Adjacent Normal Tissue Samples Research Articles

Maternal embryonic leucine zipper kinase: A novel biomarker and a potential therapeutic target in lung adenocarcinoma.

Maternal embryonic leucine zipper kinase (MELK), is an adenosine monophosphate-activated protein kinase-related kinase that serves important roles in tumourigenesis in multiple malignant tumours. However, to the best of our knowledge, the effect of MELK in lung adenocarcinoma (LUAD) has not been elucidated. The present study aimed to explore the clinical significance of MELK in the prognosis of LUAD. Data from Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA) and The Cancer Genome Atlas (TCGA) were selected to predict the differential mRNA expression levels of MELK mRNA in LUAD and normal tissues. Subsequently, LUAD and adjacent normal tissue samples were collected from 75 patients with the disease, and immunohistochemistry was used to detect the protein expression of MELK. In addition, the Kaplan-Meier Plotter database, GEPIA and TCGA were used to verify the effect of MELK expression on clinical prognosis in patients with LUAD. MELK was significantly upregulated in LUAD tissues compared with that in normal tissues based on Oncomine, GEPIA and TCGA data (P<0.05). In addition, the results from immunohistochemistry demonstrated that the MELK protein level in LUAD tissues was significantly higher compared with that in matched normal tissues (P<0.05). Prognostic analysis performed using the Kaplan-Meier plotter, GEPIA and TCGA suggested that the expression of MELK was negatively associated with the overall survival time of patients with LUAD (P<0.05). In conclusion, MELK was highly expressed in LUAD based on bioinformatics and immunohistochemistry analysis, and increased expression of MELK was associated with a poor patient prognosis. MELK may serve as a potential diagnostic marker and therapeutic target for LUAD.

Abstract 4266: Proteomics for precision oncology: Profiling the proteome of matching tumor and adjacent normal tissue using data-independent acquisition

Abstract Precision oncology requires a deep understanding of the molecular mechanism in cancer biology. The predominant approach today focuses on genome structure and gene expression data, which have become available with the rise of next-gen sequencing technology. On a phenotypic level, however, protein expression and activation are arguably more directly related to cellular function. The availability of combined genome and proteome level data from the same tumor is therefore expected to provide a much more complete picture of a tumor in a particular state. Until recently, proteomics technology could not match the scale of next-gen sequencing and consequently, precision medicine research has almost exclusively been based on gene-level data. Here we show the first truly large-scale data set for protein expression and protein phosphorylation for a large collection of biospecimens derived from the IndivuType cohort of Indivumed, Germany. Matching fresh-frozen tumor and adjacent normal tissue samples from thousands of patients including various cancer entities were obtained from Indivumed's network of partner hospitals Enabled by the novel data-independent acquisition (DIA) workflow, a mass spectrometric method that obtains peptide fragmentation data in a highly parallelized way with high reproducibility and sensitivity, more than 7,000 proteins in the whole proteome (WP) and 20,000 phospho-peptides in the phospho-proteome (PP) workflow were analyzed. Sample processing from 5 mg of tissue per sample was performed on 96-well plates with the help of a liquid handling robot. Phospho-peptide enrichment was carried out using a Kingfisher Flex device and MagReSyn Ti-IMAC magnetic beads (ReSyn Biosciences). Data-independent acquisition (DIA) LC-MS/MS was performed on multiple platforms consisting of a Thermo Scientific Q Exactive HF-X mass spectrometer coupled to a Waters M-Class LC. Chromatography was operating at 5 µL/min, and separation was achieved using 45 min (WP) and 60 min (PP) gradients. With a throughput of 850 WP and 650 PP samples per month, thousands of samples were analyzed to date. The resulting proteome data is integrated into Indivumed's IndivuType multi-omics database, supporting the identification and validation of new molecular cancer drug targets and biomarkers. Citation Format: Jakob Vowinckel, Karel Novy, Thomas Corwin, Tobias Treiber, Roland Bruderer, Lukas Reiter, Eike von Leitner, Oliver Rinner, Claudia Escher. Proteomics for precision oncology: Profiling the proteome of matching tumor and adjacent normal tissue using data-independent acquisition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4266.

Abstract LB-232: Deep integrated proteogenomic characterization of clear cell renal cell carcinoma

