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BackgroundPatients with smoldering ATLL often present with a skin eruption due to skin infiltration of ATLL cells. Although skin eruption type is known to be associated with prognosis based on its pattern, it is unknown why different types of skin eruptions are associated with different prognoses.ObjectiveGenomic analysis of patients with skin eruptions of smoldering ATLL will be performed to determine the mechanism of ATLL development and its association with prognosis.MethodsDNA from skin biopsy specimens was used for targeted sequencing of 280 genes to examine the association between genomic variation and prognosis.ResultsDue to the small number of smoldering ATLL patients (27 cases), we could not find a clear relationship between skin eruption and prognosis in this study. Genomic analysis identified 247 genomic variants (108 genes), with an average of 9.2 variants and 3.2 variants as driver genes. Pathway analysis of the driver genes showed activation of the pathway associated with HTLV-1 infection, as well as activation of the signaling pathway observed throughout ATLL. Furthermore, multivariate analysis identified age>70 years and STAT3 mutation as prognostic risk factors and TBL1XR1 mutation as a risk factor for progression-free survival.ConclusionAlthough the small number of patient samples did not allow us to determine a prognostic association with skin eruption, STAT3 mutation was identified as a prognostic risk factor for smoldering ATLL with skin eruption. Further studies are needed to increase the number of patients with this disease.

SummaryThe proteasome has an essential role in proteostasis maintenance and is critical for cell survival under proteotoxic conditions including hyperosmotic stress. However, it is unknown how proteasome activity is linked to cell survival/death under hyperosmotic stress. We have previously reported that apoptosis signal-regulating kinase 3 (ASK3) contributes to cell survival through its inactivation under hyperosmotic stress. 19S regulatory particle subunits of the proteasome were enriched in the ASK3 inactivator candidates identified through our genome-wide small interfering RNA (siRNA) screen. In this study, we demonstrate that the proteasome regulates ASK3 inactivation through its proteolytic activity. Intriguingly, the proteasome inactivates ASK3 via degradation of not ASK3 per se but another ASK family member ASK1 which activates ASK3 in a kinase activity-dependent manner. Furthermore, the elevated ASK1 level under proteasome inhibition sensitizes cells to hyperosmotic stress. These findings suggest that ASK family maintenance links proteasome capacity to cell fate bifurcation under hyperosmotic stress.

Sequences of the primers for Reverse-transcription polymerase chain reaction (RT-PCR) and Quantitative real-time PCR analysis

Loss of the tumor suppressor NDRG2 has been implicated in the development of oral squamous cell carcinoma (OSCC), acting by modulating PI3K/AKT-mediated dephosphorylation of PTEN at S380/S382/T383 (STT). Here, we show that the majority of OSCC tumors with lymph node metastasis, a major prognostic factor, exhibit high levels of phosphorylated AKT-S473 and PTEN-STT and low levels of NDRG2 expression. In Ndrg2-deficient mice, which develop a wide range of tumors, we developed a model of OSCC by treatment with the tobacco surrogate 4-nitroquinoline-1-oxide (4-NQO). In this model, both the number and size of OSCC tumors were increased significantly by Ndrg2 deficiency, which also increased invasion of cervical lymph nodes. 4-NQO treatment of human OSCC cell lines exhibiting low NDRG2 expression induced epithelial–mesenchymal transition via activation of NF-κB signaling. Conversely, ectopic expression of NDRG2 reversed the EMT phenotype and inhibited NF-κB signaling via suppression of PTEN-STT and AKT-S473 phosphorylation. Our results show how NDRG2 expression serves as a critical determinant of the invasive and metastatic capacity of OSCC. Cancer Res; 77(9); 2363–74. ©2017 AACR.

Supplemental Figure 1. Keratin staining of cervical lymph nodes from Ndrg2-deficient mice treated with 4-NQO. Supplementary figure related to Figure 2; Supplemental Figure 2. Effect of 4-NQO treatment on an OSCC cell line was observed through the activation of the AKT signalling pathway and cell migration ability. Supplementary figure related to Figure 4; Supplemental Figure 3. Immunofluorescence staining of E-cadherin, Vimentin, and NDRG2 in OSCC cell lines for determining EMT progression by treatment with 4-NQO. Supplementary figure related to Figure 5; Supplemental Figure 4. Activation of NF-kappaB signalling in SAS/OSCC cell line treated with 4-NQO. Supplementary figure related to Figure 6; Supplemental Figure 5. Hypothetical model for the molecular mechanism of EMT progression in OSCC cells. Supplementary figure related to Figure 6 and Discussion.

