Dissertation committee:
Σκορίλας Ανδρέας (Επιβλέπων), Καθηγητής, Τμήμα Βιολογίας, Ε.Κ.Π.Α.
Βασιλακοπούλου Διδώ, Αναπλ. Καθηγήτρια, Τμήμα Βιολογίας, Ε.Κ.Π.Α.
Τρουγκάκος Ιωάννης, Καθηγητής, Τμήμα Βιολογίας, Ε.Κ.Π.Α.
Σίδερης Διαμάντης, Αναπλ. Καθηγητής, Τμήμα Βιολογίας, Ε.Κ.Π.Α.
Κοντός Χρήστος, Επικ. Καθηγητής, Τμήμα Βιολογίας, Ε.Κ.Π.Α.
Ζωιδάκης Ιερώνυμος, Επικ. Καθηγητής, Τμήμα Βιολογίας, Ε.Κ.Π.Α.
Αυγέρης Μαργαρίτης, Αναπλ. Καθηγητής, Ιατρική Σχολή, Ε.Κ.Π.Α.
Summary:
Colorectal cancer (CRC) stands as the second leading cause of cancer-related fatalities on a worldwide scale. According to global cancer statistics provided by the Global Cancer Observatory (GCO), the incidence of CRC and CRC-associated mortality are expected to surge by 30% by the year 2030. The transformation of benign colorectal incidentalomas into malignant neoplasms involves a complex, multistep process influenced by genomic instability, abnormal DNA methylation, and mutations in critical oncogenes and/or tumor-suppressor genes. Given the substantial heterogeneity in CRC, accurately assessing patient prognosis poses a challenging clinical endeavor. This incidence further results in under- and/or overtreatment phenomena during the clinical management of the disease. The in-depth molecular profiling of CRC tumors will lead to the identification of novel CRC biomarkers that assist the development of personalized therapeutic strategies.
In the context of this Ph.D. thesis, the study initially brought to light the extensive and intricate range of mRNAs produced by the three most significant RAS genes, namely KRAS, NRAS, and HRAS. This involved a comprehensive analysis of splicing events and exon/intron boundaries. RAS proteins encode small GTPases that play a crucial role in signal transduction pathways. The hyperactivation of RAS signaling is a pivotal factor in the development of human malignancies. In approximately 50% of colorectal cancer (CRC) tumors, the KRAS gene is mutated, while mutations in NRAS and HRAS are also found but at a lower frequency. Notably, RAS mutations serve as an adverse predictive biomarker for the response to anti-EGFR targeted therapy. The application of a versatile, targeted nanopore-sequencing approach led to the discovery of 39 novel RAS mRNA transcript variants and provided insights into their expression patterns across a wide range of human cell lines. While this work has uncovered previously unknown facets of the RAS gene family, further research is necessary to fully understand the biological functions of these new alternative transcript variants and their potential protein isoforms.
Subsequently, we delved into the examination and identification of differentially expressed tRNA-derived RNA fragments (tRFs) and miRNAs in non-malignant colorectal tissues and colorectal cancer (CRC) samples. These molecules were further assessed for their potential diagnostic and/or prognostic utility in CRC. These small non-coding RNA (sncRNAs) molecules play a role in regulating gene expression at the post-transcriptional level and are thus involved in carcinogenesis either as oncogenes or tumor suppressors. To discern the variations in miRNA and tRF expression between CRC samples and non-malignant tissues, we employed state-of-the-art next-generation sequencing techniques (NGS). Some of these sncRNAs were subsequently subjected to clinical evaluation within a substantial cohort of CRC tumors.
MicroRNA-28-5p (miR-28-5p) targets several cancer-related genes and is hence involved in cell proliferation, migration, invasion, and epithelial-mesenchymal transition. In this study, we investigated the potential diagnostic and prognostic significance of miR-28-5p expression in colorectal adenocarcinoma, the most frequent type of colorectal cancer (CRC). Comparison of miR-28-5p levels among 86 pairs of colorectal tumors and their adjacent non-cancerous mucosae uncovered the downregulation of miR-28-5p expression in most malignant colorectal tumors (p=0,005). More importantly, high miR-28-5p expression predicts poor disease-free survival (DFS) and overall survival (OS) of colorectal adenocarcinoma patients. Multivariate Cox regression analysis revealed that miR-28-5p overexpression is a significant predictor of poor prognosis in colorectal adenocarcinoma, independent of tumor size, histological grade, TNM staging, radiotherapy and chemotherapy. Interestingly, strong miR-28-5p expression retains its predictive potential regarding relapse among patients with negative regional lymph nodes and predicts poor OS in patients diagnosed with non-metastatic colorectal adenocarcinoma.
Furthermore, the same cohort of patients was used to assess the potential diagnostic and prognostic utility of miR-15a-5p expression in colorectal adenocarcinoma. miR-15a-5p was significantly upregulated in colorectal tumors compared to non-cancerous colorectal mucosae, while ROC analysis suggested its potential use for diagnostic purposes. Moreover, miR-15a-5p overexpression predicts poor disease-free survival (DFS) and overall survival (OS). Multivariate Cox regression analysis confirmed that miR-15a-5p overexpression is a significant unfavorable prognosticator of DFS in colorectal adenocarcinoma, independent of other established prognostic factors plus treatment of patients. Importantly, miR-15a-5p overexpression retains its unfavorable prognostic value in patients with T3 colorectal adenocarcinoma and in those without distant metastasis (M0). More importantly, the cumulative DFS probability of patients with early-stage disease was significantly lower for those with colorectal adenocarcinoma overexpressing miR-15a-5p.
Finally, the prognostic utility of 5'-tiRNA-ProTGG levels in CRC was investigated. For this purpose, total RNA was extracted from 155 malignant colorectal tumors and 74 adjacent non-cancerous tissue specimens, polyadenylated and reverse-transcribed using an oligo-dT adapter as primer. Real-time quantitative PCR (qPCR) was used to assess the levels of 5'-tiRNA-ProTGG. Kaplan-Meier survival analysis demonstrated that high 5'-tiRNA-ProTGG levels predict both poor disease-free survival (DFS) and overall survival (OS) of CRC patients. Of note, high 5'-tiRNA-ProTGG levels retain their unfavorable prognostic value in patients with rectal cancer and/or moderately differentiated CRC (grade II). More importantly, multivariate cox regression analysis highlighted that the overexpression of 5'-tiRNA-ProTGG constitutes an adverse prognostic factor predicting short-term relapse of CRC patients independently of the established prognosticators in CRC. Bioinformatics analysis unveiled a potentially critical role of 5'-tiRNA-ProTGG regarding the maintenance of cellular homeostasis, signaling, cell communication, and cellular motility.
Keywords:
colorectal cancer, RAS genes, small non-coding RNA (ncRNA), cancer biomarkers, massive parallel sequencing
