Genomic analysis in human solid tumours:Copper Homeostasis Genes in Colorectal cancer

In this thesis the following specific aims have been discussed: 1. The public database: Cancer Genome Atlas Network has been consulted in order to reveal the presence of somatic mutations in copper homeostasis genes (CHGs) in colorectal cancer, and such analysis, performed on 228 colorectal tumor samples, has revealed that inactivating mutations are extremely rare in CHGs. 2. The collection of whole transcriptome profiles by oligonucleotide microarrays represents the second aim of the thesis. CHGs mRNA levels have been measured in 37 colorectal carcinoma samples in comparison to matched normal colonic mucosae. The transcriptome analysis has been perfomed using the last Human Transcriptome Array (HTA 2.0, Affymetrix) which allows to analyze simultaneously 40.000 coding transcripts and 20.000 non-coding transcripts and to reveal variations of mRNA levels between in colorectal cancer samples respect to normal colonic mucosae. The gene expression analysis has showed an up-regulation of several transcripts involved in copper homeostasis pathway (such as SLC31A1, SLC31A2, COX11, SCO1, SOD1) in two different conditions. On the contrary, some genes did not show variations of expression between two tested conditions suggesting the idea that these genes, if experimental altered in their expression, could represent the good targets for specific drug treatments. The transcriptome analysis by the last generation on oligonucleotide microarrays gives a possibility to analyse gene expression levels and single exon expression levels. In the 37 human colorectal samples, the exon-level expression analysis has been revealed the presence of alternative transcripts prevalent in a condition respect to other. 3. In this part of thesis the experimental down-regulation, by short interfering, of a copper chaperone ATOX1, significantly reduced the tumor growth in a Caco2 colorectal cell line. It has been demonstrated that the copper addition increase the toxic effects of some copper binding compounds, in particular, the ionophore copper-ionophore 5-chloro-8-hydroxyquinoline (ClHQ) and the copper chelator (N,N,N'N',-tetrakis (2- pyridylmethyl)ethylenediamine (TPEN) in two human colorectal cancer cell lines (Caco-2 and HT29). Moreover, the Atox1 silencing has enhanced the toxic effects of copper-ClHQ complexes and TPEN in Caco-2 cells confirming that the inhibition of copper chaperone Atox1 could be a good strategy to attenuate the cancer cell proliferation and to increase the anticancer effects of some copper binding drugs.