Detection of KRAS and BRAF mutations in advanced colorectal cancer by allele-specific single-base primer extension.

Evaluation of: Magnin S, Viel E, Baraquin A et al. A multiplex SNaPshot assay as a rapid method for detecting KRAS and BRAF mutations in advanced colorectal cancers. J. Mol. Diagn. 13(5), 485-492 (2011). Since mutations in the KRAS and BRAF genes are associated with resistance to therapy with anticancer drugs targeting the EGF receptor pathway, the analysis of KRAS and BRAF mutational status has become an important tool in the clinical management of patients with advanced colorectal cancer. To be useful in the clinical setting, a diagnostic assay has to address several issues related to the sensitivity and specificity of the method, the modularity of the assay, the turnaround time and the running costs. A variety of methods have been applied to the diagnosis of KRAS and BRAF mutational status. Although there is a good concordance between different methods, differences exist regarding sensitivity, multiplexing capacity and costs. In this article, we review a recently published assay for the simultaneous detection of diagnostically relevant KRAS and BRAF mutations and discuss this work in the context of conventional diagnostic methods.

KRAS, NRAS, PIK3CA exon 20, and BRAF genotypes in synchronous and metachronous primary colorectal cancers diagnostic and therapeutic implications.

Targeted therapy of advanced colorectal carcinoma (CRC) necessitates KRAS genotyping. Because we were interested in diagnostic and therapeutic consequences, we studied the KRAS, NRAS, PIK3CA exon 20, and BRAF genotypes in synchronous and metachronous primary CRCs; in addition, we studied their available metastases. We studied 21 patients with 43 synchronous and 2 metachronous adenocarcinomas of the colorectum (n = 20) and stomach (n = 1). Five patients had liver metastases and one had a distant lymph node metastasis. Genomic DNA was extracted from microdissected tumor tissue. The DNA was analyzed by Sanger sequencing and pyrosequencing. Fifty-seven different neoplastic lesions were genotyped, showing 18 (31.6%) KRAS, 2 (3.5%) NRAS, and 7 (12.3%) BRAF mutations, distributed among 10 (47.6%), 1 (4.8%), and 5 (23.8%) of the patients. An identical genotype of all synchronous primary CRCs was found only in 7 (35%) of the patients; the remainder had dissimilar genotypes in various combinations. Interestingly, a single patient had an unknown KRAS genotype (c.37_39dupGGC). Six patients with 13 primary carcinomas had distant metastases. In three of these patients, the metastasis shared the genotype only with one of the primary tumors, because the other primary tumors had another genotype. Synchronous and metachronous primary CRCs of the same patient have variable KRAS, NRAS, and BRAF genotypes. When metastases occur in these patients, the genotype has diagnostic and therapeutic implications and should be determined from the simultaneous or metachronous distant metastases.

Roles of AP-2 transcription factors in the regulation of cartilage and skeletal development.

During embryogenesis, most of the mammalian skeletal system is preformed as cartilaginous structures that ossify later. The different stages of cartilage and skeletal development are well described, and several molecular factors are known to influence the events of this enchondral ossification, especially transcription factors. Members of the AP-2 family of transcription factors play important roles in several cellular processes, such as apoptosis, migration and differentiation. Studies with knockout mice demonstrate that a main function of AP-2s is the suppression of terminal differentiation during embryonic development. Additionally, the specific role of these molecules as regulators during chondrogenesis has been characterized. This review gives an overview of AP-2s, and discusses the recent findings on the AP-2 family, in particular AP-2alpha, AP-2beta, and AP-2epsilon, as regulators of cartilage and skeletal development.

Role of deleted in colon carcinoma in osteoarthritis and in chondrocyte migration.

OBJECTIVE: The concept of the chondrocyte as a stationary cell surrounded by an apparently impenetrable matrix has been challenged by in vitro observations in recent years. Chondrocyte migration may have a role in remodelling of the cartilage and pathological conditions. Candidate molecules are repellent factors for the regulation of chondrocyte migration, which are expressed in fetal and adult cartilage. We analysed the potential role of the receptor deleted in colon carcinoma (DCC) in chondrocytes, as this may exert attractive activities. METHODS: Gene expression was determined by quantitative RT-PCR and immunohistochemistry, and gene regulation by electro mobility shift assay and chromatin immunoprecipitation. Functional assays on migration and differentiation were done after cell treatment and transfection. RESULTS: DCC was shown to be specifically up-regulated in OA compared with normal chondrocytes in vitro and in vivo. Promoter analysis and transfection studies showed that the up-regulation of DCC in OA chondrocytes may be mediated by the transcription factors Sox9 and AP-2. Netrin-1, the ligand of DCC, was revealed to induce the migration of OA chondrocytes specifically. Expression of DCC in healthy chondrocytes by transient transfection significantly induced cell migration and chemotaxis to Netrin-1. DCC expression had no influence on cell differentiation; however, induction of MMP1 and -3 expression was observed. CONCLUSION: Strong differential expression of DCC in OA compared with normal chondrocytes hints of a possible role of DCC in the pathophysiology of OA. The strong impact of the DCC receptor on cellular mobility of chondrocytes in vitro suggests a major relevance of migratory activities in physiological and pathological conditions of cartilage. However, definite proof of chondrocyte movements in vivo still has to be established.

The cartilage-specific transcription factor Sox9 regulates AP-2epsilon expression in chondrocytes.

