Towards the identification of oncogenes by high resolution mapping of gene amplifications of the 3q25-qter region in Malignant Fibrous Histiocytoma and Squamous Cell Carcinoma
HUSSENET, Thomas (2005) Towards the identification of oncogenes by high resolution mapping of gene amplifications of the 3q25-qter region in Malignant Fibrous Histiocytoma and Squamous Cell Carcinoma. Thèses de doctorat, Université Louis Pasteur.
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Cancer cells are characterized by a certain number of hallmarks. Among these, genetic instability is admitted to be a near universal one. Consequently, most of tumour cells presents genetic alterations and particularly at the chromosomal level. Actually, interstitial deletions, low-level copy number gains and high-level copy number amplifications of genomic regions are recurrently observed in tumour genomes, and produce deregulations in the expression of tumour suppressor genes and oncogenes. We used a positional cloning strategy by mapping of chromosomal aberrations to define common genomic intervals that are altered in different tumours and identify candidate oncogenes. This thesis work specifically applied this strategy to chromosome 3 aberrations in different tumour types and particularly to identify new candidate oncogenes located at 3q, amplified and consequently over-expressed. A first part was dedicated to the study of some soft-tissue sarcomas, Malignant Fibrous Histiocytomas (MFH). In a first approach, we characterized an amplicon at 3q28 in a MFH cell line. This region was also found to be amplified in two other MFH primary tumours but no obvious candidate oncogene was isolated (Hussenet et al., submitted). From this region, we finally identified a microRNA encoding gene, hsa-miR-28 representing an oncogene candidate: its inhibition indeed slows down the proliferation of 2 MFH cell lines. We further identified three targets of miR-28 (DP2, E2F6 and CDC14A): all play roles in the regulation of the cell cycle and interestingly the hsa-miR-28 gene expression itself seems to be cell cycle regulated (Hussenet et al., in preparation). A second part was dedicated to Squamous Cell Carcinoma (SCC) of two localizations (lung & head and neck). We analyzed chromosome 3 aberrations in a series of 25 lung SCC using array CGH. This work allowed us to delineate three regions of deletions at 3p, low-level copy number gain of the 3q26-qter region in a majority of tumours, as well as a common region of high-level amplifications at 3q26.3 for 20% of the tumours. Further high-resolution mapping of these amplifications using array CGH with an array providing tiling coverage of the 3q26.3 locus pinpoint a consensus region that is approximately 2 Mb in size. Analyses of the transcriptional consequences of these high-level amplifications were carried out for 9 genes of the region. Most of them are recurrently over-expressed but two, SOX2 and SOX2OT, likely represent the 3q26.3 amplification driver genes in these tumours. Ongoing in vitro functional assays will assess the effect of over-expression of candidate oncogenes located in the consensus region of high-level amplifications at 3q26.3 (Hussenet et al., in preparation). Our positional cloning strategy also enabled us to isolate the cyclin L1 (CCNL1) gene at 3q25.3 as a candidate oncogene, amplified and over-expressed in a head and neck SCC (HNSCC) cell line, as well as over-expressed in HNSCC tumours (Redon et al., 2002). Further investigations of CCNL1 gene alterations in a larger series of HNSCC revealed consistent low-level copy number gains and over-expression in these tumours (Muller et al., submitted). Finally we also screened a series of 25 HNSCC for chromosome 3 aberrations using array CGH. We were able to delineate several recurrent sites of deletions at 3p, low-level gains and high-level amplifications at 3q. A common region of high-level amplifications in HNSCC is overlapping with the consensus region we have previously defined for lung SCC and suggests that similar genes at 3q26.3 may be involved in SCC pathogenesis independently of the localization. In conclusion, this work illustrates the power of the positional cloning strategy applied to tumour genomes through the use of high resolution methods and tools to map the recurrent sites of genomic alterations. We indeed were able to isolate several oncogene candidate located at 3q that are amplified and consequently over-expressed in different tumour types. Further investigations are still needed to assess their oncogenic status as well as putative relationships with clinical parameters.
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