CTHRC1 Induces Pancreatic Stellate Cells (PSCs) into Myofibroblast-like Cancer-Associated Fibroblasts (myCAFs).

[BACKGROUND] Collagen triple helix repeat containing-1 (CTHRC1) is a secreted protein that contributes to the progression of various cancers, including pancreatic cancer. The higher expression of CTHRC1 in tumor tissues is associated with poorer survival outcomes. However, its specific roles in tumor extracellular matrix (ECM) remodeling remain unclear. Our study aims to investigate the influences of CTHRC1 on pancreatic stellate cells (PSCs), a main source of ECM production in pancreatic cancer. [METHODS AND RESULTS] The analyses of the publicly available pancreatic cancer patient data revealed that CTHRC1 is mainly expressed in cancer stroma and highly correlated with ECM-related genes. An in vitro study showed that more than 40% of these genes can be upregulated by CTHRC1. CTHRC1 specifically activated PSC into myofibroblast-like cancer-associated fibroblasts (myCAFs), which are characterized by a significantly upregulated POSTN gene expression. Periostin (coded by the POSTN gene) has a central role in the CTHRC1-PSCs-cancer metastasis axis. Furthermore, CTHRC1 promoted pancreatic cancer cell proliferation through PSC activation to a greater extent than via direct stimulation. Proof-of-concept experiments showed that the long-term (4-week) inhibition of CTHRC1 led to significant tumor suppression and ECM reduction, and also resulted in an unexpected shift in the CAF subtype from myCAFs to inflammatory CAFs (iCAFs). [CONCLUSION] PSC activation was demonstrated to be the key molecular mechanism responsible for the tumor-promoting effects of CTHRC1, and CTHRC1 has a critical role in CAF subtype differentiation and tumor microenvironment (TME) remodeling. The inhibition of CTHRC1 as a therapeutic strategy for the treatment of pancreatic cancer warrants further investigation.

PAUF Induces Migration of Human Pancreatic Cancer Cells Exclusively via the TLR4/MyD88/NF-κB Signaling Pathway.

PAUF, a tumor-promoting protein secreted by cancer cells, exerts paracrine effects on immune cells through TLR4 receptors expressed on immune cell surfaces. This study aimed to investigate if PAUF elicits autocrine effects on pancreatic cancer (PC) cells through TLR4, a receptor that is overexpressed on PC cells. In this study, TLR4 expression was detected in PC cells only, but not normal pancreatic cells. The migration of TLR4 high-expressing PC cells (i.e., BxPC-3) was reduced by a selective TLR4 inhibitor, in a dose-dependent manner. Using TLR4 overexpressed and knockout PC cell lines, we observed direct PAUF-TLR4 binding on the PC cell surfaces, and that PAUF-induced cancer migration may be mediated exclusively through the TLR4 receptor. Further experiments showed that PAUF signaling was passed down through the TLR4/MyD88 pathway without the involvement of the TLR4/TRIF pathway. TLR4 knockout also downregulated PC membrane PD-L1 expression, which was not influenced by PAUF. To the best of our knowledge, TLR4 is the first receptor identified on cancer cells that mediates PAUF's migration-promoting effect. The results of this study enhanced our understanding of the mechanism of PAUF-induced tumor-promoting effects and suggests that TLR4 expression on cancer cells may be an important biomarker for anti-PAUF treatment.

PAUF as a Target for Treatment of High PAUF-Expressing Ovarian Cancer.

Pancreatic adenocarcinoma up-regulated factor (PAUF) plays an important role in tumor growth, metastasis, and immune evasion in the pancreatic tumor microenvironment, and recent studies suggest an association between PAUF expression and poor prognosis in ovarian cancer patients. The current study aimed 1) to characterize the potential tumor-promoting role of PAUF in ovarian cancer, using in vitro and in vivo models, including a PAUF-knockout OVCAR-5 cell line, and 2) to explore the potential therapeutic effects of an anti-PAUF antibody for ovarian cancer. Recombinant PAUF significantly increased tumor metastatic capacity (migration, invasion, and adhesion) in all the ovarian cancer cell lines tested, except for the OVCAR-5 cell line which expresses PAUF at a much higher level than the other cells. PAUF-knockout in the OVCAR-5 cell line led to apparently delayed tumor growth in vitro and in vivo. Furthermore, the administration of an anti-PAUF antibody exhibited notable sensitizing and synchronizing effects on docetaxel in mice bearing the OVCAR-5 xenograft tumors. Taken together, this study shows that the expression level of PAUF is an independent factor determining malignant behaviors of ovarian cancer and, for the first time, it suggests that PAUF may be a promising therapeutic target for high PAUF-expressing ovarian cancer.

