Adjacent Non-tumor Research Articles

Increased SEC14L2 expression is associated with clinicopathological features and worse prognosis in oral squamous cell carcinoma.

Abnormal expression of SEC14L2 has been implicated in many human cancers. However, the role of SEC14L2 in oral squamous cell carcinoma (OSCC) remains unclear. Therefore, this study aimed to evaluate the expression and prognostic roles of SEC14L2 in OSCC. OSCC tumors and adjacent non-tumors were collected from OSCC patients and used for SEC14L2 mRNA expression by quantitative reverse transcription PCR (RT-qPCR). Additionally, the expression of SEC14L2 was further analyzed using The Cancer Genome Atlas-Head Neck Squamous Cell Carcinoma (TCGA-HNSCC) dataset to identify its relationship with HNSCC clinical characteristics. The Kaplan-Meier plot was used to assess survival rates, and the Tumor Immune Estimation Resource (TIMER) database was used to examine the correlation between SEC14L2 expression and tumor immune cell infiltration. In silico tools also looked at SEC14L2 involvement in cancer pathways through its protein network. The mRNA and protein levels of SEC14L2 are notably higher in both OSCC and HNSCC tissues compared to adjacent normal tissues. Upregulation of SEC14L2 was associated with advanced tumor stages, grades, metastasis, HPV-negative, and TP53 mutations in cancer patients. In addition, the high expression of SEC14L2 was negatively correlated with the poor survival of cancer patients and the infiltration of diverse immune cells in cancer patients. According to the findings of this investigation, SEC14L2 is significantly elevated in OSCC/HNSCC patients and associated with a worse prognosis. More investigation and clinical studies are required to completely understand the therapeutic potential of SEC14L2 in HNSCC and convert these findings into better patient outcomes.

Identification of miRNA, lncRNA and circRNA associated with gastric cancer metabolism through sequencing and bioinformatics analysis

Gastric cancer remains a highly prevalent malignancy worldwide with its molecular features poorly understood. To gain full insight into its genetic landscape, we performed whole-transcriptome sequencing on human tumors and adjacent non-tumors to predict the function of microRNA, long coding RNA, circular RNA, and mRNA, as well as estimate their correlation with gastric cancer characteristics through construction of ceRNA, WGCNA and PPI network. Functional enrichment analysis annotated nucleic acid binding, enzyme activity and binding related to differentially expressed miRNAs (dif-miRNAs); energy binding and enzyme binding related to dif-lncRNAs; protein binding and enzyme activity related to dif-circRNAs; protein digestion and absorption related to dif-mRNAs. The expression of key miR-135a-5p, lncRNAs-MSTRG.48856.1, ENST00000569981, MSTRG.22826.1, ENST00000564492, circRNAs-CCSER2, FNDC3B, CORO1C, FAM214A were validated by real-time PCR. The ceRNA network filtered 14 miRNAs, 30 lncRNAs, and 6 mRNA in the lncRNA-ceRNA axis and 8 miRNAs, 9 circRNAs, and 3 mRNA in the circRNA-ceRNA axis. Genes involved in ceRNA were annotated to be closely related to tumor material synthesis and metabolism. The WGCNA network filtered gene clusters related to TNM traits and extracted the hub genes CLDN10, CD177, newGene_35523, newGene_51201, CEACAM7, and newGene_46634. These genes were associated with cell proliferation, metabolism, and enzyme activity regulation. The PPI network analyzed the stable interaction relationships of the hub genes. Our research provides a valuable resource for understanding the molecular mechanisms of gastric cancer from the perspective of tumor metabolism.

MitoSomatic: a tool for accurate identification of mitochondrial DNA somatic mutations without paired controls.

Mitochondrial DNA (mtDNA) somatic mutations play important roles in the initiation and progression of cancer. Although next-generation sequencing (NGS) of paired tumor and control samples has become a common practice to identify tumor-specific mtDNA mutations, the unique nature of mtDNA and NGS-associated sequencing bias could cause false-positive/-negative somatic mutation calling. Additionally, there are clinical scenarios where matched control tissues are unavailable for comparison. Therefore, a novel approach for accurately identifying somatic mtDNA variants is greatly needed, particularly in the absence of matched controls. In this study, the ground truth mtDNA variants orthogonally validated by triple-paired tumor, adjacent nontumor, and blood samples were used to develop mitoSomatic, a random forest-based machine learning tool. We demonstrated that mitoSomatic achieved area under the curve (AUC) values over 0.99 for identifying somatic mtDNA variants without paired control in three tumor types. In addition, mitoSomatic was also applicable in nontumor tissues such as adjacent nontumor and blood samples, suggesting the flexibility of mitoSomatic's classification capability. Furthermore, analysis of triple-paired samples identified a small group of variants with uncertain somatic/germline origin, whereas application of mitoSomatic significantly facilitated the prediction of their possible source. Finally, a control-free evaluation of the public pan-cancer NGS dataset with mitoSomatic revealed a substantial number of variants that were probably misclassified by conventional tumor-control comparison, further emphasizing the usefulness of mitoSomatic in application. Taken together, our study demonstrates that mitoSomatic is valuable for accurately identifying somatic mtDNA variants in mtDNA NGS data without paired controls, applicable for both tumor and nontumor tissues.

