Projects / Programmes
New prognostic and diagnostic markers in colorectal cancerogenesis
| Code |
Science |
Field |
Subfield |
| 3.04.00 |
Medical sciences |
Oncology |
|
| Code |
Science |
Field |
| B200 |
Biomedical sciences |
Cytology, oncology, cancerology |
| Code |
Science |
Field |
| 3.02 |
Medical and Health Sciences |
Clinical medicine |
colorectal cancer, colorectal adenoma, bioinformatics, new markers, functional validation
Researchers (11)
Organisations (3)
Abstract
Objectives. Colorectal cancer (CRC) is one of the most common causes of death by cancer. Despite significant progress, 40-50% of CRC patients develop metastases. The prognosis has improved significantly since the introduction of a CRC screening program which enables to detect early lesions, including adenomas and early carcinomas as well as a significant number of problematic polyps with ambiguous features. Although the molecular pathways that are responsible for transformation of the normal mucosa to adenoma are relatively well defined, diagnostic markers that would help to differentiate among ambiguous lesions are lacking. There is an urgent need to search for new diagnostic and prognostic markers.
Hypothesis. We hypothesize that:
bioinformatics analysis enables to identify new diagnostic and prognostic markers;
new markers might be used as a prognostic tool to predict features, associated with an unfavorable course of the disease (e.g., tumor budding, metastases, serosal invasion);
new markers might be used to distinguish among ambiguous lesions, e.g., adenoma, adenoma with epithelial misplacement and adenoma with early carcinoma;
many of the identified new markers are related to important pathogenetic mechanisms in CRC, e.g., epithelial-mesenchymal transition, cancer stem cell properties, organization of extracellular matrix, tumor heterogeneity;
analysis of the expression and distribution of new markers might provide new insights into pathogenesis of CRC, thus contributing to search for novel therapeutic targets.
Patients and methods. Using meta-analysis of publicly available data, microarray expression profiles and sequencing data of mRNAs, miRNAs and methylation will be compared among the normal colon mucosa, adenoma and carcinoma samples. For all projects, the original data files will be downloaded and further analyzed in R language.
For functional validation, biopsy samples will be collected retrospectively from the archives of the Institute of Pathology, Faculty of Medicine, University of Ljubljana. At least 240 biopsy samples from 135 patients will be included, with adenoma, adenoma with epithelial misplacement, adenoma with early carcinoma and CRC of different stages.
DNA and RNA will be isolated from formalin-fixed paraffin-embedded tissue samples, using microtome, punch or dissection. RNA expression will be validated using qPCR, protein expression using immunohistochemistry and methylation using Sanger or pyrosequencing. Statistical analysis will be performed with SPSS (Willcoxon Rank for paired samples, Mann-Whitney for independent samples, Spearman for correlations).
For promising identified genes, mutation status and copy number analyses will be performed using next-generation sequencing. Raw data will be analyzed using appropriate bioinformatics algorithms. Mutation status will be further confirmed using Sanger sequencing and copy number using qPCR.
Using CRC cell lines, the most encouraging pair of miRNA-mRNA will be validated using 3'-UTR reporter assay. Further, the most promising marker will be silenced using siRNA followed by testing for cell viability, proliferation, apoptosis and migration, and analysis of RNAs (RNAseq) and proteins (western blot).
Expected results. We expect to find new markers which might be used as prognostic tools to predict an unfavorable course of the disease, as well as new markers which might be used as diagnostic tools, enabling to distinguish among ambiguous lesions. This will be of great help to choose optimal treatment for each patient. We expect to provide new insights in our understanding of the pathogenetic mechanisms, e.g., epithelial-mesenchymal transition, cancer stem cells properties, extracellular matrix organization and tumor heterogeneity, which contribute to CRC development and progression. We expect that better understanding of the CRC pathogenesis will help to reveal novel therapeutic targets in the future.
Significance for science
Relevance to the development of scientific field.
