Cancer Methylation Biomarker Detection in an Automated, Portable, Multichannel Magnetofluidic Platform.

Early detection of cancer is critical to improving clinical outcomes, especially in territories with limited healthcare resources. DNA methylation biomarkers have shown promise in early cancer detection, but typical workflows require highly trained personnel and specialized equipment for manual and lengthy processing, limiting use in resource-constrained areas. As a potential solution, we introduce the Automated Cartridge-based Cancer Early Screening System (ACCESS), a compact, portable, multiplexed, automated platform that performs droplet magnetofluidic- and methylation-specific qPCR-based assays for the detection of DNA methylation cancer biomarkers. Development of ACCESS focuses on esophageal cancer, which is among the most prevalent cancers in low- and middle-income countries with extremely low survival rates. Upon implementing detection assays for two esophageal cancer methylation biomarkers within ACCESS, we demonstrated successful detection of both biomarkers from esophageal tumor tissue samples from eight esophageal cancer patients while showing specificity in paired normal esophageal tissue samples. These results illustrate ACCESS's potential as an amenable epigenetic diagnostic tool for resource-constrained areas toward early detection of esophageal cancer and potentially other malignancies.

Establishing mouse and human oral esophageal organoids to investigate the tumor immune response.

Organoid culture systems are very powerful models that recapitulate in vivo organ development and disease pathogenesis, offering great promise in basic research, drug screening and precision medicine. However, the application of organoids derived from patients with cancer to immunotherapeutic research is a relatively untapped area. Esophageal cancer is one of the most lethal malignancies worldwide, including two major pathological subtypes: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma. ESCC shares many biological and genomic features with oral squamous cell cancers. Herein, we provide a versatile protocol for the establishment and maintenance of oral and esophageal organoid cultures derived from both murine and human samples. We describe culture conditions for organoids derived from normal tongue, esophagus and gastroesophageal junction, esophageal cancer and Barrett's esophagus. In addition, we establish an ex vivo model by co-culturing patient tumor-derived organoids and autologous CD8+ T lymphocytes to assess CD8+ T cell-mediated tumor killing. Our protocol can also be modified for organoid establishment from other squamous epithelia and carcinomas. The co-culture model can serve as a template for studies of other tumor-immune cell interactions and the efficacy of immune checkpoint blockade therapy.

Generation and multiomic profiling of a TP53/CDKN2A double-knockout gastroesophageal junction organoid model.

Inactivation of the tumor suppressor genes tumor protein p53 (TP53) and cyclin-dependent kinase inhibitor 2A (CDKN2A) occurs early during gastroesophageal junction (GEJ) tumorigenesis. However, because of a paucity of GEJ-specific disease models, cancer-promoting consequences of TP53 and CDKN2A inactivation at the GEJ have not been characterized. Here, we report the development of a wild-type primary human GEJ organoid model and a CRISPR-edited transformed GEJ organoid model. CRISPR-Cas9-mediated TP53 and CDKN2A knockout (TP53/CDKN2AKO) in GEJ organoids induced morphologic dysplasia and proneoplastic features in vitro and tumor formation in vivo. Lipidomic profiling identified several platelet-activating factors (PTAFs) among the most up-regulated lipids in CRISPR-edited organoids. PTAF/PTAF receptor (PTAFR) abrogation by siRNA knockdown or a pharmacologic inhibitor (WEB2086) reduced proliferation and other proneoplastic features of TP53/CDKN2AKO GEJ organoids in vitro and tumor formation in vivo. In addition, murine xenografts of Eso26, an established human esophageal adenocarcinoma cell line, were suppressed by WEB2086. Mechanistically, TP53/CDKN2A dual inactivation disrupted both the transcriptome and the DNA methylome, likely mediated by key transcription factors, particularly forkhead box M1 (FOXM1). FOXM1 activated PTAFR transcription by binding to the PTAFR promoter, further amplifying the PTAF-PTAFR pathway. Together, these studies established a robust model system for investigating early GEJ neoplastic events, identified crucial metabolic and epigenomic changes occurring during GEJ model tumorigenesis, and revealed a potential cancer therapeutic strategy. This work provides insights into proneoplastic mechanisms associated with TP53/CDKN2A inactivation in early GEJ neoplasia, which may facilitate early diagnosis and prevention of GEJ neoplasms.

