Identification of TMEM115 as a tumor suppressor gene through TERT regulation in pancreatic cancer

Identification of TMEM115 as a tumor suppressor gene through TERT regulation in pancreatic cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Identification of TMEM115 as a tumor suppressor gene through TERT regulation in pancreatic cancer Hiroyuki Kugoh, Yu Sakano, Takahito Ohira, Haruka Ui, Takuki Yagyu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6170448/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The catalytic component of telomerase, telomerase reverse transcriptase ( TERT ), is reactivated in immortalized cells and plays a crucial role in cancer development. Pancreatic cancer (PC) is frequently associated with loss of heterozygosity on the short arm of human chromosome 3. In a previous study, chromosome engineering experiments in PC suggested that putative TERT suppressor genes (TSGs) are present on the 3p21.3 region. Here, we performed functional analysis using a human artificial chromosome (HAC) carrying only the 3p21.3 region (3p21.3-HAC) to directly clarify if TSGs are contained in the 3p21.3 region. We observed reduced TERT transcription following the introduction of 3p21.3-HAC into PC cells. Furthermore, to identify the specific TSGs in the 3p21.3 region, we performed RNA sequencing analysis using mouse Tert ( mTert )-expressing murine LTPA PC cells containing either 3p21.3-HAC or the empty HAC vector. Through this analysis, we identified transmembrane protein 115 ( TMEM115 ) as a novel TSG. Furthermore, both human TERT ( hTERT ) and mTert transcription can be suppressed by TMEM115 . Thus, TMEM115 may contribute to PC development by functioning as a novel telomerase regulating factor via controlling TERT expression. Biological sciences/Cancer/Gastrointestinal cancer/Pancreatic cancer Biological sciences/Cell biology/Chromosomes Biological sciences/Cancer/Oncogenes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Pancreatic cancer (PC) is one of the most lethal malignant diseases. According to the 2020 World Cancer Statistics, PC is the seventh leading cause of cancer-related deaths worldwide for both men and women, with approximately 496 000 new cases and 466 000 deaths [ 1 ]. This disease is characterized by late diagnosis from insidious onset, early metastasis, limited response to chemotherapy, and poor prognosis [ 2 ]. Despite recent improvements in therapeutic strategies, the difficulties associated with early detection have resulted in PC patients often being diagnosed at an advanced stage or with distant metastases and a 5-year survival rate of less than 10% [ 3 ]. Because the molecular mechanisms involved in PC progression remain unclear, further research is urgently required to identify novel diagnostic biomarkers and therapeutic targets for this disease and develop new treatment strategies for patients. Telomerase is a ribonucleoprotein ribonucleic enzyme that catalyzes the elongation of telomere repeat sequences at the end of chromosomes, contributing to cellular immortalization [ 4 ]. The catalytic component of telomerase, telomerase reverse transcriptase ( TERT ), is stringently repressed in most human somatic cells, consequently resulting in telomerase inactivation [ 5 ]. However, telomerase is activated in most cancer cells, including PC, and supports the unlimited proliferative ability and tumorigenicity of malignant tumor cells [ 6 , 7 ]. Recent studies have revealed that, independently of its telomere-lengthening function, TERT can stimulate the epithelial-mesenchymal transition and stemness of cancer cells, indicating that TERT also contributes to cancer invasiveness and metastasis [ 8 – 10 ]. Although it is known that human TERT (h TERT ) expression is strictly regulated by multiple transcriptional activators and repressors, as well as epigenetic modifications, the exact regulatory mechanism has not been completely elucidated [ 11 , 12 ]. Loss of heterozygosity (LOH) is a common genetic alteration that occurs in cancer genomes, with LOH of tumor suppressor genes serving as a significant factor that promotes cancer progression [ 13 ]. LOH at the 3p, 9p, 17p, and 18q chromosomes are highly prevalent somatic genetic regions in PC. The tumor suppressor genes cyclin-dependent kinase inhibitor 2A ( CDKN2A ), tumor protein 53 ( TP53 ), and suppressor of mothers against decapentaplegic homolog 4 ( SMAD4 ) have been identified in the 9p, 17p, and 18q LOH regions, respectively [ 14 – 16 ]. Chromosome 3p has also been shown to contain many tumor suppressor genes, such as von Hippel-Lindau ( VHL ), Ras association domain family member 1 ( RASSF1 ), fragile histidine triad ( FHIT ), and roundabout guidance receptor 1 ( ROBO1 ), but no tumor suppressor genes have been identified that are involved in the regulation of TERT [ 17 – 20 ]. In a previous study, we reported that introducing normal human chromosome 3 using microcell-mediated chromosome transfer (MMCT) could induce the inhibition of mouse Tert ( mTert ) transcription in murine LTPA PC cells. Furthermore, we revealed that the 3p21.3 region may encode the mTert repressor gene(s) by transferring chromosomes that were truncated at arbitrary regions into LTPA cells [ 21 ]. Human artificial chromosomes (HACs) are mini-chromosome vectors with the endogenous genes removed from a normal human chromosome 21 using chromosome engineering techniques [ 22 ]. HACs are useful gene delivery vectors because they exist stably and independently in host cells and can carry an unlimited number of genome sizes, having the great advantage of facilitating genome functional analysis under more physiologically relevant conditions [ 23 , 24 ]. In addition, HAC vectors can be transferred into any cell line via MMCT [ 25 ]. We have previously demonstrated the presence of a TERT suppressor gene (TSG) through the functional analysis of a HAC carrying the 3p21.3 region in oral squamous cell carcinoma cells [ 26 ]. Transmembrane protein 115 (TMEM115), which is encoded by a gene in the 3p21.3 chromosome, is a multi-transmembrane domain protein that is localized at the medial and trans-Golgi cisternae [ 27 ]. TMEM115 helps regulate retrograde transport from the Golgi to the endoplasmic reticulum. TMEM115 is highly expressed in a wide range of epithelial cells in normal tissues, including the gastrointestinal, respiratory, genitourinary, and breast systems. Although loss of TMEM115 expression has been reported in renal clear cell carcinoma and other VHL-deficient tumors, its involvement in oncogenesis is unclear [ 28 ]. In this study, we identified TMEM115 as a novel TSG on the 3p21.3 region in PC through functional analysis using a HAC. Furthermore, we revealed that TMEM115 expression levels are positively correlated with PC patient prognosis. Our results provide important information that supports TMEM115 as a new potential therapeutic target in PC. Results Suppression of mTert expression by introducing a HAC with only the 3p21.3 region into murine LTPA PC cells To investigate the presence of TERT regulatory factors within the 3p21.3 region in PC cells, we used MMCT to establish LTPA microcell hybrid clones containing a HAC without any gene coding regions (empty-HAC; LTPA control) or a HAC with only the 3p21.3 region (LTPA 3p21.3-HAC). To confirm the presence of each transferred HAC in the LTPA calls, we performed two-color fluorescence in situ hybridization (FISH) analysis using human COT-1 DNA (rhodamine) and 234N4 PAC (FITC) located at the 3p21.3 region as probes. The results showed that only the rhodamine signal was detected in the LTPA control cells (Fig. 1 A), while both the rhodamine and FITC signals were detected in the LTPA 3p21.3-HAC cells (Fig. 1 B). This suggested that we obtained microcell hybrid clones with empty-HAC and 3p21.3-HAC that segregated as an independent chromosome in LTPA cells. Next, we investigated the mTert mRNA expression patterns in the LTPA control and LTPA 3p21.3-HAC hybrid cells using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The LTPA 3p21.3-HAC microcell hybrid cells exhibited significantly lower mTert mRNA expression levels compared with the control cells (Fig. 1 C). These results indicated the presence of a gene or genes on human chromosome 3p21.3 that can negatively regulate mTert transcription, which may play an important role in PC development and progression. Inhibition of cell proliferation and invasion after introducing 3p21.3-HAC into LTPA PC cells Next, we examined if introducing the 3p21.3 region into the LTPA cells affected cell proliferation. LTPA microcell hybrids with the 3p21.3 region displayed significantly reduced cell proliferation rates compared with the LTPA control cells (Fig. 2 A). In addition, telomerase activity has been reported to contribute to cell invasion and metastasis by activating the epithelial-mesenchymal transition [ 10 , 29 ]. Therefore, we examined the cell invasion potential of the LTPA control and LTPA 3p21.3-HAC cells. The LTPA 3p21.3-HAC cells showed a significantly reduced invasive ability compared with the control cells (Fig. 2 B–D). These results demonstrated that the reduced mTert transcription induced by introducing the 3p21.3 region was accompanied by suppressive effects on PC cell proliferation and invasion. RNA sequencing (RNA-seq) analysis of LTPA microcell hybrids to identify mTert repressor genes in the 3p21.3 region To identify the specific mTert suppressor gene suggested to be present in the 3p21.3 region, we comprehensively examined the gene expression patterns of the LTPA 3p21.3-HAC and LTPA control cells by RNA-seq analysis. Relative to the LTPA control cells, the expression levels of 26 genes encoded in the 3p21.3 region were significantly upregulated in the LTPA 3p21.3-HAC cells with repressed mTert transcription (Table S1 ). Seven of these genes, TMEM115 , calcium voltage-gated channel auxiliary subunit alpha2 delta 2 ( CACNA2D2 ), inositol hexakisphosphate kinase 1 ( IP6K1 ), G protein subunit alpha i2 ( GNAI2 ), interferon related developmental regulator 2 ( IFRD2 ), solute carrier family 38 member 3 ( SLC38A3 ), and zinc finger MYND-type containing 10 ( ZMYND10 ), were found to be downregulated in The Cancer Genome Atlas (TCGA) PC dataset of patients in whom hTERT is expressed (Fig. 3 A). We next individually knocked down each of these seven genes using two specific small interfering RNAs (siRNAs) to identify any relevant mTert regulatory genes. The LTPA 3p21.3-HAC cells treated with the siRNAs targeting these genes displayed a 0.16-fold ( ZMYND10 siRNA2) to 0.74-fold ( SLC38A3 siRNA1) decrease in target mRNA expression levels (Figs. 3 B and S1). As shown in Figs. 3 C and S1, subsequent examination of the effects of knocking down these genes on mTert transcription patterns revealed upregulation of TMEM115 and CACNA2D2 expression levels. CACNA2D2 , which encodes the α2δ2 auxiliary subunit of the voltage-activated calcium channel protein complex, is abundantly expressed in lung and brain tissues and has been shown to be a tumor suppressor gene that can induce apoptosis in non-small cell lung cancer cells [ 30 ]. Analysis of the human protein atlas (HPA) database showed that CACNA2D2 is expressed at very low levels in normal pancreatic tissues. Thus, no difference in CACNA2D2 gene expression patterns was found between PC and non-carcinoma tissues. However, TMEM115 , an endogenous membrane protein in the Golgi apparatus, is nonspecifically expressed in multiple organs, including the pancreas. Protein expression data from the HPA database indicated significantly decreased TMEM115 expression levels in PC tissues compared with the levels in normal tissues [ 31 ]. Thus, these findings suggested that TMEM115 was more likely to act as a TSG in PC, leading us to focus our further work on TMEM115 . TMEM115 inhibits mTert transcription, cell proliferation, and cell invasion To investigate the TMEM115 -mediated suppressive effects on mTert transcription, we established TMEM115 stable expression clones by transfecting the pCMV6 vector carrying the TMEM115 gene into LTPA cells. Higher TMEM115 expression levels were observed in all transfectant clones compared with those observed in the controls (Fig. 4 A). As shown in Fig. 4 B, the cells expressing TMEM115 (LTPA TMEM115 ) showed remarkably repressed mTert expression levels compared with the cells transfected with the empty vector. Furthermore, the cell proliferation and invasion rates were suppressed in the LTPA TMEM115 cells compared with the control cells (Fig. 4 C–F). Interestingly, the reduced mTert expression levels and cell proliferation and invasion rates in the LTPA TMEM115 clones were similar to those observed in the LTPA cells that received the normal human chromosome 3 or 3p21.3-HAC ([ 21 ], Figs. 1 C and 2 ). These data strongly implicated TMEM115 as the gene responsible for the observed mTert suppression resulting from introducing the 3p21.3 region. Suppression of hTERT expression by overexpressing TMEM115 in human PC cells Next, we aimed to clarify if TMEM115 , which could repress mTert transcription in murine PC cells, has similar functions in human PC cells. We established human MIA PaCa-2 PC cells overexpressing TMEM115 using the pCMV6 vector carrying the TMEM115 gene. The empty pCMV6 vector was used as a negative control. As shown in Fig. 5 A, TMEM115 expression was detected at the protein level using western blot analysis. Furthermore, qRT-PCR analysis indicated that the MIA PaCa-2 cells with