Comparative Transcriptome Analysis for Understanding Predator-Induced Polyphenism in the Water Flea Daphnia pulex.

The crustacean Daphnia pulex is one of the best model organisms for studying inducible defense mechanisms due to their inducible morphology in response to the predator Chaoborus larvae. In this study, multiple developmental stages of D. pulex were exposed to C. flavicans larvae and transcriptome profiles of samples from late embryo to fifth instar were sequenced by the RNA-seq technique to investigate the genetic background underlying inducible defenses. In comparison, differentially expressed genes between defensive and normal morphs were identified, including 908 genes in late embryo, 1383 genes in the first-third (1⁻3) instar, and 1042 genes in fourth-fifth (4⁻5) instar. Gene ontology enrichment analysis showed that structural constituents of the cuticle and structural molecule activity genes were prominent up-regulated genes in late embryos. Down-regulated genes in late embryos and 1⁻3 instar comprised metabolic process, hydrolase activity, and peptidase activity gene classes. Pathway analysis indicated that small molecule neurotransmitter pathways were potentially involved in the development of inducible defenses. The characterization of genes and pathways in multiple developmental stages can improve our understanding of inducible defense responses of D. pulex to predation at the molecular level.

Galangin enhances TGF-ß1-mediated growth inhibition by suppressing phosphorylation of threonine 179 residue in Smad3 linker region.

Smad3 linker phosphorylation is a candidate target for several kinases that play important roles in cancer cell initiation, proliferation and progression. Also, Smad3 is an essential intracellular mediator of TGF-ß1-induced transcriptional responses during carcinogenesis. Therefore, it is highly advantageous to identify and develop inhibitors targeting Smad3 linker phosphorylation for the treatment of cancers. Galangin (3,5,7-trihydroxyflavone) has been known to be an active flavonoid showing a cytotoxic effect on several cancer cells. However, the mechanism of action of galangin in various cancers remains unclear, and there has been no report concerning regulation of Smad3 phosphorylation by galangin. In the present study, we show that galangin significantly induced apoptosis and inhibited cell proliferation in the presence of TGF-ß1 in both human prostate and pancreatic cancer cell lines. Particularly, galangin effectively inhibits phosphorylation of the Thr-179 site at Smad3 linker region through suppression of CDK4 phosphorylation. Thus, galangin can be a promising candidate as a selective inhibitor to suppress phosphorylation of Smad3 linker region.

Metformin induces degradation of cyclin D1 via AMPK/GSK3ß axis in ovarian cancer.

Metformin, which is widely used as an anti-diabetic drug, reduces cancer related morbidity and mortality. However, the role of metformin in cancer is not fully understood. Here, we first describe that the anti-cancer effect of metformin is mediated by cyclin D1 deregulation via AMPK/GSK3ß axis in ovarian cancer cells. Metformin promoted cytotoxic effects only in the cancer cells irrespective of the p53 status and not in the normal primary-cultured cells. Metformin induced the G1 cell cycle arrest, in parallel with a decrease in the protein expressions of cyclin D1 without affecting its transcriptional levels. Using a proteasomal inhibitor, we could address that metformin-induced decrease in cyclin D1 through the ubiquitin/proteasome process. Cyclin D1 degradation by metformin requires the activation of GSK3ß, as determined based on the treatment with GSK3ß inhibitors. The activation of GSK3ß correlated with the inhibitory phosphorylation by Akt as well as p70S6K through AMPK activation in response to metformin. These findings suggested that the anticancer effects of metformin was induced due to cyclin D1 degradation via AMPK/GSK3ß signaling axis that involved the ubiquitin/proteasome pathway specifically in ovarian cancer cells. © 2016 Wiley Periodicals, Inc.

MAST4 regulates stem cell maintenance with DLX3 for epithelial development and amelogenesis.

