B Cells of Early-life Origin Defined by RAG2-based Lymphoid Cell Tracking under Native Hematopoietic Conditions.

During the perinatal period, the immune system sets the threshold to select either response or tolerance to environmental Ags, which leads to the potential to provide a lifetime of protection and health. B-1a B cells have been demonstrated to develop during this perinatal time window, showing a unique and restricted BCR repertoire, and these cells play a major role in natural Ab secretion and immune regulation. In the current study, we developed a highly efficient temporally controllable RAG2-based lymphoid lineage cell labeling and tracking system and applied this system to understand the biological properties and contribution of B-1a cells generated at distinct developmental periods to the adult B-1a compartments. This approach revealed that B-1a cells with a history of RAG2 expression during the embryonic and neonatal periods dominate the adult B-1a compartment, including those in the bone marrow (BM), peritoneal cavity, and spleen. Moreover, the BCR repertoire of B-1a cells with a history of RAG2 expression during the embryonic period was restricted, becoming gradually more diverse during the neonatal period, and then heterogeneous at the adult stage. Furthermore, more than half of plasmablasts/plasma cells in the adult BM had embryonic and neonatal RAG2 expression histories. Moreover, BCR analysis revealed a high relatedness between BM plasmablasts/plasma cells and B-1a cells derived from embryonic and neonatal periods, suggesting that these cell types have a common origin. Taken together, these findings define, under native hematopoietic conditions, the importance in adulthood of B-1a cells generated during the perinatal period.

Frequent mutations that converge on the NFKBIZ pathway in ulcerative colitis.

Chronic inflammation is accompanied by recurring cycles of tissue destruction and repair and is associated with an increased risk of cancer1-3. However, how such cycles affect the clonal composition of tissues, particularly in terms of cancer development, remains unknown. Here we show that in patients with ulcerative colitis, the inflamed intestine undergoes widespread remodelling by pervasive clones, many of which are positively selected by acquiring mutations that commonly involve the NFKBIZ, TRAF3IP2, ZC3H12A, PIGR and HNRNPF genes and are implicated in the downregulation of IL-17 and other pro-inflammatory signals. Mutational profiles vary substantially between colitis-associated cancer and non-dysplastic tissues in ulcerative colitis, which indicates that there are distinct mechanisms of positive selection in both tissues. In particular, mutations in NFKBIZ are highly prevalent in the epithelium of patients with ulcerative colitis but rarely found in both sporadic and colitis-associated cancer, indicating that NFKBIZ-mutant cells are selected against during colorectal carcinogenesis. In further support of this negative selection, we found that tumour formation was significantly attenuated in Nfkbiz-mutant mice and cell competition was compromised by disruption of NFKBIZ in human colorectal cancer cells. Our results highlight common and discrete mechanisms of clonal selection in inflammatory tissues, which reveal unexpected cancer vulnerabilities that could potentially be exploited for therapeutics in colorectal cancer.

Plant GARDEN: a portal website for cross-searching between different types of genomic and genetic resources in a wide variety of plant species.

BACKGROUND: Plant genome information is fundamental to plant research and development. Along with the increase in the number of published plant genomes, there is a need for an efficient system to retrieve various kinds of genome-related information from many plant species across plant kingdoms. Various plant databases have been developed, but no public database covers both genomic and genetic resources over a wide range of plant species. MAIN BODY: We have developed a plant genome portal site, Plant GARDEN (Genome And Resource Database Entry: https://plantgarden.jp/en/index ), to provide diverse information related to plant genomics and genetics in divergent plant species. Elasticsearch is used as a search engine, and cross-keyword search across species is available. Web-based user interfaces (WUI) for PCs and tablet computers were independently developed to make data searches more convenient. Several types of data are stored in Plant GARDEN: reference genomes, gene sequences, PCR-based DNA markers, trait-linked DNA markers identified in genetic studies, SNPs, and in/dels on publicly available sequence read archives (SRAs). The data registered in Plant GARDEN as of March 2023 included 304 assembled genome sequences, 11,331,614 gene sequences, 419,132 DNA markers, 8,225 QTLs, and 5,934 SNP lists (gvcf files). In addition, we have re-annotated all the genes registered in Plant GARDEN by using a functional annotation tool, Hayai-Annotation, to compare the orthologous relationships among genes. CONCLUSION: The aim of Plant GARDEN is to provide plant genome information for use in the fields of plant science as well as for plant-based industries, education, and other relevant areas. Therefore, we have designed a WUI that allows a diverse range of users to access such information in an easy-to-understand manner. Plant GARDEN will eventually include a wide range of plant species for which genome sequences are assembled, and thus the number of plant species in the database will continue to expand. We anticipate that Plant GARDEN will promote the understanding of genomes and gene diversity by facilitating comparisons of the registered sequences.

