Vanessa Andries

A Constitutional Translocation t(1;17)(p36.2;q11.2) in a Neuroblastoma Patient Disrupts the Human NBPF1 and ACCN1 Genes

The human 1p36 region is deleted in many different types of tumors, and so it probably harbors one or more tumor suppressor genes. In a Belgian neuroblastoma patient, a constitutional balanced translocation t(1;17)(p36.2;q11.2) may have led to the development of the tumor by disrupting or activating a gene. Here, we report the cloning of both translocation breakpoints and the identification of a novel gene that is disrupted by this translocation. This gene, named NBPF1 for Neuroblastoma BreakPoint Family member 1, belongs to a recently described gene family encoding highly similar proteins, the functions of which are unknown. The translocation truncates NBPF1 and gives rise to two chimeric transcripts of NBPF1 sequences fused to sequences derived from chromosome 17. On chromosome 17, the translocation disrupts one of the isoforms of ACCN1, a potential glioma tumor suppressor gene. Expression of the NBPF family in neuroblastoma cell lines is highly variable, but it is decreased in cell lines that have a deletion of chromosome 1p. More importantly, expression profiling of the NBPF1 gene showed that its expression is significantly lower in cell lines with heterozygous NBPF1 loss than in cell lines with a normal 1p chromosome. Meta-analysis of the expression of NBPF and ACCN1 in neuroblastoma tumors indicates a role for the NBPF genes and for ACCN1 in tumor aggressiveness. Additionally, DLD1 cells with inducible NBPF1 expression showed a marked decrease of clonal growth in a soft agar assay. The disruption of both NBPF1 and ACCN1 genes in this neuroblastoma patient indicates that these genes might suppress development of neuroblastoma and possibly other tumor types.

Chibby interacts with NBPF1 and clusterin, two candidate tumor suppressors linked to neuroblastoma

The NBPF genes are members of a gene family that underwent a remarkable increase in their copy number during recent primate evolution. The NBPF proteins contain 5 to 40 copies of a domain known as the NBPF repeat or DUF1220. Very little is known about the function of these domains or about the NBPF proteins. We performed a yeast two-hybrid screening with the aminoterminal domain of NBPF11 and found that Chibby, a documented repressor of Wnt signaling, interacts with multiple NBPF proteins. More specifically, a coiled-coil region in the NBPF proteins interacts with the coiled-coil domain in the carboxyterminal region of Chibby. Nonetheless, this interaction did not influence the repressor function of Chibby in a TOPFLASH reporter assay. Using Chibby as bait in a new yeast two-hybrid screening, we identified clusterin as a binding protein. Chibby and clusterin were co-immunoprecipitated with NBPF1, suggesting the formation of a tri-molecular complex. Although we have not pinpointed the role of these mutual interactions, the possible formation of a macromolecular complex of three candidate tumor suppressor proteins, including the enigmatic NBPF1, points at important functional implications.

The NBPF1 Promoter Has Been Recruited from the Unrelated EVI5 Gene Before Simian Radiation

Most new genes arise through the duplication of existing genes. In most cases, the duplication is not limited to the coding sequence but encompasses the regulatory region as well. The NBPF gene family has expanded during recent primate evolution, and it has no known mouse ortholog. One of its members, NBPF1, was found to be disrupted by a constitutional translocation in a neuroblastoma patient. Here, we show that the ancestral NBPF gene copied the regulatory region from an unrelated gene, EVI5, after the split between simians and prosimians but before simian radiation. Phylogenetic analysis points to the possible involvement of positive selection acting on the NBPF1 promoter in the simian lineage. We previously showed decreased NBPF1 expression in certain neuroblastoma cell lines. Here, we show that this expression pattern is mimicked by the EVI5 gene, but partly by different mechanisms. Epigenetic regulation of the EVI5 promoter is common in neuroblastoma cell lines, but it is not for the NBPF promoters. Here, we describe the recent acquisition of the NBPF1 promoter from an unrelated gene, and remarkably, both the donor (EVI5) and acceptor (NBPF1) genes are disrupted by constitutional translocations in patients with neuroblastoma, suggesting a functional link between these genes and the disease.

NBPF1, a tumor suppressor candidate in neuroblastoma, exerts growth inhibitory effects by inducing a G1 cell cycle arrest

