Integrative genomic approaches identify IKBKE as a breast cancer oncogene

The karyotypic chaos exhibited by human epithelial cancers complicates efforts to identify mutations critical for malignant transformation. Here we integrate complementary genomic approaches to identify human oncogenes. We show that activation of the ERK and phosphatidylinositol 3-kinase (PI3K) signaling pathways cooperate to transform human cells. Using a library of activated kinases, we identify several kinases that replace PI3K signaling and render cells tumorigenic. Whole genome structural analyses reveal that one of these kinases, IKBKE (IKKepsilon), is amplified and overexpressed in breast cancer cell lines and patient-derived tumors. Suppression of IKKepsilon expression in breast cancer cell lines that harbor IKBKE amplifications induces cell death. IKKepsilon activates the nuclear factor-kappaB (NF-kappaB) pathway in both cell lines and breast cancers. These observations suggest a mechanism for NF-kappaB activation in breast cancer, implicate the NF-kappaB pathway as a downstream mediator of PI3K, and provide a framework for integrated genomic approaches in oncogene discovery.     

Discovery of biclonal origin and a novel oncogene SLC12A5 in colon cancer by single-cell sequencing

Single-cell sequencing is a powerful tool for delineating clonal relationship and identifying key driver genes for personalized cancer management. Here we performed single-cell sequencing analysis of a case of colon cancer. Population genetics analyses identified two independent clones in tumor cell population. The major tumor clone harbored APC and TP53 mutations as early oncogenic events, whereas the minor clone contained preponderant CDC27 and PABPC1 mutations. The absence of APC and TP53 mutations in the minor clone supports that these two clones were derived from two cellular origins. Examination of somatic mutation allele frequency spectra of additional 21 whole-tissue exome-sequenced cases revealed the heterogeneity of clonal origins in colon cancer. Next, we identified a mutated gene SLC12A5 that showed a high frequency of mutation at the single-cell level but exhibited low prevalence at the population level. Functional characterization of mutant SLC12A5 revealed its potential oncogenic effect in colon cancer. Our study provides the first exome-wide evidence at single-cell level supporting that colon cancer could be of a biclonal origin, and suggests that low-prevalence mutations in a cohort may also play important protumorigenic roles at the individual level.     

Molecular cloning of a novel mouse testis-specific spermatogenic cell apoptosis inhibitor gene mTSARG7 as a candidate oncogene

A novel mouse gene, mTSARG7 (GenBank accession No. AY489184), with a full cDNA length of 2279 bp and containing 12 exons and.ll introns, was cloned from a mouse expressed sequence tag (GenBank accession No. BE644543) that was significantly up-regulated in cryptorchidism. The gene was located in mouse chromosome 8A1.3 and encoded a protein containing 403 amino acid residues that was a new member of the acyltransferase family because the sequence contained the highly conserved phosphate acyltransferase (PlsC) domain existing in all acyltransferase-like proteins. The mTSARG7 protein and AU041707 protein shared 83.9% identity in 402 amino acid residues. Expression of the mTSARG7 gene was restricted to the mouse testis. The results of the in situ hybridization analysis revealed that the mTSARG7 mRNA was expressed in mouse spermatogonia and spermatocytes. Subcellular localization studies showed that the EGFPtagged mTSARG7 protein was localized in the cytoplasm of GC-1 spg cells. The mTSARG7 mRNA expression was initiated in the mouse testis in the second week after birth, and the expression level increased steadily with spermatogenesis and sexual maturation of the mouse. The results of the heat stress experiment showed that the mTSARG7 mRNA expression gradually decreased as the heating duration increased. The pcDNA3.1 Hygro(-)/mTSARG7 plasmid was constructed and introduced into GC- 1 spg cells by liposome transfection. The mTSARG7 can accelerate GC-1 spg cells, causing them to traverse the S-phase and enter the G2-phase, compared with the control group where this did not occur as there was no transfection of mTSARG7. In conclusion, our results suggest that this gene may play an important role in spermatogenesis and the development of cryptorchid testes, and is a testis-specific apoptosis candidate oncogene.     