Downregulation of Odd-Skipped Related 2, a Novel Regulator of Epithelial-Mesenchymal Transition, Enables Efficient Somatic Cell Reprogramming.

Somatic cell reprogramming proceeds through a series of events to generate induced pluripotent stem cells (iPSCs). The early stage of reprogramming of mouse embryonic fibroblasts is characterized by rapid cell proliferation and morphological changes, which are accompanied by downregulation of mesenchyme-associated genes. However, the functional relevance of their downregulation to reprogramming remains poorly defined. In this study, we have screened transcriptional regulators that are downregulated immediately upon reprogramming, presumably through direct targeting by reprogramming factors. To test if these transcriptional regulators impact reprogramming when expressed continuously, we generated an expression vector that harbors human cytomegalovirus upstream open reading frame 2 (uORF2), which reduces translation to minimize the detrimental effect of an expressed protein. Screening of transcriptional regulators with this expression vector revealed that downregulation of (odd-skipped related 2 [Osr2]) is crucial for efficient reprogramming. Using a cell-based model for epithelial-mesenchymal transition (EMT), we show that Osr2 is a novel EMT regulator that acts through induction of transforming growth factor-ß (TGF-ß) signaling. During reprogramming, Osr2 downregulation not only diminishes TGF-ß signaling but also allows activation of Wnt signaling, thus promoting mesenchymal-epithelial transition (MET) toward acquisition of pluripotency. Our results illuminate the functional significance of Osr2 downregulation in erasing the mesenchymal phenotype at an early stage of somatic cell reprogramming.

Clinical application of long-read nanopore sequencing in a preimplantation genetic testing pre-clinical workup to identify the junction for complex Xq chromosome rearrangement-related disease.

OBJECTIVE: Xq chromosome duplication with complex rearrangements is generally acknowledged to be associated with neurodevelopmental disorders, such as Pelizaeus-Merzbacher disease (PMD) and MECP2 duplication syndrome. For couples who required a PGT-M (pre-implantation genetic testing for monogenic disease) for these disorders, junction-specific PCR is useful to directly detect pathogenic variants. Therefore, pre-clinical workup for PGT-M requires the identification of the junction of duplicated segments in PMD and MECP2 duplication syndrome, which is generally difficult. METHODS: In this report, we used nanopore long-read sequencing targeting the X chromosome using an adaptive sampling method to identify breakpoint junctions in disease-causing triplications. RESULTS: By long-read sequencing, we successfully identified breakpoint junctions in one PMD case with PLP1 triplication and in another MECP2 triplication case in a single sequencing run. Surprisingly, the duplicated region involving MECP2 was inserted 45 Mb proximal to the original position. This inserted region was confirmed by FISH analysis. With the help of precise mapping of the pathogenic variant, we successfully re-established STR haplotyping for PGT-M and avoided any potential misinterpretation of the pathogenic allele due to recombination. CONCLUSION: Long-read sequencing with adaptive sampling in a PGT-M pre-clinical workup is a beneficial method for identifying junctions of chromosomal complex structural rearrangements.

Target enrichment long-read sequencing with adaptive sampling can determine the structure of the small supernumerary marker chromosomes.

