YTHDC2 Promotes Malignant Phenotypes of Breast Cancer Cells.

YTH domain-containing 2 (YTHDC2) is known to be an important regulator for RNA metabolism. Here, we show that YTHDC2 is essential for breast cancer tumorigenesis and metastasis. We examined YTHDC2 expression levels by immunohistochemistry in human breast tumor tissues from 99 patients and found a significantly positive correlation between the YTHDC2 expression level and the tumor stage. We established YTHDC2-knocked-down cell lines using four breast cancer cell lines with different subtypes. Knockdown of YTHDC2 attenuated the sphere-forming and the metastatic ability of breast cancer cells. Although stemness and EMT markers, such as SOX2, c-MYC, and NANOG, were downregulated in several YTHDC2-knocked-down breast cancer cells, a common target gene of YTHDC2 in breast cancer cells was not identified. These findings suggest that while YTHDC2 is involved in malignant progression of breast cancers, the mechanism by which YTHDC2 regulates those phenotypes is different between subtypes of breast cancers.

SQAP, an acyl sulfoquinovosyl derivative, suppresses expression of histone deacetylase and induces cell death of cancer cells under hypoxic conditions.

Sulfoglycolipid, SQAP, is a radiosensitizing agent that makes tumor cells more sensitive to radiation therapy. A previous study revealed that SQAP induced the degradation of hypoxia-inducible factor-1α (HIF-1α) and inhibited angiogenesis in a hepatoma model mouse. Herein, we examined the biological activities of SQAP against hepatocarcinoma cells under low oxygen conditions. Cell growth inhibition of SQAP under hypoxic conditions was significantly higher than that under normoxic conditions. In addition, SQAP was found to impair the expression of histone deacetylase (HDAC) under low oxygen conditions. Our present data suggested that SQAP induced the degradation of HIF-1α and then decreased the expression of HDAC1. Unlike known HDAC inhibitors, SQAP increased the acetylation level of histone in cells without inhibition of enzymatic activity of HDACs. Our data demonstrated hypoxia-specific unique properties of SQAP.

Functional analysis of CD44 variants and xCT in canine tumours.

The cell surface glycoprotein CD44 has various types of splicing variants, which contribute to its multiple distinct cellular functions. Recently, it was reported that the CD44v8-10 isoform interacts with the system Xc(-) transporter-related protein (xCT), and inhibits the accumulation of reactive oxygen species by promoting the synthesis of the antioxidant glutathione in human tumour cells. In this study, we investigated the expression and function of CD44 variants and xCT in canine tumours. From semi-quantitative reverse transcription polymerase chain reaction analysis, the mRNA expression of the CD44v8-10 isoform was observed in canine tumour tissues as well as human cases. The overexpression of CD44v8-10 may promote the synthesis of glutathione and enhance the resistance to radiation of canine breast tumour cells. Furthermore, canine xCT mRNA expression was significantly upregulated in the canine breast tumour tissues as compared to the normal tissues surrounding the tumours. To investigate the function of canine xCT, we treated canine tumour cells with the xCT inhibitor sulfasalazine. Consequently, the sulfasalazine-treated cells were more sensitive to oxidative stress than the non-treated cells. Taken together, these results suggested that CD44v8-10 and xCT play important roles in the therapy resistance of canine tumours as well as human tumours.

The Metabolic Heterogeneity and Flexibility of Cancer Stem Cells.

Numerous findings have indicated that CSCs, which are present at a low frequency inside primary tumors, are the main cause of therapy resistance and cancer recurrence. Although various therapeutic methods targeting CSCs have been attempted for eliminating cancer cells completely, the complicated characteristics of CSCs have hampered such attempts. In analyzing the biological properties of CSCs, it was revealed that CSCs have a peculiar metabolism that is distinct from non-CSCs to maintain their stemness properties. The CSC metabolism involves not only the catabolic and anabolic pathways, but also intracellular signaling, gene expression, and redox balance. In addition, CSCs can reprogram their metabolism to flexibly respond to environmental changes. In this review, we focus on the flexible metabolic mechanisms of CSCs, and highlight the new therapeutics that target CSC metabolism.

