resistance and recurrence. To develop better therapeutic strategies, it is necessary to understand the molecular mechanisms underlying ITH and their functional significance. Patient-derived organoids (PDOs) have recently been utilized in cancer research. They can also be used to study ITH as cancer cell diversity is thought to be maintained within the organoid line. However, no reports investigated intratumor transcriptomic heterogeneity in organoids derived from patients with breast cancer. This study aimed to investigate transcriptomic ITH in breast cancer PDOs. METHODS: We established PDO lines from ten patients with breast cancer and performed single-cell transcriptomic analysis. First, we clustered cancer cells for each PDO using the Seurat package. Then, we defined and compared the cluster-specific gene signature (ClustGS) corresponding to each cell cluster in each PDO. RESULTS: Cancer cells were clustered into 3-6 cell populations with distinct cellular states in each PDO line. We identified 38 clusters with ClustGS in 10 PDO lines and used Jaccard similarity index to compare the similarity of these signatures. We found that 29 signatures could be categorized into 7 shared meta-ClustGSs, such as those related to the cell cycle or epithelial-mesenchymal transition, and 9 signatures were unique to single PDO lines. These unique cell populations appeared to represent the characteristics of the original tumors derived from patients. CONCLUSIONS: We confirmed the existence of transcriptomic ITH in breast cancer PDOs. Some cellular states were commonly observed in multiple PDOs, whereas others were specific to single PDO lines. The combination of these shared and unique cellular states formed the ITH of each PDO.


Subject(s)
Breast Neoplasms , Humans , Female , Breast Neoplasms/metabolism , Transcriptome , Breast , Gene Expression Profiling , Organoids/metabolism
3.
Biochem Biophys Res Commun ; 674: 183-189, 2023 09 24.
Article in English | MEDLINE | ID: mdl-37450958

ABSTRACT

Mitochondrial one-carbon metabolism is crucial for embryonic development and tumorigenesis, as it supplies one-carbon units necessary for nucleotide synthesis and rapid cell proliferation. However, its contribution to adult tissue homeostasis remains largely unknown. To examine its role in adult tissue homeostasis, we specifically investigated mammary gland development during pregnancy, as it involves heightened cell proliferation. We discovered that MTHFD2, a mitochondrial one-carbon metabolic enzyme, is expressed in both luminal and basal/myoepithelial cell layers, with upregulated expression during pregnancy. Using the mouse mammary tumor virus (MMTV)-Cre recombinase system, we generated mice with a specific mutation of Mthfd2 in mammary epithelial cells. While the mutant mice were capable of properly nurturing their offspring, the pregnancy-induced expansion of mammary glands was significantly delayed. This indicates that MTHFD2 contributes to the rapid development of mammary glands during pregnancy. Our findings shed light on the role of mitochondrial one-carbon metabolism in facilitating rapid cell proliferation, even in the context of the adult tissue homeostasis.


Subject(s)
Epithelial Cells , Mammary Glands, Animal , Methylenetetrahydrofolate Dehydrogenase (NADP) , Animals , Female , Mice , Pregnancy , Cell Proliferation , Epithelial Cells/metabolism , Hydrolases/metabolism , Mammary Glands, Animal/metabolism , Mice, Transgenic , Methylenetetrahydrofolate Dehydrogenase (NADP)/metabolism
4.
Cancer Sci ; 113(8): 2693-2703, 2022 Aug.
Article in English | MEDLINE | ID: mdl-35585758