Structural analysis of small supernumerary marker chromosomes (sSMCs) has revealed that many have complex structures. Structural analysis of sSMCs by whole genome sequencing using short-read sequencers is challenging however because most present with a low level of mosaicism and consist of a small region of the involved chromosome. In this present study, we applied adaptive sampling using nanopore long-read sequencing technology to enrich the target region and thereby attempted to determine the structure of two sSMCs with complex structural rearrangements previously revealed by cytogenetic microarray. In adaptive sampling, simple specification of the target region in the FASTA file enables to identify whether or not the sequencing DNA is included in the target, thus promoting efficient long-read sequencing. To evaluate the target enrichment efficiency, we performed conventional pair-end short-read sequencing in parallel. Sequencing with adaptive sampling achieved a target enrichment at about a 11.0- to 11.5-fold higher coverage rate than conventional pair-end sequencing. This enabled us to quickly identify all breakpoint junctions and determine the exact sSMC structure as a ring chromosome. In addition to the microhomology and microinsertion at the junctions, we identified inverted repeat structure in both sSMCs, suggesting the common generation mechanism involving replication impairment. Adaptive sampling is thus an easy and beneficial method of determining the structures of complex chromosomal rearrangements.

Oncogenic KRAS-expressing organoids with biliary epithelial stem cell properties give rise to biliary tract cancer in mice.

Biliary tract cancer (BTC) arises from biliary epithelial cells (BECs) and includes intrahepatic cholangiocarcinoma (IHCC), gallbladder cancer (GC), and extrahepatic cholangiocarcinoma (EHCC). Although frequent KRAS mutations and epigenetic changes at the INK4A/ARF locus have been identified, the molecular pathogenesis of BTC is unclear and the development of corresponding anticancer agents remains inadequate. We isolated epithelial cell adhesion molecule (EpCAM)-positive BECs from the mouse intrahepatic bile duct, gallbladder, and extrahepatic bile duct, and established organoids derived from these cells. Introduction of activated KRAS and homozygous deletion of Ink4a/Arf in the cells of each organoid type conferred the ability to form lethal metastatic adenocarcinoma with differentiated components and a pronounced desmoplastic reaction on cell transplantation into syngeneic mice, indicating that the manipulated cells correspond to BTC-initiating cells. The syngeneic mouse models recapitulate the pathological features of human IHCC, GC, and EHCC, and they should therefore prove useful for the investigation of BTC carcinogenesis and the development of new therapeutic strategies. Tumor cells isolated from primary tumors formed organoids in three-dimensional culture, and serial syngeneic transplantation of these cells revealed that their cancer stem cell properties were supported by organoid culture, but not by adherent culture. Adherent culture thus attenuated tumorigenic activity as well as the expression of both epithelial and stem cell markers, whereas the expression of epithelial-mesenchymal transition (EMT)-related transcription factor genes and mesenchymal cell markers was induced. Our data show that organoid culture is important for maintenance of epithelial cell characteristics, stemness, and tumorigenic activity of BTC-initiating cells.

FZR1 loss increases sensitivity to DNA damage and consequently promotes murine and human B-cell acute leukemia.

FZR1 (fizzy-related protein homolog; also known as CDH1 [cell division cycle 20 related 1]) functions in the cell cycle as a specific activator of anaphase-promoting complex or cyclosome ubiquitin ligase, regulating late mitosis, G1 phase, and activation of the G2-M checkpoint. FZR1 has been implicated as both a tumor suppressor and oncoprotein, and its precise contribution to carcinogenesis remains unclear. Here, we examined the role of FZR1 in tumorigenesis and cancer therapy by analyzing tumor models and patient specimens. In an Fzr1 gene-trap mouse model of B-cell acute lymphoblastic leukemia (B-ALL), mice with Fzr1-deficient B-ALL survived longer than those with Fzr1-intact disease, and sensitivity of Fzr1-deficient B-ALL cells to DNA damage appeared increased. Consistently, conditional knockdown of FZR1 sensitized human B-ALL cell lines to DNA damage-induced cell death. Moreover, multivariate analyses of reverse-phase protein array of B-ALL specimens from newly diagnosed B-ALL patients determined that a low FZR1 protein expression level was an independent predictor of a longer remission duration. The clinical benefit of a low FZR1 expression level at diagnosis was no longer apparent in patients with relapsed B-ALL. Consistent with this result, secondary and tertiary mouse recipients of Fzr1-deficient B-ALL cells developed more progressive and radiation-resistant disease than those receiving Fzr1-intact B-ALL cells, indicating that prolonged inactivation of Fzr1 promotes the development of resistant clones. Our results suggest that reduction of FZR1 increases therapeutic sensitivity of B-ALL and that transient rather than tonic inhibition of FZR1 may be a therapeutic strategy.