PAD4-dependent citrullination of nuclear translocation of GSK3ß promotes colorectal cancer progression via the degradation of nuclear CDKN1A.

Peptidylarginine deiminase 4 (PAD4), a Ca2+-dependent enzyme, catalyzes the conversion of arginine to citrulline and has been strongly associated with many malignant tumors. However, the molecular mechanisms of PAD4 in the development and progression of colorectal cancer (CRC) remain unclearly defined. In our study, PAD4 expression was increased in CRC tissues and cells, and was closely related to tumor size, lymph node metastasis. Moreover, the transcription factor KLF9 directly bound to PADI4 gene promoter, leading to overexpression of PAD4 in CRC cells, which augmented cell growth and migration. We revealed that PAD4 interacted with and citrullinated glycogen synthase kinase-3ß (GSK3ß) in CRC cells, and GSK3ß Arg-344 was the dominating PAD4-citrullination site. Furthermore, IgL2 and catalytic domains of PAD4 directly bound to the kinase domain of GSK3ß in CRC cells. Mechanistically, PAD4 promoted the transport of GSK3ß from the cytoplasm to the nucleus, thereby increasing the ubiquitin-dependent proteasome degradation of nuclear cyclin-dependent kinase inhibitor 1 (CDKN1A). Our study is the first to reveal the details of a critical PAD4/GSK3ß/CDKN1A signaling axis for CRC progression, and provides evidence that PAD4 is a potential diagnosis biomarker and therapeutic target in CRC.

The utility of a genetic progression risk test for Barrett esophagus.

This study sought to characterize the utility of a gene methylation-based biomarker test that has been validated to predict progression towards esophageal adenocarcinoma. Barrett esophagus (BE) is a precursor condition for esophageal adenocarcinoma (EAC) with somewhat variable approaches among gastroenterologists toward managing neoplastic progression risk. Capsulomics has developed a validated multigene DNA methylation-based biomarker assay performed on BE biopsies designed to address this variability by classifying BE patients into progression risk groups. In the current study, a survey was administered to practicing gastroenterologists in order to assess the potential impact of this assay on clinical practice. In this context, 89% (95% Cl: 85.4-92.6%) of surveyed physicians felt strongly that the multigene Barrett Esophagus test helped resolve uncertainties and optimize care of patients with BE by impacting their decisions on surveillance intervals and use of active treatments, such as ablation. The assay significantly impacted surveillance intervals for both high-risk (22.0 no assay vs 12.3 months with assay; P = 1.7E-8) and low-risk (7.9 no assay vs 11.4 months with assay, P = 8.8E-4) stratified case results. Finally, the assay also significantly impacted decisions to pursue active ablation treatments in both high-risk (5% recommending ablation without assay vs 42% with assay; P = 3.7E-11) and low-risk (42% recommending ablation without assay vs 29% with assay; P = .049) stratified case results. Results demonstrated a strong effect of the assay on clinical decision making, even in conjunction with established clinical guidelines.

Vimentin binds to a novel tumor suppressor protein, GSPT1-238aa, encoded by circGSPT1 with a selective encoding priority to halt autophagy in gastric carcinoma.

Circular RNAs (circRNAs) are covalently closed, endogenous molecules that are widespread in eukaryotes. Recent evidence indicates that circRNAs play important roles in carcinogenesis. Several circRNAs have been reported to comprise translatable RNA; however, whether circRNAs encode functional proteins remains unknown. In our study, circRNA sequencing was carried out using five pathologically diagnosed gastric carcinoma (GC) samples and their paired adjacent normal tissues, we characterized the circRNA GSPT1 (circGSPT1), which is expressed at low levels in GC. Antibody detections, and mass spectrometry were used to validate active circRNA translation. The spanning junction open reading frame in circGSPT1, driven by an internal ribosome entry site (IRES), encodes a functional peptide, termed GSPT1-238aa. Interestingly, GSPT1-238aa tends to select the start codon used to initiate translation. This is the first finding of selective translation driven by IRES. CircGSPT1 and GSPT1-238aa halted the proliferation, migration, and invasion in GC cells in vitro. We also confirmed that the vimentin/Beclin1/14-3-3 complex interacts with GSPT1-238aa and modulates autophagy via the PI3K/AKT/mTOR signaling pathway in GC cells. Our study reveals that GSPT1-238aa, a novel protein encoded by circGSPT1, halts GC tumorigenesis. We also provide insights into the function and underlying molecular mechanisms of GSPT1-238aa in GC and suggest that this protein represents a novel target for GC treatment.