PU.1 plays a pivotal role in dendritic cell migration from the periphery to secondary lymphoid organs via regulating CCR7 expression.

C-C chemokine receptor type 7 (CCR7) is essential for migration of dendritic cells (DCs) to draining lymph nodes. PU.1/Spi1 is a transcription factor playing a critical role in the gene regulation of DCs. PU.1 knockdown decreased the expression of CCR7 in bone marrow-derived DCs and subsequently attenuated migration in vitro and in vivo. Reporter assays, EMSA, and chromatin immunoprecipitation assays revealed that PU.1 binds to the most proximal Ets motif of the Ccr7 promoter, which is involved in transcriptional activation. The CCR7 expression level, which was higher in the programmed cell death 1 ligand 2 (PD-L2)+ population than in the PD-L2- population and was markedly suppressed by TGF-ß treatment, coincided with the binding level of PU.1 to the Ccr7 promoter. The PU.1 binding level in CCR7high mesenteric lymph nodes DCs was higher than in other DC subtypes. The involvement of PU.1 in the expression of the CCR7 gene was also observed in human DCs. We conclude that PU.1 plays a pivotal role in DC migration by transactivating the CCR7 gene via the Ets motif in the promoter in both humans and mice.-Yashiro, T., Takeuchi, H., Nakamura, S., Tanabe, A., Hara, M., Uchida, K., Okumura, K., Kasakura, K., Nishiyama, C. PU.1 plays a pivotal role in dendritic cell migration from the periphery to secondary lymphoid organs via regulating CCR7 expression.

Angiogenesis-related gene expression profile in clinical cases of canine cancer.

The balance between pro- and anti-angiogenic signalling is tightly regulated in normal tissues to maintain the functions of the vasculature. In contrast, the overproduction of angiogenic factors and enhanced angiogenesis are frequently observed in several types of tumours. Although there have been many reports on the correlation between tumour progression and angiogenesis in humans, little is known about tumour angiogenesis in canines. Hence, we attempted to clarify whether angiogenesis contributes to tumour progression in canines as well as humans. In this study, we investigated the expression of several angiogenesis-related genes, including CD34, VEGF-A, VEGFR-1, VEGFR-2, Ang-1, Ang-2, Tie1, and Tie2, in 66 canine tumour tissues and in the normal tissues surrounding the tumours by quantitative real-time PCR analysis. Our comparative analysis between canine tumour tissues and normal tissues revealed that several angiogenesis-related genes, such as vascular endothelial growth factor (VEGF) and VEGF-receptor genes, were significantly upregulated in canine tumour tissues when compared to the normal tissues. We also found that the angiopoietin (Ang)-1/Ang-2 gene expression ratio was lower in canine tumour tissues than in the normal tissues, suggesting less association between vascular endothelial cells and perivascular cells in the canine tumour tissues. Taken together, our results suggest that several angiogenesis-related genes may contribute to the malignant progression of canine tumours via tumour angiogenesis.

Nonbacterial thrombotic endocarditis involving all four cardiac valves.

This report describes a case of nonbacterial thrombotic endocarditis caused by Waldenström macroglobulinemia, with diffuse endocardial lesions and involvement of all 4 cardiac valves. A 77-year-old man presented with heart failure due to severe regurgitation of all 4 cardiac valves; surgical repair using bioprosthetic valves was indicated. A pathological study revealed fibrin-triggered thrombus formation that confirmed the diagnosis of nonbacterial thrombotic endocarditis. In cases of nonbacterial thrombotic endocarditis, the underlying cause should be investigated.

RNA helicase YTHDC2 promotes cancer metastasis via the enhancement of the efficiency by which HIF-1α mRNA is translated.