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.

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].

Pancreatic cancer induces muscle wasting by promoting the release of pancreatic adenocarcinoma upregulated factor.

Cancer cachexia is a highly debilitating condition characterized by weight loss and muscle wasting that contributes significantly to the morbidity and mortality of pancreatic cancer. The factors that induce cachexia in pancreatic cancer are largely unknown. We previously showed that pancreatic adenocarcinoma upregulated factor (PAUF) secreted by pancreatic cancer cells is responsible for tumor growth and metastasis. Here, we analyzed the relation between pancreatic cancer-derived PAUF and cancer cachexia in mice and its clinical significance. Body weight loss and muscle weight loss were significantly higher in mice with Panc-1/PAUF tumors than in those with Panc-1/Mock tumors. Direct administration of rPAUF to muscle recapitulated tumor-induced atrophy, and a PAUF-neutralizing antibody abrogated tumor-induced muscle wasting in Panc-1/PAUF tumor-bearing mice. C2C12 myotubes treated with rPAUF exhibited rapid inactivation of Akt-Foxo3a signaling, resulting in Atrogin1/MAFbx upregulation, myosin heavy chain loss, and muscle atrophy. The neutrophil-to-lymphocyte ratio and body weight loss were significantly higher in pancreatic cancer patients with high PAUF expression than in those with low PAUF expression. Analysis of different pancreatic cancer datasets showed that PAUF expression was significantly higher in the pancreatic cancer group than in the nontumor group. Analysis of The Cancer Genome Atlas data found associations between high PAUF expression or a high DNA copy number and poor overall survival. Our data identified tumor-secreted circulating PAUF as a key factor of cachexia, causing muscle wasting in mice. Neutralizing PAUF may be a useful therapeutic strategy for the treatment of pancreatic cancer-induced cachexia.

Monocarboxylate transporter 9 (MCT9) is down-regulated in renal cell carcinoma.

BACKGROUND: The renal cell carcinoma (RCC) incidences are continuously increasing, however, their proper characterization remains difficult. Mammalian kidneys require large amounts of energy, and monocarboxylate transporter (MCT) gene family is responsible for the transport of monocarboxylic compounds across plasma membranes. OBJECTIVE: A total of 14 MCT members have been identified in humans, which show highly distinct substrate affinities and tissue distributions. To understand the yet-uncharacterized renal cancer-specific role of MCTs, we identified MCT members that are differentially regulated during the renal tumor progression. METHODS: We examined the expression level of MCT members in renal cell tumors and their relationship with survival rate of patients using a public database. Quantitative RT-PCR and northern blotting were performed to validate the expression of MCTs. Anti-MCT9 antiserum was raised in rabbit and used to examine MCT9 expression in normal and tumor tissue arrays. Effect of MCT9 overexpression on cell proliferation was measured using renal cancer cell lines. RESULTS: MCT9 was found to be abundantly and exclusively expressed in human kidney cells, and was highly downregulated in renal cancers. Kaplan-Meier plotter analysis revealed an increased survival rate of MCT9 high-expressing RCC patients. MCT9 proteins were detected in normal kidney tissue sections and their overexpression clearly attenuated renal cell proliferation. CONCLUSIONS: MCT9 was identified as a novel highly downregulated gene in renal cell cancer, and its overexpression clearly attenuated RCC cell proliferation. Thus, functional analysis of MCT9 may help in deciphering a yet-undiscovered kidney-specific energy metabolism during renal tumor progression.

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.

Phosphofructokinase 1 Platelet Isoform Promotes ß-Catenin Transactivation for Tumor Development.

