👤 Koichi Hagiwara

Large cell neuroendocrine carcinoma (LCNEC) is a neuroendocrine carcinoma (NEC) of the lung that is characterized by its heterogeneous morphology, diverse immunophenotypes, and complex genomic profiles. Among LCNECs, a subset expressing the transcription factor POU2F3 (LCNEC-P) has been suggested to share similarities with small cell lung carcinoma (SCLC)-P, a subtype of SCLC defined by POU2F3 expression. However, the specific characteristics of LCNEC-P have not been fully elucidated. Therefore, the aim of the present study is to clarify the clinicopathological, immunohistochemical, and genetic characteristics of LCNEC-P. Fifty-six LCNEC cases were analyzed, including 12 LCNEC-P and 44 LCNEC-non-P cases. Morphologically, LCNEC-P exhibited significantly lower cytomorphology scores, indicating a resemblance to SCLC. Immunohistochemically, LCNEC-P showed the lower expression of neuroendocrine markers (SYP, CHGA, and INSM1), but the higher expression of C-MYC than LCNEC-non-P. A strong mutually exclusive expression pattern was observed between POU2F3 and ASCL1/NEUROD1. Whole-genome sequencing of 20 cases revealed that LCNEC-P harbored RB1 mutations in 100 % of cases, which was significantly higher than in LCNEC-non-P (40 %). FGFR1 amplification was observed in 60 % of LCNEC-P cases, representing a higher prevalence than previously reported for LCNEC. In addition, LCNEC-P showed a distinct copy number alteration profile, including frequent 20q13 amplification, compared with LCNEC-non-P. These results demonstrate that LCNEC-P represents a distinct subgroup of LCNEC that is characterized by a specific morphological, immunohistochemical, and genetic profile, closely resembling SCLC-P. This study provides insights into the biology of LCNEC-P and supports its classification as a unique entity within LCNEC. Show less

Previously, we advocated the importance of classifying hepatocellular carcinoma (HCC) based on physiological functions. This study aims to classify HCC by focusing on liver-intrinsic metabolism and glycolytic pathway in cancer cells. Comprehensive RNA/DNA sequencing, immunohistochemistry, and radiological evaluations were performed on HCC tissues from the training cohort (n=136) and validated in 916 public samples. HCC was classified using hierarchical clustering and compared with previous molecular, histopathological, and hemodynamic classifications. Liver-specific metabolism and glycolysis are mutually exclusive and were divided into two major subclasses: The "rich metabolism" subclass (60.3%) is characterized by enhanced bile acid and fatty acid metabolism, wellto-moderate differentiation, microtrabecular or pseudoglandular pattern, and homogeneous arterial-phase hyperenhancement (APHE), corresponding to Hoshida S3 with favorable prognosis. In IL6-JAK-STAT3-high (25.0%) conditions, upregulated ALB expression, enhanced gluconeogenesis and urea cycle activity, and an inflammatorymicroenvironment are observed. Conversely, the Wnt/β-catenin-high environment (19.9%) features elevated GLUL, APOB and CYP3A4 expression, frequent CTNNB1 (D32-S37) mutations, and an immune-desert/excluded phenotype. The "glycolysis" subclass (39.7%), characterized by histopathological dedifferentiation and downregulated liver-specific metabolism, encompasses subclasses with PI3K/mTOR (20.6%) and NOTCH/TGF-β (19.1%) signaling. These often exhibit TP53 mutations, macrotrabecular massive or compact patterns, inhomogeneous/rim-APHE, and high expression of hypoxia-inducible factors and glucose transporters, corresponding to Hoshida S1/2 with poor prognosis. The loss of liver-specific metabolism correlates with morphological dedifferentiation, indicating cellular dedifferentiation may exhibit both physiological and pathological duality. Key signaling pathways involved in the maturation process from fetal to adult liver and zonation program may play a critical role in defining HCC diversity. Show less

Apigenin, found in a variety of vegetables and fruits, exhibits anti-oxidant, anti-inflammatory and anticancer effects. Recently, we reported the possibility that apigenin induces apoptosis in human lung adenocarcinoma A549 cells through the miR-34a-5p/SNAI1/caspase-3/-7 pathway. Understanding how apigenin triggers apoptosis in cancer cells will help lay the groundwork for developing effective cancer treatments. The lung adenocarcinoma A549 Cell line was used. To determine whether caspase-8 or caspase-9 is activated, we performed a caspase activity assay. Real-time qRT-PCR was performed to identify mRNAs that may stimulate the upstream pathways, including tumor necrosis factor-a (TNF-a), spondin-2 (SPON2), and interferon-a2 (IFNA2), which are known to be involved in apoptosis in various cancer cell lines. In apigenin-treated cells, early-stage apoptosis was observed at 24 h, with increased activity of caspase-8 at 18 h and again at 24 h, and caspase-9 at 24 h and further at 48 h. However, mRNA levels of caspase-8 and caspase-9 significantly decreased after 24 h. Real-time RT-qPCR analysis revealed increased mRNA levels of TNF-a, spondin-2, and interferon-a2 after 24 h of apigenin treatment in A549 cells, whereas treatment for 48 h led to decreased expression of SPON2 and IFNA2. Apigenin promotes apoptosis in A549 cells by modulating various signaling pathways at different time points. Show less

