Andrew Taylor: Preliminary Findings and Next Steps to Better Understand Colon Cancer

Preliminary Findings

Project research as of the first few weeks has consisted of crossing DNA repair gene knockdown constructs into a multigenic fruit fly colon cancer model to look for any phenotypic changes in tumor size and cell activity, such as senescence, double stranded DNA breaks, apoptosis, and p21 activity (a cell cycle inhibitor).

Andrew Taylor, Helen Louise Lee Research Award Winner

Several early findings requiring further verification have emerged, namely the absence of the double strand break marker gamma-H2AV in cancer models with knocked down cell cycle checkpoint gene activity. These fly genes include mei-41, tefu, and grapes, which directly correspond to 3 essential regulators in human DNA damage response (ATR, ATM, CHK1 respectively).

For further background, imaging data is collected through antibody staining, with antibody structures being repurposed to bind to specific molecules of interest in cancer and emit colored light upon imaging. Gamma-H2AV is an antibody that binds to the phosphorylated (chemically activated) H2AV histone, a protein involved in flagging double stranded breaks for repair.

What is interesting about these findings is that they run counter to the worsened phenotype I expected from knockdown of cell cycle checkpoint genes. In the native multigenic RPPA cancer model used in the Bangi lab, hindguts quickly deteriorate, characterized by rapid tumor growth and the visibility of the Gamma-H2AV damage marker. Antibody imaging shows the opposite phenotype in knockdown samples, with an absence of double stranded breaks and a smaller tumor size.

As mentioned earlier, these same results are observed in experimental groups with mei-41 (ATR) and grapes (CHK1) knockdown. This data suggests that a targeted reduction in checkpoint signaling results in a reduced cancer phenotype, despite other tumor mutations being identical. Based on this preliminary data alone and in the absence of other informative markers, there are several potentially compatible conjectures that come to mind: Cells deficient in checkpoint activity become senescent and stop dividing via failsafe mechanisms, reduced checkpoint activity results in less H2AV activation and recruitment to damage sites, DNA damage is being repaired through more accurate pathways, or repair is occurring in a less active environment that reduces replication stress and improves functionality.

Moving Forward

To construct a higher resolution picture of what is happening in these tumor cells, antibody staining for other markers will be performed. These markers will further resolve ambiguities in disease states by revealing details surrounding cell cycle arrest and DNA expression activity, such as the subcellular localization of the cell cycle inhibitor p21 and heterochromatin staining associated with senescence.

Quantification of tumor sizes will also be recorded using imaging software, and statistically analyzed to look for differences in gene knockdown backgrounds when compared to the basal cancer model.

Furthermore, the activity of subsidiary genes such as RPA, ATRIP, Claspin, TopBP1 and the MRE, NBS, Rad protein complex could be investigated. These genes are further along in the DNA damage response and directly interact with DNA and each other to carry out specific repair functions; therefore, their modulation might be helpful in specifying exactly where switches in cell fates occur along signaling networks, fates that modify tumor progression and ultimately the activity of the cancer being studied.

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