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

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 ß-catenin and YAP1, not ß-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-ß 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-ß 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-ß signaling.

Caged-xanthone from Cratoxylum formosum ssp. pruniflorum inhibits malignant cancer phenotypes in multidrug-resistant human A549 lung cancer cells through down-regulation of NF-κB.

Our recent study reported that multidrug-resistant (MDR) human A549 lung cancer cells (A549RT-eto) with the elevated expression of NF-κB showed epithelial-mesenchymal transition (EMT), increasing spheroid formation and elevating the expression levels of stemness-related factors, including Oct4, Nanog, Sox2, Bmi1, and Klf4. Therefore, when new therapeutic agents targeting these malignant cancer cells were explored, we found that caged-xanthone (CX) isolated from the roots of Cratoxylum formosum ssp. pruniflorum diminished the expression of NF-κB, P-glycoprotein (P-gp) protein levels, cell migration and invasion, and sphere-forming ability of A549RT-eto cells. To address the role of NF-κB in these malignant cancer features, we treated A549RT-eto cells with NF-κB siRNAs in the present work. We found that the knockdown of NF-κB inhibited EMT and sphere formation. Furthermore, co-treatment with CX and NF-κB siRNA accelerated the death of apoptotic cells through the decrease of P-gp protein levels. These results suggest that NF-κB was involved in malignant cancer phenotypes and MDR in A549RT-eto cells. Taken together, our findings suggest that CX can be a potential therapeutic agent for the treatment of malignant tumor cells.

Andrographolide targets EGFR to impede epithelial-mesenchymal transition in human breast cancer cells.

Despite the primary surgical treatment for breast cancer patients, malignant invasiveness and metastasis remain threatening factors for women with breast cancer. As chemotherapy yields unsatisfactory results, it prompted us to search for effective natural agents with few side-effects. Although andrographolide (ADGL), a natural diterpenoid lactone isolated from Andrographis paniculata, presents anticancer effects, the molecular mechanism remains unknown. Initially, on comparing the expression of proteins related to epithelial-mesenchymal transition (EMT) between nonmetastatic cancer MCF7 cells and highly metastatic cancer MDA-MB-231 cells, we found that MDA-MB-231 cells exhibit higher protein levels of N-cadherin and vimentin and lower protein levels of E-cadherin when compared to MCF7 cells. Moreover, MDA-MB-231 cells also exhibited higher EGFR expression and activity, higher STAT1 activity and abundant HDAC4 expression. To elucidate whether these proteins are closely associated with EMT, EGFR, STAT1 or HDAC4, the proteins were silenced in MDA-MB-231 breast cancer cells by their specific siRNAs. We found that silencing these proteins reduced EMT, indicating an important role of EGFR, STAT1 and HDAC4 in EMT progression. When we treated MDA-MB-231 cells with ADGL as a potential therapeutic drug, we found that ADGL treatment inhibited cell migration and invasion. Furthermore, it also recovered E-cadherin expression and decreased N-cadherin and vimentin protein levels. ADGL treatment reduced EGFR expression at a lower concentration (1 µg/mL); however, STAT1 activity and HDAC4 expression was reduced by a higher concentration (5 µg/mL) of ADGL. Moreover, we observed that the combined treatment with ADGL and siRNAs against these proteins highly sensitized the MDA-MB-231 cells to apoptosis compared to that with ADGL and control siRNA. Collectively, our results suggest that ADGL targets EGFR, thereby inhibiting EMT in human breast cancer cells.

N-benzyl-N-methyldecan-1-amine and its derivative mitigate 2,4- dinitrobenzenesulfonic acid-induced colitis and collagen-induced rheumatoid arthritis.