Supplemental figure legends

supplementary tables: Supplemental Table 1. Expression rates of immunohistochemical staining of NDRG2 versus p-Akt or p-PTEN in tumors from OSCC patients. Supplementary table related to Figure 1; Supplemental Table 2. Incidence of tumour formation in the tongue of Ndrg2-deficient mice treated with 4-NQO for 16 weeks. Supplementary table related to Figure 2; Supplemental Table 3. Frequency of SCC (squamous cell carcinoma) tumour formation in the tongue of Ndrg2-deficient mice treated with 4-NQO for 16 weeks. Supplementary table related to Figure 2; Supplemental Table 4. Incidence of tumour formation in wild type and Ndrg2-deficient mice after treatment with 4-NQO for 30 weeks. Supplementary table related to Figure 3; Supplemental Table 5. Incidence of tumour development in wild-type and Ndrg2-deficient mice treated with 4-NQO for 30 weeks. Supplementary table related to Figure 3; Supplemental Table 6. Comparison of the frequency of cervical lymph node metastasis between wild-type control and Ndrg2-deficient mice. Supplementary table related to Figure 3.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common cancers worldwide, with a poor prognosis. Owing to the difficulty of early diagnosis, the aim of this study was to isolate biomarkers from extracellular vesicles (EVs) that can lead to early diagnosis.EVs in the culture supernatant were isolated from a pancreatic cancer cell line (PK-1) and expanded by using two-dimensional gel electrophoresis, and protein identification from each spot was performed by using matrix-assisted laser desorption ionization mass spectrometry. The identified proteins were classified and compared with previously reported results for EVs from murine pancreatic cancer PAN02 cells, and their expression specificity was examined using PDAC cell lines and patient-derived PDAC tissues. In addition, the significance of selected biomarker(s) was examined based on the changes in biomarkers in the blood EVs of PDAC patients after surgery.We found that the ITGA6A splice variant was predominantly expressed in several pancreatic cancer cell lines and blood EVs from patients with PDAC, whereas the ITGA6B splice variant was predominantly expressed in EVs from the blood of normal volunteers. In the expression pattern of ITGA6 in EVs from blood samples of two PDAC patients before and after resection surgery, the expression of ITGA6A in EVs significantly decreased after surgery and increased several months before clinical recurrence. Furthermore, the increased expression of ITGA6A in EVs occurred much earlier than that of CA19-9.Determination of ITGA6A expression in blood EVs in PDAC patients could be a useful blood marker for the early diagnosis of PDAC recurrence.

We have previously reported that the proanthocyanidin (PAC) fraction of blueberry leaf extract (BB-PAC) inhibits the proliferation of HTLV-1-infected adult T-cell leukemia (ATL) by inducing apoptosis. In the present study, we further analyzed the structure of BB-PAC and elucidated the molecular mechanism underlying the inhibitory function of HTLV-1-infected and ATL cells. After hot water extraction with fractionation with methanol-acetone, BB-PAC was found to be concentrated in fractions 4 to 7 (Fr7). The strongest inhibition of ATL cell growth was observed with Fr7, which contained the highest BB-PAC polymerization degree of 14. The basic structure of BB-PAC is mainly B-type bonds, with A-type bonds (7.1%) and cinchonain I units as the terminal unit (6.1%). The molecular mechanism of cytotoxicity observed around Fr7 against ATL cells was the degradation of JAK1 to 3 and the dephosphorylation of STAT3/5, which occurs by proteasome-dependent proteolysis, confirming that PAC directly binds to heat shock protein 90 (HSP90). JAK degradation was caused by proteasome-dependent proteolysis, and we identified the direct binding of PAC to HSP90. In addition, the binding of cochaperone ATPase homolog 1 (AHA1) to HSP90, which is required for activation of the cofactor HSP90, was inhibited by BB-PAC treatment. Therefore, BB-PAC inhibited the formation of the HSP90/AHA1 complex and promoted the degradation of JAK protein due to HSP90 dysfunction. These results suggest that the highly polymerized PAC component from blueberry leaves has great potential as a preventive and therapeutic agent against HTLV-1-infected and ATL cells.