Abstract Clear cell renal cell carcinoma (ccRCC) is the predominant histology of renal cell carcinoma (RCC), representing 75% of all cases and accounting for the majority of associated deaths. Towards understanding the impact of genomic alterations on the functional modules that drive ccRCC tumorigenesis, the Clinical Proteomics Tumor Analysis Consortium (CPTAC) leveraged a proteogenomic approach to characterize 110 RCC tumors and 84 paired normal adjacent tissue (NAT) samples. Pairing state-of-the-art mass spectrometry based proteomic and phosphoproteomic strategies with comprehensive genomic and transcriptomic analyses, we integrated global and phosphoproteomic measurements with genomic and transcriptomic data to elucidate the dysregulated cellular mechanism resulting from genomic alterations. In addition to delineating chromosomal translocations involving chromosome 3p as a possible mechanism for 3p loss in ccRCC and the altered cellular pathways between tumors and NATs, we also identified several novel proteomic, phosphoproteomic, and immune ccRCC subtypes for personalized, precision-based care. Investigation of the phosphoproteome in our cohort revealed the activation of cell signaling pathways associated with ERK/MAPK, PI3K/AKT, metabolic reprogramming, and G2/M cell cycle regulation. Leveraging FDA-approved kinase inhibitors, prioritized by our phosphoproteomic results, allowed for the examination of the functional consequences of these therapies respective to mechanisms of action in ccRCC cells. Independently, the immunogenicity of ccRCC tumors prompted our exploration of the degree of immune infiltration in our cohort, wherein we identified four subtypes based on immune and stromal tumor microenvironment cell compositions: CD8+ Inflamed, CD8- Inflamed, VEGF Immune Desert, and Metabolic Immune Desert. These subtypes were further discriminated by proteomic and transcriptomic features, as well as displaying distinct predicted outcomes related to patient survival and response to targeted therapeutic intervention. We verified the protein features of these subtypes via orthogonal methodologies, including data-independent acquisition (DIA) and selective reaction monitoring (SRM) mass spectrometry, finding a high degree of correlation between the datasets. Further examination of these proteomic features in an independent cohort found the subtypes robustly identified; providing further evidence of ccRCC inter-tumor heterogeneity and rationale to stratify ccRCC patients for treatment selection. Overall, our study highlights the unique insight that can be gained when integrating multi-level “omics” analyses to broaden our understanding of ccRCC biology, as well as leading to the identification of several new hypothesis-driven investigations for therapeutic intervention in ccRCC. Citation Format: David J. Clark, Clinical Proteomic Tumor Analysis Consortium. Deep integrated proteogenomic characterization of clear cell renal cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-232.

Abstract 2158: ART1 tumor expression mediates immune resistance in non-small cell lung cancer by elimination of P2 × 7R+ CD8 tissue resident memory T cells and conventional type I dendritic cells

Abstract Extracellular NAD+ (eNAD) is released from stressed and dying cells where it acts as a proinflammatory mediator. However, in the presence of mono-ADP-ribosyltransferases (ARTs) eNAD serves as a substrate for ADP-ribosylation of the P2 × 7 receptor (P2RX7), resulting in NAD-induced cell death (NICD). P2RX7 is expressed on several immune cells including CD103+ conventional type I dendritic cells (cDC1s) which are essential for initiation of tumor-specific immune responses as well as on tissue resident memory T cells (TRMs), of which high tumor infiltration has been associated with improved survival in non-small cell lung cancer (NSCLC) [1]. NICD of P2 × 7R+ TRMs was recently identified as a regulator of TRM tissue homeostasis [2]. CD38 is expressed on activated immune cells and can reduce eNAD levels by converting eNAD into ADPR. We tested the hypothesis that ART1 expression in NSCLC constitutes a mechanism of immune resistance by mediating NICD of P2 × 7R+ TRMs and P2 × 7R+ cDC1s. ART1 expression in human NSCLC samples was determined by immunohistochemistry scoring of NSCLC tumors using a tissue microarray (TMA) (n=493). In addition, matched tumor and adjacent normal lung tissue samples were analyzed for ART1 expression by qPCR (n=40) and for infiltration of TRM and cDC1s by flow cytometry. A mouse KP1 lung tumor cell line with high ART1 expression was established from KRASG12D/P53−/− mice and transduced with a doxycycline-inducible shRNA targeting ART1 (ART1KD). KP1 cells and their derivatives were intravenously injected into syngeneic C57BL/6 mice to generate orthotopic lung tumors. Lung tumor nodules were enumerated two weeks after tumor inoculation using H&amp;E-stained FFPE lung sections and lung-derived cell suspensions were analyzed by flow cytometry for infiltration of T cell and DC subsets. Increased expression of ART1 in tumor compared to normal lung was observed in 55% of NSCLC samples by TMA analysis, and confirmed by qPCR. The frequency of P2RX7+ CD8 TRMs (p&amp;lt;0.05) and P2 × 7R+ cDC1s (p&amp;lt;0.01) were reduced in ART1+ tumors compared to matched normal lung tissue. In line with the hypothesis that CD38 protects P2 × 7R+ cells from ART1-mediated NICD, P2 × 7R+ CD8 TRMs and P2 × 7R+ cDC1s present within ART1+ tumors had elevated expression of CD38. In mice, knockdown of ART1 in KP1 tumors resulted in significantly delayed growth in immunocompetent mice (p&amp;lt;0.001) but not in nude or CD8-depeleted mice. In an orthotopic lung model, ART1KD resulted in a 75% decrease in lung metastatic nodules (p&amp;lt;0.01) and was associated with increased infiltration of CD8 T cells (P&amp;lt;0.0001) and cDC1s (p&amp;lt;0.01). In vitro experiments confirmed that CD38- P2RX7+ CD8 T cells were susceptible to ADP-ribosylation and NICD when co-cultured with ART1high lung tumor cells, while P2 × 7R+ CD8 T cells that co-expressed CD38 were resistant to NICD. In conclusion, we show that ART1 is overexpressed in NSCLC and identify ART1-mediated NICD of P2 × 7R+ TRMs and cDC1s as a possible novel immune escape mechanism in NSCLC. Furthermore, we describe a novel role of CD38 expression on immune cells in its protection against NICD. 1. Nizard, M., et al., Induction of resident memory T cells enhances the efficacy of cancer vaccine. Nat Commun, 2017. 8: p. 15221. 2. Stark, R., et al., T RM maintenance is regulated by tissue damage via P2RX7. Sci Immunol, 2018. 3(30). Citation Format: Erik Wennerberg, Clarey Hung, Amanda Valeta, Timothy McGraw, Sandra Demaria, Brendon Stiles. ART1 tumor expression mediates immune resistance in non-small cell lung cancer by elimination of P2 × 7R+ CD8 tissue resident memory T cells and conventional type I dendritic cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2158.

