YWHAZ amplification/overexpression defines aggressive bladder cancer and contributes to chemo-/radio-resistance by suppressing caspase-mediated apoptosis.

The objective of this study was to characterize the oncogenic actions of a recently identified cancer-associated gene YWHAZ (also named as 14-3-3 ζ/δ) in urothelial carcinomas of the urinary bladder (UCUB). A genome-wide study revealed YWHAZ to be involved in the amplicon at 8q22.3, and its genetic amplification was detected predominantly in muscle-invasive bladder cancer (MIBC). Immunohistochemical staining confirmed the association of YWHAZ overexpression with higher tumor stages, lymph node/vascular invasion, and mitotic activity. Univariate and multivariate analyses further indicated the prognostic potential of YWHAZ for more aggressive cancer types. Both gene set enrichment analysis and STRING network studies suggested involvement of YWHAZ in regulating caspase-mediated apoptosis. Ectopic expression of YWHAZ in bladder cells with low endogenous YWHAZ levels boosted cell resistance to doxorubicin and cisplatin, as well as to ionizing radiation. Conversely, YWHAZ-knockdown using specific shRNA in cells with high endogenous YWHAZ levels diminished survival activity, suppressing cell growth and increasing cell death. Our findings confirm the essential role played by YWHAZ in sustaining cell proliferation during chemo/radiotherapy. Treatments based on anti-YWHAZ strategies may thus be beneficial for UCUB patients overexpressing YWHAZ. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Suppression of Cell Growth, Migration and Drug Resistance by Ethanolic Extract of Antrodia cinnamomea in Human Lung Cancer A549 Cells and C57BL/6J Allograft Tumor Model.

The purpose of this study was to investigate the inhibitory activities of ethanolic extracts from Antrodia cinnamomea (EEAC) on lung cancer. Cell proliferation and cell cycle distribution were analyzed using (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay and flow cytometry, respectively. Wound-healing assay, Western blotting, and a murine tumor model were separately used to examine cell migration, protein expression, and tumor repression. Our results showed that EEAC induced cell cycle arrest at the G0/G1 phase resulting decreased cell viability in A549 cells. Moreover, EEAC up-regulated the growth-suppressing proteins, adenosine 5'-monophosphate-activated protein kinase (AMPK), p21 and p27, but down-regulated the growth-promoting proteins, protein kinase B (Akt), mammalian tarfet of rapamycin (mTOR), extracellular signal-regulating kinase 1/2 (ERK1/2), retinoblastoma protein (Rb), cyclin E, and cyclin D1. EEAC also inhibited A549 cell migration and reduced expression of gelatinases. In addition, our data showed that tumor growth was suppressed after treatment with EEAC in a murine allograft tumor model. Some bioactive compounds from EEAC, such as cordycepin and zhankuic acid A, were demonstrated to reduce the protein expressions of matrix metalloproteinase (MMP)-9 and cyclin D1 in A549 cells. Furthermore, EEAC enhanced chemosensitivity of A549 to paclitaxel by reducing the protein levels of caveolin-1. Our data suggests that EEAC has the potential to be an adjuvant medicine for the treatment of lung cancer.

Exploration of anti-cancer effects and mechanisms of Zuo-Jin-Wan and its alkaloid components in vitro and in orthotopic HepG2 xenograft immunocompetent mice.

BACKGROUND: Zuo-Jin-Wan (ZJW), a two-herb formula consisting of Coptis chinensis (CC) and Evodia rutaecarpa (ER), is commonly used in traditional Chinese medicine for the treatment of cancers. However, the efficacies and mechanisms of ZJW and its alkaloid components on cancers are still unclear. METHODS: Here we investigated the anti-cancer effects and mechanisms of ZJW, CC, ER, berberine, and evodiamine in cells and in intrahepatic xenograft mice. RESULTS: Treatment of HepG2 cells with ZJW, CC, ER, berberine, and evodiamine significantly displayed cytotoxic effects in a dose- and time-dependent manner. Hierarchical cluster analysis of gene expression profiles showed that CC and ZJW shared a similar mechanism for the cytotoxic effects, suggesting that CC was the active ingredient of ZJW for anti-cancer activity. Network analysis further showed that c-myc was the likely key molecule involved in the regulation of ZJW-affected gene expression. A human hepatoma xenograft model was established by intrahepatic injection of HepG2 cells containing nuclear factor-κB-driven luciferase genes in immunocompetent mice. In vivo bioluminescence imaging showed that cells had been successfully transplanted in mouse liver. Oral administration of ZJW for 28 consecutive days led to a significant decrease in the accumulation of ascites, the ratio of tumor-to-liver, and the number of transplanted cells in livers. CONCLUSIONS: In conclusion, our findings suggested for the first time that ZJW significantly suppressed human cancer cell growth in orthotopic HepG2 xenograft-bearing immunocompetent mice. Moreover, c-myc might play a potent role in the cytotoxic mechanisms of ZJW, CC, ER, berberine, and evodiamine.

