UBE2C Research Articles

Enhancing risk stratification models in localized prostate cancer by novel validated tissue biomarkers.

Localized prostate cancer (PCa) is a largely heterogeneous disease regarding its clinical behavior. Current risk stratification relies on clinicopathological parameters and distinguishing between indolent and aggressive cases remains challenging. To improve risk stratification, we aimed to identify new prognostic markers for PCa. We performed an in silico analysis on publicly available PCa transcriptome datasets. The top 20 prognostic genes were assessed in PCa tissue samples of our institutional cohort (n = 92) using the NanoString nCounter technology. The three most promising candidates were further assessed by immunohistochemistry (IHC) in an institutional (n = 121) and an independent validation cohort from the EMPACT consortium (n = 199). Cancer-specific survival (CSS) and progression-free survival (PFS) were used as endpoints. Our in silico analysis identified 113 prognostic genes. The prognostic values of seven of the top 20 genes were confirmed in our institutional radical prostatectomy (RPE) cohort. Low CENPO, P2RX5, ABCC5 as well as high ASF1B, NCAPH, UBE2C, and ZWINT gene expressions were associated with shorter CSS. IHC analysis confirmed the significant associations between NCAPH and UBE2C staining and worse CSS. In the external validation cohort, higher NCAPH and ZWINT protein expressions were associated with shorter PFS. The combination of the newly identified tissue protein markers improved standard risk stratification models, such as D'Amico, CAPRA, and Cambridge prognostic groups. We identified and validated high tissue levels of NCAPH, UBE2C, and ZWINT as novel prognostic risk factors in clinically localized PCa patients. The use of these markers can improve routinely used risk estimation models.

The mechanism underlying B-cell developmental dysfunction in Kawasaki disease based on single-cell transcriptomic sequencing.

Kawasaki disease (KD) is an acute systemic vasculitis that can lead to acquired heart disease in children mostly from in developed countries. The previous research showed that B cells in KD patients underwent a profound change in both the cell numbers and types after intravenous immunoglobulin (IVIG) therapy. We performed the single-cell RNA-sequencing for the peripheral blood mononuclear cells (PBMCs) from three febrile patients and three KD patients to investigate the possible mechanism underlying B cell developmental dysfunction in KD. The pseudo-time analysis was employed to study the developmental trajectories of the PBMCs in febrile control and KD patients. Overall single-cell expression profiles show that the biological processes of immunity, B cell activation pathway and their related biological entities are repressed in KD patients before IVIG treatment compared to febrile patient and KD patients after IVIG treatment. The differentially expressed gene analyses further demonstrate that B cell signaling pathway is downregulated in B cells and plasma blast cells of KD patients before treatment while cell cycle genes and MYC gene are upregulated in dendritic cells (DCs) and hematopoietic stem and progenitor cells (HSPCs) of KD patients before treatment. The biological process of immune response is upregulated in the HSPCs of KD patients before treatment in our dataset while the biological process of inflammatory response is upregulated in the HSPCs of KD patients before treatment in GSE168732 dataset. Single-cell trajectory analyses demonstrate that KD patients before treatment have a shortened developmental path in which B cells and T cells are failed to differentiate into separate lineages. HSPD1 and HSPE1 genes show an elevated expression level in the early cell development stage of KD patients before treatment accompanied with the repression of MYC, SPI1, MT2A and UBE2C genes. Our analyses of all B cells from KD patients before treatment show most of B cells are arrested in a transitional state with an ill developmental path compared with febrile patients and KD patients after treatment. Our results indicate that the immune premature HSPCs accompanied with the abnormal expression dynamics of cell cycle and SPI1 genes are the mechanism underlying B cell developmental dysfunction in KD patients.

Therapeutic Potential of PLK1, KIF4A, CDCA5, UBE2C, CDT1, SKA3, AURKB, and PTTG1 Genes in Triple-Negative Breast Cancer: Correlating Their Expression with Sensitivity to GSK 461364 and IKK 16 Drugs.

The treatment of triple-negative breast cancer (TNBC) has been associated with challenges due to the lack of expression of ER, PR, and HER2 receptors in tumor cells. This study aimed to identify genes with potential therapeutic targets in TNBC. Data from the cancer genome atlas regarding breast cancer (BC) were downloaded. After initial preprocessing, cancer samples were categorized into four groups: TNBC, HER2-positive, luminal A, and luminal B. Gene expression differences between these groups were calculated, focusing on genes that showed differential expression in TNBC. A protein-protein interaction network was conducted to identify hub genes among the candidate genes related to TNBC. The protein expression of candidate genes was assessed using immunohistochemistry data from the human protein atlas. Drug resistance and sensitivity associated with hub genes were identified using data from PharmacoDB. TNBC samples and the RT-qPCR method were used to confirm the results. Our findings revealed that eight genes, namely PLK1, KIF4A, CDCA5, UBE2C, CDT1, SKA3, AURKB, and PTTG1, had significant upregulation at the RNA level in TNBC subgroup compared to other subgroups and could be considered hub genes in TNBC. Compared to other subgroups, their expression level in TNBC samples had high sensitivity and specificity. RT-qPCR results also demonstrated a significant increase in levels of SKA3 and PTTG1 in the TNBC compared to healthy tissue and other subgroups. The protein expression of these genes was notably high in some BC samples. PharmacoDB data showed that some candidate genes were closely linked to drug sensitivity of GSK 461364 and IKK 16. The results of this study showed a significant increase in the expression level of PLK1, KIF4A, CDCA5, UBE2C, CDT1, SKA3, AURKB, and PTTG1 in TNBC compared to other BC subgroups. These genes show considerable promise as therapeutic targets for the TNBC subgroup.

