Functional genetic approaches identify tumorigenic small RNAs
With the discovery in the last few years of thousands of genes that produce small, non-coding RNA (small RNAs) transcripts, it has become evident that the complexity of gene expression regulation is far greater than expected. These small RNAs have been implicated in critical processes including differentiation, apoptosis, proliferation, and their misexpression has been linked to different pathologies including cancer.
The finding that interference RNA (iRNA) and micro RNAs (miRNAs) can be exploited to suppress gene expression has led to the rapid identification of genes involved in many different biological processes through powerful loss-of function screens.
A central theme of research in our laboratory focuses on the development of new strategies that allow the identification of small RNAs involved in tumorigenesis. One approach has been the generation of improved and inexpensive iRNAs expression libraries targeting whole transcriptomes. These libraries are generated from 19-mer random oligonucleotides flanked by fixed sequences and enriched for specific oligonucleotides by hybridization with cDNAs (1c) derived from specific transcriptomes. After retrotranscription, the duplex oligonucleotides are cloned in a lentiviral vector between the H1 and U6 RNA polymerase III promoters. Lentivirus are integrated into the genome of the cells and molecules corresponding to each mature iRNAs are generated resulting in activation of the RNA-induced silencing complex (RISC), which recruits the correct RNA strand and degrades homologous mRNA molecules. While each iRNA knocks down a single gene, a miRNA may target numerous mRNAs, which represent an extraordinary tool to look for phenotypes implicating several genes. At present, miRNAs libraries have been only used to identify miRNAs and to assay their expression pattern and thus, have not been suitable for functional assays. In this regard, we have used a novel approach to develop functional genetic screens using lentiviral-based miRNAs libraries, following standards procedures for miRNAs cloning. This strategy presents two novelties. On one hand, the small RNAs are expressed as active processed molecules (Patents P200703258, EPO9382003) and on the other hand may be used to clone any small RNA molecule of the cell. These unique libraries of small RNAs are being used to screen for genes involved in different processes such as in the mitotic spindle checkpoint signalling, in apoptosis resulting from loss of cell-matrix interactions (anoikis) and in tumour cell invasion.
Role of the pttg1/securin gene in cell cycle and tumorigenesis
The Ptt1/securin gene is frequently overexpressed in tumours of different aetiologies and its expression is closely correlated with the aggressiveness of the tumour. We investigate different non-excluding mechanisms behind PTTG1 tumorigenic function. PTTG1 inhibits sister-chromatid separation in vertebrates, and may thereby mediate chromosome missegregation leading to an increase in proto-oncogene dose or to loss of heterozygosity of tumour suppressors. Also, PTTG1 binds to Ku, the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). This association is disrupted and prevented by double-strand breaks suggesting that PTTG1 connects the DNA damage-response pathway with sister chromatid separation. Furthermore, PTTG1 interacts with and inhibits p53’s transcriptional activity, indicating that the oncogenic effect of PTTG1 could result from modulation of p53 functions. Finally, we have proposed that PTTG1 acts as a transcriptional activator, playing a role in the transcriptional regulation of genes involved in a variety of cellular processes. We are currently identifying the PTTG1 target genes and studying the mechanisms by which these genes could participate in the PTTG1 biological role. We have focused our attention on the target genes coding for the chemokines CXCL12 and CCL2. Cells overexpressing these chemokines attract monocytes, macrophages and lymphocytes, which are potent factories producing proteases, growth factors and angiogenic and lymphogenic factors. These proteins could be closely implicated in PTTG1 tumorigenic function, providing a growth advantage in the primary tumour and mediating metastasis. Furthermore, we anticipate that Pttg1 plays an important role in the preadipocite differentiation, through the induction of dlk-1 gene, a potent inhibitor of the differentiation process. Relevant tools in these studies are the pttg1 knockdown mice and the bitransgenic mice, generated in our lab, which express PTTG1 both in mammary and salivary glands under a doxycycline-inducible promoter.