A novel protein AXIN1-295aa encoded by circAXIN1 activates the Wnt/ß-catenin signaling pathway to promote gastric cancer progression.

BACKGROUND: Circular RNA (circRNA), a subclass of non-coding RNA, plays a critical role in cancer tumorigenesis and metastasis. It has been suggested that circRNA acts as a microRNA sponge or a scaffold to interact with protein complexes; however, its full range of functions remains elusive. Recently, some circRNAs have been found to have coding potential. METHODS: To investigate the role of circRNAs in gastric cancer (GC), parallel sequencing was performed using five paired GC samples. Differentially expressed circAXIN1 was proposed to encode a novel protein. FLAG-tagged circRNA overexpression plasmid construction, immunoblotting, mass spectrometry, and luciferase reporter analyses were applied to confirm the coding potential of circAXIN1. Gain- and loss-of-function studies were conducted to study the oncogenic role of circAXIN1 and AXIN1-295aa on the proliferation, migration, invasion, and metastasis of GC cells in vitro and in vivo. The competitive interaction between AXIN1-295aa and adenomatous polyposis coli (APC) was investigated by immunoprecipitation analyses. Wnt signaling activity was observed using a Top/Fopflash assay, real-time quantitative RT-PCR, immunoblotting, immunofluorescence staining, and chromatin immunoprecipitation. RESULTS: CircAXIN1 is highly expressed in GC tissues compared with its expression in paired adjacent normal gastric tissues. CircAXIN1 encodes a 295 amino acid (aa) novel protein, which was named AXIN1-295aa. CircAXIN1 overexpression enhances the cell proliferation, migration, and invasion of GC cells, while the knockdown of circAXIN1 inhibits the malignant behaviors of GC cells in vitro and in vivo. Mechanistically, AXIN1-295aa competitively interacts with APC, leading to dysfunction of the "destruction complex" of the Wnt pathway. Released ß-catenin translocates to the nucleus and binds to the TCF consensus site on the promoter, inducing downstream gene expression. CONCLUSION: CircAXIN1 encodes a novel protein, AXIN1-295aa. AXIN1-295aa functions as an oncogenic protein, activating the Wnt signaling pathway to promote GC tumorigenesis and progression, suggesting a potential therapeutic target for GC.

EpiPanGI Dx: A Cell-free DNA Methylation Fingerprint for the Early Detection of Gastrointestinal Cancers.

PURPOSE: DNA methylation alterations have emerged as front-runners in cell-free DNA (cfDNA) biomarker development. However, much effort to date has focused on single cancers. In this context, gastrointestinal (GI) cancers constitute the second leading cause of cancer-related deaths worldwide; yet there is no blood-based assay for the early detection and population screening of GI cancers. EXPERIMENTAL DESIGN: Herein, we performed a genome-wide DNA methylation analysis of multiple GI cancers to develop a pan-GI diagnostic assay. By analyzing DNA methylation data from 1,781 tumor and adjacent normal tissues, we first identified differentially methylated regions (DMR) between individual GI cancers and adjacent normal, as well as across GI cancers. We next prioritized a list of 67,832 tissue DMRs by incorporating all significant DMRs across various GI cancers to design a custom, targeted bisulfite sequencing platform. We subsequently validated these tissue-specific DMRs in 300 cfDNA specimens and applied machine learning algorithms to develop three distinct categories of DMR panels RESULTS: We identified three distinct DMR panels: (i) cancer-specific biomarker panels with AUC values of 0.98 (colorectal cancer), 0.98 (hepatocellular carcinoma), 0.94 (esophageal squamous cell carcinoma), 0.90 (gastric cancer), 0.90 (esophageal adenocarcinoma), and 0.85 (pancreatic ductal adenocarcinoma); (ii) a pan-GI panel that detected all GI cancers with an AUC of 0.88; and (iii) a multi-cancer (tissue of origin) prediction panel, EpiPanGI Dx, with a prediction accuracy of 0.85-0.95 for most GI cancers. CONCLUSIONS: Using a novel biomarker discovery approach, we provide the first evidence for a cfDNA methylation assay that offers robust diagnostic accuracy for GI cancers.

microRNA-196b promotes esophageal squamous cell carcinogenesis and chemoradioresistance by inhibiting EPHA7, thereby restoring EPHA2 activity.

