ASCL1 regulates super-enhancer-associated miRNAs to define molecular subtypes of small cell lung cancer.

Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine tumor with dismal prognosis. Recently, molecular subtypes of SCLC have been defined by the expression status of ASCL1, NEUROD1, YAP1, and POU2F3 transcription regulators. ASCL1 is essential for neuroendocrine differentiation and is expressed in the majority of SCLC. Although previous studies investigated ASCL1 target genes in SCLC cells, ASCL1-mediated regulation of miRNAs and its relationship to molecular subtypes remain poorly explored. Here, we performed genome-wide profiling of chromatin modifications (H3K27me3, H3K4me3, and H3K27ac) by CUT&Tag assay and ASCL1 knockdown followed by RNA sequencing and miRNA array analyses in SCLC cells. ASCL1 could preferentially regulate genes associated with super-enhancers (SEs) defined by enrichment of H3K27ac marking. Moreover, ASCL1 positively regulated several SE-associated miRNAs, such as miR-7, miR-375, miR-200b-3p, and miR-429, leading to repression of their targets, whereas ASCL1 suppressed miR-455-3p, an abundant miRNA in other molecular subtypes. We further elucidated unique patterns of SE-associated miRNAs in different SCLC molecular subtypes, highlighting subtype-specific miRNA networks with functional relevance. Notably, we found apparent de-repression of common target genes of different miRNAs following ASCL1 knockdown, suggesting combinatorial action of multiple miRNAs underlying molecular heterogeneity of SCLC (e.g., co-targeting of YAP1 by miR-9 and miR-375). Our comprehensive analyses provide novel insights into SCLC pathogenesis and a clue to understanding subtype-dependent phenotypic differences.

ASCL1 promotes tumor progression through cell-autonomous signaling and immune modulation in a subset of lung adenocarcinoma.

The master regulator of neuroendocrine differentiation, achaete-scute complex homolog 1 (ASCL1) defines a subgroup of lung adenocarcinoma. However, the mechanistic role of ASCL1 in lung tumorigenesis and its relation to the immune microenvironment is principally unknown. Here, the immune landscape of ASCL1-positive lung adenocarcinomas was characterized by immunohistochemistry. Furthermore, ASCL1 was transduced in mouse lung adenocarcinoma cell lines and comparative RNA-sequencing and secretome analyses were performed. The effects of ASCL1 on tumorigenesis were explored in an orthotopic syngeneic transplantation model. ASCL1-positive lung adenocarcinomas revealed lower infiltration of CD8+, CD4+, CD20+, and FOXP3+ lymphocytes and CD163+ macrophages indicating an immune desert phenotype. Ectopic ASCL1 upregulated cyclin transcript levels, stimulated cell proliferation, and enhanced tumor growth in mice. ASCL1 suppressed secretion of chemokines, including CCL20, CXCL2, CXCL10, and CXCL16, indicating effects on immune cell trafficking. In accordance with lower lymphocytes infiltration, ASCL1-positive lung adenocarcinomas demonstrated lower abundance of CXCR3-and CCR6-expressing cells. In conclusion, ASCL1 mediates its tumor-promoting effect not only through cell-autonomous signaling but also by modulating chemokine production and immune responses. These findings suggest that ASCL1-positive tumors represent a clinically relevant lung cancer entity.

Patency Capsule Aspiration.

A 77-year-old man with anemia who had undergone 2 abdominal surgeries for colon and gastric cancer experienced dyspnea after swallowing a patency capsule before endoscopy for investigating the cause of anemia. Chest radiography and computed tomography revealed that the patency capsule was located within the bronchus intermedius. It was successfully removed by flexible bronchoscopy. The balloon was placed over the capsule and inflated. Subsequently, the catheter was pulled, while thus dragging the capsule with it and preventing its destruction. In cases of patency capsule aspiration, the capsule must be removed without deformity, before it causes inflammation by releasing barium into the airway.

Pulmonary Adenocarcinoma, Harboring Both an EGFR Mutation and ALK Rearrangement, Presenting a Stable Disease to Erlotinib and a Partial Response to Alectinib.

