Unveiling the Role of DPYS: A New Prognostic Biomarker in Sarcoma

BackgroundThe transmembrane 9 superfamily protein member 4 (TM9SF4) is a transmembrane protein upregulated in multiple cancers; however, its role in hepatocellular carcinoma (HCC) remains unknown.MethodsThe Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx) and International Cancer Genome Consortium (ICGC) databases were utilized to investigate the differential expression of TM9SF4 in HCC and tumor tissues. The prognostic and value of TM9SF4 in HCC was evaluated using Kaplan–Meier analysis, Cox regression, and receiver operating characteristic (ROC) curve analyses. The expression pattern and prognostic value of TM9SF4 was further verified using immunohistochemical (IHC) examination of 87 pairs of HCC clinical specimens. A nomogram was constructed by combining TM9SF4 expression and clinicopathological parameters to predict prognosis for individual patient. Additionally, gene set enrichment analysis (GSEA) was performed to identify key pathways related to TM9SF4.ResultsTM9SF4 expression was upregulated in the HCC tissues. High expression of TM9SF4 was significantly associated with advanced T stage, histological grade, and worse survival. Multivariable Cox analysis revealed that TM9SF4 expression was an independent factor for overall survival. The nomogram by incorporating the TM9SF4 and T stage showed good performance in predicting prognosis. Moreover, GSEA analysis revealed that TM9SF4 was functionally involved in pathways associated with the cell cycle.ConclusionsThese findings suggest that TM9SF4 is a promising biomarker with prognostic potential and functional significance in HCC.

Colorectal cancer (CRC) remains a significant global health challenge due to its high incidence and mortality rates. The disease's complexity and heterogeneity impede early diagnosis and effective treatment. The study aims to investigate the role of Tetraspanin 11 (TSPAN11) in CRC, exploring its potential as a prognostic biomarker and immunotherapy target through bioinformatics analysis and experimental validation. Pan-cancer patient data were obtained from The Cancer Genome Atlas (TCGA) and the GSE71187 dataset, including 672 CRC tissues and 51 adjacent normal tissues. Differential expression analysis, Kaplan-Meier survival analysis, gene set enrichment analysis (GSEA), and immune infiltration assessment were performed. TSPAN11 expression was validated in CRC cell lines using quantitative reverse transcription PCR (qRT-PCR). TSPAN11 was significantly downregulated in CRC tissues compared to normal tissues (p < 0.001), with lower expression associated with poorer overall survival (OS; p = 0.011) and disease- specific survival (DSS; p = 0.038). Multivariate analysis identified TSPAN11 as an independent prognostic factor (p = 0.045). TSPAN11 expression was linked to key pathways such as ECM receptor interaction and TGF-β signaling, and correlated with immune infiltration, immune checkpoint genes, tumor mutational burden (TMB), microsatellite instability (MSI), and drug sensitivity. The findings suggest that TSPAN11 may influence CRC progression through multiple biological pathways and immune-related mechanisms. Its downregulation is associated with poorer prognosis and immune evasion, highlighting its potential as a biomarker and therapeutic target. However, validation in larger cohorts and elucidation of underlying mechanisms are needed to confirm these results and translate them into clinical practice. TSPAN11 may serve as a promising prognostic biomarker and immunotherapy target in CRC. Its associations with clinical outcomes, immune features, and drug sensitivity underscore its potential for improving CRC diagnosis and treatment strategies.

