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// Xu Zhou 1, * , Hui-Ying Wang 1, * , Bin Wu 1, * , Cai-Yi Cheng 1 , Wei Xiao 2 , Zhen-Zhong Wang 2 , Yu-Yu Yang 1 , Ping Li 1 and Hua Yang 1 1 State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, China 2 State Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Process, Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, China * These authors have contributed equally to this work Correspondence to: Ping Li, email: liping2004@126.com Hua Yang, email: 104yang104@163.com Keywords: ginkgolide K, mitochondrial fission, mitochondrial permeability transition pore, neuron apoptosis, ischemic stroke Received: December 22, 2016 Accepted: April 27, 2017 Published: May 18, 2017 ABSTRACT Ginkgolide K (GK) belongs to the ginkgolide family of natural compounds found in Ginkgo biloba leaves, which have been used for centuries to treat cerebrovascular and cardiovascular diseases. We evaluated the protective effects of GK against neuronal apoptosis by assessing its ability to sustain mitochondrial integrity and function. Co-immunoprecipitation showed that Drp1 binding to GSK-3β was increased after an oxygen-glucose deprivation/reperfusion (OGD/R) insult in cultured neuroblastoma cells. This induced Drp1 and GSK-3β translocation to mitochondria and mitochondrial dysfunction, which was attenuated by GK. GK also reduced mitochondrial fission by increasing Drp1 phosphorylation at Ser637 and inhibiting mitochondrial Drp1 recruitment. In addition, GK exposure induced GSK-3β phosphorylation at Ser9 and enhanced the interaction between adenine nucleotide translocator (ANT) and p-GSK-3β. This interaction suppressed the interaction between ANT and cyclophilin D (CypD), which inhibited mitochondrial permeability transition pore (mPTP) opening. Similarly, suppression of mitochondrial fission by Mdivi-1 also inhibited GSK-3β-induced mPTP opening. Treating mice with GK prevented GSK-3β and Drp1 translocation to mitochondria and attenuated mitochondrial dysfunction after middle cerebral artery occlusion. We therefore propose that by inhibiting mitochondrial fission and attenuating mPTP opening, GK exerts neuroprotective effects that mitigate or prevent neuronal damage secondary to ischemic stroke.

Although mesenchymal stem cells (MSC) have been shown to be safe in preclinical studies of cardiovascular disease, multiple meta-analyses have debated whether functional improvement is significant or not. The cardiac differentiation from MSC is achievable using cardiogenic factors, however, the high cost and long culture period may limit the applications. Here, we developed a novel method to optimize the therapeutic outcome for myocardial infarction (MI). Treatment of MSC with apicidin, a histone deacetylase inhibitor, dramatically increased the expressions of cardiac markers such as GATA4, Nkx2.5, and cardiac troponin I (cTnI). In AC/MSC, stemness-related genes and yes-associated protein (YAP), a potent oncogene that drives cell proliferation, were significantly suppressed. Furthermore apicidin treatment or YAP knockdown downregulated miR-130a expression followed by induction of cardiac markers in MSC. In the comparison study, we found that both cardiac gene induction and angiogenesis were most prominent in the mixture of non-treated MSC and AC/MSC (Mix). Using mouse MI model, we show that application of Mix was strongly associated with cardiac differentiation of injected MSC and improved cardiac performance. Our results suggest that suppression of YAP/miR-130a shifts MSC cell fate toward cardiac lineage and identify apicidin as a potential pharmacological target for therapeutic development.

// Xiao-Yu Chen 1 , De-Fang Li 2 , Ji-Chun Han 2 , Bo Wang 3 , Zheng-Ping Dong 2 , Li-Na Yu 2 , Zhao-Hai Pan 2 , Chuan-Jun Qu 2 , Ying Chen 2 , Shi-Guo Sun 1 , Qiu-Sheng Zheng 2 1 College of Chemistry & Pharmacy, Northwest A&F University, Yangling, Shaanxi, 712100, China 2 Binzhou Medical University, Yantai, Shandong, 264003, China 3 Key Laboratory of Xinjiang Endemic Phytomedicine Resources of Ministry of Education, School of Pharmacy, Shihezi University, Shihezi, 832002, China Correspondence to: Qiu-Sheng Zheng, email: zqsyt@sohu.com Shi-Guo Sun, email: sunsg@nwsuaf.edu.cn Keywords: reprogramming, melanoma, cachexia, isoliquiritigenin, mTORC2-AKT-GSK3β signaling Received: December 08, 2016 Accepted: March 15, 2017 Published: March 29, 2017 ABSTRACT Isoliquiritigenin (ISL), a member of the flavonoids, is known to have anti-tumor activity in vitro and in vivo . The effect of ISL on reprogramming in cancer cells, however, remains elusive. In this study, we investigated the effect of ISL on reprogramming in human melanoma A375 cells. ISL (15 μg/ml) significantly inhibited A375 cell proliferation, anchorage independent cell proliferation and G2/M cell cycle arrest after ISL exposure for 24 h. However, there were no significant changes in apoptosis rate. Terminal differentiation indicators (melanin content, melanogenesis mRNA expression, tyrosinase (TYR) activity) were all up-regulated by ISL treatment. In ISL-treated cells, glucose uptake, lactate levels and mRNA expression levels of GLUT1 and HK2 were significantly decreased, and accompanied by an increase in O 2 consumption rate (OCR) and adenosine triphosphate (ATP) deficiency. Protein expression levels of mTORC2-AKT-GSK3β signaling pathway components (mTOR, p-mTOR, RICTOR, p-AKT, p-GSK3β) decreased significantly after ISL treatment. Co-treatment of ISL and the mTOR-specific inhibitor Ku-0063794 had a synergistic effect on the inhibition of proliferation, and increased melanin content and TYR activity. Glucose uptake and lactate levels decreased more significantly than treatment with ISL alone. These findings indicate that ISL induced reprogramming in A375 melanoma cells by activating mTORC2-AKT-GSK3β signaling.

// Xiupeng Zhang 1 , Haijing Zhou 1 , Yong Zhang 2 , Lin Cai 1 , Guiyang Jiang 1 , Ailin Li 3 , Yuan Miao 1 , Qingchang Li 1 , Xueshan Qiu 1 and Enhua Wang 1 1 Department of Pathology, College of Basic Medicine Science and First Affiliated Hospital of China Medical University, Shenyang, China 2 Department of Pathology, Cancer Hospital of China Medical University, Shenyang, China 3 Department of Radiotherapy, First Affiliated Hospital of China Medical University, Shenyang, China Correspondence to: Yuan Miao, email: cmumiaoyuan@163.com Keywords: ZNF452, NSCLC, AKT signaling, proliferation, invasion and metastasis Received: January 17, 2017 Accepted: February 24, 2017 Published: March 21, 2017 ABSTRACT ZNF452 is a zinc-finger protein family member which contains an isolated SCAN (SRE-ZBP, CTfin51, AW-1 and Number 18 cDNA) zinc-finger domain. Despite the SCAN N-terminus domain is known to play a role in transcriptional regulation of genes involved in cell survival and differentiation, there are no precise cellular functions that have been assigned to ZNF452. In the present study, we found that either endogenous or exogenous ZNF452 was overexpressed in the cytoplasm of NSCLC cells and positive ratio of ZNF452 in NSCLC samples (50.8%, 93/183) was significantly higher than that in normal lung tissues (22.4%, 13/58, P

Alcohol is a leading risk factor for osteonecrosis of the femoral head (ONFH). We explored the molecular mechanisms underlying alcohol-induced ONFH and investigated the protective effect of the novel Akt activator SC-79 against this disease. We found that ethanol inhibited expression of the osteogenic genes RUNX2 and OCN, downregulated osteogenic differentiation, impaired the recruitment of Akt to the plasma membrane, and suppressed Akt phosphorylation at Ser473, thereby inhibiting the Akt/GSK3β/β-catenin signaling pathway in bone mesenchymal stem cells. To assess SC-79's ability to counteract the inhibitory effect of ethanol on Akt-Ser73 phosphorylation, we performed micro-computerized tomography and immunofluorescent staining of osteopontin, osteocalcin and collagen type 1 in a rat model of alcohol-induced ONFH. We found that SC-79 injections inhibited alcohol-induced osteonecrosis. These results show that alcohol-induced ONFH is associated with suppression of p-Akt-Ser473 in the Akt/GSK3β/β-catenin signaling pathway in bone mesenchymal stem cells. We propose that SC-79 treatment to rescue Akt activation could be tested in the clinic as a potential therapeutic approach to preventing the development of alcohol-induced ONFH.

// Jing Ma 1,2 , Yuan Gao 3 , Lin Jiang 1,2 , Feng-lei Chao 1,2 , Wei Huang 1,2 , Chun-ni Zhou 1,2 , Wei Tang 1,2 , Lei Zhang 1,2 , Chun-xia Huang 4 , Yi Zhang 1,2 , Yan-min Luo 1,2 , Qian Xiao 1,2 , Hua-rong Yu 4 , Rong Jiang 1,2 and Yong Tang 1,2 1 Department of Histology and Embryology, Chongqing Medical University, Chongqing, P. R. China 2 Laboratory of Stem Cells and Tissue Engineering, Chongqing Medical University, Chongqing, P. R. China 3 Department of Geriatrics, The First Affiliated Hospital, Chongqing Medical University, Chongqing, P. R. China 4 Department of Physiology, Chongqing Medical University, Chongqing, P. R. China Correspondence to: Yong Tang, email: // Keywords : fluoxetine, Alzheimer’s disease, cognition, neuron, APP/PS1 mice, Gerotarget Received : July 04, 2016 Accepted : January 31, 2017 Published : February 16, 2017 Abstract Selective serotonin reuptake inhibitors (SSRIs) have been reported to increase cognitive performance in some clinical studies of Alzheimer’s disease (AD). However, there is a lack of evidence supporting the efficacy of SSRIs as cognition enhancers in AD, and the role of SSRIs as a treatment for AD remains largely unclear. Here, we characterized the impact of fluoxetine (FLX), a well-known SSRI, on neurons in the dentate gyrus (DG) and in CA1 and CA3 of the hippocampus of middle-aged (16 to 17 months old) APPswe/PSEN1dE9 (APP/PS1) transgenic AD model mice. We found that intraperitoneal (i.p.) injection of FLX (10 mg/kg/day) for 5 weeks effectively alleviated the impairment of spatial learning ability in middle-aged APP/PS1 mice as evaluated using the Morris water maze. More importantly, the number of neurons in the hippocampal DG was significantly increased by FLX. Additionally, FLX reduced the deposition of beta amyloid, inhibited GSK-3β activity and increased the level of β-catenin in middle-aged APP/PS1 mice. Collectively, the results of this study indicate that FLX delayed the progression of neuronal loss in the hippocampal DG in middle-aged AD mice, and this effect may underlie the FLX-induced improvement in learning ability. FLX may therefore serve as a promising therapeutic drug for AD.