Associating transcriptomics data with inflammatory markers to understand tumour microenvironment in hepatocellular carcinoma.

Liver cancer is the fourth leading cause of cancer-related death globally which is estimated to reach more than 1 million deaths a year by 2030. Among liver cancer types, hepatocellular carcinoma (HCC) accounts for approximately 90% of the cases and is known to have a tumour promoting inflammation regardless of its underlying aetiology. However, current promising treatment approaches, such as immunotherapy, are partially effective for most of the patients due to the immunosuppressive nature of the tumour microenvironment (TME). Therefore, there is an urgent need to fully understand TME in HCC and discover new immune markers to eliminate resistance to immunotherapy. We analyse three microarray datasets, using unsupervised and supervised methods, in an effort to discover signature genes. First, univariate, and multivariate, feature selection methods, such as the Boruta algorithm, are applied. Subsequently, an optimisation procedure, which utilises random forest algorithm with three dataset pairs combinations, is performed. The resulting optimal gene sets are then combined and further subjected to network analysis and pathway enrichment analysis so as to obtain information related to their biological relevance. The microarray datasets were analysed via the MCP-counter, CIBERSORT, TIMER, EPIC, and quanTIseq deconvolution methods and an estimation of cell type abundances for each dataset sample were identified. The differences in the cell type abundances, between the adjacent and tumour sample groups, were then assessed using a Wilcoxon Rank Sum test (p-value < 0.05). The optimal gene signature sets, derived from each of the data pairs combination, achieved AUC values ranging from 0.959 to 0.988 in external validation sets using Random Forest model. CLEC1B and PTTG1 genes are retrieved across each optimal set. Among the signature genes, PTTG1, AURKA, and UBE2C genes are found to be involved in the regulation of mitotic sister chromatid separation and anaphase-promoting complex (APC) dependent catabolic process (adjusted p-value < 0.001). Additionally, the application of deconvolution algorithms revealed significant changes in cell type abundances of Regulatory T (Treg) cells, M0 and M1 macrophages, and T CD8+ cells between adjacent and tumour samples. We identified ECM1 gene as a potential immune-related marker acting through immune cell migration and macrophage polarisation. Our results indicate that macrophages, such as M0 macrophage and M1 macrophage cells, undergo significant changes in HCC TME. Moreover, our immune deconvolution approach revealed significant infiltration of Treg cells and M0 macrophages, and a significant decrease in T CD8+ cells and M1 macrophages in tumour samples.

Functions of three ubiquitin-conjugating enzyme 2 genes in hepatocellular carcinoma diagnosis and prognosis.

BACKGROUNDLiver cancer ranks the third cause of cancer-related death worldwide. The most common type of liver cancer is hepatocellular carcinoma (HCC). The survival time for HCC patients is very limited by years due to the lack of efficient treatment, failure of early diagnosis, and poor prognosis. Ubiquitination plays an essential role in the biochemical processes of a variety of cellular functions. AIMTo investigate three ubiquitination-associated genes in HCC. METHODSHerein, the expression levels of ubiquitin-conjugating enzymes 2 (UBE2) including UBE2C, UBE2T, and UBE2S in tumor samples of HCC patients and non-tumor controls at the Cancer Genome Atlas (TCGA) database, was comprehensively analyzed. The relationship of UBE2 gene expression level with cancer stage, prognostic outcome, and TP53 mutant status was studied.RESULTSOur results showed that UBE2C, UBE2T, and UBE2S genes were overexpressed in HCC samples compared to non-tumor tissues. Dependent on the cancer progression stage, three UBE2 genes showed higher expression in tumor tissues at all four stages compared to non-tumor control samples. Furthermore, a significantly higher expression of these genes was found in stage 2 and stage 3 cancers compared to stage 1 cancer. Additionally, overexpression of those genes was negatively associated with prognostic outcome and overall survival time. Patients with TP53 mutation showed a higher expression level of three UBE2 genes, indicating an association between UBE2 expression with p53 function.CONCLUSIONIn summary, this study shed light on the potential roles of UBE2C, UBE2T, UBE2S on diagnostic and prognostic biomarkers for HCC. Moreover, based on our findings, it is appealing to further explore the correlation of those genes with TP53 mutation in HCC and the related mechanisms.