Glioblastoma (GBM) is the most aggressive form of adult brain tumors with a poor median survival of less than 15 months. The alkylating agent temozolomide is used for chemotherapy for GBM. Regardless of the rigorous therapeutic regime, almost all cases develop resistance and recur. The mechanisms of the inherent and acquired resistance are less understood and demand in-depth research. We performed a pooled and bar-coded shRNA screen (5045 shRNAs) in a glioma cell line to identify mediators of temozolomide resistance in GBM. The data was analyzed by the HitSelect algorithm, and the top ranking genes imparting resistance (n=257) and sensitivity (n=256) were further subjected to gene set enrichment analysis (GSEA), Gene Ontology (GO) annotation and survival analysis in The Cancer Genome Atlas (TCGA) GBM cohort. An enrichment of translation and protein synthesis-related pathways was found in sensitivity genes, while enrichment of proliferation, metabolic processes, cytokine-cytokine receptor interaction and response to stress was found in resistance genes. Suppressor of Lin-12-Like Protein 1 (SEL1L) was identified as one of the top ranking genes in our screen that caused resistance to temozolomide. SEL1L is a component gene of the endoplasmic-reticulum-associated protein degradation (ERAD). It is known to play an important role in clearing misfolded proteins from cells, thus maintaining proteostasis. SEL1L was found to be a poor prognostic marker in the TCGA GBM cohort treated with temozolomide, as identified by univariate and Kaplan- Meier analysis. In vitro SEL1L knockdown resulted in a significant reduction of proliferation and in an increased sensitivity towards temozolomide. Upon temozolomide treatment of cells in the SEL1L knockdown condition, an increase in apoptosis was observed as measured by Annexin V staining. In vivo tumor showed increased SEL1L protein levels as compared to control brain and mice tumor that responded to temozolomide. We subjected to GSEA, (i) the genes that exhibited survival significance in the TCGA GBM cohort with temozolomide, as well as (ii) the genes that showed differential expression in the SEL1L low and high expression groups. Interestingly, both these analyses identified significant enrichment of overlapping pathways like TNFA mediated NF-kappaB signaling, EMT, IL6 STAT3 signaling, G2M checkpoint and E2F target genes, further highlighting the role of SEL1L in temozolomide resistance. This underscores that SEL1L is an important mediator for temozolomide resistance and is a poor prognosticator in GBM. SEL1L high expression correlates with various resistance-related signaling pathways. Our study emphasizes on the interplay between these pathways and the ERAD machinery in maintaining proteostasis in response to chemotherapeutic stress. Citation Format: Anjali Arora, Vivek Singh Tomar, Sannu Thomas, Vikas Patil, Jörg Hoheisel, Kumaravel Somasundaram. Pooled shRNA screen uncovered the role of SEL1L, an ERAD (endoplasmic reticulum associated degradation) gene, in modulating temozolomide resistance in GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-098.

Introduction: Much has been learned about the molecular basis of melanoma in recent years. However, the role of epigenetic alterations in melanoma genesis is poorly understood. Here we report the identification and characterization of MLL2, which encodes a histone lysine methyltransferase, as a novel tumor suppressor in melanoma. Methods: We performed an in vivo shRNA screen using 475 shRNAs targeting 95 epigenetic enzymes to identify novel tumor suppressors of melanoma genesis. Immortalized human melanocytic cell lines expressing BrafV600E (HMEL/PMEL) were transduced with pooled shRNAs and injected subcutaneously in nude mice. Candidate genes were identified from orthotopic tumors formed using PCR and Sanger sequencing and independently validated using multiple independent shRNAs and human cell lines. Results: Among 11 candidate genes identified from tumors formed in the in vivo shRNA screen, MLL2 knockdown was associated with the shortest time to tumor appearance (6 weeks) and highest penetrance (tumor formation at 80% of sites injected). In validation experiments, loss of MLL2 in human melanocytic and melanoma cell lines resulted in worse tumor-free survival when injected orthotopically in mice. Analysis of human melanoma data from The Cancer Genome Atlas (TCGA) project revealed that MLL2 is mutated in 15% of TCGA cases and is not associated with either BRAF or NRAS mutations. We found that MLL2 knockdown in human cell lines was associated with decreased expression of transcription factor TCF3 by qRT-PCR as well as decreased expression of TCF3 gene targets by gene set enrichment analysis (GSEA) of microarray data. Furthermore, GSEA analysis of data from TCGA samples with MLL2 mutations across tumor types revealed decreased expression of genes with TCF3 binding sites. Finally, we found that MLL2 knockdown in a human melanocytic cell line resulted in decreased H3K4 di- and tri-methylation at the promoter region of the TCF3 gene as well as at transcriptional start sites of TCF3 gene targets in ChIP-Seq experiments, suggesting a possible mechanism by which MLL2 loss may contribute to melanoma genesis and progression through dysregulation of the transcription factor TCF3. Conclusions: MLL2 is a novel tumor suppressor in melanoma identified through an in vivo shRNA tumorigenesis screen targeting 95 epigenetic enzymes. MLL2, which encodes for an epigenetic enzyme (a histone methyltransferase), is significantly mutated in human melanoma as represented in the TCGA and loss of MLL2 results in enhanced tumorigenesis in vivo. Studies to elucidate the mechanisms by which MLL2 loss contributes to melanoma genesis and progression and which focus on the transcription factor TCF3 are ongoing. Citation Format: Emily Z. Keung, Kunal Rai, Kadir C. Akdemir, Liren Li, Sneha Sharma, Bryce Axelrad, Lynda Chin. The identification and characterization of MLL2, an epigenetic enzyme, as a novel tumor suppressor in melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-134. doi:10.1158/1538-7445.AM2014-LB-134