// Qiong Xu 1, 2, * , Hai-Xu Xu 1, * , Jin-Ping Li 2 , Song Wang 2 , Zheng Fu 1, 2 , Jing Jia 2 , Li Wang 1, 2 , Zhi-Feng Zhu 2 , Rong Lu 1, 2 , Zhi Yao 1 1 Department of Immunology, Tianjin Medical University, Tianjin, China 2 Tianjin Kangzhe Pharmaceutical Company, Ltd., Tianjin, China * These authors contributed equally to this work Correspondence to: Zhi Yao, email: yaozhi@tijmu.edu.cn Rong Lu, email: lu_rong@vip.sina.com Keywords: cancer stem cells, hepatocellular carcinoma, GDF15, metastasis, tumorigenesis Received: November 25, 2016 Accepted: January 27, 2017 Published: February 09, 2017 ABSTRACT Cancer stem cells in liver cancer are thought to be responsible for tumor recurrence and metastasis. However, the factors that mediate this mechanism have yet to be completely elucidated. In this study, we isolated CD13 + CD44 + sphere cells (SCs) derived from liver cancer tissues and SK-Hep-1 cells, which possessed cancer stem cell-like properties. Through cytokine array analysis, growth differentiation factor 15 (GDF15) was significantly increased in SCs. Clinical data showed GDF15 was overexpressed in liver cancer tissues and was positively related to pathological grading. GDF15 knockdown significantly inhibited the growth and metastasis of SCs through AKT/GSK-3β/β-catenin pathway suppression. Moreover, a PI3K inhibitor LY294002 inhibited AKT/GSK-3β/β-catenin pathway activated by GDF15 and attenuated GDF15-induced proliferation, colony formation and invasion of SCs. Conclusion: Our studies suggest that CD13 + CD44 + SCs may represent a subset of LCSCs. GDF15 promotes the growth and metastasis of SCs by activating AKT/GSK-3β/β-catenin signaling pathway. Promisingly, GDF15 could be considered as a potential therapeutic target in liver cancer.

// Qingmei Kang 1, 2, * , Yue Xiang 1, 2, * , Dan Li 1, 2 , Jie Liang 1, 2 , Xiong Zhang 2 , Fanlin Zhou 1, 2 , Mengyuan Qiao 1, 2 , Yingling Nie 1, 2 , Yurong He 1, 2 , Jingyi Cheng 1, 2 , Yubing Dai 3 , Yu Li 1, 2 1 Department of Pathology, Chongqing Medical University, Chongqing, 400016, China 2 Center for Molecular Medicine Testing, Chongqing Medical University, Chongqing, 400016, China 3 Department of Neurosurgery, The Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou, 550004, China * These authors contributed equally to this work Correspondence to: Yu Li, email: liyu100@163.com Keywords: miR-124-3p, tau, caveolin-1, PI3K/Akt/GSK3β, Alzheimer's disease Received: November 02, 2016 Accepted: January 24, 2017 Published: February 07, 2017 ABSTRACT Hyperphosphorylation of Tau forming neurofibrillary tangles has been considered as a crucial event in the pathogenesis of Alzheimer's disease (AD). MiR-124-3p belongs to microRNA (miRNA) family and was markedly decreased in AD, however, the functions of miR-124-3p in the pathogenesis of AD remain unknown. We observed that the expression of miR-124-3p was significantly decreased in N2a/APP695swe cells; and transfection of miR-124-3p mimics not only attenuated cell apoptosis and abnormal hyperphosphorylation of Tau protein without any changes of total Tau protein, but also increased expression levels of Caveolin-1, phosphoinositide 3-kinase (PI3K), phospho-Akt (Akt-Ser473)/Akt, phospho-glycogen synthase kinase-3 beta (GSK-3β-Ser9)/GSK-3β in N2a/APP695swe cells. We further found that miR-12-3p directly targeted Caveolin-1; miR-124-3p inhibited abnormal hyperphosphorylation of Tau by regulating Caveolin-1-PI3K/Akt/GSK3β pathway in AD. This study reveals that miR-124-3p may play a neuroprotective role in AD, which may provide new ideas and therapeutic targets for AD.

// Biying Liu 1 , Haili Zhang 1 , Xiao Tan 1 , Daqian Yang 1 , Zhanjun Lv 1 , Huijie Jiang 1 , Jingjing Lu 1 , Ruiqi Baiyun 1 and Zhigang Zhang 1 1 College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China Correspondence to: Zhigang Zhang, email: zhangzhigang@neau.edu.cn Keywords: grape seed procyanidin extract, kidney injury, oxidative stress, Nrf2, nephroprotective effect Received: September 24, 2016 Accepted: January 16, 2017 Published: February 02, 2017 ABSTRACT Lead (Pb) is a global environmental health hazard that leads to nephrotoxicity. However, the effective treatment of Pb-induced nephrotoxicity remains elusive. Grape seed procyanidin extract (GSPE) has beneficial properties for multiple biological functions. Therefore, the present study investigated whether GSPE reduced Pb-induced nephrotoxicity as well as the protective mechanism of GSPE in a well-established 35-day Pb induced nephrotoxicity rat model. The results showed that GSPE normalized Pb-induced oxidative stress, histological damage, inflammatory, apoptosis, and changes of miR153 and glycogen synthase kinase 3β (GSK-3β) levels in rat kidney. Moreover, GSPE enhanced the induction of phase II detoxifying enzymes (heme oxygenase-1 and NAD(P)H quinone oxidoreductase 1) by increasing nuclear factor-erythroid-2-related factor 2 (Nrf2) expression. This study identifies for the first time that Pb-induced oxidative stress in rat kidney is attenuated by GSPE treatment via activating Nrf2 signaling pathway and suppressing miR153 and GSK-3β. Nrf2 signaling provides a new therapeutic target for renal injury induced by Pb, and GSPE could be a potential natural agent to protect against Pb-induced nephrotoxicity.

miR-27a regulates cell differentiation in a variety of diseases. However, whether and how miR-27a participates in laryngeal cancer cell differentiation remains unknown. Therefore, we explored role and molecular mechanism of miR-27a in laryngeal cancer differentiation in the study. We found that miR-27a expression was inversely correlated with laryngeal cancer differentiation degree based on the clinical pathological diagnosis of each patient. miR-27 asignificantly rescued differentiation and inhibited β-catenin, LEF1, OCT4 and SOX2 in Wnt/β-catenin pathway in all-trans-retinoic acid (ATRA)-induced laryngeal cancer cells. Bindings of RARα to miR-27a and miR-27a to GSK-3β were confirmed by ChIP and Luciferase reporter assays, respectively. In conclusion, miR-27a is a negative regulator in laryngeal cancer differentiation. RARα-mediated miR-27a transcriptional inactivation releases the inhibition of miR-27a on GSK-3β leading to laryngeal cancer differentiation through GSK-3β-involved Wnt/β-catenin pathway, suggesting that miR-27a is a usefully therapeutic target at least in ATRA-induced laryngeal cancer differentiation.

Evidence for long-term use of Chinese herbal medicine (CHM) as an adjuvant treatment in patients with type 2 diabetes (T2D) remains limited. This study aimed to assess the frequency of use, utilization patterns, and therapeutic effects of adjuvant CHM for ischemic heart disease (IHD) in patients with T2D in Taiwan. We identified 4620 IHD patients with T2D. After matching for age, gender, and insulin use, 988 subjects each were allocated to a CHM group and a non-CHM group. There were no differences in baseline characteristics except for comorbidities. The CHM group contained more cases with chronic obstructive pulmonary disease, hepatitis, ulcer disease, and hyperlipidemia. The cumulative survival probability was higher in CHM users than in matched non-CHM users aged 60 years or older (P < .0001, log rank test) regardless of gender (P = .0046 for men, P = .0010 for women, log rank test). Among the top 12 CHM combinations, Shu-Jing-Huo-Xue-Tang and Shao-Yao-Gan-Cao-Tang (13.6%) were the most common. This dual combination improved antiapoptotic activity in H2O2-exposed H9C2 cells by enhancing phosphorylation of glycogen synthase kinase-3β and p38 mitogen-activated protein kinase and could increase the survival of myocardial cells. Our study suggests that adjuvant CHM therapy may increase the survival probability and provides a comprehensive list for future investigations of the safety and efficacy of CHM for IHD patients with T2D.

// Youngmi Kim 1, * , Hyuna Kim 1, * , Deokbum Park 1 , Hansoo Lee 2 , Yun Sil Lee 3 , Jongseon Choe 4 , Young Myeong Kim 4 , Doyong Jeon 5 , Dooil Jeoung 1 1 Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chunchon 24341, Korea 2 Department of Biological Sciences, College of Natural Sciences, Kangwon National University, Chunchon 24341, Korea 3 College of Pharmacy, Ewha Womans University, Seoul 03760, Korea 4 Graduate School of Medicine, Kangwon National University, Chunchon 24341, Korea 5 L-Base Company, Seoul 01062, Korea * These authors contributed equally to this work Correspondence to: Dooil Jeoung, email: jeoungd@kangwon.ac.kr Keywords: anti-cancer drug-resistance, CAGE, cyclinD1, GSK3β, peptides Received: February 12, 2016 Accepted: January 03, 2017 Published: January 13, 2017 ABSTRACT We previously reported the role of cancer/testis antigen CAGE in the response to anti-cancer drugs. CAGE increased the expression of cyclinD1, and pGSK3β Ser9 , an inactive GSK3β, while decreasing the expression of phospho-cyclinD1Thr 286 . CAGE showed binding to GSK3β and the domain of CAGE (amino acids 231–300) necessary for binding to GSK3β and for the expression regulation of cyclinD1 was determined. 269 GTGKT 273 peptide, corresponding to the DEAD box helicase domain of CAGE, decreased the expression of cyclinD1 and pGSK3β Ser9 while increasing the expression of phospho-cyclinD1 Thr286 . GTGKT peptide showed the binding to CAGE and prevented CAGE from binding to GSK3β. GTGKT peptide changed the localization of CAGE and inhibited the binding of CAGE to the promoter sequences of cyclin D1. GTGKT peptide enhanced the apoptotic effects of anti-cancer drugs and decreased the migration, invasion, angiogenic, tumorigenic and metastatic potential of anti-cancer drug-resistant cancer cells. We found that Lys 272 of GTGKT peptide was necessary for conferring anti-cancer activity. Peptides corresponding to the DEAD box helicase domain of CAGE, such as AQTGTGKT, QTGTGKT and TGTGKT, also showed anti-cancer activity by preventing CAGE from binding to GSK3β. GTGKT peptide showed ex vivo tumor homing potential. Thus, peptides corresponding to the DEAD box helicase domain of CAGE can be developed as anti-cancer drugs in cancer patients expressing CAGE.