ABSTRACT

Colorectal cancer (CRC) is a heterogenous disease, and patients have differences in therapeutic response. However, the mechanisms underlying interpatient heterogeneity in the response to chemotherapeutic agents remain to be elucidated, and molecular tumor characteristics are required to select patients for specific therapies. Patient-derived organoids (PDOs) established from CRCs recapitulate various biological characteristics of tumor tissues, including cellular heterogeneity and the response to chemotherapy. Patient-derived organoids established from CRCs show various morphologies, but there are no criteria for defining these morphologies, which hampers the analysis of their biological significance. Here, we developed an artificial intelligence (AI)-based classifier to categorize PDOs based on microscopic images according to their similarity in appearance and classified tubular adenocarcinoma-derived PDOs into six types. Transcriptome analysis identified differential expression of genes related to cell adhesion in some of the morphological types. Genes involved in ribosome biogenesis were also differentially expressed and were most highly expressed in morphological types showing CRC stem cell properties. We identified an RNA polymerase I inhibitor, CX-5641, to be an upstream regulator of these type-specific gene sets. Notably, PDO types with increased expression of genes involved in ribosome biogenesis were resistant to CX-5461 treatment. Taken together, these results uncover the biological significance of the morphology of PDOs and provide novel indicators by which to categorize CRCs. Therefore, the AI-based classifier is a useful tool to support PDO-based cancer research.


Subject(s)
Adenocarcinoma , Antineoplastic Agents , Colorectal Neoplasms , Adenocarcinoma/drug therapy , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Antineoplastic Agents/pharmacology , Artificial Intelligence , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Humans , Organoids/metabolism
5.
J Pathol ; 255(2): 177-189, 2021 10.
Article in English | MEDLINE | ID: mdl-34184756

ABSTRACT

Genetically engineered mice have been the gold standard in modeling tumor development. Recent studies have demonstrated that genetically engineered organoids can develop subcutaneous tumors in immunocompromised mice, at least for organs that prefer predominant driver mutations for tumorigenesis. To further substantiate this concept, the fallopian tube (FT), a major cell of origin of ovarian high-grade serous carcinoma (HGSC), which almost invariably carries TP53 mutations, was investigated for p53 inactivation-driven tumorigenesis. Murine FT organoids subjected to lentiviral Cre-mediated Trp53 deletion did not develop tumors. However, subsequent suppression of Pten and simultaneous induction of mutant Pik3ca led to the development of carcinoma in situ and HGSC-like tumors, respectively, whereas concurrent deletion of Apc resulted in the development of benign cysts, mirroring frequent activation of the PI3K/AKT axis and the marginal impact of Wnt pathway activation in HGSC. Consistent with the frequent activation of the RAS pathway in HGSC, mutant Kras cooperated with Trp53 deletion for the development of tumors, which unexpectedly contained sarcoma cells in addition to carcinoma cells, despite the epithelial origin of the inoculated organoids. This finding is in sharp contrast with the exclusive adenocarcinoma development from gastrointestinal organoids with the same genotype reported in previous studies, suggesting a tissue-specific epithelial-mesenchymal transition program. In tumor-derived organoids, the Cre-mediated recombination rate reached 100% for Trp53 but not for the other genes, highlighting the advantage of p53 inactivation in FT tumorigenesis. The Trp53 wildtype FT organoids expressing the mutant Kras developed sarcoma and carcinoma upon Cdkn2a suppression and Tgfbr2 deletion, respectively, revealing novel pro-tumorigenic genetic cooperation and critical roles of TGF-ß signaling for epithelial-mesenchymal transition in FT-derived tumorigenesis. Collectively, the organoid-based approach represents a shortcut to tumorigenesis and provides novel insights into the relationships among genotype, cell type, and tumor phenotype underlying tumorigenesis. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Subject(s)
Carcinogenesis/pathology , Fallopian Tubes/pathology , Neoplasms, Experimental/pathology , Organoids/pathology , Precancerous Conditions/pathology , Animals , Carcinogenesis/genetics , Carcinoma, Ovarian Epithelial , Cystadenocarcinoma, Serous/genetics , Cystadenocarcinoma, Serous/pathology , Female , Mice , Neoplasms, Experimental/genetics , Precancerous Conditions/genetics , Tumor Suppressor Protein p53/genetics
6.
Cancer Sci ; 110(4): 1293-1305, 2019 Apr.
Article in English | MEDLINE | ID: mdl-30724425