Abstract 3623: Differences in the frequency of CRC-associated pathogen variants in colorectal adenomas and cancers from the Hungarian population

Abstract Background: Colorectal cancer (CRC) has high incidence in Eastern-Europe, especially in Hungary. In addition to environmental factors, the genetic and epigenetic background of this phenomenon still remains unknown. Aims: We aimed to perform whole exome sequencing along the colorectal adenoma-carcinoma sequence on tissue samples in order to identify characteristic germline and somatic variants. Materials &amp; methods: Genomic DNA was isolated from 111 colonic tissue samples including 20 adenomas (AD) with matched normal adjacent tissue (NAT) pairs, 18 CRC with matched NAT pairs, 3 normal (N) young donors, 1 CRC and 1 AD and from 2 colorectal cancer cell lines (HT29, SW480), furthermore, 20 buffy coat (from 10 healthy, 5 adenoma and CRC patients). Exome enrichment was done with the Nextera DNA Exome Kit and sequencing was performed with the NextSeq 500/550 High Output v2 kit using the NextSeq 500 Instrument. Raw data analysis and demultiplexing was completed on BaseSpace Sequence Hub. Germline and somatic variants were determined by Genome Analysis Toolkit (GATK) and MuTect2 algorithms by using the default filter settings and were confirmed by ddPCR. Results: The total number of detected variants increased along the adenoma-carcinoma sequence and NAT samples showed higher number of variants compared to healthy tissues. On the basis of the somatic variant analysis, certain genes with the most frequent CRC-associated pathogenic mutations could be detected in relatively higher [e.g. APC (89% of AD samples, 74% of CRC samples)], with relatively lower [e.g. KRAS (47% of ADs, 33% CRC), TP53 (26% of ADs, 14% of CRCs), or with average frequencies [PIK3CA (4.8% of AD samples, 11% of CRC samples), NRAS (5% of ADs, 4.7% of CRCs)]. One adenoma sample (4.7%) exerted BRAF mutation. On the basis of the COSMIC database, the expected variant profile of the CRC cell lines could be confirmed (HT-29: BRAF, TP53; SW480: KRAS, TP53). Among the germline variants, MTHFR C677T (rs1801133) were detected in 4 N (40% CT) 8 AD (37% CT, 5% TT) 9 CRC samples (37% CT, 11% TT), and MTHFR A1298C (rs1801131) were detected in 7 N (50% AC, 20% CC) 12 AD (47% AC, 16% CC) and 10 CRC samples (42% AC, 11% CC) representing a relatively higher frequency in the Hungarian population compared to the European and worldwide statistics. Conclusion: Identification of CRC-associated pathogen variant patterns in the Hungarian population revealed a higher tumor mutation burden already in pre-malignant colorectal adenoma samples, as well as in CRC. Alterations in the frequencies of CRC-associated somatic variants compared to the European averages might contribute to the high CRC incidence observed in the Hungarian population. The germline variants of MTHFR gene were observed with a relatively higher frequency in our cohort having an effect on the DNA methylation regulation system that might be a link between the genetic and epigenetic alterations of the CRC development. Citation Format: Alexandra Kalmar, Orsolya Galamb, Barnabas Wichmann, Barbara B Bartak, Zsofia B Nagy, Sara Zsigrai, Krisztina Szigeti, Orsolya Pipek, Anna Medgyes-Horváth, Istvan Csabai, Zsolt Tulassay, Peter Igaz, Bela Molnar. Differences in the frequency of CRC-associated pathogen variants in colorectal adenomas and cancers from the Hungarian population [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3623.

LncRNA DLG2-AS1 as a Novel Biomarker in Lung Adenocarcinoma.