Genetic variations of MUC17 are associated with endometriosis development and related infertility.

BACKGROUND: Genetic alterations of mucin genes, such as MUC2 and MUC4, were previously identified to be associated with endometriosis and related infertility. Additionally, gene expression profiling has confirmed MUC17 to be overexpressed in mucinous ovarian carcinoma; however, its associated risk for endometriosis remains unclear. This study was focused on the potential impact of genetic variations in MUC17 on endometriosis development and associated clinical features. METHODS: The study subjects included 189 female Taiwanese patients with pathology-proven endometriosis and 191 healthy Taiwanese women as controls. Five single-nucleotide polymorphisms (rs4729645, rs10953316, rs74974199, rs4729655, and rs4729656) within the MUC17 gene were selected and genotyped using the Taqman genotyping assay to examine the allele frequency and genotype distributions of MUC17 polymorphisms. RESULTS: Genotyping revealed that the A allele at rs10953316 in MUC17 was a protective genetic factor in endometriosis development (p = 0.008; OR = 0.53; 95% CI: 0.36-0.79). Genetic variation of rs4729655 protected patients against endometriosis-induced infertility, but was associated with a higher cancer antigen 125 (CA125) level. Base-pairing analysis, called MaxExpect, predicted an additional loop in the mRNA structure caused by rs10953316 polymorphism, possibly influencing ribosome sliding and translation efficiency. Such predictions were confirmed by immunohistochemistry that patients with AA genotype at rs10953316 showed low MUC17 levels in their endometrium, patients with GA genotype showed moderate levels, and strong staining could be found in patients with GG genotype. CONCLUSIONS: MUC17 polymorphisms are involved in endometriosis development and the associated infertility in the Taiwanese population.

2'-hydroxyflavanone induces apoptosis of human osteosarcoma 143 B cells by activating the extrinsic TRAIL- and intrinsic mitochondria-mediated pathways.

Flavanones demonstrate a propensity to antiproliferation and induce apoptosis of malignant cells. Among the 4 flavanones under study, 2'-hydroxyflavanone exhibited the greatest potency to reduce the cell viability of 143 B cells in 4 osteosarcoma cells. Flow cytometry analysis showed that 2'-hydroxyflavanone increased the hypodiploid cells in the sub-G1 phase but resulted in the reduced DNA content in the G0/G1 phase in 143 B cells. The 2'-hydroxyflavanone-induced apoptosis in 143 B cells was confirmed by 4'-6-diamidino-2-phenylindole staining and mitochondrial membrane potential (Δψm) assay. Increasing expressions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and death receptor 5 (DR5) were found in 2'-hydroxyflavanone-treated cells. Moreover, 2'-hydroxyflavanone increased the expressions of B-cell lymphoma-extra small, cytochrome c, and cleavage poly (ADP-ribose) polymerase but downregulated B-cell lymphoma/leukemia-2expressions in 143 B cells. Furthermore, in vivo experiments showed that 2'-hydroxyflavanone inhibited the tumor growth of 143 B cells. 2'-hydroxyflavanone induced the apoptosis of 143 B cells via the extrinsic TRAIL- and intrinsic mitochondrial-dependent pathways, indicating its potential for inducing cancer apoptosis in osteosarcoma.

Rsf-1, a chromatin remodelling protein, interacts with cyclin E1 and promotes tumour development.