ABSTRACT

Colorectal cancer (CRC) is caused by genetic alterations, and comprehensive sequence analyses have revealed the mutation landscapes. In addition to somatic changes, genetic variations are considered important factors contributing to tumor development; however, our knowledge on this subject is limited. Familial adenomatous polyposis coli (FAP) is an autosomal-dominant inherited disease caused by germline mutations in the adenomatous polyposis coli (APC) gene. FAP patients are classified into two major groups based on clinical manifestations: classical FAP (CFAP) and attenuated FAP (AFAP). In this study, we established 42 organoids from three CFAP patients and two AFAP patients. Comprehensive gene expression analysis demonstrated a close association between IFN/STAT signaling and the phenotypic features of FAP patients. Genetic disruption of Stat1 in the mouse model of FAP reduced tumor formation, demonstrating that the IFN/STAT pathway is causally associated with the tumor-forming potential of APC-deficient tumors. Mechanistically, STAT1 is downstream target of KRAS and is phosphorylated by its activating mutations. We found that enhanced IFN/STAT signaling in CFAP conferred resistance to MEK inhibitors. These findings reveal the crosstalk between RAS signaling and IFN/STAT signaling, which contributes to the tumor-forming potential and drug response. These results offer a rationale for targeting of IFN/STAT signaling and for the stratification of CRC patients.


Subject(s)
Cell Transformation, Neoplastic/metabolism , Colorectal Neoplasms/etiology , Colorectal Neoplasms/metabolism , Interferons/metabolism , STAT Transcription Factors/metabolism , Signal Transduction , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cell Transformation, Neoplastic/drug effects , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Disease Models, Animal , Drug Resistance, Neoplasm , Gene Expression Profiling , Humans , Immunohistochemistry , Mice , Models, Biological , Organoids , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , Tissue Culture Techniques , Xenograft Model Antitumor Assays
7.
Cancer Sci ; 107(2): 189-202, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26919617

ABSTRACT

Non-clinical studies are necessary at each stage of the development of oncology drugs. Many experimental cancer models have been developed to investigate carcinogenesis, cancer progression, metastasis, and other aspects in cancer biology and these models turned out to be useful in the efficacy evaluation and the safety prediction of oncology drugs. While the diversity and the degree of engagement in genetic changes in the initiation of cancer cell growth and progression are widely accepted, it has become increasingly clear that the roles of host cells, tissue microenvironment, and the immune system also play important roles in cancer. Therefore, the methods used to develop oncology drugs should continuously be revised based on the advances in our understanding of cancer. In this review, we extensively summarize the effective use of those models, their advantages and disadvantages, ranges to be evaluated and limitations of the models currently used for the development and for the evaluation of oncology drugs.


Subject(s)
Disease Models, Animal , Drug Discovery/methods , Medical Oncology/methods , Neoplasms , Animals , Antineoplastic Agents , Humans
8.
Cancer Sci ; 106(4): 475-9, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25898792

ABSTRACT

To integrate and discuss the cutting-edge science and revolutionized therapeutics of cancer in Japan and the United States, JCA (Japanese Cancer Association)-AACR (American Association for Cancer Research) Joint Symposia were held on 25th (Symposium 2) and 26th (Symposium 1) in September 2014 as a part of the 73rd Annual Meeting of the Japanese Cancer Association at Pacifico Yokohama in Yokohama, Japan. The symposia focused on mouse genetics and human genomics in cancer research. Eight prominent scientists from JCA and AACR discussed their own research in the symposia. They provided substantial fruitful information not only for identification of novel target molecules and pathways in cancer therapeutics but also for direct translation of cancer genomics into clinics.