Recent studies have shown that adipose tissue is an immunological organ. While inflammation in energy-storing white adipose tissues has been the focus of intense research, the regulatory mechanisms of inflammation in heat-producing brown adipose tissues remain largely unknown. We previously identified apoptosis signal-regulating kinase 1 (ASK1) as a critical regulator of brown adipocyte maturation; the PKA-ASK1-p38 axis facilitates uncoupling protein 1 (UCP1) induction cell-autonomously. Here, we show that ASK1 suppresses an innate immune pathway and contributes to maintenance of brown adipocytes. We report a novel chemical pull-down method for endogenous kinases using analog sensitive kinase allele (ASKA) technology and identify an ASK1 interactor in brown adipocytes, receptor-interacting serine/threonine-protein kinase 2 (RIPK2). ASK1 disrupts the RIPK2 signaling complex and inhibits the NOD-RIPK2 pathway to downregulate the production of inflammatory cytokines. As a potential biological significance, an in vitro model for intercellular regulation suggests that ASK1 facilitates the expression of UCP1 through the suppression of inflammatory cytokine production. In parallel to our previous report on the PKA-ASK1-p38 axis, our work raises the possibility of an auxiliary role of ASK1 in brown adipocyte maintenance through neutralizing the thermogenesis-suppressive effect of the NOD-RIPK2 pathway.

Adhesion GPCRs (aGPCRs) are a subfamily of GPCRs that are involved in cell adhesion, cell proliferation, and cell migration in various tissues. G protein-coupled receptor proteolytic site (GPS) of aGPCR is required to cleave the extracellular domain autocatalytically, generating two fragments; a N-terminal fragment (NTF) and a C-terminal fragment (CTF) containing seven transmembrane structure. NTF can interact with CTF non-covalently after cleavage, however the physiological significance of the cleavage of aGPCR at GPS, and also the interaction between NTF and CTF have not been fully clarified yet. In this study, we first investigated the expression profiles of two aGPCRs, GPR56/ADGRG1, and LPHN1/ADGRL1 in mouse brain, and found that the NTF and CTF of GPR56 independently expressed in different brain region at different developmental stages. Immunoprecipitation of GPR56CTF co-immunoprecipitated LPHN1NTF from mouse brain and HEK293T cells expressing both fragments. Stimulation with LPHN1 ligand, α-Latrotoxin N4C (αLTXN4C), to cells expressing LPHN1NTF and GPR56CTF increased intracellular Ca

SummaryBiomolecular condensates are membraneless structures formed through phase separation. Recent studies have demonstrated that the material properties of biomolecular condensates are crucial for their biological functions and pathogenicity. However, the phase maintenance of biomolecular condensates in cells remains elusive. Here, we show that sodium ion (Na+) influx regulates the condensate liquidity under hyperosmotic stress. The fluidity of ASK3 condensates increases at the high intracellular Na+ concentration derived from extracellular hyperosmotic solution. Moreover, we identified TRPM4 as a cation channel that allows Na+ influx under hyperosmotic stress. TRPM4 inhibition causes the liquid-to-solid phase transition of ASK3 condensates, leading to impairment of the ASK3 osmoresponse. In addition to ASK3 condensates, intracellular Na+ widely regulates the condensate liquidity and aggregate formation of biomolecules, including DCP1A, TAZ and polyQ-protein, under hyperosmotic stress. Our findings demonstrate that changes in Na+ contribute to the cellular stress response via liquidity maintenance of biomolecular condensates.

N‐myc downstream‐regulated gene 2 (NDRG2) is a candidate tumor suppressor in various cancers, including adult T‐cell leukemia/lymphoma (ATLL). NDRG2, as a stress‐responsive protein, is induced by several stress‐related signaling pathways and NDRG2 negatively regulates various signal transduction pathways. Although it has not been found to function alone, NDRG2 binds serine/threonine protein phosphatase 2A (PP2A), generating a complex that is involved in the regulation of various target proteins. The main function of NDRG2 is to maintain cell homeostasis by suppressing stress‐induced signal transduction; however, in cancer, genomic deletions and/or promoter methylation may inhibit the expression of NDRG2, resulting in enhanced tumor development through overactivated signal transduction pathways. A wide variety of tumors develop in Ndrg2‐deficient mice, including T‐cell lymphoma, liver, lung and other tumors, the characteristics of which are similar to those in Pten‐deficient mice. In particular, PTEN is a target molecule of the NDRG2/PP2A complex, which enhances PTEN phosphatase activity by dephosphorylating residues in the PTEN C‐terminal region. In ATLL cells, loss of NDRG2 expression leads to the failed recruitment of PP2A to PTEN, resulting in the inactivation of PTEN phosphatase with phosphorylation, ultimately leading to the activation of PI3K/AKT. Thus, NDRG2, as a PP2A adaptor, regulates the global phosphorylation of important signaling molecules. Moreover, the downregulation of NDRG2 expression by long‐term stress‐induced methylation is directly correlated with the development of ATLL and other cancers. Thus, NDRG2 might be important for the development of stress‐induced leukemia and other cancers and has become an important target for novel molecular therapies., In tumor cells, the loss of NDRG2 expression induces constitutive phosphorylation of PTEN C‐terminal, which maintains PTEN in an inactive closed conformation and leads to constitutive AKT activation.