SUZ12 is a novel putative oncogene promoting tumorigenesis in head and neck squamous cell carcinoma

The suppressor of zest 12 (SUZ12), one of the core polycomb repressive complex 2 (PRC2) components, has increasingly appreciated as a key mediator during human tumorigenesis. However, its expression pattern and oncogenic roles in head and neck squamous cell carcinoma (HNSCC) remain largely unexplored yet. Here, we sought to determine its expression pattern, clinicopathological significance and biological roles in HNSCC. Through data mining and interrogation from multiple publicly available databases, our bioinformatics analyses revealed that SUZ12 mRNA was significantly overexpressed in multiple HNSCC patient cohorts. Moreover, SUZ12 protein was markedly up‐regulated in primary HNSCC samples from our patient cohort as assessed by immunohistochemical staining and its overexpression significantly associated with cervical node metastasis and reduced overall and disease‐free survival. In the 4‐nitroquinoline 1‐oxide (4NQO)‐induced HNSCC mouse model, increased SUZ12 immunostaining was observed along with disease progression from epithelial hyperplasia to squamous cell carcinoma in tongue. Furthermore, shRNA‐mediated SUZ12 knock‐down significantly inhibited cell proliferation, migration and invasion in HNSCC cells, and resulted in compromised tumour growth in vivo. Collectively, our data reveal that SUZ12 might serve as a putative oncogene by promoting cell proliferation, migration and invasion, and also a novel biomarker with diagnostic and prognostic significance for HNSCC.     

WD Repeat Protein WDR48 in Complex with Deubiquitinase USP12 Suppresses Akt-dependent Cell Survival Signaling by Stabilizing PH Domain Leucine-rich Repeat Protein Phosphatase 1 (PHLPP1)

PHLPP1 (PH domain leucine-rich repeat protein phosphatase 1) is a protein-serine/threonine phosphatase and a negative regulator of the PI3-kinase/Akt pathway. Although its function as a suppressor of tumor cell growth has been established, the mechanism of its regulation is not completely understood. In this study, by utilizing the tandem affinity purification approach we have identified WDR48 and USP12 as novel PHLPP1-associated proteins. The WDR48·USP12 complex deubiquitinates PHLPP1 and thereby enhances its protein stability. Similar to PHLPP1 function, WDR48 and USP12 negatively regulate Akt activation and thus promote cellular apoptosis. Functionally, we show that WDR48 and USP12 suppress proliferation of tumor cells. Importantly, we found a WDR48 somatic mutation (L580F) that is defective in stabilizing PHLPP1 in colorectal cancers, supporting a WDR48 role in tumor suppression. Together, our results reveal WDR48 and USP12 as novel PHLPP1 regulators and potential suppressors of tumor cell survival.     

KLRC3, a Natural Killer receptor gene,is a key factor involved in glioblastoma tumourigenesis and aggressiveness

Glioblastoma is the most lethal brain tumour with a poor prognosis. Cancer stem cells (CSC) were proposed to be the most aggressive cells allowing brain tumour recurrence and aggressiveness. Current challenge is to determine CSC signature to characterize these cells and to develop new therapeutics. In a previous work, we achieved a screening of glycosylation‐related genes to characterize specific genes involved in CSC maintenance. Three genes named CHI3L1, KLRC3 and PRUNE2 were found overexpressed in glioblastoma undifferentiated cells (related to CSC) compared to the differentiated ones. The comparison of their roles suggest that KLRC3 gene coding for NKG2E, a protein initially identified in NK cells, is more important than both two other genes in glioblastomas aggressiveness. Indeed, KLRC3 silencing decreased self‐renewal capacity, invasion, proliferation, radioresistance and tumourigenicity of U87‐MG glioblastoma cell line. For the first time we report that KLRC3 gene expression is linked to glioblastoma aggressiveness and could be a new potential therapeutic target to attenuate glioblastoma.     