Subject(s)
Neoplasms/genetics , Neoplasms/pathology , PTEN Phosphohydrolase/genetics , Tumor Suppressor Proteins/genetics , Animals , Genomics , High-Throughput Nucleotide Sequencing , Humans , Mice
9.
Cancer Sci ; 105(10): 1360-8, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25088905

ABSTRACT

Mutant mouse models are indispensable tools for clarifying gene functions and elucidating the pathogenic mechanisms of human diseases. Here, we describe novel cancer models bearing point mutations in the retinoblastoma gene (Rb1) generated by N-ethyl-N-nitrosourea mutagenesis. Two mutations in splice sites reduced Rb1 expression and led to a tumor spectrum and incidence similar to those observed in the conventional Rb1 knockout mice. The missense mutant, Rb1(D326V/+) , developed pituitary tumors, but thyroid tumors were completely suppressed. Immunohistochemical analyses of thyroid tissue revealed that E2F1, but not E2F2/3, was selectively inactivated, indicating that the mutant Rb protein (pRb) suppressed thyroid tumors by inactivating E2F1. Interestingly, Rb1(D326V/+) mice developed pituitary tumors that originated from the intermediate lobe of the pituitary, despite selective inactivation of E2F1. Furthermore, in the anterior lobe of the pituitary, other E2F were also inactivated. These observations show that pRb mediates the inactivation of E2F function and its contribution to tumorigenesis is highly dependent on the cell type. Last, by using a reconstitution assay of synthesized proteins, we showed that the D326V missense pRb bound to E2F1 but failed to interact with E2F2/3. These results reveal the effect of the pRb N-terminal domain on E2F function and the impact of the protein on tumorigenesis. Thus, this mutant mouse model can be used to investigate human Rb family-bearing mutations at the N-terminal region.


Subject(s)
E2F1 Transcription Factor/physiology , E2F2 Transcription Factor/physiology , E2F3 Transcription Factor/physiology , Mutation , Retinoblastoma Protein/genetics , Thyroid Neoplasms/genetics , Animals , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Thyroid Neoplasms/etiology
10.
Brain Res ; 1841: 149094, 2024 Oct 15.
Article in English | MEDLINE | ID: mdl-38914219

ABSTRACT

Neuroinflammation has been gaining attention as one of the potential causes of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis in recent years. The suppression of excessive proinflammatory responses is expected to be a target for the treatment and prevention of neurodegenerative diseases. Collapsin response mediator protein 4 (CRMP4) is involved in cytoskeleton-associated axonal guidance in the developing brain. Recently, the involvement of CRMP4 in several pathological conditions, including inflammation induced by lipopolysaccharide (LPS), a widely used inflammatory molecule, has been reported. However, the role of CRMP4 in LPS-induced inflammation in vivo remains largely unknown. In this study, we generated microglia-specific CRMP4 knockout mice for the first time and examined the role of CRMP4 in an LPS-induced brain inflammation model. We found that microglia after LPS injection in substantia nigra was significantly reduced in Crmp4-/- mice compared to Crmp4+/+mice. The increased expression of IL-10 in striatum samples was downregulated in Crmp4-/- mice. A significant reduction in Iba1 expression was also observed in microglia-specific Crmp4 knockout mice compared with that in control mice. In contrast, the expression of IL-10 did not change in these mice, whereas arginase 1 (Arg1) expression was significantly suppressed. These results demonstrate the involvement of CRMP4 in LPS-induced inflammation in vivo, that CRMP4 suppresses microglial proliferation in a cell-autonomous manner.


Subject(s)
Lipopolysaccharides , Mice, Knockout , Microglia , Nerve Tissue Proteins , Neuroinflammatory Diseases , Animals , Lipopolysaccharides/pharmacology , Microglia/metabolism , Microglia/drug effects , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Mice , Neuroinflammatory Diseases/metabolism , Neuroinflammatory Diseases/chemically induced , Inflammation/metabolism , Inflammation/chemically induced , Interleukin-10/metabolism , Substantia Nigra/metabolism , Substantia Nigra/drug effects , Mice, Inbred C57BL , Corpus Striatum/metabolism , Corpus Striatum/drug effects , Male , Microfilament Proteins/metabolism , Arginase/metabolism
11.
JCI Insight ; 9(11)2024 Jun 10.
Article in English | MEDLINE | ID: mdl-38855864