Metabolism plays a critical role in direct regulation of a variety of cellular activities via metabolic enzymes and metabolites. Here, we demonstrate that phosphofructokinase 1 platelet isoform (PFKP), which catalyzes a rate-limiting reaction in glycolysis, promotes EGFR activation-induced nuclear translocation and activation of ß-catenin, thereby enhancing the expression of its downstream genes CCND1 and MYC in human glioblastoma cells. Importantly, we showed that EGFR-phosphorylated PFKP Y64 has a critical role in AKT activation and AKT-mediated ß-catenin S552 phosphorylation and subsequent ß-catenin transactivation and promotion of tumor cell glycolysis, migration, invasion, proliferation, and brain tumor growth. These findings highlight a novel mechanism underlying a glycolytic enzyme-mediated ß-catenin transactivation and underscore the integrated and reciprocal regulation of metabolism and gene expression, which are two fundamental biological processes in tumor development.

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

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-ß transcriptional activity and signaling. TGF-ß increased stemness-like phenotypes in the control cells whereas TGF-ß inhibitor blocked induction of the phenotypes, indicating that NPM1 is required for CUG2-mediated stemness-like phenotypes through TGF-ß signaling. Furthermore, the suppression of Smad- and non-Smad-dependent TGF-ß 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-ß signaling.

CGK062, a small chemical molecule, inhibits cancer upregulated gene 2­induced oncogenesis through NEK2 and ß­catenin.

The mechanisms through which cancer­upregulated gene 2 (CUG2), a novel oncogene, affects Wnt/ß­catenin 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 ß­catenin were evaluated by western blot analysis and luciferase assay. To examine a biological consequence of ß­catenin under CUG2 overexpression, cell migration, invasion and sphere formation assay were performed. The upregulation of ß­catenin induced by CUG2 overexpression was also accessed by xenotransplantation in mice. We first found that CUG2 overexpression increased ß­catenin expression and activity. The suppression of ß­catenin decreased cancer stem cell (CSC)­like phenotypes, indicating that ß­catenin is involved in CUG2­mediated CSC­like phenotypes. Notably, CUG2 overexpression increased the phosphorylation of ß­catenin at Ser33/Ser37, which is known to recruit E3 ligase for ß­catenin degradation. Moreover, CUG2 interacted with and enhanced the expression and kinase activity of never in mitosis gene A­related kinase 2 (NEK2). Recombinant NEK2 phosphorylated ß­catenin at Ser33/Ser37, while NEK2 knockdown decreased the phosphorylation of ß­catenin, suggesting that NEK2 is involved in the phosphorylation of ß­catenin at Ser33/Ser37. Treatment with CGK062, a small chemical molecule, which promotes the phosphorylation of ß­catenin at Ser33/Ser37 through protein kinase C (PKC)α to induce its degradation, reduced ß­catenin levels and inhibited the CUG2­induced features of malignant tumors, including increased cell migration, invasion and sphere formation. Furthermore, CGK062 treatment suppressed CUG2­mediated tumor formation in nude mice. Taken together, the findings of this study suggest that CUG2 enhances the phosphorylation of ß­catenin at Ser33/Ser37 by activating NEK2, thus stabilizing ß­catenin. CGK062 may thus have potential for use as a therapeutic drug against CUG2­overexpressing lung cancer cells.

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.

STAT1­HDAC4 signaling induces epithelial­mesenchymal transition and sphere formation of cancer cells overexpressing the oncogene, CUG2.

Our previous studies have shown that the novel oncogene, cancer upregulated gene 2 (CUG2), activates STAT1, which is linked to anticancer drug resistance, induces epithelial­mesenchymal transition (EMT) and cancer stem cell­like 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 N­cadherin 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 E­cadherin expression. STAT1 siRNA also diminished CUG2­induced TGF­ß signaling, which is critical in EMT, and TGF­ß transcriptional activity. Conversely, inhibition of TGF­ß signaling reduced phosphorylation of STAT1, indicating a crosstalk between STAT1 and TGF­ß signaling. Furthermore, STAT1 silencing diminished sphere formation, which was supported by downregulation of stemness­related 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 TGF­ß signaling and decreased expression of Sox2 and Nanog. Taken together, we suggest that STAT1­HDAC4 signaling induces malignant tumor features such as EMT and sphere formation in CUG2­overexpressing cancer cells.