// Xiupeng Zhang 1 , Guiyang Jiang 1 , Mingfang Sun 1 , Haijing Zhou 1 , Yuan Miao 1 , Mengyuan Liang 1 , Enhua Wang 1 , Yong Zhang 2 1 Department of Pathology, First Affiliated Hospital and College of Basic Medical Sciences, China Medical University, Shenyang, China 2 Department of Pathology, Cancer Hospital of China Medical University, Shenyang, China Correspondence to: Yong Zhang, email: zhangyong@cancerhosp-ln-cmu.com Keywords: THUMPD1, breast cancer, AKT signaling, snail, GSK3β Received: October 05, 2016 Accepted: December 27, 2016 Published: January 05, 2017 ABSTRACT Human THUMP domain-containing protein 1 (THUMPD1) is a specific adaptor protein that modulates tRNA acetylation through interaction with NAT10. Immunohistochemical analysis of 146 breast cancer specimens (82 triple-negative and 64 non-triple-negative cases) indicated THUMPD1 expression is higher in breast cancer tissues (60.9%, 89/146) than normal breast tissues (28.3%, 15/53; p < 0.001). Overall and cytosolic, but not nuclear, THUMPD1 expression in breast cancer correlated with advanced TNM stage ( p = 0.003 and p < 0.001, respectively), lymph node metastasis ( p = 0.001 and p < 0.001, respectively), and poor patient prognosis ( p = 0.001 and p < 0.001, respectively). THUMPD1 interacted and co-localized with YAP, but did not affect Hippo pathway activity. THUMPD1 overexpression enhanced breast cancer cells invasion and migration in vivo and in vitro , possibly through activation of AKT, GSK3β and Snail, and inhibition of E-cadherin. Treatment with the AKT inhibitor, LY294002, reduced the effects of THUMPD1 overexpression in breast cancer cells. These results indicate that THUMPD1 promotes breast cancer cells invasion and migration via the AKT-GSK3β-Snail pathway.

// Mamoru Takada 1, * , Ming Zhuang 2, * , Hiroyuki Inuzuka 3 , Jing Zhang 1 , Giada Zurlo 1 , Jinfang Zhang 3 , Qing Zhang 1, 4 1 Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA 2 Department of General Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China 3 Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA 4 Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA * These authors have contributed equally to this work Correspondence to: Qing Zhang, email: Qing_Zhang@med.unc.edu Keywords: EglN2, triple negative breast cancer, FBW7 Received: September 01, 2016 Accepted: December 13, 2016 Published: December 27, 2016 ABSTRACT EglN2 contributes to ERα-positive breast tumorigenesis by acting as an estrogen-inducible gene. However, the detailed molecular mechanism(s) underlying the post-transcriptional regulation of EglN2 and its potential role in Triple Negative Breast Cancer (TNBC) remains largely unclear. By using C3Tag transgenic mice and tumor-derived C3Tag cell line, here we report that EglN2 contributes to TNBC tumor progression and genetic knockout of EglN2 improves C3Tag mice survival from tumor progression. Mechanistically, we further show that FBW7, an E3 ligase complex component that is frequently downregulated in TNBC, negatively regulates EglN2 protein stability. As such, depletion of FBW7 in breast cell lines leads to upregulation of EglN2 and other canonical FBW7 substrates. Conversely, FBW7 overexpression leads to EglN2 downregulation in a GSK3β-dependent manner. Furthermore, we identified some potential serine or threonine residues on the C-terminal of EglN2 that may mediate its binding and potential regulation by FBW7. Together, our study reveals that EglN2 might act as an FBW7 ubiquitin ligase substrate contributing to TNBC.

// Tingting Cheng 1 , Kui Zhai 2 , Yan Chang 2 , Guidong Yao 1 , Jiahuan He 1 , Fang Wang 1 , Huijuan Kong 1 , Hang Xin 1 , Huiwen Wang 2 , Meng Jin 2 , Bing Gong 3 , Lei Gu 2 , Zhiguang Yang 2 , Yanyun Wu 2 , Guangju Ji 2 , Yingpu Sun 1 1 Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China 2 National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China 3 Department of Cardiac Surgery, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Disease, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Correspondence to: Guangju Ji, email: gj28@ibp.ac.cn Yingpu Sun, email: syp2008@vip.sina.com Keywords: CHIR99021, retinoic acid, primordial germ cells, human embryonic stem cells, β-catenin Received: September 24, 2016 Accepted: December 01, 2016 Published: December 15, 2016 ABSTRACT Primordial germ cells (PGCs) derived from human embryonic stem cells (hESCs) represent as a desirable experimental model as well as a potential strategy for treating male infertility. Here, we developed a simple and feasible method for differentiation of PGCs from hESCs by using CHIR99021 (an inhibitor of glycogen synthase kinase 3) and retinoic acid (RA). We firstly found that the deleted in azoospermia-like (DAZL) protein can be detected in 3 d CHIR99021 plus 9 d retinoic acid treated cultures and 12 d CHIR99021 plus retinoic acid co-treated cultures, but not expressed in single CHIR99021 treated cultures, single retinoic acid treated cultures, as well as 3 d retinoic acid plus 9 d CHIR99021 treated cultures. Next, we showed that several PGCs’ markers were expressed in the 12 d CHIR99021 and retinoic acid co-treated cultures or 3 d CHIR99021 plus 9 d retinoic acid treated cultures. Moreover, meiosis was initiated in CHIR99021 and retinoic acid co-treated cultures as evidenced by a significant expression of the punctate synaptonemal complex protein 3 (SCP3). Fluorescent in situ hybridization (FISH) analysis indicated that a small percentage of putative 1N populations were formed. Mechanically, we found that β-catenin relocated into nucleus after the treatment of 3 d CHIR99021 suggesting that Wnt signaling pathway was activated. Furthermore, blockade of Wnt signaling pathway by IWR-1 can reverse CHIR99021 and retinoic acid mediated-effects. Taken together, our results indicate that CHIR99021 combined with retinoic acid can effectively differentiate hESCs into PGCs via activating Wnt signaling pathway.

// Shida Yang 1 , Zhiyong Zhu 3 , Xiaobing Zhang 1 , Ning Zhang 4 , Zhicheng Yao 2 1 Department of Laboratory Medicine, The People’s Hospital of Liaoning Province, Shenyang, China 2 Department of Neurology, The People’s Hospital of Liaoning Province, Shenyang, China 3 Department of Orthopedics, The People’s Hospital of Liaoning Province, Shenyang, China 4 Department of Laboratory Medicine, The First Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang, China Correspondence to: Zhicheng Yao, email: yangshidasy1@gmail.com Keywords: idelalisib, colon cancer, PUMA, apoptosis, GSK3β Received: July 29, 2016 Accepted: December 12, 2016 Published: December 20, 2016 ABSTRACT Idelalisib, a PI3K inhibitor, specifically targeting p110δ, has been approved for the treatment of chronic lymphocytic leukemia/small lymphocytic lymphoma and follicular lymphoma. However, the mechanisms of action of idelalisib in colon cancer cells are not well understood. We investigated how idelalisib suppresses colon cancer cells growth and potentiates effects of other chemotherapeutic drugs. In this study, we found that idelalisib treatment induces PUMA in colon cancer cells irrespective of p53 status through the p65 pathway following AKT inhibition and glycogen synthase kinase 3β (GSK3β) activation. PUMA is necessary for idelalisib-induced apoptosis in colon cancer cells. Idelalisib also synergized with 5-FU or regorafenib to induce marked apoptosis via PUMA in colon cancer cells. Furthermore, PUMA deficiency suppressed apoptosis and antitumor effect of idelalisib in xenograft model. These results demonstrate a critical role of PUMA in mediating the anticancer effects of idelalisib in colon cancer cells and suggest that PUMA induction can be used as an indicator of idelalisib sensitivity, and also have important implications for it clinical applications.

// Feng He 1, 2, * , Huacui Chen 1, * , Ping Yang 1, * , Qianlong Wu 1 , Tong Zhang 1 , Chengxing Wang 1 , Jianchang Wei 1 , Zhuanpeng Chen 1 , He Hu 1 , Wanglin Li 1 , Jie Cao 1 1 Department of General Surgery, Guangzhou Digestive Disease Center, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou 510180, China 2 Department of Pathology, Interdepartmental Program in Vascular Biology and Therapeutics, Yale University School of Medicine, New Haven, Connecticut 06520, USA * These authors contributed equally to this work Correspondence to: Wanglin Li, email: 421255910@qq.com Jie Cao, email: czhongt@126.com Keywords: gankyrin, colorectal cancer, metastasis, PI3K/Akt signaling, Wnt/β-catenin signaling Received: July 06, 2016 Accepted: November 02, 2016 Published: November 08, 2016 ABSTRACT High levels of angiogenesis, metastasis and chemoresistance are major clinical features of colorectal cancer (CRC), a lethal disease with a high incidence worldwide. Aberrant activation of Wnt/β-catenin pathway contributes to CRC progression. However, little is known about regulatory mechanisms of the β-catenin activity in cancer progression. Here we report that Gankyrin was markedly upregulated in primary tumor tissues from CRC patients and was associated with poor survival. Moreover, we demonstrated that overexpressing Gankyrin promoted, while knockdown of Gankyrin impaired, the aggressive phenotype of proliferation, angiogenesis, chemoresistance and metastasis of CRC cells both in vitro and in vivo . Importantly, we found a unique molecular mechanism of Gankyrin in CRC cells signaling transduction, that regulated the cross-talk between PI3K/Akt and Wnt/β-catenin signaling pathways, sustaining PI3K/GSK-3β/β-catenin signal activation in CRC. Therefore, these findings not only reveal a mechanism that promotes aggressiveness and progression in CRC, but also provide insight into novel molecular targets for antitumor therapy in CRCs.

