STAT1-HDAC4 signaling induces epithelial-mesenchymal transition and sphere formation of cancer cells overexpressing the oncogene, <i>CUG2</i>

Kaowinn, Sirichat,Kaewpiboon, Chutima,Koh, Sang Seok,Krä,mer, Oliver H.,Chung, Young-Hwa D.A. Spandidos 2018 ONCOLOGY REPORTS Vol.40 No.5

<P>Our previous studies have shown that the novel oncogene, cancer upregulated gene 2 (CUG2), activates STAT1, which is linked to anticancer drug resistance, induces epithelial-mesenchymal transition (EMT) and cancer stem cell-like phenotypes as determined by MTT, migration and sphere formation assays. We thus aimed to ascertain whether the activation of STAT1 by CUG2 is involved in these malignant phenotypes besides drug resistance. Here, we showed that STAT1 suppression decreased the expression of N-cadherin and vimentin, biomarkers of EMT, which led to inhibition of the migration and invasion of human lung A549 cancer cells stably expressing CUG2, but did not recover E-cadherin expression. STAT1 siRNA also diminished CUG2-induced TGF-β signaling, which is critical in EMT, and TGF-β transcriptional activity. Conversely, inhibition of TGF-β signaling reduced phosphorylation of STAT1, indicating a crosstalk between STAT1 and TGF-β signaling. Furthermore, STAT1 silencing diminished sphere formation, which was supported by downregulation of stemness-related factors such as Sox2, Oct4, and Nanog. Constitutive suppression of STAT1 also inhibited cell migration, invasion and sphere formation. As STAT1 acetylation counteracts STAT1 phosphorylation, acetylation of STAT1 by treatment with trichostatin A, an inhibitor of histone deacetylases (HDACs), reduced cell migration, invasion, and sphere formation. As HDAC4 is known to target STAT1, its role was investigated under CUG2 overexpression. HDAC4 suppression resulted in inhibition of cell migration, invasion, and sphere formation as HDAC4 silencing hindered TGF-β signaling and decreased expression of Sox2 and Nanog. Taken together, we suggest that STAT1-HDAC4 signaling induces malignant tumor features such as EMT and sphere formation in CUG2-overexpressing cancer cells.</P>

Pancreatic adenocarcinoma upregulated factor (PAUF) confers resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNA receptor-mediated signaling

Kaowinn, Sirichat,Cho, Il-Rae,Moon, Jeong,Jun, Seung Won,Kim, Chang Seok,Kang, Ho Young,Kim, Manbok,Koh, Sang Seok,Chung, Young-Hwa Elsevier 2015 Biochemical and biophysical research communication Vol.459 No.2

<P><B>Abstract</B></P> <P>Pancreatic adenocarcinoma upregulated factor (PAUF), a novel oncogene, plays a crucial role in the development of pancreatic cancer, including its metastasis and proliferation. Therefore, PAUF-expressing pancreatic cancer cells could be important targets for oncolytic virus-mediated treatment. Panc-1 cells expressing PAUF (Panc-PAUF) showed relative resistance to parvovirus H-1 infection compared with Panc-1 cells expressing an empty vector (Panc-Vec). Of interest, expression of type I IFN-α receptor (IFNAR) was higher in Panc-PAUF cells than in Panc-Vec cells. Increased expression of IFNAR in turn increased the activation of Stat1 and Tyk2 in Panc-PAUF cells compared with that in Panc-Vec cells. Suppression of Tyk2 and Stat1, which are important downstream molecules for IFN-α signaling, sensitized pancreatic cancer cells to parvovirus H-1-mediated apoptosis. Further, constitutive suppression of PAUF sensitized Bxpc3 pancreatic cancer cells to parvovirus H-1 infection. Taken together, these results suggested that PAUF conferred resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNAR-mediated signaling.</P> <P><B>Highlights</B></P> <P> <UL> <LI> PAUF confers resistance against oncolytic parvovirus H-1 infection. </LI> <LI> PAUF enhances the expression of IFNAR in Panc-1 cells. </LI> <LI> Increased activation of Tyk2 or Stat1 by PAUF provides resistance to parvovirus H-1-mediated apoptosis. </LI> <LI> Constitutive inhibition of PAUF enhances parvovirus H-1-mediated oncolysis of Bxpc3 pancreatic cancer cells. </LI> </UL> </P>