The prognostic value and mechanisms of centromere protein M in patients with lung adenocarcinoma.

Centromere protein M (CENPM) has been reported to exert important roles in promoting tumor initiation and progression. However, the expression, effect, impact on prognosis and underlying mechanism of CENPM in lung adenocarcinoma (LUAD) remain unclear. Seventy-eight paired clinical samples of LUAD and corresponding adjacent non-tumor (ANT) tissues were obtained. The clinical pathological data and clinical outcome were tested, including univariate and multivariate Cox regression model. The relationship between CENPM expression and LUAD prognosis were identified according to the data obtained from the Cancer Genome Atlas (TCGA) database. Then, we explored the protein and mRNA levels of CENPM in LUAD and paired ANT tissues, and analyzed the correlation between CENPM and LUAD overall survival in our patients. In vitro studies, LUAD cell lines were treated with CENPM-short hairpin RNA (shRNA) (shCENPM), or transfected with CENPM overexpression plasmids with or without LY294002 (PI3K inhibitor) treatment. Cell proliferation ability was determined through cell counting kit-8 (CCK-8) assays. Cell cycle and apoptosis were detected by flow cytometer. The migration and invasion ability were assessed through Transwell assay. In vivo studies, the growth of xenografts in nude mice were evaluated after shCENPM stimulated cells injection, and the proliferation and apoptosis of xenografts were also analyzed. CENPM was significantly upregulated in LUAD patients compared with healthy controls, and CENPM upregulation was relevant to the higher pathological stages and poor survival rates in our LUAD patients. The bioinformatics analysis also revealed similar trends. CENPM could promote cell proliferation, cause alterations in cell cycle progression, enhance cell migration and invasion capacity, promote apoptosis in LUAD cell lines and promote the growth of xenografts in nude mice via regulation of AKT1/mTOR signaling pathway. CENPM was upregulated in LUAD patients, and it correlated with higher pathological stages and poor survival rates. CENPM could affect cell proliferation, cell cycle, cell migration and invasion capacity, and apoptosis in LUAD cell lines via regulation of AKT1/mTOR signaling pathway.

Novel prognostic model predicts overall survival in colon cancer based on RNA splicing regulation gene expression

Colon cancer is the third most common cancer and the second leading cause of cancer‐related death worldwide. Dysregulated RNA splicing factors have been reported to be associated with tumorigenesis and development in colon cancer. In this study, we interrogated clinical and RNA expression data of colon cancer patients from The Cancer Genome Atlas (TCGA) dataset and the Gene Expression Omnibus (GEO) database. Genes regulating RNA splicing correlated with survival in colon cancer were identified and a risk score model was constructed using Cox regression analyses. In the risk model, RNA splicing factor peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PPARGC1) is correlated with a good survival outcome, whereas Cdc2‐like kinase 1(CLK1), CLK2, and A‐kinase anchor protein 8‐like (AKAP8L) with a bad survival outcome. The risk model has a good performance for clinical prognostic prediction both in the TCGA cohort and the other two validation cohorts. In the tumor microenvironment (TME) analysis, the immune score was higher in the low‐risk group, and TME‐related pathway gene expression was also higher in low‐risk group. We further verified the mRNA and protein expression levels of these four genes in the adjacent nontumor, tumor, and liver metastasis tissues of colon cancer patients, which were consistent with bioinformatics analysis. In addition, knockdown of AKAP8L can suppress the proliferation and migration of colon cancer cells. Animal studies have also shown that AKAP8L knockdown can inhibit tumor growth in colon cancer in vivo. We established a prognostic risk model for colon cancer based on genes related to RNA splicing regulation and uncovered the role of AKAP8L in promoting colon cancer progression.

Identification of Novel Subtypes in Lung Adenocarcinoma: Evidence from Gene Set Variation Analysis in Tumor and Adjacent Nontumor Samples.