Esophageal cancer (EC) is extremely aggressive and has a very poor survival rate. Esophageal squamous cell carcinoma (ESCC) accounts for 80% of all ECs worldwide, with the majority of the remaining 20% being esophageal adenocarcinoma (EAC). Due to its occult and insidious presentation, ESCC is typically diagnosed and treated in its advanced stages, thereby limiting the success of present therapeutic modalities. microRNAs (miRNAs) can function as tumor suppressors or oncogenes, playing critical roles in cancer initiation and progression by regulating target genes in oncogenic pathways. In the current study, we demonstrated that microRNA-196b (miR-196b) is one of the most upregulated miRNAs in both ESCC and EAC. miR-196b was overexpressed in ESCC and EAC cell lines, cellular exosomal RNAs, and ESCC tissue samples. Functional studies revealed that miR-196b acted as an oncomiR by directly targeting a tumor suppressor, ephrin type-A receptor 7 (EPHA7). EPHA7 abrogates the activity of ephrin type-A receptor 2 (EPHA2), a key molecule involved in the epithelial-to-mesenchymal transition (EMT) and MAPK/ERK pathways, mediating resistance to UV and chemoradiotherapy in both ESCC and EAC. Taken together, these findings suggest that miR-196b is a strong candidate molecular target for EC treatment.

Interplay and cooperation between SREBF1 and master transcription factors regulate lipid metabolism and tumor-promoting pathways in squamous cancer.

Squamous cell carcinomas (SCCs) comprise one of the most common histologic types of human cancer. Transcriptional dysregulation of SCC cells is orchestrated by tumor protein p63 (TP63), a master transcription factor (TF) and a well-researched SCC-specific oncogene. In the present study, both Gene Set Enrichment Analysis (GSEA) of SCC patient samples and in vitro loss-of-function assays establish fatty-acid metabolism as a key pathway downstream of TP63. Further studies identify sterol regulatory element binding transcription factor 1 (SREBF1) as a central mediator linking TP63 with fatty-acid metabolism, which regulates the biosynthesis of fatty-acids, sphingolipids (SL), and glycerophospholipids (GPL), as revealed by liquid chromatography tandem mass spectrometry (LC-MS/MS)-based lipidomics. Moreover, a feedback co-regulatory loop consisting of SREBF1/TP63/Kruppel like factor 5 (KLF5) is identified, which promotes overexpression of all three TFs in SCCs. Downstream of SREBF1, a non-canonical, SCC-specific function is elucidated: SREBF1 cooperates with TP63/KLF5 to regulate hundreds of cis-regulatory elements across the SCC epigenome, which converge on activating cancer-promoting pathways. Indeed, SREBF1 is essential for SCC viability and migration, and its overexpression is associated with poor survival in SCC patients. Taken together, these data shed light on mechanisms of transcriptional dysregulation in cancer, identify specific epigenetic regulators of lipid metabolism, and uncover SREBF1 as a potential therapeutic target and prognostic marker in SCC.

Protein synthesis inhibitor omacetaxine is effective against hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the sixth most common and the fourth most deadly cancer worldwide. The development cost of new therapeutics is a major limitation in patient outcomes. Importantly, there is a paucity of preclinical HCC models in which to test new small molecules. Herein, we implemented potentially novel patient-derived organoid (PDO) and patient-derived xenografts (PDX) strategies for high-throughput drug screening. Omacetaxine, an FDA-approved drug for chronic myelogenous leukemia (CML), was found to be a top effective small molecule in HCC PDOs. Next, omacetaxine was tested against a larger cohort of 40 human HCC PDOs. Serial dilution experiments demonstrated that omacetaxine is effective at low (nanomolar) concentrations. Mechanistic studies established that omacetaxine inhibits global protein synthesis, with a disproportionate effect on short-half-life proteins. High-throughput expression screening identified molecular targets for omacetaxine, including key oncogenes, such as PLK1. In conclusion, by using an innovative strategy, we report - for the first time to our knowledge - the effectiveness of omacetaxine in HCC. In addition, we elucidate key mechanisms of omacetaxine action. Finally, we provide a proof-of-principle basis for future studies applying drug screening PDOs sequenced with candidate validation in PDX models. Clinical trials could be considered to evaluate omacetaxine in patients with HCC.

