Author ORCID Identifier
https://orcid.org/0000-0002-4242-2127
Date of Graduation
5-2024
Document Type
Dissertation (PhD)
Program Affiliation
Cancer Biology
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Pawel Mazur, PhD, Chair
Committee Member
Boyi Gan, PhD
Committee Member
Haoqiang Ying, MD, PhD
Committee Member
Pamela Wenzel, PhD
Committee Member
Di Zhao, PhD
Abstract
Abstract
More than 100 lysine methyltransferases (KMTs) were predicted to be present in the human proteome, and many were implicated in cancer etiology. However, the catalytic activity and substrate specificity for many of these enzymes remain unknown. Our work aimed to explore the role of Methyltransferase like 21A (METTL21A), amember of the little-studied seven β- strand family of candidate lysine methyltransferases (KMTs), and its role in regulating pancreatic ductal adenocarcinoma (PDAC) and lung adenocarcinoma (LUAC) tumorigenesis. Through the meta-analysis of publicly available gene expression datasets, I found that METTL21A is significantly downregulated in PDAC and LUAC versus normal tissue, and the reduced expression predicts poor patient survival. Thus, there are intriguing correlations connecting METTL21A to PDAC and LUAC pathogenesis; however, to date, there is no data on the role of METTL21A in cancer, and the enzymatic function of METTL21A is poorly recognized. To test the hypothesis that METTL21A plays a role in PDAC and LUAC initiation and progression, I generated conditional Mettl21a knockout mice and crossed to PDAC models: p48Cre/+ KrasG12D/+(Kras vs. Kras; Mettl21a) and p48Cre/+; KrasG12D/+; p53fl/fl (Kras; p53 vs. Kras;p53; Mettl21a), and LUAC models:KrasG12D/+ (Kras vs. Kras; Mettl21a) and KrasG12D/+; p53fl/fl (Kras; p53 vs. Kras;p53; Mettl21a). Histopathological analysis of tissues of Kras and Kras; Mettl21a mice at six months of age revealed that loss of Mettl21a significantly accelerates pancreatic and lung cancer initiation. Additional studies using Kras; p53 and Kras; p53; Mettl21a mice demonstrated that METTL21A depletion accelerates PDAC and LUAC progression, resulting in a significantly shorter overall survival. Furthermore, I studied the impact of METTL21A depletion on human PDACand LUAC cell lines and corresponding PDX growth in vivo and in vitro. In congruence with previousobservations, the knockdown of
Mettl21a in human PDAC and LUAC cell lines leads to a robust increase in cell proliferation and colony formation ability in vitro and xenograft growth in vivo. In contrast, cells rescued with ectopic expression of wild-typeMETTL21A but not with enzyme-dead mutant METTL21A showed significant growth impairment in vitro and in vivo. To identify the substrate of METTL21A enzymatic activity, we performed methylation assays followed by mass spectrometry analysis. Our in vivo methylation assays identified HSPA1 and HSPA8 (members of theHSP70 protein family) as substrates of METTL21A in PDAC and LUAC cells. Our subsequent analysis showedthat METTL21A mainly tri-methylates HSPA1 at lysine 561 (K561me3), but not HSPA8. Next, to directly evaluate the role of METTL21A-mediated methylation of HSPA1 at K561 in regulating cancer phenotype, we knocked out HSPA1 and then complemented cells with wild-type or K561A mutant HSPA1 in PDAC and LUAC cell lines. We found that only methylation-resistant HSPA1 increased cell proliferation and tumor growth in vivo. Furthermore,we analyzed the changes in protein aggregation on METTL21A- depleted cells and found that un-methylatedHSPA1 correlated with lower protein aggregation levels. It indicates that un-methylated HSPA1 has increased disaggregation ability. Additionally, we screened available HSP70 inhibitors and identified that Pifithrin-μ couldinhibit METTL21A-depleted cells compared with the control. We further investigated the sensitivity of METTL21A depletion cells to the KrasG12C inhibitor, AMG510, treatment. The results suggest that METTL21A-depleted cells were more sensitive to AMG510. Interestingly, there is a synergistic effect between Pifithrin-μ and AMG510 for METTL21A depletion cells. Together, these results support that in PDAC and LUAC, the principal physiologic activity of METTL21A is the generation of HSPA1/A8 K561me3, which suppresses cancer growth. Pifithrin-μ and AMG510 could be the target therapy for METTL21A depletion tumors.
Keywords
Methyltransferases; PDAC; LUAC; HSP70 proteins