1189P EGFR mutation p.747_749del enhances anlotinib sensitivity by upregulating EGR1 and DUSP6 in NSCLC cells

Lung cancer is very common and one of the leading causes of cancer mortality worldwide. Anlotinib has been widely applied to treat many types of cancers in the clinic, including NSCLC. In clinic, we found the effect of treatment with anlotinib was more significant in NSCLC with EGFR mutation p.747_749del. In this study, we have verified the hypothesis and further study its specific path which is mainly achieved by increasing the expression of the EGR1 and UBE2C genes.

Chromenopyrimidinone Controls Stemness and Malignancy by suppressing CD133 Expression in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a highly malignant human cancer that has increasing mortality rates worldwide. Because CD133+ cells control tumor maintenance and progression, compounds that target CD133+ cancer cells could be effective in combating HCC. We found that the administration of chromenopyrimidinone (CPO) significantly decreased spheroid formation and the number of CD133+ cells in mixed HCC cell populations. CPO not only significantly inhibited cell proliferation in HCC cells exhibiting different CD133 expression levels, but also effectively induced apoptosis and increased the expression of LC3-II in HCC cells. CPO also exhibits in vivo therapeutic efficiency in HCC. Specifically, CPO suppressed the expression of CD133 by altering the subcellular localization of CD133 from the membrane to lysosomes in CD133+ HCC cells. Moreover, CPO treatment induced point mutations in the ADRB1, APOB, EGR2, and UBE2C genes and inhibited the expression of these proteins in HCC and the expression of UBE2C is particularly controlled by CD133 expression among those four proteins in HCC. Our results suggested that CPO may suppress stemness and malignancies in vivo and in vitro by decreasing CD133 and UBE2C expression in CD133+ HCC. Our study provides evidence that CPO could act as a novel therapeutic agent for the effective treatment of CD133+ HCC.

UBE2C promotes the progression of head and neck squamous cell carcinoma

The development of head and neck squamous cell carcinoma (HNSCC) is a complex pathological process and many cellular and molecular events may occur. The ubiquitin conjugating enzyme E2 (UBE2C) was found to play an oncogenic role in several human cancers. However, its functional role in HNSCC tumorigenesis remains unknown. In this study, UBE2C gene expression in HNSCC was first evaluated using the data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. The connection between UBE2C gene expression and patients’ survival rates of HNSCC and other human cancers was also investigated. Liquid chromatography with tandem mass spectrometry was used to identify differentially expressed proteins, including UBE2C, between UMSCC1 oral cancer cells and normal human oral keratinocytes (NHOKs). Immunohistochemistry (IHC) was used to verify the differential expression of UBE2C protein between HNSCC and adjacent control tissues. Cell cycle analysis, MTT, colony formation, Transwell migration, and Matrigel invasion assays were used to study the effect of UBE2C downregulation on the malignant phenotypes of HNSCC cells. The bioinformatic analysis of the proteins interacting with UBE2C in HNSCC cells was also performed. Based on the data obtained from the cancer databases and our in vitro studies, we found that UBE2C was overexpressed in HNSCC and patients with high UBE2C expression suffered a remarkably worse overall survival rate than those with low UBE2C expression, and a similar observation was found in a number of other human cancers. UBE2C was also found to be overexpressed in HNSCC cells versus normal human oral keratinocytes and inhibition of UBE2C expression significantly suppressed the malignant phenotypes of HNSCC cells in vitro. The bioinformatic analysis indicated that UBE2C may be involved in head and neck tumorigenesis through the mediation of important pathways such as ubiquitin mediated proteolysis, proteasome, and cell cycle. In conclusion, our results suggest that UBE2C is consistently upregulated in many human solid tumors. It promotes HNSCC progression and may serve as a potential prognostic biomarker in HNSCC. Future studies are warranted to unveil the underlying molecular pathways of UBE2C in HNSCC.

Grade-specific diagnostic and prognostic biomarkers in breast cancer

An integrative approach is presented to identify grade-specific biomarkers for breast cancer. Grade-specific molecular interaction networks were constructed with differentially expressed genes (DEGs) of cancer grade 1, 2, and 3. We observed that the molecular network of grade3 is predominantly associated with cancer-specific processes. Among the top ten connected DEGs in the grade3, the increase in the expression of UBE2C and CCNB2 genes was statistically significant across different grades. Along with UBE2C and CCNB2 genes, the CDK1, KIF2C, NDC80, and CCNB2 genes are also profoundly expressed in different grades and reduce the patient's survival. Gene set enrichment analysis of these six genes reconfirms their role in metastatic phenotype. Moreover, the coexpression network shows a strong association of these six genes promotes cancer specific biological processes and possibly drives cancer from lower to a higher grade. Collectively the identified genes can act as potential biomarkers for breast cancer diagnosis and prognosis.