TMEM115 overexpression displayed significantly suppressed hTERT transcription compared with the control cells with the empty vector (Fig. 5 B). The MIA PaCa-2 cells with TMEM115 overexpression also showed decreased cell proliferation and invasion rates (Fig. 5 C–F). Interestingly, the TERT expression levels were 1.9-fold more strongly suppressed in the human PC cells than in the murine cells following TMEM115 overexpression, with a corresponding greater suppression of the cell proliferative and invasive capacities (Figs. 4 and 5 ). This consequentially provided crucial evidence that TMEM115 acts as a TERT repressor gene in both mouse and human PC cells. Low TMEM115 expression is a poor prognostic factor in PC patients Using the TCGA database, we analyzed the association between TMEM115 mRNA expression levels and survival time in 181 PC patients to elucidate the clinical impact of this gene. The receiver operating characteristic curve analysis for overall survival (OS) revealed the optimal cutoff value for TMEM115 expression (Fig. S2), which was used to divide the patients into high ( TMEM115 -high, n = 116) and low ( TMEM115 -low, n = 65) TMEM115 expression groups. Kaplan-Meier survival analysis demonstrated that the TMEM115 -low patients had significantly shorter OS than the TMEM115 -high patients ( P = 0.006; Fig. 6 A). Furthermore, we used immunohistochemistry (IHC) assays to examine tissues samples from 100 PC patients who underwent pancreatectomy to determine the prognostic significance of TMEM115 expression in these individuals. TMEM115 was expressed generally without cell specificity in the pancreatic tissues, with the islets of Langerhans cells, which displayed a more consistent expression intensity in all cases, used as internal positive controls (Fig. 6 ). Positive cells were defined as those with a TMEM115 staining intensity equal to or greater than that of the islets of Langerhans cells in noncancerous pancreatic tissues. Tumors with < 50% positive cells were classified as low TMEM115 expression. As shown in Fig. 6 C/D, IHC analysis of the PC tissue samples indicated low TMEM115 expression in 22 of the 100 samples. When evaluating the relationships between TMEM115 expression and clinicopathological factors, the local tumor progression, specifically the pathological T (pT) stage, was found to be significantly higher in the PC patients with low TMEM115 expression than in those with high TMEM115 expression (Table 1). Kaplan-Meier survival analysis examining the relationship between TMEM115 expression and prognosis showed that the OS, disease-specific survival, and recurrence-free survival rates were all significantly lower in the patients with low TMEM115 expression compared with those in patients with high TMEM115 expression (Fig. 6 E–G). We subsequently investigated if TMEM115 expression was an independent prognostic risk factor for PC in these patients. The univariate analysis indicated that the pathological N (pN) stage and levels of serum carcinoembryonic antigen, duodenal pancreatic cancer antigen 2, and TMEM115 expression were risk factors for OS after surgery in PC patients. The multivariate analysis using the Cox proportional hazards model revealed that the pN stage and TMEM115 expression levels were independent prognostic factors for PC patient OS (Table 2). The presence and number of metastatic lymph nodes are well-established strong prognostic factors for PC [ 32 , 33 ]. Therefore, identifying the pN stage as an independent prognostic factor in this analysis emphasizes the validity of the postoperative PC patient cohort used in this study. Overall, these results suggested that TMEM115 expression, identified as a prognostic factor along with pN stage, is an important indicator for PC patients. Discussion The combination of chromosome transfer and gene expression profile analysis is an effective approach for identifying the genes responsible for recessive genetic diseases, including tumor suppressor genes [ 34 ]. In fact, we previously found that the paired-like homeodomain 1 ( PITX1 ) gene, which is encoded on chromosome 5, can suppress TERT in melanoma, further analyzing the function of the gene using chromosome transfer technology [ 35 , 36 ]. Furthermore, the use of HAC vectors is considered a more effective approach to identify such genes for several reasons: 1) HAC vectors are not limited by the size of the target genomic region they can carry, 2) they are independently maintained in the host cells, and 3) they support gene functional analysis under physiological conditions using their own native promoters [ 37 ]. The 3p21.3 region has a high frequency of LOH in several carcinomas, including lung, renal, and gastrointestinal cancers, as well as PC, suggesting the presence of tumor suppressor genes in the region [ 38 , 39 ]. In fact, several genes, such as RASSF1 , ZMYND10 , LIM domain containing 1 ( LIMD1 ), and nitrogen permease regulator like-2 ( NPRL2 ), have been reported as tumor suppressors [ 18 , 40 – 43 ]. We previously reported that chromosome 3 is involved in tumor suppression through TERT regulation in renal cell carcinoma and oral squamous cell carcinoma [ 44 , 45 ]. In addition, we recently suggested that the 3p21.3 region in PC may encode TSG(s) by functional analysis of truncated chromosome 3 fragments at arbitrary regions [ 21 ]. However, no specific gene has been identified in this region that can regulate TERT transcription. In the current study, we showed that TMEM115 encoded in the 3p21.3 region may be a novel TSG in PC by introducing HAC vectors carrying the 3p21.3 region and RNA-seq analysis. To the best of our knowledge, this is the first report to refer to TMEM115 as a potential tumor suppressor gene in PC. Furthermore, in other carcinomas where TMEM115 expression levels are significantly decreased in cancerous tissues compared with the levels in normal tissues, as in PC [ 31 ], TMEM115 dysfunction may involve tumorigenesis through TERT regulation. This suggests that TMEM115 possibly plays an important role as a TSG not only in PC, but also in other cancer types. In this study, the suppressive effects on mTert transcription were comparable in both LTPA 3p21.3-HAC cells and TMEM115 -overexpressing LTPA cells (Figs. 1 C and 4 B). It is possible that there may be other important genes in the 3p21.3 region that cooperate with TMEM115 . The human MIA PaCa-2 cells overexpressing TMEM115 showed markedly reduced the hTERT expression levels and cell proliferation and invasion rates compared with the murine LTPA cells. There are some significant differences in telomere regulation between human and mouse biology. Mouse telomere length is longer than that of humans, suggesting that telomere length does not significantly affect the regulation of mouse lifespan, which is much shorter relative to humans [ 46 , 47 ]. Therefore, the functions of genes involved in TERT regulation may also be less strictly controlled in mice, which may explain why the suppressive effects of TMEM115 on TERT were significantly less robust in the mouse PC cells. To investigate the clinical impact of TMEM115 expression in PC, we performed IHC assays on specimens from 100 PC patients. In general, telomeres in cells are known to be shortened in pancreatic intraepithelial neoplasia and intraluminal papillary mucinous neoplasms, the precursor lesions of PC [ 48 , 49 ]. The repression of TERT expression in precancerous lesions that reach the limit of telomere shortening is released, and telomerase reactivation occurs, leading to immortalization. This process promotes the PC transition from early stage to advanced stage. Thus, TERT may contribute to the late-stage portion of multi-stage PC carcinogenesis. The results of these previous reports are consistent with those of this study, where we observed significantly higher local progression of tumors in the low TMEM115 expression group because TMEM115 can suppress TERT transcription. In addition, we found that the patients with low TMEM115 expression levels displayed significantly lower survival rates than those with high TMEM115 expression levels. Further analysis indicated that low TMEM115 expression was an independent prognostic factor for OS in these PC patients. Our results also demonstrated that the pT factor, which was significantly different between the two TMEM115 expression groups, was not a prognostic factor. These data suggest that TMEM115 may be used as a prognostic biomarker independent of TNM classification. Furthermore, analyzing the relationship between TMEM115 mRNA expression levels and prognosis in PC patients using public datasets also indicated a positive correlation. Thus, these results provide clinical evidence that TMEM115 is crucially involved in PC development and progression. The Golgi apparatus plays a central role in protein modifications, such as glycosylation. TMEM115, present in the Golgi complex, is also involved in glycoprotein modifications. Previous work has suggested that TMEM115 knockdown can alter the O-linked glycosylation profile on the cell surface [ 27 ]. O-linked N-acetylglucosamine (O-GlcNAc), one type of O-glycosylation, has been shown to affect cancer cell metabolism, with hyper O-GlcNAcylation being observed in various carcinomas, including PC [ 50 – 52 ]. O-GlcNAcylation can also reportedly lead to the overexpression of oncogenic transcription factors, such as c-MYC, hypoxia-inducible factor 1, and nuclear factor kappa B [ 53 , 54 ]. In addition, c-MYC is known to be one of the major TERT transcriptional activators [ 12 ]. Considering these findings, it is possible that disordered glycosylation from dysfunctional TMEM115 can activate c-MYC-mediated TERT transcription, which promotes PC tumor development and progression. Further examination of the detailed role of TMEM115 in the mechanism of tumorigenesis via TERT regulation could significantly support the discovery of novel PC inhibitory pathways. In conclusion, this study used chromosome engineering technology with HAC vectors to demonstrate that the 3p21.3 region encodes gene(s) that can regulate TERT in PC. Furthermore, we identified TMEM115 as a novel TERT repressor gene. In addition, we reported for the first time that TMEM115 expression is an important prognostic factor for PC patients. Targeting TMEM115 may therefore lead to the development of novel therapeutic strategies against PC. Further studies analyzing the specific TMEM115 functions will be required to define its significance in PC oncogenesis and progression. Materials and Methods Cell culture The LTPA cells and MIA PaCa-2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Wako, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaillé, France). Transfected LTPA cells were selected with 4 µg/mL blasticidin S hydrochloride (Wako) or 600 µg/mL G418 (Calbiochem, La Jolla, CA, USA). Transfected MIA PaCa-2 cells were selected with 7 µg/mL blasticidin S hydrochloride or 1000 µg/mL G418. A9 cells containing a 3p21.3-HAC or empty-HAC vector were cultured in DMEM supplemented with 10% FBS and 8 µg/mL blasticidin S hydrochloride. These microcell hybrid clones were established as previously described (26). All cells were cultured in a humidified incubator at 37°C with 5% CO 2 . MMCT MMCT was performed as previously described [ 26 ]. Briefly, the A9 cells containing the HAC vector were incubated with 0.05 µg/mL colcemid (Sigma-Aldrich, St. Louis, MO, USA) in DMEM containing 20% FBS. Micronuclei were collected by treatment with 10 mg/mL cytochalasin B (Sigma-Aldrich) and centrifugation with sequential filtering through polycarbonate filters (Whatman Nuclepore, Kent, UK). The fusion was mediated by adding 47% polyethylene-glycol 1000 (Wako) and washing with serum-free DMEM. After incubation for 24 h in DMEM containing 10% FBS, the cells with transferred HAC vectors were selected in the presence of blasticidin S hydrochloride, as described above. FISH analysis FISH analysis was performed using cells in metaphase. Biotin-labeled RP-6 234N4 PAC vector containing the 3p21.3 genomic DNA region and digoxigenin-labeled human Cot-1 DNA (Life Technologies, Carlsbad, CA, USA) were used as probe DNAs. Chromosome and probe preparation, hybridization, washing, signal detection, and analysis were performed as previously reported [ 21 ]. qRT-PCR RNA isolation and qRT-PCR were performed as described previously [ 21 ]. The amplification of cDNA was performed using an Applied Biosystems StepOne thermal cycler system and a SYBR Green PCR kit (Foster City, CA, USA). The primers used in this study are listed in the Supplementary Information (Table S2). Cell proliferation assay Cell proliferation assays were performed as described previously [ 21 ]. Briefly, cells were seeded in 60-mm cell culture dishes (1×10 5 cells/dish) and counted daily using a Coulter Counter Z2 (Beckman Coulter, Woerden, the Netherlands). The average cell number was calculated. Cell invasion assay Cell invasion assays were performed as described previously [ 21 ]. These assays were conducted using Corning BioCoat Matrigel Invasion Chambers with 8.0 µm PET membrane (BD Biosciences, Bedford, MA, USA) following the manufacturer's protocol. The invading cells that passed through the filter were stained with Diff-Quik rapid stain (Sysmex Corporation, Hyogo, Japan) and counted using an ECLIPSE Ti-U (Nikon, Tokyo, Japan) at 200× magnification. RNA-seq analysis The transcriptomic analysis was conducted at GENEWIZ from Azenta Life Sciences (Tokyo, Japan). The detailed methods