Cancer upregulated gene 2 (CUG2), a novel oncogene, promotes stemness-like properties via the NPM1-TGF-β signaling axis

Kaowinn, Sirichat,Seo, Eun Jin,Heo, Woong,Bae, Jae-Ho,Park, Eun-Jung,Lee, Soojin,Kim, Yeon Jeong,Koh, Sang Seok,Jang, Il Ho,Shin, Dong Hoon,Chung, Young-Hwa Elsevier 2019 Biochemical and biophysical research communication Vol.514 No.4

<P><B>Abstract</B></P> <P>Our previous study reported that cancer upregulated gene (CUG)2, a novel oncogene, induces both faster cell migration and anti-cancer drug resistance. We thus wonder whether CUG2 also induces stemness, a characteristic of cancer stem cells (CSCs) and further examine the molecular mechanism of this phenotype. To test that CUG2 induces stemness, we examined expression of stemness-related factors. Overexpression of CUG2 enhanced expression levels of stemness-related factors in human lung carcinoma A549 and immortalized bronchial BEAS-2B cells. Consequently, CUG2 increased cellular spherical cluster forming ability. Overexpression of CUG2 also induced tumor formation in xenotransplanted nude mice whereas transplantation of control cells failed to, implying that CUG2 possesses malignant tumorigenic potential. We paid attention to nucleophosmin (NPM1) for its known interaction with CUG2. Suppression of NPM1 hindered the CUG2-mediated stemness-like phenotypes and diminished TGF-β transcriptional activity and signaling. TGF-β increased stemness-like phenotypes in the control cells whereas TGF-β inhibitor blocked induction of the phenotypes, indicating that NPM1 is required for CUG2-mediated stemness-like phenotypes through TGF-β signaling. Furthermore, the suppression of Smad- and non-Smad-dependent TGF-β signaling pathways also prevented CUG2 from inducing stemness-like phenotypes. Altogether, we suggest that the novel CUG2 oncogene promotes cellular transformation and stemness, mediated by nuclear NPM1 protein and TGF-β signaling.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Overexpression of CUG2 promotes stemness-like properties. </LI> <LI> NPM1 is involved in CUG2-mediated stemness-like phenotypes. </LI> <LI> NPM1 utilizes TGF-β signaling for CUG2-mediated stemness-like phenotypes. </LI> <LI> TGF-β signaling plays a crucial role in CUG2-mediated stemness-like phenotypes. </LI> </UL> </P>

Increased EGFR expression induced by a novel oncogene, CUG2, confers resistance to doxorubicin through Stat1-HDAC4 signaling

Kaowinn, S.,Jun, S. W.,Kim, C. S.,Shin, D. M.,Hwang, Y. H.,Kim, K.,Shin, B.,Kaewpiboon, C.,Jeong, H. H.,Koh, S. S. Springer Science + Business Media 2017 CELLULAR ONCOLOGY Vol.40 No.6

<i>N</i>-Benzyl-<i>N</i>-methyl-dodecan-1-amine, a novel compound from garlic, exerts anti-cancer effects on human A549 lung cancer cells overexpressing cancer upregulated gene (CUG)2

Kaowinn, Sirichat,Kaewpiboon, Chutima,Kim, Ji Eun,Lee, Mi Rim,Hwang, Dae Youn,Choi, Young Whan,Kim, Hong Won,Park, Jin Kyoon,Song, Kyung-Mo,Lee, Nam Hyouck,Maeng, Jin-Soo,Chung, Young-Hwa Elsevier 2018 european journal of pharmacology Vol.841 No.-