Identification of CDKN3 and UBE2C mRNAs as Prognostic Biomarkers in Early-Stage Lung Adenocarcinoma Using Bioinformatics Strategy

Background: Lung adenocarcinoma (LUAD) is the most common histological subtype of non-small cell lung cancer with very poor 5-year overall survival (OS) rate. It is histopathologically difficult to predict clinical outcome in early-stage LUAD. Identifying reliable prognostic biomarker is absolutely critical to benefit from early additional treatment for early-stage LUAD patients. Objectives: The purpose of the current study was to identify critical genes as prognostic biomarkers in early-stage LUAD using gene expression profiles based on the microarray. Methods: In this bioinformatics-based cross-study, gene expression profiles from early-stage LUAD, including GSE10072 and GSE19804 genes were integrated using bioinformatics methods, including differentially expressed gene analysis (DEGA), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and protein-protein interaction (PPI) network construction. Subsequently, the survival analysis of key genes was performed using The Cancer Genome Atlas (TCGA) database and was validated using online Gene Expression Profiling Interactive Analysis (GEPIA) database. Results: A total of 89 up-regulated and 214 down-regulated genes were identified in early-stage LUAD, and the functional changes of 303 differentially expressed genes (DEGs) were mainly related to cell cycle. A PPI network was established by online STRING database with 207 nodes and 775 edges. Centrality analysis showed that CDKN3 and UBE2C genes were identified as key genes implicated in early-stage LUAD. Survival analysis revealed that low mRNA expressions of CDKN3 and UBE2C were significantly associated with longer OS of early-stage LUAD patients. Conclusions: This cross-study found key dysregulated genes involved in early-stage LUAD, which might provide insights into the pathogenesis of early-stage LUAD, and identified UBE2C and CDKN3 might serve as potential diagnostic and prognostic biomarkers and therapeutic targets for early-stage LUAD.

Integrated Bioinformatics Analysis of Hub Genes and Pathways in Anaplastic Thyroid Carcinomas.

Anaplastic thyroid carcinoma (ATC) is a very rare malignancy; the pathogenesis of which is still not fully understood. The aim of the present study was to identify hub genes and pathways in ATC by microarray expression profiling. Two independent datasets (GSE27155 and GSE53072) were downloaded from GEO database. The differentially expressed genes (DEGs) between ATC tissues and normal thyroid tissues were screened out by the limma package and then enriched by gene ontology (GO) and KEGG pathway analysis. The hub genes were selected by protein-protein interaction (PPI) analysis. A total of 141 common upregulated and 87 common downregulated genes were screened out. These DEGs were significantly enriched in the phagosome and NF-kappa B signaling pathway. Through PPI analysis, TOP2A, TYMS, CCNB1, RACGAP1, FEN1, PRC1, and UBE2C were selected as hub genes, which were highly expressed in ATC tissues. TCGA data suggested that the expression levels of TOP2A, TYMS, FEN1, and PRC1 genes were also upregulated in other histological subtypes of thyroid carcinoma. High expression of TOP2A, TYMS, FEN1, PRC1, or UBE2C gene significantly decreased disease-free survival of patients with other thyroid carcinomas. In conclusion, the present study identified several hub genes and pathways, which will contribute to elucidating the pathogenesis of ATC and providing therapeutic targets for ATC.

Abstract 3991: Novel small molecule inhibitors of p300/CBP down-regulate androgen receptor (AR) and c-Myc for the treatment of prostate cancer and beyond

Abstract Background: E1A binding protein (p300) and CREB binding protein (CBP), two paralogue histone acetyl transferase proteins, act as transcriptional co-activators of a variety of cancer related genes. We have developed CCS1477, a potent, selective and orally active small molecule inhibitor of the bromodomain of p300/CBP and investigated its role in regulating AR and c-Myc expression and function, for the treatment of prostate cancer and haematological malignancies. We have also examined the role of p300/CBP in driving synthetic lethality in tumours with loss of function mutations (eg. bladder cancer). Methods: Potency and functional activity was evaluated in a panel of prostate cells lines representing hormone responsive, hormone independent and castration resistant disease. Effects of CCS1477 on AR, AR-V7 splice variant and c-Myc protein, as well as KLK3, c-Myc, UBE2C, CCNA2 and TMPRSS2 gene expression, were assessed. Inhibition of proliferation and function by CCS1477 was also examined in acute myeloid leukaemia cell lines and patient derived primary AML cells. In addition, potency was determined in bladder cell lines possessing a loss of function mutation in p300/CBP and compared to wild type. Results: CCS1477 is a potent inhibitor of cell proliferation in castration resistant prostate cell lines (IC50 = 96nM 22Rv1; 49nM VCaP) with minimal effect in AR-ve lines (PC3 and DU145). Treatment of 22Rv1 and VCaP cells with CCS1477 significantly reduced expression of KLK3, UBE2C and CCNA2 in the presence and absence of enzalutamide indicating compromised signalling via AR and AR-SV. Furthermore, AR and AR-SV protein levels were inhibited in response to CCS1477 treatment. Utilising an enzalutamide-resistant cell line (LNCaP-ARF876L), CCS1477 treatment down-regulated both androgen and enzalutamide-stimulated KLK3 and TMPRSS2 gene expression. CCS1477 dosed at 20mg/kg qd caused complete tumour growth inhibition in a 22Rv1 xenograft model. CCS1477 is also a potent inhibitor of proliferation in AML cell lines (IC50 ~ 100nM; THP-1; MV4-11), with effects mediated by G1 cell cycle arrest and accompanied by myeloid differentiation. Comparable results are observed on patient derived primary AML cells. In bladder cancer cell lines, we observed differential sensitivity to CCS1477 with p300/CBP deficient lines (IC50 = 300nM VM-CUB-2 and 647V) compared with wild type (no activity at 30uM, RT112). Conclusions: Taken together these data support the clinical testing of p300/CBP inhibition in patients in three settings; (i) castration resistant prostate cancer through down-regulating AR, AR-SV and c-Myc expression and function; (ii) haematological cancers by effects on cell cycle arrest and myeloid differentiation, and (iii) patients with loss of function mutations in p300 or CBP by driving synthetic lethality. Citation Format: Neil Pegg, Jenny Worthington, Barbara Young, Amy Prosser, Luke Gaughan, Gary Spencer, Tim Somervaille, Julie Burns, Margaret Knowles, Nigel Brooks. Novel small molecule inhibitors of p300/CBP down-regulate androgen receptor (AR) and c-Myc for the treatment of prostate cancer and beyond [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 3991.

