Summer Research with Undergraduates (Grinnell College 2017)

My previous research demonstrated that a SUMO-targeted ubiquitin ligase complex, Slx5/Slx8 (RNF4 in mammalian), supports cell survival despite the damaged genome in budding yeast (Saccharomyces cerevisiae). We reason that this finding in yeast can be translated to mammalian cells since the pathways involved in the process are functionally conserved. In fact, understanding the tolerance pathway for genome instability has a significant implication in cancer. Indeed, genome instability is one inherent characteristics of malignant cells. How cancer cells withstand the burden of unstable genome is poorly understood.

The experimental evidence led us to pursue RNF4 function in the tolerance of genome instability. We asked two research questions during this summer. First, do mutations of RNF4 identified in cancer tissues contribute to genome stability? Second, does RNF4 confer cancer cells resistance to replication stress?

For the first project, we took advantage of the functional conservation of RNF4 between Saccharomyces cerevisiae and human cells. We introduced wild-type RNF4 or mutant RNF4 carrying one of the cancer-associated mutations into yeast and tested their ability to grow in the presence of genotoxic chemicals.

For the second project, we examined if overexpression of RNF4 changes the response of cancer cells to replication stress. To this end, we used breast cancer cell lines and cell biology techniques to observe cells in mitosis or cells with micronuclei.

Mitosis and Micronuclei

 

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Post Doctoral Research

Slx5_Slx8 model.jpg

This study was published in Thu et al., Cell Reports, 2016.

One essential process of life, known as DNA replication, is to accurately pass on the inheritable genetic information to the future generation.  Intriguingly, replication is an orchestrated process involving multiple molecular players. Disrupting the function of any of these players result in a form of cellular stress known as replication stress. This condition, if remained unresolved, can introduce errors in the genome and therefore be deleterious to cells. Replication stress is in fact a forerunner of cancer. Fortunately, cells evolve multiple cellular mechanisms that are set in motion to counter-balance the compromise. My post-doctoral research effort was to understand some of these mechanisms.

We used a mutant of Mcm10, a replication protein which is essential for cell survival. A mutation that we used in MCM10 gene compromised cells’ ability to accurately copy the genome, leading to replication stress. We were interested in genes that were specifically necessary when MCM10 was absent. In other words, we were on the hunt for genes required to protect cells from replication stress. From this effort, we identified SLX5 and SLX8. When SLX5 or SLX8 gene was absent, cells with the MCM10 mutation grew poorly. Intrigued by the data,  we wanted to find out how Slx5 and Slx8 proteins might protect cells from replication stress.

We had some clues from previous studies which have looked into these proteins’ function. It turns out that Slx5 and Slx8 work together as a protein complex. They are enzymes that mark other proteins for degradation. Protein degradation is one strategy cells use to terminate a biological process. If the participant proteins are destroyed, the biological process will grind to a halt. Therefore, we ask the question: what biological process or pathway is being turned off by Slx5/Slx8 complex under stress?

This question was answered by isolating all potential target proteins that Slx5/Slx8 may mark for degradation and identifying them what they were by proteomics. We found approximately a hundred proteins that may be targeted by Slx5/Slx8 in cells experiencing replication stress. Which ones did we think were involved in a process, when interrupted, would rescue cells from replication stress-induced death?

We used bioinformatics tool and a large body of knowledge from the literature to answer this question. We decided to hone in on a group of proteins involved in checkpoint signaling. This makes sense because checkpoint signaling pathways are surveillance forces of a cell. If, for example, the genome is damaged, checkpoint pathways stop cells from growing or dividing until the problem has been resolved. In other words, checkpoint mechanisms prevent propagation of faulty materials. However, this genome integrity check comes at a price. Cells that cannot escape the checkpoint due to persistent DNA damage will eventually die. We reasoned that checkpoint proteins were perhaps degraded by Slx5/Slx8, relieving the break which would otherwise stop the cell cycle. Our data from both genetics and biochemical experiments support this model.

In essence, we demonstrated the existence of a pathway that provides another chance for cells to repair the damaged genome. When cells are faced with the burden of genome instability, checkpoint signaling may prevail. Alternatively, replication stress resistance mechanisms such as one mediated by Slx5/Slx8 may ensue, allowing cells to repair the damage during the next round of cell cycle.

Intriguingly, the balance between replication stress tolerance and checkpoint-mediated cell cycle arrest/death is intricately linked to cancer biology. Due to uncontrolled proliferation, cancer cells are constantly hampered by replication stress. Yet, they somehow manage to be alive. Understanding the mechanism underlying replication stress resistance will give us clues on how to specifically target cancer cells.

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Thesis Research

 

Part of this research has been published in Thu et al., Oncogene. 2011.

Melanoma is a deadly malignancy which develops from melanocytes, a cell type in the skin which produces melanin. Previous research from my graduate research lab demonstrated that a transcription factor NF-kB activity is elevated in melanoma cells. However, how this activity is up-regulated in these malignant cells was unclear. My dissertation research was to understand the upstream signaling mechanisms that are likely to contribute to NF-kB activation.

I set out to explore the upstream protein kinase of the NF-kB pathway called NIK (NF-kB inducing kinase). We found that NIK expression was much higher in melanoma patient tissues and cell lines compared to the normal counterparts. This clue led us to propose that NIK fueled the growth of cancer cells. To test this idea, we experimentally reduced NIK expression levels by RNA interference. As we predicted, NIK depletion substantially attenuated the tumor growth  both in culture and in mice! Along the way, we also discovered a novel connection between NIK and another cancer signaling pathway known to be active in melanoma. These findings have therapeutic values in treating melanoma since NIK is a “druggable target”.

In addition, we wanted to understand if targeting NF-kB activity is a viable approach for cancer therapy. This signaling pathway does not evolve to be the contributor of cancer cell growth: it has normal physiological functions. For example, NF-kB signaling is a master regulator of the host immune system. Thus, the question is whether targeting elevated activity in NF-kB in cancer cells will have undesirable side-effects for patients. Part of my thesis research was to understand the consequences of using NF-kB inhibitors on the host immune system as a whole and its defense against cancer.

 

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