Long non-coding RNAs (lncRNAs) are a heterogeneous class of non-coding RNAs whose biological roles are still poorly understood. LncRNAs serve as gene expression regulators, frequently interacting with epigenetic factors to shape the outcomes of crucial biological processes, and playing roles in different pathologies including cancer. Over the last years, growing scientific evidence supports the key role of some lncRNAs in tumor development and proposes them as valuable biomarkers for the clinic. In this study, we aimed to characterize lncRNAs whose expression is altered in tumor samples from patients with lung adenocarcinoma (LUAD) compared to adjacent normal tissue samples. On an RT-qPCR survey of 90 cancer-related lncRNAs, we found one lncRNA, DLG2-AS1, which was consistently downregulated in 70 LUAD patients. To gain insight into its biological function, DLG2-AS1 was cloned and successfully re-expressed in LUAD cancer cell lines. We determined that DLG2-AS1 is not a cis-regulatory element of its overlapping gene DLG2, as their transcription levels were not correlated, nor did DLG2-AS1 restoration modify the expression of DLG2 protein. Furthermore, after generating a receiver operating curve (ROC) and calculating the area under curve (AUC), we found that DLG2-AS1 expression showed high sensitivity and specificity (AUC = 0.726) for the classification of LUAD and normal samples, determining its value as a potential lung cancer biomarker.

RNA-Seq Profiling of Circular RNAs and the Oncogenic Role of circPVT1 in Cutaneous Squamous Cell Carcinoma.

BackgroundCutaneous squamous cell carcinoma (CSCC) is associated with a poor 5-year survival rate. circRNAs have an important role in a number of physiological and pathological processes. However, the relationship between circRNAs and cutaneous squamous cell carcinoma (CSCC) is unclear.PurposeThe aim of the present study was to investigate the expression of circRNAs in cutaneous squamous cell carcinoma (CSCC) and its effect on CSCC proliferation and metastasis.MethodsWe used high-throughput sequencing (RNA-seq) to identify circRNAs that were differentially in CSCC tissue and their paracarcinoma tissue. Quantitative real-time PCR results confirm deep-sequencing findings in CSCC tissue and cell lines. CCK-8 assay and flow cytometry were used to detect the effect of circPVT1 on the proliferation and migration of CSCC cells.ResultsWe identified 449 circRNAs that were differentially expressed between CSCC and normal adjacent tissue samples. circPVT1 (hsa_circ_0001821) was further researched to confirm its oncogene role in CSCC.ConclusionDifferentially expressed circular RNA plays an important role in the development of CSCC, and circPVT1 may be an important target for the treatment of CSCC.

BRAFV600E mutation, BRAF-activated long non-coding RNA and miR-9 expression in papillary thyroid carcinoma, and their association with clinicopathological features

BackgroundThe incidence of thyroid cancer is increasing worldwide. This study investigated the association of B-type RAF kinase (BRAF)V600E mutation status, the expression of BRAF-activated long non-coding RNA (BANCR) and microRNA miR-9, and the clinicopathological features of papillary thyroid carcinoma (PTC).MethodsClinicopathological data for PTC patients (n = 51) diagnosed and treated between 2018 and 2019 were collected. Carcinoma and adjacent normal tissue samples were analyzed for the presence of the BRAFV600E mutation and/or expression of BANCR and miR-9.ResultsLarger tumor, higher rate of bilateral tumors and multifocality, extracapsular invasion, and lateral lymph node metastasis (LNM) were observed in PTC patients with BRAF V600E mutation. Patients with higher BANCR expression had a higher rate of extracapsular invasion and lateral LNM in carcinoma tissue and a lower frequency of bilateral tumors and multifocality in normal adjacent tissue. Patients with higher miR-9 expression had a lower rate of central and lateral LNM in carcinoma tissue and higher rates of bilateral tumor location and multifocality in normal adjacent tissue. Patients with BRAFV600E mutation have a higher rate of BANCR overexpression and tended to have a lower rate of miR-9 overexpression (P = 0.057), and a negative association was observed between BANCR and miR-9 expression in carcinoma tissue.ConclusionsBRAFV600E mutation and the BANCR and miR-9 expression were closely associated with the tumor size, bilateral tumor location, multifocality, extracapsular invasion, and lateral LNM. PTC patients with these clinicopathological characteristics, BRAFV600E mutation, and high BANCR expression and low miR-9 expression needed earlier surgical treatment and are recommended for total thyroidectomy in primary surgery for reducing the risk of recurrence. These findings provide new insight into the molecular basis for PTC and can inform strategies for the management of PTC.

TIPE1-mediated autophagy suppression promotes nasopharyngeal carcinoma cell proliferation via the AMPK/mTOR signalling pathway.

Recent studies have shown that tumour necrosis factor‐α–induced protein 8 like‐1(TIPE1) plays distinct roles in different cancers. TIPE1 inhibits tumour proliferation and metastasis in a variety of tumours but acts as an oncogene in cervical cancer. The role of TIPE1 in nasopharyngeal carcinoma (NPC) remains unknown. Interestingly, TIPE1 expression was remarkably increased in NPC tissue samples compared to adjacent normal nasopharyngeal epithelial tissue samples in our study. TIPE1 expression was positively correlated with that of the proliferation marker Ki67 and negatively correlated with patient lifespan. In vitro, TIPE1 inhibited autophagy and induced cell proliferation in TIPE1‐overexpressing CNE‐1 and CNE‐2Z cells. In addition, knocking down TIPE1 expression promoted autophagy and decreased proliferation, whereas overexpressing TIPE1 increased the levels of pmTOR, pS6 and P62 and decreased the level of pAMPK and the LC3B. Furthermore, the decrease in autophagy was remarkably rescued in TIPE1‐overexpressing CNE‐1 and CNE‐2Z cells treated with the AMPK activator AICAR. In addition, TIPE1 promoted tumour growth in BALB/c nude mice. Taken together, results indicate that TIPE1 promotes NPC progression by inhibiting autophagy and inducing cell proliferation via the AMPK/mTOR signalling pathway. Thus, TIPE1 could potentially be used as a valuable diagnostic and prognostic biomarker for NPC.