ABSTRACT

The transcription factor SRY-related HMG box 9 (Sox9) is essential for chondrogenesis. Mutations in and around SOX9 cause campomelic dysplasia (CD) characterized by skeletal malformations. Although the function of Sox9 in this context is well studied, the mechanisms that regulate Sox9 expression in chondrocytes remain to be elucidated. Here, we have used genome-wide profiling to identify 2 Sox9 enhancers located in a proximal breakpoint cluster responsible for CD. Enhancer activity of E308 (located 308 kb 5' upstream) and E160 (located 160 kb 5' upstream) correlated with Sox9 expression levels, and both enhancers showed a synergistic effect in vitro. While single deletions in mice had no apparent effect, simultaneous deletion of both E308 and E160 caused a dwarf phenotype, concomitant with a reduction of Sox9 expression in chondrocytes. Moreover, bone morphogenetic protein 2-dependent chondrocyte differentiation of limb bud mesenchymal cells was severely attenuated in E308/E160 deletion mice. Finally, we found that an open chromatin region upstream of the Sox9 gene was reorganized in the E308/E160 deletion mice to partially compensate for the loss of E308 and E160. In conclusion, our findings reveal a mechanism of Sox9 gene regulation in chondrocytes that might aid in our understanding of the pathophysiology of skeletal disorders.


Subject(s)
Campomelic Dysplasia , Cell Differentiation , Chondrocytes , Chondrogenesis , SOX9 Transcription Factor , SOX9 Transcription Factor/metabolism , SOX9 Transcription Factor/genetics , Animals , Chondrocytes/metabolism , Mice , Campomelic Dysplasia/genetics , Campomelic Dysplasia/pathology , Campomelic Dysplasia/metabolism , Chondrogenesis/genetics , Cell Differentiation/genetics , Enhancer Elements, Genetic/genetics , Chromatin/metabolism , Chromatin/genetics , Gene Expression Regulation, Developmental , Mice, Knockout , Bone Morphogenetic Protein 2/metabolism , Bone Morphogenetic Protein 2/genetics , Humans , Bone Development/genetics
12.
Commun Biol ; 7(1): 27, 2024 01 05.
Article in English | MEDLINE | ID: mdl-38182890

ABSTRACT

Tumor tissues consist of heterogeneous cells that originate from stem cells; however, their cell fate determination program remains incompletely understood. Using patient-derived organoids established from patients with advanced colorectal cancer (CRC), we evaluated the potential of olfactomedin 4 (OLFM4)+ stem cells to produce a bifurcated lineage of progenies with absorptive and secretory properties. In the early phases of organoid reconstruction, OLFM4+ cells preferentially gave rise to secretory cells. Additionally, we found that Paneth-like cells, which do not exist in the normal colon, were induced in response to Notch signaling inhibition. Video recordings of single OLFM4+ cells revealed that organoids containing Paneth-like cells were effectively propagated and that their selective ablation led to organoid collapse. In tumor tissues, Paneth-like cells were identified only in the region where tumor cells lost cell adhesion. These findings indicate that Paneth-like cells are directly produced by OLFM4+ stem cells and that their interaction contributes to tumor formation by providing niche factors. This study reveals the importance of the cell fate specification program for building a complete tumor cellular ecosystem, which might be targeted with novel therapeutics.


Subject(s)
Colorectal Neoplasms , Ecosystem , Humans , Stem Cells , Cell Proliferation , Organoids , Granulocyte Colony-Stimulating Factor
13.
Cancer Sci ; 104(7): 937-44, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23551873

ABSTRACT

Mutant mouse models are indispensable tools for clarifying the functions of genes and elucidating the underlying pathogenic mechanisms of human diseases. We carried out large-scale mutagenesis using the chemical mutagen N-ethyl-N-nitrosourea. One specific aim of our mutagenesis project was to generate novel cancer models. We screened 7012 animals for dominant traits using a necropsy test and thereby established 17 mutant lines predisposed to cancer. Here, we report on a novel cancer model line that developed osteoma, trichogenic tumor, and breast cancer. Using fine mapping and genomic sequencing, we identified a point mutation in the adenomatous polyposis coli (Apc) gene. The Apc1576 mutants bear a nonsense mutation at codon 1576 in the Apc gene. Although most Apc mutant mice established thus far have multifocal intestinal tumors, mice that are heterozygous for the Apc1576 mutation do not develop intestinal tumors; instead, they develop multifocal breast cancers and trichogenic tumors. Notably, the osteomas that develop in the Apc1576 mutant mice recapitulate the lesion observed in Gardner syndrome, a clinical variant of familial adenomatous polyposis. Our Apc1576 mutant mice will be valuable not only for understanding the function of the Apc gene in detail but also as models of human Gardner syndrome.


