The Colorectal cancer disease-specific transcriptome may facilitate the discovery of more biologically and clinically relevant information

<p>Abstract</p> <p>Background</p> <p>To date, there are no clinically reliable predictive markers of response to the current treatment regimens for advanced colorectal cancer. The aim of the current study was to compare and assess the power of transcriptional profiling using a generic microarray and a disease-specific transcriptome-based microarray. We also examined the biological and clinical relevance of the disease-specific transcriptome.</p> <p>Methods</p> <p>DNA microarray profiling was carried out on isogenic sensitive and 5-FU-resistant HCT116 colorectal cancer cell lines using the Affymetrix HG-U133 Plus2.0 array and the Almac Diagnostics Colorectal cancer disease specific Research tool. In addition, DNA microarray profiling was also carried out on pre-treatment metastatic colorectal cancer biopsies using the colorectal cancer disease specific Research tool. The two microarray platforms were compared based on detection of probesets and biological information.</p> <p>Results</p> <p>The results demonstrated that the disease-specific transcriptome-based microarray was able to out-perform the generic genomic-based microarray on a number of levels including detection of transcripts and pathway analysis. In addition, the disease-specific microarray contains a high percentage of antisense transcripts and further analysis demonstrated that a number of these exist in sense:antisense pairs. Comparison between cell line models and metastatic CRC patient biopsies further demonstrated that a number of the identified sense:antisense pairs were also detected in CRC patient biopsies, suggesting potential clinical relevance.</p> <p>Conclusions</p> <p>Analysis from our <it>in vitro </it>and clinical experiments has demonstrated that many transcripts exist in sense:antisense pairs including <it>IGF2BP2</it>, which may have a direct regulatory function in the context of colorectal cancer. While the functional relevance of the antisense transcripts has been established by many studies, their functional role is currently unclear; however, the numbers that have been detected by the disease-specific microarray would suggest that they may be important regulatory transcripts. This study has demonstrated the power of a disease-specific transcriptome-based approach and highlighted the potential novel biologically and clinically relevant information that is gained when using such a methodology.</p

Synthetic anticancer gene medicine exploits intrinsic antitumor activity of cationic vector to cure established tumors

The systemic delivery of genetic therapies required for the treatment of inaccessible tumors and metastases remains a challenge despite the development of various viral and synthetic vector systems. Here we show that a synthetic vector system based on polypropylenimine dendrimers has the desired properties of a systemic delivery vehicle and mediates efficient transgene expression in tumors after i.v. administration. The systemic tumor necrosis factor alpha (TNFalpha) gene therapy was efficacious in the experimental treatment of established A431 epidermoid carcinoma, C33a cervix carcinoma, and LS174T colorectal adenocarcinoma. Specifically, the systemic injection of dendrimer nanoparticles containing a TNFalpha expression plasmid regulated by telomerase gene promoters (hTR and hTERT) leads to transgene expression, regression of remote xenograft murine tumors, and long-term survival of up to 100% of the animals. Interestingly, these dendrimers and, to a lesser extent, other common polymeric transfection agents also exhibit plasmid-independent antitumor activity, ranging from pronounced growth retardation to complete tumor regression. The genetic therapy as well as treatment with dendrimer alone was well tolerated with no apparent signs of toxicity in the animals. The combination of intrinsic dendrimer activity and transcriptionally targeted TNFalpha when complexed was significantly more potent than either treatment alone or when both were administered in sequence. The combination of pharmacologically active synthetic transfection agent and transcriptionally targeted antitumor gene creates an efficacious gene medicine for the systemic treatment of experimental solid tumors

Identification of Galanin and Its Receptor GalR1 as Novel Determinants of Resistance to Chemotherapy and Potential Biomarkers in Colorectal Cancer

PURPOSE: A major factor limiting the effective clinical management of colorectal cancer (CRC) is resistance to chemotherapy. Therefore, the identification of novel, therapeutically targetable mediators of resistance is vital. EXPERIMENTAL DESIGN: We used a CRC disease-focused microarray platform to transcriptionally profile chemotherapy-responsive and non-responsive pre-treatment metastatic CRC liver biopsies and in vitro samples, both sensitive and resistant to clinically relevant chemotherapeutic drugs (5-FU and oxaliplatin). Pathway and gene set enrichment analyses (GSEA) identified candidate genes within key pathways mediating drug resistance. Functional RNAi screening identified regulators of drug resistance. RESULTS: MAPK signalling, focal adhesion, cell cycle, insulin signalling and apoptosis were identified as key pathways involved in mediating drug resistance. The G-protein coupled receptor galanin receptor 1 (GalR1) was identified as a novel regulator of drug resistance. Notably, silencing either GalR1 or its ligand galanin, induced apoptosis in drug-sensitive and resistant cell lines and synergistically enhanced the effects of chemotherapy. Mechanistically, GalR1/galanin silencing resulted in down-regulation of the endogenous caspase 8 inhibitor FLIP(L), resulting in induction of caspase 8-dependent apoptosis. Galanin mRNA was found to be overexpressed in colorectal tumours, and importantly, high galanin expression correlated with poor disease-free survival of early stage CRC patients. CONCLUSION: This study demonstrates the power of systems biology approaches to identify key pathways and genes that are functionally involved in mediating chemotherapy resistance. Moreover, we have identified a novel role for the GalR1/galanin receptor-ligand axis in chemo-resistance, providing evidence to support its further evaluation as a potential therapeutic target and biomarker in CRC

A Systems Biology Approach Identifies SART1 as a Novel Determinant of Both 5-Fluorouracil and SN38 Drug Resistance in Colorectal Cancer

Chemotherapy response rates for advanced colorectal cancer remain disappointingly low, primarily due to drug resistance, so there is an urgent need to improve current treatment strategies. In order to identify novel determinants of resistance to the clinically relevant drugs 5-Fluorouracil (5-FU) and SN38 (the active metabolite of irinotecan), transcriptional profiling experiments were carried out on pre-treatment metastatic colorectal cancer biopsies and HCT116 parental and chemotherapy-resistant cell line models using a disease-specific DNA microarray. To enrich for potential chemo-resistance-determining genes, an unsupervised bioinformatics approach was employed, and 50 genes were selected and then functionally assessed using custom-designed siRNA screens. In the primary siRNA screen, silencing of 21 genes sensitised HCT116 cells to either 5-FU or SN38 treatment. Three genes (RAPGEF2, PTRF and SART1) were selected for further analysis in a panel of 7 CRC cell lines. Silencing SART1 sensitised all 7 cell lines to 5-FU treatment and 4/7 cell lines to SN38 treatment. However, silencing of RAPGEF2 or PTRF had no significant effect on 5-FU or SN38 sensitivity in the wider cell line panel. Further functional analysis of SART1 demonstrated that its silencing induced apoptosis that was caspase 8-dependent. Furthermore, silencing of SART1 led to a down-regulation of the caspase 8 inhibitor, c-FLIP, which we have previously demonstrated is a key determinant of drug resistance in colorectal cancer. This study demonstrates the power of systems biology approaches for identifying novel genes that regulate drug resistance and identifies SART1 as a previously unidentified regulator of c-FLIP and drug-induced activation of caspase 8