Periostin antisense oligonucleotide suppresses bleomycin-induced formation of a lung premetastatic niche for melanoma.

Metastasis is the leading cause of cancer death. A tumor-supportive microenvironment, or premetastatic niche, at potential secondary tumor sites plays an important role in metastasis, especially in tumor cell colonization. Although a fibrotic milieu is known to promote tumorigenesis and metastasis, the underlying molecular contributors to this effect have remained unclear. Here we show that periostin, a component of the extracellular matrix that functions in tissue remodeling, has a key role in formation of a fibrotic environment that promotes tumor metastatic colonization. We found that periostin was widely expressed in fibrotic lesions of mice with bleomycin-induced lung fibrosis, and that up-regulation of periostin expression coincided with activation of myofibroblasts positive for α-smooth muscle actin. We established a lung metastasis model for B16 murine melanoma cells and showed that metastatic colonization of the lung by these cells was markedly promoted by bleomycin-induced lung fibrosis. Inhibition of periostin expression by giving an intratracheal antisense oligonucleotide targeting periostin mRNA was found to suppress bleomycin-induced lung fibrosis and thereby to attenuate metastatic colonization of the lung by melanoma cells. Our results indicate that periostin is a key player in the development of bleomycin-induced fibrosis and consequent enhancement of tumor cell colonization in the lung. Our results therefore implicate periostin as a potential target for prevention or treatment of lung metastasis.

Development of a functional thyroid model based on an organoid culture system.

The low turnover rate of thyroid follicular cells and the lack of a long-term thyroid cell culture system have hampered studies of thyroid carcinogenesis. We have now established a thyroid organoid culture system that supports thyroid cell proliferation in vitro. The established mouse thyroid organoids performed thyroid functions including thyroglobulin synthesis, iodide uptake, and the production and release of thyroid hormone. Furthermore, transplantation of the organoids into recipient mice resulted in the formation of normal thyroid-like tissue capable of iodide uptake and thyroglobulin production in vivo. Finally, forced expression of oncogenic NRAS (NRASQ61R) in thyroid organoids established from p53 knockout mice and transplantation of the manipulated organoids into mouse recipients generated a model of poorly differentiated thyroid cancer. Our findings suggest that this newly developed thyroid organoid culture system is a potential research tool for the study of thyroid physiology and pathology including thyroid cancer.

CD44 variant 9 is a potential biomarker of tumor initiating cells predicting survival outcome in hepatitis C virus-positive patients with resected hepatocellular carcinoma.

This study investigated whether the expression of CD44 variant 9 (CD44v9) might be a functional marker of tumor-initiating stem-like cells in primary hepatocellular carcinomas (HCCs) of hepatitis C virus (HCV)(+) patients and provide an indicator of patient survival, as well as associated mechanisms. A total of 90 HCV(+) HCC patients who underwent surgery from 2006 to 2011 were enrolled and monitored for 2-8 years. Expression of CD44v9 was validated immunohistochemically in all HCCs, followed by comparative proteome, survival, and clinicopathological analyses. CD44 variant 8--10 was further evaluated in diethylnitrosamine-induced HCCs of C57Bl/6J mice. Focally localized CD44v(+) cells with a membranous staining pattern were detected in human HCV(+) and mouse HCCs. CD44v9(+) cells of HCCs were predominantly negative for Ki67 and P-p38, indicating decrease of cell proliferation in the CD44v9(+) tumor cell population, likely to be related to suppression of intracellular oxidative stress due to activation of Nrf2-mediated signaling, DNA repair, and inhibition of xenobiotic metabolism. CD44v9 IHC evaluation in 90 HCV(+) HCC cases revealed that positive expression was significantly associated with poor overall and recurrence-free survival, a younger age, poor histological differentiation of HCCs, and high alkaline phosphatase levels compared with patients with negative expression. CD44v9 is concluded to be a potential biomarker of tumor-initiating stem-like cells and a prognostic marker in HCV(+) HCC patients associated with Nrf2-mediated resistance to oxidative stress.