The asymmetric division of stem cells permits the maintenance of the cell population and differentiation for harmonious progress. Developing mouse incisors allows inspection of the role of the stem cell niche to provide specific insights into essential developmental phases. Microtubule-associated serine/threonine kinase family member 4 (Mast4) knockout (KO) mice showed abnormal incisor development with low hardness, as the size of the apical bud was decreased and preameloblasts were shifted to the apical side, resulting in amelogenesis imperfecta. In addition, Mast4 KO incisors showed abnormal enamel maturation, and stem cell maintenance was inhibited as amelogenesis was accelerated with Wnt signal downregulation. Distal-Less Homeobox 3 (DLX3), a critical factor in tooth amelogenesis, is considered to be responsible for the development of amelogenesis imperfecta in humans. MAST4 directly binds to DLX3 and induces phosphorylation at three residues within the nuclear localization site (NLS) that promotes the nuclear translocation of DLX3. MAST4-mediated phosphorylation of DLX3 ultimately controls the transcription of DLX3 target genes, which are carbonic anhydrase and ion transporter genes involved in the pH regulation process during ameloblast maturation. Taken together, our data reveal a novel role for MAST4 as a critical regulator of the entire amelogenesis process through its control of Wnt signaling and DLX3 transcriptional activity.

Tm4sf19 deficiency inhibits osteoclast multinucleation and prevents bone loss.

BACKGROUND: Multinucleation is a hallmark of osteoclast formation and has a unique ability to resorb bone matrix. During osteoclast differentiation, the cytoskeleton reorganization results in the generation of actin belts and eventual bone resorption. Tetraspanins are involved in adhesion, migration and fusion in various cells. However, its function in osteoclast is still unclear. In this study, we identified Tm4sf19, a member of the tetraspanin family, as a regulator of osteoclast function. MATERIALS AND METHODS: We investigate the effect of Tm4sf19 deficiency on osteoclast differentiation using bone marrow-derived macrophages obtained from wild type (WT), Tm4sf19 knockout (KO) and Tm4sf19 LELΔ mice lacking the large extracellular loop (LEL). We analyzed bone mass of young and aged WT, KO and LELΔ mice by µCT analysis. The effects of Tm4sf19 LEL-Fc fusion protein were accessed in osteoclast differentiation and osteoporosis animal model. RESULTS: We found that deficiency of Tm4sf19 inhibited osteoclast function and LEL of Tm4sf19 was responsible for its function in osteoclasts in vitro. KO and LELΔ mice exhibited higher trabecular bone mass compared to WT mice. We found that Tm4sf19 interacts with integrin αvß3 through LEL, and that this binding is important for cytoskeletal rearrangements in osteoclast by regulating signaling downstream of integrin αvß3. Treatment with LEL-Fc fusion protein inhibited osteoclast function in vitro and administration of LEL-Fc prevented bone loss in an osteoporosis mouse model in vivo. CONCLUSION: We suggest that Tm4sf19 regulates osteoclast function and that LEL-Fc may be a promising drug to target bone destructive diseases caused by osteoclast hyper-differentiation.

Combination treatment of T1-44, a PRMT5 inhibitor with Vactosertib, an inhibitor of TGF-ß signaling, inhibits invasion and prolongs survival in a mouse model of pancreatic tumors.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal type of cancer and the third leading cause of cancer death with the lowest 5-year survival rate. Heterogeneity, difficulty in diagnosis, and rapid metastatic progression are the causes of high mortality in pancreatic cancer. Recent studies have shown that Protein arginine methyltransferase 5 (PRMT5) is overexpressed in pancreatic cancers, and these patients have a worse prognosis. Recently, PRMT5 as an anti-cancer target has gained considerable interest. In this study, we investigated whether inhibition of PRMT5 activity was synergistic with blockade of TGF-ß1 signaling, which plays an important role in the construction of the desmoplastic matrix in pancreatic cancer and induces therapeutic vulnerability. Compared with T1-44, a selective inhibitor of PRMT5 activity, the combination of T1-44 with the TGF-ß1 signaling inhibitor Vactosertib significantly reduced tumor size and surrounding tissue invasion and significantly improved long-term survival. RNA sequencing analysis of mouse tumors revealed that the combination of T1-44 and Vactosertib significantly altered the expression of genes involved in cancer progression, such as cell migration, extracellular matrix, and apoptotic processes. In particular, the expression of Btg2, known as a tumor suppressor factor in various cancers, was markedly induced by combination treatment. Ectopic overexpression of Btg2 inhibited the EMT response, blocking cell migration, and promoted cancer cell death. These data demonstrate that the combination therapy of T1-44 with Vactosertib is synergistic for pancreatic cancer, suggesting that this novel combination therapy has value in the treatment strategy of patients with pancreatic cancer.

