School of Medicine and Health Sciences Poster Presentations

Title

The Impact of ATR Mutations in Melanoma on Genomic Stability, Tumor Invasion Potential, and Metastasis

Poster Number

145

Document Type

Poster

Publication Date

3-2016

Abstract

Throughout the day, the skin is constantly exposed to harmful UV rays. The body has various defense mechanisms to protect the skin from detrimental DNA damage, such as melanin production by melanocytes in the epidermis, as well as intracellular DNA damage repair proteins. When these defense mechanism fail, cells can accumulate multiple DNA breaks which in turn leads to genomic instability and the potential for cancer development.

This summer, I studied the impact of ATR, a kinase involved in the UV-induced ssDNA damage response, on melanoma development and progression. We propose that defects in the DNA damage response mechanism involving ATR leads to the ability to accumulate multiple mutations and speeds up melanoma progression. Dr. Ganesan’s lab has already discovered that loss of ATR activity increases melanoma tumor volume, number of tumors, and number of metastases. For my project, I sought to further validate this hypothesis by studying the downstream effects of loss of ATR activity. We used RT-PCR to confirm a mouse model with the kinase domain of ATR floxed out. Additionally, using western blot analysis, we studied the level of CHK1 phosphorylation, a downstream target of the ATR DNA damage response mechanism, in ATR mutant versus ATR wild-type human melanoma cultures. Lastly, we genotyped 100 mice using PCR to collect at least 5 mice each with a ATR wt/wt, ATR fl/wt, or ATR fl/fl genotype for use in future experiments on immune cell infiltration into the tumor microenvironment. A preliminary study of T cell, B cell, and macrophage infiltration into the tumors was performed using flow cytometry.

While we were able to confirm that the ATR kinase domain had successfully by floxed out of the mouse models, we were unable to come to any further conclusions during my summer experience. More protocol troubleshooting was needed for the western blot analysis of CHK1 phosphorylation, as well as for the flow cytometry analysis of the immune cell infiltration. We successfully collected at least 5 mice in each ATR genotype category for later use in immune cell infiltration studies with flow cytometry, as well as gene expression investigation using a Nanostring immune profiling panel. While I was unable to complete the experiments for this project, positive results would suggest that ATR mutation plays a vital role in the progression of melanoma, and could be a potential therapeutic target to slow expansion of disease.

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Presented at: GW Research Days 2016

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The Impact of ATR Mutations in Melanoma on Genomic Stability, Tumor Invasion Potential, and Metastasis

Throughout the day, the skin is constantly exposed to harmful UV rays. The body has various defense mechanisms to protect the skin from detrimental DNA damage, such as melanin production by melanocytes in the epidermis, as well as intracellular DNA damage repair proteins. When these defense mechanism fail, cells can accumulate multiple DNA breaks which in turn leads to genomic instability and the potential for cancer development.

This summer, I studied the impact of ATR, a kinase involved in the UV-induced ssDNA damage response, on melanoma development and progression. We propose that defects in the DNA damage response mechanism involving ATR leads to the ability to accumulate multiple mutations and speeds up melanoma progression. Dr. Ganesan’s lab has already discovered that loss of ATR activity increases melanoma tumor volume, number of tumors, and number of metastases. For my project, I sought to further validate this hypothesis by studying the downstream effects of loss of ATR activity. We used RT-PCR to confirm a mouse model with the kinase domain of ATR floxed out. Additionally, using western blot analysis, we studied the level of CHK1 phosphorylation, a downstream target of the ATR DNA damage response mechanism, in ATR mutant versus ATR wild-type human melanoma cultures. Lastly, we genotyped 100 mice using PCR to collect at least 5 mice each with a ATR wt/wt, ATR fl/wt, or ATR fl/fl genotype for use in future experiments on immune cell infiltration into the tumor microenvironment. A preliminary study of T cell, B cell, and macrophage infiltration into the tumors was performed using flow cytometry.

While we were able to confirm that the ATR kinase domain had successfully by floxed out of the mouse models, we were unable to come to any further conclusions during my summer experience. More protocol troubleshooting was needed for the western blot analysis of CHK1 phosphorylation, as well as for the flow cytometry analysis of the immune cell infiltration. We successfully collected at least 5 mice in each ATR genotype category for later use in immune cell infiltration studies with flow cytometry, as well as gene expression investigation using a Nanostring immune profiling panel. While I was unable to complete the experiments for this project, positive results would suggest that ATR mutation plays a vital role in the progression of melanoma, and could be a potential therapeutic target to slow expansion of disease.