are described in the Supplementary Information. The RNA-seq data have been deposited to DNA Data Bank of Japan (DDBJ) (accession number: DRA018313). Gene knockdown by RNA interference Cells were transfected with the AccuTarget Predesigned TMEM115 siRNA#1 or #2 (Bioneer, Yuseong-gu, Korea) to knock down TMEM115 expression or a negative control siRNA (Bioneer). LTPA 3p21.3-HAC cells were seeded in 6-well plates (2.0×10 5 cells/well). Following the manufacturer's protocol, each siRNA was transfected at a concentration of 20 pmol/mL using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA). Cells were harvested 48 h after transfection, followed by qRT-PCR analysis. The siRNA duplexes of the seven genes, including TMEM115 , are described in the Supplementary Information (Table S3). Plasmid construction The TMEM115 overexpression pCMV6 vectors were purchased from Origene (RC203956, Rockville, MD, USA). The control vectors were constructed by deleting the TMEM115 cDNA sequence from the TMEM115 overexpression vectors using the KOD Plus mutagenesis kit (Toyobo, Osaka, Japan) according to the manufacturer's protocol. Overexpression Parental LTPA or MIA PaCa-2 cells were seeded in 6-well plates (2.0×10 5 cells/well). Following the manufacturer's protocol, pCMV6 vectors were transfected using Lipofectamine LTX Reagent (Invitrogen). Twenty-four h after transfection, the cells were cultured in their respective medium, as described above. After 1 to 2 weeks, drug-resistant colonies were selected and expanded for further analysis following the procedures described above. Western blot analysis Western blot analysis was performed as described previously [ 36 ]. The membranes were blocked and incubated with a rabbit polyclonal anti-TMEM115 antibody (1:2500; PACO30462, AssayGenie, Dublin, Ireland) for 60 min at room temperature, then incubated with an anti-rabbit IgG secondary antibody (1:5000; 7074P2, Cell Signaling Technology, Danvers, MA, USA) for 45 min at room temperature according to the manufacturer's instructions. Immunoreactive bands were visualized using the ECL detection system (Pierce, Rockford, IL, USA). Data acquisition TCGA data of PC patient mRNA expression profiles were obtained from the UCSC Xena platform ( https://xena.ucsc.edu/ ) on 9.6.2024. The protein expression data of pancreatic and PC tissues were obtained from the HPA database ( https://www.proteinatlas.org/ ) on 9.6.2024. IHC staining Between January 2013 and December 2020, 100 consecutive patients underwent pancreatomy at Tottori University Hospital (Yonago, Japan) for PC. This protocol was approved by the Tottori University Ethical Board (approval number: 24A017), and all patients provided written informed consent for pathological analysis. The clinicopathological and laboratory data of all patients were extracted from their electronic medical records. IHC staining was performed with Histostainer-36A (Nichirei Biosciences, Tokyo, Japan), as described in the Supplementary Information. The rabbit polyclonal anti-TMEM115 antibody (AssayGenie) was used as the primary antibody. The islets of Langerhans cells were used as internal positive controls. For analysis, a two-tiered classification using high and low expression was used. The classification method is described in the results of this article. All slides were evaluated independently by Y.S. and Y.U., with consensus reached in all cases. Statistical analysis Data from at least three separate experiments are presented as the mean ± standard deviation. P -values < 0.05 were considered statistically significant. Categorical variables were compared using Fisher’s exact test and continuous variables were compared using two-tailed t-tests. Survival curves were plotted using the Kaplan-Meier method, with the differences between survival curves examined using the log-rank test. Receiver operating characteristic curve analysis was used to determine the optimal cutoff values. SPSS for Windows version 28 (IBM, Armonk, NY, USA) was used for all statistical analyses. Declarations Competing Interests The authors declare no competing financial interests. Acknowledgements This work was supported by Japan Society for the Promotion of Science KAKENHI (Grant Number 23K08112). This research was partly performed at the Tottori Bio Frontier managed by Tottori prefecture. We thank the staff of the tissue analysis section, Technical Department, Tottori University for technical assistance. We thank J. Iacona, Ph.D., from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. Author Contributions YS, TO, and HK designed the experiments and analyzed the data. YS, TO, and HU performed the experiments and contributed to the discussion. YS and HK wrote the manuscript. TY, ToS, TeS, YU, and YF contributed to data analysis and discussion. HK conceived and managed the project. All authors revised and edited the manuscript. Competing Interests The authors declare no competing financial interests. Data Availability Statement The datasets generated and analyzed during the current study are available in the DDBJ repository (accession number: DRA018313). References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021; 71:209–249. Kolbeinsson HM, Chandana S, Wright GP, Chung M. Pancreatic cancer: a review of current treatment and novel therapies. J Invest Surg. 2023; 36:2129884. Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin. 2024; 74:12–49. Bernardes de Jesus B, Blasco MA. Telomerase at the intersection of cancer and aging. Trends Genet. 2013; 29:513–520. Meyerson M, Counter CM, Eaton EN, Ellisen LW, Steiner P, Caddle SD, et al. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell. 1997; 90:785–795. Collins K, Mitchell JR. Telomerase in the human organism. Oncogene. 2002; 21:564–579. Shay JW, Bacchetti S. A survey of telomerase activity in human cancer. European journal of cancer (Oxford, England : 1990). 1997; 33:787–791. Flores I, Cayuela ML, Blasco MA. Effects of telomerase and telomere length on epidermal stem cell behavior. Science. 2005; 309:1253–1256. Hannen R, Bartsch JW. Essential roles of telomerase reverse transcriptase hTERT in cancer stemness and metastasis. FEBS Lett. 2018; 592:2023–2031. Liu Z, Li Q, Li K, Chen L, Li W, Hou M, et al. Telomerase reverse transcriptase promotes epithelial-mesenchymal transition and stem cell-like traits in cancer cells. Oncogene. 2013; 32:4203–4213. Ramlee MK, Wang J, Toh WX, Li S. Transcription regulation of the human telomerase reverse transcriptase (hTERT) gene. Genes (Basel). 2016; 7:50. Yuan X, Larsson C, Xu D. Mechanisms underlying the activation of TERT transcription and telomerase activity in human cancer: old actors and new players. Oncogene. 2019; 38:6172–6183. Baker DJ, Jin F, Jeganathan KB, van Deursen JM. Whole chromosome instability caused by Bub1 insufficiency drives tumorigenesis through tumor suppressor gene loss of heterozygosity. Cancer Cell. 2009; 16:475–486. Calhoun ES, Hucl T, Gallmeier E, West KM, Arking DE, Maitra A, et al. Identifying allelic loss and homozygous deletions in pancreatic cancer without matched normals using high-density single-nucleotide polymorphism arrays. Cancer Res. 2006; 66:7920–7928. Hahn SA, Schutte M, Hoque AT, Moskaluk CA, da Costa LT, Rozenblum E, et al. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science. 1996; 271:350–353. Mahlamäki EH, Höglund M, Gorunova L, Karhu R, Dawiskiba S, Andrén-Sandberg A, et al. Comparative genomic hybridization reveals frequent gains of 20q, 8q, 11q, 12p, and 17q, and losses of 18q, 9p, and 15q in pancreatic cancer. Genes Chromosomes Cancer. 1997; 20:383–391. Gnarra JR, Tory K, Weng Y, Schmidt L, Wei MH, Li H, et al. Mutations of the VHL tumour suppressor gene in renal carcinoma. Nat Genet. 1994; 7:85–90. Agathanggelou A, Cooper WN, Latif F. Role of the Ras-association domain family 1 tumor suppressor gene in human cancers. Cancer Res. 2005; 65:3497–3508. Ji L, Fang B, Yen N, Fong K, Minna JD, Roth JA. Induction of apoptosis and inhibition of tumorigenicity and tumor growth by adenovirus vector-mediated fragile histidine triad (FHIT) gene overexpression. Cancer Res. 1999; 59:3333–3339. Dallol A, Forgacs E, Martinez A, Sekido Y, Walker R, Kishida T, et al. Tumour specific promoter region methylation of the human homologue of the Drosophila Roundabout gene DUTT1 (ROBO1) in human cancers. Oncogene. 2002; 21:3020–3028. Yagyu T, Ohira T, Shimizu R, Morimoto M, Murakami Y, Hanaki T, et al. Human chromosome 3p21.3 carries TERT transcriptional regulators in pancreatic cancer. Sci Rep. 2021; 11:15355. Kazuki Y, Hoshiya H, Takiguchi M, Abe S, Iida Y, Osaki M, et al. Refined human artificial chromosome vectors for gene therapy and animal transgenesis. Gene Ther. 2011; 18:384–393. Kouprina N, Larionov V. Recent advances in chromosome engineering. Chromosome Res. 2015; 23:1–5. Uno N, Abe S, Oshimura M, Kazuki Y. Combinations of chromosome transfer and genome editing for the development of cell/animal models of human disease and humanized animal models. J Hum Genet. 2018; 63:145–156. Fournier RE, Ruddle FH. Microcell-mediated transfer of murine chromosomes into mouse, Chinese hamster, and human somatic cells. Proc Natl Acad Sci U S A. 1977; 74:319–323. Ohira T, Yoshimura K, Kugoh H. Human artificial chromosome carrying 3p21.3-p22.2 region suppresses hTERT transcription in oral cancer cells. Chromosome Res. 2023; 31:17. Ong YS, Tran TH, Gounko NV, Hong W. TMEM115 is an integral membrane protein of the Golgi complex involved in retrograde transport. J Cell Sci. 2014; 127:2825–2839. Ivanova AV, Vortmeyer A, Ivanov SV, Nickerson ML, Maher ER, Lerman MI. Loss of PL6 protein expression in renal clear cell carcinomas and other VHL-deficient tumours. J Pathol. 2008; 214:46–57. Park YJ, Kim EK, Bae JY, Moon S, Kim J. Human telomerase reverse transcriptase (hTERT) promotes cancer invasion by modulating cathepsin D via early growth response (EGR)-1. Cancer Lett. 2016; 370:222–231. Carboni GL, Gao B, Nishizaki M, Xu K, Minna JD, Roth JA, et al. CACNA2D2-mediated apoptosis in NSCLC cells is associated with alterations of the intracellular calcium signaling and disruption of mitochondria membrane integrity. Oncogene. 2003; 22:615–626. The Human Protein Atlas. https://www.proteinatlas.org/. Basturk O, Saka B, Balci S, Postlewait LM, Knight J, Goodman M, et al. Substaging of lymph node status in resected pancreatic ductal adenocarcinoma has strong prognostic correlations: proposal for a revised N classification for TNM staging. Ann Surg Oncol. 2015; 22:S1187–S1195. Strobel O, Hinz U, Gluth A, Hank T, Hackert T, Bergmann F, et al. Pancreatic adenocarcinoma: number of positive nodes allows to distinguish several N categories. Ann Surg. 2015; 261:961–969. Kugoh H, Ohira T, Oshimura M. Studies of tumor suppressor genes via chromosome engineering. Cancers (Basel). 2015; 8:4. Qi DL, Ohhira T, Fujisaki C, Inoue T, Ohta T, Osaki M, et al. Identification of PITX1 as a TERT suppressor gene located on human chromosome 5. Mol Cell Biol. 2011; 31:1624–1636. Ohira T, Naohiro S, Nakayama Y, Osaki M, Okada F, Oshimura M, et al. miR-19b regulates hTERT mRNA expression through targeting PITX1 mRNA in melanoma cells. Sci Rep. 2015; 5:8201. Kazuki Y, Kobayashi K, Aueviriyavit S, Oshima T, Kuroiwa Y, Tsukazaki Y, et al. Trans-chromosomic mice containing a human CYP3A cluster for prediction of xenobiotic metabolism in humans. Hum Mol Genet. 2013; 22:578–592. Hung J, Kishimoto Y, Sugio K, Virmani A, McIntire DD, Minna JD, et al. Allele-specific chromosome 3p deletions occur at an early stage in the pathogenesis of lung carcinoma. JAMA. 1995; 273:1908. Kok K, Naylor SL, Buys CH. Deletions of the short arm of chromosome 3 in solid tumors and the search for suppressor genes. Adv Cancer Res. 1997; 71:27–92. Hesson LB, Cooper WN, Latif F. Evaluation of the 3p21.3 tumour-suppressor gene cluster. Oncogene. 2007; 26:7283–7301. Agathanggelou A, Dallol A, Zöchbauer-Müller S, Morrissey C, Honorio S, Hesson L, et al. Epigenetic inactivation of the candidate 3p21.3 suppressor gene BLU in human cancers. Oncogene. 2003; 22:1580–1588. Sharp TV, Al-Attar A, Foxler DE, Ding L, de AVTQ, Zhang Y, et al. The chromosome 3p21.3-encoded gene, LIMD1, is a critical tumor suppressor involved in human lung cancer development. Proc Natl Acad Sci U S A. 2008; 105:19932–19937. Li J, Wang F, Haraldson K, Protopopov A, Duh FM, Geil L, et al. Functional characterization of the candidate tumor suppressor gene NPRL2/G21 located in 3p21.3C. Cancer Res. 2004; 64:6438–6443. Abe S, Tanaka H, Notsu T, Horike S, Fujisaki C, Qi DL, et al. Localization of an hTERT repressor region on human chromosome 3p21.3 using chromosome engineering. Genome Integr. 2010; 1:6. Nishio S, Ohira T, Sunamura N, Oshimura M, Ryoke K, Kugoh H. Repression of hTERT transcription by the introduction of chromosome 3 into human oral squamous cell carcinoma. Biochem Biophys Res Commun. 2015; 466:755–759. Gomes NM, Ryder OA, Houck ML, Charter SJ, Walker W, Forsyth NR, et al. Comparative biology of mammalian telomeres: hypotheses on ancestral states and the roles of telomeres in longevity determination. Aging Cell. 2011; 10:761–768. Guterres AN, Villanueva J. Targeting telomerase for cancer therapy. Oncogene. 