Immunotherapy is becoming a promising approach for unresectable-hepatocellular carcinoma (HCC); the anti-tumor response is affected by the tumor microenvironment (TME). Although Wnt/β-catenin mutations are reported to cause non-inflamed phenotype, their role on TME remains controversial. We aimed to clarify the heterogeneity of immunophenotype in HCC with Wnt/β-catenin mutations. This study includes 152 resected HCCs; mutations in the Forty of 152 (26.3%) HCCs carried the Wnt/β-catenin mutations. Of these, 33 were classified as non-inflamed (33/40, 82.5%) and 7 as inflamed (7/40, 17.5%). Non-inflamed class was characterized by low number of CD3+, CD4+, and CD8+ cells on immunostaining, and high mRNA expressions of Heterogeneity of tumor traits and TME was observed in HCC with Wnt/β-catenin mutation. The potential was indicated that tumor traits and TME are determined not only by the activation of the Show less

Apigenin is a flavonoid with antioxidant and anticancer effects. It has been reported that apigenin inhibits proliferation, migration, and invasion and induces apoptosis in cultured lung cancer cells. However, there is little information on the involvement of microRNAs (miRNAs) in its effects. miRNA microarray analysis and polymerase-chain-reaction analysis of miRNAs revealed that treatment of human lung cancer A549 cells with apigenin up-regulated the level of miR-34a-5p. Furthermore, mRNA microarray analysis and the results of three microRNA target prediction tools showed that Snail Family Transcriptional Repressor 1 (SNAI1), which inhibits the induction of apoptosis, had its mRNA expression down-regulated in A549 cells treated with apigenin. Our findings suggest that apigenin might induce apoptosis by down-regulation of SNAI1 through up-regulation of miR-34a-5p in A549 cells. Show less

Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are genetically and phenotypically heterogeneous. Cardiac function is improved after treatment in some cardiomyopathy patients, but little is known about genetic predictors of long-term outcomes and myocardial recovery following medical treatment. To elucidate the genetic basis of cardiomyopathy in Japan and the genotypes involved in prognosis and left ventricular reverse remodeling (LVRR), we performed targeted sequencing on 120 DCM (70 sporadic and 50 familial) and 52 HCM (15 sporadic and 37 familial) patients and integrated their genotypes with clinical phenotypes. Among the 120 DCM patients, 20 (16.7%) had TTN truncating variants and 13 (10.8%) had LMNA variants. TTN truncating variants were the major cause of sporadic DCM (21.4% of sporadic cases) as with Caucasians, whereas LMNA variants, which include a novel recurrent LMNA E115M variant, were the most frequent in familial DCM (24.0% of familial cases) unlike Caucasians. Of the 52 HCM patients, MYH7 and MYBPC3 variants were the most common (12 (23.1%) had MYH7 variants and 11 (21.2%) had MYBPC3 variants) as with Caucasians. DCM patients harboring TTN truncating variants had better prognosis than those with LMNA variants. Most patients with TTN truncating variants achieved LVRR, unlike most patients with LMNA variants. Show less

Preparing targeted cells for medical applications from human induced pluripotent stem cells (hiPSCs) using growth factors, compounds, or gene transfer has been challenging. Here, we report that human induced hepatic lineage-oriented stem cells (hiHSCs) were generated and expanded as a new type of hiPSC under non-typical coculture with feeder cells in a chemically defined hiPSC medium at a very high density. Self-renewing hiHSCs expressed markers of both human embryonic stem cells (hESCs) and hepatocytes. Those cells were highly expandable, markedly enhancing gene expression of serum hepatic proteins and cytochrome P450 enzymes with the omission of FGF-2 from an undefined hiPSC medium. The hepatic specification of hiHSCs was not attributable to the genetic and epigenetic backgrounds of the starting cells, as they were established from distinct donors and different types of cells. Approximately 90% of hiHSCs autonomously differentiated to hepatocyte-like cells, even in a defined minimum medium without any of the exogenous growth factors necessary for hepatic specification. After 12 days of this culture, the differentiated cells significantly enhanced gene expression of serum hepatic proteins (ALB, SERPINA1, TTR, TF, FABP1, FGG, AGT, RBP4, and AHSG), conjugating enzymes (UGT2B4, UGT2B7, UGT2B10, GSTA2, and GSTA5), transporters (SULT2A1, SLC13A5, and SLCO2B1), and urea cycle-related enzymes (ARG1 and CPS1). In addition, the hepatocyte-like cells performed key functions of urea synthesis, albumin secretion, glycogen storage, indocyanine green uptake, and low-density lipoprotein uptake. The autonomous hepatic specification of hiHSCs was due to their culture conditions (coculture with feeder cells in a defined hiPSC medium at a very high density) in self-renewal rather than in differentiation. These results suggest the feasibility of preparing large quantities of hepatocytes as a convenient and inexpensive hiPSC differentiation. Our study also suggests the necessity of optimizing culture conditions to generate other specific lineage-oriented hiPSCs, allowing for a very simple differentiation. Show less

