Publication Date

2018-11-15

Availability

Embargoed

Embargo Period

2020-11-14

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2018-08-22

First Committee Member

Fangliang Zhang

Second Committee Member

Sandra Lemmon

Third Committee Member

Murray Deutscher

Fourth Committee Member

Priyamvada Rai

Fifth Committee Member

Justin Percival

Sixth Committee Member

Marcus Cooke

Abstract

Prostate cancer is a major disease affecting millions of men every year, with hundreds of thousands of new diagnoses and tens of thousands of deaths per year. Most deaths from prostate cancer result from metastasis of the disease. However, the causes for metastasis of prostate cancer (PC) remain poorly understood, making its prediction and treatment a major challenge. Recently, a significant reduction of Arginyltranferase1 (Ate1) was observed in prostate cancer remote metastatic sites, suggesting Ate1 may be involved in the progression of PC. However, the exact role of Ate1 in PC remains unknown. Ate1 is the enzyme that mediates arginylation, the posttranslational addition of one extra arginine to proteins. Previous evidence suggested arginylation is involved in the physiological response to many stressing factors that are commonly associated with cancer risk, therefore providing a potential connection between Ate1 and prostate cancer. I hypothesize that a reduction of Ate1 allows cells to better survive stress, acquire more mutations, and eventually promote the metastasis of prostate cancer cells. To test this hypothesis, I used cellular and biochemical models to examine the role of Ate1 on cellular pathways including the effect on the stress response and mutagenesis, followed by validation of relevant hypotheses in cell-based and animal-based test models, as well as human patient samples. I found that chronic stress leads to decreased Ate1 level in prostate cancer cells. I showed that a loss or reduction of Ate1 increases cellular resistance to a variety of cancer-related stressors, including cellular oxidant, heavy metal, apoptosis-inducing toxins, and radiations. Furthermore, I observed that the role of Ate1 in stress-induced cell death appears to be dependent on Ate1’s arginylation activity. I then found that deletion of Ate1 in mouse fibroblasts leads to disruption of contact inhibition and cell cycle checkpoint activity, which is often a sign of genomic instability. To test whether Ate1 is relevant to mutagenesis in mammalian cells, I developed a novel mutation-activated fluorescent reporter CherryOFF-GFP, which enables fast and sensitive detection of mutagenesis in mammalian cells. With this reporter, I found that a reduction of Ate1 in mouse cell or human prostate cancer cells increases mutation frequency in both non-stressed and stressed conditions using radiation and chemical mutagens. Therefore, I concluded that a reduction of Ate1 leads to an increase of stress tolerance as well as mutation frequency, both risk factors for metastasis. To directly test whether a reduction of Ate1 is a driving force for prostate cancer metastasis, I used both cell-based and animal-based assays, in addition to bioinformatics analysis of available databases of prostate cancer metastasis. I found that a reduction of Ate1 leads to increased cell migration, increased anchorage-independent growth, and enhanced invasion. By using prostate-orthotopic xenografts in immunocompromised mice, I found that a reduction of Ate1 in a prostate cancer cell line PC-3 was sufficient to promote its local invasion and remote metastasis, to a level similar to the metastasis-competent cell line PC3-ML. Finally, by immunostaining human prostate cancer tissues, and by bioinformatics analysis of prostate cancer databases, I found that a reduction of Ate1 expression in the primary prostate tumor is correlated with current and future remote metastasis risks. In conclusion, this study suggests that a reduction of Ate1 is a direct driving force for metastasis in prostate cancer, likely through the role of Ate1 in stress response and mutagenesis suppression.

Keywords

Arginylation; Prostate Cancer; Metastasis; Post-translational modification; Ate1

Available for download on Saturday, November 14, 2020

Share

COinS