2020; 39:5811–5824. Shou S, Li Y, Chen J, Zhang X, Zhang C, Jiang X, et al. Understanding, diagnosing, and treating pancreatic cancer from the perspective of telomeres and telomerase. Cancer Gene Ther. 2024; 31:1292–1305. Hashimoto Y, Murakami Y, Uemura K, Hayashidani Y, Sudo T, Ohge H, et al. Telomere shortening and telomerase expression during multistage carcinogenesis of intraductal papillary mucinous neoplasms of the pancreas. J Gastrointest Surg. 2008; 12:17–28; discussion 28–29. Ma Z, Vocadlo DJ, Vosseller K. Hyper-O-GlcNAcylation is anti-apoptotic and maintains constitutive NF-κB activity in pancreatic cancer cells. J Biol Chem. 2013; 288:15121–15130. Caldwell SA, Jackson SR, Shahriari KS, Lynch TP, Sethi G, Walker S, et al. Nutrient sensor O-GlcNAc transferase regulates breast cancer tumorigenesis through targeting of the oncogenic transcription factor FoxM1. Oncogene. 2010; 29:2831–2842. Mi W, Gu Y, Han C, Liu H, Fan Q, Zhang X, et al. O-GlcNAcylation is a novel regulator of lung and colon cancer malignancy. Biochim Biophys Acta. 2011; 1812:514–519. Makwana V, Ryan P, Patel B, Dukie SA, Rudrawar S. Essential role of O-GlcNAcylation in stabilization of oncogenic factors. Biochim Biophys Acta Gen Subj. 2019; 1863:1302–1317. Singh JP, Zhang K, Wu J, Yang X. O-GlcNAc signaling in cancer metabolism and epigenetics. Cancer Lett. 2015; 356:244–250. Tables Tables are available in the Supplementary Files section. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files Supplementaryinformation.pdf Supplementary information table1.xlsx table2.xlsx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6170448","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":428452734,"identity":"c193a71f-7a0f-4a60-818f-784502bbbd8f","order_by":0,"name":"Hiroyuki Kugoh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYBACCSBmZqiA8hKgtAFhLWcMGHjYSNLC2AbVQhSQnJF+8XHhvD9y9vLNxz48YDjMwN9+gKG4AI8WaYmcYuOZ2wyMedjYkmckALVInElgMJ6BR4ucRE6aNO82g8QeNh5jhsR/hxkYbjAwGPMQ1DIHqgVkizwhLdIS6cekeRuQtBgQ0iLZ84bZmOeYMRCnJQO1pPMYnklswOsXiePpDx/z1MjJsTcfPsz4g8FaTu744WPG+EKMgYEHNd6ATmJsM8arg4H9AYYQ82P8WkbBKBgFo2CEAQDOuT1n0+6KCQAAAABJRU5ErkJggg==","orcid":"","institution":"Tottori University","correspondingAuthor":true,"prefix":"","firstName":"Hiroyuki","middleName":"","lastName":"Kugoh","suffix":""},{"id":428452735,"identity":"681281cd-ebe6-4199-b163-56c7f0325fcc","order_by":1,"name":"Yu Sakano","email":"","orcid":"https://orcid.org/0009-0000-7871-8221","institution":"Tottori University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Sakano","suffix":""},{"id":428452736,"identity":"c8962c4d-1c1e-426f-8102-e17c656896aa","order_by":2,"name":"Takahito Ohira","email":"","orcid":"","institution":"Tottori University","correspondingAuthor":false,"prefix":"","firstName":"Takahito","middleName":"","lastName":"Ohira","suffix":""},{"id":428452737,"identity":"0b5c43b6-c311-4d03-9277-5f0d917bd990","order_by":3,"name":"Haruka Ui","email":"","orcid":"","institution":"Tottori University","correspondingAuthor":false,"prefix":"","firstName":"Haruka","middleName":"","lastName":"Ui","suffix":""},{"id":428452738,"identity":"31126bdd-10b6-483c-a646-b49f4322005e","order_by":4,"name":"Takuki Yagyu","email":"","orcid":"","institution":"Tottori University","correspondingAuthor":false,"prefix":"","firstName":"Takuki","middleName":"","lastName":"Yagyu","suffix":""},{"id":428452739,"identity":"6cd8961d-373f-465f-a608-bd524c9c62f3","order_by":5,"name":"Tomohiko Sakabe","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Tomohiko","middleName":"","lastName":"Sakabe","suffix":""},{"id":428452740,"identity":"cd9b8a88-bfbb-4093-bfee-85911b0ad119","order_by":6,"name":"Teruhisa Sakamoto","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Teruhisa","middleName":"","lastName":"Sakamoto","suffix":""},{"id":428452741,"identity":"2085cfe7-e353-4c1d-9812-d81af79b1f69","order_by":7,"name":"Yoshihisa Umekita","email":"","orcid":"https://orcid.org/0000-0002-2846-390X","institution":"Tottori University, 86 Nishicho, Yonago, Tottori","correspondingAuthor":false,"prefix":"","firstName":"Yoshihisa","middleName":"","lastName":"Umekita","suffix":""},{"id":428452742,"identity":"24d746f7-f6ca-4bb6-8b2a-45951329d26b","order_by":8,"name":"Yoshiyuki Fujiwara","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yoshiyuki","middleName":"","lastName":"Fujiwara","suffix":""}],"badges":[],"createdAt":"2025-03-06 12:20:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6170448/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6170448/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83166996,"identity":"4c3cf4af-eb30-44ff-8846-2b694a190181","added_by":"auto","created_at":"2025-05-20 16:23:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":806417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuppression of mouse telomerase reverse transcriptase (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003emTert\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) expression by introducing a human artificial chromosome (HAC) with only the 3p21.3 region into murine LTPA pancreatic cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFISH analysis of the (A) LTPA control cells and (B) LTPA 3p21.3-HAC cells. Rhodamine-labeled human COT-1 DNA and FITC-labeled 234N4 PAC were used as probe DNA. The gray arrowhead indicates the HAC2 with only the rhodamine signal. The orange arrowhead indicates 3p21.3-HAC, which has both rhodamine and FITC signals. (C) Quantitative RT-PCR analysis of relative\u003cem\u003e mTert\u003c/em\u003e mRNA expression levels in the LTPA control and LTPA 3p21.3-HAC cells. The \u003cem\u003emGapdh\u003c/em\u003emRNA expression levels were used as the internal control. Data are presented as the mean ± standard deviation of three independent experiments (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6170448/v1/f180aaee15b51a65aa699fc8.png"},{"id":83166407,"identity":"20c9edc3-75b7-4b84-8d76-cf0056651289","added_by":"auto","created_at":"2025-05-20 16:15:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1300414,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of cell proliferation and invasion by introducing 3p21.3-HAC into LTPA pancreatic cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Cell numbers for the LTPA control and LTPA 3p21.3-HAC cells over a 5-day period. The bars correspond to the mean ± standard deviation (SD) of three independent experiments (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). Representative images of the invasion assay for the (B) LTPA control and (C) LTPA 3p21.3-HAC cells. Scale bar = 200 µm. (D) Counts of invading cells per five microscopic fields for the LTPA control and LTPA 3p21.3-HAC cells. The bars correspond to the mean ± SD of three independent experiments (***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6170448/v1/96645346a2b9094abea95676.png"},{"id":83166403,"identity":"45b6dfc5-f95c-4737-8698-a27f3eca1659","added_by":"auto","created_at":"2025-05-20 16:15:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":325166,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of transmembrane protein 115\u003c/strong\u003e \u003cstrong\u003e(\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTMEM115\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) as a mouse telomerase reverse transcriptase (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003emTert\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003esuppressor gene in the 3p21.3 region.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Flow chart of our screening strategy. Gene expression patterns in the LTPA control and LTPA 3p21.3-HAC cells were analyzed by RNA sequencing analysis. Overall, 26 genes were identified with expression levels that were significantly upregulated. The relationships between the expression patterns of these 26 genes and \u003cem\u003ehTERT \u003c/em\u003eexpression levels in pancreatic cancer patients in The Cancer Genome Atlas database were examined. Further work focused on seven genes with r values lower than -0.15. (B) Quantitative RT-PCR analysis of the relative \u003cem\u003eTMEM115 \u003c/em\u003emRNA expression levels in the LTPA 3p21.3-HAC cells transfected with the negative control small interfering RNA (siRNA) or \u003cem\u003eTMEM115\u003c/em\u003esiRNA 1 or 2. (C) Quantitative RT-PCR analysis of the relative \u003cem\u003emTert \u003c/em\u003emRNA expression levels in these cells. The \u003cem\u003emGapdh\u003c/em\u003e mRNA expression levels were used as the internal control. Data are presented as the mean ± standard deviation of three independent experiments (***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6170448/v1/92ee89660d38a3365123021f.png"},{"id":83167707,"identity":"4411482a-5da9-4aa3-8b82-ff459dd2f4f8","added_by":"auto","created_at":"2025-05-20 16:31:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1652585,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuppression of mouse telomerase reverse transcriptase (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003emTert\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression and inhibition of cell proliferation and invasion by transmembrane protein 115\u003c/strong\u003e (\u003cem\u003e\u003cstrong\u003eTMEM115\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) overexpression in LTPA cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot analysis of the TMEM115 protein expression levels in the LTPA control and LTPA \u003cem\u003eTMEM115\u003c/em\u003e overexpression cells. The TMEM115 expression levels were normalized to those of α-tubulin. (B) Quantitative RT-PCR analysis of the relative \u003cem\u003emTert\u003c/em\u003e mRNA expression levels in these clones. The \u003cem\u003emGapdh \u003c/em\u003emRNA expression levels were used as the internal control. Data are presented as the mean ± standard deviation (SD) of three independent experiments (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). (C) Cell numbers for the LTPA control and LTPA \u003cem\u003eTMEM115\u003c/em\u003e cells over a 5-day period. The bars correspond to the mean ± SD of three independent experiments (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). Representative images of the invasion assay for the (D) LTPA control and (E) LTPA \u003cem\u003eTMEM115\u003c/em\u003ecells. Scale bar = 200 µm. (F) Counts of invading cells per five microscopic fields for the LTPA control and LTPA \u003cem\u003eTMEM115 \u003c/em\u003ecells. The bars correspond to the mean ± SD of three independent experiments (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6170448/v1/6318b3034de4b1b5633dc98e.png"},{"id":83167001,"identity":"b02d22e0-2637-43e8-a83d-dc4538d85e4c","added_by":"auto","created_at":"2025-05-20 16:23:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2069368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuppression of human telomerase reverse transcriptase\u003c/strong\u003e \u003cstrong\u003e(\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehTERT\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression by transmembrane protein 115 (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTMEM115\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) overexpression in human pancreatic cancer cell lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot analysis of the TMEM115 protein expression levels in MIA PaCa-2 control and MIA PaCa-2 \u003cem\u003eTMEM115\u003c/em\u003e overexpression cells. The TMEM115 expression levels were normalized to those of α-tubulin. (B) Quantitative RT-PCR analysis of the relative \u003cem\u003ehTERT\u003c/em\u003e mRNA expression levels in these clones. The \u003cem\u003eGAPDH \u003c/em\u003emRNA expression levels were used as the internal control. Data are presented as the mean ± standard deviation (SD) of three independent experiments (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). (C) Cell numbers for the MIA PaCa-2 control and MIA PaCa-2 \u003cem\u003eTMEM115\u003c/em\u003e cells over a 5-day period. The bars correspond to the mean ± SD of three independent experiments (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). Representative images of the invasion assay for the (D) MIA PaCa-2 control and (E) MIA PaCa-2 \u003cem\u003eTMEM115\u003c/em\u003e cells. Scale bar = 200 µm. (F) Counts of invading cells per five microscopic fields for the MIA PaCa-2 control and MIA PaCa-2 \u003cem\u003eTMEM115\u003c/em\u003ecells. The bars correspond to the mean ± SD of three independent experiments (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6170448/v1/1eab2459e11e9cb965d90e60.png"},{"id":83166413,"identity":"7d20961f-2c6b-42c1-a0da-4ed3808a5b23","added_by":"auto","created_at":"2025-05-20 16:15:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4168177,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLow transmembrane protein 115\u003c/strong\u003e(\u003cstrong\u003eTMEM115) expression is a poor prognostic factor in pancreatic cancer (PC) patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Overall survival (OS) curves of PC patients with high (n = 116) and low (n = 65) \u003cem\u003eTMEM115\u003c/em\u003e mRNA expression levels from The Cancer Genome Atlas database. (B) Image of an islet of Langerhans cells in noncancerous pancreatic tissue used as an internal positive control. Representative TMEM115 immunohistochemistry images of PC patient tissue samples from the (C) TMEM115 high group and (D) TMEM115 low group. Scale bar = 100 µm. Survival curves of PC patients with high (n = 78) and low (n = 22) TMEM115 expression for (E) OS, (F) disease-specific survival, and (G) recurrence-free survival.