<P><B>Abstract</B></P> <P>Dietary garlic has been suggested to possess anticancer properties, and several attempts have been made to isolate the anticancer compounds. In this study, we efficiently synthesized <I>N</I>-benzyl-<I>N</I>-methyl-dodecan-1-amine (BMDA) by the reductive amination method. We evaluated the potential anticancer activities of BMDA against A549 lung cancer cells with cancer stem cell-like phenotypes due to the overexpression of cancer upregulated gene (CUG)2. <I>N</I>-Benzyl-<I>N</I>-methyl-dodecan-1-amine treatment sensitized A549 cells overexpressing CUG2 (A549-CUG2) to apoptosis and autophagy compared with those of the control cells. The treatment with BMDA also reduced tumor development in xenografted nude mice. Furthermore, BMDA inhibited cell migration, invasion, and sphere formation in A549-CUG2 cells, in which TGF-β signaling is involved. Further analysis showed that BMDA hindered TGF-β promoter activity, protein synthesis, and phosphorylation of Smad2, thus decreasing the expression of TGF-β-targeted proteins, including Snail and Twist. <I>N</I>-Benzyl-<I>N</I>-methyl-dodecan-1-amine also decreased Twist expression <I>in vivo</I>. In addition, BMDA inhibited Akt-ERK activities, β-catenin expression, and its transcriptional activity. These results suggest that BMDA can be a promising anticancer agent against cancer cells overexpressing CUG2.</P>

Cancer upregulated gene (CUG)2 elevates YAP1 expression, leading to enhancement of epithelial-mesenchymal transition in human lung cancer cells

Kaowinn, Sirichat,Yawut, Natpaphan,Koh, Sang Seok,Chung, Young-Hwa Elsevier 2019 Biochemical and biophysical research communication Vol.511 No.1

<P><B>Abstract</B></P> <P>Although our previous studies have showed that a novel oncogene, cancer upregulated gene (CUG)2 induced epithelial-mesenchymal transition (EMT), the detailed molecular mechanism remains unknown. Because several lines of evidence documented that Yes-Associated Protein (YAP)1 is closely associated with cancer stem cell (CSC)-like phenotypes including EMT, stemness, and drug resistance, we wondered if YAP1 is involved in CUG2-induced EMT. We herein found that the overexpression of CUG2 increased YAP1 expression at the transcriptional as well as protein levels. Chromatin immunoprecipitation assay revealed that the elevated YAP1 transcripts are attributed to c-Jun and AP2 bindings to the YAP1 promoter. Akt and MAPK kinases including ERK, JNK, and p38 MAPK enhanced the level of YAP1 protein. In spite of a close relationship between β-catenin and YAP1, not β-catenin but NEK2 played the role in increasing YAP1 expression. Silencing YAP1 inhibited CUG2-induced cell migration and invasion. N-cadherin and vimentin expressions were decreased during YAP1 knockdown. The suppression of YAP1 diminished TGF-β transcriptional activity and expression as well as phosphorylation level of Smad2 and Twist protein. Conversely, LY2109761 or Smad2 siRNA treatment reduced YAP1 protein levels, indicating a close interplay between YAP1 and TGF-β signaling. Taken together, we suggest that CUG2 induces up-regulation of YAP1 expression, leading to enhancing CUG2-induced EMT via a close crosstalk between YAP1 and TGF-β signaling.</P> <P><B>Highlights</B></P> <P> <UL> <LI> CUG2 enhances expression of YAP1 protein. </LI> <LI> Akt and MAPK kinases are involved in the increase of YAP1 protein expression under overexpression of CUG2. </LI> <LI> Not β-catenin but NEK2 is involved in increase of YAP1 protein expression under overexpression of CUG2. </LI> <LI> YAP1 is involved in CUG2-induced EMT. </LI> </UL> </P>

CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

Kaowinn, Sirichat,Oh, Sangtaek,Moon, Jeong,Yoo, Ah Young,Kang, Ho Young,Lee, Mi Rim,Kim, Ji Eun,Hwang, Dae Youn,Youn, So Eun,Koh, Sang Seok,Chung, Young-Hwa D.A. Spandidos 2019 International journal of oncology Vol.54 No.4

<P>The mechanisms through which cancer-upregulated gene 2 (CUG2), a novel oncogene, affects Wnt/β-catenin signaling, essential for tumorigenesis, are unclear. In this study, we aimed to elucidate some of these mechanisms in A549 lung cancer cells. Under the overexpression of CUG2, the protein levels and activity of β-catenin were evaluated by western blot analysis and luciferase assay. To examine a biological consequence of β-catenin under CUG2 overexpression, cell migration, invasion and sphere formation assay were performed. The upregulation of β-catenin induced by CUG2 overexpression was also accessed by xenotransplantation in mice. We first found that CUG2 overexpression increased β-catenin expression and activity. The suppression of β-catenin decreased cancer stem cell (CSC)-like phenotypes, indicating that β-catenin is involved in CUG2-mediated CSC-like phenotypes. Notably, CUG2 overexpression increased the phosphorylation of β-catenin at Ser33/Ser37, which is known to recruit E3 ligase for β-catenin degradation. Moreover, CUG2 interacted with and enhanced the expression and kinase activity of never in mitosis gene A-related kinase 2 (NEK2). Recombinant NEK2 phosphorylated β-catenin at Ser33/Ser37, while NEK2 knockdown decreased the phosphorylation of β-catenin, suggesting that NEK2 is involved in the phosphorylation of β-catenin at Ser33/Ser37. Treatment with CGK062, a small chemical molecule, which promotes the phosphorylation of β-catenin at Ser33/Ser37 through protein kinase C (PKC)α to induce its degradation, reduced β-catenin levels and inhibited the CUG2-induced features of malignant tumors, including increased cell migration, invasion and sphere formation. Furthermore, CGK062 treatment suppressed CUG2-mediated tumor formation in nude mice. Taken together, the findings of this study suggest that CUG2 enhances the phosphorylation of β-catenin at Ser33/Ser37 by activating NEK2, thus stabilizing β-catenin. CGK062 may thus have potential for use as a therapeutic drug against CUG2-overexpressing lung cancer cells.</P>

Hepatitis C virus core protein enhances hepatocellular carcinoma cells to be susceptible to oncolytic vesicular stomatitis virus through down-regulation of HDAC4

Moon, J.,Kaowinn, S.,Cho, I.R.,Min, D.S.,Myung, H.,Oh, S.,Kaewpiboon, C.,Kraemer, O.H.,Chung, Y.H. Academic Press 2016 Biochemical and biophysical research communication Vol.474 No.3

Since hepatitis C virus (HCV) core protein is known to possess potential oncogenic activity, we explored whether oncolytic vesicular stomatitis virus (VSV) could efficiently induce cytolysis in hepatocellular carcinoma cells stably expressing HCV core protein (Hep3B-Core). We found that Hep3B-Core cells were more susceptible to VSV as compared to control (Hep3B-Vec) cells owing to core-mediated inactivation of STAT1 and STAT2 proteins. Core expression induced lower phosphorylation levels of type I IFN signaling proteins such as Tyk2 and Jak1, and a reduced response to exogenous IFN-α, which resulted in susceptibility to VSV. Furthermore, as STAT1 acetylation by switching phosphorylation regulated its activity, the role of STAT1 acetylation in susceptibility of Hep3B-Core cells to VSV was investigated. Treatment with trichostatin A, an inhibitor of histone deacetylase (HDAC), increased STAT1 acetylation but blocked IFN-α-induced phosphorylation of STAT1, leading to increase of susceptibility to VSV. Interestingly, the core protein decreased HDCA4 transcript levels, leading to down-regulation of HDAC4 protein. However, ectopic expression of HDAC4 conversely enforced phosphorylation of STAT1 and hindered VSV replication, indicating that core-mediated reduction of HDAC4 provides a suitable intracellular circumstance for VSV replication. Collectively, we suggest that VSV treatment will be a useful therapeutic strategy for HCV-infected hepatocellular carcinoma cells because HCV core protein suppresses the anti-viral threshold by down-regulation of the STAT1-HDAC4 signaling axis.