In patients with lung adenocarcinoma (LUAD), the prognostic role of adjacent nontumor tissues is still unknown. Alterations in the activity of immunologic and hallmark gene sets in adjacent nontumor tissues may have a potential influence on cell proliferation of normal lung cell after pulmonary lobectomy. We sought to discover LUAD subgroups and prognostic gene sets based on changes in gene set activity in tumor and adjacent nontumor tissues. Firstly, we used gene set variation analysis (GSVA) to characterize the activity changes of 4922 gene sets in LUAD and nontumor samples. Luckily, we identified three novel LUAD subtypes using the nonnegative matrix factorization (NMF) algorithm. In detailed, patients with subtype-3 had a favorable prognosis, but subtypes 1 and 2 had a bad prognosis. In addition, patients with subtype-3 in the validation cohort also lived longer. Meanwhile, using the LASSO-Cox algorithm, we discovered 15 prognostic gene sets in tumors (T gene sets) and two prognostic gene sets in adjacent nontumors (N gene sets). Interestingly, genes from N gene sets were related with immune response in nontumor tissues, but genes from T gene sets were correlated with DNA damaging and repairing in tumor tissues. These findings highlighted the possibility of a stronger immune response in nearby nontumor tissues. In conclusion, our study established a theoretical foundation for selecting therapy strategy for LUAD patients that should be guided by changes in activity in tumor and adjacent nontumor tissues, particularly after pulmonary lobectomy.

Identification of novel hub genes and lncRNAs related to the prognosis and progression of pancreatic cancer by microarray and integrated bioinformatics analysis.

Pancreatic cancer (PC) is one of the most invasive and metastatic neoplasms among the fatal malignancies of the digestive system. Abnormal expression of genes and long non-coding RNAs (lncRNAs) are reportedly linked to multiple cancers. However, the lncRNA-mRNA expression profiles and their molecular mechanisms in PC progression are poorly known. This study aimed to map the hub genes and lncRNAs which might play core roles in the development of PC. This study used microarray expression analysis to screen for both differentially expressed genes (DEGs) and differentially expressed lncRNAs (DElncRNAs) between PC and matched adjacent non-tumor (AN) tissues. In order to clarify the functional classification of DEGs, we conducted GO and KEGG pathway enrichment analyses via the Enrichr database. LncRNA-mRNA co-expressed networks were also constructed to explore the probable core regulating DEGs and DElncRNAs. Subsequently, the hub genes and lncRNAs were validated via the ONCOMINE and GEPIA databases and the co-expressed networks. By analyzing an mRNA-lncRNA microarray, we identified 943 mRNAs and 1,138 lncRNAs differentially expressed in PC tumors compared with the matched AN tissues. GO analysis confirmed that both up-regulated and down-regulated DEGs were enriched in multiple terms. The KEGG pathways enrichment analyses revealed that DEGs were mostly enriched in the focal adhesion and glutathione metabolism pathways, amongst others. Co-expressed networks were established to reveal the differential interactions between DEGs and DElncRNAs, and to indicate the core regulatory factors located at the core nodes of the co-expressed networks. The expression levels of potential core-regulating DEGs were validated by the GEPIA and ONCOMINE databases, and the relationship between overall survival and tumor stage and the potential core-regulating DEGs was analyzed using the GEPIA database. As a result, five genes and sixteen lncRNAs were finally considered as the hub transcripts in PC. This study identified DEGs and DElncRNAs between PC tumors and matched AN tissues, and these transcripts were connected with malignant phenotypes in PC through different BPs and signaling pathways. Furthermore, five hub genes and sixteen lncRNAs were identified, which are expected to represent candidate diagnostic biomarkers or potential therapeutic targets for PC.

GOLM1 Drives Colorectal Cancer Metastasis by Regulating Myeloid-derived Suppressor Cells

Colorectal cancer (CRC) is the most common digestive neoplasms worldwide, metastasis and recurrence still account for the leading cause for the high mortality rate, but the exact mechanisms remain unclear. More and more evidence has indicated that the deregulation of GOLM1 plays a crucial role in cancer progression. Here, we reported a novel role of GOLM1 in promoting CRC metastasis. In this study, the expression of GOLM1 was detected in human CRC cohort. The function of GOLM1 in CRC metastasis was analyzed by in vivo cecum orthotopic model. We found that the expression of GOLM1 was significantly increased in CRC tissues than adjacent nontumor. Overexpression GOLM1 can promote CRC immune escape and metastasis by recruiting of myeloid-derived suppressor cells (MDSCs) at the same time. PF-04136309, a small molecule and specific inhibitor of CCR2 can largely suppressed GOLM1-mediated CRC metastasis. These results suggest that GOLM1 can promote CRC metastasis and is a prognostic biomarker in human CRC.

