DNA damage response functionality and possible impacts on epigenetic regulation

• Thesis title in Czech: Funkce opravných drah poškozeni DNA a dopady na epigenetickou regulaci.
• Thesis title in English: DNA damage response functionality and possible impacts on epigenetic regulation
• English key words: DNA damage, DNA repair, signaling, methylation, therapy response; cancer cell lines
• Academic year of topic announcement: 2022/2023
• Thesis type: dissertation
• Thesis language: angličtina
• Department: Department of Genetics and Microbiology (31-140)
• Supervisor: MUDr. Pavel Vodička, CSc., DrSc.
• Author: hidden - assigned by the advisor
• Date of registration: 18.08.2023
• Date of assignment: 18.08.2023

Preliminary scope of work

The project will be focused on the effects of DNA damage responses on cancer cells and identify the possible epigenetic changes triggered by DNA damage. DNA damage will be induced either by direct treatment or the transfection of the damaging agents to colorectal cancer cells. Cell lines will be selected in order to represent main cancer phenotypes (i.e. right-sided tumor, right-sided tumor with microsatellite instability/mismatch repair deficient, left-sided tumor). Regulations of these responses will be compared with healthy colon cells. Cell lines from human precancerous adenomas may also be obtained.
In earlier studies (Slyskova et al. Clinical Cancer Research 2012; Farkas et al. Epigenomics 2014; Vymetalkova et al. Epigenomics 2016), an aberrant methylation was observed in colorectal cancer patients with an absence or scarcity of DNA repair genes. Further, the base excision repair (BER) pathway (responsible for removing for oxidative DNA damage) works differently in tumor tissue and adjacent mucosa, BER capacity is additionally connected with the disease prognosis and treatment prediction. During the completion of the review article on DNA repair role in ovary cancer (Tomasova et al. Cancers 2020) it is learned that aberrant DNA repair in malignant tissue may alter the function of DNA methyltransferases and affect DNMT1, DNMT3A, and DNMT3B. These changes may drive aberrant methylation in carcinogenesis and contribute to epithelial-to-mesenchymal transition (EMT). The EMT transition will be induced in colorectal cell lines treated with TGF-beta, E-cadherin, occluding (epithelial character, less-invasive), N-cadherin, vimentin and fibronectin (mesenchymal, invasive) will be assayed. The DNA repair will be suppressed either pharmacologically (mirin, olaparib for instance) or by shRNA or in knock-out of a given pathway. DNA repair genes and DNA methyltransferases will be assayed for mRNA expression in candidate genes, and western blot analysis for protein expression will be applied.
If the in vitro section will proceed satisfactorily, we may contemplate an additional intervention by major chemotherapeutics used in the cure of colorectal cancer (5-Fluorouracil, Oxaliplatin).
The results will help to understand the mechanisms behind the cancer progression upon DNA damage response and provide a basis for preclinical data. Enlightening epigenetic regulations and transcriptomic changes may be used to generate new therapeutic approaches for the prevention of tumorigenesis or better prognosis in cancer treatments. To accomplish this stage, fresh biopsies of tumor tissues and adjacent mucosa from 10 patients with right-sided colon cancer, TNM II-III, and from 10 patients with left-sided colon cancer (same TNM stage) will be received from collaborating surgical department of the Medical Faculty of Charles University and Biomedical Center in Pilsen. The level of DNA repair, EMT stage, and DNA methyltransferases will be assayed for in this discovery set by large platforms (e.g: RNAseq). Obtained results will be validated on 50 tissue pairs from right-sided colon cancer and 50 left-sided colon cancer patients. The above aspects also will be implemented in tissue biopsies from colorectal cancer patients with an early onset of the disease (below 50 years of age), these tumors may be expected to be more invasive.

Preliminary scope of work in English

The project will be focused on the effects of DNA damage responses on cancer cells and identify the possible epigenetic changes triggered by DNA damage. DNA damage will be induced either by direct treatment or the transfection of the damaging agents to colorectal cancer cells. Cell lines will be selected in order to represent main cancer phenotypes (i.e. right-sided tumor, right-sided tumor with microsatellite instability/mismatch repair deficient, left-sided tumor). Regulations of these responses will be compared with healthy colon cells. Cell lines from human precancerous adenomas may also be obtained.
In earlier studies (Slyskova et al. Clinical Cancer Research 2012; Farkas et al. Epigenomics 2014; Vymetalkova et al. Epigenomics 2016), an aberrant methylation was observed in colorectal cancer patients with an absence or scarcity of DNA repair genes. Further, the base excision repair (BER) pathway (responsible for removing for oxidative DNA damage) works differently in tumor tissue and adjacent mucosa, BER capacity is additionally connected with the disease prognosis and treatment prediction. During the completion of the review article on DNA repair role in ovary cancer (Tomasova et al. Cancers 2020) it is learned that aberrant DNA repair in malignant tissue may alter the function of DNA methyltransferases and affect DNMT1, DNMT3A, and DNMT3B. These changes may drive aberrant methylation in carcinogenesis and contribute to epithelial-to-mesenchymal transition (EMT). The EMT transition will be induced in colorectal cell lines treated with TGF-beta, E-cadherin, occluding (epithelial character, less-invasive), N-cadherin, vimentin and fibronectin (mesenchymal, invasive) will be assayed. The DNA repair will be suppressed either pharmacologically (mirin, olaparib for instance) or by shRNA or in knock-out of a given pathway. DNA repair genes and DNA methyltransferases will be assayed for mRNA expression in candidate genes, and western blot analysis for protein expression will be applied.
If the in vitro section will proceed satisfactorily, we may contemplate an additional intervention by major chemotherapeutics used in the cure of colorectal cancer (5-Fluorouracil, Oxaliplatin).
The results will help to understand the mechanisms behind the cancer progression upon DNA damage response and provide a basis for preclinical data. Enlightening epigenetic regulations and transcriptomic changes may be used to generate new therapeutic approaches for the prevention of tumorigenesis or better prognosis in cancer treatments. To accomplish this stage, fresh biopsies of tumor tissues and adjacent mucosa from 10 patients with right-sided colon cancer, TNM II-III, and from 10 patients with left-sided colon cancer (same TNM stage) will be received from collaborating surgical department of the Medical Faculty of Charles University and Biomedical Center in Pilsen. The level of DNA repair, EMT stage, and DNA methyltransferases will be assayed for in this discovery set by large platforms (e.g: RNAseq). Obtained results will be validated on 50 tissue pairs from right-sided colon cancer and 50 left-sided colon cancer patients. The above aspects also will be implemented in tissue biopsies from colorectal cancer patients with an early onset of the disease (below 50 years of age), these tumors may be expected to be more invasive.