YTH domain containing 2 (YTHDC2) is a member of the DExD/H-box family of ATP-dependent RNA helicases. We previously found that YTHDC2 expression is up-regulated in several human cancer cells. In this study, we demonstrate novel roles for YTHDC2 in metastasis of colon tumor cells via translation-dependent pathway. Knockdown of YTHDC2 attenuated protein expression of metastasis-related genes, such as hypoxia-inducible factor-1alpha (HIF-1α), and inhibited metastasis of colon tumor cells in vitro and in vivo. To confirm that YTHDC2 promotes translation initiation by unwinding the 5'-untranslated region (5'UTR) of mRNA, we constructed a firefly luciferase reporter containing the 5'UTR of the HIF-1α mRNA and showed reduction in luciferase activity in YTHDC2-silenced cells. Furthermore, we examined expression levels of YTHDC2 by immunohistochemical staining in human colon cancer tissues from 72 patients and found a significantly positive correlation between YTHDC2 expression and the tumor stage, including metastasis. In conclusion, these results suggest that the RNA helicase YTHDC2 contributes to colon tumor metastasis by promoting translation of HIF-1α and that YTHDC2 is potentially a diagnostic marker and target gene for treating colon cancer patients.

Novel anticancer agent, SQAP, binds to focal adhesion kinase and modulates its activity.

SQAP is a novel and promising anticancer agent that was obtained by structural modifications from a natural compound. SQAP inhibits angiogenesis in vivo resulting in increased hypoxia and reduced tumor volume. In this study, the mechanism by which SQAP modifies the tumor microenvironment was revealed through the application of a T7 phage display screening. This approach identified five SQAP-binding proteins including sterol carrier protein 2, multifunctional enzyme type 2, proteasomal ubiquitin receptor, UV excision repair protein and focal adhesion kinase (FAK). All the interactions were confirmed by surface plasmon resonance analysis. Since FAK plays an important role in cell turnover and angiogenesis, the influence of SQAP on FAK was the principal goal of this study. SQAP decreased FAK phosphorylation and cell migration in human umbilical vein endothelial cells and A549 cancer cells. These findings suggest that inhibition of FAK phosphorylation works as the mechanism for the anti-angiogenesis activity of SQAP.

Risk factors for bleeding evaluated using the Forrest classification in Japanese patients after endoscopic submucosal dissection for early gastric neoplasm.

BACKGROUND/AIMS: Bleeding remains a serious complication after endoscopic submucosal dissection (ESD). Second-look endoscopy for hemostasis helps prevent post-ESD bleeding. We investigated the relationships between patient characteristics, tumor characteristics, and the Forrest classification for exposed vessels on artificial ulcers after ESD and evaluated whether hemostasis during second-look endoscopy was useful for preventing post-ESD bleeding. PATIENTS AND METHODS: We analyzed 250 patients (265 lesions) who underwent ESD for gastric neoplasms. Vessels classified by Forrest classifications during scheduled second-look endoscopy were analyzed for associations with patient characteristics, tumor characteristics, and recurrent bleeding. RESULTS: Two of 250 patients (0.8%) underwent emergency hemostatic endoscopy before scheduled second-look endoscopy. The remaining 248 patients (99.2%) underwent scheduled second-look endoscopy on the day after ESD. Patients with Forrest classification Ia, Ib, or IIa vessels had a significantly higher risk for recurrent bleeding after scheduled second-look endoscopy compared with patients with IIb or III vessels according to univariate analysis (P<0.05) and multivariate logistic regression analysis (odds ratio: 3.45; 95% confidence interval: 1.04-11.41; P=0.042). Univariate analysis indicated that hemodialysis correlated significantly with the presence of Ia, Ib, or IIa vessels compared with that of IIb or III vessels found during second-look endoscopy (P<0.05). Multivariate analysis indicated a significant relationship between hemodialysis and recurrent bleeding after second-look endoscopy (odds ratio: 10.05; 95% confidence interval: 1.97-51.26; P=0.006). CONCLUSION: Hemodialysis is a risk factor for post-ESD bleeding. Proper classification of exposed vessels within post-ESD ulcers according to the Forrest classification using second-look endoscopy might help predict or prevent recurrent bleeding.

