Abstract
Cancer metastases cause 90% of cancer deaths. Many proteins can prevent cancer, amongst these, the best characterized is tumour protein 53 (p53) known as the ‘guardian of the genome’. Other variants of p53 exist, including p53 isoforms, and these may promote cancer. The Δ133p53 isoforms (Δ133p53a, Δ133p53β or Δ133p53γ) result from the use of an alternative promoter and alternative 3’ splicing and have functions that promote invasion, proliferation, and inflammation. This project investigated if Δ133p53 isoforms promoted cancer by increasing cancer-promoting proteins on their cell surface. Secondary aims determined if the proteins altered on the Δ133p53 cell surface could be directly explained by changes to gene transcription, or the cell cycle. Investigations used five stable clonal cell lines overexpressing Δ133p53α (Δ133p53α1, Δ133p53α2, Δ133p53α8) or Δ133p53β (Δ133p53β5 and Δ133p53β10) isoforms, derived from the p53 null H1299 cell line along with the original H1299 cell line (p53 null). Fluorescence-activated cell sorting (FACS) flow cytometry (FC) determined if a panel of candidate proteins (ITGβ4, IL6Ra, c-MET, EGFR CSF2, CCL2, and CXCR7) were increased at the cell surface. A discovery proteomic based approach identified a cell surface protein “finger-print” and an endocytosis “finger-print” by biotin labelling cell surface proteins and L-glutathione protection assays, respectively. Altered proteins were identified using mass spectrometry (ESI LC-MS/MS) and bioinformatic analyses. RNA sequencing identified differentially expressed genes. Three candidate proteins (ITGβ4, c-MET, and EGFR) were increased on the Δ133p53b and/or Δ133p53a cell surface, and mass spectrometry results identified several cancer-promoting proteins increased at the Δ133p53 cell surface including ACACA, RSP6, EGFR, ENO1, PLXNβ2, AXL, TFRC, c-MET, ITGα6, and ITGβ4. The endocytosis analysis confirmed an increase in ITGB1, ENO1, and RPS6, and identified a higher number of ribosomal proteins increased (RLPs and RPSs) or decreased from the Δ133p53 cell surface (HnRNPs). Comparison of the mass spectrometry data with transcriptional data showed little overlap, suggesting an alternate or indirect transcriptional based mechanism was responsible for changes to proteins at the cell surface. Live cell imaging revealed more Δ133p53β was expressed in the M phase. To conclude, Δ133p53 isoforms may enhance carcinogenesis by upregulating several key cancer proteins at the cell surface, suggesting Δ133p53 cancers may be better treated with cell surface protein targeted treatments. Future directions of this project will focus on identifying the new core mechanism that led to increased proteins on the cell Δ133p53 surface, and strategies to treat Δ133p53 cancers.