BackgroundLung adenocarcinoma (LUAD), as the most common histological subtype of lung cancer, is a high-grade malignancy and a leading cause of cancer-related death globally. Identification of biomarkers with prognostic value is of great significance for the diagnosis and treatment of LUAD. Heterogeneous nuclear ribonucleoprotein C (HNRNPC) is an RNA-binding protein “reader” of N6-methyladenosine (m6A) methylation, and is related to the progression of various cancers; however, its role in LUAD is unclear. The aims of this study aims were to study the expression and prognostic value of HNRNPC in LUAD.MethodsThe Oncomine database and gene expression profiling interactive analysis (GEPIA) were used for preliminary exploration of HNRNPC expression and prognostic value in LUAD. LUAD cases from The Cancer Genome Atlas (TCGA) (n = 416) and the Kaplan-Meier plotter database (n = 720) were extracted to study the differential expression and prognostic value of HNRNPC. HNRNPC expression in the National Cancer Center of China (NCC) cohort was analyzed by immunohistochemical staining, and the relationship between HNRNPC expression and survival rate evaluated using the Kaplan-Meier method and log-rank test. Univariate and multivariate Cox regression analyses were used to identify independent prognostic factors. Several pathways that were significantly enriched in the HNRNPC high expression group were identified by Gene Set Enrichment Analysis (GSEA).ResultsFive data sets from the Oncomine and GEPIA databases all supported that HNRNPC expression is significantly higher in LUAD than in normal lung tissue. In TCGA cohort, HNRNPC was highly expressed in LUAD tissues and significantly related to age, sex, smoking history, ethnicity, lymph node metastasis, and TNM staging (P < 0.001). High HNRNPC expression was significantly correlated with poor prognosis in the three cohorts (NCC, TCGA, and K-M plotter) (P < 0.05). Multivariate Cox regression analysis showed that HNRNPC expression was an independent prognostic factor in both TCGA and NCC cohorts (P < 0.05). Further, 10 significantly enriched pathways were identified from TCGA data and 118 lung cancer cell lines in CCLE, respectively.ConclusionsHigh HNRNPC expression is significantly related to poor overall survival in patients with LUAD, suggesting that HNRNPC may be a cancer-promoting factor and a potential prognostic biomarker in LUAD.