Chromosome 11q13.5 containing RSF1 (HBXAP), a gene involved in chromatin remodelling, is amplified in several human cancers including ovarian carcinoma. Our previous studies demonstrated requirement of Rsf-1 for cell survival in cancer cells, which contributed to tumour progression; however, its role in tumourigenesis has not yet been elucidated. In this study, we co-immunoprecipitated proteins with Rsf-1 followed by nanoelectrospray mass spectrometry and identified cyclin E1, besides SNF2H, as one of the major Rsf-1 interacting proteins. Like RSF1, CCNE1 is frequently amplified in ovarian cancer, and both Rsf-1 and cyclin E1 were found co-up-regulated in ovarian cancer tissues. Ectopic expression of Rsf-1 and cyclin E1 in non-tumourigenic TP53(mut) RK3E cells led to an increase in cellular proliferation and tumour formation by activating cyclin E1-associated kinase (CDK2). Tumourigenesis was not detected if either cyclin E1 or Rsf-1 was expressed, or they were expressed in a TP53(wt) background. Domain mapping showed that cyclin E1 interacted with the first 441 amino acids of Rsf-1. Ectopic expression of this truncated domain significantly suppressed G1/S-phase transition, cellular proliferation, and tumour formation of RK3E-p53(R175H) /Rsf-1/cyclin E1 cells. The above findings suggest that Rsf-1 interacts and collaborates with cyclin E1 in neoplastic transformation and TP53 mutations are a prerequisite for tumour-promoting functions of the RSF/cyclin E1 complex.

High cytoplasmic expression of Krüppel-like factor 4 is an independent prognostic factor of better survival in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality in the world. Hepatocarcinogenesis is complex, with an extraordinary molecular heterogeneity. Krüppel-like factor 4 (KLF4) plays an important role in cell proliferation and differentiation, and it can function as a tumor suppressor or an oncoprotein, depending on tissue type. The role of KLF4 in HCC remains controversial. The aim of this study was to explore the clinical significance of KLF4 expression in HCC. The study included 205 patients with surgical resection. We performed immunostaining for KLF4 and Ki-67 to investigate the correlations of the clinicopathological parameters of HCC and to examine the proliferative index. KLF4 staining was observed in the cytoplasm of non-tumorous hepatocytes and tumor cells. We subdivided the immunohistological staining results for KLF4 into low expression (Staining 0 and 1+) and high expression (Staining 2+ and 3+) subgroups. The expression of KLF4 was significantly correlated with tumor differentiation (p = 0.001). The Ki-67 proliferative index was significantly lower in well-differentiated HCCs (0.781% ± 1.02% vs. 2.16% ± 3.14%, p = 0.012), but not significantly different between low-KLF4 expression and high-KLF4 expression (1.87% ± 2.93% vs. 2.51% ± 3.28%, p = 0.32). Kaplan-Meier analysis showed that a high expression of KLF4 was significantly correlated with a longer disease-specific survival (p = 0.019). Univariate and multivariate analyses showed that high KLF4 expression was an independent predictor of a better disease-specific survival (p = 0.017; hazard ratio = 0.398; 95% confidence interval: 0.19-0.85). High cytoplasmic expression of KLF4 was associated with better disease-specific survival and was an independently favorable prognostic factor in hepatocellular carcinoma. These promising results suggest that KLF4 may play an anti-oncogenic role in hepatocarcinogenesis.

Tocilizumab Exerts Anti-Tumor Effects on Colorectal Carcinoma Cell Xenografts Corresponding to Expression Levels of Interleukin-6 Receptor.

The use of tocilizumab against the interleukin-6 receptor (IL-6R) has been demonstrated as inhibiting the progression of diverse cancers in vitro and in vivo. Nonetheless, evidence regarding the anti-tumor effects of tocilizumab on human colorectal carcinoma (CRC) corresponding to IL-6R expression levels remains scarce. To investigate the influence of IL-6R expression, SW480 and HT-29 cells inoculated subcutaneously into NU/NU mice were used as human CRC xenograft models with anti-IL-6R antibody (tocilizumab) therapy. The IL-6R expression levels, histology of CRC growth/invasiveness, and tumor growth-related signaling pathway were estimated by H&E and immunohistochemical staining. SW480 tumor cells with higher IL-6R expression levels showed better responsiveness in tocilizumab therapy than in the treated HT-29 group. Likewise, therapeutic effects of tocilizumab on the proliferative ability with mitotic index and Ki-67 expressions, invasiveness with MMP-9 proteinase expressions, and ERK 1/2 and STAT3 signaling transduction in the SW480 treatment group were superior to the HT-29 treatment group. In light of our results, IL-6R is the key indicator for the efficacy of tocilizumab treatment in CRC xenografts. From the perspective of precision medicine, tumor response to anti-IL-6R antibody therapy could be predicted on the basis of IL-6R expression levels. In this manner, tocilizumab may serve as a targeted and promising anti-CRC therapy.