BackgroundNBPF1 (Neuroblastoma Breakpoint Family, member 1) was originally identified in a neuroblastoma patient on the basis of its disruption by a chromosomal translocation t(1;17)(p36.2;q11.2). Considering this genetic defect and the frequent genomic alterations of the NBPF1 locus in several cancer types, we hypothesized that NBPF1 is a tumor suppressor. Decreased expression of NBPF1 in neuroblastoma cell lines with loss of 1p36 heterozygosity and the marked decrease of anchorage-independent clonal growth of DLD1 colorectal carcinoma cells with induced NBPF1 expression further suggest that NBPF1 functions as tumor suppressor. However, little is known about the mechanisms involved.MethodsExpression of NBPF was analyzed in human skin and human cervix by immunohistochemistry. The effects of NBPF1 on the cell cycle were evaluated by flow cytometry. We investigated by real-time quantitative RT-PCR the expression profile of a panel of genes important in cell cycle regulation. Protein levels of CDKN1A-encoded p21CIP1/WAF1 were determined by western blotting and the importance of p53 was shown by immunofluorescence and by a loss-of-function approach. LC-MS/MS analysis was used to investigate the proteome of DLD1 colon cancer cells with induced NBPF1 expression. Possible biological interactions between the differentially regulated proteins were investigated with the Ingenuity Pathway Analysis tool.ResultsWe show that NBPF is expressed in the non-proliferative suprabasal layers of squamous stratified epithelia of human skin and cervix. Forced expression of NBPF1 in HEK293T cells resulted in a G1 cell cycle arrest that was accompanied by upregulation of the cyclin-dependent kinase inhibitor p21CIP1/WAF1 in a p53-dependent manner. Additionally, forced expression of NBPF1 in two p53-mutant neuroblastoma cell lines also resulted in a G1 cell cycle arrest and CDKN1A upregulation. However, CDKN1A upregulation by NBPF1 was not observed in the DLD1 cells, which demonstrates that NBPF1 exerts cell-specific effects. In addition, proteome analysis of NBPF1-overexpressing DLD1 cells identified 32 differentially expressed proteins, of which several are implicated in carcinogenesis.ConclusionsWe demonstrated that NBPF1 exerts different tumor suppressive effects, depending on the cell line analyzed, and provide new clues into the molecular mechanism of the enigmatic NBPF proteins.

p120 Catenin-mediated stabilization of E-cadherin is essential for primitive endoderm specification

E-cadherin-mediated cell-cell adhesion is critical for naive pluripotency of cultured mouse embryonic stem cells (mESCs). E-cadherin-depleted mESC fail to downregulate their pluripotency program and are unable to initiate lineage commitment. To further explore the roles of cell adhesion molecules during mESC differentiation, we focused on p120 catenin (p120ctn). Although one key function of p120ctn is to stabilize and regulate cadherin-mediated cell-cell adhesion, it has many additional functions, including regulation of transcription and Rho GTPase activity. Here, we investigated the role of mouse p120ctn in early embryogenesis, mESC pluripotency and early fate determination. In contrast to the E-cadherin-null phenotype, p120ctn-null mESCs remained pluripotent, but their in vitro differentiation was incomplete. In particular, they failed to form cystic embryoid bodies and showed defects in primitive endoderm formation. To pinpoint the underlying mechanism, we undertook a structure-function approach. Rescue of p120ctn-null mESCs with different p120ctn wild-type and mutant expression constructs revealed that the long N-terminal domain of p120ctn and its regulatory domain for RhoA were dispensable, whereas its armadillo domain and interaction with E-cadherin were crucial for primitive endoderm formation. We conclude that p120ctn is not only an adaptor and regulator of E-cadherin, but is also indispensable for proper lineage commitment.

The human NANOS3 gene contributes to lung tumour invasion by inducing epithelial–mesenchymal transition

We have explored the role of the human NANOS3 gene in lung tumour progression. We show that NANOS3 is over‐expressed by invasive lung cancer cells and is a prognostic marker for non‐small cell lung carcinomas (NSCLCs). NANOS3 gene expression is restricted in testis and brain and is regulated by epigenetic events. It is up‐regulated in cultured cells undergoing epithelial − mesenchymal transition (EMT). NANOS3 over‐expression in human NSCLC cell lines enhances their invasiveness by up‐regulating EMT, whereas its silencing induces mesenchymal − epithelial transition. NANOS3 represses E‐cadherin at the transcriptional level and up‐regulates vimentin post‐transcriptionally. Also, we show that NANOS3 binds mRNAs encoding vimentin and regulates the length of their poly(A) tail. Finally, NANOS3 can also protect vimentin mRNA from microRNA‐mediated repression. We thus demonstrate a role for NANOS3 in the acquisition of invasiveness by human lung tumour cells and propose a new mechanism of post‐transcriptional regulation of EMT. Copyright

The NBPF Gene Family

Neuroblastoma is one of the most intensely studied solid malignancies that affect children (Maris & Matthay, 1999). These tumours are heterogeneous biologically and clinically. One subset of neuroblastoma is susceptible to spontaneous apoptosis with little or no therapy and another subset differentiates over time, but most of these tumours are difficult to cure with current treatments. A relatively high proportion of affected children die due to resistance of the neuroblastoma to therapy (Van Roy et al., 2009). Every step in the identification or functional understanding of the genes that play important roles in this cancer could bring us closer to understanding the molecular mechanism and could help to develop more effective therapy. We will describe the novel NBPF gene family and its role in neuroblastoma. The NBPF gene family was originally identified by the disruption of one of its members in a neuroblastoma patient (Vandepoele et al., 2008). This gene was named NBPF1, for Neuroblastoma Breakpoint Family, member 1. Several reports indicate that the NBPF gene family might play an important role in neuroblastoma and possibly in other cancers as well. Additionally, this evolutionarily recent gene family has been involved as an important player in human evolution.