BackgroundGNL2, a nuclear protein, is involved in ribosome production and cell cycle regulation. However, its expression and function in different types of tumors are not well understood. Comprehensive studies across multiple cancer types are needed to assess the potential of GNL2 as a diagnostic, prognostic, and immunological marker.MethodsmRNA expression data, copy number alteration threshold data, masked copy number segmentation data, and DNA methylation 450 K data from The Cancer Genome Atlas (TCGA) pan-cancer cohort were obtained from the Firehose database. Additional data, including miRNA, The Cancer Proteome Atlas (TCPA), mutation data, and clinical information, were sourced from the University of California Santa Cruz (UCSC) Xena database. The cBioPortal database facilitates the examination of GNL2 mutation frequency, location, and 3D structure in the TCGA database. Gene Expression Omnibus (GEO) data verified the transcriptome level expression in the TCGA cohort. Protein expression levels were analyzed via the Human Protein Atlas (HPA) database and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database. Gene set enrichment analysis (GSEA) was employed to investigate the biological role of GNL2 across cancers. Multiple immune infiltration algorithms from the TIMER2.0 database were utilized to examine the correlation between GNL2 expression and the tumor immune microenvironment. The transcriptome-wide immune infiltration results were validated using 72 single-cell datasets from the Tumor Immune Single-cell Hub (TISCH) database. Pan-cancer survival maps were constructed, and GNL2 expression in different molecular subtypes across cancers was examined. The relationship between GNL2 and drug resistance was investigated using data from CellMiner, GDSC, and CTRP. The Comparative Toxicogenomics Database (CTD) was used to identify chemicals affecting GNL2 expression.ResultsGNL2 is located primarily in the nucleus, and its expression is regulated mainly through somatic copy number alteration (SCNA) and aberrant DNA methylation, according to TCGA data. Database analysis and immunohistochemical results from clinical samples revealed high GNL2 expression in most tumors, which was correlated with diagnostic significance. High GNL2 expression often indicates a poor prognosis with pan-cancer prognostic value. Gene set enrichment analysis (GSEA) suggested that GNL2 is involved in tumor development through cell proliferation-related pathways. GNL2 expression is correlated with the expression of immune-related genes and the infiltration levels of multiple immune cells. The relationships between GNL2 and various drugs and chemicals were examined, revealing its influence on drug sensitivity and identifying five chemicals countering GNL2-mediated pro-cancer effects.ConclusionComprehensive bioinformatics analysis of GNL2 in pan-cancer tissues, combined with experimental validation, elucidated the pan-cancer expression pattern of GNL2, determined its diagnostic and prognostic value, and explored the biological functions of GNL2. GNL2 may be involved in the regulation of cell cycle progression and remodeling of the tumor microenvironment and is associated with poor prognosis as a risk factor in most tumors. The potential of GNL2-based cancer therapies is emphasized, assisting in predicting the response to chemotherapy.

The involvement of HBXIP in cancer development and cancer cell survival is well known. This work probed the potential of HBXIP as a prognostic biomarker in hepatic cell cancer (HCC). First, pan-cancer analysis of HBXIP expression was conducted using The Cancer Genome Atlas (TCGA) database to validate the expression of HBXIP in different cancers. The GSE14520 (GPL3721 Subset) database was used to validate HBXIP in HCC. The association between survival outcomes and prognostic factors was assessed employing univariate and multivariate survival analyses for TCGA Liver Hepatocellular Carcinoma. The biological function of the HBXIP Gene was annotated by gene set enrichment analysis. The relationship between HBXIP expression and immune cells and immune markers was analyzed from the Gene Expression Profiling Interactive Analysis (GEPIA) database. Malignant tissues demonstrated evident upregulation of HBXIP at transcriptional and protein levels over normal tissues (p < 0.05) with this elevated expression linked to an advanced tumor stage in HCC cohorts. Univariate analysis revealed an evident correlation emerged between prognosis and HBXIP for GSE14520 databases (p < 0.05). The disease-free survival (DFS) and overall survival (OS) (five-year values) were lower in samples demonstrating elevated HBXIP (HR: 2.413; 95% CI 1.601, 3.638; p < 0.001) and (HR: 1.613; 95% CI 1.446, 1.844; p = 0.003), respectively vs. lower HBXIP expression. HBXIP emerged as an independent factor in OS prognosis (HR 2.184; 95% CI 1.495, 3.196; p < 0.001) and DFS (HR 1.764; 95% CI 1.261, 2.466; p < 0.001), respectively according to multivariate analysis. Further, multiple Cox analyses in the validation cohort revealed that independent factors for OS were HBXIP, AJCC T stage, vascular invasion, and tumor status with the C-index score of 0.727 (95% CI, 0.704 to 0.750). HBXIP level showed a significantly positive association with tumor immune cell infiltration, and biomarkers of immune cells; besides, the rectum Rho GTPase effectors signaling pathway was also identified. HCC advancement and survival involves HBXIP, which also emerged as a functional biomarker for HCC survival prediction.