As our previous study revealed that N-benzyl-N-methyldecan-1-amine (BMDA), a new molecule originated from Allium sativum, exhibits anti-neoplastic activities, we herein explored other functions of the compound and its derivative [decyl-(4-methoxy-benzyl)-methyl-amine; DMMA] including anti-inflammatory and anti-oxidative activities. Pretreatment of THP-1 cells with BMDA or DMMA inhibited tumor necrosis factor (TNF)-α and interleukin (IL)-1ß production, and blocked c-jun terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), MAPKAP kinase (MK)2 and NF-κΒ inflammatory signaling during LPS stimulation. Rectal treatment with BMDA or DMMA reduced the severity of colitis in 2,4-dinitrobenzenesulfonic acid (DNBS)-treated rat. Consistently, administration of the compounds decreased myeloperoxidase (MPO) activity (representing neutrophil infiltration in colonic mucosa), production of inflammatory mediators such as cytokine-induced neutrophil chemoattractant (CINC)-3 and TNF-α, and activation of JNK and p38 MAPK in the colon tissues. In addition, oral administration of these compounds ameliorated collagen-induced rheumatoid arthritis (RA) in mice. The treatment diminished the levels of inflammatory cytokine transcripts, and protected connective tissues through the expression of anti-oxidation proteins such as nuclear factor erythroid-related factor (Nrf)2 and heme oxygenase (HO)1. Additionally, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels did not differ between the BMDA- or DMMA-treated and control animals, indicating that the compounds do not possess liver toxicity. Taken together, these findings propose that BMDA and DMMA could be used as new drugs for curing inflammatory bowel disease (IBD) and RA.

Formoxanthone C Inhibits Malignant Tumor Phenotypes of Human A549 Multidrug Resistant-cancer Cells through Signal Transducer and Activator of Transcription 1-Histone Deacetylase 4 Signaling.

Considering that presence of cancer stem cell (CSC) subpopulation in tumor tissues confers anticancer drug resistance, we investigated whether human A549 lung cancer cells resistant to etoposide possess CSC-like phenotypes. Furthermore, it is known that these malignant tumor features are the leading cause of treatment failure in cancer. We have thus attempted to explore new therapeutic agents from natural products targeting these malignancies. We found that formoxanthone C (XanX), a 1,3,5,6-tetraoxygenated xanthone from Cratoxylum formosum ssp. pruniflorum, at a non-cytotoxic concentration reduced the expression of the signal transducer and activator of transcription 1 (STAT1) and histone deacetylase 4 (HDAC4) proteins, leading to inhibition of CSC-like phenotypes such as cell migration, invasion, and sphere-forming ability. Moreover, we found that treatment with STAT1 or HDAC4 small interfering RNAs significantly hindered these CSC-like phenotypes, indicating that STAT1 and HDAC4 play a role in the malignant tumor features. Taken together, our findings suggest that XanX may be a potential new therapeutic agent targeting malignant lung tumors.

Translocalization of enhanced PKM2 protein into the nucleus induced by cancer upregulated gene 2 confers cancer stem cell-like phenotypes.

Increased mRNA levels of cancer upregulated gene (CUG)2 have been detected in many different tumor tissues using Affymetrix microarray. Oncogenic capability of the CUG2 gene has been further reported. However, the mechanism by which CUG2 overexpression promotes cancer stem cell (CSC)-like phenotypes remains unknown. With recent studies showing that pyruvate kinase muscle 2 (PKM2) is overexpressed in clinical tissues from gastric, lung, and cervical cancer patients, we hypothesized that PKM2 might play an important role in CSC-like phenotypes caused by CUG2 overexpression. The present study revealed that PKM2 protein levels and translocation of PKM2 into the nucleus were enhanced in CUG2-overexpressing lung carcinoma A549 and immortalized bronchial BEAS-2B cells than in control cells. Expression levels of c-Myc, CyclinD1, and PKM2 were increased in CUG2-overexpressing cells than in control cells. Furthermore, EGFR and ERK inhibitors as well as suppression of Yap1 and NEK2 expression reduced PKM2 protein levels. Interestingly, knockdown of ß-catenin expression failed to reduce PKM2 protein levels. Furthermore, reduction of PKM2 expression with its siRNA hindered CSC-like phenotypes such as faster wound healing, aggressive transwell migration, and increased size/number of sphere formation. The introduction of mutant S37A PKM2-green fluorescence protein (GFP) into cells without ability to move to the nucleus did not confer CSC-like phenotypes, whereas forced expression of wild-type PKM2 promoted such phenotypes. Overall, CUG2-induced increase in the expression of nuclear PKM2 contributes to CSC-like phenotypes by upregulating c-Myc and CyclinD1 as a co-activator. [BMB Reports 2022;55(2): 98-103].

