Abstract
In New Zealand melanoma is the third most common cancer in both men and women and it has the highest reported incidence and mortality rates worldwide. While early-stage melanoma is often curable, no available treatments are curative for late-stage disease. Unlike many cancers, melanoma typically retains wild-type p53; however, its tumour-suppressive function is often inactivated through alternative mechanisms rather than mutation. There are published studies describing interactions occurring between p53 and Δ133p53, a truncated isoform encoded by TP53, and how Δ133p53 may exert a dominant-negative effect on p53. Drug-tolerant persisters (DTPs) are a rare subpopulation of cancer cells that arise following drug treatment and serve as an intermediate step in the acquisition of drug resistance. Previous observations in DTPs from the WT-TP53 A375 melanoma cell line, showed upregulation of Δ133p53 mRNA while Δ133p53 protein remained undetectable. We hypothesised that upregulation of Δ133p53 mRNA and the absence of Δ133p53 protein may be indicative of Δ133p53 acting as a noncoding RNA to inhibit p53 function in DTPs. Additionally we sought to determine if this phenomena occurred in other WT-TP53 cell-lines such as lung cancer, and we attempted to knockdown Δ133p53 expression using siRNA.
We successfully replicated Δ133p53 mRNA increases in the WT-TP53 Malme-3M melanoma cell line without any corresponding increases in Δ133p53 protein expression . This supports our hypothesis that Δ133p53 may function as a non-coding RNA to inhibit p53 activity, potentially contributing to functional inactivation of p53 in DTPs. We established that Malme- 3M melanoma cells form DTPs following 1 μM vemurafenib treatment for at least three days, confirmed by upregulation of CD36 and NGFR, both known DTP marker genes. We attempted to replicate the findings from the Malme-3M cell line in a different WT-TP53 cell lines but we were unable to reliably induce DTPs in the WT-TP53 SW1573 lung cancer cell line using 3.33 μM adagrasib, likely due to the cell line’s low drug sensitivity. Sotorasib was also ineffective, aligning with prior reports of partial resistance. Δ133p53 mRNA upregulation was not observed through RT-qPCR; however, without clear DTP detection, its role in WT-TP53 lung cancer remains uncertain. Attempts to knock down Δ133p53 in A375 cells using siRNA were unsuccessful, likely due to its nuclear localization, which may limit siRNA accessibility. Futurestudies should explore CRISPR interference or antisense oligonucleotides to effectively target Δ133p53 and assess its role in p53 suppression.
As many cancers retain WT-TP53 but exhibit functional inactivation, understanding Δ133p53’s role in DTPs may provide critical insights into drug resistance acquisition mechanisms. Our findings support growing evidence that Δ133p53 plays a key role in p53 suppression, potentially offering a therapeutic target to prevent DTP formation in WT-TP53 cancers and the onset of drug resistance. Future research should focus on defining Δ133p53’s molecular mechanisms and exploring strategies to prevent DTPs progressing into drug resistance.