Talin-1 overexpression defines high risk for aggressive oral squamous cell carcinoma and promotes cancer metastasis.

Oral squamous cell carcinoma (OSCC) is highly invasive and is associated with frequent tumour recurrences and lymph node metastases. Identification of genes involved in the aggressiveness of OSCC may provide new targets for clinical intervention. A genome-wide study based on the Sty1 250K SNP array indicated the involvement of the Talin-1 (TLN1) gene in the 9p13.3 amplicon, which was further validated by dual colour fluorescence in situ hybridization (FISH). Comparative analyses revealed that TLN1 was the most highly expressed integrin-cytoskeleton cross-linker that can trigger integrin activation. IHC analyses and mouse study also revealed an association between TLN1 overexpression and advanced OSCC with invasion to adjacent tissues. Survival analyses indicated a significant association between TLN1 genetic gain/overexpression and a reduced overall survival in patients. Functional knockdown by a dominant negative TLN1 fragment reduced cell growth and invasiveness in TLN1-overexpressing cells via inactivation of downstream oncogenic signalling. The present study suggests an important role for TLN1 in oral cancer development. TLN1 overexpression could serve as a diagnostic marker for aggressive phenotypes and a potential target for treating OSCC.

Protective effect of Flos carthami extract against ethylene glycol-induced urolithiasis in rats.

Flos carthami (FC), also known as Carthamus tinctorius, is a traditional Chinese herbal plant that has been prescribed since centuries for treating various symptoms related to blood circulation improvement. This study aimed to investigate the effects of FC on calcium oxalate (CaOx) formation in ethylene glycol (EG)-fed rats. A total of 50 male Sprague-Dawley rats were divided into the following 6 groups: group 1, as the normal control (n = 5); group 2 received gastric gavages of starch and 0.75% EG (placebo, n = 5) as a stone inducer; group 3 (n = 10) received EG and potassium citrate as positive controls; group 4 (n = 10) received 0.75% EG and 300 mg/day FC; group 5 (n = 10) was treated with EG and 600 mg/day FC; group 6 (n = 10) received with EG and 1,200 mg/day FC. For all experimental animals, 24-h urine and blood samples were analyzed at the beginning and end of the experiment. Kidney tissue was histopathologically examined using a polarized light microscope, and crystal deposits were evaluated by a semi-quantitative scoring method; these scores were significantly lower in the FC groups (600 and 1,200 mg/day) than in the placebo group. Thus, FC administration appeared to inhibit the deposition of CaOx crystal EG-fed rats. We, therefore, consider that FC may be effective for preventing stone disease, albeit with certain side effects, such as a bleeding tendency. Further clinical trials are needed for evaluating its benefits and possible side effects.

Genetic variations in UCA1, a lncRNA functioning as a miRNA sponge, determine endometriosis development and the potential associated infertility via regulating lipogenesis.

Endometriosis is a hormone-associated disease which has been considered as the precursor for certain types of ovarian cancer. In recent years, emerging evidence demonstrated potent roles of lncRNA in regulating cancer development. Since endometriosis shares several features with cancer, we investigated the possible involvement of cancer-related lncRNAs in endometriosis, including UCA1, GAS5 and PTENP1. By using massARRAY system, we investigated certain genetic variations in cancer-related lncRNAs that can change the thermo-stability, leading to up-regulation or down-regulation of those lncRNAs. Our data indicated three risk genetic haplotypes in UCA1 which can stabilize the RNA structure and increase the susceptibility of endometriosis. Of note, such alterations were found to be associated with long-term pain and infertility in patients. It has been known that UCA1 can function as a ceRNA to sponge and inhibit miRNAs, resulting in loss-of-control on downstream target genes. Gene network analyses revealed fatty acid metabolism and mitochondria beta-oxidation as the major pathways associated with altered UCA1 expression in endometriosis patients. Our study thus provides evidence to highlight functional/epigenetic roles of UCA1 in endometriosis development via regulating fatty acid metabolism in women.

Alterations of Cytoskeleton Networks in Cell Fate Determination and Cancer Development.