With the current worldwide pandemic of COVID-19, there is an urgent need to develop effective treatment and prevention methods against SARS-CoV-2 infection. We have previously reported that the proanthocyanidin (PAC) fraction in blueberry (BB) leaves has strong antiviral activity against hepatitis C virus (HCV) and human T-lymphocytic leukemia virus type 1 (HTLV-1). In this study, we used Kunisato 35 Gou (K35) derived from the rabbit eye blueberry (Vaccinium virgatum Aiton), which has a high PAC content in the leaves and stems. The mean of polymerization (mDP) of PAC in K35 was the highest of 7.88 in Fraction 8 (Fr8) from the stems and 12.28 of Fraction 7 (Fr7) in the leaves. The composition of BB-PAC in K35 is that most are B-type bonds with a small number of A-type bonds and cinchonain I as extension units. A strong antiviral effect was observed in Fr7, with a high polymerized PAC content in both the leaves and stems. Furthermore, when we examined the difference in the action of BB-PAC before and after SARS-CoV-2 infection, we found a stronger inhibitory effect in the pre-infection period. Moreover, BB-PAC Fr7 inhibited the activity of angiotensin II converting enzyme (ACE2), although no effect was observed in a neutralization test of pseudotyped SARS-CoV-2. The viral chymotrypsin-like cysteine protease (3CLpro) of SARS-CoV-2 was also inhibited by BB-PAC Fr7 in leaves and stems. These results indicate that BB-PAC has at least two different inhibitory effects, and that it is effective in suppressing SARS-CoV-2 infection regardless of the time of infection.

Adult T-cell leukemia/leukemia (ATLL) is an aggressive peripheral T-cell malignancy, caused by infection with the human T-cell leukemia virus type 1 (HTLV-1). We have recently shown that cell adhesion molecule 1 (CADM1), a member of the immunoglobulin superfamily, is specifically and consistently overexpressed in ATLL cells, and functions as a novel cell surface marker. In this study, we first show that a soluble form of CADM1 (sCADM1) is secreted from ATLL cells by mainly alternative splicing. After developing the Alpha linked immunosorbent assay (AlphaLISA) for sCADM1, we showed that plasma sCADM1 concentrations gradually increased during disease progression from indolent to aggressive ATLL. Although other known biomarkers of tumor burden such as soluble interleukin-2 receptor α (sIL-2Rα) also increased with sCADM1 during ATLL progression, multivariate statistical analysis of biomarkers revealed that only plasma sCADM1 was selected as a specific biomarker for aggressive ATLL, suggesting that plasma sCADM1 may be a potential risk factor for aggressive ATLL. In addition, plasma sCADM1 is a useful marker for monitoring response to chemotherapy as well as for predicting relapse of ATLL. Furthermore, the change in sCADM1 concentration between indolent and aggressive type ATLL was more prominent than the change in the percentage of CD4+CADM1+ ATLL cells. As plasma sCADM1 values fell within normal ranges in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients with higher levels of serum sIL-2Rα, a measurement of sCADM1 may become a useful tool to discriminate between ATLL and other inflammatory diseases, including HAM/TSP.