Granulomatosis with Polyangiitis (Wegener's Granulomatosis) Complicated with Ruptured Posteromedial Papillary Muscle in the Absence of Coronary Angiographic Findings.

Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis, associated with antineutrophil cytoplasmic autoantibody-associated systemic vasculitis, and it can affect many organ systems via the inflammation of small-to-medium-sized vessels. Cardiac involvements in GPA are relatively rare. We report a 75-year-old woman who was diagnosed with GPA and rapid progressive glomerulonephritis that resulted in a partial posteromedial papillary muscle rupture, but with no coronary angiographic findings. The surgical and pathological findings with regard to the ruptured papillary muscle revealed necrotic muscle and acute ischemic change. The mechanism of papillary muscle rupture in GPA is coronary vasculitis leading to myocardial infarction. The ischemic change is not always detected on coronary angiography, so assessment using an echocardiogram is important.

A case of diffuse esophageal spasm successfully treated by steroid therapy.

A 53-year-old man was admitted to our hospital with anterior chest pain and difficulty swallowing. Computed tomography revealed significant esophageal wall thickening. Esophageal intraluminal manometry revealed uncoordinated contraction and strong peristaltic pressure associated with the chest pain. The patient was subsequently diagnosed with diffuse esophageal spasm (DES). His serum immunoglobulin E level was high, and peripheral blood eosinophilia was observed. No eosinophilic infiltration was detected in the esophageal mucosa on endoscopic biopsy. It was presumed that this case of DES was induced by allergic disease. Treatment with 30 mg of oral prednisolone led to a prompt resolution of symptoms;the thickness of the esophageal wall decreased, and the simultaneous contractions disappeared. However, given the presence of a strong peristaltic wave, nutcracker esophagus (NE) was also suspected. This was a rare case of atypical DES induced by allergic disease and associated with NE.

Critical Roles for PU.1, GATA1, and GATA2 in the expression of human FcεRI on mast cells: PU.1 and GATA1 transactivate FCER1A, and GATA2 transactivates FCER1A and MS4A2.

The high-affinity IgE receptor, FcεRI, which is composed of α-, ß-, and γ-chains, plays an important role in IgE-mediated allergic responses. In the current study, involvement of the transcription factors, PU.1, GATA1, and GATA2, in the expression of FcεRI on human mast cells was investigated. Transfection of small interfering RNAs (siRNAs) against PU.1, GATA1, and GATA2 into the human mast cell line, LAD2, caused significant downregulation of cell surface expression of FcεRI. Quantification of the mRNA levels revealed that PU.1, GATA1, and GATA2 siRNAs suppressed the α transcript, whereas the amount of ß mRNA was reduced in only GATA2 siRNA transfectants. In contrast, γ mRNA levels were not affected by any of the knockdowns. Chromatin immunoprecipitation assay showed that significant amounts of PU.1, GATA1, and GATA2 bind to the promoter region of FCER1A (encoding FcεRIα) and that GATA2 binds to the promoter of MS4A2 (encoding FcεRIß). Luciferase assay and EMSA showed that GATA2 transactivates the MS4A2 promoter via direct binding. These knockdowns of transcription factors also suppressed the IgE-mediated degranulation activity of LAD2. Similarly, all three knockdowns suppressed FcεRI expression in primary mast cells, especially PU.1 siRNA and GATA2 siRNA, which target FcεRIα and FcεRIß, respectively. From these results, we conclude that PU.1 and GATA1 are involved in FcεRIα transcription through recruitment to its promoter, whereas GATA2 positively regulates FcεRIß transcription. Suppression of these transcription factors leads to downregulation of FcεRI expression and IgE-mediated degranulation activity. Our findings will contribute to the development of new therapeutic approaches for FcεRI-mediated allergic diseases.

Transcriptional machinery of TNF-α-inducible YTH domain containing 2 (YTHDC2) gene.