Degradation of DRAK1 by CUL3/SPOP E3 Ubiquitin ligase promotes tumor growth of paclitaxel-resistant cervical cancer cells.

Despite favorable responses to initial chemotherapy, drug resistance is a major cause limiting chemotherapeutic efficacy in many advanced cancers. However, mechanisms that drive drug-specific resistance in chemotherapy for patients with advanced cancers are still unclear. Here, we report a unique role of death-associated protein kinase-related apoptosis-inducing kinase 1 (DRAK1) associated with paclitaxel resistance in cervical cancer cells. Interestingly, DRAK1 protein level was markedly decreased in paclitaxel-resistant cervical cancer cells without affecting its mRNA expression, which resulted in an increase in tumor necrosis factor receptor-associated factor 6 (TRAF6) expression, as well as an activation of TRAF6-mediated nuclear factor-kappa B (NF-κB) signaling cascade, thereby promoting tumor progression. DRAK1 depletion markedly increased the chemotherapeutic IC50 values of paclitaxel in cervical cancer cells. Ectopic expression of DRAK1 inhibited growth of paclitaxel-resistant cervical cancer cells in vitro and in vivo. Furthermore, DRAK1 was markedly underexpressed in chemoresistant cervical cancer patient tissues compared with chemosensitive samples. We found that DRAK1 protein was destabilized through K48-linked polyubiquitination promoted by the Cullin scaffold protein 3 (CUL3) / speckle-type POZ (poxvirus and zinc finger protein) protein (SPOP) E3 ubiquitin ligase in paclitaxel-resistant cells. Collectively, these findings suggest that DRAK1 may serve as a potential predictive biomarker for overcoming paclitaxel resistance in cervical cancer.

Tetraarsenic hexoxide enhances generation of mitochondrial ROS to promote pyroptosis by inducing the activation of caspase-3/GSDME in triple-negative breast cancer cells.

Although tetraarsenic hexoxide is known to exert an anti-tumor effect by inducing apoptosis in various cancer cells, its effect on other forms of regulated cell death remains unclear. Here, we show that tetraarsenic hexoxide induces the pyroptotic cell death through activation of mitochondrial reactive oxygen species (ROS)-mediated caspase-3/gasdermin E (GSDME) pathway, thereby suppressing tumor growth and metastasis of triple-negative breast cancer (TNBC) cells. Interestingly, tetraarsenic hexoxide-treated TNBC cells exhibited specific pyroptotic characteristics, including cell swelling, balloon-like bubbling, and LDH releases through pore formation in the plasma membrane, eventually suppressing tumor formation and lung metastasis of TNBC cells. Mechanistically, tetraarsenic hexoxide markedly enhanced the production of mitochondrial ROS by inhibiting phosphorylation of mitochondrial STAT3, subsequently inducing caspase-3-dependent cleavage of GSDME, which consequently promoted pyroptotic cell death in TNBC cells. Collectively, our findings highlight tetraarsenic hexoxide-induced pyroptosis as a new therapeutic strategy that may inhibit cancer progression of TNBC cells.

Inhibition of TGF-ß signalling in combination with nal-IRI plus 5-Fluorouracil/Leucovorin suppresses invasion and prolongs survival in pancreatic tumour mouse models.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies. TGF-ß is strongly expressed in both the epithelial and stromal compartments of PDAC, and dysregulation of TGF-ß signalling is a frequent molecular disturbance in PDAC progression and metastasis. In this study, we investigated whether blockade of TGF-ß signalling synergizes with nal-IRI/5-FU/LV, a chemotherapy regimen for malignant pancreatic cancer, in an orthotopic pancreatic tumour mouse model. Compared to nal-IRI/5-FU/LV treatment, combining nal-IRI/5-FU/LV with vactosertib, a TGF-ß signalling inhibitor, significantly improved long-term survival rates and effectively suppressed invasion to surrounding tissues. Through RNA-sequencing analysis, we identified that the combination treatment results in robust abrogation of tumour-promoting gene signatures and positive enrichment of tumour-suppressing and apoptotic gene signatures. Particularly, the expression of tumour-suppressing gene Ccdc80 was induced by vactosertib and further induced by vactosertib in combination with nal-IRI/5-FU/LV. Ectopic expression of CCDC80 suppressed migration and colony formation concomitant with decreased expression of epithelial-to-mesenchymal transition (EMT) markers in pancreatic cancer cells. Collectively, these results indicate that combination treatment of vactosertib with nal-IRI/5-FU/LV improves overall survival rates in a mouse model of pancreatic cancer by suppressing invasion through CCDC80. Therefore, combination therapy of nal-IRI/5-FU/LV with vactosertib could provide clinical benefits to pancreatic cancer patients.

