All cellular life forms have evolved mechanisms to faithfully duplicate their genetic material and repair damaged DNA. These processes pose a remarkable barrier against cancer in multicellular organisms. Yet, genome integrity is constantly at risk due to external factors such as exposure to toxic chemicals or radiation. In parallel, endogenous sources such as replication stress can be equally deleterious. Replication stress refers to any condition, which compromises the process of normal replication during S-phase. If remained unresolved, replication stress may result in mutations, DNA double-strand breaks or chromosome rearrangements and pave the way for tumorigenesis. Replication stress is not only a prelude to cancer but also a chronic condition experienced by highly proliferative cancer cells. Uncontrolled proliferation perturbs the precise timing of S-phase and compromises the progression of normal replication. This phenomenon, known as “oncogene-induced replication stress,” has been widely documented (Gaillard et al., 2015).
Under normal circumstances, insults to the genome will activate checkpoints – surveillance mechanisms that postpone cell cycle transitions when cellular integrity is compromised – and thus, allowing time for the repair of damaged DNA (Fig. 1). Cells may enter the next phase of the cell cycle after errors have been corrected. Yet, failure to complete this step may result in cell death (Fig. 1). This is to ensure that the faulty genome is not propagated from one generation to the next. Despite these mechanisms, cells may overcome checkpoint activation and resume cell cycle even in the presence of replication stress or damaged DNA (Fig. 1). How cells tolerate the burden of genome instability is poorly understood. This is a topic of interest especially in cancer cells since they continue to proliferate in the midst of replication stress. This phenotype can be partially attributed to defective checkpoint signaling in some cancer cells. In parallel, it is highly probable that cancer cells upregulate replication stress tolerance mechanisms to accommodate less optimal growth conditions.
My previous research demonstrated that yeast heterodimeric protein complex Slx5/Slx8 (RNF4 in mammalian cells) confers cellular tolerance for replication stress. Both Slx5/Slx8 and RNF4 belong to a family of SUMO (small ubiquitin-like modifier) targeted ubiquitin ligases (STUbLs), which coordinate signaling mechanisms between two types of post-translational modification, ubiquitin and SUMO (Fig. 2). Similar to ubiquitin, SUMO is a small peptide that can be covalently conjugated to target proteins in a process known as sumoylation. However, unlike ubiquitin, the primary purpose of SUMO conjugation is not proteasomal destruction. Instead, sumoylation serves as a binding platform for protein-protein interaction and thus, orchestrates the formation of protein complexes (Sarangi and Zhao, 2015). The Slx5/Slx8 complex recognizes sumoylated proteins and conjugates ubiquitin chains to target proteins for subsequent proteasomal destruction (Fig. 2). The working model is that sumoylation promotes formation of protein complexes necessary for genome stability, whereas, ubiquitination by a STUbL counterbalances the effect of SUMO after genome integrity has been restored.
My post-doctoral research demonstrated a novel mechanism by which Slx5/Slx8 enables cells to tolerate replication stress in Saccharomyces cerevisiae. Using quantitative mass spectrometry, we identified a mitotic checkpoint protein Bir1 to be a target of Slx5/Slx8. By degrading sumoylated Bir1, Slx5/Slx8 attenuates mitotic checkpoint activation, allowing cells to complete cell cycle in the presence of replication stress (Thu et al., 2016). Based on this data, the next logical step is to explore the contribution of its human homolog RNF4 to cancer. I hypothesize that oncogene-induced replication stress activates mitotic checkpoint as a tumor suppressor mechanism (Fig. 3A and B). To overcome this barrier, cancer cells might up-regulate RNF4 (Fig. 3C). The specific goal of my research is to further understand whether a change in RNF4’s activity contributes to tumorigenesis. I use S. cerevisiae and human breast cancer cell lines as model systems to address my research questions.
Carmena, M., Wheelock, M., Funabiki, H., and Earnshaw, W.C. (2012). The chromosomal passenger complex (CPC): from easy rider to the godfather of mitosis. Nat. Rev. Mol. Cell Biol., 13, 789-803.
Gaillard, H., Garcia-Muse, T., and Aguilera, A. (2015). Replication stress and cancer. Nat. Rev. Cancer., 15, 276-289.
Hills, S., and Diffley, J. (2014). DNA Replication and Oncogene-Induced Replicative Stress. Current Biology., 24, R435-444.
Mankouri, H., Huttner, D., and Hickson, I. (2013). How unfinished business from S-phase affects mitosis and beyond. EMBO J., 32, 2661-2671.
Maya-Mendoza, A., Ostrakova, J., Kosar, M., Hall, A., Duskova, P., Mistrik, M., Merchut-Maya, J., Hodny, Z., Bartkova, J., Christensen, C., Bartek, J. (2015). Myc and Ras oncogenes engage different energy metabolism programs and evoke distinct patterns of oxidative and DNA replication stress. Mol. Oncol., 3, 601-616.
Neiman, P.E., Kimmel, R., Icreverzi, A., Elsaesser, K., Bowers, S.J., Burnside, J., and Delrow, J. (2006). Genomic instability during Myc-induced lymphomagenesis in the bursa of Fabricius. Oncogene, 25, 6325-6335.
Sarangi, P. and Zhao, X. (2015). SUMO-mediated regulation of DNA damage repair and responses. Trends in Biochem. Sci., 40, 233-242.
Thomas, J., Abed, M., Heuberger, J., Novak, R., Zohar, Y., Beltran Lopez, A., Trausch-Azar, J., Ilagan, M., Benhamou, D., Dittmar, G., Kopan, R., Birchmeier, W., Schwartz, A., Orian, A. (2016). RNF4-Dependent Oncogene Activation by Protein Stabilization. Cell Reports, 12, 3388-3400.
Thu, Y.M., Van Riper, S., Higgins, L., Zhang, T., Becker, J., Markowski, T., Nguyen, H.D., Griffin, T., and Bielinsky, A. (2016). Slx5/Slx8 promotes replication stress tolerance by facilitating mitotic progression. Cell Reports, 15, 1254-1265.
Wei, L., and Zhao, X. (2016). A new MCM modification cycle regulates DNA replication initiation. Nat. Stru. and Mol. Bio., 23, 209–216.