Arcyptera fusca and Arcyptera tornosi repetitive DNA families: whole-comparative genomic hybridization (W-CGH) as a novel approach to the study of satellite DNA libraries

Whole-comparative genomic hybridization (W-CGH) has been used to exemplify a simple methodology which allows identifying and mapping whole genome differences for highly repetitive DNA sequences between two related species of unknown genomic background. The use of this technique to the species binomy Arcyptera fusca/Arcyptera tornosi has allowed the identification of different DNA families mainly concentrated within the para-/peri-centromeric and distal heterochromatic regions of different chromosomes, which are differentially expanded in both genomes. Additionally, W-CGH allowed chromosome mapping of particular euchromatic regions immersed in the chromosome arms which have been affected by processes of DNA amplification and losses. A molecular approach was also conducted to analyse satellite DNA families in these species. We have found three different families showing an unequal representation in both species. Two of these families showed a centromeric location (EcoRV-390CEN and Sau3A-419CEN), whereas the last one was located at distal heterochromatic regions (Sau3A-197TEL). As A. fusca is a widely distributed species represented in most European high mountains, whereas A. tornosi is an endemic species represented in the Iberian Peninsula, the differences and resemblances reported here offer a good basis to support a close evolutionary relationship between both of the actually isolated species. Finally, W-CGH allowed identification of an asynchronic pattern of heterochromatin condensation through early prophase (characteristic in both species) which is uncommon or probably has been poorly analysed within classical early condensing chromosome domains through meiosis. The congruence of the obtained cytological and molecular results is analysed in light of the ancestral genome relationship between both species.     

Prediction of a competing endogenous RNA co‐expression network as a prognostic marker in glioblastoma

Due to its high proliferation capacity and rapid intracranial spread, glioblastoma (GBM) has become one of the least curable malignant cancers. Recently, the competing endogenous RNAs (ceRNAs) hypothesis has become a focus in the researches of molecular biological mechanisms of cancer occurrence and progression. However, there is a lack of correlation studies on GBM, as well as a lack of comprehensive analyses of GBM molecular mechanisms based on high‐throughput sequencing and large‐scale sample sizes. We obtained RNA‐seq data from The Cancer Genome Atlas (TCGA) and Genotype‐Tissue Expression (GTEx) databases. Further, differentially expressed mRNAs were identified from normal brain tissue and GBM tissue. The similarities between the mRNA modules with clinical traits were subjected to weighted correlation network analysis (WGCNA). With the mRNAs from clinical‐related modules, a survival model was constructed by univariate and multivariate Cox proportional hazard regression analyses. Thereafter, we carried out Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Finally, we predicted interactions between lncRNAs, miRNAs and mRNAs by TargetScan, miRDB, miRTarBase and starBase. We identified 2 lncRNAs (NORAD, XIST), 5 miRNAs (hsa‐miR‐3613, hsa‐miR‐371, hsa‐miR‐373, hsa‐miR‐32, hsa‐miR‐92) and 2 mRNAs (LYZ, PIK3AP1) for the construction of a ceRNA network, which might act as a prognostic biomarker of GBM. Combined with previous studies and our enrichment analysis results, we hypothesized that this ceRNA network affects immune activities and tumour microenvironment variations. Our research provides novel aspects to study GBM development and treatment.     

RDH12, a retinol dehydrogenase causing Leber's congenital amaurosis, is also involved in steroid metabolism

Three retinol dehydrogenases (RDHs) were tested for steroid converting abilities: human and murine RDH 12 and human RDH13. RDH12 is involved in retinal degeneration in Leber's congenital amaurosis (LCA). We show that murine Rdh12 and human RDH13 do not reveal activity towards the checked steroids, but that human type 12 RDH reduces dihydrotestosterone to androstanediol, and is thus also involved in steroid metabolism. Furthermore, we analyzed both expression and subcellular localization of these enzymes.     

Regression as a method to predict copy numbers in comparative genomic hybridization studies on bacteria

Comparative genomic hybridizations (CGH) using microarrays are performed with bacteria in order to determine the level of genomic similarity between various strains. The microarrays applied in CGH experiments are constructed on the basis of the genome sequence of one strain, which is used as a control, or reference, in each experiment. A strain being compared with the known strain is called the unknown strain. The ratios of fluorescent intensities obtained from the spots on the microarrays can be used to determine which genes are divergent in the unknown strain, as well as to predict the copy number of actual genes in the unknown strain. In this paper, we focus on the prediction of gene copy number based on data from CGH experiments. We assumed a linear connection between the log2 of the copy number and the observed log2-ratios, then predictors based on the factor analysis model and the linear random model were proposed in an attempt to identify the copy numbers. These predictors were compared to using the ratio of the intensities directly. Simulations indicated that the proposed predictors improved the prediction of the copy number in most situations. The predictors were applied on CGH data obtained from experiments with Enterococcus faecalis strains in order to determine copy number of relevant genes in five different strains.