Mutant p53 reactivation by prima-1 : molecular mechanism and biological effects

Cancer has become one of the leading causes of death worldwide. It is now clear that tumors arise due to the malfunction of a number of genes, thus emphasising the importance of targeted therapies directed to crucial types of genetic alterations. The tumor suppressor gene, p53, is such a target as it is a key regulator of life and death for cells exposed to diverse forms of stress such as oncogene activation, hypoxia and DNA damaging agents. Moreover it is inactivated by mutation in about half of all human cancers, or by alternative mechanisms in the remaining cases. Loss of p53 tumor suppressor function often counteracts effective cancer treatment, since most of anti-cancer drugs attack tumors by activating p53-dependent apoptosis. Mutant p53 rescue has been demonstrated with low molecular weight compounds including substances found in our group: MIRA-1, STIMA-1 and PRIMA-1. Previous studies have shown that PRIMA-1 (p53 reactivation and induction of massive apoptosis) and its more potent analogue PRIMA-1MET restore wild type properties to mutant p53, transactivation of several p53 target genes and induction of apoptosis in a mutant p53-dependent manner.

In papers I and II we investigated the in vitro and in vivo effect of PRIMA-1 on mouse models. At first we showed the ability of PRIMA-1 to trigger apoptosis by targeting the p53 mutant ser249 often found in hepatocellular carcinoma patients exposed to aflatoxin B1. Mice receiving i.v. injections of PRIMA-1 developed tumors more slowly than the control group. Next we demonstrated that PRIMA-1-induced apoptosis in situ in tumors grafted into immunocompetent mice.

In our transcriptomics study, paper III, we confirmed the impact of PRIMA-1 on mRNA upregulation of several genes involved in cell cycle and cell death, including p53 target genes. Furthermore it revealed a new insight into PRIMA-1-induced apoptosis by indicating the participation of the endoplasmic reticulum stress response following PRIMA-1 treatment in a mutant p53-dependent manner. This newly identified pathway not only reconciliates previous divergent studies with regards to the involvment of the Bax-dependent or JNK-dependent pathways, but also gives a strong advantage to PRIMA-1 as a candidate drug capable of targeting multiple pathways, thus increasing the odds to avoid drug resistance.

As shown in paper IV, PRIMA-1 is rapidly degraded, both in vitro and in vivo, into active compounds which are prompt to react with thiol groups. While PRIMA-1 covalently binds to cysteines in the p53 core domain, it is not excluded that it also binds to several other proteins with exposed thiol groups. However, as shown by transfer of PRIMA-1-treated recombinant mutant p53 into null p53 cells, the modification of p53 per se is sufficient to induce p53-dependent transactivation of target genes and trigger apoptosis. The same experiments with PRIMA-1-treated BSA failed to induce apoptosis. The preference for binding to mutant p53 can be due to the unstable structure of mutant p53 that enhances accessability to thiol groups. Moreover thiol-alkylation has probably a stronger impact for mutant p53 than for other proteins due to p53 s central role in determining cell fate.

In conclusion, this thesis sheds new light on the mechanism of mutant p53 reactivation by PRIMA-1, as well as the downstream cascade of activated pathways which converge towards the induction of apoptosis. This work validates the principle of reactivation of mutant p53 as a working strategy for anticancer drug development.

List of scientific papers

I. Shi H, Lambert JM, Hautefeuille A, Bykov VJ, Wiman KG, Hainaut P, de Fromentel CC (2008). "In vitro and in vivo cytotoxic effects of PRIMA-1 on hepatocellular carcinoma cells expressing mutant p53ser249." Carcinogenesis 29(7): 1428-34. Epub 2007 Nov 28
https://pubmed.ncbi.nlm.nih.gov/18048389

II. Zache N, Lambert JM, Wiman KG, Bykov VJ (2008). "PRIMA-1MET inhibits growth of mouse tumors carrying mutant p53." Cell Oncol 30(5): 411-8
https://pubmed.ncbi.nlm.nih.gov/18791272

III. Lambert JMR, Moshfegh A, Hainaut P, Wiman KG, Bykov VJN (2008). "Mutant p53 rescue by PRIMA-1MET induces endoplasmic reticulum stress mediated apoptosis." (Submitted)

IV. Lambert JMR, Gorzov P, Veprintsev DB , Westman J, Söderqvist M, Segerbäck D, Bergman J, Fersht AR, Hainaut P, Wiman KG, Bykov VJN (2008). "PRIMA-1 reactivates mutant p53 by covalent binding to the core domain." (Submitted)