Activating enhancer-binding protein (AP)-2epsilon was previously described as a new regulator of integrin alpha(10) expression in cartilage. In this study, we analyzed the expression of AP-2epsilon in differentiated chondrocytes and in human mesenchymal stem cells (HMSCs), which have been differentiated into chondrocytes in vitro. AP-2epsilon is predominantly expressed during the late stages of chondrocyte differentiation, mainly in early hypertrophic cartilage, consistent with immunohistochemical stainings of mouse embryo sections. Furthermore, osteoarthritic chondrocytes, resembling a hypertrophic phenotype, have high AP-2epsilon levels. The AP-2epsilon promoter harbors binding sites for the transcription factors AP-2alpha and Sox9. Both transcription factors strongly activate AP-2epsilon expression in a cooperative manner in the chondrosarcoma cell line SW1353. The inhibition of Sox9 expression by small interfering RNA resulted in decreased AP-2epsilon expression. In addition, direct interaction of Sox9 with the AP-2epsilon promoter could be confirmed by chromatin immunoprecipitation and electromobility shift assays. This is the first study to prove the direct regulation of AP-2epsilon by the transcription factor Sox9, and to indicate that AP-2epsilon potentially has an important role as a modulator of hypertrophic cartilage.

Identification of novel sense and antisense transcription at the TRPM2 locus in cancer.

It has been proposed that in cancer, where the bulk of the genome becomes hypomethylated, there is an increase in transcriptional noise that might lead to the generation of antisense transcripts that could affect the function of key oncosuppressor genes, ultimately leading to malignant transformation. Here, we describe the computational identification of a melanoma-enriched antisense transcript, TRPM2-AS, mapped within the locus of TRPM2, an ion channel capable of mediating susceptibility to cell death. Analysis of the TRPM2-AS genomic region indicated the presence in the same region of another tumor-enriched TRPM2 transcript, TRPM2-TE, located across a CpG island shared with TRPM2-AS. Quantitative PCR experiments confirmed that TRPM2-AS and TRPM2-TE transcripts were up-regulated in melanoma, and their activation was consistent with the methylation status of the shared CpG island. Functional knock-out of TRPM2-TE, as well as over-expression of wild-type TRPM2, increased melanoma susceptibility to apoptosis and necrosis. Finally, expression analysis in other cancer types indicated that TRPM2-AS and TRPM2-TE over-expression might have an even wider role than anticipated, reinforcing the relevance of our computational approach in identifying new potential therapeutic targets.

Expression of integrin alpha10 is induced in malignant melanoma.

Recently, integrin alpha10 was described as a collagen type II-binding integrin expressed mainly in chondrocytes. However, by array studies we detected integrin alpha10 also to be upregulated in malignant melanoma compared to primary melanocytes. Subsequent analysis of melanoma cell lines and melanoma tumor samples confirmed this finding. Further, we demonstrated that expression of integrin alpha10 is controlled by AP-2 and Ets-1, two transcription factors known to be involved in melanoma development and progression. To investigate the functional relevance of integrin alpha10, expression was downregulated via stable antisense transfection. Proliferation assays and colony forming assays revealed no differences comparing antisense integrin alpha10 cell clones with control and wild type melanoma cells, respectively. However, antisense integrin alpha10 cell clones and Mel Im cells treated with an inhibitory antibody against integrin alpha10 showed a reduced migratory potential. In summary, these data indicate that AP-2 and Ets-1 regulated expression of integrin alpha10 plays a role in migration of malignant melanoma cells.

Crystal structure of the archaeal heat shock regulator from Pyrococcus furiosus: a molecular chimera representing eukaryal and bacterial features.

We report here the crystal structure of a protein from Pyrococcus furiosus (Phr) that represents the first characterized heat shock transcription factor in archaea. Phr specifically represses the expression of heat shock genes at physiological temperature in vitro and in vivo but is released from the promoters upon heat shock response. Structure analysis revealed a stable homodimer, each subunit consisting of an N-terminal winged helix DNA-binding domain (wH-DBD) and a C-terminal antiparallel coiled coil helical domain. The overall structure shows as a molecular chimera with significant folding similarity of its DBD to the bacterial SmtB/ArsR family, while its C-terminal part was found to be a remote homologue of the eukaryotic BAG domain. The dimeric protein recognizes a palindromic DNA sequence. Molecular docking and mutational analyses suggested a novel binding mode in which the major specific contacts occur at the minor groove interacting with the strongly basic wing containing a cluster of three arginine residues.

Regulation of integrin alpha10 expression in chondrocytes by the transcription factors AP-2epsilon and Ets-1.

Expression of integrin alpha10 is initiated at the beginning of chondrogenesis and continues throughout cartilage development in adult cartilage. In our study, we aim to identify regulatory sequences that control the cell-type specific expression of the human integrin alpha10 gene. Therefore, promoter constructs harboring 1139bp 5' of the transcriptional start site of the human integrin alpha10 gene were analyzed. Our experiments localized a promoter region that directs high levels of expression specifically in chondrocytes. A sequence analysis detected three consensus AP-2 binding sites within this functional domain. Functionality of these sites was tested and confirmed by cotransfection of AP-2 in a luciferase reporter assay. Interestingly, EMSA identified AP-2epsilon as the major AP-2 protein binding to the AP-2 consensus sequences. Additionally, Ets-1 was shown to be a positive regulator of the integrin alpha10 expression whereas Sox9 was irrelevant. Taken together, these results suggest that AP-2epsilon and Ets-1 are involved in the regulation of integrin alpha10 transcription in chondrocytes.