Graduation Year
2022
Document Type
Dissertation
Degree
Ph.D.
Degree Name
Doctor of Philosophy (Ph.D.)
Degree Granting Department
Biology (Cell Biology, Microbiology, Molecular Biology)
Major Professor
Florian A. Karreth, Ph.D.
Committee Member
Elsa R. Flores, Ph.D.
Committee Member
Keiran S. M. Smalley, Ph.D.
Committee Member
Eric K. Le-Lau, Ph.D.
Committee Member
Sheri L. Holmen, Ph.D.
Keywords
Embryonic Stem Cell, Genetically Engineered Mouse Models
Abstract
The cumbersome and time-consuming process of generating new mouse strains and multiallelic experimental animals often hinders the use of genetically engineered mouse models (GEMM) in cancer research. In chapter 2, we describe the development and validation of an embryonic stem cell (ESC)-GEMM platform for rapid modeling of melanoma in mice. The platform incorporates 12 clinically relevant genotypes composed of combinations of four driver alleles (LSL-BrafV600E, LSL-NrasQ61R, PtenFlox, and Cdkn2aFlox) and regulatory alleles to spatiotemporally control the perturbation of genes of interest. The ESCs produce high-contribution chimeras, which recapitulate the melanoma phenotypes of conventionally bred mice. Using the ESC-GEMM platform to modulate Pten expression in melanocytes in vivo, we highlighted the utility and advantages of gene depletion by CRISPR-Cas9, RNAi, or conditional knockout for melanoma modeling. Moreover, complementary genetic methods demonstrated the impact of Pten restoration on the prevention and maintenance of Pten-deficient melanomas. In addition, we showed in part that chimera-derived melanoma cell lines retain regulatory allele competency and are a powerful resource to complement ESC-GEMM chimera experiments in vitro and in syngeneic grafts in vivo. In chapter 3, we reported establishment of a total of 21 cell lines from ESC-GEMM chimera melanomas driven by LSL-BrafV600E; PtenFlox, LSL-BrafV600E; Cdkn2aFlox, LSL-NrasQ61R; PtenFlox, and LSL-NrasQ61R; Cdkn2aFlox. We reported a comprehensive characterization of these cell lines; specifically, we validated melanocytic origin, driver allele recombination and expression, and activation of the oncogenic MAPK and AKT pathways. We further tested tumor formation in syngeneic immunocompetent recipients as well as functionality of the integrated Tet-ON system and recombination-mediated cassette exchange (RMCE) homing cassette. Finally, by deleting the transcription factor Mafg with an inducible CRISPR/Cas9 approach we demonstrate the utility of the regulatory alleles for candidate gene modulation. These cell lines will be a valuable resource for studying melanoma biology and therapy. Thus, when combined with sophisticated genetic tools, the ESC-GEMM platform enables rapid, high-throughput, and versatile studies aimed at addressing outstanding questions in melanoma biology. The significance is that this study presents a high-throughput and versatile ES cell-based mouse modeling platform that can be combined with state-of-the-art genetic tools to address unanswered questions in melanoma in vivo.
Loss of PTEN frequently occurs in melanoma and leads to hyperactivation of the PI3K/AKT pathway. However, targeting AKT in melanoma patients and mouse models provoked limited therapeutic effects. In addition to opposing PI3K/AKT activation as a lipid phosphatase, PTEN has protein phosphatase activity and scaffold functions and I found that reactivating endogenous PTEN in PTEN-deficient melanomas halted tumor growth. Since PTEN reactivation restores all its functions, I hypothesize that PTEN suppresses melanoma through both its canonical and non-canonical functions. To determine whether PTEN suppresses melanoma through its canonical pathway, I expressed activated AKT (AKT1E17K) in PTEN-restored PTEN-null melanoma cells. Activated AKT didn’t rescue the tumor suppression by PTEN, adding to previous evidence suggesting that AKT activation alone is insufficient to promote melanoma upon PTEN loss. Thus, PTEN deficiency may promote melanoma through PIP3-dependent effectors other than AKT and/or through lipid phosphatase-independent functions. To test the role of PTEN protein phosphatase activity in melanoma suppression I compared the tumor suppressive potential of PTENWT, lipid- and protein-phosphatase-dead PTENC124S, lipid-phosphatase-dead PTENG129E, and protein-phosphatase-dead PTENY138L in Pten-deficient mouse melanoma cells. Both PTENY138L and PTENG129E decreased melanoma cell growth in vitro and tumor formation but neither was as potent as PTENWT. Thus, both lipid and protein phosphatase activities suppress melanoma. My findings suggest that PTEN suppresses melanoma through non-canonical pathways, involving AKT-independent lipid phosphatase as well as protein phosphatase functions. Thus, combinatorial inhibition of PTEN-regulated pathways may be a promising treatment strategy for PTEN-deficient melanoma.
Scholar Commons Citation
Bok, Ilah, "Establishment of a Melanoma ESC-GEMM Platform and Its Use to Study PTEN Tumor Suppressor Functions" (2022). USF Tampa Graduate Theses and Dissertations.
https://digitalcommons.usf.edu/etd/10282