// Chen-Xu Ni 1, * , Yang Qi 1, * , Jin Zhang 1, 2, * , Ying Liu 2 , Wei-Heng Xu 1 , Jing Xu 2 , Hong-Gang Hu 1 , Qiu-Ye Wu 1 , Yan Wang 1 , Jun-Ping Zhang 1 1 School of Pharmacy, Second Military Medical University, Shanghai 200433, China 2 Department of Pharmacy, Shanghai East Hospital, Tongji University, Shanghai 310000, China * These authors have contributed equally to this work Correspondence to: Jun-Ping Zhang, email: jpzhang08@163.com Yan Wang, email: wangyansmmu@126.com Keywords: matrine derivative, hepatocellular carcinoma, cancer stem-like cells, GSK3β, AKT Received: January 18, 2016 Accepted: October 14, 2016 Published: October 22, 2016 ABSTRACT The eradication of cancer stem cells (CSCs) is significant for cancer therapy and prevention. In this study, we evaluated WM130, a novel derivative of matrine, for its effect on CSCs using human hepatocellular carcinoma (HCC) cell lines, their sphere cells, and sorted EpCAM + cells. We revealed that WM130 could not only inhibit proliferation and colony formation of HCC cells, but also suppress the expression of some stemness-related genes and up-regulate some mature hepatocyte marker genes, indicating a promotion of differentiation from CSCs to hepatocytes. WM130 also suppressed the proliferation of doxorubicin-resistant hepatoma cells, and markedly reduced the cells with CSC biomarker EpCAM. Moreover, WM130 suppressed HCC spheres, not only primary spheres but also subsequent spheres, indicating an inhibitory effect on self-renewal capability of CSCs. Interestingly, WM130 exhibited a remarkable inhibitory preference on HCC spheres and EpCAM + cells rather than their parental HCC cells and EpCAM - cells respectively. In vivo , WM130 inhibited HCC xenograft growth, decreased the number of sphere-forming cells, and remarkably decreased the levels of EpCAM mRNA and protein in tumor xenografts. Better inhibitory effect was achieved by WM130 in combination with doxorubicin. Further mechanism study revealed that WM130 inhibited AKT/GSK3β/β-catenin signaling pathway. Collectively, our results suggest that WM130 remarkably inhibits hepatic CSCs, and this effect may via the down-regulation of the AKT/GSK3β/β-catenin pathway. These findings provide a strong rationale for the use of WM130 as a novel drug candidate in HCC therapy.

// Shingo Shimozaki 1, 4, * , Norio Yamamoto 1 , Takahiro Domoto 4, * , Hideji Nishida 1 , Katsuhiro Hayashi 1 , Hiroaki Kimura 1 , Akihiko Takeuchi 1 , Shinji Miwa 1, 3, 4 , Kentaro Igarashi 1 , Takashi Kato 1 , Yu Aoki 1 , Takashi Higuchi 1 , Mayumi Hirose 4 , Robert M Hoffman 2, 3 , Toshinari Minamoto 4 , Hiroyuki Tsuchiya 1 1 Department of Orthopedic Surgery, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Japan 2 Department of Surgery, University of California, San Diego, CA, U.S.A 3 AntiCancer Incorporated, San Diego, CA, U.S.A 4 Division of Translational and Clinical Oncology, Cancer Research Institute, Kanazawa University, Kanazawa, Japan * These authors contributed equally to this work Correspondence to: Robert M. Hoffman, email: all@anticancer.com Norio Yamamoto, email: norinori@med.kanazawa-u.ac.jp Keywords: glycogen synthase kinase-3β, molecular target, treatment, osteosarcoma, orthotopic nude mice Received: September 09, 2016 Accepted: October 05, 2016 Published: October 20, 2016 ABSTRACT Development of innovative more effective therapy is required for refractory osteosarcoma patients. We previously established that glycogen synthase kinase-3β (GSK- 3β) is a therapeutic target in various cancer types. In the present study, we explored the therapeutic efficacy of GSK-3β inhibition against osteosarcoma and the underlying molecular mechanisms in an orthotopic mouse model. Expression and phosphorylation of GSK-3β in osteosarcoma and normal osteoblast cell lines was examined, together with efficacy of GSK-3β inhibition on cell survival, proliferation and apoptosis and on the growth of orthotopically-transplanted human osteosarcoma in nude mice. We also investigated changes in expression, phosphorylation and co-transcriptional activity of β-catenin in osteosarcoma cells following GSK-3β inhibition. Expression of the active form of GSK- 3β (tyrosine 216-phosphorylated) was higher in osteosarcoma than osteoblast cells. Inhibition of GSK-3β activity by pharmacological inhibitors or of its expression by RNA interference suppressed proliferation of osteosarcoma cells and induced apoptosis. Treatment with GSK-3β-specific inhibitors attenuated the growth of orthotopic osteosaroma in mice. Inhibition of GSK-3β reduced phosphorylation at GSK- 3β-phospho-acceptor sites in β-catenin and increased β-catenin expression, nuclear localization and co-transcriptional activity. These results suggest the efficacy of GSK-3β inhibitors is associated with activation of β-catenin, a putative tumor suppressor in bone and soft tissue sarcoma and an important component of osteogenesis. Our study thereby demonstrates a critical role for GSK-3β in sustaining survival and proliferation of osteosarcoma cells, and identifies this kinase as a potential therapeutic target against osteosarcoma.

Airway smooth muscle (ASM) hyperplasia is a key feature of airway remodeling in development of lung diseases such as asthma. Anomalous proliferation of ASM cells directly contributes to ASM hyperplasia. However, the molecular mechanisms controlling ASM cell proliferation are not completely understood. Semaphorins are versatile regulators of various cellular processes including cell growth and proliferation. The role of semaphorins in ASM cell proliferation has remained to be addressed. Here, we report that semaphorin 3A (Sema3A) receptor, neuropilin 1 (Nrp1), is expressed on human ASM cells (HASMC) isolated from healthy and asthmatic donors and treatment of these cells with exogenous Sema3A inhibits growth factor-induced proliferation. Sema3A inhibitory effect on HASMC proliferation is associated with decreased tyrosine phosphorylation of PDGFR, downregulation of Rac1 activation, STAT3 and GSK-3β phosphorylation. Bronchial sections from severe asthmatics displayed immunoreactivity of Nrp1, suggestive of functional contribution of Sema3A-Nrp1 axis in airway remodeling. Together, our data suggest Sema3A-Nrp1 signaling as a novel regulatory pathway of ASM hyperplasia.

// Sreevidya Santha 1 , Gantulga Davaakhuu 1 , Aninda Basu 1 , Rong Ke 1 , Subhasis Das 1 , Ajay Rana 1, 2, 3 , Basabi Rana 1, 2, 3 1 Department of Surgery, Division of Surgical Oncology, University of Illinois at Chicago, Chicago, IL 60612, USA 2 University of Illinois Hospital and Health Sciences System Cancer Center, University of Illinois at Chicago, Chicago, IL 60612, USA 3 Jesse Brown VA Medical Center, Chicago, IL 60612, USA Correspondence to: Basabi Rana, email: basrana@uic.edu Keywords: GSK3β, AKT, PPARγ, AMPK, apoptosis-resistance Received: July 26, 2016 Accepted: August 29, 2016 Published: September 02, 2016 ABSTRACT Glycogen Synthase Kinase-3β (GSK3β) is a serine/threonine kinase, known to regulate various cellular processes including proliferation, differentiation, survival, apoptosis as well as TRAIL-resistance. Thus pathways that can modulate GSK3β axis are important targets for cancer drug development. Our earlier studies have shown that combinatorial treatment with Troglitazone (TZD) and TRAIL can induce apoptosis in TRAIL-resistant cancer cells. The current studies were undertaken to investigate whether GSK3β pathway was modulated during this apoptosis. Our results indicated an increase in inhibitory GSK3β Ser9 phosphorylation during apoptosis, mediated via AKT. At a later time, however, TZD alone and TRAIL-TZD combination produced a dramatic reduction of GSK3β expression, which was abolished by cycloheximide. Luciferase assays with GSK3β-luc promoter reporter showed that TZD can effectively antagonize GSK3β promoter activity. Since TZD is a ligand for transcription factor PPARγ and can activate AMPK, we determined their roles on antagonism of GSK3β. Knockdown of PPARγ was unable to restore GSK3β expression or antagonize GSK3β Ser9 phosphorylation. Although pretreatment with Compound C (pharmacological inhibitor of AMPK) partially rescued GSK3β expression, knockdown of AMPKα1 or α2 alone or in combination were ineffective. These studies suggested a novel PPARγ-AMPK-independent mechanism of targeting GSK3β by TZD, elucidation of which might provide newer insights to improve our understanding of TRAIL-resistance.

// Ye Li 1, * , Shu-Xiang Cui 2, * , Shi-Yue Sun 3 , Wen-Na Shi 3 , Zhi-Yu Song 3 , Shu-Qing Wang 3 , Xin-Feng Yu 3 , Zu-Hua Gao 4 , Xian-Jun Qu 3 1 Department of Pharmacology, School of Chemical Biology & Pharmaceutical Sciences, Capital Medical University, Beijing, China 2 Beijing Key Laboratory of Environmental Toxicology, Department of Toxicology and Sanitary Chemistry, School of Public Health, Capital Medical University, Beijing, China 3 Department of Pharmacology, Capital Medical University School of Basic Medical Sciences, Beijing, China 4 Department of Pathology, McGill University, Montreal, Quebec, Canada * These authors have contributed equally to this work Correspondence to: Xian-Jun Qu, email: quxj@ccmu.edu.cn Zu-Hua Gao, email: zu-hua.gao@mcgill.ca Keywords: myricetin, APC Min/+ mouse model, intestinal adenomatous polyps, Wnt/β-catenin pathway, chronic inflammation Received: December 22, 2015 Accepted: July 26, 2016 Published: August 06, 2016 ABSTRACT Myricetin is a natural dietary flavonoid compound. We evaluated the efficacy of myricetin against intestinal tumorigenesis in adenomatous polyposis coli multiple intestinal neoplasia ( APC Min/+ ) mice. Myricetin was given orally once a day for 12 consecutive weeks. APC Min/+ mice fed with myricetin developed fewer and smaller polyps without any adverse effects. Histopathological analysis showed a decreased number of dysplastic cells and degree of dysplasia in each polyp. Immunohistochemical and western blot analysis revealed that myricetin selectively inhibits cell proliferation and induces apoptosis in adenomatous polyps. The effects of myricetin were associated with a modulation the GSK-3β and Wnt/β-catenin pathways. ELISA analysis showed a reduced concentration of pro-inflammatory cytokines IL-6 and PGE2 in blood, which were elevated in APC Min/+ mice. The effect of myricetin treatment was more prominent in the adenomatous polyps originating in the colon. Further studies showed that myricetin downregulates the phosphorylated p38 MAPK/Akt/mTOR signaling pathways, which may be the mechanisms for the inhibition of adenomatous polyps by myricetin. Taken together, our data show that myricetin inhibits intestinal tumorigenesis through a collection of biological activities. Given these results, we suggest that myricetin could be used preventatively to reduce the risk of developing colon cancers.