The Clinical and Biological Effects of the Expression of Dedicator of Cytokinesis 1 (DOCK1) in Acute Myeloid Leukemia

Neuronal guanine nucleotide exchange factor (NGEF) plays a key role in several cancers; however, its role in lung adenocarcinoma (LUAD) remains unclear. The aim of this study was to evaluate the efficacy of NGEF as a prognostic biomarker and potential therapeutic target for LUAD. NGEF expression data for multiple cancers and LUAD were downloaded from multiple databases. The high- and low-NGEF expression groups were constructed based on median NGEF expression in LUAD samples, and then performed Kaplan-Meier survival analysis. Differentially expressed genes (DEGs) from the two NGEF expression groups were screened and applied to construct a protein-protein interaction network. The primary pathways were obtained using gene set enrichment analysis. The associations between NGEF expression and clinical characteristics, immune infiltration, immune checkpoint inhibitors (ICIs), sensitivity to chemotherapy, and tumor mutation burden (TMB) were investigated using R. Levels of NGEF expression in the lung tissue was validated using single-cell RNA sequencing, quantitative polymerase chain reaction (qPCR), immunohistochemical staining, and western blot analysis. The expression of NGEF mRNA was upregulated in multiple cancers. mRNA and protein expression levels of NGEF were higher in patients with LUAD than in controls, as validated using qPCR and western blot. High NGEF expression was an independent prognostic factor for LUAD and was associated with advanced tumor stage, large tumor size, more lymph node metastasis, and worse overall survival (OS). A total of 182 overlapping DEGs were screened between The Cancer Genome Atlas and GSE31210, among which the top 20 hub genes were identified. NGEF expression was mainly enriched in the pathways of apoptosis, cell cycle, and DNA replication. Moreover, elevated NGEF expression were associated with a high fraction of activated memory CD4+ T cells and M0 macrophages; elevated expression levels of the ICIs: programmed cell death 1 and programmed cell death 1 ligand 1 expression; higher TMB; and better sensitivity to bortezomib, docetaxel, paclitaxel, and parthenolide, but less sensitivity to axitinib and metformin. NGEF expression is upregulated in LUAD and is significantly associated with tumor stages, OS probability, immune infiltration, immunotherapy response, and chemotherapy response. NGEF may be a potential diagnostic and prognostic biomarker and therapeutic target in LUAD.

Alternative splicing plays a vital role in cancer development and progression. The splicing C complex is involved in alternative splicing. However, the role of PRKR-interacting protein 1 (PRKRIP1), a component of the splicing C complex, in colorectal cancer (CRC) remains unclear. This study aimed to determine the clinicopathological, biological and prognostic significance of PRKRIP1 expression in CRC. We used a bioinformatics approach to screen for oncogenes using The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) datasets and identified PRKRIP1 as a driver gene on chromosome 7q. The mRNA expression of PRKRIP1 was measured using reverse transcription-quantitative PCR in 165 surgically resected CRC samples in our hospital, and its localization was determined using immunohistochemical staining. Gene Set Enrichment Analysis (GSEA) was performed using TCGA dataset. High PRKRIP1 expression was significantly associated with poor prognosis in both the samples and TCGA dataset. A positive correlation was observed between copy number variation and PRKRIP1 expression in TCGA and CCLE datasets, and the frequency of PRKRIP1 mutations was less than 5%. Immunohistochemistry revealed that PRKRIP1 was located in the cytoplasm of tumor cells. GSEA revealed that PRKRIP1 expression was correlated with apoptosis-related gene sets. PRKRIP1 overexpression may be a poor prognostic biomarker for CRC. Although it is known that PRKRIP1, a spliceosome factor, is essential for splicing, we now revealed the way by which its expression accelerates CRC progression.