Elevated expression of pancreatic adenocarcinoma upregulated factor (PAUF) is associated with poor prognosis and chemoresistance in epithelial ovarian cancer.

Pancreatic adenocarcinoma upregulated factor (PAUF) is a ligand of toll-like receptors (TLRs) and has been reported to be involved in pancreatic tumor development. However, the significance of PAUF expression in epithelial ovarian cancer remains unclear. We aimed to investigate the possible clinical significance of PAUF in epithelial ovarian cancer. We examined the link between PAUF and TLR4 in ovarian cancer cell lines. Recombinant PAUF induced cell activation and proliferation in ovarian cancer cell lines, whereas PAUF knockdown inhibited these properties. Subsequently, we assessed PAUF and TLR4 expression by immunohistochemistry on tissue microarray of 408 ovarian samples ranging from normal to metastatic. PAUF expression positively correlated with TLR4 expression. Overexpression of PAUF was associated with high-grade tumor (p = 0.014) and chemoresistant tumor (p = 0.017). Similarly, high expression of TLR4 correlated with advanced tumor stage (p = 0.002) and chemoresistant tumor (p = 0.001). Multivariate analysis indicated that PAUFhigh, TLR4high, and PAUFhigh/TLR4high expression are independent prognostic factor for progression-free survival, while TLR4high and PAUFhigh/TLR4high expression were independent prognostic factors for overall survival. Our results suggest that PAUF has a role in ovarian cancer progression and is a potential prognostic marker and novel chemotherapeutic target for ovarian cancer.

DCPP1 is the mouse ortholog of human PAUF that possesses functional analogy in pancreatic cancer.

Pancreatic adenocarcinoma upregulated factor (PAUF) overexpressed in pancreatic ductal adenocarcinoma (PDAC) plays a major role in tumor progression and metastasis by autocrine and paracrine manners. However, underlying molecular mechanism of PAUF functioning in pancreatic cancer are not fully understood yet. The objective of this study was to evaluate the potential of demilune cell and parotid protein 1 (DCPP1) as a putative mouse ortholog of human PAUF by sequence alignment and functional studies. Overexpression of mouse DCPP1 in Chinese hamster ovary (CHO) cells or pancreatic cancer cells increased cell proliferation, migration, invasion, and adhesion ability in vitro. Treatment of human pancreatic cancer cells with recombinant mouse DCPP1 elevated cell growth, motility, invasiveness, and adhesiveness. Mouse DCPP1 exerted its function on pancreatic cancer cells by activating intracellular signaling pathways involved in aggressive cancer phenotype of human pancreatic cancer cells. Moreover, subcutaneous injection of mice with DCPP1-overexpressing CHO cells increased tumor sizes. Taken together, we conclude that mouse DCPP1 is a multifunctional promoter of tumor growth through functional activation of pancreatic cancer cells, suggesting it to be an ortholog of human PAUF.