Cytoskeleton proteins have been long recognized as structural proteins that provide the necessary mechanical architecture for cell development and tissue homeostasis. With the completion of the cancer genome project, scientists were surprised to learn that huge numbers of mutated genes are annotated as cytoskeletal or associated proteins. Although most of these mutations are considered as passenger mutations during cancer development and evolution, some genes show high mutation rates that can even determine clinical outcomes. In addition, (phospho)proteomics study confirms that many cytoskeleton-associated proteins, e.g., ß-catenin, PIK3CA, and MB21D2, are important signaling mediators, further suggesting their biofunctional roles in cancer development. With emerging evidence to indicate the involvement of mechanotransduction in stemness formation and cell differentiation, mutations in these key cytoskeleton components may change the physical/mechanical properties of the cells and determine the cell fate during cancer development. In particular, tumor microenvironment remodeling triggered by such alterations has been known to play important roles in autophagy, metabolism, cancer dormancy, and immune evasion. In this review paper, we will highlight the current understanding of how aberrant cytoskeleton networks affect cancer behaviors and cellular functions through mechanotransduction.

A More Diverse Cervical Microbiome Associates with Better Clinical Outcomes in Patients with Endometriosis: A Pilot Study.

Infection-induced chronic inflammation is common in patients with endometriosis. Although microbial communities in the reproductive tracts of patients have been reported, little was known about their dynamic profiles during disease progression and complication development. Microbial communities in cervical mucus were collected by cervical swabs from 10 healthy women and 23 patients, and analyzed by 16S rRNA amplicon sequencing. The abundance, ecological relationships and functional networks of microbiota were characterized according to their prevalence, clinical stages, and clinical features including deeply infiltrating endometriosis (DIE), CA125, pain score and infertility. Cervical microbiome can be altered during endometriosis development and progression with a tendency of increased Firmicutes and decreased Actinobacteria and Bacteroidetes. Distinct from vaginal microbiome, upregulation of Lactobacillus, in combination with increased Streptococcus and decreased Dialister, was frequently associated with advanced endometriosis stages, DIE, higher CA125 levels, severe pain, and infertility. Significantly, reduced richness and diversity of cervical microbiome were detected in patients with more severe clinical symptoms. Clinical treatments against infertility can partially reverse the ecological balance of microbes through remodeling nutrition metabolism and transport and cell-cell/cell-matrix interaction. This study provides a new understanding on endometriosis development and a more diverse cervical microbiome may be beneficial for patients to have better clinical outcomes.

Ribosome Biogenesis Serves as a Therapeutic Target for Treating Endometriosis and the Associated Complications.

Ribosome biogenesis is a cellular process critical for protein homeostasis during cell growth and multiplication. Our previous study confirmed up-regulation of ribosome biogenesis during endometriosis progression and malignant transition, thus anti-ribosome biogenesis may be effective for treating endometriosis and the associated complications. A mouse model with human endometriosis features was established and treated with three different drugs that can block ribosome biogenesis, including inhibitors against mTOR/PI3K (GSK2126458) and RNA polymerase I (CX5461 and BMH21). The average lesion numbers and disease frequencies were significantly reduced in treated mice as compared to controls treated with vehicle. Flow cytometry analyses confirmed the reduction of small peritoneal macrophage and neutrophil populations with increased large versus small macrophage ratios, suggesting inflammation suppression by drug treatments. Lesions in treated mice also showed lower nerve fiber density which can support the finding of pain-relief by behavioral studies. Our study therefore suggested ribosome biogenesis as a potential therapeutic target for treating endometriosis.

Safrole-2',3'-oxide induces cytotoxic and genotoxic effects in HepG2 cells and in mice.