Ubiquitin-conjugating enzyme E2T (UBE2T) plays a significant role in carcinogenesis. Previous studies have demonstrated that UBE2T promotes the development and progression of numerous types of cancer. However, the association between UBE2T expression and colorectal cancer (CRC) remains unclear. In the present study, UBE2T protein expression was examined in the tissues of patients with CRC via immunohistochemistry. In addition, UBE2T expression data and corresponding clinical information were obtained from The Cancer Genome Atlas (TCGA). In the clinical samples, the associations between UBE2T expression and clinicopathological factors were evaluated by the χ2 or Fisher's exact tests. In TCGA data, associations between UBE2T expression and clinical characteristics were evaluated using a logistic regression model. Overall survival was analyzed using Kaplan-Meier and Cox regression analyses. Reverse transcription-quantitative PCR (RT-qPCR) and western blotting assays were used to examine UBE2T expression in normal and CRC cell lines. Gene set enrichment analysis (GSEA) was performed on the dataset from TCGA. UBE2T protein was highly expressed in the cytoplasm of tumor cells in 29/50 clinical samples, whereas in the adjacent normal tissues, it was only highly expressed in 2/50 samples. Furthermore, UBE2T expression was associated with the N classification (P<0.001), clinical TNM stage (P<0.001) and histological grade of tumors (P=0.010). Survival analysis showed an association between high UBE2T expression and poor survival rate in patients with CRC (P=0.002). Cox regression analysis also revealed that UBE2T expression was an independent prognostic factor for these patients (P=0.006). RT-qPCR and western blotting showed that UBE2T was expressed in CRC cell lines at higher levels than that in a normal colon cell line. Analysis of TCGA data revealed that UBE2T was highly expressed in tumor samples compared with normal samples, but was not associated with prognosis. GSEA showed that high expression of UBE2T was associated with the Kyoto Encyclopedia of Genes and Genomes pathways ‘cell cycle’, ‘oxidative phosphorylation’, ‘DNA replication’, ‘p53 signaling pathway’, ‘ubiquitin mediated proteolysis’ and ‘pentose phosphate pathway’. These results indicate that UBE2T may play an important role in the progression of CRC and serve as a potential prognostic factor during the treatment of cancer.

Lung adenocarcinoma (LUAD), a form of lung cancer, is reported to cause first and second-order cancer morbidity to men and women in China, respectively. We assessed the mRNA expression of GJB2 in LUAD patients in our study, based on data acquired from the cancer genome atlas (TCGA) and so as to increase further knowledge into the biological pathways involved in LUAD pathogenesis related to GJB2.Information on gene expression and comparing clinical data were recognized and downloaded from TCGA. Gene set enrichment analysis (GSEA) created an arranged list of all genes is indicated by their connection with GJB2 expression.Our study cohort included 265 (54.5%) female and 221 (36.0%) male patients. The scatter plot and paired plot showed the difference of GJB2 expression between normal and tumor samples (P < .01). Overall survival (OS) analysis demonstrated that LUAD with GJB2 -high had a more terrible prognosis than that with GJB2 -low (P < .01). Multivariate analysis with the cox proportional hazards model indicated that the expression of Cx26 (HR: 1.00; 95%CI: 1.00–1.01; P = .041) and stage (HR: 1.95; 95%CI: 1.23–3.09; P = .003) were independent prognostic factors for patients with LUAD. The GSEA results showed that cytosolic DNA sensing pathway, apoptosis, cytokine-cytokine receptor interaction, natural killer cell mediated cytotoxicity, regulation of actin cytoskeleton, toll-like receptor signaling pathway, small cell lung cancer and pathways in cancer are differentially enriched in GJB2 high expression phenotype.Our study confirmed the significantly high levels of Cx26 expression in LUAD patients with several observed clinical features. GJB2 may be a potentially useful prognostic molecular biomarker of bad survival in LUAD, while further experimental ought to be performed to demonstrate the biologic effect of GJB2.

Abnormal spindle-like microcephaly-associated (ASPM) protein is essential for mitotic spindle function during cell replication. The present study aimed to evaluate the hypothesis that ASPM serves a critical role in cancer invasiveness and may act as a prognostic biomarker in bladder cancer. In total, 6 independent worldwide bladder cancer microarray mRNA expression datasets (n=1,355) with clinical and follow-up annotations were collected from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Reverse transcription-quantitative polymerase chain reaction analysis revealed that ASPM mRNA expression was higher in bladder cancer tissue compared with adjacent normal bladder mucosae in 10 paired human tissue samples (P=0.004). ASPM overexpression in human bladder cancer samples was consistent with the mRNA expression datasets from GEO and TCGA. Bioinformatics analysis indicated that ASPM mRNA expression was significantly associated with grade and tumor node metastasis (TNM) stage in bladder cancer, based on pooled GEO and TCGA datasets (P<0.05). Stratification analysis indicated that the clinical significance of ASPM was particularly pronounced in low-grade or papillary subtypes of bladder cancer. Individual Cox and pooled Kaplan-Meier analyses suggested that ASPM expression was significantly directly correlated with poor overall (OS) and progression-free survival (PFS) in bladder cancer. Multivariate and stratification analyses demonstrated that the prognostic significance of ASPM was evident in low-grade or papillary bladder cancers, yet not in high-grade or non-papillary subgroups. Increased expression of ASPM was associated with poor OS in muscle-invasive bladder cancer and with poor PFS in non-muscle-invasive bladder cancer (P<0.05). Bioinformatics analysis identified the top 11 ASPM-related genes on STRING-DB.org. The expression of the majority of these genes was associated with poor outcomes of bladder cancer with statistical significance. Gene set enrichment analysis indicated that the high expression of ASPM could enrich gene signatures involved in mitosis, differentiation and metastasis in bladder cancer. Further analysis of TCGA datasets indicated that increased ASPM expression was significantly associated with higher Gleason score, T stage, N stage and poor clinical outcome in prostate cancer. It was also significantly associated with late TNM stage and poor PFS in renal cell carcinoma. In summary, ASPM may serve as a novel prognostic biomarker for low-grade or papillary bladder cancer.