CD44 variant-dependent redox status regulation in liver fluke-associated cholangiocarcinoma: A target for cholangiocarcinoma treatment.

Expression of CD44, especially the variant isoforms (CD44v) of this major cancer stem cell marker, contributes to reactive oxygen species (ROS) defense through stabilizing xCT (a cystine-glutamate transporter) and promoting glutathione synthesis. This enhances cancer development and increases chemotherapy resistance. We investigate the role of CD44v in the regulation of the ROS defense system in cholangiocarcinoma (CCA). Immunohistochemical staining of CD44v and p38(MAPK) (a major ROS target) expression in Opisthorchis viverrini-induced hamster CCA tissues (at 60, 90, 120, and 180 days) reveals a decreased phospho-p38(MAPK) signal, whereas the CD44v signal was increased during bile duct transformation. Patients with CCA showed CD44v overexpression and negative-phospho-p38(MAPK) patients a significantly shorter survival rate than the low CD44v signal and positive-phospho-p38(MAPK) patients (P = 0.030). Knockdown of CD44 showed that xCT and glutathione levels were decreased, leading to a high level of ROS. We examined xCT-targeted CD44v cancer stem cell therapy using sulfasalazine. Glutathione decreased and ROS increased after the treatment, leading to inhibition of cell proliferation and induction of cell death. Thus, the accumulation of CD44v leads to the suppression of p38(MAPK) in transforming bile duct cells. The redox status regulation of CCA cells depends on the expression of CD44v to contribute the xCT function and is a link to the poor prognosis of patients. Thus, an xCT inhibitor could inhibit cell growth and activate cell death. This suggests that an xCT-targeting drug may improve CCA therapy by sensitization to the available drug (e.g. gemcitabine) by blocking the mechanism of the cell's ROS defensive system.

Synergistic antiproliferative effect of imatinib and adriamycin in platelet-derived growth factor receptor-expressing osteosarcoma cells.

Osteosarcoma (OS) is the most frequent primary solid malignant tumor of bone. Its prognosis remains poor in the substantial proportion of patients who do not respond to chemotherapy and novel therapeutic options are therefore needed. We previously established a mouse model that mimics the aggressive behavior of human OS. Enzyme-linked immunosorbent assay-based screening of such mouse tumor lysates identified platelet-derived growth factor-BB (PDGF-BB) as an abundant soluble factor, the gene for which was expressed dominantly in surrounding non-malignant cells of the tumor, whereas that for the cognate receptor (PDGF receptor ß) was highly expressed in OS cells. Platelet-derived growth factor-BB induced activation of both MEK-ERK and phosphatidylinositol 3-kinase-protein kinase B signaling pathways and promoted survival in OS cells deprived of serum, and these effects were blocked by the PDGF receptor inhibitor imatinib. However, these actions of PDGF-BB and imatinib were mostly masked in the presence of serum. Whereas imatinib alone did not manifest an antitumor effect in mice harboring OS tumors, combined treatment with imatinib and adriamycin exerted a synergistic antiproliferative effect on OS cells in vivo. These results suggest that treatment of OS with imatinib is effective only when cell survival is dependent on PDGF signaling or when imatinib is combined with another therapeutic intervention that renders the tumor cells susceptible to imatinib action, such as by inducing cellular stress.

The Arf/p53 protein module, which induces apoptosis, down-regulates histone H2AX to allow normal cells to survive in the presence of anti-cancer drugs.