The clinicopathological significance of UBE2C in breast cancer: a study based on immunohistochemistry, microarray and RNA-sequencing data

BackgroundUbiquitin-conjugating enzyme E2C (UBE2C) has been previously reported to correlate with the malignant progression of various human cancers, however, the exact molecular function of UBE2C in breast carcinoma (BRCA) remained elusive. We aimed to investigate UBE2C expression in BRCA and its clinical significance.MethodsThe expression of UBE2C in 209 BRCA tissue samples and 53 adjacent normal tissue samples was detected using immunohistochemistry. The clinical role of UBE2C was analyzed. Public databases including the human protein atlas and Oncomine were used to assess UBE2C expression in BRCA. Moreover, the cancer genome atlas (TCGA) database was employed to investigate the prognostic value of UBE2C in BRCA.ResultsThe positive expression rate of UBE2C in BRCA was 70.8% (148/209), and UBE2C expression in the adjacent breast tissue was negative. The expression of UBE2C was positively correlated with tumor size (r = 0.32, P < 0.001), histological grade (r = 0.237, P = 0.001), clinical stage (r = 0.198, P = 0.004), lymph node metastasis (r = 0.155, P = 0.026), HER2 expression level (r = 0.356, P < 0.001), Ki-67 expression level (r = 0.504, P < 0.001), and P53 expression level (r = 0.32, P = 0.001). Negative correlations were found between UBE2C expression and the ER (r = − 0.403, P < 0.001) and PR (r = − 0.468, P < 0.001) status. UBE2C gene expression data from the public databases all proved that UBE2C was overexpressed in BRCA. According to the TCGA data analysis, a higher positive expression of UBE2C was associated with worse survival of BRCA patients (P = 0.0428), and data from cBioPortal indicated that 11% of all sequenced BRCA patients possessed a gene alteration of UBE2C, predominately gene amplification and mRNA regulation.ConclusionUbiquitin-conjugating enzyme E2C might pose an oncogenic effect on the progression of BRCA.

DNA copy number alterations, gene expression changes and disease-free survival in patients with colorectal cancer: a 10 year follow-up.

DNA copy number alterations (CNAs) and gene expression changes have amply been encountered in colorectal cancers (CRCs), but the extent at which CNAs affect gene expression, as well as their relevance for tumor development, are still poorly defined. Here we aimed at assessing the clinical relevance of these parameters in a 10year follow-up study. Tumors and normal adjacent colon mucosa, obtained at primary surgery from 21 CRC patients, were subjected to (i) high-resolution array CGH (a-CGH) for the detection of CNAs and (ii) microarray-based transcriptome profiling for the detection of gene expression (GE) changes. Correlations between these genomic and transcriptomic changes and their associations with clinical and histopathological parameters were assessed with the aim to identify molecular signatures associated with disease-free survival of the CRC patients during a 10year follow-up. DNA copy number gains were frequently detected in chromosomes 7, 8q, 13, 19, 20q and X, whereas DNA copy number losses were frequently detected in chromosomes 1p, 4, 8p, 15, 17p, 18, 19 and 22q. None of these alterations were observed in all samples. In addition, we found that 2,498 genes were up- and that 1,094 genes were down-regulated in the tumor samples compared to their corresponding normal mucosa (p < 0.01). The expression of 65 genes was found to be significantly associated with prognosis (p < 0.01). Specifically, we found that up-regulation of the IL17RA, IGF2BP2 and ABCC2 genes, and of genes acting in the mTOR and cytokine receptor pathways, were strongly associated with a poor survival. Subsequent integrated analyses revealed that increased expression levels of the MMP9, BMP7, UBE2C, I-CAM, NOTCH3, NOTCH1, PTGES2, HMGB1 and ERBB3 genes were associated with copy number gains, whereas decreased expression levels of the MUC1, E2F2, HRAS and SIRT3 genes were associated with copy number losses. Pathways related to cell cycle progression, eicosanoid metabolism, and TGF-β and apoptosis signaling, were found to be most significantly affected. Our results suggest that CNAs in CRC tumor tissues are associated with concomitant changes in the expression of cancer-related genes. In other genes epigenetic mechanism may be at work. Up-regulation of the IL17RA, IGF2BP2 and ABCC2 genes, and of genes acting in the mTOR and cytokine receptor pathways, appear to be associated with a poor survival. These alterations may, in addition to Dukes' staging, be employed as new prognostic biomarkers for the prediction of clinical outcome in CRC patients.