VP2 capsid domain of the H-1 parvovirus determines susceptibility of human cancer cells to H-1 viral infection

Cho, I-R,Kaowinn, S,Song, J,Kim, S,Koh, S S,Kang, H-Y,Ha, N-C,Lee, K H,Jun, H-S,Chung, Y-H Nature America, Inc. 2015 Cancer gene therapy Vol.22 No.5

Although H-1 parvovirus is used as an antitumor agent, not much is known about the relationship between its specific tropism and oncolytic activity. We hypothesize that VP2, a major capsid protein of H-1 virus, determines H-1-specific tropism. To assess this, we constructed chimeric H-1 viruses expressing Kilham rat virus (KRV) capsid proteins, in their complete or partial forms. Chimeric H-1 viruses (CH1, CH2 and CH3) containing the whole KRV VP2 domain could not induce cytolysis in HeLa, A549 and Panc-1 cells. However, the other chimeric H-1 viruses (CH4 and CH5) expressing a partial KRV VP2 domain induced cytolysis. Additionally, the significant cytopathic effect caused by CH4 and CH5 infection in HeLa cells resulted from preferential viral amplification via DNA replication, RNA transcription and protein synthesis. Modeling of VP2 capsid protein showed that two variable regions (VRs) (VR0 and VR2) of H-1 VP2 protein protrude outward, because of the insertion of extra amino-acid residues, as compared with those of KRV VP2 protein. This might explain the precedence of H-1 VP2 protein over KRV in determining oncolytic activity in human cancer cells. Taking these results together, we propose that the VP2 protein of oncolytic H-1 parvovirus determines its specific tropism in human cancer cells.

Formoxanthone C, isolated from <i>Cratoxylum formosum</i> ssp. <i>pruniflorum</i>, reverses anticancer drug resistance by inducing both apoptosis and autophagy in human A549 lung cancer cells

Kaewpiboon, Chutima,Boonnak, Nawong,Kaowinn, Sirichat,Chung, Young-Hwa Elsevier 2018 Bioorganic & medicinal chemistry letters Vol.28 No.4

<P><B>Abstract</B></P> <P>Multidrug resistance (MDR) cancer toward cancer chemotherapy is one of the obstacles in cancer therapy. Therefore, it is of interested to use formoxanthone C (1,3,5,6-tetraoxygenated xanthone; XanX), a natural compound, which showed cytotoxicity against MDR human A549 lung cancer (A549RT-eto). The treatment with XanX induced not only apoptosis- in A549RT-eto cells, but also autophagy-cell death. Inhibition of apoptosis did not block XanX-induced autophagy in A549RT-eto cells. Furthermore, suppression of autophagy by beclin-1 small interfering RNAs (siRNAs) did not interrupt XanX-induced apoptosis, indicating that XanX can separately induce apoptosis and autophagy. Of interest, XanX treatment reduced levels of histone deacetylase 4 (HDAC4) protein overexpressed in A549RT-etocells. The co-treatment with XanX and HDAC4 siRNA accelerated both autophagy and apoptosis more than that by XanX treatment alone, suggesting survival of HDAC4 in A549RT-eto cells. XanX reverses etoposide resistance in A549RT-eto cells by induction of both autophagy and apoptosis, and confers cytotoxicity through down-regulation of HDAC4.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>