Activation of the LINC00242/miR-141/FOXC1 axis underpins the development of gastric cancer

BackgroundLong non-coding RNAs (LncRNAs) are a class of newly identified transcripts recognized as critical governors of gene expression during human carcinogenesis, whereas their tumor-suppressive or tumor-promoting effects on gastric cancer (GC) are required for further investigation. In the study, we identify the expression pattern of a novel lncRNA LINC00242 in GC and its possible permissive role in the development of GC.MethodsThe study included 68 pairs of GC and adjacent normal gastric tissue samples. The viability, migration, and invasion of cultured human GC cells HGC27 were evaluated by CCK-8 and Transwell chamber assays. In vitro tube formation of human brain microvascular endothelial cells (HBMVECs) in HGC27 cell coculture was detected. The regulatory network of LINC00242/miR-141/FOXC1 was verified using dual luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay. Subcutaneous xenografts of HGC27 cells were performed in nude mice.ResultsLINC00242 was highly expressed in GC tissues and cells and contributed to poor prognosis. LINC00242 knockdown inhibited HGC27 cell viability, migration and invasion, and tube formation of HBMVECs. LINC00242 interacted with miR-141 and positively regulated FOXC1, a target gene of miR-141. LINC00242 knockdown was partially lost in HGC27 cells upon miR-141 inhibition or FOXC1 overexpression. The tumor-promoting effect of LINC00242 on GC was demonstrated in nude mice.ConclusionTaken together, the present study demonstrates the oncogenic role of the LINC00242/miR-141/FOXC1 axis in GC, highlighting a theoretical basis for GC treatment.

SPANXN2 functions a cell migration inhibitor in testicular germ cell tumor cells.

BackgroundSPANX family members are thought to play an important role in cancer progression. The SPANXN2 is a gene expressed mainly in normal testis, but its role in testicular germ cell tumors (TGCTs) has yet to be investigated. TGCT is one of the most common solid tumors in young men and is associated with poor prognosis; however, effective prognostic indicators remain elusive. Therefore, we investigated the role of SPANXN2 in TGCT development.MethodsSPANXN2 expression levels were validated by quantitative real-time polymerase chain reaction (qRT-PCR) analyses of 14 TGCT samples and five adjacent normal tissue samples. SPANXN2 was transiently overexpressed in TGCT cells to study the consequences for cell function. The effects of SPANXN2 on cell migration were evaluated in transwell and wound healing assays. The effects on cloning ability were evaluated in colony formation assays. MTT assays and cell cycle analysis were used to detect the effects of SPANXN2 on cell proliferation. The expression levels of EMT- and AKT-related proteins in cells overexpressing SPANXN2 were analyzed by Western blotting.ResultsCompared with adjacent normal tissues, the Gene Expression Profiling Interactive Analysis database showed SPANXN2 expression was downregulated in TGCTs which was consistent with the qRT-PCR analysis. SPANXN2 overexpression reduced cell migration and colony formation capability and downregulated expression of EMT- and AKT-related proteins, Vimentin, Snail, AKT, and p-AKT.ConclusionOur results suggest that SPANXN2 regulates TGCT cell migration via EMT- and AKT-related proteins although its role in the occurrence and development of TGCT remains to be fully elucidated.

The Expression of Dynamin 1, 2, and 3 in Human Hepatocellular Carcinoma and Patient Prognosis.

BackgroundThe classical dynamin family consists of dynamin 1, 2, and 3, which have different expression levels in different tissues to regulate cell membrane fission and endocytosis. Recent studies have reported increased expression of dynamins in human cancer, but their expression in hepatocellular carcinoma (HCC) remains to be determined. This study aimed to investigate the expression of dynamin 1, 2, and 3 in tissue sections of human HCC using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry.Material/MethodsThe expression of dynamin 1, 2, and 3 were investigated in 192 cases of HCC and 14 paired samples of HCC and adjacent normal liver tissue by qRT-PCR and immunohistochemistry. The clinical significance of dynamin 1, 2, and 3 were determined by correlating their expression levels with patient clinicopathological factors and survival rates. Independent prognostic factors were determined using the Cox regression hazard model.ResultsIn tissue samples from 192 patients with HCC, the expression of dynamin 1, 2, and 3 were upregulated in 41.15%, 29.69%, and 8.33% of cases, respectively. Dynamin 1 had a significantly increased mRNA expression level in HCC compared with adjacent normal liver tissues and was significantly correlated with alpha fetoprotein (AFP) levels, T stage, and TNM stage. Only dynamin 1 expression was correlated with the reduced overall survival (OS), and was identified as an independent prognostic biomarker of human HCC.ConclusionsUpregulation of dynamin 1 at the protein and mRNA level was an independent prognostic biomarker of reduced OS in patients with HCC.