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6170448/v1/3b9bef3b058de6b586f0ecbd.png"},{"id":83168594,"identity":"0979f400-ce34-4e72-9349-2eccef7baf76","added_by":"auto","created_at":"2025-05-20 16:39:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14298988,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6170448/v1/6f29b3d2-2d3a-44d5-a4e4-b10f0d07e570.pdf"},{"id":83166994,"identity":"d5412f68-cd1c-41c8-b174-7940ea69ea46","added_by":"auto","created_at":"2025-05-20 16:23:21","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":355886,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"Supplementaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6170448/v1/5309712ccfdf7636c3f359a5.pdf"},{"id":83167706,"identity":"17415862-bcc7-4280-99ca-255f5e927573","added_by":"auto","created_at":"2025-05-20 16:31:21","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":22454,"visible":true,"origin":"","legend":"","description":"","filename":"table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6170448/v1/f2284e12db99a7da7c60ddc6.xlsx"},{"id":83166411,"identity":"45b624bd-d21a-4525-b9ee-ad4fb2379208","added_by":"auto","created_at":"2025-05-20 16:15:21","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14410,"visible":true,"origin":"","legend":"","description":"","filename":"table2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6170448/v1/6e0591413b65d0ca97792cec.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"\u003cp\u003eIdentification of \u003cem\u003eTMEM115\u003c/em\u003e as a tumor suppressor gene through \u003cem\u003eTERT\u003c/em\u003e regulation in pancreatic cancer\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePancreatic cancer (PC) is one of the most lethal malignant diseases. According to the 2020 World Cancer Statistics, PC is the seventh leading cause of cancer-related deaths worldwide for both men and women, with approximately 496 000 new cases and 466 000 deaths [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This disease is characterized by late diagnosis from insidious onset, early metastasis, limited response to chemotherapy, and poor prognosis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite recent improvements in therapeutic strategies, the difficulties associated with early detection have resulted in PC patients often being diagnosed at an advanced stage or with distant metastases and a 5-year survival rate of less than 10% [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Because the molecular mechanisms involved in PC progression remain unclear, further research is urgently required to identify novel diagnostic biomarkers and therapeutic targets for this disease and develop new treatment strategies for patients.\u003c/p\u003e \u003cp\u003eTelomerase is a ribonucleoprotein ribonucleic enzyme that catalyzes the elongation of telomere repeat sequences at the end of chromosomes, contributing to cellular immortalization [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The catalytic component of telomerase, telomerase reverse transcriptase (\u003cem\u003eTERT\u003c/em\u003e), is stringently repressed in most human somatic cells, consequently resulting in telomerase inactivation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, telomerase is activated in most cancer cells, including PC, and supports the unlimited proliferative ability and tumorigenicity of malignant tumor cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Recent studies have revealed that, independently of its telomere-lengthening function, \u003cem\u003eTERT\u003c/em\u003e can stimulate the epithelial-mesenchymal transition and stemness of cancer cells, indicating that \u003cem\u003eTERT\u003c/em\u003e also contributes to cancer invasiveness and metastasis [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Although it is known that human \u003cem\u003eTERT\u003c/em\u003e (h\u003cem\u003eTERT\u003c/em\u003e) expression is strictly regulated by multiple transcriptional activators and repressors, as well as epigenetic modifications, the exact regulatory mechanism has not been completely elucidated [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLoss of heterozygosity (LOH) is a common genetic alteration that occurs in cancer genomes, with LOH of tumor suppressor genes serving as a significant factor that promotes cancer progression [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. LOH at the 3p, 9p, 17p, and 18q chromosomes are highly prevalent somatic genetic regions in PC. The tumor suppressor genes cyclin-dependent kinase inhibitor 2A (\u003cem\u003eCDKN2A\u003c/em\u003e), tumor protein 53 (\u003cem\u003eTP53\u003c/em\u003e), and suppressor of mothers against decapentaplegic homolog 4 (\u003cem\u003eSMAD4\u003c/em\u003e) have been identified in the 9p, 17p, and 18q LOH regions, respectively [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Chromosome 3p has also been shown to contain many tumor suppressor genes, such as von Hippel-Lindau (\u003cem\u003eVHL\u003c/em\u003e), Ras association domain family member 1 (\u003cem\u003eRASSF1\u003c/em\u003e), fragile histidine triad (\u003cem\u003eFHIT\u003c/em\u003e), and roundabout guidance receptor 1 (\u003cem\u003eROBO1\u003c/em\u003e), but no tumor suppressor genes have been identified that are involved in the regulation of \u003cem\u003eTERT\u003c/em\u003e [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In a previous study, we reported that introducing normal human chromosome 3 using microcell-mediated chromosome transfer (MMCT) could induce the inhibition of mouse \u003cem\u003eTert\u003c/em\u003e (\u003cem\u003emTert\u003c/em\u003e) transcription in murine LTPA PC cells. Furthermore, we revealed that the 3p21.3 region may encode the \u003cem\u003emTert\u003c/em\u003e repressor gene(s) by transferring chromosomes that were truncated at arbitrary regions into LTPA cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHuman artificial chromosomes (HACs) are mini-chromosome vectors with the endogenous genes removed from a normal human chromosome 21 using chromosome engineering techniques [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. HACs are useful gene delivery vectors because they exist stably and independently in host cells and can carry an unlimited number of genome sizes, having the great advantage of facilitating genome functional analysis under more physiologically relevant conditions [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In addition, HAC vectors can be transferred into any cell line via MMCT [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. We have previously demonstrated the presence of a \u003cem\u003eTERT\u003c/em\u003e suppressor gene (TSG) through the functional analysis of a HAC carrying the 3p21.3 region in oral squamous cell carcinoma cells [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTransmembrane protein 115 (TMEM115), which is encoded by a gene in the 3p21.3 chromosome, is a multi-transmembrane domain protein that is localized at the medial and trans-Golgi cisternae [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. TMEM115 helps regulate retrograde transport from the Golgi to the endoplasmic reticulum. TMEM115 is highly expressed in a wide range of epithelial cells in normal tissues, including the gastrointestinal, respiratory, genitourinary, and breast systems. Although loss of TMEM115 expression has been reported in renal clear cell carcinoma and other VHL-deficient tumors, its involvement in oncogenesis is unclear [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we identified \u003cem\u003eTMEM115\u003c/em\u003e as a novel TSG on the 3p21.3 region in PC through functional analysis using a HAC. Furthermore, we revealed that TMEM115 expression levels are positively correlated with PC patient prognosis. Our results provide important information that supports \u003cem\u003eTMEM115\u003c/em\u003e as a new potential therapeutic target in PC.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eSuppression of\u003c/b\u003e \u003cb\u003emTert\u003c/b\u003e \u003cb\u003eexpression by introducing a HAC with only the 3p21.3 region into murine LTPA PC cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the presence of \u003cem\u003eTERT\u003c/em\u003e regulatory factors within the 3p21.3 region in PC cells, we used MMCT to establish LTPA microcell hybrid clones containing a HAC without any gene coding regions (empty-HAC; LTPA control) or a HAC with only the 3p21.3 region (LTPA 3p21.3-HAC). To confirm the presence of each transferred HAC in the LTPA calls, we performed two-color fluorescence in situ hybridization (FISH) analysis using human COT-1 DNA (rhodamine) and 234N4 PAC (FITC) located at the 3p21.3 region as probes. The results showed that only the rhodamine signal was detected in the LTPA control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), while both the rhodamine and FITC signals were detected in the LTPA 3p21.3-HAC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This suggested that we obtained microcell hybrid clones with empty-HAC and 3p21.3-HAC that segregated as an independent chromosome in LTPA cells. Next, we investigated the \u003cem\u003emTert\u003c/em\u003e mRNA expression patterns in the LTPA control and LTPA 3p21.3-HAC hybrid cells using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The LTPA 3p21.3-HAC microcell hybrid cells exhibited significantly lower \u003cem\u003emTert\u003c/em\u003e mRNA expression levels compared with the control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These results indicated the presence of a gene or genes on human chromosome 3p21.3 that can negatively regulate \u003cem\u003emTert\u003c/em\u003e transcription, which may play an important role in PC development and progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eInhibition of cell proliferation and invasion after introducing 3p21.3-HAC into LTPA PC cells\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eNext, we examined if introducing the 3p21.3 region into the LTPA cells affected cell proliferation. LTPA microcell hybrids with the 3p21.3 region displayed significantly reduced cell proliferation rates compared with the LTPA control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In addition, telomerase activity has been reported to contribute to cell invasion and metastasis by activating the epithelial-mesenchymal transition [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, we examined the cell invasion potential of the LTPA control and LTPA 3p21.3-HAC cells. The LTPA 3p21.3-HAC cells showed a significantly reduced invasive ability compared with the control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;D). These results demonstrated that the reduced \u003cem\u003emTert\u003c/em\u003e transcription induced by introducing the 3p21.3 region was accompanied by suppressive effects on PC cell proliferation and invasion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA sequencing (RNA-seq) analysis of LTPA microcell hybrids to identify\u003c/b\u003e \u003cb\u003emTert\u003c/b\u003e \u003cb\u003erepressor genes in the 3p21.3 region\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo identify the specific \u003cem\u003emTert\u003c/em\u003e suppressor gene suggested to be present in the 3p21.3 region, we comprehensively examined the gene expression patterns of the LTPA 3p21.3-HAC and LTPA control cells by RNA-seq analysis. Relative to the LTPA control cells, the expression levels of 26 genes encoded in the 3p21.3 region were significantly upregulated in the LTPA 3p21.3-HAC cells with repressed \u003cem\u003emTert\u003c/em\u003e transcription (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Seven of these genes, \u003cem\u003eTMEM115\u003c/em\u003e, calcium voltage-gated channel auxiliary subunit alpha2 delta 2 (\u003cem\u003eCACNA2D2\u003c/em\u003e), inositol hexakisphosphate kinase 1 (\u003cem\u003eIP6K1\u003c/em\u003e), G protein subunit alpha i2 (\u003cem\u003eGNAI2\u003c/em\u003e), interferon related developmental regulator 2 (\u003cem\u003eIFRD2\u003c/em\u003e), solute carrier family 38 member 3 (\u003cem\u003eSLC38A3\u003c/em\u003e), and zinc finger MYND-type containing 10 (\u003cem\u003eZMYND10\u003c/em\u003e), were found to be downregulated in The Cancer Genome Atlas (TCGA) PC dataset of patients in whom \u003cem\u003ehTERT\u003c/em\u003e is expressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next individually knocked down each of these seven genes using two specific small interfering RNAs (siRNAs) to identify any relevant \u003cem\u003emTert\u003c/em\u003e regulatory genes. The LTPA 3p21.3-HAC cells treated with the siRNAs targeting these genes displayed a 0.16-fold (\u003cem\u003eZMYND10\u003c/em\u003e siRNA2) to 0.74-fold (\u003cem\u003eSLC38A3\u003c/em\u003e siRNA1) decrease in target mRNA expression levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and S1). As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and S1, subsequent examination of the effects of knocking down these genes on \u003cem\u003emTert\u003c/em\u003e transcription patterns revealed upregulation of \u003cem\u003eTMEM115\u003c/em\u003e and \u003cem\u003eCACNA2D2\u003c/em\u003e expression levels.