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

BACKGROUND: Previously, it has been found that the cancer upregulated gene 2 (CUG2) and the epidermal growth factor receptor (EGFR) both contribute to drug resistance of cancer cells. Here, we explored whether CUG2 may exert its anticancer drug resistance by increasing the expression of EGFR. METHODS: EGFR expression was assessed using Western blotting, immunofluorescence and capacitance assays in A549 lung cancer and immortalized bronchial BEAS-2B cells, respectively, stably transfected with a CUG2 expression vector (A549-CUG2; BEAS-CUG2) or an empty control vector (A549-Vec; BEAS-Vec). After siRNA-mediated EGFR, Stat1 and HDAC4 silencing, antioxidant and multidrug resistance protein and mRNA levels were assessed using Western blotting and RT-PCR. In addition, the respective cells were treated with doxorubicin after which apoptosis and reactive oxygen species (ROS) levels were measured. Stat1 acetylation was assessed by immunoprecipitation. RESULTS: We found that exogenous CUG2 overexpression induced EGFR upregulation in A549 and BEAS-2B cells, whereas EGFR silencing sensitized these cells to doxorubicin-induced apoptosis. In addition, we found that exogenous CUG2 overexpression reduced the formation of ROS during doxorubicin treatment by enhancing the expression of antioxidant and multidrug resistant proteins such as MnSOD, Foxo1, Foxo4, MRP2 and BCRP, whereas EGFR silencing congruently increased the levels of ROS by decreasing the expression of these proteins. We also found that EGFR silencing and its concomitant Akt, ERK, JNK and p38 MAPK inhibition resulted in a decreased Stat1 phosphorylation and, thus, a decreased activation. Since also acetylation can affect Stat1 activation via a phospho-acetyl switch, HDAC inhibition may sensitize cells to doxorubicin-induced apoptosis. Interestingly, we found that exogenous CUG2 overexpression upregulated HDAC4, but not HDAC2 or HDAC3. Conversely, we found that HDAC4 silencing sensitized the cells to doxorubicin resistance by decreasing Stat1 phosphorylation and EGFR expression, thus indicating an interplay between HDAC4, Stat1 and EGFR. CONCLUSION: Taken together, we conclude that CUG2-induced EGFR upregulation confers doxorubicin resistance to lung (cancer) cells through Stat1-HDAC4 signaling.

Characterization of VNTRs Within the Entire Region of SLC6A3 and Its Association with Hypertension.

The dopamine transporter SLC6A3 (DAT1) mediates uptake of dopamine into presynaptic terminals. In addition, in previous reports, hypertensive rats were associated with DAT gene, but the genetic association with SLC6A3 and hypertension is still unknown. We examined the distribution of variable number of tandem repeats (VNTRs) and conducted polymorphic analysis of the entire region of SLC6A3. Ten VNTR regions (MS1-10) were revealed throughout the intronic and UTRs; seven VNTR regions were newly isolated and three VNTRs were previously reported. Four VNTR regions (SLC6A3-MS1, -MS4, -MS8 [rs3836790], and -MS9 [rs28363170]) showed polymorphism and these loci were found to be transmitted through meiosis following Mendelian inheritance. These VNTR polymorphisms may be useful markers for paternity mapping and DNA fingerprinting. Furthermore, we also conducted a case-control study between the controls and essential hypertensive cases. Analysis of the genotypes of SLC6A3-MS8 (rs3836790) revealed that having an 8/6-repeat allele, which was only detected in hypertensive cases, was associated with hypertension (p < 0.05). Additional significant association was identified between the short 7-repeat allele of SLC6A3-MS9 (rs28363170) and the occurrence of hypertension (odds ratio 2.02; p < 0.05). These results revealed the genetic association between SLC6A3 and hypertension, and the specific VNTR alleles of SLC6A3 may be a risk factor for hypertension.

Cancer upregulated gene 2 induces epithelial-mesenchymal transition of human lung cancer cells via TGF-ß signaling.

Cancer upregulated gene 2 (CUG2) enhances cell migration and invasion, but the underlying mechanism has not been revealed. Herein, CUG2 decreased the expression of E-cadherin and increased the expression of N-cadherin and vimentin, characteristics of the epithelial-mesenchymal transition (EMT). A CUG2 deletion mutant, lacking interaction with nucleophosmin 1 (NPM1), or suppression of NPM1 reduced wound healing and cell invasion, indicating that CUG2-mediated EMT requires NPM1. CUG2 enhanced activation of Smad2/3 and expression of Snail and Twist, while the CUG2 silence decreased these TGF-ß signaling pathways, leading to suppression of EMT. NPM silence also inhibited the CUG2-induced TGF-ß signaling. These results suggest that TGF-ß signaling is involved in CUG2-induced EMT. Treatment with EW-7197, a novel inhibitor of TGF-ß signaling, diminished CUG2-mediated EMT and inhibition of Akt, ERK, JNK, and p38 MAPK, non-canonical TGF-ß signaling molecules, also decreased expression of Smad2/3, Snail and Twist, leading to inhibition of EMT. The results confirm that TGF-ß signaling is essential for CUG2-mediated EMT. Interestingly, TGF-ß enhanced CUG2 expression. We further found that both CUG2-induced TGF-ß production and TGF-ß-induced CUG2 up-regulation required a physical interaction between Sp1 and Smad2/3 in the CUG2 and TGF-ß promoter, as demonstrated by a promoter reporter assay, immunoprecipitation, and ChIP assay. These results indicated close crosstalk between CUG2 and TGF-ß. Conversely, suppression of CUG2 or NPM1 did not completely inhibit TGF-ß-induced EMT, indicating that the effect of TGF-ß on EMT is dominant over the effect of CUG2 on EMT. Collectively, our findings suggest that CUG2 induces the EMT via TGF-ß signaling.