Blood-derived DNA methylation predictors of mortality discriminate tumor and healthy tissue in multiple organs.

Evidence has shown that certain methylation markers derived from blood can mirror corresponding methylation signatures in internal tissues. In the current study, we aimed to investigate two strong epigenetic predictors for life span, derived from blood DNA methylation data, in tissue samples of solid cancer patients. Using data from the Cancer Genome Atlas (TCGA) and the German DACHS study, we compared a mortality risk score (MRscore) and DNAmPhenoAge in paired tumor and adjacent normal tissue samples of patients with lung (N = 69), colorectal (n = 299), breast (n = 90), head/neck (n = 50), prostate (n = 50), and liver (n = 50) cancer. To explore the concordance across tissue and blood, we additionally assessed the two markers in blood samples of colorectal cancer (CRC) cases and matched controls (n = 93) in the DACHS+ study. The MRscore was significantly elevated in tumor tissues compared to normal tissues of all cancers except prostate cancer, for which an opposite pattern was observed. DNAmPhenoAge was consistently higher in all tumor tissues. The MRscore discriminated lung, colorectal, and prostate tumor tissues from normal tissues with very high accuracy [AUCs of 0.87, 0.99 (TCGA) /0.94 (DACHS), and 0.92, respectively]. DNAmPhenoAge accurately discriminated five types of tumor tissues from normal tissues (except prostate cancer), with AUCs of 0.82–0.93. The MRscore was also significantly higher in blood samples of CRC cases than in controls, with areas under the curve (AUC) of 0.74, whereas DNAmPhenoAge did not distinguish cases from controls, with AUC of 0.54. This study provides compelling evidence that blood‐derived DNAm markers could reflect methylation changes in less accessible tissues. Further research should explore the potential use of these findings for cancer diagnosis and early detection.

DNA methylation signatures predicting liver metastasis of colorectal cancer: A proof-of-concept pilot study.

e16080 Background: Liver metastasis (LIM) is the leading cause of death in colorectal cancer (CRC) patients. Early detection of LIM may improve outcome in CRC patients. The aim of this study was to evaluate the feasibility of predicting LIM of CRC using methylation profiles. Methods: We performed Roche targeted (~5.5 million methylation sites) bisulfite sequencing of matched primary, metastatic and their adjacent normal tissue samples from 5 CRC patients with LIM, 5 patients with lung metastasis (LUM) and 8 patients without metastasis in the training cohort (n = 48 samples). Differential methylation regions (DMR) of LUM were identified and a predictive model was developed. The model was further validated in primary tumor sample from nine patients (6 with LIM). Results: By comparing primary tumor vs adjacent normal tissues and metastatic tumor vs adjacent normal tissues in CRC patients with LIM, we identified 28954 common DMRs which indicating the methylation characteristic of CRC with LIM. Similarly, 16187 DMRs were identified in patients with LUM. 9179 DMRs are shared in both LIM and LUM comparisons which should be the common characteristic of CRC tumor tissue regardless of the location of metastasis. 7008 DMRs are LUM specific and 19775 DMRs are LIM specific. In order to predict LIM in primary, early changes in LIM specific DMRs should be identified. Hence, we further selected 4134 DMRs by chossing significantly differentically methylated regions between LIM primary tissues and LUM primary tissues. To increase the ability of distinguishing LIM from other normal tissues and non-matastasis CRC tumors, 1215 DMRs were finally selected which also showed increasing or decreasing trend of methylation level through the progression of CRC. The final 1215 biomarkers were used to construct a random forest model using methlylation profile of 5 CRC patients with LIM as positive training data and 5 CRC patients with LUM as well as 8 patients without metastasis as negative training data. Through the feature recursive elimination method, one methylation site (chr8.72468901-72469000) was identified with ROC of 0.9 in the training dataset. The predictive model was validated in an independent dataset which is composed of 6 patients with LIM and 3 patients without metastasis, and achieved an AUC of 0.87. Conclusions: Our findings demonstrate the utility of methylation biomarkers for the molecular characterization of metastatic precursors, with implications for prediction and early detection of liver metastasis in CRC.

XPD inhibits cell growth and invasion and enhances chemosensitivity in esophageal squamous cell carcinoma by regulating the PI3K/AKT signaling pathway.