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCACNA2D2\u003c/em\u003e, which encodes the α2δ2 auxiliary subunit of the voltage-activated calcium channel protein complex, is abundantly expressed in lung and brain tissues and has been shown to be a tumor suppressor gene that can induce apoptosis in non-small cell lung cancer cells [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Analysis of the human protein atlas (HPA) database showed that CACNA2D2 is expressed at very low levels in normal pancreatic tissues. Thus, no difference in CACNA2D2 gene expression patterns was found between PC and non-carcinoma tissues. However, \u003cem\u003eTMEM115\u003c/em\u003e, an endogenous membrane protein in the Golgi apparatus, is nonspecifically expressed in multiple organs, including the pancreas. Protein expression data from the HPA database indicated significantly decreased TMEM115 expression levels in PC tissues compared with the levels in normal tissues [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Thus, these findings suggested that \u003cem\u003eTMEM115\u003c/em\u003e was more likely to act as a TSG in PC, leading us to focus our further work on \u003cem\u003eTMEM115\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTMEM115\u003c/b\u003e \u003cb\u003einhibits\u003c/b\u003e \u003cb\u003emTert\u003c/b\u003e \u003cb\u003etranscription, cell proliferation, and cell invasion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the \u003cem\u003eTMEM115\u003c/em\u003e-mediated suppressive effects on \u003cem\u003emTert\u003c/em\u003e transcription, we established \u003cem\u003eTMEM115\u003c/em\u003e stable expression clones by transfecting the pCMV6 vector carrying the \u003cem\u003eTMEM115\u003c/em\u003e gene into LTPA cells. Higher TMEM115 expression levels were observed in all transfectant clones compared with those observed in the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, the cells expressing \u003cem\u003eTMEM115\u003c/em\u003e (LTPA \u003cem\u003eTMEM115\u003c/em\u003e) showed remarkably repressed \u003cem\u003emTert\u003c/em\u003e expression levels compared with the cells transfected with the empty vector. Furthermore, the cell proliferation and invasion rates were suppressed in the LTPA \u003cem\u003eTMEM115\u003c/em\u003e cells compared with the control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026ndash;F). Interestingly, the reduced \u003cem\u003emTert\u003c/em\u003e expression levels and cell proliferation and invasion rates in the LTPA \u003cem\u003eTMEM115\u003c/em\u003e clones were similar to those observed in the LTPA cells that received the normal human chromosome 3 or 3p21.3-HAC ([\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These data strongly implicated \u003cem\u003eTMEM115\u003c/em\u003e as the gene responsible for the observed \u003cem\u003emTert\u003c/em\u003e suppression resulting from introducing the 3p21.3 region.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSuppression of\u003c/b\u003e \u003cb\u003ehTERT\u003c/b\u003e \u003cb\u003eexpression by overexpressing\u003c/b\u003e \u003cb\u003eTMEM115\u003c/b\u003e \u003cb\u003ein human PC cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNext, we aimed to clarify if \u003cem\u003eTMEM115\u003c/em\u003e, which could repress \u003cem\u003emTert\u003c/em\u003e transcription in murine PC cells, has similar functions in human PC cells. We established human MIA PaCa-2 PC cells overexpressing \u003cem\u003eTMEM115\u003c/em\u003e using the pCMV6 vector carrying the \u003cem\u003eTMEM115\u003c/em\u003e gene. The empty pCMV6 vector was used as a negative control. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, TMEM115 expression was detected at the protein level using western blot analysis. Furthermore, qRT-PCR analysis indicated that the MIA PaCa-2 cells with \u003cem\u003eTMEM115\u003c/em\u003e overexpression displayed significantly suppressed \u003cem\u003ehTERT\u003c/em\u003e transcription compared with the control cells with the empty vector (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The MIA PaCa-2 cells with \u003cem\u003eTMEM115\u003c/em\u003e overexpression also showed decreased cell proliferation and invasion rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;F). Interestingly, the \u003cem\u003eTERT\u003c/em\u003e expression levels were 1.9-fold more strongly suppressed in the human PC cells than in the murine cells following \u003cem\u003eTMEM115\u003c/em\u003e overexpression, with a corresponding greater suppression of the cell proliferative and invasive capacities (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This consequentially provided crucial evidence that \u003cem\u003eTMEM115\u003c/em\u003e acts as a \u003cem\u003eTERT\u003c/em\u003e repressor gene in both mouse and human PC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLow TMEM115 expression is a poor prognostic factor in PC patients\u003c/h3\u003e\n\u003cp\u003eUsing the TCGA database, we analyzed the association between \u003cem\u003eTMEM115\u003c/em\u003e mRNA expression levels and survival time in 181 PC patients to elucidate the clinical impact of this gene. The receiver operating characteristic curve analysis for overall survival (OS) revealed the optimal cutoff value for \u003cem\u003eTMEM115\u003c/em\u003e expression (Fig. S2), which was used to divide the patients into high (\u003cem\u003eTMEM115\u003c/em\u003e-high, n\u0026thinsp;=\u0026thinsp;116) and low (\u003cem\u003eTMEM115\u003c/em\u003e-low, n\u0026thinsp;=\u0026thinsp;65) \u003cem\u003eTMEM115\u003c/em\u003e expression groups. Kaplan-Meier survival analysis demonstrated that the \u003cem\u003eTMEM115\u003c/em\u003e-low patients had significantly shorter OS than the \u003cem\u003eTMEM115\u003c/em\u003e-high patients (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, we used immunohistochemistry (IHC) assays to examine tissues samples from 100 PC patients who underwent pancreatectomy to determine the prognostic significance of TMEM115 expression in these individuals. TMEM115 was expressed generally without cell specificity in the pancreatic tissues, with the islets of Langerhans cells, which displayed a more consistent expression intensity in all cases, used as internal positive controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Positive cells were defined as those with a TMEM115 staining intensity equal to or greater than that of the islets of Langerhans cells in noncancerous pancreatic tissues. Tumors with \u0026lt;\u0026thinsp;50% positive cells were classified as low TMEM115 expression. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC/D, IHC analysis of the PC tissue samples indicated low TMEM115 expression in 22 of the 100 samples. When evaluating the relationships between TMEM115 expression and clinicopathological factors, the local tumor progression, specifically the pathological T (pT) stage, was found to be significantly higher in the PC patients with low TMEM115 expression than in those with high TMEM115 expression (Table\u0026nbsp;1). Kaplan-Meier survival analysis examining the relationship between TMEM115 expression and prognosis showed that the OS, disease-specific survival, and recurrence-free survival rates were all significantly lower in the patients with low TMEM115 expression compared with those in patients with high TMEM115 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE\u0026ndash;G).\u003c/p\u003e \u003cp\u003eWe subsequently investigated if TMEM115 expression was an independent prognostic risk factor for PC in these patients. The univariate analysis indicated that the pathological N (pN) stage and levels of serum carcinoembryonic antigen, duodenal pancreatic cancer antigen 2, and TMEM115 expression were risk factors for OS after surgery in PC patients. The multivariate analysis using the Cox proportional hazards model revealed that the pN stage and TMEM115 expression levels were independent prognostic factors for PC patient OS (Table\u0026nbsp;2). The presence and number of metastatic lymph nodes are well-established strong prognostic factors for PC [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Therefore, identifying the pN stage as an independent prognostic factor in this analysis emphasizes the validity of the postoperative PC patient cohort used in this study. Overall, these results suggested that TMEM115 expression, identified as a prognostic factor along with pN stage, is an important indicator for PC patients.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe combination of chromosome transfer and gene expression profile analysis is an effective approach for identifying the genes responsible for recessive genetic diseases, including tumor suppressor genes [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In fact, we previously found that the paired-like homeodomain 1 (\u003cem\u003ePITX1\u003c/em\u003e) gene, which is encoded on chromosome 5, can suppress \u003cem\u003eTERT\u003c/em\u003e in melanoma, further analyzing the function of the gene using chromosome transfer technology [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Furthermore, the use of HAC vectors is considered a more effective approach to identify such genes for several reasons: 1) HAC vectors are not limited by the size of the target genomic region they can carry, 2) they are independently maintained in the host cells, and 3) they support gene functional analysis under physiological conditions using their own native promoters [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe 3p21.3 region has a high frequency of LOH in several carcinomas, including lung, renal, and gastrointestinal cancers, as well as PC, suggesting the presence of tumor suppressor genes in the region [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In fact, several genes, such as \u003cem\u003eRASSF1\u003c/em\u003e, \u003cem\u003eZMYND10\u003c/em\u003e, LIM domain containing 1 (\u003cem\u003eLIMD1\u003c/em\u003e), and nitrogen permease regulator like-2 (\u003cem\u003eNPRL2\u003c/em\u003e), have been reported as tumor suppressors [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. We previously reported that chromosome 3 is involved in tumor suppression through \u003cem\u003eTERT\u003c/em\u003e regulation in renal cell carcinoma and oral squamous cell carcinoma [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In addition, we recently suggested that the 3p21.3 region in PC may encode TSG(s) by functional analysis of truncated chromosome 3 fragments at arbitrary regions [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, no specific gene has been identified in this region that can regulate \u003cem\u003eTERT\u003c/em\u003e transcription. In the current study, we showed that \u003cem\u003eTMEM115\u003c/em\u003e encoded in the 3p21.3 region may be a novel TSG in PC by introducing HAC vectors carrying the 3p21.3 region and RNA-seq analysis. To the best of our knowledge, this is the first report to refer to \u003cem\u003eTMEM115\u003c/em\u003e as a potential tumor suppressor gene in PC. Furthermore, in other carcinomas where \u003cem\u003eTMEM115\u003c/em\u003e expression levels are significantly decreased in cancerous tissues compared with the levels in normal tissues, as in PC [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], \u003cem\u003eTMEM115\u003c/em\u003e dysfunction may involve tumorigenesis through \u003cem\u003eTERT\u003c/em\u003e regulation. This suggests that \u003cem\u003eTMEM115\u003c/em\u003e possibly plays an important role as a TSG not only in PC, but also in other cancer types.\u003c/p\u003e \u003cp\u003eIn this study, the suppressive effects on \u003cem\u003emTert\u003c/em\u003e transcription were comparable in both LTPA 3p21.3-HAC cells and \u003cem\u003eTMEM115\u003c/em\u003e-overexpressing LTPA cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). It is possible that there may be other important genes in the 3p21.3 region that cooperate with \u003cem\u003eTMEM115\u003c/em\u003e. The human MIA PaCa-2 cells overexpressing \u003cem\u003eTMEM115\u003c/em\u003e showed markedly reduced the \u003cem\u003ehTERT\u003c/em\u003e expression levels and cell proliferation and invasion rates compared with the murine LTPA cells. There are some significant differences in telomere regulation between human and mouse biology. Mouse telomere length is longer than that of humans, suggesting that telomere length does not significantly affect the regulation of mouse lifespan, which is much shorter relative to humans [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Therefore, the functions of genes involved in \u003cem\u003eTERT\u003c/em\u003e regulation may also be less strictly controlled in mice, which may explain why the suppressive effects of \u003cem\u003eTMEM115\u003c/em\u003e on \u003cem\u003eTERT\u003c/em\u003e were significantly less robust in the mouse PC cells.\u003c/p\u003e \u003cp\u003eTo investigate the clinical impact of TMEM115 expression in PC, we performed IHC assays on specimens from 100 PC patients. In general, telomeres in cells are known to be shortened in pancreatic intraepithelial neoplasia and intraluminal papillary mucinous neoplasms, the precursor lesions of PC [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The repression of \u003cem\u003eTERT\u003c/em\u003e expression in precancerous lesions that reach the limit of telomere shortening is released, and telomerase reactivation occurs, leading to immortalization. This process promotes the PC transition from early stage to advanced stage. Thus, \u003cem\u003eTERT\u003c/em\u003e may contribute to the late-stage portion of multi-stage PC carcinogenesis. The results of these previous reports are consistent with those of this study, where we observed significantly higher local progression of tumors in the low TMEM115 expression group because TMEM115 can suppress \u003cem\u003eTERT\u003c/em\u003e transcription. In addition, we found that the patients with low TMEM115 expression levels displayed significantly lower survival rates than those with high TMEM115 expression levels. Further analysis indicated that low TMEM115 expression was an independent prognostic factor for OS in these PC patients. Our results also demonstrated that the pT factor, which was significantly different between the two TMEM115 expression groups, was not a prognostic factor. These data suggest that TMEM115 may be used as a prognostic biomarker independent of TNM classification. Furthermore, analyzing the relationship between \u003cem\u003eTMEM115\u003c/em\u003e mRNA expression levels and prognosis in PC patients using public datasets also indicated a positive correlation. Thus, these results provide clinical evidence that TMEM115 is crucially involved in PC development and progression.