We previously reported that rabbit-eye blueberry (

B-Cell leukemia/lymphoma 11B (BCL11B) is a transcription factor important for T-cell development and acts as a tumor suppressor gene in T-cell acute lymphoblastic leukemia. Here, we identified BCL11B as a candidate leukemia-associated gene in human T-cell leukemia virus type 1 (HTLV-1)-induced adult T-cell leukemia/lymphoma (ATLL). Interestingly, the short form lacking exon 3 (BCL11B/S) protein was more highly expressed than the full-length BCL11B (BCL11B/L) in leukemic cells from most of the ATLL patients, although expression ratios of BCL11B/L to BCL11B/S were almost equal in control CD4

ATL (adult T-cell leukemia/lymphoma) is a highly chemoresistant malignancy of peripheral T lymphocytes caused by the HTLV-1 (human T-cell leukemia virus type 1) infection and there is an urgent need for more effective therapeutic options. The molecular chaperone HSP90 (heat shock protein 90) plays a crucial role in NF-κB (nuclear factor κB)-mediated anti-apoptosis in ATL cells, and HSP90 inhibitors are new candidate therapeutics for ATL. Accordingly, we investigated the anti-ATL effects of a novel oral HSP90 inhibitor TAS-116 (pimitespib) and the mechanisms involved in ex vivo and in vivo preclinical models. TAS-116 achieved IC50 (half maximal inhibitory concentration) values of < 0.5 μM in ten ATL-related cell lines and < 1 μM in primary peripheral blood cells of nine ATL patients; no toxicity was observed toward CD4+ lymphocytes from healthy donors, indicating the safety of this agent. Orally administered TAS-116 also exhibited significant inhibitory effects against tumor cell growth in ATL cell-xenografted mice. Furthermore, gene expression profiling of TAS-116-treated Tax-positive or -negative cell lines and primary ATL cells using DNA microarray and multiple pathway analysis revealed the significant downregulation of the NF-κB pathway in Tax-positive cells and the cell-cycle arrest in Tax-negative cells and primary ATL cells. TAS-116 suppressed the activator protein-1 and tumor necrosis factor pathways in all examined cells. These findings strongly demonstrate the efficacy of TAS-116, regardless of the stage of ATL progression, and its potential application as a novel clinical anti-ATL therapeutic agent.

MEL1 (MDS1/EVI1-like gene 1/PRDM16), a zinc finger protein, is located near the chromosomal breakpoint at 1p36 in human acute myeloid leukemia (AML) cells with the t (1; 3) (p36; q21) translocation. Mel1/Prdm16 is not only a causative gene of leukemia, but also has multiple regulatory functions, such as the regulation of fat metabolism. To investigate the function of Mel1/Prdm16, we generated Mel1/Prdm16-deficient mice, but homozygous deficiency (Mel1/Prdm16

Cell adhesion molecule 1 (CADM1) is aberrantly expressed by T-cell neoplasms such as adult T-cell leukemia/lymphoma (ATLL) and mycosis fungoides (MF). We studied the expression of CADM1 and its splicing variants in Sézary syndrome (SS), MF, other cutaneous T-cell lymphoma (CTCL), and cell lines derived from T- and B-cell lymphomas. Soluble CADM1 was measured in the patients' sera. CADM1+ cells in the blood and skin lesions were examined by flow cytometry and immunostaining, respectively. Soluble CADM1 was measured by ELISA, and the splicing variants of CADM1 transcripts were determined by reverse transcriptase-polymerase chain reaction, followed by sequencing. As a result, circulating CADM1+ cells were significantly increased in seven out of 10 patients with SS, ranging from 7.9% to 74.5% of the CD3+CD4+ fractions (median 33.7%; cut-off value 6.5%). The percentages of CADM1+ cells were usually less than those of circulating Sézary cells. CADM1 was expressed, to various degrees, in six of nine T-cell lines derived from SS, MF, ATLL, and anaplastic large cell lymphoma (ALCL), but negative in B-cell lymphoma-derived cell lines. CADM1+ cells were present in the skin infiltrates of MF, SS, ATLL and ALCL. Serum levels of soluble CADM1 were not significantly elevated in SS/MF. Three major splicing variants of CADM1 expressed by neoplastic T-cells contained different combinations of the exons 7, 8, 9 and 11, including a putative oncogenic variant composed of exons 7-8-9-11. In conclusion, CADM1 is frequently expressed in Sézary cells and cell lines from CTCL.