A 63-year-old woman with pulmonary adenocarcinoma (stage IIIB) that was positive for an epidermal growth factor receptor (EGFR) mutation and an anaplastic lymphoma kinase (ALK) rearrangement was treated with erlotinib as the first-line treatment, resulting in a stable disease. Due to skin rashes, fatigue and anorexia, erlotinib was suspended on erlotinib day 44. Alectinib was administered as the second-line treatment, exhibiting a partial response. On alectinib day 56, drug-induced lung injury forced suspension of alectinib, which was cured with corticosteroid therapy. ALK-tyrosine kinase inhibitors may be more effective for patients positive for both EGFR mutation and ALK rearrangement than other agents.

Women with Subclinical Hypothyroidism Are at Low Risk of Poor Pregnancy Outcome in Japan.

Maternal subclinical hypothyroidism may be associated with adverse pregnancy outcomes, although not consistently across regions. Here, we sought to determine the effect of elevated thyroid-stimulating hormone (TSH) on pregnancy outcomes in Japanese women without known medical complications. TSH was determined by dried blood spots at 8-20 weeks of gestation, and 3.0-10.0 µU/mL of TSH was considered as elevated TSH (eTSH). A retrospective study involving 167 cases of eTSH was conducted. Five hundred and seventy eight of controls with normal TSH and without thyroid antibodies were selected. We compared a composite adverse maternal outcome comprised of spontaneous abortion, premature delivery, gestational diabetes mellitus (GDM), placental abruption, and pregnancy-induced hypertension, as well as composite adverse neonatal outcome including stillbirths, heavy for date, light for date, and a low Apgar score (< 7) at 5 minutes between two groups. The incidence of GDM was significantly higher in eTSH (p < 0.01); however, composite adverse maternal and neonatal outcome did not differ between groups (p = 0.19 and p = 0.50, respectively). Although 27 out of 167 cases in eTSH have antibodies, composite adverse outcome did not differ between eTSH with antibodies and controls (p = 0.64 and p = 0.50, respectively). Additionally, composite adverse maternal and neonatal outcome did not differ between the group larger than the median of TSH in eTSH (n = 81) and controls (p = 0.43 and p = 0.98, respectively). Thus, elevated TSH is not associated with overall adverse pregnancy outcomes in women without known medical complications.

Age-related expression analysis of mouse liver nuclear protein binding to 3'-untranslated region of Period2 gene.

In mammals, both circadian rhythm and aging play important roles in regulating time-dependent homeostasis. We previously discovered an age-related increase element binding protein, hnRNP A3, which binds to the 3'-untranslated region (UTR) of blood coagulation factor IX (FIX). Here, we describe other members of this protein family, hnRNP C and hnRNP H, which bind to the 3'-UTR of the mouse circadian clock gene Period 2 (mPer2). RNA electrophoretic mobility shift assays using a (32)P-labeled Per2 RNA probe coupled with two-dimensional gel electrophoresis followed by MALDI-TOF/MS peptide mass fingerprint analysis was used to analyze these proteins. Western blotting suggested that the total expression of these proteins in mouse liver cell nuclei does not increase with age. Two-dimensional gel electrophoresis analysis of age-related protein expression showed that many isoforms of these proteins exist in the liver and that each protein exhibits a complex age-related expression pattern. These results suggest that many isoforms of proteins are regulated by different aging systems and that many age regulation systems function in the liver.

ORL1 receptor-mediated down-regulation of mPER2 in the suprachiasmatic nucleus accelerates re-entrainment of the circadian clock following a shift in the environmental light/dark cycle.

The circadian pacemaker in the suprachiasmatic nucleus (SCN) generates the near 24-h period of the circadian rhythm and is entrained to the 24-h daily cycle by periodic environmental signals, such as the light/dark cycle (photic signal), and can be modulated by various drugs (non-photic signals). The mechanisms by which non-photic signals modulate the circadian clock are not well understood in mice. In mice, many reportedly non-photic stimuli have little effect on the circadian rhythm in vivo. Herein, we investigated the molecular mechanism in W-212393-induced phase advance using mice. W-212393 caused a significant phase advance of locomotor activity rhythm in mice at subjective day. Injection of W-212393 during subjective day elicited down-regulation of mPER2 protein in the SCN shell region, but not mPer2 mRNA. Administration of W-212393 during subjective day failed to produce phase advance in mPer2-mutant mice as well as in ORL1 receptor deficient mice. Furthermore, we show that such inhibition of mPER2 accelerates re-entrainment of the circadian clock following an abrupt shift in the environmental light/dark cycle, such as occurs with transmeridian flight. The present results suggest that post-translational down-regulation of mPER2 protein in the shell region of mouse SCN may be involved in W-212393-induced non-photic phase advance.