We previously demonstrated that a cellular factor, cyclosporin A (CsA) associated helicase-like protein (CAHL) that is identical to YTH domain containing 2 (YTHDC2), forms trimer complex with cyclophilin B and NS5B of hepatitis C virus (HCV) and facilitates HCV genome replication. Gene expression of YTHDC2 was shown in tumor cell lines and tumor necrosis factor (TNF)-α-treated hepatocytes, but not in untreated. However, the function of YTHDC2 in the tumor cells and the mechanism by which the YTHDC2 gene is transcribed in these cells is largely unknown. We first evaluated that the role of YTHDC2 in the proliferation of hepatocellular carcinoma (HCC) cell line Huh7 using RNA interference and found that YTHDC2-downregulated Huh7 were significantly decreased cell growth as compared to control. We next demonstrated that the cAMP response element (CRE) site in the promoter region of the YTHDC2 gene is critical for YTHDC2 transcription. To further investigate the transcription factors bound to the CRE site, we performed chromatin immunoprecipitation assays. Our findings demonstrate that c-Jun and ATF-2 bind to the CRE site in Huh7, and that TNF-α induces the biological activity of these transcription factors in hepatocytes as well as Huh7. Moreover, treatment with the HDAC inhibitor, trichostatin A (TSA), reduces YTHDC2 expression in Huh7 and in TNF-α-stimulated hepatocytes. Collectively, these data show that YTHDC2 plays an important role in tumor cells growth and activation/recruitment of c-Jun and ATF-2 to the YTHDC2 promoter is necessary for the transcription of YTHDC2, and that HDAC activity is required for the efficient expression of YTHDC2 in both of hepatocyte and HCC cells.

G protein-coupled receptor 43 moderates gut inflammation through cytokine regulation from mononuclear cells.

BACKGROUND: Short-chain fatty acids (SCFAs), which are produced by the fermentation of dietary fiber by intestinal microbiota, may positively influence immune responses and protect against gut inflammation. SCFAs bind to G protein-coupled receptor 43 (GPR43). Here, we show that SCFA-GPR43 interactions profoundly affect the gut inflammatory response. METHODS: Colitis was induced by adding dextran sulfate sodium to the drinking water of GPR43 knockout (-/-) and wild-type mice. RESULTS: Dextran sulfate sodium-treated GPR43 mice exhibited weight loss, increased disease activity index (a combined measure of weight loss, rectal bleeding, and stool consistency), decreased hematocrit, and colon shortening, resulting in significantly worse colonic inflammation than in wild-type mice. Tumor necrosis factor alpha and interleukin 17 protein levels in the colonic mucosa of GPR43 mice were significantly higher than in wild-type mice. Treatment of wild-type mice with 150 mM acetate in their drinking water markedly improved these disease indices, with an increase in colon length and decrease in the disease activity index; however, it had no effect on GPR43 mice. Mononuclear cell production of tumor necrosis factor alpha after lipopolysaccharide stimulation was suppressed by acetate. This effect was inhibited by anti-GPR43 antibody. CONCLUSIONS: SCFA-GPR43 interactions modulate colitis by regulating inflammatory cytokine production in mononuclear cells.

Gastric calcifying fibrous tumor removed by endoscopic submucosal dissection.

The World Health Organization describes calcifying fibrous tumors (CFTs) as rare, benign lesions characterized by hypocellular, densely hyalinized collagenization with lymphoplasmacytic infiltration. These tumors rarely involve the gastrointestinal (GI) tract. A routine endoscopic upper gastrointestinal screen detected a 10-mm submucosal tumor (SMT) in the lesser curvature of the lower corpus of the stomach of an apparently healthy, 37-year-old woman with no history of Helicobacter pylori infection. Endoscopic ultrasonography (EUS) localized the internally isoechoic, homogeneous SMT mainly within the submucosa. Malignancy was ruled out using endoscopic submucosal dissection (ESD). A pathological examination confirmed complete resection of the SMT, and defined a hypocellular, spindle-cell tumor with a densely hyalinized, collagenous matrix, scattered lymphoplasmacytic aggregates as well as a few psammomatous, dystrophic calcified foci. The mass was immunohistochemically positive for vimentin and negative for CD117 (c-kit protein), CD34, desmin, smooth muscle actin (SMA) and S100. Therefore, the histological findings were characteristic of a CFT. To date, CFT resection by ESD has not been described. This is the first case report of a gastric calcifying fibrous tumor being completely resected by ESD after endoscopic ultrasonography.