During the process of tumorigenesis, inactivation of tumor suppressors is a critical step. EZH2, a histone methyltransferase, promotes cell growth and migration through catalyzing trimethylation of histone H3 at Lys 27 (H3K27me3) and plays an important role in tumorigenesis. Its expression can be controlled by phosphorylation. However, the regulation of EZH2 activity by tumor suppressor kinase is not well understood. In this study, we show that glycogen synthase kinase 3 beta (GSK3β) negatively regulates H3K27 trimethylation. We also validate that GSKβ physically interacts with EZH2, and their interaction occurs in the cytosol. GSK3β phosphorylates EZH2 at Ser363 and Thr367 in vitro, and activating GSK3β upregulates Thr367 phosphorylationin vivo. Cells expressing GSK3β-non-phosphorylatable mutant EZH2 have higher H3K27 trimethylation and enhanced ability of cell migration and anchorage-independent growth. Inactivation of GSK3β as measured by its phosphorylation at Ser9 is positively correlated with higher level of H3K27 trimethylation in tumor tissues from breast cancer patients. Our study indicated that GSK3β phosphorylates EZH2 at Ser363 and Thr367, resulting in reduced H3K27 trimethylation and biological activity of EZH2 in breast cancer.

// Anwen Wei 1, * , Bo Fan 2, * , Yujie Zhao 1 , Han Zhang 1 , Liping Wang 1 , Xiao Yu 3 , Qingmin Yuan 1 , Deyong Yang 2 , Shujing Wang 1 1 Department of Biochemistry and Molecular Biology, Institute of Glycobiology, Dalian Medical University, Dalian, 116044, Liaoning Province, China 2 Department of Urology, First Affiliated Hospital of Dalian Medical University, Dalian, 116011, Liaoning Province, China 3 Department of Pathology, Dalian Medical University, Dalian, 116044, Liaoning Province, China * These authors contributed equally to this work Correspondence to: Shujing Wang, email: wangshujing@dlmedu.edu.cn Deyong Yang, email: yangdeyong@dlmedu.edu.cn Keywords: ST6Gal-I, α2,6-sialic acid, prostate cancer, prognosis, proliferation and invasion Received: March 01, 2016 Accepted: August 24, 2016 Published: August 30, 2016 ABSTRACT ST6Gal-I sialyltransferase adds α2,6-linked sialic acids to the terminal ends of glycan chains of glycoproteins and glycolipids. ST6Gal-I is reportedly upregulated in many cancers, including hepatocellular carcinoma, ovarian cancer and breast cancer. However, the expression and function of ST6Gal-I in prostate cancer (PCa) and the mechanism underlying this function remain largely unknown. In this study, we observed that ST6Gal-I expression was upregulated in human PCa tissues compared to non-malignant prostate tissues. High ST6Gal-I expression was positively correlated with Gleason scores, seminal vesicle involvement and poor survival in patients with PCa. ST6Gal-I knockdown in aggressive prostate cancer PC-3 and DU145 cells significantly inhibited the proliferation, growth, migration and invasion capabilities of these cells. ST6Gal-I knockdown decreased the levels of several PI3K/Akt/GSK-3β/ β-catenin pathway components, such as p-PI3K, (Ser473)p-Akt, (Ser9)p-GSK-3β and β-catenin. Furthermore, targeting this pathway with a PI3K inhibitor or Akt RNA interference decreased p-Akt, p-GSK-3β and β-catenin expression, resulting in decreased PC-3 and DU145 proliferation, migration and invasion. Taken together, these results indicate that ST6Gal-I plays a critical role in cell proliferation and invasion via the PI3K/Akt/GSK-3β/β-catenin signaling pathway during PCa progression and that it might be a promising target for PCa prognosis determination and therapy.

Colorectal cancer (CRC) with microsatellite instability (MSI) may exhibit impaired epithelial-mesenchymal transition (EMT), but little is known about the underlying mechanisms of this phenomenon. In this study, we investigated the role of Twist1 and its downstream signaling cascades in EMT induction according to MSI status. To investigate the effects of Twist1 on EMT induction according to MSI status, MSS LS513 and MSI LoVo colon cancer cell lines, which overexpress human Twist1, were generated. Twist1-induced EMT and its downstream signaling pathways were evaluated via in vitro and in vivo experiments. We found that Twist1 induced EMT markers and stem cell-like characteristics via AKT signaling pathways. Twist1 induced activation of AKT and suppression of glycogen synthase kinase (GSK)-3β, which resulted in the activation of β-catenin, increasing CD44 expression. In addition, Twist1 activated the AKT-induced NF-κB pathway, increasing CD44 and CD166 expression. Activation of both the AKT/GSK-3β/β-catenin and AKT/NF-κB pathways occurred in MSS LS513 cells, while only the AKT/GSK-3β/β-catenin pathway was activated in MSI LoVo cells. In conclusion, Twist1 induces stem cell-like characteristics in colon cancer cell lines related to EMT via AKT signaling pathways, and those pathways depend on MSI status.

// Zhaoming Zhong 1, 2, * , Zedong Hu 1, * , Yue Jiang 1 , Ruimei Sun 1 , Xue Chen 1 , Hongying Chu 1 , Musheng Zeng 3 , Chuanzheng Sun 1 1 Department of Head and Neck Surgery, The Third Affiliated Hospital of Kunming Medical University, Kunming, China 2 Department of Medical Oncology, The First Affiliated Hospital of Kunming Medical University, Kunming, China 3 State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, China * These authors have contributed equally to this work Correspondence to: Musheng Zeng, email: mushengzeng@163.com Chuanzheng Sun, email: scz008@126.com Keywords: anaplastic thyroid carcinoma, interleukin-11, epithelial-mesenchymal transition, metastasis, hypoxia-inducible factor-1α Received: September 13, 2015 Accepted: June 29, 2016 Published: July 25, 2016 ABSTRACT Metastasis is the major cause of treatment failure in anaplastic thyroid carcinoma (ATC) patients. In the preliminary study, we demonstrated that interleukin (IL)-11 expression is positively correlated with distant metastasis in ATC. However, the mechanisms underlying remain largely unknown. Here, we found that cobalt chloride (a hypoxia mimetic) promoted IL-11 expression via HIF-1α activation. Furthermore, the resultant increase in IL-11 expression significantly induced epithelial-mesenchymal transition (EMT) in ATC cells, accompanied by Akt/GSK3β pathway activation and increased invasive and migratory abilities. Conversely, HIF-1α or IL-11 knockdown, or treating cells with a neutralizing antibody against IL-11, a PI3K inhibitor, or Akt inhibitor V, significantly suppressed the induction of EMT and counteracted the enhancements in invasive and migratory abilities. These results indicate that hypoxia increases IL-11 secretion in ATC cells via HIF-1α induction and that IL-11 then induces EMT in these cells via the PI3K/Akt/GSK3β pathway, ultimately improving their invasive and migratory potential. This study elucidates the prometastatic role played by IL-11 in ATC metastasis and indicates it as a potential target for the treatment of cancer metastasis. However, many questions remain to be explored.

Cholangiocarcinoma (CCA) invasion and metastasis are the primary causes of poor survival rates in patients. The epithelial-mesenchymal transition (EMT) is a crucial step in cancer invasion and metastasis. However, it is still unclear of the molecular mechanism. In this study, the expression of 14-3-3ζ and atypical protein kinase C-ι (aPKC-ι) was further detected in CCA tissues and cell lines. Meanwhile, we established the EMT model of CCA cells and investigated 14-3-3ζ and aPKC-ι co-regulatory effect on the EMT in vitro and in vivo. Further, we identified the downstream molecular glycogen synthase kinase 3 beta (GSK-3β)/Snail signalling pathway that contribute to regulating the EMT. Our data showed that the expression of 14-3-3ζ and aPKC-ι was synergistically increased in CCA tissues compared with adjacent noncancerous tissues and was intimately associated with differentiation and the tumour-node-metastasis (TNM) stage. Multivariate Cox regression analysis indicated that high 14-3-3ζ and aPKC-ι expression separately predicted a poor prognosis and were independent prognostic indicators in patients with CCA. The CO-IP experiment confirmed that the mutual binding relationship between 14-3-3ζ and aPKC-ι. Small interfering RNAs and siRNA rescue experiment demonstrated that 14-3-3ζ and aPKC-ι regulated each other. In addition, 14-3-3ζ and aPKC-ι pretreatment by si-RNA inhibit the phosphorylated GSK-3β and Snail expression during EMT. Meanwhile, silence of 14-3-3ζ or aPKC-ι suppressed CCA cells migration, metastasis and proliferation in vitro and in vivo. Our study demonstrates that 14-3-3ζ and aPKC-ι synergistically facilitate EMT of CCA via GSK-3β/Snail signalling pathway, and may be potential therapeutic target for CCA.

Mutations that deregulate protein degradation lead to human malignancies. The SCF ubiquitin E3 ligase complex degrades key oncogenic regulators, thereby limiting their oncogenic potential. FBW7 is a substrate recognition subunit of SCFFBW7 and is among the most commonly mutated ubiquitin-proteasome system proteins in cancer. FBW7-mutated cancer cells display increased genome instability, but the molecular mechanism by which FBW7 preserves genome integrity remains elusive. Here, we demonstrate that SCFFBW7 regulates the stability of FAAP20, a critical component of the Fanconi anemia (FA) DNA interstrand cross-link (ICL) repair pathway. Phosphorylation of the FAAP20 degron motif by GSK3β provides a platform for recognition and polyubiquitination of FAAP20 by FBW7, and its subsequent degradation by the proteasome. Accordingly, enhanced GSK3β-FBW7 signaling disrupts the FA pathway. In cells expressing non-phosphorylatable FAAP20 mutant, the turnover of its binding partner, FANCA, is deregulated in the chromatin during DNA ICL repair, and the FA pathway is compromised. We propose that FAAP20 degradation, which is prompted by its phosphorylation, controls the dynamics of the FA core complex required for completing DNA ICL repair. Together, this study provides insights into how FBW7-mediated proteolysis regulates genome stability and how its deregulation is associated with tumorigenesis.