Using bioinformatics, new mechanisms could be identified leading to better understanding of progression of colorectal carcinoma (CRC) and the identification of new prognostic markers related to tumour budding, serosal invasion, metastases etc.
Identified new markers would be a useful tool to help to distinguish among ambiguous lesions to avoid unnecessary treatment. Determination of the set of appropriate markers would be helpful during routine histological examination of removed lesions.
New insights into the significance of EMT (epithelial-mesenchymal transition), ECM (extracellular matrix) and CSCs (carcinoma stem cells) would be another step towards understanding CRC cancerogenesis and intra-tumor heterogeneity.
Furthermore, all this knowledge might lead to the development of new treatment modalities. For example, prevention or reversing the EMT and CSC process in CRC might decrease the possibility of metastases development, prevent recurrence of tumour and resistance to therapy. Additionally, miRNAs are feasible therapeutic targets. The introduction of down-regulated miRNA or miRNA inhibitors of up-regulated miRNAs would restore the miRNA function leading to normal expression of target genes. There are already numerous studies using miRNAs as therapeutic targets.
Relevance to the development of science
We expect for the majority of our results to be original. Meta-analysis of different data on mRNA expression in relation to miRNA expression and methylation status of the normal colon mucosa, adenoma and carcinoma to the best of our knowledge has not be performed yet. Extensive research has focused on the development of metastases and tumour budding in CRC; however, reliable prognostic markers are still lacking. There is still much to discover about mechanisms leading to serosal invasion in CRC and intra-tumour heterogeneity. There are only few published studies on adenoma as ambiguous lesions, epithelial misplacement in adenoma and malignant transformation of adenoma.
Moreover, we were not able to find any systematically analysis of well-established markers or analysis of newly identified markers of the EMT, ECM and CSC to investigate possible contribution of these mechanisms to the malignant transformation of adenoma, serosal invasion and intra-tumor heterogeneity.
Significance for the country
Relevance to the development of scientific field.
Using bioinformatics, new mechanisms could be identified leading to better understanding of progression of colorectal carcinoma (CRC) and the identification of new prognostic markers related to tumour budding, serosal invasion, metastases etc.
Identified new markers would be a useful tool to help to distinguish among ambiguous lesions to avoid unnecessary treatment. Determination of the set of appropriate markers would be helpful during routine histological examination of removed lesions.
New insights into the significance of EMT (epithelial-mesenchymal transition), ECM (extracellular matrix) and CSCs (carcinoma stem cells) would be another step towards understanding CRC cancerogenesis and intra-tumor heterogeneity.
Furthermore, all this knowledge might lead to the development of new treatment modalities. For example, prevention or reversing the EMT and CSC process in CRC might decrease the possibility of metastases development, prevent recurrence of tumour and resistance to therapy. Additionally, miRNAs are feasible therapeutic targets. The introduction of down-regulated miRNA or miRNA inhibitors of up-regulated miRNAs would restore the miRNA function leading to normal expression of target genes. There are already numerous studies using miRNAs as therapeutic targets.
Relevance to the development of science
We expect for the majority of our results to be original. Meta-analysis of different data on mRNA expression in relation to miRNA expression and methylation status of the normal colon mucosa, adenoma and carcinoma to the best of our knowledge has not be performed yet. Extensive research has focused on the development of metastases and tumour budding in CRC; however, reliable prognostic markers are still lacking. There is still much to discover about mechanisms leading to serosal invasion in CRC and intra-tumour heterogeneity. There are only few published studies on adenoma as ambiguous lesions, epithelial misplacement in adenoma and malignant transformation of adenoma.
Moreover, we were not able to find any systematically analysis of well-established markers or analysis of newly identified markers of the EMT, ECM and CSC to investigate possible contribution of these mechanisms to the malignant transformation of adenoma, serosal invasion and intra-tumor heterogeneity.
Most important scientific results
Interim report
Most important socioeconomically and culturally relevant results
Interim report