MiR-140 and miR-196a as Potential Biomarkers in Breast Cancer Patients.

Objective: MiR-140 and miR-196a were known to be correlated with cancer diagnosis and prognosis. The current study aimed at the analysis of miR-140 and miR-196a expression patterns and their clinical significance for breast cancer (BC) patients. Methods:Differentially expressed miR-140 and miR-196a were examined via quantitative PCR in 110 cases of BC and their adjacent non-tumor (ANT) tissues. Results:The results indicated that miR-140 and miR-196a, respectively, notably decreased and increased expression in BC samples in comparison with ANT (p<0.001). Reduced miR-140 expression was also related to Lymph node metastasis (LNM, P= 0.023) and stage (P = 0.009). Additionally, Receiver Operating Characteristics (ROC) analysis illustrated that miR-140 had a significant diagnostic accuracy for stage and LNM of BC patients. We also discovered a strong negative correlation between miR-196a expression with histological grade (P = 0.038), LNM (P = 0.012) and stage (P = 0.001). Conclusion:Overall, exploring the miR-140 and miR-196a profiles not only can statistically different among BC and ANT samples, but it is also expected to become potential BC biomarkers.

Exosome-Derived Differentiation Antagonizing Non-Protein Coding RNA Promotes Tumor Recurrences in Hepatitis C-Related Hepatocellular Carcinoma

Background & Aims: Differentiation antagonizing non-protein coding RNA (DANCR) associated with various types of neoplasms. Hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) (HCV-HCC) has a high risk of recurrence. We explored the role of DANCR in HCV-related hepatocarcinogenesis and identified potential therapeutic targets and non-invasive prognostic markers for long-term outcome of HCV-HCC after surgical resection. Methods: DANCR relevant to HCV-HCC were identified by comparative RNA-sequencing of tumor (T) and adjacent non-tumor (ANT) tissues in a screening set, validated using real-time PCR Target lncRNAs in tissues and serum exosomes were used to predict HCC recurrence of HCV-HCC after curative surgical resection in a large application cohort. Exosome-Derived DANCR was explored using JFH1-infected cells and macrophages. Results: We confirmed that DANCR was upregulated by HCV infection and identified as the lncRNA most relevant to HCV-HCC in tumor as compared to ANT tissues. In 183 HCV-HCC patients followed for 10 years after curative HCC resection, the expression level of circulating exosomal DANCR was positively associated with HCC recurrence and was the most predictive factor associated with HCC recurrence and mortality (hazard ratio/95% confidence intervals: 7.0/4.3–11.6 and 2.7/1.5–5.1, respectively). In vitro study further demonstrated that exosomal DANCR was positively associated with mesenchymal-associated β-catenin expression in HCV infectious hepatocyte and macrophages, which might consequently promote tumor cell migration and invasion abilities in the tumor microenvironment. Conclusions: The lncRNA DANCR is highly relevant to disease progression of HCV-HCC Furthermore, circulating exosomal DANCR might serve as a non-invasive prognostic biomarker for HCV-HCC. Funding Statement: This study supported by the grants from the Ministry of Science Technology (MOST) grant (108-2314-B-037-079-MY3) (106-2221-E-039-011-MY3); China medical University (CMU108-MF-93), (CMU108-Z-02), (CMU108-S-22); Kaohsiung Medical University (KMU-Q108009), (KMUQ109005), (108-CCH-KMU-001); partially by Kaohsiung Medical University Research Grant KMU-TC108A04 and KMU-TC108B06. This study was partially supported by of Liquid Biopsy and of Cancer Research, Kaohsiung Medical University, and the Center for Intelligent Drug Systems and Smart Bio-devices (IDS2B) from The Featured Areas Research Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. Declaration of Interests: All authors declare no support from any organization other than the below mentioned ones for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years; and no other relationships or activities that could appear to have influenced the submitted work. Hence, all authors declare themselves to be independent from funders with respect to this manuscript. Ethics Approval Statement: The study protocol was approved by the Institutional Review Board at each hospital and the TLCN User Committee and was carried out in accordance with the Declaration of Helsinki and the guidelines of the International Conference on Harmonization for Good Clinical Practice.