Dual activation of Hedgehog and Wnt/ß-catenin signaling pathway caused by downregulation of SUFU targeted by miRNA-150 in human gastric cancer.

Mounting evidence has shown that miRNA-150 expression is upregulated in gastric cancer (GC) and is associated with gastric carcinogenesis, but the underlying oncogenic mechanism remains elusive. Here, we discovered that miRNA-150 targets the tumor suppressor SUFU to promote cell proliferation, migration, and the epithelial-mesenchymal transition (EMT) via the dual activation of Hedgehog (Hh) and Wnt signaling. MiRNA-150 was highly expressed in GC tissues and cell lines, and the level of this miRNA was negatively related to that of SUFU. In addition, both the miRNA-150 and SUFU levels were associated with tumor differentiation. Furthermore, miRNA-150 activated GC cell proliferation and migration in vitro. We found that miRNA-150 inhibitors repressed not only Wnt signaling by promoting cytoplasmic ß-catenin localization, but also repressed Hh signaling and EMT. MiRNA-150 inhibition also resulted in significant tumor volume reductions in vivo, suggesting the potential application of miRNA-150 inhibitors in GC therapy. The expression of genes downstream of Hh and Wnt signaling was also reduced in tumors treated with miRNA-150 inhibitors. Notably, anti-SUFU siRNAs rescued the inhibitory effects of miRNA-150 inhibitors on Wnt signaling, Hh activation, EMT, cell proliferation, cell migration, and colony formation. Taken together, these findings indicate that miRNA-150 is oncogenic and promotes GC cell proliferation, migration, and EMT by activating Wnt and Hh signaling via the suppression of SUFU expression.

MiRNA-20b/SUFU/Wnt axis accelerates gastric cancer cell proliferation, migration and EMT.

Previous research has found that miRNA-20b is highly expressed in gastric cancer (GC), however, its function and underlying mechanism are not clear. Wnt signaling pathway, implicated in tumorigeneisis, is activated in more than 30% of GC. We would like to characterize the biological behavior of miRNA-20b in terms of modulating Wnt/ß-catenin signaling and EMT. We showed that miRNA-20b inhibitors suppressed Topflash/Fopflash dependent luciferase activity and the ß-catenin nuclear translocation, resulting in inhibition of Wnt pathway activity and EMT. SUFU, negatively regulating Wnt and Hedgehog signaling pathway, was proved to be targeted by miRNA-20b. Moreover, additional knockdown of SUFU alleviated the inhibitory effect on Wnt pathway activity, EMT, cell proliferation/migration and colony formation caused by miRNA-20b inhibition. In summary, miRNA-20b is an oncogenic miRNA and promoted cell proliferation, migration and EMT in GC partially by activating Wnt pathway via targeting SUFU.

Novel Long Noncoding RNA miR205HG Functions as an Esophageal Tumor-Suppressive Hedgehog Inhibitor.

Barrett's esophagus (BE) is a precursor to esophageal adenocarcinoma (EAC). Recently, long noncoding RNAs (lncRNAs) have been identified as key regulators of biological pathways. However, involvement of lncRNAs in the development of BE and EAC has not been well-studied. The aims of the current study were: (1) to study involvement of the lncRNA, miR205HG, in the development of BE and EAC; (2) to clarify the role of miR205HG in in vitro and in vivo experiments; and (3) to investigate the mechanism of miR205HG involving the Hedgehog (Hh) signaling pathway. These experiments revealed that miR205HG was downregulated in EAC vs. normal esophageal epithelia (NE) as well as in EAC cell lines, and its forced overexpression inhibited EAC cell proliferation and cell cycle progression in vitro. Similarly, overexpression of miR205HG inhibited xenograft tumor growth in mice In vivo. Finally, we show that one mechanism of action of miR205HG involves the Hh signaling pathway: miR205HG and Hh expression levels were inversely correlated in both EAC (r = -0.73) and BE (r = -0.83) tissues, and in vitro studies revealed details of Hh signaling inhibition induced by miR205HG. In conclusion, these findings establish that lncRNA miR205HG functions as a tumor suppressor in the development of BE and EAC, at least in part through its effect on the Hh signaling pathway.