E2 Ubiquitin-conjugating Enzyme, UBE2C Gene, Is Reciprocally Regulated by Wild-type and Gain-of-Function Mutant p53

Spindle assembly checkpoint governs proper chromosomal segregation during mitosis to ensure genomic stability. At the cellular level, this event is tightly regulated by UBE2C, an E2 ubiquitin-conjugating enzyme that donates ubiquitin to the anaphase-promoting complex/cyclosome. This, in turn, facilitates anaphase-onset by ubiquitin-mediated degradation of mitotic substrates. UBE2C is an important marker of chromosomal instability and has been associated with malignant growth. However, the mechanism of its regulation is largely unexplored. In this study, we report that UBE2C is transcriptionally activated by the gain-of-function (GOF) mutant p53, although it is transcriptionally repressed by wild-type p53. We showed that wild-type p53-mediated inhibition of UBE2C is p21-E2F4-dependent and GOF mutant p53-mediated transactivation of UBE2C is NF-Y-dependent. We further explored that DNA damage-induced wild-type p53 leads to spindle assembly checkpoint arrest by repressing UBE2C, whereas mutant p53 causes premature anaphase exit by increasing UBE2C expression in the presence of 5-fluorouracil. Identification of UBE2C as a target of wild-type and GOF mutant p53 further highlights the contribution of p53 in regulation of spindle assembly checkpoint.

Abstract 5159: Identification of ARv567es expression profile in the prostate cancer clinical samples with a newly developed antibody

Abstract Background: Androgen receptor splice variants (AR-Vs) have emerged as an important diagnostic biomarker and potential therapeutic oncotarget in castration-resistant prostate cancer. Extensive RNA sequencing of a large cohort of metastases from men treated with androgen deprivation therapy (ADT) including abiraterone and enzalutamide, revealed a marked increase in the amount and variety of AR-Vs when compared to primary tumor samples. These and other data suggest that the AR-Vs should have a selective growth advantage over the full-length AR (AR-FL). However, this growth advantage only occurs after ADT even though AR-FL and AR-V RNA expression are both present in the tumor. Methods: In order to determine how this progression to AR-V predominance occurs, we used the LuCaP 86.2 xenograft, which has a genomic rearrangement of the AR gene so that tumor cells either express AR-FL or ARv567es. Xenografts were implanted sc into the flanks of SCID mice. When tumors reached a volume of 200 mm3 cohorts of mice were castrated and followed then euthanized at 0, 1 day, 1 week, or 3 weeks post-castration. Intact control mice bearing LuCaP 86.2 xenografts were euthanized at similar time points. Tumors were analyzed for AR-FL and ARv567es mRNA and protein. In addition canonical and AR-V regulated gene expression was analyzed by quantitative PCR. We then used an ARv567es antibody to examine the presence of ARv567es in 155 metastases. Results: The ARv567es antibody is a rabbit monoclonal antibody against the unique exon-9 amino acid sequence expressed by ARv567es (CERAASVHF). This antibody does not react with AR-FL. As previously described, there is no difference in growth rates for castration-resistant LuCaP 86.2 xenografts grown in intact and castrate mice. Following castration there was a marked increase in ARv567es nuclear staining (p &amp;lt; 0.01), with no significant change in ARv567es mRNA. AR-amino terminal staining was unchanged following castration; however, consistent with the loss of expression of AR-FL protein and increase in ARv567es protein, there was no detectable carboxy-terminal AR protein staining. PSA gene expression did not change following castration but a marked increase in TMPRSS2, UGT2B17, and UBE2C gene expression was seen, consistent with a shift from ligand-mediated AR-FL activity to AR-V activity. Finally, immunohistochemistry on a tissue microarray (TMA) of 155 metastases from 55 patients in the University of Washington rapid autopsy program demonstrated a significant increase in AR-V staining compared to a primary tumor TMA. Summary: This study demonstrates that expression of ARv567es in development of a castrate resistant xenograft may be regulated at the level of protein translation and that the presence of an AR gene rearrangement for ARv567es prior to castration may be a marker that the tumor can become resistant to castration by an AR-V mechanism. Citation Format: Gang Liu, Aihua Li, Shihua Sun, Cynthia Sprenger, Eva Corey, Colm Morrisey, Scott Dehm, Stephen Plymate. Identification of ARv567es expression profile in the prostate cancer clinical samples with a newly developed antibody. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5159. doi:10.1158/1538-7445.AM2015-5159

