Abstract
The Δ133p53 family of p53 isoforms, characterised by the absence of the first 132 N-terminal amino acids of full-length p53, have tumour-promoting properties. Many of these properties affect the tumour-microenvironment. This study hypothesized that Δ133p53 plays a role in facilitating a central mechanism in regulating protein trafficking to the cell surface and as such Δ133p53 will have increased proteins that promote cancer on the cell surface. To investigate this hypothesis, candidate proteins were investigated on the cell surface (chapter 3), and cell surface proteins were labelled and identified (chapter 4). To investigate if proteins altered on the cell surface were directly explained by transcription, a transcriptome analysis was performed (chapter 5). Lastly, cell surface trafficking was inhibited in a syngraft mouse tumour model to determine if this would reduce Δ133p53 tumour growth (chapter 6). A series of experiments utilising flow cytometry, mass spectrometry, RNA sequencing, immunohistochemistry, and immunofluorescence techniques were conducted on clonal H1299 and LN229 cell lines engineered to express different Δ133p53 isoform variants, namely Δ133p53α, Δ133p53β, and Δ133p53γ, or murine pancreatic adenocarcinoma (PDAC), melanoma (B16F10), and embryonic fibroblast (MEF10.1) cells engineered to express a murine mimic of Δ133p53α (Δ122p53).
Flow cytometry analysis of nine candidate proteins found Δ133p53β cells exhibited significantly higher expression of Programmed Death Ligand 1 (PDL1) and Activin A receptor like Type 1 (ALK1) on their cell surfaces (p ≤ 0.0001), while Δ133p53α (p ≤ 0.05) and PDAC Δ122p53 cells displayed increased levels of Aquaporin 4 (AQP4)(p ≤ 0.001). Additionally, Mannose Receptor C Type 2 (MRC2) showed increased cell surface expression for both Δ133p53β and Δ133p53γ cells (p ≤ 0.0001), and Vascular Endothelial Growth Factor Receptor 1 (VEGFR1) demonstrated higher expression for all Δ133p53 variants on the cell surface compared to p53-null cells (p ≤ 0.05) and it was increased on B16F10 cells with Δ122p53 (p ≤ 0.001). TFRC expression was elevated on LN229 Δ133p53β (p ≤ 0.0001) and B16F10 cells expressing Δ122p53 (p ≤ 0.05) while CCL2 (MCP1) was increased on PDAC and MEF10.1 Δ122p53 expressed cells (p ≤ 0.0001). These findings were further confirmed through immunocytochemistry and immunofluorescence microscopy techniques on cells. A broader analysis of cell surface proteins used biotin labelling the cell surface proteins followed by mass spectrometry revealed increased expression of Api5, Hspa5, Ppia, Sqstm1, Parp1, Sf3b1, Hadha, Hnrnpc, Baz1b, Top1, Csnk2a1, Smc3, and Drg1 on the cell surfaces of all three murine cell lines expressing Δ122p53 compared to the control. Many of these proteins have been reported to be increased with mutant p53 and have enhanced cancer-promoting characteristics. The transcriptome analysis using RNA sequencing found few proteins altered on the cell surface of PDAC, B16F10, and MEF10.1 Δ122p53 expressing cells that could be explained directly by altered gene expression.
To determine if inhibiting cell surface could limit tumour growth PDAC and B16F10 cell lines with and without Δ122p53 expression were syngrafted onto C57BL/6 mice, and mice treated with monensin or brefeldin A (cell surface trafficking inhibitors). The Δ122p53 tumours demonstrated accelerated tumour development and heightened tumour metastasis compared to control tumours. Treatment with monensin and brefeldin A led to reduced tumour growth as evidenced by an extended time to reach the tumour-size euthanasia endpoint. For PDAC Δ122p53 tumours, the median survival increased from 18.5 days to 30 days with monensin treatment (p = 0.0066) and further extended to 38 days with brefeldin A treatment (p < 0.0001). Brefeldin A was more effective at reducing tumour growth than monensin (p = 0.0015). No difference in tumour growth was observed with monensin administration for the PDAC control tumours. For B16F10 Δ122p53 tumours, the median survival increased from 15 to 19 days with monensin treatment (p = 0.0009) and to 24 days with brefeldin A treatment (p < 0.0001). Brefeldin A was once again more effective at reducing tumour growth than monensin (p = 0.0432). No difference in tumour growth was observed with the administration of monensin, while brefeldin A improved the survival for the B16F10 control tumours.
In summary, this research highlighted the involvement of the Δ133p53 or Δ122p53 isoform in upregulating the expression of cell surface cancer-promoting proteins, consequently promoting metastasis. The regulatory mechanism that leads to an increase in cell surface proteins still needs to be elucidated as many of these proteins increased on the Δ122p53 cell surface were not directly explained by increased gene expression. Furthermore, inhibiting cell surface trafficking proves to be an effective approach for reducing the tumour growth advantage of Δ122p53 (a mimic of Δ133p53). This study advances our comprehension of the role of Δ133p53 in metastasis, offering insights into potential cell type-specific functions of Δ133p53 or Δ122p53 in regulating cell surface trafficking.