Metabolic reprogramming of leukemia cells is important for survival, proliferation, and drug resistance under conditions of metabolic stress in the bone marrow. Deregulation of cellular metabolism, leading to development of leukemia, occurs through abnormally high expression of transcription factors such as MYC and Ecotropic Virus Integration site 1 protein homolog (EVI1). Overexpression of EVI1 in adults and children with mixed lineage leukemia-rearrangement acute myeloid leukemia (MLL-r AML) has a very poor prognosis. To identify a metabolic inhibitor for EVI1-induced metabolic reprogramming in MLL-r AML, we used an XFp extracellular flux analyzer to examine metabolic changes during leukemia development in mouse models of AML expressing MLL-AF9 and Evi1 (Evi1/MF9). Oxidative phosphorylation (OXPHOS) in Evi1/MF9 AML cells accelerated prior to activation of glycolysis, with a higher dependency on glutamine as an energy source. Furthermore, EVI1 played a role in glycolysis as well as driving production of metabolites in the tricarboxylic acid cycle. L-asparaginase (L-asp) exacerbated growth inhibition induced by glutamine starvation and suppressed OXPHOS and proliferation of Evi1/MF9 both in vitro and in vivo; high sensitivity to L-asp was caused by low expression of asparagine synthetase (ASNS) and L-asp-induced suppression of glutamine metabolism. In addition, samples from patients with EVI1+MF9 showed low ASNS expression, suggesting that it is a sensitive marker of L-asp treatment. Clarification of metabolic reprogramming in EVI1+ leukemia cells may aid development of treatments for EVI1+MF9 refractory leukemia.

PurposeEcotropic viral integration site-1 (EVI1) is a transcription factor that contributes to the unfavorable prognosis of leukemia, some epithelial cancers, and glial tumors. However, the biological function of EVI1 in glioblastoma multiforme (GBM) remains unclear. Based on microarray experiments, EVI1 has been reported to regulate epidermal growth factor receptor (EGFR) transcription. Signal transduction via EGFR plays an essential role in glioblastoma. Therefore, we performed this study to clarify the importance ofEVI1in GBM by focusing on the regulatory mechanism between EVI1 andEGFRtranscription.MethodsWe performed immunohistochemical staining and analyzed the EVI1-expression in glioma tissue. To determine the relationship betweenEVI1andEGFR, we induced siRNA-mediated knockdown ofEVI1in GBM cell lines. To investigate the region that was essential for the EVI1 regulation ofEGFRexpression, we conducted promoter reporter assays. We performed WST-8 assay to investigate whether EVI1 affected on the proliferation of GBM cells or not.ResultsIt was observed that 22% of GBM tissues had over 33% of tumor cells expressing EVI1, whereas no lower-grade glioma tissue had over 33% by immunohistochemistry. In A172 and YKG1 cells, the expression levels of EGFR and EVI1 correlated. Analysis of theEGFRpromoter region revealed that the EGFR promoter (from − 377 to − 266 bp) was essential for the EVI regulation ofEGFRexpression. We showed that EVI1 influenced the proliferation of A172 and YKG1 cells.ConclusionThis is the first study reporting the regulation ofEGFRtranscription by EVI1 in GBM cells.

N-myc downstream-regulated gene 2 (NDRG2) is a candidate tumor suppressor that is frequently downregulated in adult T-cell leukemia/lymphoma (ATLL) and functions to negatively regulate several cellular signaling pathways as PP2A recruiter. To clarify the molecular mechanisms of suppression of NDRG2 expression, we initially determined the expression pattern of NDRG2 in various types of T-cells and ATLL cells. NDRG2 expression was significantly upregulated in HTLV-1/Tax-immortalized T-cells, which was mediated by NF-κB activation through Tax expression. On the other hand, NDRG2 expression was suppressed in HTLV-1-infected cell lines and various types of ATLL cells, which was dependent on the DNA methylation of the NDRG2 promoter. We found that the expression of enhancer of zeste homolog 2 (EZH2), a member of the polycomb family, is increased in ATLL, and that EZH2 directly binds to the NDRG2 promoter and induces DNA methylation of the NDRG2 promoter. Since the expression of EZH2 were anti-parallelly regulated with the NDRG2 expression, EZH2 might be one of the most important regulators of the downregulation of NDRG2, contributing to enhanced activation of signaling pathways during ATLL development.

Oncogenic mutations confer on cells the ability to propagate indefinitely, but whether oncogenes alter the cell fate of these cells is unknown. Here, we show that the transcriptional regulator PRDM16s causes oncogenic fate conversion by transforming cells fated to form platelets and erythrocytes into myeloid leukemia stem cells (LSCs). Prdm16s expression in megakaryocyte-erythroid progenitors (MEPs), which normally lack the potential to generate granulomonocytic cells, caused AML by converting MEPs into LSCs. Prdm16s blocked megakaryocytic/erythroid potential by interacting with super enhancers and activating myeloid master regulators, including PU.1. A CRISPR dropout screen confirmed that PU.1 is required for Prdm16s-induced leukemia. Ablating PU.1 attenuated leukemogenesis and reinstated the megakaryocytic/erythroid potential of leukemic MEPs in mouse models and human AML with PRDM16 rearrangement. Thus, oncogenic PRDM16s expression gives MEPs an LSC fate by activating myeloid gene regulatory networks.