Preconditioning of surgical pedicle flaps with DNA plasmid expressing hypoxia-inducible factor-1α (HIF-1α) promotes tissue viability.

Ischemic necrosis of surgical flaps after reconstruction is a major clinical problem. Hypoxia-inducible factor-1α (HIF-1α) is considered the master regulator of the adaptive response to hypoxia. Among its many properties, it regulates the expression of genes encoding angiogenic growth factors, which have a short half-life in vivo. To achieve a continuous application of the therapeutic, we utilized DNA plasmid delivery. Transcription of the DNA plasmid confirmed by qRT-PCR showed significantly increased mRNA for HIF-1α in the transfected tissue compared to saline control tissue. Rats were preconditioned by injecting with either HIF-1α DNA plasmid or saline intradermally in the designated flap region on each flank. Seven days after preconditioning, each rat had two isolated pedicle flaps raised with a sterile silicone sheet implanted between the skin flap and muscle layer. The flaps preconditioned with HIF-1α DNA plasmid had significantly less necrotic area. Angiogenesis measured by CD31 staining showed a significant increase in the number of vessels per high powered field in the HIF-1α group (p < 0.05). Our findings offer a potential therapeutic strategy for significantly promoting the viability of surgical pedicle flaps by ischemic preconditioning with HIF-1α DNA plasmid.

Targeting the Hedgehog Pathway Using Itraconazole to Prevent Progression of Barrett's Esophagus to Invasive Esophageal Adenocarcinoma.

OBJECTIVE: The aim of the study was to investigate whether inhibition of Sonic Hedgehog (SHH) pathway would prevent progression of Barrett's Esophagus (BE) to esophageal adenocarcinoma. BACKGROUND: The hedgehog signaling pathway is a leading candidate as a molecular mediator of BE and esophageal adenocarcinoma (EAC). Repurposed use of existing off-patent, safe and tolerable drugs that can inhibit hedgehog, such as itraconazole, could prevent progression of BE to EAC. METHODS: The efficacy of itraconazole was investigated using a surgical rat reflux model of Barrett's Metaplasia (BM). Weekly intraperitoneal injections of saline (control group) or itraconazole (treatment group; 200 mg/kg) were started at 24 weeks postsurgery. Esophageal tissue was harvested at 40 weeks. The role of the Hh pathway was also evaluated clinically. Esophageal tissue was harvested after 40 weeks for pathological examination and evaluation of the SHH pathway by immunohistochemistry. RESULTS: BM was present in control animals 29 of 31 (93%) versus itraconazole 22 of 24 (91%). EAC was significantly lower in itraconazole 2 of 24 (8%) versus control 10 of 31 (32%), respectively (P = 0.033). Esophageal SHH levels were lower in itraconazole vs control (P = 0.12). In esophageal tissue from humans with recurrent or persistent dysplastic BE within 24 months of ablative treatment, strong SHH and Indian Hedgehog expression occurred in distal BE versus proximal squamous epithelium, odds ratio = 6.1 (95% confidence interval: 1.6, 23.4) and odds ratio = 6.4 (95% confidence interval: 1.2, 32.8), respectively. CONCLUSION: Itraconazole significantly decreases EAC development and SHH expression in a preclinical animal model of BM. In humans, BE tissue expresses higher SHH, Indian Hedgehog, and bone morphogenic protein levels than normal squamous esophageal epithelium.

Striking heterogeneity of somatic L1 retrotransposition in single normal and cancerous gastrointestinal cells.