Subject(s)
Disease Models, Animal , Ethylnitrosourea , Gardner Syndrome/chemically induced , Gardner Syndrome/genetics , Mutagens , Animals , Codon , Female , Genes, APC , Genome , Heterozygote , Intestinal Neoplasms/chemically induced , Intestinal Neoplasms/genetics , Male , Mammary Neoplasms, Experimental/chemically induced , Mammary Neoplasms, Experimental/genetics , Mice , Mutagenesis , Mutation , Osteoma/chemically induced , Osteoma/genetics , Phenotype
14.
Nat Genet ; 36(5): 528-33, 2004 May.
Article in English | MEDLINE | ID: mdl-15107851

ABSTRACT

Spermatogenesis is a complex process that involves cooperation of germ cells and testicular somatic cells. Various genetic disorders lead to impaired spermatogenesis, defective sperm function and male infertility. Here we show that Cnot7(-/-) males are sterile owing to oligo-astheno-teratozoospermia, suggesting that Cnot7, a CCR4-associated transcriptional cofactor, is essential for spermatogenesis. Maturation of spermatids is unsynchronized and impaired in seminiferous tubules of Cnot7(-/-) mice. Transplantation of spermatogonial stem cells from male Cnot7(-/-) mice to seminiferous tubules of Kit mutant mice (Kit(W/W-v)) restores spermatogenesis, suggesting that the function of testicular somatic cells is damaged in the Cnot7(-/-) condition. The testicular phenotypes of Cnot7(-/-) mice are similar to those of mice deficient in retinoid X receptor beta (Rxrb). We further show that Cnot7 binds the AF-1 domain of Rxrb and that Rxrb malfunctions in the absence of Cnot7. Therefore, Cnot7 seems to function as a coregulator of Rxrb in testicular somatic cells and is thus involved in spermatogenesis.


Subject(s)
Chromosomal Proteins, Non-Histone/physiology , DNA-Binding Proteins/physiology , Gene Expression Regulation , Oligospermia/complications , Sperm Motility , Spermatozoa/abnormalities , Animals , COS Cells , Chromatin Assembly Factor-1 , Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins/genetics , Female , Fibroblasts , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Oligospermia/etiology , Oligospermia/genetics , Phenotype , Proto-Oncogene Proteins c-kit/genetics , Proto-Oncogene Proteins c-kit/physiology , Seminiferous Tubules/metabolism , Spermatogenesis/genetics , Stem Cell Transplantation
15.
Nat Commun ; 14(1): 4521, 2023 08 22.
Article in English | MEDLINE | ID: mdl-37607907

ABSTRACT

Oncogene-induced DNA replication stress (RS) and consequent pathogenic R-loop formation are known to impede S phase progression. Nonetheless, cancer cells continuously proliferate under such high-stressed conditions through incompletely understood mechanisms. Here, we report taurine upregulated gene 1 (TUG1) long noncoding RNA (lncRNA), which is highly expressed in many types of cancers, as an important regulator of intrinsic R-loop in cancer cells. Under RS conditions, TUG1 is rapidly upregulated via activation of the ATR-CHK1 signaling pathway, interacts with RPA and DHX9, and engages in resolving R-loops at certain loci, particularly at the CA repeat microsatellite loci. Depletion of TUG1 leads to overabundant R-loops and enhanced RS, leading to substantial inhibition of tumor growth. Our data reveal a role of TUG1 as molecule important for resolving R-loop accumulation in cancer cells and suggest targeting TUG1 as a potent therapeutic approach for cancer treatment.


