CHARACTERIZING AND TARGETING THE NON-CATALYTIC FUNCTIONS OF PHOSPHATASE OF REGENERATING LIVER 3 (PRL-3) IN ONCOGENESIS AND CANCER PROGRESSION

Author

Jeffery T. Jolly, University of KentuckyFollow

Author ORCID Identifier

https://orcid.org/0000-0002-6748-3591

Date Available

8-12-2025

Year of Publication

2025

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Medicine

Department/School/Program

Molecular and Cellular Biochemistry

Faculty

Dr. Jessica Blackburn

Faculty

Dr. Doug Andres

Abstract

Phosphatase of Regenerating Liver 3 (PRL-3) is frequently upregulated in various cancers and is associated with poor patient prognosis. Although traditionally studied for its phosphatase activity, PRL-3 also interacts with the CNNM family of magnesium transporters through its catalytic site, and these two functions are mutually exclusive at any given time. Most previous studies relied on a commonly used PRL-3 mutation that disrupts both phosphatase activity and CNNM binding, making it challenging to determine which function drives its oncogenic effects. To address this gap in the field, I utilized a panel of PRL-3 mutants that selectively disrupt either phosphatase activity or CNNM binding to assess the contribution of each function to cancer progression. These mutants were evaluated in transgenic zebrafish models of acute lymphoblastic leukemia and rhabdomyosarcoma, as well as several human cancer cell lines. I examined phenotypes associated with tumor progression and metastasis, including leukemogenesis, tumor burden, self-renewal, migration, invasion, and resistance to apoptosis. Across all models, wild-type PRL-3 and the phosphatase-inactive mutant produced similar oncogenic effects, while the CNNM-binding-deficient mutant did not enhance disease progression. These results indicate that PRL-3 promotes cancer progression independently of its phosphatase activity, likely through its interaction with CNNM proteins. To further explore this mechanism, I investigated the effects of PRL-3 in colon cancer cells. PRL-3 overexpression did not alter proliferation rates but enhanced survival under stress conditions, including hypoxia, acidification, and nutrient deprivation, in a manner that did not require phosphatase activity. PRL-3 overexpression also altered intracellular magnesium content, leading to aberrant accumulation. RNA sequencing and untargeted metabolomics of cells expressing different PRL-3 mutants revealed significant changes in metabolism, apoptosis resistance, and epithelial-to-mesenchymal transition. These alterations were present in cells expressing wild-type or phosphatase-inactive PRL-3 but not in the CNNM-binding-deficient mutant, implicating the CNNM interaction and magnesium disruption as central to PRL-3’s effects. Further metabolic profiling demonstrated that PRL-3 enhanced glycolysis, increased cystine uptake, and elevated alanine synthesis, and metabolic adaptations that support survival in hostile environments. Finally, I developed an in vitro FRET-based assay to rapidly evaluate the ability of molecules to disrupt the PRL:CNNM interaction. Using this assay, I discovered that existing PRL-3 inhibitors suppress phosphatase activity but fail to disrupt CNNM binding, which may explain their limited success in preclinical models. I adapted this assay for a high- throughput system and screened a library of drug fragments, along with a panel of in silico-docked compounds. By pairing these results with a thermal stability assay, we identified a handful of compounds that could bind to PRL-3 and significantly reduce its ability to bind CNNMs in vitro. These findings serve as promising leads for future drug development campaigns. Together, these findings indicate that the oncogenic activity of PRL-3 is not a result of its phosphatase activity. Instead, these effects are likely a consequence of its non-catalytic activity, such as binding to CNNMs. This interaction promotes cancer cell survival under stress, likely by disrupting magnesium homeostasis and rewiring cellular metabolism. These results suggest a shift in drug development strategies toward targeting the non-catalytic functions of PRL-3, providing a new platform to support the discovery of compounds that can inhibit this previously underappreciated aspect of its function.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2025.337

Funding Information

This work was funded by NCIR37CA227656 and NIH DP2CA228043 to J.S.B

Recommended Citation

Jolly, Jeffery T., "CHARACTERIZING AND TARGETING THE NON-CATALYTIC FUNCTIONS OF PHOSPHATASE OF REGENERATING LIVER 3 (PRL-3) IN ONCOGENESIS AND CANCER PROGRESSION" (2025). Theses and Dissertations--Molecular and Cellular Biochemistry. 76.
https://uknowledge.uky.edu/biochem_etds/76