BACKGROUND: It is unclear how DNA-damaging agents target cancer cells over normal somatic cells. RESULTS: Arf/p53-dependent down-regulation of H2AX enables normal cells to survive after DNA damage. CONCLUSION: Transformed cells, which harbor mutations in either Arf or p53, are more sensitive to DNA-damaging agents. SIGNIFICANCE: Cellular transformation renders cells more susceptible to some DNA-damaging agents. Anti-cancer drugs generally target cancer cells rather than normal somatic cells. However, the factors that determine this differential sensitivity are poorly understood. Here we show that Arf/p53-dependent down-regulation of H2AX induced the selective survival of normal cells after drug treatment, resulting in the preferential targeting of cancer cells. Treatment with camptothecin, a topoisomerase I inhibitor, caused normal cells to down-regulate H2AX and become quiescent, a process mediated by both Arf and p53. In contrast, transformed cells that harbor mutations in either Arf or p53 do not down-regulate H2AX and are more sensitive to drugs unless they have developed drug resistance. Such transformation-associated changes in H2AX expression rendered cancer cells more susceptible to drug-induced damage (by two orders of magnitude). Thus, the expression of H2AX and γH2AX (phosphorylated form of H2AX at Ser-139) is a critical factor that determines drug sensitivity and should be considered when administering chemotherapy.

Expression of CD24 is associated with HER2 expression and supports HER2-Akt signaling in HER2-positive breast cancer cells.

Human epidermal growth factor receptor 2 (HER2)-positive breast cancer is treated with HER2-targeted agents, such as trastuzumab and lapatinib, that suppress signaling by phosphatidylinositol 3-kinase (PI3K)-Akt and MAPK pathways. However, resistance to HER2-targeted therapy remains a major clinical problem. Overexpression of CD24 has been detected in many cancers and is associated with a poor prognosis in women with breast cancer. HER2-positive breast tumors are predominantly positive for CD24, suggesting that the expression of the two molecules is related. To investigate the relation between HER2 and CD24, we overexpressed HER2 in breast cancer cells that were triple-negative for the estrogen receptor, progesterone receptor and HER2. We found that expression of CD24 was increased by stable overexpression of HER2. Flow cytometry thus revealed that the percentage of CD24-positive cells was markedly higher in the HER2-positive fraction than in the HER2-negative fraction. Knockdown of CD24 in breast cancer cells positive for endogenous HER2 downregulated HER2 expression, whereas knockdown of HER2 did not affect the expression of CD24. Knockdown of CD24 also suppressed the phosphorylation of Akt, which functions downstream of HER2 and PI3K to promote cell survival. Moreover, knockdown of CD24 increased the sensitivity of HER2-positive breast cancer cells to lapatinib treatment. Our results thus indicate that CD24 supports both the expression of HER2 and the consequent activation of PI3K-Akt signaling. Furthermore, CD24 may contribute to resistance to HER2-targeted therapy and, therefore, is itself a potential therapeutic target in HER2-positive breast cancer.

IGF1 receptor signaling regulates adaptive radioprotection in glioma stem cells.

Cancer stem cells (CSCs) play an important role in disease recurrence after radiation treatment as a result of intrinsic properties such as high DNA repair capability and antioxidative capacity. It is unclear, however, how CSCs further adapt to escape the toxicity of the repeated irradiation regimens used in clinical practice. Here, we have exposed a population of murine glioma stem cells (GSCs) to fractionated radiation in order to investigate the associated adaptive changes, with the ultimate goal of identifying a targetable factor that regulates acquired radioresistance. We have shown that fractionated radiation induces an increase in IGF1 secretion and a gradual upregulation of the IGF type 1 receptor (IGF1R) in GSCs. Interestingly, IGF1R upregulation exerts a dual radioprotective effect. In the resting state, continuous IGF1 stimulation ultimately induces downregulation of Akt/extracellular-signal-regulated kinases (ERK) and FoxO3a activation, which results in slower proliferation and enhanced self-renewal. In contrast, after acute radiation, the abundance of IGF1R and increased secretion of IGF1 promote a rapid shift from a latent state toward activation of Akt survival signaling, protecting GSCs from radiation toxicity. Treatment of tumors formed by the radioresistant GSCs with an IGF1R inhibitor resulted in a marked increase in radiosensitivity, suggesting that blockade of IGF1R signaling is an effective strategy to reverse radioresistance. Together, our results show that GSCs evade the damage of repeated radiation not only through innate properties but also through gradual inducement of resistance pathways and identify the dynamic regulation of GSCs by IGF1R signaling as a novel mechanism of adaptive radioprotection.