Somatic LINE-1 (L1) retrotransposition has been detected in early embryos, adult brains, and the gastrointestinal (GI) tract, and many cancers, including epithelial GI tumors. We previously found numerous somatic L1 insertions in paired normal and GI cancerous tissues. Here, using a modified method of single-cell analysis for somatic L1 insertions, we studied adenocarcinomas of colon, pancreas, and stomach, and found a variable number of somatic L1 insertions in tumors of the same type from patient to patient. We detected no somatic L1 insertions in single cells of 5 of 10 tumors studied. In three tumors, aneuploid cells were detected by FACS. In one pancreatic tumor, there were many more L1 insertions in aneuploid than in euploid tumor cells. In one gastric cancer, both aneuploid and euploid cells contained large numbers of likely clonal insertions. However, in a second gastric cancer with aneuploid cells, no somatic L1 insertions were found. We suggest that when the cellular environment is favorable to retrotransposition, aneuploidy predisposes tumor cells to L1 insertions, and retrotransposition may occur at the transition from euploidy to aneuploidy. Seventeen percent of insertions were also present in normal cells, similar to findings in genomic DNA from normal tissues of GI tumor patients. We provide evidence that: 1) The number of L1 insertions in tumors of the same type is highly variable, 2) most somatic L1 insertions in GI cancer tissues are absent from normal tissues, and 3) under certain conditions, somatic L1 retrotransposition exhibits a propensity for occurring in aneuploid cells.

SUFU mediates EMT and Wnt/ß-catenin signaling pathway activation promoted by miRNA-324-5p in human gastric cancer.

The poor prognosis of late gastric carcinomas (GC) underscores the necessity to identify novel biomarkers for earlier diagnosis and effective therapeutic targets. MiRNA-324-5p has been shown to be over-expressed in GC, however the biological function of miRNA-324-5p implicated in gastric cancer and its downstream targets were not well understood. Wnt/ß-catenin signaling pathway is aberrantly regulated in GC. We sought to explore if miRNA-324-5p promotes oncogenesis through modulating Wnt signaling and EMT. MiRNA-324-5p is highly expressed in GC based on qRT-PCR and TCGA data. In addition, in vitro cell proliferation, cell migration assays and in vivo animal exenograft were executed to show that miRNA-324-5p is an oncogenic miRNA in GC. MiRNA-324-5p activates Wnt signaling and induces EMT in GC. Further, SUFU was identified as a target of miRNA-324-5p confirmed by western blotting and luciferase assays. Spearson analysis and TCGA data indicate that the expression of SUFU is negatively associated with the expression of miRNA-324-5p. Rescue experiments were performed to determine if SUFU mediates the Wnt activation, EMT and oncogenic function of miRNA-324-5p. MiRNA-324-5p inhibitors plus SUFU siRNAs rescue partially the inhibitory effect on Wnt signaling and EMT caused by miRNA-324-5p inhibitors. Finally, the suppression of cell proliferation, migration, and colony formation ability induced by miRNA-324-5p inhibitors is alleviated by addition of SUFU siRNAs. In summary, miRNA-324-5p is overexpressed in vivo and exerts cell growth and migration-promoting effects through activating Wnt signaling and EMT by targeting SUFU in GC. It represents a potential miRNA with an oncogenic role in human gastric cancer.

Histone methyltransferase SET8 is regulated by miR-192/215 and induces oncogene-induced senescence via p53-dependent DNA damage in human gastric carcinoma cells.

Gastric cancer (GC) is the most common cancer throughout the world. Despite advances of the treatments, detailed oncogenic mechanisms are largely unknown. In our previous study, we investigated microRNA (miR) expression profiles in human GC using miR microarrays. We found miR-192/215 were upregulated in GC tissues. Then gene microarray was implemented to discover the targets of miR-192/215. We compared the expression profile of BGC823 cells transfected with miR-192/215 inhibitors, and HFE145 cells transfected with miR-192/-215 mimics, respectively. SET8 was identified as a proposed target based on the expression change of more than twofold. SET8 belongs to the SET domain-containing methyltransferase family and specifically catalyzes monomethylation of H4K20me. It is involved in diverse functions in tumorigenesis and metastasis. Therefore, we focused on the contributions of miR-192/215/SET8 axis to the development of GC. In this study, we observe that functionally, SET8 regulated by miR-192/215 is involved in GC-related biological activities. SET8 is also found to trigger oncogene-induced senescence (OIS) in GC in vivo and in vitro, which is dependent on the DDR (DNA damage response) and p53. Our findings reveal that SET8 functions as a negative regulator of metastasis via the OIS-signaling pathway. Taken together, we investigated the functional significance, molecular mechanisms, and clinical impact of miR-192/215/SET8/p53 in GC.