Subject(s)
Neoplasms , R-Loop Structures , Humans , DNA Replication/genetics , Cell Proliferation/genetics , Neoplasms/genetics , Microsatellite Repeats/genetics , Taurine
16.
eNeuro ; 9(3)2022.
Article in English | MEDLINE | ID: mdl-35523582

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disorder that affects upper and lower motor neurons; however, its pathomechanism has not been fully elucidated. Using a comprehensive phosphoproteomic approach, we have identified elevated phosphorylation of Collapsin response mediator protein 1 (Crmp1) at serine 522 in the lumbar spinal cord of ALS model mice overexpressing a human superoxide dismutase mutant (SOD1G93A). We investigated the effects of Crmp1 phosphorylation and depletion in SOD1G93A mice using Crmp1S522A (Ser522→Ala) knock-in (Crmp1ki/ki ) mice in which the S522 phosphorylation site was abolished and Crmp1 knock-out (Crmp1-/-) mice, respectively. Crmp1ki/ki /SOD1G93A mice showed longer latency to fall in a rotarod test while Crmp1-/-/SOD1G93A mice showed shorter latency compared with SOD1G93A mice. Survival was prolonged in Crmp1ki/ki /SOD1G93A mice but not in Crmp1-/-/SOD1G93A mice. In agreement with these phenotypic findings, residual motor neurons and innervated neuromuscular junctions (NMJs) were comparatively well-preserved in Crmp1ki/ki /SOD1G93A mice without affecting microglial and astroglial pathology. Pathway analysis of proteome alterations showed that the sirtuin signaling pathway had opposite effects in Crmp1ki/ki /SOD1G93A and Crmp1-/-/SOD1G93A mice. Our study indicates that modifying CRMP1 phosphorylation is a potential therapeutic strategy for ALS.


Subject(s)
Amyotrophic Lateral Sclerosis , Amyotrophic Lateral Sclerosis/metabolism , Animals , Disease Models, Animal , Mice , Mice, Transgenic , Phosphorylation , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Superoxide Dismutase-1/metabolism
17.
J Cell Biol ; 172(6): 835-46, 2006 Mar 13.
Article in English | MEDLINE | ID: mdl-16533944

ABSTRACT

Separase is an evolutionarily conserved protease that is essential for chromosome segregation and cleaves cohesin Scc1/Rad21, which joins the sister chromatids together. Although mammalian separase also functions in chromosome segregation, our understanding of this process in mammals is still incomplete. We generated separase knockout mice, reporting an essential function for mammalian separase. Separase-deficient mouse embryonic fibroblasts exhibited severely restrained increases in cell number, polyploid chromosomes, and amplified centrosomes. Chromosome spreads demonstrated that multiple chromosomes connected to a centromeric region. Live observation demonstrated that the chromosomes of separase-deficient cells condensed, but failed to segregate, although subsequent cytokinesis and chromosome decondensation proceeded normally. These results establish that mammalian separase is essential for the separation of centromeres, but not of the arm regions of chromosomes. Other cell cycle events, such as mitotic exit, DNA replication, and centrosome duplication appear to occur normally. We also demonstrated that heterozygous separase-deficient cells exhibited severely restrained increases in cell number with apparently normal mitosis in the absence of securin, which is an inhibitory partner of separase.


Subject(s)
Cell Cycle Proteins/genetics , Centromere/physiology , Chromosome Segregation/physiology , Endopeptidases/genetics , Fibroblasts/metabolism , Interphase/physiology , Mitosis/physiology , Animals , Carrier Proteins/genetics , Cell Proliferation , Cells, Cultured , Centrosome/physiology , Cytokinesis/physiology , Female , Fibroblasts/cytology , Male , Mice , Mice, Knockout , Polyploidy , Securin , Separase
18.
STAR Protoc ; 2(4): 100780, 2021 12 17.
Article in English | MEDLINE | ID: mdl-34585151

ABSTRACT

Patient-derived organoids (PDOs) recapitulate the cellular heterogeneity of the original colorectal tumor tissue. Here, we describe a protocol to generate genetically modified PDOs to investigate cancer stem cells. This protocol uses the CRISPR-Cas9 system to knock-in the IRES-EGFP-P2A-iCaspase9 cassette into the 3' UTR of the potential cancer stem cell marker gene, which allows us to investigate their potential for self-replication and pluripotency. We describe the procedure for generating mutant PDOs and their application for stem cell research. For complete details on the generation and use of this protocol, please refer to Okamoto et al. Okamoto et al. (2021).