Cancer metastasis is the most frequent cause of death for patients with cancer. The main current treatment for cancer metastasis is chemotherapy targeting cancer cells’ ability to proliferate. However, some types of cancer cells show resistance to chemotherapy. Recently, cancer cell migration has become the subject of interest as a novel target of cancer therapy. Cell migration requires many factors, such as the cytoskeleton, cell‐matrix adhesion and cell volume regulation. Here, we focus on cell volume regulation and the role of ion/water transport systems in cell migration. Transport proteins, such as ion channels, ion carriers, and aquaporins, are indispensable for cell volume regulation under steady‐state conditions and during exposure to osmotic stress. Studies from the last ~25 years have revealed that cell volume regulation also plays an important role in the process of cell migration. Water flow in accordance with localized osmotic gradients generated by ion transport contributes to the driving force for cell migration. Moreover, it has been reported that metastatic cancer cells have higher expression of these transport proteins than nonmetastatic cancer cells. Thus, ion/water transport proteins involved in cell volume regulation and cell migration could be novel therapeutic targets for cancer metastasis. In this review, after presenting the importance of ion/water transport systems in cell volume regulation, we discuss the roles of transport proteins in a pathophysiological context, especially in the context of cancer cell migration.

We had previously reported that in addition to p53 inactivation, overexpression of the DNA sensor protein—absent in melanoma 2 (AIM2)—contributes to tumorigenesis of oral squamous cell carcinoma (OSCC). Given that AIM2 is highly expressed in the OSCC tumors from patients with metastasis, we investigated whether AIM2 expression contributes to the progression of OSCC metastasis. In in vitro assays using OSCC cell lines, the high migration and invasion capacity of OSCC cells were dependent on the increased expression of AIM2, resulting in enhanced epithelial-mesenchymal transition (EMT), with EMT-related gene expression. Moreover, the in vivo short-term metastasis assay using orthotopic implantation into immunodeficient mice demonstrated that OSCC cells with high levels of AIM2 expression exhibited enhanced tumor growth in the tongue, resulting in decreased survival of the mice. Further, the cells overexpressing AIM2 dominantly invaded into the tumor lymphatic vessels, unlike OSCC cells with low AIM2 expression. Thus, the high expression of AIM2 in OSCC enhances progression of tumor growth.

Ecotropic viral integration site-1 (EVI1) has a critical role in normal and malignant hematopoiesis. Since we previously identified high expression of calcitonin receptor like receptor (CRLR) in acute myeloid leukemia (AML) with high EVI1 expression, we here characterized the function of CRLR in hematopoiesis. Since higher expression of CRLR and receptor activity modifying protein 1 (RAMP1) was identified in immature hematopoietic bone marrow (BM) cells, we focused on calcitonin gene-related peptide (CGRP), a specific ligand for the CRLR/RAMP1 complex. To elucidate the role of CGRP in hematopoiesis, Ramp1-deficient (Ramp1−/−) mice were used. The steady-state hematopoiesis was almost maintained in Ramp1−/− mice; however, the BM repopulation capacity of Ramp1−/− mice was significantly decreased, and the transplanted Ramp1−/− BM mononuclear cells had low proliferation capacity with enhanced reactive oxygen species (ROS) production and cell apoptosis. Thus, CGRP is important for maintaining hematopoiesis during temporal exposures with proliferative stress. Moreover, continuous CGRP exposure to mice for two weeks induced a reduction in the number of BM immature hematopoietic cells along with differentiated myeloid cells. Since CGRP is known to be increased under inflammatory conditions to regulate immune responses, hematopoietic exhaustion by continuous CGRP secretion under chronic inflammatory conditions is probably one of the important mechanisms of anti-inflammatory responses.