Inhibition of microRNA-17/20a suppresses cell proliferation in gastric cancer by modulating UBE2C expression.

microRNAs (miRNAs) are small non-coding RNAs that potentially play a critical role in carcinogenesis. Increasing evidence indicates that the miR-17/20 cluster is upregulated in numerous types of human cancers including gastric cancer which suggests that the miR-17/20 cluster may play an important role in tumorigenesis. However, its role in gastric cancer carcinogenesis remains poorly defined due to the lack of target gene information. The aim of the present study was to investigate the target genes of the miR-17/20 cluster and their role in the tumor growth of gastric cancer. We found that both miR-17 and miR-20a (miR-17/20a) target the UBE2C gene in gastric cancer cells. Luciferase assay, qRT-PCR and western blot analysis confirmed that UBE2C is a direct target of miR‑17/20a in gastric cancer cells. Our results showed that the expression of UBE2C was positively regulated by miR-17/20a at both the mRNA and protein levels. Moreover, miR-17/20a was upregulated and positively associated with UBE2C in the gastric cancer tissues when compared to the adjacent nontumor tissues. Inhibition of miR-17/20a in gastric cancer cells was statistically correlated with a decrease in cell growth. These results demonstrate that upregulation of miR-17/20a promotes gastric cancer cell growth by targeting UBE2C and inhibition of their levels is a potentially promising therapeutic strategy for gastric cancer.

Abstract 922: Evaluating markers of cisplatin sensitivity and survival in small cell lung cancer

Abstract Background: Molecular characterization of small cell lung cancer (SCLC) can lead to improved outcome through the identification of tumor subtypes with differential clinical outcome and response to treatment. We characterized SCLC cell lines and patient samples by a combination of a genome-wide and a custom expression panel to assess expression changes associated with cisplatin sensitivity, in the case of cell lines, and overall survival, in terms of SCLC patients.Method: SCLC cell lines (H69, H128, H146, H526, H187, H209, D53, DMS153, and DMS114) and 79 archival samples of pulmonary neuroendocrine tumor samples were employed. Sensitivity to cisplatin, and PARP inhibitor, veliparib was determined by MTS assay. In order to identify expression profiles characteristic of sensitive and insensitive cell lines, gene expression for untreated and treated cells was obtained from Illumina microarray gene expression and NanoString platforms. Comparisons of expression were performed between treated cell lines and controls using a combination of ANOVA and empirical variance distributions to assess sensitivity. Prognostic impact of differentially expressed genes in cell lines was assessed in patients using gene expression obtained by NanoString and protein expression by immunohistochemistry (IHC). Kaplan Meier Method, Logrank test, and Cox proportional hazard model were employed for testing differences in survival. Additionally, a quantile survival analyses was performed to identify patient subgroups associated with early and/or late changes in expression.Results: Unsupervised analysis of the Illumina probesets identified a 23 gene panel with differential expression between cell lines with differing platinum sensitivity: Sensitive - GLS, UBEC2, HACL1, MSI2 and LOC100129585; insensitive - CENPE, CRYGS, FAM83D, FLJ44342, GNA12, LOC88523, LRDD, N4BP2L2, SLC35A3, SPC25; shared - AURKA, CENPA, DLGAP5, HMMR, KIF20B, LOC100129585, LOC100131735, RBMX, SFRS3. Unsupervised analysis of expression data from Illumina and nCounter NanoString showed considerable concordance in cluster patterns. The Cox regression models of gene expression on overall survival identified LOC100131735 as a predictive marker and, SLC35A3, SPC25, DLGAP5 and UBE2C genes as prognostic biomarkers. Concomitantly high expression of SLC35A3 and GLS genes characterized SCLC with prolonged survival (Logrank p=0.025) while a trend toward reduced risk of death (HR: 0.798; 95%CI, 0.375-1.700, p=0.559) was observed with high GLS protein expression.Conclusions: Our use of both cell line and patient SCLC data has provided evidence of markers characteristics of cisplatin insensitivity that also show a relation with poor overall survival in patients. Additional studies to fully understand the mechanisms underlying such markers are currently underway. Supported through the Georgia Cancer Coalition Distinguished Scholar Award and NIH 5K23CA164015 grant to TK Owonikoko Citation Format: Taofeek Kunle Owonikoko, Guojing Zhang, Gabriel L. Sica, Zhengjia Chen, Jeffrey M. Switchenko, Sungjin Kim, Anthony A. Gal, Suresh S. Ramalingam, Xingming Deng, Michael R. Rossi, Jeanne Kowalski, Fadlo R. Khuri. Evaluating markers of cisplatin sensitivity and survival in small cell lung cancer. [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 922. doi:10.1158/1538-7445.AM2014-922

Abstract 4361: Target and drug discovery for recalcitrant, rare and neglected cancers.