\u003c/p\u003e \u003cp\u003eThe Golgi apparatus plays a central role in protein modifications, such as glycosylation. TMEM115, present in the Golgi complex, is also involved in glycoprotein modifications. Previous work has suggested that TMEM115 knockdown can alter the O-linked glycosylation profile on the cell surface [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. O-linked N-acetylglucosamine (O-GlcNAc), one type of O-glycosylation, has been shown to affect cancer cell metabolism, with hyper O-GlcNAcylation being observed in various carcinomas, including PC [\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. O-GlcNAcylation can also reportedly lead to the overexpression of oncogenic transcription factors, such as c-MYC, hypoxia-inducible factor 1, and nuclear factor kappa B [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In addition, c-MYC is known to be one of the major \u003cem\u003eTERT\u003c/em\u003e transcriptional activators [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Considering these findings, it is possible that disordered glycosylation from dysfunctional TMEM115 can activate c-MYC-mediated \u003cem\u003eTERT\u003c/em\u003e transcription, which promotes PC tumor development and progression. Further examination of the detailed role of TMEM115 in the mechanism of tumorigenesis via \u003cem\u003eTERT\u003c/em\u003e regulation could significantly support the discovery of novel PC inhibitory pathways.\u003c/p\u003e \u003cp\u003eIn conclusion, this study used chromosome engineering technology with HAC vectors to demonstrate that the 3p21.3 region encodes gene(s) that can regulate \u003cem\u003eTERT\u003c/em\u003e in PC. Furthermore, we identified \u003cem\u003eTMEM115\u003c/em\u003e as a novel \u003cem\u003eTERT\u003c/em\u003e repressor gene. In addition, we reported for the first time that \u003cem\u003eTMEM115\u003c/em\u003e expression is an important prognostic factor for PC patients. Targeting \u003cem\u003eTMEM115\u003c/em\u003e may therefore lead to the development of novel therapeutic strategies against PC. Further studies analyzing the specific \u003cem\u003eTMEM115\u003c/em\u003e functions will be required to define its significance in PC oncogenesis and progression.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe LTPA cells and MIA PaCa-2 cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Wako, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaill\u0026eacute;, France). Transfected LTPA cells were selected with 4 \u0026micro;g/mL blasticidin S hydrochloride (Wako) or 600 \u0026micro;g/mL G418 (Calbiochem, La Jolla, CA, USA). Transfected MIA PaCa-2 cells were selected with 7 \u0026micro;g/mL blasticidin S hydrochloride or 1000 \u0026micro;g/mL G418. A9 cells containing a 3p21.3-HAC or empty-HAC vector were cultured in DMEM supplemented with 10% FBS and 8 \u0026micro;g/mL blasticidin S hydrochloride. These microcell hybrid clones were established as previously described (26). All cells were cultured in a humidified incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMMCT\u003c/h2\u003e \u003cp\u003eMMCT was performed as previously described [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Briefly, the A9 cells containing the HAC vector were incubated with 0.05 \u0026micro;g/mL colcemid (Sigma-Aldrich, St. Louis, MO, USA) in DMEM containing 20% FBS. Micronuclei were collected by treatment with 10 mg/mL cytochalasin B (Sigma-Aldrich) and centrifugation with sequential filtering through polycarbonate filters (Whatman Nuclepore, Kent, UK). The fusion was mediated by adding 47% polyethylene-glycol 1000 (Wako) and washing with serum-free DMEM. After incubation for 24 h in DMEM containing 10% FBS, the cells with transferred HAC vectors were selected in the presence of blasticidin S hydrochloride, as described above.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFISH analysis\u003c/h3\u003e\n\u003cp\u003eFISH analysis was performed using cells in metaphase. Biotin-labeled RP-6 234N4 PAC vector containing the 3p21.3 genomic DNA region and digoxigenin-labeled human Cot-1 DNA (Life Technologies, Carlsbad, CA, USA) were used as probe DNAs. Chromosome and probe preparation, hybridization, washing, signal detection, and analysis were performed as previously reported [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eqRT-PCR\u003c/h3\u003e\n\u003cp\u003eRNA isolation and qRT-PCR were performed as described previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The amplification of cDNA was performed using an Applied Biosystems StepOne thermal cycler system and a SYBR Green PCR kit (Foster City, CA, USA). The primers used in this study are listed in the Supplementary Information (Table S2).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation assay\u003c/h2\u003e \u003cp\u003eCell proliferation assays were performed as described previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Briefly, cells were seeded in 60-mm cell culture dishes (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/dish) and counted daily using a Coulter Counter Z2 (Beckman Coulter, Woerden, the Netherlands). The average cell number was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell invasion assay\u003c/h2\u003e \u003cp\u003eCell invasion assays were performed as described previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These assays were conducted using Corning BioCoat Matrigel Invasion Chambers with 8.0 \u0026micro;m PET membrane (BD Biosciences, Bedford, MA, USA) following the manufacturer's protocol. The invading cells that passed through the filter were stained with Diff-Quik rapid stain (Sysmex Corporation, Hyogo, Japan) and counted using an ECLIPSE Ti-U (Nikon, Tokyo, Japan) at 200\u0026times; magnification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq analysis\u003c/h2\u003e \u003cp\u003eThe transcriptomic analysis was conducted at GENEWIZ from Azenta Life Sciences (Tokyo, Japan). The detailed methods are described in the Supplementary Information. The RNA-seq data have been deposited to DNA Data Bank of Japan (DDBJ) (accession number: DRA018313).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGene knockdown by RNA interference\u003c/h2\u003e \u003cp\u003eCells were transfected with the AccuTarget Predesigned \u003cem\u003eTMEM115\u003c/em\u003e siRNA#1 or #2 (Bioneer, Yuseong-gu, Korea) to knock down \u003cem\u003eTMEM115\u003c/em\u003e expression or a negative control siRNA (Bioneer). LTPA 3p21.3-HAC cells were seeded in 6-well plates (2.0\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well). Following the manufacturer's protocol, each siRNA was transfected at a concentration of 20 pmol/mL using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA). Cells were harvested 48 h after transfection, followed by qRT-PCR analysis. The siRNA duplexes of the seven genes, including \u003cem\u003eTMEM115\u003c/em\u003e, are described in the Supplementary Information (Table S3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid construction\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eTMEM115\u003c/em\u003e overexpression pCMV6 vectors were purchased from Origene (RC203956, Rockville, MD, USA). The control vectors were constructed by deleting the \u003cem\u003eTMEM115\u003c/em\u003e cDNA sequence from the \u003cem\u003eTMEM115\u003c/em\u003e overexpression vectors using the KOD Plus mutagenesis kit (Toyobo, Osaka, Japan) according to the manufacturer's protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eOverexpression\u003c/h2\u003e \u003cp\u003eParental LTPA or MIA PaCa-2 cells were seeded in 6-well plates (2.0\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well). Following the manufacturer's protocol, pCMV6 vectors were transfected using Lipofectamine LTX Reagent (Invitrogen). Twenty-four h after transfection, the cells were cultured in their respective medium, as described above. After 1 to 2 weeks, drug-resistant colonies were selected and expanded for further analysis following the procedures described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eWestern blot analysis was performed as described previously [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The membranes were blocked and incubated with a rabbit polyclonal anti-TMEM115 antibody (1:2500; PACO30462, AssayGenie, Dublin, Ireland) for 60 min at room temperature, then incubated with an anti-rabbit IgG secondary antibody (1:5000; 7074P2, Cell Signaling Technology, Danvers, MA, USA) for 45 min at room temperature according to the manufacturer's instructions. Immunoreactive bands were visualized using the ECL detection system (Pierce, Rockford, IL, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eData acquisition\u003c/h2\u003e \u003cp\u003eTCGA data of PC patient mRNA expression profiles were obtained from the UCSC Xena platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://xena.ucsc.edu/\u003c/span\u003e\u003cspan address=\"https://xena.ucsc.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) on 9.6.2024. The protein expression data of pancreatic and PC tissues were obtained from the HPA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.proteinatlas.org/\u003c/span\u003e\u003cspan address=\"https://www.proteinatlas.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) on 9.6.2024.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eIHC staining\u003c/h2\u003e \u003cp\u003eBetween January 2013 and December 2020, 100 consecutive patients underwent pancreatomy at Tottori University Hospital (Yonago, Japan) for PC. This protocol was approved by the Tottori University Ethical Board (approval number: 24A017), and all patients provided written informed consent for pathological analysis. The clinicopathological and laboratory data of all patients were extracted from their electronic medical records.\u003c/p\u003e \u003cp\u003eIHC staining was performed with Histostainer-36A (Nichirei Biosciences, Tokyo, Japan), as described in the Supplementary Information. The rabbit polyclonal anti-TMEM115 antibody (AssayGenie) was used as the primary antibody. The islets of Langerhans cells were used as internal positive controls. For analysis, a two-tiered classification using high and low expression was used. The classification method is described in the results of this article. All slides were evaluated independently by Y.S. and Y.U., with consensus reached in all cases.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData from at least three separate experiments are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. \u003cem\u003eP\u003c/em\u003e-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. Categorical variables were compared using Fisher\u0026rsquo;s exact test and continuous variables were compared using two-tailed t-tests. Survival curves were plotted using the Kaplan-Meier method, with the differences between survival curves examined using the log-rank test. Receiver operating characteristic curve analysis was used to determine the optimal cutoff values. SPSS for Windows version 28 (IBM, Armonk, NY, USA) was used for all statistical analyses.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Japan Society for the Promotion of Science KAKENHI (Grant Number 23K08112). This research was partly performed at the Tottori Bio Frontier managed by Tottori prefecture. We thank the staff of the tissue analysis section, Technical Department, Tottori University for technical assistance. We thank J. Iacona, Ph.D., from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYS, TO, and HK designed the experiments and analyzed the data. YS, TO, and HU performed the experiments and contributed to the discussion. YS and HK wrote the manuscript. TY, ToS, TeS, YU, and YF contributed to data analysis and discussion. HK conceived and managed the project. All authors revised and edited the manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are available in the DDBJ repository (accession number: DRA018313).\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021; 71:209\u0026ndash;249.\u003c/li\u003e\n\u003cli\u003eKolbeinsson HM, Chandana S, Wright GP, Chung M. Pancreatic cancer: a review of current treatment and novel therapies. J Invest Surg. 2023; 36:2129884.\u003c/li\u003e\n\u003cli\u003eSiegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin. 2024; 74:12\u0026ndash;49.\u003c/li\u003e\n\u003cli\u003eBernardes de Jesus B, Blasco MA. Telomerase at the intersection of cancer and aging. Trends Genet. 2013; 29:513\u0026ndash;520.\u003c/li\u003e\n\u003cli\u003eMeyerson M, Counter CM, Eaton EN, Ellisen LW, Steiner P, Caddle SD, et al. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell. 1997; 90:785\u0026ndash;795.\u003c/li\u003e\n\u003cli\u003eCollins K, Mitchell JR. Telomerase in the human organism. Oncogene. 