[Mitral valve replacement for a severely calcified mitral annulus].

An 80-year-old woman was referred to our institution because of acute heart failure due to moderate mitral stenosis and severe regurgitation. After medical treatment of heart failure, she underwent mitral valve surgery. Intraoperatively severe calcification of the posterior mitral annulus was revealed. We excised only the anterior mitral leaflet and preserved the posterior mitral leaflet to prevent a fatal complication such as left ventricular rupture, injury of the coronary artery or embolism. Partial resection of the calcified annulus was performed using Ultrasonic Surgical System (SonoSurg), after 2-0 polyester mattress sutures were placed through the anterior and posterior annuli from the left ventricle to the left atrium. Then, mitral valve replacement was performed using a St. Jude Medical mechanical heart valve at the supra-annular position. The postoperative course was uneventful. We concluded that partial resection of a severely calcified posterior mitral annulus by the ultrasonic device was a safe and simple procedure.

Protein-losing enteropathy associated with collagenous colitis cured by withdrawal of a proton pump inhibitor.

A 63-year-old woman was admitted with symptoms of watery diarrhea and generalized edema lasting for five months. She had been administered 15 mg/day of lansoprazole. Laboratory findings revealed severe hypoproteinemia with normal liver, renal, thyroid and adrenal functions and no proteinuria. Colonoscopy revealed edematous mucosa, minor diminished vascular transparency and apparent longitudinal linear lacerations. The histopathological findings were compatible with a diagnosis of collagenous colitis (CC). Protein leakage from the colon was identified on (99m)Tc-human serum albumin scintigraphy. The results indicated CC associated with protein-losing enteropathy. Discontinuing lansoprazole ameliorated the watery diarrhea and generalized edema, increased the serum albumin level and improved the hypoproteinemia.

GATA2 is a critical transactivator for the human IL1RL1/ST2 promoter in mast cells/basophils: opposing roles for GATA2 and GATA1 in human IL1RL1/ST2 gene expression.

The IL1RL1/ST2 gene encodes a receptor for IL-33. Signaling from IL1RL1/ST2 induced by IL-33 binding was recently identified as a modulator of the Th2 response. The target cells for IL-33 are restricted in some hematopoietic lineages, including mast cells, basophils, eosinophils, Th2 cells, natural killer cells, and dendritic cells. To clarify the molecular mechanisms of cell type-specific IL1RL1/ST2 expression in mast cells and basophils, transcriptional regulation of the human IL1RL1/ST2 promoter was investigated using the mast cell line LAD2 and the basophilic cell line KU812. Reporter assays suggested that two GATA motifs just upstream of the transcription start site in the ST2 promoter are critical for transcriptional activity. These two GATA motifs possess the capacity to bind GATA1 and GATA2 in EMSA. ChIP assay showed that GATA2, but not GATA1, bound to the ST2 promoter in LAD2 cells and that histone H3 at the ST2 promoter was acetylated in LAD2 cells, whereas binding of GATA1 and GATA2 to the ST2 promoter was detected in KU812 cells. Knockdown of GATA2 mRNA by siRNA reduced ST2 mRNA levels in KU812 and LAD2 cells and ST2 protein levels in LAD2 cells; in contrast, GATA1 siRNA transfection up-regulated ST2 mRNA levels in KU812 cells. The ST2 promoter was transactivated by GATA2 and repressed by GATA1 in coexpression analysis. When these siRNAs were introduced into human peripheral blood basophils, GATA2 siRNA reduced ST2 mRNA, whereas GATA1 siRNA up-regulated ST2 mRNA. These results indicate that GATA2 and GATA1 positively and negatively control human ST2 gene transcription, respectively.