Role of 5-FU in DNA double strand break repair for improved targets in colorectal cancer therapy
by Upadhyayula Sai Srinivas
Date of Examination:2014-11-07
Date of issue:2015-04-14
Advisor:Prof. Dr. Matthias Dobbelstein
Referee:Prof. Dr. Matthias Dobbelstein
Referee:PD Dr. Wilfried Kramer
Persistent Address:
http://dx.doi.org/10.53846/goediss-5025
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Abstract
English
Colorectal cancer (CRC) is the second leading cause of cancer related deaths in the world, and 5-Fluorouracil (5-FU) based regimens are chiefly employed for its therapy. Modern regimens, based on 5-FU in combination with other drugs have improved the response rates in the terminal CRCs to ~50%. However, a lack of molecular understanding of the cellular response process upon 5-FU treatment has stalled development of more improved therapies. Here, it behooves to study the mechanistic aspects of the complex, but coordinated signaling pathway that gets activated upon DNA damage, the DNA damage response (DDR). Current focus is on understanding the action of chemotherapeutic drugs, especially nucleoside analogs like 5-FU, in the context of DDR to find molecular targets to enhance their efficacies. In the present study, we focus on understanding the molecular basis of 5-FU based neoadjuvant therapy and finding novel targets for improving the response rates in CRC patients. We find that 5-FU pretreatment radiosensitizes CRC cell lines to neocarzinostatin (NCS) and leads to persistent γ-H2AX, a marker for DNA double strand breaks. We further show that 5-FU reduces the ability of cells to perform homologous recombination repair (HRR) but not non-homologous end joining (NHEJ), which points towards HRR being the underlying molecular mechanism of radiosensitizaztion by 5-FU. The use of B02, an inhibitor of HRR also shows persistent γ-H2AX, further strengthening our hypothesis. Our subsequent investigations rule out impaired recruitment of repair proteins as the possible cause of 5-FU induced HRR reduction. On the other hand, ribonucleotide supplementation reduces 5-FU and NCS induced DNA damage suggesting that nucleotide pool disruption is a possible cause of 5-FU induced HRR reduction. Also the Thymidylate synthetase (TS) inhibitor raltitrexed does not synergize with NCS. Interestingly, we find complementary lethality between TS depletion and 5-FU; however, TS depletion using siRNA also does not synergize with NCS. We, therefore, postulate that 5-FU induced TS inhibition and DNA damage both are essential for reducing the HRR. We also find that Gemcitabine, another nucleoside analog, does not show any synergism with NCS, which render it unsuitable for studying DDR in SW480 cells. Interestingly, Ribonucleotide Reductase M2 (RRM2) depletion and hydroxyurea treatment also does not lead to synergism with NCS. Therefore, we conclude that Ribonucleotide reductase inhibition cannot synergize or affect the HRR in SW480 cells. Further, MK2 depletion, which rescues cells from gemcitabine-induced DNA damage does not exert such an effect in 5-FU treated cells, indicating that MK2 does not play any significant part in 5-FU induced DNA damage. In conclusion, our results strongly suggest that the molecular reason for 5-FU induced radiosensitization is a reduction in the cellular ability to carry out HRR. This leads us to postulate that HRR inhibitors like B02 can be used in future regimens to overcome 5-FU resistant colorectal cancers.
Keywords: 5-FU; colorectal cancer; DNA repair; DNA damage response