Elevated Expression of JMJD5 Protein Due to Decreased miR-3656 Levels Contributes to Cancer Stem Cell-Like Phenotypes under Overexpression of Cancer Upregulated Gene 2.

Overexpression of cancer upregulated gene (CUG) 2 induces cancer stem cell-like phenotypes, such as enhanced epithelial-mesenchymal transition, sphere formation, and doxorubicin resistance. However, the precise mechanism of CUG2-induced oncogenesis remains unknown. We evaluated the effects of overexpression of CUG2 on microRNA levels using a microRNA microarray. Levels of miR-3656 were decreased when CUG2 was overexpressed; on the basis of this result, we further examined the target proteins of this microRNA. We focused on Jumonji C domain-containing protein 5 (JMJD5), as it has not been previously reported to be targeted by miR-3656. When CUG2 was overexpressed, JMJD5 expression was upregulated compared to that in control cells. A 3' untranslated region (UTR) assay revealed that an miR-3656 mimic targeted the JMJD5 3'UTR, but the miR-3656 mimic failed to target a mutant JMJD5 3'UTR, indicating that miR-3656 targets the JMJD5 transcript. Administration of the miR-3656 mimic decreased the protein levels of JMD5 according to Western blotting. Additionally, the miR-3656 mimic decreased CUG2-induced cell migration, evasion, and sphere formation and sensitized the cells to doxorubicin. Suppression of JMJD5, with its small interfering RNA, impeded CUG2-induced cancer stem cell-like phenotypes. Thus, overexpression of CUG2 decreases miR-3656 levels, leading to upregulation of JMJD5, eventually contributing to cancer stem cell-like phenotypes.

Overexpression of Cancer Upregulated Gene 2 (CUG2) Decreases Spry2 Through c-Cbl, Leading to Activation of EGFR and ß-Catenin Signaling.

PURPOSE: The mechanism by which cancer upregulated gene 2 (CUG2) overexpression induces cancer stem cell-like phenotypes is not fully understood. Because the increased activity and expression of epidermal growth factor receptor (EGFR) kinase have been reported in A549 cancer cells overexpressing CUG2 (A549-CUG2) compared with control cells (A549-Vec), the Sprouty2 (Spry2) protein has gained attention as the downstream molecule of EGFR signaling. Therefore, we aim to identify the role of Spry2 in CUG2-overexpressing lung cancer cells. MATERIALS AND METHODS: Spry2 expression levels were examined in A549-CUG2 and A549-Vec cells by Western blotting and qRT-PCR. Cell migration, invasion, and sphere formation were examined after Spry2 suppression and overexpression. EGFR-Stat1 and Akt-ERK protein phosphorylation levels were detected via immunoblotting. NEK2 kinase and ß-catenin reporter assay were performed for downstream of Spry2 signaling. RESULTS: Although A549-CUG2 cells showed lower levels of the Spry2 protein than A549-Vec cells, no difference in levels of Spry2 transcript was observed between both cells via qRT-PCR. Furthermore, MG132 treatment enhanced the protein levels and ubiquitination of Spry2, suggesting that Spry2 protein expression can be regulated via the ubiquitin-proteasome pathway. The enforced expression of c-Cbl, known as the binding partner of Spry2, decreased the Spry2 protein levels, whereas its knockdown oppositely increased them. Epithelial-mesenchymal transition (EMT) and sphere formation were increased in A549-Vec cells during Spry2 siRNA treatment, confirming the role of Spry2 in CUG2-induced oncogenesis. Furthermore, EMT and sphere formation were determined by the Spry2 protein levels through the regulation of EGFR-Stat1 and ß-catenin-NEK2-Yap1 signaling pathways. CONCLUSION: CUG2 reduces Spry2 protein levels, the negative signaling molecule of cell proliferation, via c-Cbl, possibly activating the EGFR and ß-catenin signaling pathways and, in turn, contributing to the induction of cancer stem cell-like phenotypes.