Circulating exosome DANCR correlated to the recurrence of hepatitis C virus-related hepatocellular carcinoma.

e13688 Background: Long non-coding RNAs (lncRNAs) have been associated with various types of neoplasms. Hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) has high risk of recurrence. Methods: Differentially-expressed candidate lncRNAs relevant to HCV-HCC were identified by RNA-seq in Screening set, validated by qPCR in Validation set, and confirmed in external Test cohort. Target lncRNAs in tissue and serum exosome were applied to predict HCC recurrence of HCV-HCC after curative surgical resection in a large Application cohort. Pathogenetic mechanisms of target lncRNA were explored by using JFH1-infectious cells. Results: Of the top ten differentially-expressed lncRNAs relevant to HCV-related HCC identified in Screening set, differentiation antagonizing non-protein coding RNA (DANCR), which was upregulated by HCV infection and identified as the most relevant lncRNA to HCV-HCC, was overexpressed in tumor than in adjacent non-tumor (ANT) tissues. Of 183 HCV-HCC patients followed for 10 years after curative HCC resection, tumor DANCR, tumor/ANT DANCR ratio and circulating exosome DANCR levels were positively associated with HCC recurrence. Circulating exosome DANCR levels by droplet digital PCR provided the best predictive power of HCC recurrence with area under the curve of 0.88, when compared to tumor DANCR levels, tumor/ANT DANCR ratio, and alfa-fetoprotein. Exosome DANCR &gt; 50 copies/ml was the most predictive factor associated with HCC recurrence and mortality (hazard ratio/95% confidence intervals: 7.0/4.3-11.6 and 2.7/1.5-5.1 respectively, Cox regression model). Cell experiments showed that DANCR, positively associated with mesenchymal-associated β-catenin expression, promoted cell migration and invasion ability. Conclusions: lncRNA-DANCR was highly relevant to disease progression of HCV-HCC. Circulating exosome DANCR could serve as a non-invasive prognostic biomarker for HCV-HCC.

Viral integration drives multifocal HCC during the occult HBV infection

Background & AimsAlthough the prognosis of patients with occult hepatitis B virus (HBV) infection (OBI) is usually benign, a small portion may undergo cirrhosis and subsequently hepatocellular carcinoma (HCC). We studied the mechanism of life-long Integration of virus DNA into OBI host’s genome, of which may induce hepatocyte transformation.MethodsWe applied HBV capture sequencing on single cells from an OBI patient who, developed multiple HCC tumors and underwent liver resection in May 2013 at Tongji Hospital in China. Despite with the undetectable virus DNA in serum, we determined the pattern of viral integration in tumor cells and adjacent non-tumor cells and obtained the details of the viral arrangement in host genome, and furthermore the HBV integrated region in cancer genome.ResultsHBV captured sequencing of tissues and individual cells revealed that samples from multiple tumors shared two viral integration sites that could affect three host genes, including CSMD2 on chr1 and MED30/EXT1 on chr8. Whole genome sequencing further indicated one hybrid chromosome formed by HBV integrations between chr1 and chr8 that was shared by multiple tumors. Additional 50 poorly differentiated liver tumors and the paired adjacent non-tumors were evaluated and functional studies suggested up-regulated EXT1 expression promoted HCC growth. We further observed that the most somatic mutations within the tumor cell genome were common among the multiple tumors, suggesting that HBV associated, multifocal HCC is monoclonal in origin.ConclusionThrough analyzing the HBV integration sites in multifocal HCC, our data suggested that the tumor cells were monoclonal in origin and formed in the absence of active viral replication, whereas the affected host genes may subsequently contribute to carcinogenesis.

HnRNPL promotes Wilms tumor progression by regulating the p53 and Bcl2 pathways.

Background: Wilms tumor (WT) is the most common renal tumor in children with diffusely anaplastic or unfavorable histology, indicative of a poor prognosis. Heterogeneous nuclear ribonucleoprotein L (hnRNPL) is an RNA-binding protein (RBP) and a regulator of alternative RNA splicing that plays an important role in the occurrence and development of several cancers.Methods: Next generation sequencing technologies was used to discovery differentially expressed genes between WT and adjacent nontumors. The gene ontology (GO) analysis was performed to uncover the biological functions of differentially expressed genes, and the kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis was applied to find out the related signal pathways. Expression levelsof hnRNPL with WT tissues and cells were determined by RT-qPCR.After silencing hnRNPL, the expression of hnRNPL, p53 and Bcl-2 were detected by RT-qPCR and Western blot in WT cell line. The regulatory effects of hnRNPLon proliferative and apoptotic potentials of WT cells were evaluated by MTT and flow cytometry, respectively. RNA-binding protein immuno-precipitation was used to confirm the direct interaction of hnRNPL with p53 mRNA. Mouse xenograft models ofhnRNPL knockdown were established to test the functions in the growth of WT in vivo.Results: High levels of hnRNPL were expressed in WT tissues and cells. Functional analysis revealed that hnRNPL silencing suppressed cell proliferation and promoted cell apoptosis in WT. Molecular mechanism exploration indicated that hnRNPL directly targeted p53. Moreover, knockdown of hnRNPL inhibited the expression of p53 and Bcl2 in WT. Additionally, hnRNPL silencing inhibited the growth of xenograft tumors in vivo.Conclusion: HnRNPL act as p53 mRNA-binding protein, which plays an important role in the proliferation and apoptosis of WT through p53 and Bcl2 pathways and these findings provide new insights into the mechanism of WT pathogenesis.