Subject(s)
CRISPR-Cas Systems/genetics , Colorectal Neoplasms , Gene Editing/methods , Gene Knock-In Techniques/methods , Organoids/pathology , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Humans , Neoplastic Stem Cells/cytology , Tumor Cells, Cultured
19.
Stem Cell Reports ; 16(4): 954-967, 2021 04 13.
Article in English | MEDLINE | ID: mdl-33711267

ABSTRACT

Metastasis is the major cause of cancer-related death, but whether metastatic lesions exhibit the same cellular composition as primary tumors has yet to be elucidated. To investigate the cellular heterogeneity of metastatic colorectal cancer (CRC), we established 72 patient-derived organoids (PDOs) from 21 patients. Combined bulk transcriptomic and single-cell RNA-sequencing analysis revealed decreased gene expression of markers for differentiated cells in PDOs derived from metastatic lesions. Paradoxically, expression of potential intestinal stem cell markers was also decreased. We identified OLFM4 as the gene most strongly correlating with a stem-like cell cluster, and found OLFM4+ cells to be capable of initiating organoid culture growth and differentiation capacity in primary PDOs. These cells were required for the efficient growth of primary PDOs but dispensable for metastatic PDOs. These observations demonstrate that metastatic lesions have a cellular composition distinct from that of primary tumors; patient-matched PDOs are a useful resource for analyzing metastatic CRC.


Subject(s)
Colorectal Neoplasms/pathology , Granulocyte Colony-Stimulating Factor/metabolism , Organoids/metabolism , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Colorectal Neoplasms/genetics , Colorectal Neoplasms/surgery , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Neoplasm Metastasis , Organoids/pathology
20.
Hum Reprod ; 25(11): 2734-44, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20823117

ABSTRACT

BACKGROUND: Recently we reported that an oocyte activation ability in human and mouse sperm is associated with head flatness or the presence of perinuclear theca (PT) substance, MN13, which is an oocyte activation-related protein localized on the post-acrosomal sheath (PAS). As such, we hypothesize that the appearance of oocyte activation ability is stage-specifically regulated and depends on the formation of the acrosome or PAS/PT in spermatids. METHODS: We monitored the appearance and movement of MN13 as a PT-specific molecule during spermatogenesis and analysed how the MN13 localization is affected in mouse and human globozoospermic acrosomeless sperm. RESULTS: MN13 was first detected on the surface of acrosomic vesicles, i.e. on the nascent outer acrosomal membrane of step 5-6 round spermatids (Sb1 spermatids in human), and it was then translocated via the outer acrosomal membrane surface to the most distal region of the acrosome in step 7 round spermatids (Sb2 spermatids). As spermatids elongated, MN13 was translocated via the cytoplasmic space between the nuclear envelope and the overlying plasma membrane towards the post-acrosomal region, and it was organized on the top of the nascent PAS that was typically found in step 14 elongated spermatids (Sd1 spermatids). In contrast, MN13 was not found in any GOPC-deficient spermatids, which completely lack the acrosome but have manchettes (microtubule bundles), nor in mouse and human acrosomeless sperm. CONCLUSIONS: The MN13 appearance or the MN13-related PAS/PT formation is highly dependant on acrosome formation; the MN13-related oocyte activation factor/ability is stage-specifically acquired in elongating/elongated spermatids.


Subject(s)
Antibodies, Monoclonal/metabolism , Oocytes/physiology , Spermatogenesis/immunology , Animals , Humans , Male , Mice , Mice, Inbred ICR , Microtubules/metabolism , Middle Aged , Spermatids/metabolism , Spermatozoa/abnormalities , Testis/metabolism
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