Esophageal squamous cell carcinoma (ESCC) is a lethal disease due to its high aggressiveness. The aim of the present study was to investigate the role of xeroderma pigmentosum complementation group D (XPD) in the growth and invasion of ESCC and to elucidate the potential underlying molecular mechanisms. Western blot analysis and RT-qPCR were used to detect the expression level of XPD in ESCC tissue samples and adjacent normal esophageal tissue samples. The pEGFP-N2/XPD plasmid was transfected into human ESCC cell lines (EC9706 and EC109). The proliferation, apoptosis, migration and invasion of EC9706 or EC109 cells were assessed following transfection with the XPD overexpression plasmid. The chemosensitivity of EC9706 or EC109 cells to cisplatin or fluorouracil was evaluated by CCK-8 assay. The expression levels of phosphoinositide 3-kinase (PI3K)/AKT, nuclear factor (NF)-κB, Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK) signaling pathway-related genes were detected by RT-qPCR and western blot analysis. The results demonstrated that the expression level of XPD was markedly lower in ESCC tissue samples than in adjacent normal esophageal tissue samples. The pEGFP-N2/XPD plasmid was successfully transfected into EC9706 or EC109 cells, inducing XPD overexpression. A High XPD expression markedly suppressed cell proliferation, migration and invasion, and increased the apoptotic rate of EC9706 and EC109 cells. Furthermore, the overexpression of XPD significantly increased the chemosensitivity of EC9706 and EC109 cells to cisplatin or fluorouracil. Following XPD overexpression, the expression levels of PI3K, p-AKT, c-Myc, Cyclin D1, Bcl-2, vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 were markedly downregulated, while the expression level of p21 was markedly upregulated. On the whole, the findings of the present study demonstrate that XPD inhibits the growth and invasion of EC9706 and EC109 cells, whilst also enhancing the chemosensitivity of EC9706 and EC109 cells to cisplatin or fluorouracil by regulating the PI3K/AKT signaling pathway. XPD may thus be an underlying target for ESCC treatment and drug resistance.

High long non-coding LIFR-AS1 expression correlates with poor survival in gastric carcinoma.

Abundant evidence has demonstrated that long non-coding RNAs (lncRNAs) play key roles in the development of human neoplasms. A novel cancer-related lncRNA, leukemia inhibitory factor receptor antisense RNA 1 (LIFR-AS1), has been reported to be under-expressed in breast cancer and associated with poor prognosis, but its significance in gastric cancer (GC) remains to be determined. Therefore, we assessed the prognostic and diagnostic value of LIFR-AS1 in GC. Quantitative RT-PCR assay was used to detect the expression levels of LIFR-AS1 in GC tissues and adjacent normal tissues. The correlation between LIFR-AS1 expression and clinicopathological features was analyzed by Pearson's χ2-test. The disease-free survival and overall survival rates of GC patients were calculated by the Kaplan-Meier method. Cox regression analysis was used to assess factors related to survival. In this study, levels of LIFR-AS1 were significantly higher in GC tumor samples relative to adjacent normal tissue samples. A ROC analysis suggested LIFR-AS1 expression could be reliably used to differentiate between normal and GC tumor tissue. In addition, elevated LIFR-AS1 expression was positively correlated with more advanced and aggressive GC features, such as larger tumor size, lymphatic metastasis, and more advanced TNM stage. Survival analyses revealed that elevated LIFR-AS1 expression was correlated with worse overall survival and disease-free survival. Multivariate analysis further confirmed the relevance of LIFR-AS1 as an independent predictor of GC patient outcomes. In summary, these results indicate that the lncRNA LIFR-AS1 is a promising prognostic indicator in GC patients.

The landscape of CD28, CD80, CD86, CTLA4, and ICOS DNA methylation in head and neck squamous cell carcinomas

ABSTRACT CTLA-4 blocking therapeutic antibodies are currently under investigation in head and neck squamous cell carcinoma (HNSCC). A better understanding of the epigenetic regulation of the CD28 superfamily members CD28, CTLA-4, and ICOS and their B7 ligands, CD80 and CD86, could support the development of biomarkers for response prediction to anti-CTLA-4 immunotherapy. We investigated methylation of the encoding genes CD28, CTLA4, ICOS, CD80, and CD86 at single CpG resolution (51 CpG sites) in a cohort of HNSCC (N = 528) and normal adjacent tissue samples (N = 50) provided by The Cancer Genome Research Atlas, in isolated blood leukocytes from healthy individuals (N = 28), and HNSCC cell lines (N = 39). We analysed methylation levels with regard to mRNA expression, overall survival, mutational load, interferon-γ signature, and signatures of immune cell infiltrates. Depending on the location of the CpG sites (promoter, promoter flank, gene body, and intergenic sites), we found significant differences in methylation levels among isolated leukocytes, between leukocytes and HNSCC cell lines, and among HNSCCs. Methylation of all analysed genes correlated inversely or positively with mRNA expression, depending on the CpG site. CD28, CTLA4, and ICOS revealed almost identical correlation patterns. Furthermore, we found significant correlations with survival and features of response to immunotherapy, i.e. interferon-γ signature, signatures of tumour infiltrating immune cells, and mutational load. Our results suggest CD28, CTLA4, ICOS, CD80, and CD86 expression levels are epigenetically co-regulated by DNA methylation. This study provides rationale to test their DNA methylation as potential biomarker for prediction of response to CTLA-4 immune checkpoint inhibitors.