Significant predictors of overall survival in patients with hepatocellular carcinoma after surgical resection.

BackgroundThe predictive factors of overall survival after hepatectomy for HCC remain controversial and need to be investigated.MethodsIn total, 535 consecutive HCC patients undergoing resection were included and their clinicopathological data and overall survival were recorded. Both the tumor and adjacent non-tumor (ANT) tissues were subjected to immunohistochemistry analysis for the expression of autophagy-related markers.ResultsDeath was observed for 219 patients, and the cumulative overall survival rates at 1, 3, 5 and 7 years were 91.0%, 72.3%, 58.8%, and 27.7%, respectively. In the multivariate analysis, mortality was significantly associated with the following: diminished LC3 expression in both the tumor and ANT tissues, in the HCC tissues alone and in the ANT tissues alone (hazard ratio/95% confidence interval: 6.74/2.052–22.19, 6.70/1.321–33.98 and 2.58/1.499–4.915, respectively); recurrent HCC (5.11/3.136–8.342); HBV infection (2.75/1.574–4.784); cirrhosis (1.78/1.059–2.974); and antiviral therapy (0.42/0.250–0.697). The 5-year overall survival rates were 70.2%, 57.3%, 49.6% and 10.7% for patients with positive LC3 expression in both tissue types, in the HCC tissues alone, in the ANT tissues alone, and in neither tissue type, respectively. The 5-year overall survival rates were 56.7%, 47.3%, 51.2% and 38.7% for patients with HBV-related HCC, cirrhosis, no antiviral therapy, and recurrent HCC, respectively, and these rates were significantly lower than those in their counterparts.ConclusionsPatients with recurrent HCC, HBV-related HCC, cirrhosis, and the absence of antiviral therapy showed significantly lower overall survival rates. Furthermore, LC3 expression in both the tumor and liver microenvironments were significantly predictive of overall survival after resection for HCC.

Microarray expression profile of long non-coding RNAs in human lung adenocarcinoma.

BackgroundLong non‐coding RNAs (lncRNAs) participate in many biological dynamics and play significant roles in gene regulation. LncRNA expression is altered in many cancers; however, the expressions and functions of lncRNA genes in lung adenocarcinoma (LAD) remain unknown.MethodsLncRNA and messenger RNA (mRNA) expression in LAD without lymphatic metastasis versus paired adjacent non‐tumor (ANT) lung tissues and LAD with versus without lymphatic metastasis were analyzed using Human LncRNA Arraystar V3.0. The expression levels of four downregulated and four upregulated lncRNAs were verified using quantitative real‐time PCR in cells and tissue specimens.ResultsIn this study, 949 lncRNAs and 681 mRNAs had differential expression in LAD without lymphatic metastasis compared to ANT lung tissues, while 2740 lncRNAs and 1714 mRNAs were differentially expressed in LAD with lymphatic metastasis compared to LAD without lymphatic metastasis. The expression patterns of selected lncRNAs (LINC00113, AC005009.1, ARHGAP22‐IT1, AC009411.1, SRGAP3‐AS2, EGFEM1P, FAM66E, and HLA‐F‐AS1) were consistent with microarray data. Differentially expressed mRNA genes were enriched in crucial Gene Ontology terms and pathways.ConclusionOur results revealed differentially expressed lncRNAs in LAD, suggesting lncRNAs may be potential indicators for LAD diagnosis and therapy.