The ectopic expression of the glucose-dependent insulinotropic polypeptide receptor (GIPR) in the human adrenal gland causes significant hypercortisolemia after ingestion of each meal and leads to Cushing's syndrome, implying that human GIPR activation is capable of robustly activating adrenal glucocorticoid secretion. In this study, we transiently transfected the human GIPR expression vector into cultured human adrenocortical carcinoma cells (H295R) and treated them with GIP to examine the direct link between GIPR activation and steroidogenesis. Using quantitative RT-PCR assay, we examined gene expression of steroidogenic related proteins, and carried out immunofluorescence analysis to prove that forced GIPR overexpression directly promotes production of steroidogenic enzymes CYP17A1 and CYP21A2 at the single cell level. Immunofluorescence showed that the transfection efficiency of the GIPR gene in H295R cells was approximately 5%, and GIP stimulation enhanced CYP21A2 and CYP17A1 expression in GIPR-introduced H295R cells (H295R-GIPR). Interestingly, these steroidogenic enzymes were also expressed in the GIPR (-) cells adjacent to the GIPR (+) cells. The mRNA levels of a cholesterol transport protein required for all steroidogenesis, StAR, and steroidogenic enzymes, HSD3β2, CYP11A1, CYP21A2, and CYP17A1 increased 1.2-2.1-fold in GIP-stimulated H295R-GIPR cells. These changes were reflected in the culture medium in which 1.5-fold increase in the cortisol concentration was confirmed. Furthermore, the levels of adenocorticotropic hormone (ACTH) receptor and ACTH precursor proopiomelanocortin (POMC) mRNA were upregulated 2- and 1.5-fold, respectively. Immunofluorescence showed that ACTH expression was detected in GIP-stimulated H295R-GIPR cells. An ACTH-receptor antagonist significantly inhibited steroidogenic gene expression and cortisol production. Immunostaining for both CYP17A1 and CYP21A2 was attenuated in cells treated with ACTH receptor antagonists as well as with POMC siRNA. These results demonstrated that GIPR activation promoted production and release of ACTH, and that steroidogenesis is activated by endogenously secreted ACTH following GIP administration, at least in part, in H295R cells. Show less

The sleep-deprivation-induced changes in delta power, an electroencephalographical correlate of sleep need, and brain transcriptome profiles have importantly contributed to current hypotheses on sleep function. Because sleep deprivation also induces stress, we here determined the contribution of the corticosterone component of the stress response to the electrophysiological and molecular markers of sleep need in mice. N/A SETTINGS: Mouse sleep facility. C57BL/6J, AKR/J, DBA/2J mice. Sleep deprivation, adrenalectomy (ADX). Sleep deprivation elevated corticosterone levels in 3 inbred strains, but this increase was larger in DBA/2J mice; i.e., the strain for which the rebound in delta power after sleep deprivation failed to reach significance. Elimination of the sleep-deprivation-associated corticosterone surge through ADX in DBA/2J mice did not, however, rescue the delta power rebound but did greatly reduce the number of transcripts affected by sleep deprivation. Genes no longer affected by sleep deprivation cover pathways previously implicated in sleep homeostasis, such as lipid, cholesterol (e.g., Ldlr, Hmgcs1, Dhcr7, -24, Fkbp5), energy and carbohydrate metabolism (e.g., Eno3, G6pc3, Mpdu1, Ugdh, Man1b1), protein biosynthesis (e.g., Sgk1, Alad, Fads3, Eif2c2, -3, Mat2a), and some circadian genes (Per1, -3), whereas others, such as Homer1a, remained unchanged. Moreover, several microRNAs were affected both by sleep deprivation and ADX. Our findings indicate that corticosterone contributes to the sleep-deprivation-induced changes in brain transcriptome that have been attributed to wakefulness per se. The study identified 78 transcripts that respond to sleep loss independent of corticosterone and time of day, among which genes involved in neuroprotection prominently feature, pointing to a molecular pathway directly relevant for sleep function. Show less