// Tie-qiu Yin 1 , Xuan Ou-yang 1 , Fang-yan Jiao 1 , Lu-ping Huang 1 , Xu-dong Tang 1 , Bi-qiong Ren 1 1 The Second People’s Hospital of Hunan Province, Clinical Medical College of Hunan University of Chinese Medicine, Changsha 410007, China Correspondence to: Tie-qiu Yin, email: ytqstar@163.com Keywords: cervical cancer, PA-MSHA, PTEN, p-AKT, p-GSK3β Received: September 30, 2015 Accepted: April 24, 2016 Published: May 19, 2016 ABSTRACT We investigated the effects of Pseudomonas aeruginosa mannose-sensitive hemagglutinin (PA-MSHA) on the proliferation and invasion of human cervical cancer cell lines, as well as the molecular pathways underlying these effects. MTT cell proliferation assays revealed a time- and concentration-dependent cytotoxic effect of PA-MSHA on HeLa cells but not H8 cells. Flow cytometry with propidium iodide and annexin-V-fluorescein isothiocyanate labeling (FITC) indicated that various concentrations of PA-MSHA could induce apoptosis and G2-M cell cycle arrest in HeLa cells. PA-MSHA also impaired the migration and invasion abilities of HeLa cells in Wound healing and Transwell invasion assays. Western blot results demonstrated that PA-MSHA reduced the expression of p-AKT, p-GSK3β, BCL-2, Vimentin and β-catenin, but increased the levels of PTEN, BAD, BAX and E-cadherin in HeLa cells. Importantly, PTEN siRNA induced the activity of p-AKT, while PA-MSHA partly inhibited this induction, indicating that PA-MSHA may reduce the cell proliferation and invasion potential by activating PTEN and thus inhibiting the AKT pathway in vitro . These data suggest the potential application of PA-MSHA to the treatment of human cervical cancer.

// Jing Yang 1, * , Kun Zhang 1, * , Haixing Song 1, * , Mingbo Wu 1, * , Jingyi Li 1 , Ziyi Yong 2 , Sheng Jiang 3 , Xi Kuang 4 , Tao Zhang 1 1 School of Biomedical Sciences, Chengdu Medical College, Chengdu, China 2 School of Pharmacy, Chengdu Medical College, Chengdu, China 3 School of Basic Medical Sciences, Chengdu Medical College, Chengdu, China 4 Department of Pharmacology, Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, Sichuan, China * These authors have contributed equally to this work Correspondence to: Xi Kuang, email: kuangxi56@163.com Tao Zhang, email: ztbillcmc@163.com Keywords: visfatin, colorectal cancer, EMT, Snail, Akt/GSK-3β Received: December 10, 2015 Accepted: March 28, 2016 Published: April 6, 2016 ABSTRACT Increasing evidences suggested visfatin, a newly discovered obesity-induced adipocytokine, is involved in promotion of cancer malignancy and correlated with worse clinical prognosis. While its effects and mechanisms on progression of colorectal cancer (CRC) remain unclear. Our clinical data show that visfatin protein is over expressed, positive associated with lymph node metastasis, high-grade tumor, and poor prognosis in 87 CRC patients. The levels of plasma visfatin are significantly upregulated in Stage IV colon cancer. Visfatin can significantly promote the in vitro migration and invasion of CRC cells via induction epithelial mesenchymal transition (EMT). It can increase the expression and nuclear translocation of Snail, a key transcription factor in regulating EMT. While silencing of Snail attenuates visfatin induced EMT. Further studies reveal visfatin can inhibit the association of Snail with GSK-3β and subsequently suppress ubiquitylation of Snail. In addition, visfatin can increase the expression and nuclear translocation of β-catenin, elevate its binding with Snail promoter, and then increase the transcription of Snail. While inhibitor of PI3K/Akt, LY294002, abolishes visfatin induced up regulation of Snail, Vimentin (Vim), β-catenin, and phosphorylated GSK-3β. In summary, our data suggest that increased expression of visfatin are associated with a more aggressive phenotype of CRC patients. It can trigger the EMT of CRC cells via Akt/GSK-3β/β-catenin signals.

// Shuo Chen 1 , Kai-Xuan Sun 1 , Bo-Liang Liu 1 , Zhi-Hong Zong 2 , Yang Zhao 1 1 Department of Gynecology, The First Affiliated Hospital of China Medical University, Shenyang 110001, China 2 Department of Biochemistry and Molecular Biology, College of Basic Medicine, China Medical University, Shenyang 100013, China Correspondence to: Yang Zhao, e-mail: yida.zhaoyang@163.com Keywords: endometrial carcinoma, GSK-3β, microRNA-129, AZD 1080, tumorigenesis and progression Received: December 18, 2015 Accepted: March 16, 2016 Published: March 30, 2016 ABSTRACT Purpose: Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase involved in cancer development. Herein, we demonstrated the role of GSK-3β in endometrial cancer (EC) and identified new therapeutic targets. Results: GSK-3β was overexpressed in EC tissues, and was positively correlated with International Federation of Gynecology and Obstetrics (FIGO) staging, dedifferentiation, and myometrial infiltration depth. Besides, GSK-3β overexpression predicted lower cumulative and relapse-free survival rate. si-GSK-3β transfection suppressed cell proliferation, migration, invasion, and promoted cell apoptosis through downregulating NF-kB, Cyclin D1 and MMP9 expression whereas upregulating P21 expression. Bioinformatic predictions and dual-luciferase reporter assays showed that GSK-3β was a possible target of miR-129. MiR-129 transfection reduced GSK-3β expression, and exhibited the same trend as si-GSK-3β transfection in cell function experiments. The nude mouse xenograft assay showed that miR-129 overexpression may suppress tumor growth through downregulating GSK-3β expression. Further studies showed that AZD1080, a GSK-3β inhibitor, could also inhibit EC cell proliferation, migration and invasion, while induced cell apoptosis through modulating relevant genes downstream of GSK-3β signaling. Experimental Design: GSK-3β expression was determined in EC tissue and normal endometrial tissues by immunohistochemistry. After GSK-3β down-regulation by si-GSK-3β, microRNA-129 mimic transfection or GSK-3β inhibitor exposure, EC cell phenotypes and related molecules were examined. Conclusions: Our results demonstrate for the first time that GSK-3β may be a novel and important therapeutic target for the treatment of endometrial carcinoma. GSK-3β inhibitor AZD1080 may be an effective drug for treating endometrial carcinoma.

// Senlin Zhao 1, * , Huamei Tang 2, * , Dongwang Yan 1, * , Junwei Fan 1, * , Hongcheng Sun 1 , Yugang Wen 1 , Fudong Yu 1 , Feifei Cui 1 , Dongyuan Zhang 1 , Yingming Xue 1 , Chenchen Liu 1 , Ben Yue 1 , Jian Chen 1 , Jingtao Wang 1 , Xiao Wang 1 , Meng Zhang 3 , Yang Yu 1 , Weiliang Jiang 4 , Xisheng Liu 1 , Yushuai Mi 1 , Zongguang Zhou 5 , Xuebin Qin 6 , Zhihai Peng 1 1 Department of General Surgery, Shanghai First People’s Hospital, Affiliated to Shanghai Jiao Tong University, Shanghai, China 2 Department of Pathology, Shanghai First People’s Hospital, Affiliated to Shanghai Jiao Tong University, Shanghai, China 3 Department of Pathology, Fudan University Affiliated Shanghai Cancer Center, Shanghai, China 4 Department of Gastroenterology, Shanghai First People’s Hospital, Affiliated to Shanghai Jiaotong University, Shanghai, China 5 Department of Gastrointestinal Surgery, West China Hospital, Sichuan University, Chengdu, China 6 Department of Neuroscience, Temple University School of Medicine, Philadelphia, PA, USA * These authors contributed equally to this work Correspondence to: Zhihai Peng, e-mail: pengpzhh@hotmail.com Xuebin Qin, e-mail: xuebin.qin@temple.edu Zongguang Zhou, e-mail: zhouzzghuaxi@163.com Keywords: DDA1, colon cancer, tumor recurrence, prognosis, NFκB Received: September 01, 2015 Accepted: February 06, 2016 Published: March 02, 2016 ABSTRACT Conventional high-recurrence risk factors are not sufficient to predict post-operative risk of tumor recurrence or sensitivity to 5-fluorouracil (5-FU)-based chemotherapy for stage II colon cancer. DDA1, an evolutionarily conserved gene located at 19p13.11, may be involved in the activation of nuclear factor kappaB (NFκB). This study aimed to investigate whether DDA1 contributes to tumorigenesis and progression of stage II colon cancer via activation of the NFκB pathway. We found that positive expression of DDA1 alone or in combination with p65 nuclear translocation correlated with increased risk of tumor recurrence in patients with stage IIB–IIC colon cancer. DDA1 overexpression in colon cancer lines promoted cell proliferation, facilitated cell cycle progression, inhibited 5-FU-induced apoptosis, enhanced invasion, and induced the epithelial-mesenchymal transition. Suppression of DDA1 inhibited tumor progression, and reduced tumor growth in vivo . We also demonstrated that DDA1-mediated tumor progression is associated with the activation of the NFκB/COP9 signalosome 2(CSN2)/glycogen synthase kinase3β (GSK3β) pathway. These results indicate that DDA1 promotes colon cancer progression through activation of NFκB/CSN2/GSK3β signaling. DDA1, together with NFκB activation status, may serve as a sensitive biomarker for tumor recurrence risk and prognosis in patients with stage IIB–IIC colon cancers.

Increasing evidence has shown that UBE2T plays an important role in genomic integrity and carcinogenesis; however, its role in nasopharyngeal carcinoma (NPC) has not been investigated. Here, we evaluated the clinicopathological significance of UBE2T in NPC and its underlying mechanisms. Using immunohistochemical analysis of UBE2T expression in NPC samples, we demonstrated that UBE2T is highly expressed in NPC tissues, which correlated with the T/M classification, skull invasion, and poor prognosis. The in vitro assay showed that UBE2T overexpression promoted proliferation, migration, and invasion of NPC cells, while UBE2T knockdown inhibited these processes. Consistent with our in vitro results, in vivo studies indicated that UBE2T overexpression promoted the growth of NPC xenografts and NPC cell metastasis. We found that UBE2T overexpression activated, whereas UBE2T knockdown inhibited, the AKT/GSK3β/β-catenin pathway. Moreover, the pathway-activation and in vitro pro-metastasis effects of UBE2T were blocked by the AKT inhibitor, MK-2206 2HCl. Additionally, UBE2T and p-GSK3 β co-expressed in NPC samples by serial section, and their expressions are correlated. Collectively, our findings demonstrated that UBE2T is a possible diagnostic/prognostic biomarker for NPC and may promote the development and progression of NPC by activating the AKT/GSK3β/β-catenin pathway. Thus, UBE2T could serve as an alternative target for the treatment of NPC.