CTHRC1 promotes angiogenesis by recruiting Tie2-expressing monocytes to pancreatic tumors.

CTHRC1 (collagen triple-helix repeat-containing 1), a protein secreted during the tissue-repair process, is highly expressed in several malignant tumors, including pancreatic cancer. We recently showed that CTHRC1 has an important role in the progression and metastasis of pancreatic cancer. Although CTHRC1 secretion affects tumor cells, how it promotes tumorigenesis in the context of the microenvironment is largely unknown. Here we identified a novel role of CTHRC1 as a potent endothelial activator that promotes angiogenesis by recruiting bone marrow-derived cells to the tumor microenvironment during tumorigenesis. Recombinant CTHRC1 (rCTHRC1) enhanced endothelial cell (EC) proliferation, migration and capillary-like tube formation, which was consistent with the observed increases in neovascularization in vivo. Moreover, rCTHRC1 upregulated angiopoietin-2 (Ang-2), a Tie2 receptor ligand, through ERK-dependent activation of AP-1 in ECs, resulting in recruitment of Tie2-expressing monocytes (TEMs) to CTHRC1-overexpressing tumor tissues. Treatment with a CTHRC1-neutralizing antibody-abrogated Ang-2 expression in the ECs in vitro. Moreover, administration of a CTHRC1-neutralizing antibody to a xenograft mouse model reduced the tumor burden and infiltration of TEMs in the tumor tissues, indicating that blocking the CTHRC1/Ang-2/TEM axis during angiogenesis inhibits tumorigenesis. Collectively, our findings support the hypothesis that CTHRC1 induction of the Ang-2/Tie2 axis mediates the recruitment of TEMs, which are important for tumorigenesis and can be targeted to achieve effective antitumor responses in pancreatic cancers.

A polymorphic minisatellite region of BORIS regulates gene expression and its rare variants correlate with lung cancer susceptibility.

Aberrant expression of BORIS/CTCFL (Brother of the Regulator of Imprinted Sites/CTCF-like protein) is reported in different malignancies. In this study, we characterized the entire promoter region of BORIS/CTCFL, including the CpG islands, to assess the relationship between BORIS expression and lung cancer. To simplify the construction of luciferase reporter cassettes with various-sized portions of the upstream region, genomic copies of BORIS were isolated using TAR cloning technology. We analyzed three promoter blocks: the GATA/CCAAT box, the CpG islands and the minisatellite region BORIS-MS2. Polymorphic minisatellite sequences were isolated from genomic DNA prepared from the blood of controls and cases. Of the three promoter blocks, the GATA/CCAAT box was determined to be a critical element of the core promoter, while the CpG islands and the BORIS-MS2 minisatellite region were found to act as regulators. Interestingly, the polymorphic minisatellite region BORIS-MS2 was identified as a negative regulator that repressed the expression levels of luciferase reporter cassettes less effectively in cancer cells compared with normal cells. We also examined the association between the size of BORIS-MS2 and lung cancer in a case-control study with 590 controls and 206 lung cancer cases. Rare alleles of BORIS-MS2 were associated with a statistically significantly increased risk of lung cancer (odds ratio, 2.04; 95% confidence interval, 1.02-4.08; and P=0.039). To conclude, our data provide information on the organization of the BORIS promoter region and gene regulation in normal and cancer cells. In addition, we propose that specific alleles of the BORIS-MS2 region could be used to identify the risk for lung cancer.