Both natural viral infections and therapeutic interventions using viral vectors pose significant risks of malignant transformation. Monitoring for clonal expansion of infected cells is important for detecting cancer. Here we developed a novel method of tracking transgene integration sites. RAISING (Rapid Amplification of Integration Sites without Interference by Genomic DNA contamination) is a sensitive, inexpensive alternative to established methods. Its compatibility with Sanger sequencing combined with our CLOVA (Clonality Value) software is critical for those without access to expensive next-generation sequencing. To model our method, we analyzed samples from 698 patients infected with the retrovirus HTLV-1, which causes adult T-cell leukemia/lymphoma (ATL). We defined a clonality value identifying ATL patients with 100% sensitivity and 95.3% specificity, and our preliminary longitudinal analysis suggests this may also be useful for ATL risk assessment. We anticipate future studies will confirm the broad applicability of our technology, especially in the emerging gene therapy sector.

Both natural viral infections and therapeutic interventions using viral vectors pose significant risks of malignant transformation. Monitoring for clonal expansion of infected cells is important for detecting cancer. Here we developed a novel method of tracking clonality via the detection of transgene integration sites. RAISING (Rapid Amplification of Integration Sites without Interference by Genomic DNA contamination) is a sensitive, inexpensive alternative to established methods. Its compatibility with Sanger sequencing combined with our CLOVA (Clonality Value) software is critical for those without access to expensive high throughput sequencing. We analyzed samples from 688 individuals infected with the retrovirus HTLV-1, which causes adult T-cell leukemia/lymphoma (ATL) to model our method. We defined a clonality value identifying ATL patients with 100% sensitivity and 94.8% specificity, and our longitudinal analysis also demonstrates the usefulness of ATL risk assessment. Future studies will confirm the broad applicability of our technology, especially in the emerging gene therapy sector.

Aims Cell adhesion molecule 1 (CADM1) mediates interepithelial adhesion and is upregulated in crowded epithelial monolayers. This study aimed to examine CADM1 expression in the human endometrium of proliferative and secretory phases, and its transcriptional regulation in terms of estrogen stimuli and higher cellularity. Main methods CADM1 immunohistochemistry was conducted on endometrial tissues from women in their 40s and adult mice subcutaneously injected with estradiol following ovariectomy. Dual-luciferase reporter assays were conducted using human endometrial HEC-50B and HEC-1B cells and reporter plasmids harboring the human CADM1 3.4-kb promoter and its deleted and mutated forms. Cells were transfected with estrogen receptor α cDNA and reporter plasmids, and treated with estradiol before luciferase activity measurement. Key findings Immunohistochemistry revealed that CADM1 was clearly expressed on the lateral membranes of the simple columnar glandular cells in the proliferative phase, but not in the secretory phase, from both women and the mouse model. The glandular cell density increased two-fold in the proliferative phase. Reporter assays identified three Sp1-binding sites as estradiol-responsive elements in the proximal region (from −223 to −84) of the transcription start site (+1) in HEC-50B cells. When the cell culture was started at eight-fold higher cell density, the CADM1 3.4-kb promoter was transactivated at a two-fold higher level in HEC-50B cells. This cell density effect was not detected for the CADM1 2.3-kb or 1.6-kb promoter. Significance Two (proximal and distal) promoter regions are suggested to function additively to transactivate CADM1 in endometrial glandular cells that crowd in the proliferative phase.

Cells are under threat of osmotic perturbation; cell volume maintenance is critical in cerebral edema, inflammation and aging, in which prominent changes in intracellular or extracellular osmolality emerge. After osmotic stress-enforced cell swelling or shrinkage, the cells regulate intracellular osmolality to recover their volume. However, the mechanisms recognizing osmotic stress remain obscured. We previously clarified that apoptosis signal-regulating kinase 3 (ASK3) bidirectionally responds to osmotic stress and regulates cell volume recovery. Here, we show that macromolecular crowding induces liquid-demixing condensates of ASK3 under hyperosmotic stress, which transduce osmosensing signal into ASK3 inactivation. A genome-wide small interfering RNA (siRNA) screen identifies an ASK3 inactivation regulator, nicotinamide phosphoribosyltransferase (NAMPT), related to poly(ADP-ribose) signaling. Furthermore, we clarify that poly(ADP-ribose) keeps ASK3 condensates in the liquid phase and enables ASK3 to become inactivated under hyperosmotic stress. Our findings demonstrate that cells rationally incorporate physicochemical phase separation into their osmosensing systems., Cells experience various osmotic perturbation, but cellular osmosensing mechanisms remain obscure. Here, the authors report that cells recognize osmotic stress from the inside through macromolecular crowding-driven and poly(ADP-ribose)-conditioned liquid–liquid phase separation.