Identification of S100 calcium binding protein A9 as a prognostic biomarker in gallbladder cancer

BackgroundGallbladder cancer (GBC) is the most lethal orphan malignancy of biliary tract, with poor prognosis and varied global incidences, predominately reported from, Northern India. Moreover, early diagnostic biomarker(s) of GBC is still undetermined. Earlier studies have demonstrated that S100A9 is associated with the prognosis of various malignancies. S100A9 (calgranulin B or Myeloid Related Protein-14) is a calcium binding protein which has wide range of possible intracellular as well as extracellular functions. Previous reports showed that S100A9 stimulates malignant development and cancer progression, and is also involved in other inflammatory diseases. So far, no study has been made on the expression and epigenetic regulation of S100A9 in GBC. Therefore, the present study aimed to investigate the expression and promoter methylation status of S100A9 in GBC to evaluate its utility as a diagnostic biomarker for GBC. Materials and methodMethylation specific PCR (MS-PCR) was done to determine the methylation status of S100A9 gene promoter. Semi quantitative and quantitative RT-PCR were performed to assess its expression level in tumor and non tumor tissues. Immunohistochemistry (IHC) was carried out to understand its cellular distribution. Statistical analysis was performed using student’s t-test for paired case and control tissue samples, and one way ANOVA for multiple comparison procedures. ResultsMS-PCR of S100A9 showed methylation (p =0.022) in the promoter region of about i.e.,75% (9/12) in adjacent non tumor (ANT) tissues and about 25% (3/12) in GBC cases. RT-PCR analyses revealed upregulation of S100A9 in GBC at transcriptional level. IHC analysis further supported the change in the transcriptional level, scored positive for S100A9 (62%; 36/58), in GBC and about (40%; 12/30) in non tumor (p <0.048). ConclusionThe study shows that elevated expression and hypomethylation in the S100A9 gene promoter suggesting that DNA methylation plays a crucial role in the regulation of S100A9 expression in GBC. However, further studies are warranted to confirm this in larger pool of samples before concluding its usefulness as a biomarker of GBC.

Autophagy-related gene LC3 expression in tumor and liver microenvironments significantly predicts recurrence of hepatocellular carcinoma after surgical resection.

BackgroundThe role of autophagy-related markers as the prognostic factor of post-operative hepatocellular carcinoma (HCC) recurrence remained controversial.MethodsOverall, 535 consecutive HCC patients undergoing curative resection from 2010 to 2014 were followed and classified with early (ER, <2 years) or late recurrence (LR). Autophagy-related markers, LC3, Beclin-1, and p62 expression was immunohistochemically assessed in HCC and adjacent non-tumor (ANT) tissues.ResultsHCC recurred in 245 patients: 116 with ER and 129 with LR. The cumulative incidence of recurrence at 1, 3, 5, and 7 years was 9.7%, 33.9%, 53.3%, and 66.3%, respectively. In multivariate analysis, HCC recurrence was significantly associated with low LC3 expression in tumor and ANT tissues, HCC tissues only and ANT tissues only (hazard ratio/95% confidence interval: 6.12/2.473–17.53, 4.18/1.285–13.61, and 1.89/1.299–2.757) and macrovascular invasion (1.63/1.043–2.492) and cirrhosis (1.59/1.088–2.326). ER was significantly associated with low LC3 expression in tumor and ANT tissues, HCC tissues only and ANT tissues only (6.54/2.934–15.81, 3.26/1.034–10.27, and 2.09/1.313–3.321) and macrovascular and microvascular invasion (2.65/1.306–5.343 and 2.55/1.177–5.504). LR was significantly associated with low LC3 expression in tumor and ANT tissues, HCC tissues only and ANT tissues only (5.02/1.372–18.83, 3.19/1.13–12.09, and 1.66/1.051–2.620) and cirrhosis (1.66/1.049–2.631). Patients with low and high LC3 expression in tumor and ANT tissues showed a 5-year cumulative recurrence of 94.3% and 41.7%, respectively (p < 0.001).ConclusionsThe high LC3 expression in the tumor and liver microenvironments is significantly associated with lower HCC recurrence. Furthermore, tumor characteristics and liver microenvironment were also significantly associated with ER and LR, respectively.Translational impactThe analysis for LC3 expression in both the HCC and ANT tissues could identify patients at risk of HCC recurrence.