Complexity of cancer stem cells.

Heterogeneity of tumor tissue has been accounted for in recent years by a hierarchy-based model in which cancer stem cells (CSCs) have the ability both to self-renew and to give rise to differentiated tumor cells and are responsible for the overall organization of a tumor. Research into CSCs has progressed rapidly and concomitantly with recent advances in the biology of normal tissue stem cells, resulting in the identification of CSCs in a wide range of human tumors. Studies of mouse models of human cancer have provided further insight into the characteristics of CSCs as well as a basis for the development of novel therapies targeted to these cells. However, recent studies have revealed complexities, such as plasticity of stem cell properties and clonal diversity of CSCs, in certain tumor types that have led to revision of the original CSC model. In this review, we summarize the history of the discovery and characterization of CSCs, as well as address recent advances that have revealed the complexity of these cells and their therapeutic implications.

Twist2 functions as a tumor suppressor in murine osteosarcoma cells.

The epithelial-mesenchymal transition (EMT) contributes to the malignant progression of cancer cells including acquisition of the ability to undergo metastasis. However, whereas EMT-related transcription factors (EMT-TF) are known to play an important role in the malignant progression of epithelial tumors, their role in mesenchymal tumors remains largely unknown. We show that expression of the gene for Twist2 is downregulated in human osteosarcoma and correlates inversely with tumorigenic potential in mouse osteosarcoma. Forced expression of Twist2 in highly tumorigenic murine osteosarcoma cells induced a slight inhibition of cell growth in vitro but markedly suppressed tumor formation in vivo. Conversely, knockdown of Twist2 in osteosarcoma cells with a low tumorigenic potential promoted tumor formation in vivo, suggesting that Twist2 functions as a tumor suppressor in osteosarcoma cells. Furthermore, Twist2 induced expression of fibulin-5, which has been reported as a tumor suppressor. Medium conditioned by mouse osteosarcoma cells overexpressing Twist2 inhibited expression of the MMP9 gene as well as invasion in mouse embryonic fibroblasts, and forced expression of Twist2 in osteosarcoma cells suppressed MMP9 gene expression in tumor tissue. Data from the present study suggest that Twist2 inhibits formation of a microenvironment conducive to tumor growth and thereby attenuates tumorigenesis in osteosarcoma.

Arf and p53 act as guardians of a quiescent cellular state by protecting against immortalization of cells with stable genomes.

Normal cells undergo a growth-arrested status that is produced by p53-dependent down-regulation of histone H2AX. Immortality is developed after abrogation of the H2AX-diminished state, which is associated with genomic instability (often with tetraploidy) and the induction of mutations in either the Arf or p53 gene. However, the role of Arf in control of H2AX expression and genome stability is still unclear. Here, we show that both Arf and p53 are required for the down-regulation of H2AX and formation of the growth-arrested state. Wild-type (WT) mouse embryonic fibroblasts (MEFs) subjected to tetraploidization with DNA lesions did not undergo mitotic catastrophe-associated cell death and stayed in a growth-arrested state, until immortality was attained with mutations in the Arf/p53 module and recovery of H2AX expression. Whereas tetraploidization was essential for immortalization of WT MEFs, this event was not required for immortalization of MEFs containing mutations in Arf/p53 and these cells still underwent mitotic catastrophe-associated cell death. Thus, WT MEFs are protected from immortalization with genome stability, which is abrogated with tetraploidization and mutation of either Arf or p53.