Integrin Subunit Alpha 5 (ITGA5) Gene Circular RNA Sponges microRNA-107 in Colorectal Carcinoma Cells and Tissues and Regulates the Expression of the Forkhead Box J3 (FOXJ3) Gene.

BackgroundCircular RNAs (circRNAs) can function as sponges for microRNA (miRNA) in carcinogenesis. This study aimed to investigate the role of the circRNA of the integrin subunit alpha 5 (ITGA5) gene and microRNA-107 (miR-107) in human colorectal carcinoma (CRC) cells in vitro and tissue samples from patients with CRC and the expression of forkhead box J3 (FOXJ3) protein.Material/MethodsThirty paired CRC tissue samples and adjacent normal colon tissue samples were studied. Human CRC cell lines, including HT29, SW480, LoVo, and HIEC cells, were studied for cell proliferation using the cell counting kit-8 (CCK-8) assay. Cell migration was studied using a transwell assay, and cell apoptosis was determined using flow cytometry. The luciferase reporter assay was used to study the interactions between ITGA5 circRNA, FOXJ3, and miR-107 in human CRC cells. The expression of ITGA5 circRNA and miR-107 was determined using quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of FOXJ3 were measured by Western blot.ResultsThe expression of ITGA5 circRNA was significantly reduced in CRC tissues and CRC cell lines. High ITGA5 circRNA expression inhibited the proliferation and cell migration of CRC cells and promoted the apoptosis of CRC cells. The luciferase reporter assay confirmed that ITGA5 circRNA bound to miR-107, which directly targeted FOXJ3.ConclusionsITGA5 circRNA may act as a sponge for miR-107 to upregulate FOXJ3 expression and act as a tumor suppressor in CRC cells.

Association of lncRNA-p53 regulatory network (lincRNA-p21, lincRNA-ROR and MALAT1) and p53 with the clinicopathological features of colorectal primary lesions and tumors.

Colorectal cancer (CRC) is a common intestinal cancer with a high mortality rate. Early detection of this type of cancer is fundamental to the prevention of the disease, which results in improved survival rates. In the human colon tissue, transition from normal epithelium to adenoma is considered to be caused by unknown molecular incidents occurring over 5–10 years. The detection of CRC has proved problematic when in the early stages of disease. In addition, identifying suitable biomarkers for the detection of CRC progress in patients remains one of the most significant challenges. Long non-coding RNAs have been demonstrated to contribute to the promotion of CRC. The aim of the present study was to investigate the clinical and biological significance of long intergenic non-coding (linc)RNA-p21, lincRNA-regulator of reprogramming (ROR) and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in the colon tumor and polyp tissue, and the association that these have with the expression of p53 at the mRNA level. Neoplastic and paired adjacent normal tissue samples were obtained from 72 patients (46 polyps and 26 tumors). Reverse transcription-quantitative PCR was performed to determine the relative fold changes in the expression of lincRNA-p21, lincRNA-RoR, MALAT1 and p53 in the samples. A significant association was observed between the levels of MALAT1 and p53 in neoplasm tissues (R=0.073; P<0.05). The relative expression of the MALAT1 gene revealed a statistically significant difference between the different polyp types and number of polyps (P=0.0028 and 0.022, respectively). Adjuvant therapy in patients with tumors revealed an association between the levels of lincRNA-ROR and lincRNA-p21 expression (P=0.015 and 0.038, respectively). MALAT1 may be selected as an early detection biomarker for CRC. Furthermore, lincRNA-ROR and lincRNA-p21 may serve as prognostic and therapeutic biomarkers in patients with CRC.

Association of mutation and low expression of the CTCF gene with breast cancer progression

BackgroundCTCF encodes 11-zinc finger protein which is implicated in multiple tumors including the carcinoma of the breast. The Present study investigates the association of CTCF mutations and their expression in breast cancer cases. MethodsA total of 155 breast cancer and an equal number of adjacent normal tissue samples from 155 breast cancer patients were examined for CTCF mutation(s) by PCR-SSCP and automated DNA sequencing. Immunohistochemistry (IHC) method was used to analyze CTCF expression. Molecular findings were statistically analyzed with various clinicopathological features to identify associations of clinical relevance. ResultsOf the total, 16.1% (25/155) cases exhibited mutation in the CTCF gene. Missense mutations Gln > His (G > T) in exon 1 and silent mutations Ser > Ser (C > T) in exon 4 of CTCF gene were analyzed. A significant association was observed between CTCF mutations and some clinicopathological parameters namely menopausal status (p = 0.02) tumor stage (p = 0.03) nodal status (p = 0.03) and ER expression (p = 0.04). Protein expression analysis showed 42.58% samples having low or no expression (+), 38.0% with moderate (++) expression and 19.35% having high (+++) expression for CTCF. A significant association was found between CTCF protein expression and clinicopathological parameters include histological grade (p = 0.04), tumor stage (p = 0.04), nodal status (p = 0.03) and ER status (p = 0.04). ConclusionsThe data suggest that CTCF mutations leading to its inactivation significantly contribute to the progression of breast cancer.