Abstract Efforts are underway to improve the treatment of recalcitrant, rare and neglected cancers through the discovery of potential therapeutic targets, the identification of possible therapeutic combinations, and the identification of genomic vulnerabilities, using state-of-the-art drug discovery, molecular characterization, and mechanism-of-action techniques. Our current focus is on sarcoma and small cell lung cancer. Sarcoma comprises approximately 1% of cancers inclusive of 50 subtypes and occurs in patients of all ages with a frequency spread evenly over the human age range. Small cell lung cancer (SCLC) constitutes approximately 15% of lung cancers, is extremely aggressive, has a high mortality rate and frequently recurs after conventional cytotoxic chemotherapy. We have established comprehensive panels of human sarcoma and small cell lung cancer cell lines (approximately 80 per tumor type). Cell lines are being screened for response to all of the FDA approved anticancer agents and to a library of investigational agents with a goal of identifying existing agents that may be suitable for novel clinical trials, plus identifying potential points of vulnerability for drug discovery. Analysis of selected somatic mutations showed that ATM is frequently mutated in both soft tissue and bone sarcoma. In addition, gene expression profiles were measured in sarcoma cell lines using Affymetrix Exon 1.0 ST arrays in an effort to facilitate identification of splice variants and fusion genes. The gene-level data from the Exon ST1 array compares well with published U133plus 2 expression profiles. Principal components analysis of genes in 48 sarcoma lines indicates that expression varies primarily by disease subtype. For instance, Ewing's Sarcoma overexpressed genes including PRKCB, NPY5R and NPY1R, ITM2A plus adrenergic receptors B1, B3 and A1D, while ACTC1, IGF2 and CHRNA1 were dysregulated in rhabdomyosarcoma. Predicted associations between gene and miRNA expression included CSF1 with miR-128, thioredoxin reductase with miR-324-5p, MDM4 with miR-152, and PODXL with miR-199a-5p, suggesting potential regulatory relationships that might be exploited as cancer targets. In these lines, exome expression was found to be altered more than 10% in 232 genes, including known splice variant genes such as KLK11, UBE2C and sarcoma related fusion genes EWSR1 and ETV. Calculated fusion scores based on EWSR1-FLI1 expression profiles successfully segregated Ewing's sarcoma from all other sarcoma cell lines. Association analysis between gene and microRNA expression and sensitivity to specific anticancer agents is ongoing. Our mission is to improve the treatment of these neglected cancers by providing a comprehensive public database of molecular signatures and sensitivities that can be leveraged by the scientific community at large. Funded by NCI Contract no. HHSN261200800001E. Citation Format: Anne Monks, Annamaria Rapisarda, Kazimierz O. Wrzeszczynski, E. Michael August, Eric C. Polley, Sudhir B. Kondapaka, Gurmeet Kaur, Dianne Newton, Beverley A. Teicher. Target and drug discovery for recalcitrant, rare and neglected cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4361. doi:10.1158/1538-7445.AM2013-4361

Modeling a lethal prostate cancer variant with small-cell carcinoma features.

Small-cell prostate carcinoma (SCPC) morphology predicts for a distinct clinical behavior, resistance to androgen ablation, and frequent but short responses to chemotherapy. We sought to develop model systems that reflect human SCPC and can improve our understanding of its biology. We developed a set of castration-resistant prostate carcinomas xenografts and examined their fidelity to their human tumors of origin. We compared the expression and genomic profiles of SCPC and large-cell neuroendocrine carcinoma (LCNEC) xenografts to those of typical prostate adenocarcinoma xenografts. Results were validated immunohistochemically in a panel of 60 human tumors. The reported SCPC and LCNEC xenografts retain high fidelity to their human tumors of origin and are characterized by a marked upregulation of UBE2C and other mitotic genes in the absence of androgen receptor (AR), retinoblastoma (RB1), and cyclin D1 (CCND1) expression. We confirmed these findings in a panel of samples of CRPC patients. In addition, array comparative genomic hybridization of the xenografts showed that the SCPC/LCNEC tumors display more copy number variations than the adenocarcinoma counterparts. Amplification of the UBE2C locus and microdeletions of RB1 were present in a subset, but none displayed AR nor CCND1 deletions. The AR, RB1, and CCND1 promoters showed no CpG methylation in the SCPC xenografts. Modeling human prostate carcinoma with xenografts allows in-depth and detailed studies of its underlying biology. The detailed clinical annotation of the donor tumors enables associations of anticipated relevance to be made. Future studies in the xenografts will address the functional significance of the findings.