Erratum: Identification of Epithelial-Mesenchymal Transition-related Target Genes Induced by the Mutation of Smad3 Linker Phosphorylation.

[This corrects the article on p. 1 in vol. 23, PMID: 29629343.].

Identification of Epithelial-Mesenchymal Transition-related Target Genes Induced by the Mutation of Smad3 Linker Phosphorylation.

BACKGROUND: Smad3 linker phosphorylation plays essential roles in tumor progression and metastasis. We have previously reported that the mutation of Smad3 linker phosphorylation sites (Smad3-Erk/Pro-directed kinase site mutant constructs [EPSM]) markedly reduced the tumor progression while increasing the lung metastasis in breast cancer. METHODS: We performed high-throughput RNA-Sequencing of the human prostate cancer cell lines infected with adenoviral Smad3-EPSM to identify the genes regulated by Smad3-EPSM. RESULTS: In this study, we identified genes which are differentially regulated in the presence of Smad3-EPSM. We first confirmed that Smad3-EPSM strongly enhanced a capability of cell motility and invasiveness as well as the expression of epithelial-mesenchymal transition marker genes, CDH2, SNAI1, and ZEB1 in response to TGF-ß1 in human pancreatic and prostate cancer cell lines. We identified GADD45B, CTGF, and JUNB genes in the expression profiles associated with cell motility and invasiveness induced by the Smad3-EPSM. CONCLUSIONS: These results suggested that inhibition of Smad3 linker phosphorylation may enhance cell motility and invasiveness by inducing expression of GADD45B, CTGF, and JUNB genes in various cancers.

Differential Tissue-specific and Pathway-specific Anti-obesity Effects of Green Tea and Taeumjowitang, a Traditional Korean Medicine, in Mice.

BACKGROUND: Traditional medicines have been leveraged for the treatment and prevention of obesity, one of the fastest growing diseases in the world. However, the exact mechanisms underlying the effects of traditional medicine on obesity are not yet fully understood. METHODS: We produced the transcriptomes of epididymal white adipose tissue (eWAT), liver, muscle, and hypothalamus harvested from mice fed a normal diet, high-fat-diet alone, high-fat-diet together with green tea, or a high-fat-diet together with Taeumjowitang, a traditional Korean medicine. RESULTS: We found tissue-specific gene expression patterns as follows: (i) the eWAT transcriptome was more significantly altered by Taeumjowitang than by green tea, (ii) the liver transcriptome was similarly altered by Taeumjowitang and green tea, and (iii) both the muscle and hypothalamus transcriptomes were more significantly altered by green tea than Taeumjowitang. We then applied integrated network analyses, which revealed that functional networks associated with lymphocyte activation were more effectively regulated by Taeumjowitang than by green tea in the eWAT. In contrast, green tea was a more effective regulator of functional networks associated with glucose metabolic processes in the eWAT. CONCLUSIONS: Taeumjowitang and green tea have a differential tissue-specific and pathway-specific therapeutic effect on obesity.

The complete mitochondrial genome of Aplysia kurodai (Anaspidea: Aplysiidae).

Complete mitochondrial genome is sequenced from an opisthobranch gastropod Aplysia kurodai. Mitochondrial genome size of the species is 14,113 bp. The mitochondrial genome of A. kurodai contains 13 protein coding genes, two ribosomal RNA genes, and 22 tRNA genes like mitochondrial genomes of congeneric species. The gene order of protein coding genes is identical to that of other Aplysia species. A+T content (65.9%) of the mitochondrial genome is included in the range for A+T content of congeneric species. This genome data provides evolutionary and systematic implications for the related species.