The role of Clock in the plasticity of circadian entrainment.

The mammalian circadian clock lying in suprachiasmatic nucleus (SCN) is synchronized to about 24 h by the environmental light-dark cycle (LD). The circadian clock exhibits limits of entrainment above and below 24 h, beyond which it will not entrain. Little is known about the mechanisms regulating the limits of entrainment. In this study, we show that wild-type mice entrain to only an LD 24 h cycle, whereas Clock mutant mice can entrain to an LD 24, 28, and 32 h except for LD 20 h and LD 36 h cycle. Under an LD 28 h cycle, Clock mutant mice showed a clear rhythm in Per2 mRNA expression in the SCN and behavior. Light response was also increased. This is the first report to show that the Clock mutation makes it possible to adapt the circadian oscillator to a long period cycle and indicates that the clock gene may have an important role for the limits of entrainment of the SCN to LD cycle.

Effect of lithium on the circadian rhythms of locomotor activity and glycogen synthase kinase-3 protein expression in the mouse suprachiasmatic nuclei.

A circadian clock located in the suprachiasmatic nucleus (SCN) regulates the period of physiological and behavioural rhythms to approximately 24 h. Lithium can lengthen the period of circadian rhythms in most organisms although little is known about the underlying mechanism. In the present study, we examined Drosophila shaggy ortholog glycogen synthase kinase-3 (GSK-3) protein expression in the SCN after lithium treatment. When locomotor activity was assessed, we found an association between the effect of lithium and the period of circadian oscillation as well as the level of GSK-3 protein expression. The decreased expression of GSK-3 and increased expression of phosphorylated GSK-3 (pGSK-3) resulted in an antiphasic circadian rhythm between the two in the SCN of lithium-treated mice housed under both light-dark and constant dark conditions. The enzyme activity of GSK-3 in the SCN was low when the level of pGSK-3 protein was high, as examined by immunoblotting analysis. Thus, GSK-3 enzyme activity has a correlation with the expression of GSK-3 protein in the SCN. Although both GSK-3 and pGSK-3 proteins are also expressed in the arcuate nucleus, lithium did not affect their expression. Based on the association that we found between lengthened circadian period and GSK-3 protein and GSK-3 activity in the SCN, we suggest that GSK-3 plays a role in regulating the period of the mammalian circadian pacemaker.

Secretion of FGF-16 requires an uncleaved bipartite signal sequence.

Fibroblast growth factor (FGF)-16 is one of the rare secreted proteins that do not possess a cleavable signal sequence. Here we describe our examination of the mechanism and structural requirements for the secretion of FGF-16 from COS-1 transfectants. Inhibition of its secretion by brefeldin A and identification of an N-glycan on the secreted form confirmed that FGF-16 is secreted by means of the endoplasmic reticulum and Golgi apparatus, as are secreted proteins having a conventional cleavable signal sequence. Deletion of its N terminus abolished secretion of FGF-16. When chimerized with prolactin, however, the N-terminal sequence of FGF-16 was not able to mediate secretion of the chimera. Point mutations that made the N terminus less hydrophobic had little effect on secretion of FGF-16, whereas making the central hydrophobic region less hydrophobic abolished secretion. Within cells, an unsecretable FGF-16 N-terminal deletion mutant was distributed in the perinuclear region and overlapped the distribution of the Golgi apparatus. Mutants with less hydrophobic central regions were distributed evenly throughout the cytosol. Collectively, these results indicate that FGF-16 employs a unique bipartite signal sequence (i.e. both the N-terminal region and central hydrophobic region) that is not cleaved, although it shares the same secretory machinery used by secreted proteins with cleavable signal sequences.