Tau hyperphosphorylation is an important pathological feature of Alzheimer's disease (AD). To investigate whether escitalopram could inhibit amyloid-β (Aβ)-induced tau hyperphosphorylation and the underlying mechanisms, we treated the rat primary hippocampal neurons with Aβ1-42 and examined the effect of escitalopram on tau hyperphosphorylation. Results showed that escitalopram decreased Aβ1–42-induced tau hyperphosphorylation. In addition, escitalopram activated the Akt/GSK-3β pathway, and the PI3K inhibitor LY294002 blocked the attenuation of tau hyperphosphorylation induced by escitalopram. Moreover, the 5-HT1A receptor agonist 8-OH-DPAT also activated the Akt/GSK-3β pathway and decreased Aβ1-42-induced tau hyperphosphorylation. Furthermore, the 5-HT1A receptor antagonist WAY-100635 blocked the activation of Akt/GSK-3β pathway and the attenuation of tau hyperphosphorylation induced by escitalopram. Finally, escitalopram improved Aβ1–42 induced impairment of neurite outgrowth and spine density, and reversed Aβ1–42 induced reduction of synaptic proteins. Our results demonstrated that escitalopram attenuated Aβ1–42-induced tau hyperphosphorylation in primary hippocampal neurons through the 5-HT1A receptor mediated Akt/GSK-3β pathway.

// Yaochen Wang 1, 2 , Jing Liu 1, 2 , Yuling Qiu 1 , Meihua Jin 1 , Xi Chen 1 , Guanwei Fan 3 , Ran Wang 1 , Dexin Kong 1, 2 1 Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmaceutical Sciences, Tianjin Medical University, Tianjin 300070, China 2 Research Center of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China 3 State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Nankai Dsitrict, Tianjin 300193, China Correspondence to: Dexin Kong, e-mail: kongdexin@tmu.edu.cn Ran Wang, e-mail: wangran@tmu.edu.cn Keywords: ZSTK474, PI3K inhibitor, MCF-7, G1 arrest, autophagy Received: November 10, 2015 Accepted: February 16, 2016 Published: February 24, 2016 ABSTRACT Multifaceted activities of class I phosphatidylinositol 3-kinase (PI3K) inhibitor ZSTK474 were investigated on human breast cancer cell MCF-7. ZSTK474 inhibited proliferation of MCF-7 cells potently. Flow cytometric analysis indicated that ZSTK474 induced cell cycle arrest at G1 phase, but no obvious apoptosis occurred. Western blot analysis suggested that blockade of PI3K/Akt/GSK-3β/cyclin D1/p-Rb pathway might contribute to the G1 arrest induced. Moreover, we demonstrated that ZSTK474 induced autophagy in MCF-7 cells by use of various assays including monodansylcadaverine (MDC) staining, transmission electron microscopy (TEM), tandem mRFP-GFP-LC3 fluorescence microscopy, and western blot detection of the autophagy protein markers of LC3B II, p62 and Atg 5. Inhibition of class I PI3K and the downstream mTOR might be involved in the autophagy-inducing effect. Combinational use of ZSTK474 and autophagy inhibitors enhanced cell viability, suggesting ZSTK474-induced autophagy might contribute to the antitumor activity. Our report supports the application of ZSTK474, which is being evaluated in clinical trials, for breast cancer therapy.

Obg-like ATPase 1 (OLA1) belongs to the Obg family of P-loop NTPases, and may serve as a “molecular switch” regulating multiple cellular processes. Aberrant expression of OLA1 has been observed in several human malignancies. However, the role of OLA1 in cancer progression remains poorly understood. In this study, we used the Kaplan-Meier plotter search tool to show that increased expression of OLA1 mRNA was significantly associated with shorter overall survival in lung cancer patients. By immunohistochemical analysis we discovered that levels of OLA1 protein in lung cancer tissues were positively correlated with TNM stage and lymph node metastasis, but negatively correlated with the epithelial-mesenchymal transition (EMT) marker E-cadherin. Knockdown of OLA1 in a lung adenocarcinoma cell line rendered the cells more resistant to TGF- β-induced EMT and the accompanied repression of E-cadherin. Furthermore, our results demonstrated that OLA1 is a GSK3 β-interacting protein and inhibits GSK3 β activity by mediating its Ser9 phosphorylation. During EMT, OLA1 plays an important role in suppressing the GSK3 β-mediated degradation of Snail protein, which in turn promotes downregulation of E-cadherin. These data suggest that OLA1 contributes to EMT by modulating the GSK3 β/Snail/E-cadherin signaling, and its overexpression is associated with clinical progression and poor survival in lung cancer patients.

// Feng Wen 1, * , Yongmei Liu 1, * , Wei Wang 2 , Meng Li 2 , Fuchun Guo 1 , Yaxiong Sang 2 , Qing Qin 1 , Yongsheng Wang 1 , Qiu Li 1 1 The Department of Medical Oncology, Cancer Center, State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, University of Sichuan, Sichuan, China 2 State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, University of Sichuan, Sichuan, China * These authors have contributed equally to this work Correspondence to: Yongsheng Wang, e-mail: wangys75@gmail.com Qiu Li, e-mail: fbqiu9@163.com Keywords: adenomatous polyposis coli, NF- κ B, β -catenin, colon cancer Received: July 30, 2015 Accepted: January 01, 2016 Published: January 8, 2016 ABSTRACT Toll-like receptors (TLRs)/NF-κB activation stimulated by lipopolysaccharide (LPS) was associated with diverse biological response in colon cancer, but the underlying mechanism was largely unknown. In the current study, we reported cell proliferation was elevated in adenomatous polyposis coli (APC) mutated- and APC knockdown cell lines, while the proliferation was inhibited in APC wild-type cell lines. Besides, in vivo experiments showed that LPS promoted APC knockdown tumor growth while inhibited proliferation of APC wild type. Further study confirmed that activation of TLRs/NF-κB signaling pathway by LPS cross regulated with APC/GSK-3β/β-catenin pathway, which were depend on APC status of cell lines. Taken together, APC genotypes play a key role in LPS induced different colon cancer biological response by cross-regulating β-catenin and NF-κB, which may provide a novel strategy for carcinogenesis prevention.

OLA1 is an Obg family P-loop NTPase that possesses both GTP- and ATP-hydrolyzing activities. Here we report that OLA1 is a GSK3β interacting protein, and through its ATPase activity, inhibits the GSK3β-mediated activation of protein serine/threonine phosphatase 1 (PP1). It is hypothesized that GSK3β phosphorylates inhibitor 2 (I-2) of PP1 at Thr-72 and activates the PP1 · I-2 complex, which in turn dephosphorylates and stimulates GSK3β, thus forming a positive feedback loop. We revealed that the positive feedback loop is normally suppressed by OLA1, and becomes over-activated under OLA1 deficiency, resulting in increased cellular PP1 activity and dephosphorylation of multiple Ser/Thr phosphoproteins, and more strikingly, decreased global protein threonine phosphorylation. Furthermore, using xenograft models of colon cancer (H116) and ovarian cancer (SKOV3), we established a correlation among downregulation of OLA1, over-activation of the positive feedback loop as indicated by under-phosphorylation of I-2, and more aggressive tumor growth. This study provides the first evidence for the existence of a GSK3β-I-2-PP1 positive feedback loop in human cancer cells, and identifies OLA1 as an endogenous suppressor of this signaling motif.

Type XIX collagen is a minor collagen associated with basement membranes. It was isolated for the first time in a human cDNA library from rhabdomyosarcoma and belongs to the FACITs family (Fibril Associated Collagens with Interrupted Triple Helices). Previously, we demonstrated that the NC1 domain of collagen XIX (NC1(XIX)) exerts anti-tumor properties on melanoma cells by inhibiting their migration and invasion. In the present work, we identified for the first time the integrin αvβ3 as a receptor of NC1(XIX). Moreover, we demonstrated that NC1(XIX) inhibits the FAK/PI3K/Akt/mTOR pathway, by decreasing the phosphorylation and activity of the major proteins involved in this pathway. On the other hand, NC1(XIX) induced an increase of GSK3β activity by decreasing its degree of phosphorylation. Treatments targeting this central signaling pathway in the development of melanoma are promising and new molecules should be developed. NC1(XIX) seems to have the potential for the design of new anti-cancer drugs.

// Changbin Li 1,2 , Yan Ge 2 , Ai Peng 1 and Rujun Gong 2 1 Department of Nephrology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, China 2 Division of Kidney Disease and Hypertension, Department of Medicine, Rhode Island Hospital, Brown University School of Medicine, Providence, Rhode Island, USA Correspondence to: Rujun Gong, email: // Keywords : conditional gene knockout, glycogen synthase kinase 3β, podocyte, glycogen, antioxidant defense, Pathology Section Received : May 12, 2015 Accepted : October 30, 2015 Published : November 11, 2015 Abstract The redox sensitive glycogen synthase kinase (GSK) 3 has been recently implicated in the pathogenesis of proteinuric glomerulopathy. However, prior studies are less conclusive because they relied solely on chemical inhibitors of GSK3, which provide poor discrimination between the isoforms of GSK3 apart from potential off target activities. In murine kidneys, the β rather than the α isoform of GSK3 was predominantly expressed in glomeruli and distributed intensely in podocytes. By employing the doxycycline-activated Cre- lox P site specific gene targeting system, GSK3β was successfully knocked out (KO) selectively in podocytes in adult mice, resulting in a phenotype no different from control littermates. Electron microscopy of glomeruli in KO mice demonstrated more glycogen accumulation in podocytes but otherwise normal ultrastructures. Upon oxidative glomerular injury induced by protein overload, KO mice excreted significantly less albuminuria and had much attenuated podocytopathy and glomerular damage. The anti-proteinuric and glomerular protective effect was concomitant with diminished accumulation of reactive oxygen species in glomeruli in KO mice, which was likely secondary to a reinforced Nrf2 antioxidant response in podocytes. Collectively, our data suggest that GSK3β is dispensable for glomerular function and histology under normal circumstances but may serve as a therapeutic target for protecting from oxidative glomerular injuries.

Despite progress in clinical cancer medicine in multiple fields, the prognosis of pancreatic cancer has remained dismal. Recently, chemopreventive strategies using phytochemicals have gained considerable attention as an alternative in the management of cancer. The present study aimed to evaluate the chemopreventive effects of resveratrol (RV) and apocynin (AC) in N-Nitrosobis(2-oxopropyl)amine-induced pancreatic carcinogenesis in hamster. RV- and AC-treated hamsters showed significant reduction in the incidence of pancreatic cancer with a decrease in Ki-67 labeling index in dysplastic lesions. RV and AC suppressed cell proliferation of human and hamster pancreatic cancer cells by inhibiting the G1 phase of the cell cycle with cyclin D1 downregulation and inactivation of AKT-GSK3β and ERK1/2 signaling. Further, decreased levels of GSK3β(Ser9) and ERK1/2 phosphorylation and cyclin D1 expression in the nuclear fraction were observed in cells treated with RV or AC. Nuclear expression of phosphorylated GSK3β(Ser9) was also decreased in dysplastic lesions and adenocarcinomas of hamsters treated with RV or AC in vivo. These results suggest that RV and AC reduce phosphorylated GSK3β(Ser9) and ERK1/2 in the nucleus, resulting in inhibition of the AKT-GSK3β and ERK1/2 signaling pathways and cell cycle arrest in vitro and in vivo. Taken together, the present study indicates that RV and AC have potential as chemopreventive agents for pancreatic cancer.