BackgroundThe serine peptidase inhibitor Kazal type 13 (SPINK13) gene has tumor suppressor activity, but its role in renal cell carcinoma (RCC) remains unknown. This study aimed to investigate mRNA expression of SPINK13 in clear cell renal cell carcinoma (CCRCC) in human tissue and to use bioinformatics data to investigate the role of SPINK13 expression as a clinicopathological and prognostic biomarker for patients with CCRCC.Material/MethodsPatients with CCRCC (N=533) with available RNA sequence data from The Cancer Genome Atlas (TCGA)-CCRCC database were analyzed with patients who had a tissue diagnosis of CCRCC (N=305) at the Fudan University Shanghai Cancer Center (FUSCC). Differential transcriptional and proteome expression profiles were obtained from the ONCOMINE cancer microarray database, TCGA, and the Human Protein Atlas (HPA) database. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) measured SPINK13 mRNA expression in 305 samples of CCRCC tissue from the FUSCC. The effects of clinicopathological parameters on progression-free survival (PFS) and overall survival (OS) were analyzed using the Kaplan-Meier and log-rank test.ResultsTranscriptional and proteome expression of SPINK13 were significantly increased CCRCC tissue samples. Increased SPINK13 mRNA expression was significantly associated with reduced PFS and OS in 838 patients with CCRCC patients from the two independent cohorts, the FUSCC and the TCGA-CCRCC cohorts (p<0.01). Gene set enrichment analysis (GSEA) showed that SPINK13 expression was involved in complement, apical junction, epithelial-mesenchymal transition (EMT), glycolysis, hypoxia, and inflammation signaling pathways.ConclusionsIncreased expression of SPINK13 was associated with poor prognosis in patients with CCRCC.

In gastric cancer (GC), peritoneal dissemination (PD) occurs frequently and is incurable. In this study, we aimed to identify PD-associated genes in GC. We identified a PD-associated gene using three GC datasets: highly disseminated peritoneal GC cell lines, the Singapore dataset and The Cancer Genome Atlas (TCGA) dataset. We assessed the clinicopathological significance of the gene expression using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and performed immunohistochemical analysis for the gene in our patient cohort. We also performed survival analyses of the gene in our patient cohort, the Singapore dataset and the GSE62254 datasets. Moreover, gene set enrichment analysis (GSEA) was performed using the Singapore and TCGA datasets. Finally, in vitro experiments such as invasion/migration assays, immunofluorescence staining of actin filaments, epidermal growth factor (EGF) treatment analysis, and gene expression analysis were conducted using three gene-knockdown GC cell lines (AGS, 58As9, MKN45). ADP-ribosylation factor-like 4c (ARL4C) was identified as a PD-associated gene, and immunohistochemical analysis showed that ARL4C was overexpressed in GC cells. High ARL4C expression was associated with the depth of invasion (p<0.01) and PD (p<0.05) and was a poor prognostic factor (p<0.05) in our patient cohort, the Singapore dataset and the GSE62254 dataset. ARL4C expression positively correlated with the epithelial-mesenchymal transition (EMT) gene set in GSEA. Moreover, ARL4C knockdown reduced invasion/migration capacity, SLUG expression, and the formation of lamellipodia or filopodia in AGS and 58As9 cells. Finally, EGF treatment increased ARL4C expression in MKN45 cells. ARL4C was associated with PD and was a poor prognostic factor in GC, possibly through promoting invasive capacity by activation of both EMT and motility.