Safrole-2',3'-oxide (SAFO) is a reactive electrophilic metabolite of the hepatocarcinogen safrole, the main component of sassafras oil. Safrole occurs naturally in a variety of spices and herbs, including the commonly used Chinese medicine Xi xin (Asari Radix et Rhizoma) and Dong quai (Angelica sinensis). SAFO is the most mutagenic metabolite of safrole tested in the Ames test. However, little or no data are available on the genotoxicity of SAFO in mammalian systems. In this study, we investigated the cytotoxicity and genotoxicity of SAFO in human HepG2 cells and male FVB mice. Using MTT assay, SAFO exhibited a dose- and time-dependent cytotoxic effect in HepG2 cells with TC(50) values of 361.9µM and 193.2µM after 24 and 48h exposure, respectively. In addition, treatment with SAFO at doses of 125µM and higher for 24h in HepG2 cells resulted in a 5.1-79.6-fold increase in mean Comet tail moment by the alkaline Comet assay and a 2.6-7.8-fold increase in the frequency of micronucleated binucleated cells by the cytokinesis-block micronucleus assay. Furthermore, repeated intraperitoneal administration of SAFO (15, 30, 45, and 60mg/kg) to mice every other day for a total of twelve doses caused a significant dose-dependent increase in mean Comet tail moment in peripheral blood leukocytes (13.3-43.4-fold) and in the frequency of micronucleated reticulocytes (1.5-5.8-fold). Repeated administration of SAFO (60mg/kg) to mice caused liver lesions manifested as a rim of ballooning degeneration of hepatocytes immediately surrounding the central vein. Our data clearly demonstrate that SAFO significantly induced cytotoxicity, DNA strand breaks, micronuclei formation both in human cells in vitro and in mice. More studies are needed to explore the role SAFO plays in safrole-induced genotoxicity.

Genetic impacts on thermostability of onco-lncRNA HOTAIR during the development and progression of endometriosis.

HOTAIR is a well-known long non-coding RNA (lncRNA) involved in various cellular signaling, whereas its functional impacts on endometriosis development are still largely unknown. To this end, six potential functional single nucleotide polymorphisms (SNPs) in HOTAIR, with minor allele frequencies more than 10% in Han population and altered net energy of RNA structures larger than 0.5 kcal/mol, were selected for genotyping study. The study included 207 endometriosis patients and 200 healthy women. Genetic substitutions at rs1838169 and rs17720428 were frequently found in endometriosis patients, and rs1838169 showed statistical significance (p = 0.0174). The G-G (rs1838169-rs17720428) haplotype showed the most significant association with endometriosis (p < 0.0001) with enhanced HOTAIR stability, and patients who harbor such haplotype tended to show higher CA125. Data mining further revealed higher mRNA HOTAIR levels in the endometria of patients with severe endometriosis which consistently showed reduced HOXD10 and HOXA5 levels. HOTAIR knockdown with specific shRNAs down-regulated cell proliferation and migration with the induction of HOXD10 and HOXA5 expression in human ovarian clear cancer cells. Our study therefore provided evidence to indicate a prominent role of HOTAIR in promoting endometriosis, which could be used as a potential target for clinical applications.

Overexpression of wild type or a Q311E mutant MB21D2 promotes a pro-oncogenic phenotype in HNSCC.

Cadherin-mediated cell-cell contacts regulated by intracellular binders play critical roles in tissue homeostasis and tumorigenesis. Here, we screened mutational profiles of 312 annotated genes involved in cadherin binding in human squamous cell carcinomas and found MB21D2 to carry a unique recurrent Q311E mutation. MB21D2 overexpression was also frequently found in head and neck cancer (HNSCC) and was associated with poor clinical outcomes. Cell-based characterizations revealed pro-oncogenic roles for MB21D2 wild-type (WT) and its Q311E mutant (Q311E) in cell proliferation, colony formation, sphere growth, and migration/invasion by promoting epithelial-mesenchymal transition. Conversely, MB21D2 knockdown in MB21D2-overexpressing cells resulted in cell growth arrest and apoptosis. Xenograft tumor models with Q311E-expressing cells formed larger and more aggressive lesions, compared to models with WT-MB21D2-expressing cells or an empty vector. Transcriptome and protein interactome analyses revealed enrichment of KRAS signaling by MB21D2 expression. Immunoblotting confirmed RAS elevation, along with upregulation/phosphorylation of PI3K, AKT, and CREB. Blocking RAS signaling in MB21D2-expressing cells by manumycin significantly reduced cell growth and survival. Our study thus defined RAS signaling-dependent pro-oncogenic roles for MB21D2 overexpression and Q311E MB21D2 expression in HNSCC development.

Somatic mutational landscapes of adherens junctions and their functional consequences in cutaneous melanoma development.

