Abstract
Colorectal cancer (CRC) is proposed to originate through three major molecular pathways, chromosomal instability (CIN), microsatellite instability (MSI) and the CpG island methylator phenotype (CIMP). Many studies have characterized these three pathways but with the advent of, “omic” technologies they have been investigated in a more refined way.
The current project was started by the generation of DNA copy number profiles using comparative genomic hybridization (CGH) and matched with whole transcriptomic profiles for 84 stage II and stage III CRC tumors. The tumors samples were classified into the three major pathways; the DNA copy number profiles of the tumors were used to assess CIN, while microsatellite instability was assessed using the two-microsatellite markers BAT-26 and NR-24. CIMP status for the tumors was assigned using the MethyLight assay and a well-established panel of five markers. The well-known molecular features influencing the outcome of the patients, KRAS mutation and BRAF mutation were also examined in these tumors.
The present project dealt with two major themes. The first focused on molecular mechanisms resulting from CIN, MSI and CIMP as well as investigating the influence of the immune response on outcome in CIN tumors. The second theme dealt with the identification of oncogenes with prognostic potential by integrating copy number profiles with expression microarray profiles of the tumors.
The third chapter comprised an investigation of molecular mechanisms in triple negative tumors. Triple negative tumors are negative for all the three major pathways responsible for CRC origin and have been suggested to be distinct molecular pathway. Triple negative tumors were identified and molecular mechanisms were investigated by considering the differentially expressed genes between triple negative tumors and remaining subtypes. Interestingly only one gene, RAB27A was found to be up regulated in triple negative tumors. RAB27A is a member of the Ras related small GTPases superfamily involved in vesicle trafficking, a process, which regulates multiple signaling mechanisms and has been linked to tumorigenesis in previous studies. Pathways associated with RAB27A were then investigated, and an association was found with focal adhesion, glycosphingolipid metabolism and regulation of actin cytoskeleton pathways. These pathways have been implicated to have a role in cell-cell, cell-ECM interaction and cell migration by controlling the focal complex assembly and disassembly at the leading edge of lamellipodia of invading cells. This suggests that RAB27A driven pathways may contribute to tumorigenesis and pathogenesis of the triple negative tumors. RAB27A has been associated with proliferation in previous studies; in this study the proliferation phenotype in triple negative tumors was investigated using two proliferation metagenes from the literature. Interestingly triple negative tumors had low proliferation and poor disease free survival compared to the subtypes with higher proliferation, suggesting triple negative tumors might have aggressive behavior.
The fourth chapter investigated molecular mechanisms in CIMP(+) and CIMP(-) tumor with and without CIN. The most interesting observation was the presence of CIN in CIMP(+) tumors, which was mostly contributed by MSS tumors. There was no measurable significant difference in the molecular mechanisms between CIMP(+) tumors with and without CIN. In the case of CIMP(-) tumors, the proliferation genes were up regulated in CIMP(-)CIN(+) tumors compared to CIMP(-)CIN(-) tumors. This observation was confirmed by assessing the proliferation phenotype using two proliferation metagenes from the literature. The proliferation genes up regulated in CIMP(-)CIN(+) tumors were significantly correlated to DNA copy number and expression levels, suggesting enhanced proliferation could be due to amplification of proliferation genes in CIMP(-)CIN(+) tumors. Interestingly even CIMP(+) tumors with CIN and without CIN had enhanced proliferation when assessed with the proliferation metagene suggesting proliferation is independent of CIN in CIMP(+) tumors. In CRC the tumors with enhanced proliferation have previously been shown to be associated with better survival while CIN tumors were associated with poor outcome. In this study CIN tumors with higher proliferation were associated with better outcome with near to significance, suggesting high proliferation could provide prognostic marker for better survival independent of CIN status.
The fifth chapter involved an investigation of the interaction of immune response and outcome in CIN tumors. Increased immune response has previously shown to be associated with better survival while CIN has been associated with poor survival of the patients. In this study the role of the immune response in determining outcome in CIN tumors was investigated. CIN(+) and CIN(-) tumors were clustered based on immune response signatures from the literature. Two of four signatures from literature clustered the samples into distinct groups with high immune response and low immune response. Survival analysis was performed between these two groups; the group with high immune response was associated with increased overall survival of the patients and was independent of CIN. To confirm this finding an immune response signature was generated from the current dataset by performing a T-test between MSI and MSS tumors. Tumors were then clustered into high immune response and low immune response groups. Survival analysis was performed between the groups. Even in this case, the high immune response group had a significant increase in overall survival. The prognostic significance of the immune response signature was validated and confirmed on three independent datasets. These findings suggested that the immune response determines better survival irrespective of CIN status of the tumors.
The sixth chapter aimed to identify oncogenes, which could be potential prognostic markers and therapeutic targets, by integrating the DNA copy number profiles and microarray expression profiles. As a result, among the significantly correlated genes, C17orf37 was significantly associated with disease free survival, and this was validated on independent datasets followed by pathway analysis. C17orf37 was found to be significantly associated with oxidative phosphorylation suggesting a link between C17orf37 overexpression, oxidative phosphorylation and recurrence.
Overall this thesis dealt with investigating the molecular mechanisms involved in tumorigenesis and pathogenesis of triple negative tumors, mechanisms modified due to CIN in CIMP(+) and CIMP(-) tumors, interactions of CIN, immune response and outcome of patients, and identification of biomarkers to predict recurrence in CRC.