PHD-finger domain protein 5A functions as a novel oncoprotein in lung adenocarcinoma

BackgroundPHD-finger domain protein 5A (PHF5A) is a highly conserved small transcriptional regulator also involved in pre-mRNA splicing; however, its biological functions and molecular mechanisms in non-small cell lung cancer (NSCLC) have not yet been investigated. The purpose of this study was to determine the functional relevance and therapeutic potential of PHF5A in lung adenocarcinoma (LAC).MethodsThe expression of PHF5A in LAC tissues and adjacent non-tumor (ANT) tissues was investigated using immunohistochemistry of a tissue microarray, qRT-PCR, western blot and bioinformatics. The function of PHF5A was determined using several in vitro assays and also in vivo assay by lentiviral vector-mediated PHF5A depletion in LAC cell lines.ResultsPHF5A was highly upregulated in LAC tissues compared with the ANT counterparts, and closely associated with tumor progression and poor patient prognosis. These results were further confirmed by findings of the TCGA database. Moreover, functional studies demonstrated that PHF5A knockdown not only resulted in reduced cell proliferation, increased cell apoptosis, and cell cycle arrest, but also suppressed migration and invasion in LAC cells. PHF5A silencing was also found to inhibit LAC tumor growth in nude mice. Microarray and bioinformatics analyses revealed that PHF5A depletion led to dysregulation of multiple tumor signaling pathways; selected factors in key signaling pathways were verified in vitro.ConclusionsThe data suggest for the first time that PHF5A is an oncoprotein that contributes to LAC progression by regulating multiple signaling pathways, and may constitute a prognostic factor and potential new therapeutic target in NSCLC.

Abstract A14: Downregulation of desmin involves gene promoter hypermethylation in gallbladder cancer

Abstract Gallbladder cancer (GBC) is a fatal malignancy with widespread geographical variations in incidence worldwide. There are limited studies on systematic molecular profiling of gallbladder cancer in literature. Previously, we carried out differential proteomic and methylome-based analysis in gallbladder tumors and adjacent non-tumors to identify differentially expressed proteins and global methylation status of genes involved in gallbladder cancer. Quantitative tissue proteomics analysis using Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) based Liquid Chromatography-Mass Spectrometry (LC-MS/MS) showed that desmin protein was significantly downregulated (~3 folds) in tumors. Global methylation profiling using Illumina's Infinium Human Methylation 450 BeadChip array revealed hypermethylation in desmin in gallbladder tumors compared to non-tumor tissue. These findings lead us to further investigate the epigenetic regulation of desmin gene expression in gallbladder cancer. Real-time PCR analysis revealed significant downregulation of desmin (p=0.001) expression in tumors (n=12) as compared to the non-tumor tissue (n=12). Immunohistochemical analysis also showed significantly (p&amp;lt;0.0001) decreased level of desmin in 95% (26/27) of tumor cells in comparison to non-tumors 35.71% (5/14). This was again validated by Western blotting, showing downregulation of desmin (p=0.0081) at protein level in tumor tissues compared to the non-tumors. Methylation specific polymerase chain reaction analysis showed 86.67% methylation of desmin in corresponding gallbladder tumors compared to non-tumor tissue (p=0.004). Taken together, our observations showing decreased expression and hypermethylation of desmin gene suggest desmin as a potential epigenetically controlled biomarker for GBC. However, further studies are warranted to explore its role in tumorogenesis of gallbladder cancer. Citation Format: Shushruta Bhunia, Mustafa Ahmed Barbhuiya, Sanjiv Gupta, Braj Raj Shrivastava, Pramod Kumar Tiwari. Downregulation of desmin involves gene promoter hypermethylation in gallbladder cancer [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A14.

Preliminary findings for metabolite profiles of papillary thyroid carcinoma and laryngeal squamous cell carcinoma

Objective:To identify distinct metabolite profiles of papillary thyroid cancer (PTC) and laryngeal squamous cell carcinoma (LSCC).Method:Tumor and adjacent non-tumor specimens were collected from 57 PTC and 33 LSCC patients. Distinct metabolite profiles of tissues were examined using a combination of gas chromatography-time-of-flight mass spectrometry and ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. The data were analyzed with multivariate statistical analysis to compare the distinct metabolite profiles and related pathways of these three tumors.Result:A panel of 46 and 41 differentially expressed metabolites were identified in tumor and adjacent non-tumor tissues of PTC and LSCC, respectively. Increased glycolysis, amino acids metabolism, one carbon metabolism and tryptophan metabolism were found in these two types of tumor tissues compared to adjacent non tumor tissues. Purine and pyrimidine metabolism was significantly elevated in PTC and LSCC tumor tissues, while taurine and hypotaurine were only higher in PTC tumor tissues. The fatty acid metabolism was detected at lower level in both PTC and LSCC tumor tissue.Conclusion:PTC and LSCC tumor tissues not only have common metabolic signatures characterized by increased glycolysis, amino acids metabolism, one carbon metabolism and tryptophan metabolism, but also have distinct metabolic signatures. It is helpful to understand the nature of these two tumors.