Hes1 is a transcription factor that influences cell proliferation and differentiation. However, the effect of Hes1 on invasiveness and the underlying mechanism remain unknown. In the current study, we found that Hes1 suppressed cell apoptosis, promoted cell growth, induced EMT phenotype and cytoskeleton reconstruction, and enhanced the metastatic potential of colon cancer cells in vitro and in vivo. Furthermore, we indicated that Bmi-1 mediated Hes1-induced cell proliferation and migration, downregulated PTEN and activated the Akt/GSK3β pathway, consequently induced EMT and cytoskeleton reconstruction, ultimately leading to enhanced invasiveness of cancer cells. In addition, we also found that both Hes1 and Bmi-1 could directly regulate PTEN by associating at the PTEN locus, and played important roles in regulating PTEN/Akt/GSK3β pathway. Our results provide functional and mechanistic links between Hes1 and Bmi-1/PTEN/Akt/GSK3β signaling in the development and progression of colon cancer.

The Wnt/β-catenin signaling pathway, commonly hyperactivated in pancreatic cancer, has been reported to play an important role in the maintenance of stemness of cancer stem cells (CSCs), which is closely related to the progression of pancreatic cancer. Therefore, exploring the regulatory mechanism in Wnt/β-catenin signaling may provide valuable clinical targets for cancer therapy. In the current study, we demonstrated that upregulation of miR-744 in pancreatic cancer promoted Wnt/β-catenin signaling by directly targeting secreted frizzled-related protein 1 (SFRP1), glycogen synthase kinase 3β (GSK3β), and transducin-like enhancer of split 3 (TLE3), important negative modulators of Wnt/β-catenin signaling. Expression of miR-744 was markedly upregulated in pancreatic cancer and positively correlated with poor patient survival. Furthermore, we found that overexpressing miR-744 enhanced, while inhibiting miR-744 reduced, the stem cell-like phenotype of pancreatic cancer cells in vitro. Importantly, in vivo model of human-derived pancreatic xenografts showed that miR-744 upregulation enhanced the tumorigenicity of pancreatic cancer cells. These findings suggest that miR-744 plays a vital role in promoting the stem cell-like phenotype of pancreatic cancer cells, and may represent a novel prognostic biomarker and therapeutic target.

Compelling evidence suggests that the epithelial-mesenchymal transition (EMT) correlates with aggressiveness of tumors and poor survival. FGF19 has been shown to be involved in EMT in cholangiocarcinoma and colorectal cancer, however, molecular mechanisms underlying FGF19-induced EMT process in hepatocellular carcinoma (HCC) remain largely unknown. Here, we show the expression of FGF19 is significantly elevated and negatively associated with the expression of E-cadherin in HCC tissues and cell lines. Ectopic FGF19 expression promotes EMT and invasion in epithelial-like HCC cells through repression of E-cadherin expression, whereas FGF19 knockdown enhances E-cadherin expression and hence diminishes EMT traits in mesenchymal-like HCC cells, suggesting FGF19 exerts its tumor progressing functions as an EMT inducer. Interestingly, depletion of FGF19 cannot abrogate EMT traits in the presence of GSK3β inhibitors. Furthermore, FGF19-induced EMT can be markedly attenuated when FGFR4 is knocked out. These observations clearly indicate that FGFR4/GSK3β/β-catenin axis may play a pivotal role in FGF19-induced EMT in HCC cells. As FGF19 and its specific receptor FGFR4 are frequently amplified in HCC cells, selective targeting this signaling node may lend insights into a potential effective therapeutic approach for blocking metastasis of HCC.

Insulinoma associated-1 (IA-1/INSM1) gene is exclusively expressed during early embryonic development, but has been found to be re-expressed at high levels in neuroendocrine tumors including neuroblastoma. Using over-expression and knockdown experiments in neuroblastoma cells, we showed that INSM1 is critical for cell proliferation, BME-coated invasion, and soft agar colony formation. Here, we identified INSM1 as a novel target gene activated by N-myc in N-myc amplified neuroblastoma cells. The Sonic hedgehog signaling pathway induced INSM1 by increasing N-myc expression. INSM1 activated PI3K/AKT/GSK3β pathways to suppress N-myc phosphorylation (Thr-58) and inhibited degradation of N-myc. Inversely, N-myc protein bound to the E2-box region of the INSM1 promoter and activated INSM1 expression. The invasion assay and the xenograft nude mouse tumor model revealed that the INSM1 factor facilitated growth and oncogenesis of neuroblastoma. The current data supports our hypothesis that a positive-feedback loop of sonic hedgehog signaling induced INSM1 through N-myc and INSM1 enhanced N-myc stability contributing to the transformation of human neuroblastoma.

Proliferation of bronchioalveolar stem cells (BASCs) is essential for epithelial repair. XB130 is a novel adaptor protein involved in the regulation of epithelial cell survival, proliferation and migration through the PI3K/Akt pathway. To determine the role of XB130 in airway epithelial injury repair and regeneration, a naphthalene-induced airway epithelial injury model was used with XB130 knockout (KO) mice and their wild type (WT) littermates. In XB130 KO mice, at days 7 and 14, small airway epithelium repair was significantly delayed with fewer number of Club cells (previously called Clara cells). CCSP (Club cell secreted protein) mRNA expression was also significantly lower in KO mice at day 7. At day 5, there were significantly fewer proliferative epithelial cells in the KO group, and the number of BASCs significantly increased in WT mice but not in KO mice. At day 7, phosphorylation of Akt, GSK-3β, and the p85α subunit of PI3K was observed in airway epithelial cells in WT mice, but to a much lesser extent in KO mice. Microarray data also suggest that PI3K/Akt-related signals were regulated differently in KO and WT mice. An inhibitory mechanism for cell proliferation and cell cycle progression was suggested in KO mice. XB130 is involved in bronchioalveolar stem cell and Club cell proliferation, likely through the PI3K/Akt/GSK-3β pathway.

The N-myc downstream regulated gene 1 (NDRG1) is significantly associated with advanced tumor stages and poor survival of hepatocellular carcinoma (HCC), thereby implicating it as a potential target for HCC treatment. We aim to further understand its biological roles in hepatocarcinogenesis, as a means to exploit it for therapeutic purposes. By screening using the ProtoArray® Human Protein Microarrays, we identified glycogen synthase kinase 3β (GSK-3β) and the orphan nuclear receptor (Nur77) as potential interaction partners of NDRG1. These interactions were confirmed in HCC cell lines in vitro by co-immunoprecipitation; and co-localizations of NDRG1 with GSK-3β and Nur77 were observed by immunofluorescence staining. Additionally, high levels of NDRG1 competitively bind to GSK-3β and Nur77 to allow β-catenin to escape degradation, with consequent elevated levels of downstream oncogenic genes. In vivo, we consistently observed that NDRG1 suppression in HCC xenografts decreased β-catenin levels and its downstream target Cyclin D1, with concomitant tumor growth inhibition. Clinically, the over-expression of NDRG1 in HCC patient samples is positively correlated with GSK-3β-9ser (|”‚ R | = 0.28, p = 0.01), Nur77 (|”‚ R | = 0.42, p < 0.001), and β-catenin (| R |= 0.32, p = 0.003) expressions. In conclusion, we identified GSK-3β and Nur77 as novel interaction partners of NDRG1. These protein-protein interactions regulate the turnover of β-catenin and subsequent downstream signaling mediated by β-catenin in HCC cells, and provides potential targets for future therapeutic interventions.

Glioma cells release glutamate through expression of system xc-, which exchanges intracellular glutamate for extracellular cysteine. Lack of the excitatory amino acid transporter 2 (EAAT2) expression maintains high extracellular glutamate levels in the glioma microenvironment, causing excitotoxicity to surrounding parenchyma. Not only does this contribute to the survival and proliferation of glioma cells, but is involved in the pathophysiology of tumour-associated epilepsy (TAE). We investigated the role of the peroxisome proliferator activated receptor gamma (PPARγ) agonist pioglitazone in modulating EAAT2 expression in glioma cells. We found that EAAT2 expression was increased in a dose dependent manner in both U87MG and U251MG glioma cells. Extracellular glutamate levels were reduced with the addition of pioglitazone, where statistical significance was reached in both U87MG and U251MG cells at a concentration of ≥ 30 μM pioglitazone (p < 0.05). The PPARγ antagonist GW9662 inhibited the effect of pioglitazone on extracellular glutamate levels, indicating PPARγ dependence. In addition, pioglitazone significantly reduced cell viability of U87MG and U251MG cells at ≥ 30 μM and 100 μM (p < 0.05) respectively. GW9662 also significantly reduced viability of U87MG and U251MG cells with 10 μM and 30 μM (p < 0.05) respectively. The effect on viability was partially dependent on PPARγ activation in U87MG cells but not U251MG cells, whereby PPARγ blockade with GW9662 had a synergistic effect. We conclude that PPARγ agonists may be therapeutically beneficial in the treatment of gliomas and furthermore suggest a novel role for these agents in the treatment of tumour associated seizures through the reduction in extracellular glutamate.

Here we demonstrated that chemotherapy induced 14-3-3σ expression in tongue cancer (TC) cells and overexpressed 14-3-3σ sensitized TC cells to chemotherapy especially in multidrug resistant TC (MDR-TC) cells. In agreement, 14-3-3σ knockdown enhanced resistance of TC cells to chemotherapy. Mechanically, we found 14-3-3σ physically bound to GSK3β in protein level and the binding inhibited β-catenin signaling. Coincidentally, chemotherapy as well as 14-3-3σ overexpression led to increase of GSK3β protein level. Increased GSK3β protein sensitized TC cells to chemotherapy. Moreover, deregulation of 14-3-3σ/GSK3β/β-catenin axis led to overexpressed ZEB1 in TC cells, especially in MDR-TC cells. As a negative feedback loop, ZEB1 bond to 14-3-3σ promoter to enhance promoter hypermethylation in TC cells. Promoter hypermethylation resulted into the decrease of 14-3-3σ expression. Importantly, a positive correlation was observed between 14-3-3σ and GSK3β protein expression in TC tissues from patients receiving chemotherapy. High levels of 14-3-3σ and GSK3β were associated with better prognosis in TC patients.