Cell-cell interaction in skin homeostasis is tightly controlled by adherens junctions (AJs). Alterations in such regulation lead to melanoma development. However, mutations in AJs and their functional consequences are still largely unknown. Methods: Cadherin mutations in skin cutaneous melanoma were identified using sequencing data from TCGA dataset, followed by cross-validation with data from non-TCGA cohorts. Mutations with significant occurrence were subjected to structural prediction using MODELLER and functional protein simulation using GROMACS software. Neo-antigen prediction was carried out using NetMHCpan tool. Cell-based fluorescence reporter assay was used to validate ß-catenin activity in the presence of cadherin mutations. Clinical significance was analyzed using datasets from TCGA and other non-TCGA cohorts. Targeted gene exon sequencing and immunofluorescence staining on melanoma tissues were performed to confirm the in silico findings. Results: Highly frequent mutations in type-II classical cadherins were found in melanoma with one unique recurrent mutation (S524L) in the fifth domain of CDH6, which potentially destabilizes Ca2+-binding and cell-cell contacts. Mutational co-occurrence and physical dynamics analyses placed CDH6 at the center of the top-four mutated cadherins (core CDHs; all type-II), suggesting altered heterophilic interactions in melanoma development. Mutations in the intracellular domains significantly disturbed CDH6/ß-catenin complex formation, resulting in ß-catenin translocation into cytosol or nucleus and dysregulation of canonical Wnt/ß-catenin signaling. Although mutations in core CDH genes correlated with advanced cancer stages and lymph node invasion, the overall and disease-free survival times in those patients were longer in patients with wild-type. Peptide/MHC-I binding affinity predictions confirmed overall increased neo-antigen potentials of mutated cadherins, which associated with T-lymphocyte infiltration and better clinical outcomes after immunotherapy. Conclusion: Changes in cell-cell communications by somatic mutations in AJ cadherins function as one of mechanisms to trigger melanoma development. Certain mutations in AJs may serve as potential neo-antigens which conversely benefit patients for longer survival times.

Antioxidative and hepatoprotective effects of magnolol on acetaminophen-induced liver damage in rats.

Acute liver failure (ALF), an often fatal condition characterized by massive hepatocyte necrosis, is frequently caused by drug poisoning, particularly with acetaminophen (N-acetyl-p-aminophenol/APAP). Hepatocyte necrosis is consecutive to glutathione (GSH) depletion and mitochondrial damage caused by reactive oxygen species (ROS) overproduction. Magnolol, one major phenolic constituent of Magnolia officinalis, have been known to exhibit potent antioxidative activity. In this study, the anti-hepatotoxic activity of magnolol on APAP-induced toxicity in the Sprague-Dawley rat liver was examined. After evaluating the changes of several biochemical parameters in serum, the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) were elevated by APAP (500 mg/kg) intraperitoneal administration (8 and 24 h) and reduced by treatment with magnolol (0.5 h after APAP administration; 0.01, 0.1, and 1 mug/kg). Histological changes around the hepatic central vein, lipid peroxidation (thiobarbituric acid-reactive substance/TBARS), and GSH depletion in liver tissue induced by APAP were also recovered by magnolol treatment. The data show that oxidative stress followed by lipid peroxidation may play a very important role in the pathogenesis of APAP-induced hepatic injury; treatment with lipid-soluble antioxidant, magnolol, exerts anti-hepatotoxic activity. Our study points out the potential interest of magnolol in the treatment of toxic ALF.

Novel K6-K14 keratin fusion enhances cancer stemness and aggressiveness in oral squamous cell carcinoma.

Keratin intermediate filament (IF) is one component of cellular architectures, which provides necessary mechanical support to conquer environmental stresses. Recent findings reveal its involvement in mechano-transduction and the associated stem cell reprogramming, suggesting the possible roles in cancer development. Here, we report t(12;17)(q13.13;q21.2) chromosomal rearrangement as the most common fusion event in OSCC, resulting in a variety of inter-keratin fusions. Junction site mapping verified 9 in-frame K6-K14 variants, three of which were correlated with lymph node invasion, late tumor stages (T3/T4) and shorter disease-free survival times. When expressed in OSCC cells, those fusion variants disturbed wild-type K14 organization through direct interaction or aggregate formation, leading to perinuclear structure loss and nuclear deformation. Protein array analyses showed the ability of K6-K14 variant 7 (K6-K14/V7) to upregulate TGF-ß and G-CSF signaling, which contributed to cell stemness, drug tolerance, and cell aggressiveness. Notably, K6-K14/V7-expressing cells easily adapted to a soft 3-D culture condition in vitro and formed larger, less differentiated tumors in vivo. In addition to the anti-mechanical-stress activity, our data uncover oncogenic functionality of novel keratin filaments caused by gene fusions during OSCC development.