2002; 21:564\u0026ndash;579.\u003c/li\u003e\n\u003cli\u003eShay JW, Bacchetti S. A survey of telomerase activity in human cancer. European journal of cancer (Oxford, England : 1990). 1997; 33:787\u0026ndash;791.\u003c/li\u003e\n\u003cli\u003eFlores I, Cayuela ML, Blasco MA. Effects of telomerase and telomere length on epidermal stem cell behavior. Science. 2005; 309:1253\u0026ndash;1256.\u003c/li\u003e\n\u003cli\u003eHannen R, Bartsch JW. Essential roles of telomerase reverse transcriptase hTERT in cancer stemness and metastasis. FEBS Lett. 2018; 592:2023\u0026ndash;2031.\u003c/li\u003e\n\u003cli\u003eLiu Z, Li Q, Li K, Chen L, Li W, Hou M, et al. Telomerase reverse transcriptase promotes epithelial-mesenchymal transition and stem cell-like traits in cancer cells. Oncogene. 2013; 32:4203\u0026ndash;4213.\u003c/li\u003e\n\u003cli\u003eRamlee MK, Wang J, Toh WX, Li S. Transcription regulation of the human telomerase reverse transcriptase (hTERT) gene. Genes (Basel). 2016; 7:50.\u003c/li\u003e\n\u003cli\u003eYuan X, Larsson C, Xu D. Mechanisms underlying the activation of TERT transcription and telomerase activity in human cancer: old actors and new players. Oncogene. 2019; 38:6172\u0026ndash;6183.\u003c/li\u003e\n\u003cli\u003eBaker DJ, Jin F, Jeganathan KB, van Deursen JM. Whole chromosome instability caused by Bub1 insufficiency drives tumorigenesis through tumor suppressor gene loss of heterozygosity. Cancer Cell. 2009; 16:475\u0026ndash;486.\u003c/li\u003e\n\u003cli\u003eCalhoun ES, Hucl T, Gallmeier E, West KM, Arking DE, Maitra A, et al. Identifying allelic loss and homozygous deletions in pancreatic cancer without matched normals using high-density single-nucleotide polymorphism arrays. Cancer Res. 2006; 66:7920\u0026ndash;7928.\u003c/li\u003e\n\u003cli\u003eHahn SA, Schutte M, Hoque AT, Moskaluk CA, da Costa LT, Rozenblum E, et al. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science. 1996; 271:350\u0026ndash;353.\u003c/li\u003e\n\u003cli\u003eMahlam\u0026auml;ki EH, H\u0026ouml;glund M, Gorunova L, Karhu R, Dawiskiba S, Andr\u0026eacute;n-Sandberg A, et al. Comparative genomic hybridization reveals frequent gains of 20q, 8q, 11q, 12p, and 17q, and losses of 18q, 9p, and 15q in pancreatic cancer. Genes Chromosomes Cancer. 1997; 20:383\u0026ndash;391.\u003c/li\u003e\n\u003cli\u003eGnarra JR, Tory K, Weng Y, Schmidt L, Wei MH, Li H, et al. Mutations of the VHL tumour suppressor gene in renal carcinoma. Nat Genet. 1994; 7:85\u0026ndash;90.\u003c/li\u003e\n\u003cli\u003eAgathanggelou A, Cooper WN, Latif F. Role of the Ras-association domain family 1 tumor suppressor gene in human cancers. Cancer Res. 2005; 65:3497\u0026ndash;3508.\u003c/li\u003e\n\u003cli\u003eJi L, Fang B, Yen N, Fong K, Minna JD, Roth JA. Induction of apoptosis and inhibition of tumorigenicity and tumor growth by adenovirus vector-mediated fragile histidine triad (FHIT) gene overexpression. Cancer Res. 1999; 59:3333\u0026ndash;3339.\u003c/li\u003e\n\u003cli\u003eDallol A, Forgacs E, Martinez A, Sekido Y, Walker R, Kishida T, et al. Tumour specific promoter region methylation of the human homologue of the Drosophila Roundabout gene DUTT1 (ROBO1) in human cancers. Oncogene. 2002; 21:3020\u0026ndash;3028.\u003c/li\u003e\n\u003cli\u003eYagyu T, Ohira T, Shimizu R, Morimoto M, Murakami Y, Hanaki T, et al. Human chromosome 3p21.3 carries TERT transcriptional regulators in pancreatic cancer. Sci Rep. 2021; 11:15355.\u003c/li\u003e\n\u003cli\u003eKazuki Y, Hoshiya H, Takiguchi M, Abe S, Iida Y, Osaki M, et al. Refined human artificial chromosome vectors for gene therapy and animal transgenesis. Gene Ther. 2011; 18:384\u0026ndash;393.\u003c/li\u003e\n\u003cli\u003eKouprina N, Larionov V. Recent advances in chromosome engineering. Chromosome Res. 2015; 23:1\u0026ndash;5.\u003c/li\u003e\n\u003cli\u003eUno N, Abe S, Oshimura M, Kazuki Y. Combinations of chromosome transfer and genome editing for the development of cell/animal models of human disease and humanized animal models. J Hum Genet. 2018; 63:145\u0026ndash;156.\u003c/li\u003e\n\u003cli\u003eFournier RE, Ruddle FH. Microcell-mediated transfer of murine chromosomes into mouse, Chinese hamster, and human somatic cells. Proc Natl Acad Sci U S A. 1977; 74:319\u0026ndash;323.\u003c/li\u003e\n\u003cli\u003eOhira T, Yoshimura K, Kugoh H. Human artificial chromosome carrying 3p21.3-p22.2 region suppresses hTERT transcription in oral cancer cells. Chromosome Res. 2023; 31:17.\u003c/li\u003e\n\u003cli\u003eOng YS, Tran TH, Gounko NV, Hong W. TMEM115 is an integral membrane protein of the Golgi complex involved in retrograde transport. J Cell Sci. 2014; 127:2825\u0026ndash;2839.\u003c/li\u003e\n\u003cli\u003eIvanova AV, Vortmeyer A, Ivanov SV, Nickerson ML, Maher ER, Lerman MI. Loss of PL6 protein expression in renal clear cell carcinomas and other VHL-deficient tumours. J Pathol. 2008; 214:46\u0026ndash;57.\u003c/li\u003e\n\u003cli\u003ePark YJ, Kim EK, Bae JY, Moon S, Kim J. Human telomerase reverse transcriptase (hTERT) promotes cancer invasion by modulating cathepsin D via early growth response (EGR)-1. Cancer Lett. 2016; 370:222\u0026ndash;231.\u003c/li\u003e\n\u003cli\u003eCarboni GL, Gao B, Nishizaki M, Xu K, Minna JD, Roth JA, et al. CACNA2D2-mediated apoptosis in NSCLC cells is associated with alterations of the intracellular calcium signaling and disruption of mitochondria membrane integrity. Oncogene. 2003; 22:615\u0026ndash;626.\u003c/li\u003e\n\u003cli\u003eThe Human Protein Atlas. https://www.proteinatlas.org/. \u003c/li\u003e\n\u003cli\u003eBasturk O, Saka B, Balci S, Postlewait LM, Knight J, Goodman M, et al. Substaging of lymph node status in resected pancreatic ductal adenocarcinoma has strong prognostic correlations: proposal for a revised N classification for TNM staging. Ann Surg Oncol. 2015; 22:S1187\u0026ndash;S1195.\u003c/li\u003e\n\u003cli\u003eStrobel O, Hinz U, Gluth A, Hank T, Hackert T, Bergmann F, et al. Pancreatic adenocarcinoma: number of positive nodes allows to distinguish several N categories. Ann Surg. 2015; 261:961\u0026ndash;969.\u003c/li\u003e\n\u003cli\u003eKugoh H, Ohira T, Oshimura M. Studies of tumor suppressor genes via chromosome engineering. Cancers (Basel). 2015; 8:4.\u003c/li\u003e\n\u003cli\u003eQi DL, Ohhira T, Fujisaki C, Inoue T, Ohta T, Osaki M, et al. Identification of PITX1 as a TERT suppressor gene located on human chromosome 5. Mol Cell Biol. 2011; 31:1624\u0026ndash;1636.\u003c/li\u003e\n\u003cli\u003eOhira T, Naohiro S, Nakayama Y, Osaki M, Okada F, Oshimura M, et al. miR-19b regulates hTERT mRNA expression through targeting PITX1 mRNA in melanoma cells. Sci Rep. 2015; 5:8201.\u003c/li\u003e\n\u003cli\u003eKazuki Y, Kobayashi K, Aueviriyavit S, Oshima T, Kuroiwa Y, Tsukazaki Y, et al. Trans-chromosomic mice containing a human CYP3A cluster for prediction of xenobiotic metabolism in humans. Hum Mol Genet. 2013; 22:578\u0026ndash;592.\u003c/li\u003e\n\u003cli\u003eHung J, Kishimoto Y, Sugio K, Virmani A, McIntire DD, Minna JD, et al. Allele-specific chromosome 3p deletions occur at an early stage in the pathogenesis of lung carcinoma. JAMA. 1995; 273:1908.\u003c/li\u003e\n\u003cli\u003eKok K, Naylor SL, Buys CH. Deletions of the short arm of chromosome 3 in solid tumors and the search for suppressor genes. Adv Cancer Res. 1997; 71:27\u0026ndash;92.\u003c/li\u003e\n\u003cli\u003eHesson LB, Cooper WN, Latif F. Evaluation of the 3p21.3 tumour-suppressor gene cluster. Oncogene. 2007; 26:7283\u0026ndash;7301.\u003c/li\u003e\n\u003cli\u003eAgathanggelou A, Dallol A, Z\u0026ouml;chbauer-M\u0026uuml;ller S, Morrissey C, Honorio S, Hesson L, et al. Epigenetic inactivation of the candidate 3p21.3 suppressor gene BLU in human cancers. Oncogene. 2003; 22:1580\u0026ndash;1588.\u003c/li\u003e\n\u003cli\u003eSharp TV, Al-Attar A, Foxler DE, Ding L, de AVTQ, Zhang Y, et al. The chromosome 3p21.3-encoded gene, LIMD1, is a critical tumor suppressor involved in human lung cancer development. Proc Natl Acad Sci U S A. 2008; 105:19932\u0026ndash;19937.\u003c/li\u003e\n\u003cli\u003eLi J, Wang F, Haraldson K, Protopopov A, Duh FM, Geil L, et al. Functional characterization of the candidate tumor suppressor gene NPRL2/G21 located in 3p21.3C. Cancer Res. 2004; 64:6438\u0026ndash;6443.\u003c/li\u003e\n\u003cli\u003eAbe S, Tanaka H, Notsu T, Horike S, Fujisaki C, Qi DL, et al. Localization of an hTERT repressor region on human chromosome 3p21.3 using chromosome engineering. Genome Integr. 2010; 1:6.\u003c/li\u003e\n\u003cli\u003eNishio S, Ohira T, Sunamura N, Oshimura M, Ryoke K, Kugoh H. Repression of hTERT transcription by the introduction of chromosome 3 into human oral squamous cell carcinoma. Biochem Biophys Res Commun. 2015; 466:755\u0026ndash;759.\u003c/li\u003e\n\u003cli\u003eGomes NM, Ryder OA, Houck ML, Charter SJ, Walker W, Forsyth NR, et al. Comparative biology of mammalian telomeres: hypotheses on ancestral states and the roles of telomeres in longevity determination. Aging Cell. 2011; 10:761\u0026ndash;768.\u003c/li\u003e\n\u003cli\u003eGuterres AN, Villanueva J. Targeting telomerase for cancer therapy. Oncogene. 2020; 39:5811\u0026ndash;5824.\u003c/li\u003e\n\u003cli\u003eShou S, Li Y, Chen J, Zhang X, Zhang C, Jiang X, et al. Understanding, diagnosing, and treating pancreatic cancer from the perspective of telomeres and telomerase. Cancer Gene Ther. 2024; 31:1292\u0026ndash;1305.\u003c/li\u003e\n\u003cli\u003eHashimoto Y, Murakami Y, Uemura K, Hayashidani Y, Sudo T, Ohge H, et al. Telomere shortening and telomerase expression during multistage carcinogenesis of intraductal papillary mucinous neoplasms of the pancreas. J Gastrointest Surg. 2008; 12:17\u0026ndash;28; discussion 28\u0026ndash;29.\u003c/li\u003e\n\u003cli\u003eMa Z, Vocadlo DJ, Vosseller K. Hyper-O-GlcNAcylation is anti-apoptotic and maintains constitutive NF-\u0026kappa;B activity in pancreatic cancer cells. J Biol Chem. 2013; 288:15121\u0026ndash;15130.\u003c/li\u003e\n\u003cli\u003eCaldwell SA, Jackson SR, Shahriari KS, Lynch TP, Sethi G, Walker S, et al. Nutrient sensor O-GlcNAc transferase regulates breast cancer tumorigenesis through targeting of the oncogenic transcription factor FoxM1. Oncogene. 2010; 29:2831\u0026ndash;2842.\u003c/li\u003e\n\u003cli\u003eMi W, Gu Y, Han C, Liu H, Fan Q, Zhang X, et al. O-GlcNAcylation is a novel regulator of lung and colon cancer malignancy. Biochim Biophys Acta. 2011; 1812:514\u0026ndash;519.\u003c/li\u003e\n\u003cli\u003eMakwana V, Ryan P, Patel B, Dukie SA, Rudrawar S. Essential role of O-GlcNAcylation in stabilization of oncogenic factors. Biochim Biophys Acta Gen Subj. 2019; 1863:1302\u0026ndash;1317.\u003c/li\u003e\n\u003cli\u003eSingh JP, Zhang K, Wu J, Yang X. O-GlcNAc signaling in cancer metabolism and epigenetics. Cancer Lett. 2015; 356:244\u0026ndash;250.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6170448/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6170448/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe catalytic component of telomerase, telomerase reverse transcriptase (\u003cem\u003eTERT\u003c/em\u003e), is reactivated in immortalized cells and plays a crucial role in cancer development. Pancreatic cancer (PC) is frequently associated with loss of heterozygosity on the short arm of human chromosome 3. In a previous study, chromosome engineering experiments in PC suggested that putative \u003cem\u003eTERT\u003c/em\u003e suppressor genes (TSGs) are present on the 3p21.3 region. Here, we performed functional analysis using a human artificial chromosome (HAC) carrying only the 3p21.3 region (3p21.3-HAC) to directly clarify if TSGs are contained in the 3p21.3 region. We observed reduced \u003cem\u003eTERT\u003c/em\u003e transcription following the introduction of 3p21.3-HAC into PC cells. Furthermore, to identify the specific TSGs in the 3p21.3 region, we performed RNA sequencing analysis using mouse \u003cem\u003eTert\u003c/em\u003e (\u003cem\u003emTert\u003c/em\u003e)-expressing murine LTPA PC cells containing either 3p21.3-HAC or the empty HAC vector. Through this analysis, we identified transmembrane protein 115 (\u003cem\u003eTMEM115\u003c/em\u003e) as a novel TSG. Furthermore, both human \u003cem\u003eTERT\u003c/em\u003e (\u003cem\u003ehTERT\u003c/em\u003e) and \u003cem\u003emTert\u003c/em\u003e transcription can be suppressed by \u003cem\u003eTMEM115\u003c/em\u003e. Thus, \u003cem\u003eTMEM115\u003c/em\u003e may contribute to PC development by functioning as a novel telomerase regulating factor via controlling \u003cem\u003eTERT\u003c/em\u003e expression.\u003c/p\u003e","manuscriptTitle":"Identification of TMEM115 as a tumor suppressor gene through TERT regulation in pancreatic cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-20 16:15:16","doi":"10.21203/rs.3.rs-6170448/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1d492e43-2984-4f79-a9b2-47b491335da6","owner":[],"postedDate":"May 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":48809084,"name":"Biological sciences/Cancer/Gastrointestinal cancer/Pancreatic cancer"},{"id":48809085,"name":"Biological sciences/Cell biology/Chromosomes"},{"id":48809086,"name":"Biological sciences/Cancer/Oncogenes"}],"tags":[],"updatedAt":"2025-05-20T16:15:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-20 16:15:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6170448","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6170448","identity":"rs-6170448","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}