Functional Assessment on the Differential Effect of Mutant p53-Plakoglobin Interaction in Carcinoma Cells

  • Author / Creator
    Lo, Chu Shiun
  • p53 transcriptional factor and tumor suppressor protein is mutated in over 50% of all cancers and most metastatic tumors. Majority of p53 mutations are single missense mutation and occur in the DNA-binding domain (DBD). The six most common missense mutations in the p53 DBD are known as “hot spots” and are classified into “conformational” and “contact” mutants. Both mutant types interfere with the interaction of p53 with DNA either by altering p53 conformation/structure (conformational) or affecting amino acid residues directly involved in DNA binding (contact). Conformational and contact p53 mutant oncogenic activities have been shown to be cell-context and mutation type dependent. Using carcinoma cell lines of various origins, previously, we showed that plakoglobin interacted with wild-type and several endogenous p53 mutants (e.g., R280K, R273H, S241F, S215R) and this interaction was mediated by the p53 DBD and plakoglobin C-terminus domain. We further showed that plakoglobin expression in carcinoma cell lines deficient in plakoglobin and expressing various p53 mutants restored the tumor suppressor activities of mutants in vitro.
    To compare the effects of plakoglobin expression on oncogenic activities of contact vs. conformational mutants and to avoid potential confounding cell-context dependent factors, we established an in vitro cellular model using the p53-null and plakoglobin-deficient H1299 non-small cell lung carcinoma cell line. This system allowed us to exogenously express p53-R273H contact or p53-R175H conformational mutant with or without plakoglobin to directly compare tumor suppressive effects of plakoglobin on these mutants in the same genetic background. Functional assays were performed to assess cell growth, colony formation, migration, and invasion. qPCR and immunoblotting were used to examine the expression specific genes and level and subcellular distribution of proteins that are typically regulated by or regulate p53 function and are altered in mutant p53 expressing cell lines and tumors. Plakoglobin interacted with both mutants, affected their oncogenic properties differently, and its tumor suppressive effects were significantly stronger in p53-R175H expressing cells. We further extended our studies and identified potential amino acid residues in p53-R175H and plakoglobin that mediated their interaction. Using in silico 3D molecular dynamic modeling, we identified Q167 and R248 on p53-R175H, and N690 on plakoglobin as potential amino acid residues mediating their interaction. We then validated the in silico model by generating plasmids encoding p53-R175H in which Q167 and R248 residues were substituted by alanine individually or together. Similarly, we developed a plasmid encoding plakoglobin in which N690 was substituted by alanine. Different combinations of plasmids were expressed in H1299 cells and the resulting transfectants characterized for changes in p53-R175H and plakoglobin interaction and the functional consequences of these changes was measured by the in vitro invasiveness of various transfectants. The results showed decreased p53-R175H- plakoglobin interaction when p53-R175H Q167 and R248 residue were substituted by alanine. In contrast, plakoglobin N690A substitution had no effect on its interaction with p53-R175H. Intriguingly, plakoglobin co-expression with all forms of p53-R175H reduced their invasiveness by more than 80%. These observations suggested the potential ability of plakoglobin to interact with various conformational mutants. The larger implication of these studies is the potential for exploring plakoglobin interactions with p53 conformational mutants as a useful approach to develop cancer therapeutics for tumors expressing this type of mutations.

  • Subjects / Keywords
  • Graduation date
    Spring 2023
  • Type of Item
  • Degree
    Master of Science
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.