Interplay between MDM4 and P53 in breast cancer

Abstract

Breast cancer (BC) is one of the most prevalent cancer types and second leading cause of cancer death among Australian women. BC is a genetically heterogeneous disease, divided into sub-types that differ in their aggressiveness, metastatic potential and response to treatment. Notably, relapsing triple negative BC (TNBC) have limited therapeutic options and a poor survival. A key driver of metastatic TNBC is the mutated form of p53. P53 is a major tumour suppressor, and plays a pivotal role in the cellular response to stress conditions. The function of the key tumour suppressor protein p53 is compromised frequently in cancers, either by direct mutation or by its deregulation. The latter is achieved largely by two key negative regulators: MDM2, its E3 ligase, and MDM4 a potent inhibitor of p53’s transcriptional activities. This thesis describes the interplay between p53, both wild-type (wt-p53) and mutant forms, and its regulator MDM4 in multiple subtypes of BC.

In chapter 3 of this thesis, we identified MDM4 as a potent negative regulator of wt-p53 in BC, and its depletion impedes the growth of BC cells, both in vitro and in vivo experimental models. MDM4 knockdown (KD) reduces tumour progression in vitro and in vivo by activating the cell cycle inhibitor p21 in a p53-dependent manner. Our screen of BC samples in a tissue microarray and cell lines revealed high levels of MDM4 expression not only in wt-p53 expressing BC, but also in basal-like and luminal BCs that express mutant p53 (mt-p53). This prompted us to test the contribution of MDM4 to the oncogenesis of BC that expresses mt-p53, which is described in chapter 4. Surprisingly, we have demonstrated that KD of MDM4 in a range of BC cell lines expressing mt-p53 across multiple subtypes impedes their growth in culture. The therapeutic potential of this approach was further validated using XI-011, a small molecule inhibitor of MDM4 in vitro in TNBC cell lines. In a search for the underlying mechanism of growth inhibition in response to MDM4 depletion, we have established the involvement of p27 protein, a key cell cycle inhibitor. The inverse correlation between the protein expression of p27 and MDM4 was found in our basal-like BC tissue microarray supporting the relationship between p27 and MDM4 in these patients.

Mutations in p53 not only result in loss of wt-p53’s tumour suppressive functions, but importantly certain hot spot mutations also acquire Gain Of Functions (GOFs), which drive oncogenesis, and in particular metastatic disease. These render mt-p53 expressing cells addicted to the expression and function of mt-p53. This opens an exciting therapeutic opportunity to target mt-p53 in these cancers. In chapter 5 we tested the effect of the first in class wt-p53 reactivating drug, APR-246, which is currently in multiple clinical trials. We rationalized that combining APR-246 treatment with MDM4 targeting would potentiate this treatment by protecting the ‘reactivated wt p53’ to suppress tumour growth. A combinatorial therapeutic strategy using APR-246 to reactivate mt-p53 and XI-011, which targets MDM4, was very effective in the inhibition of TNBC growth in vitro. We also demonstrated the potency of XI-011 and APR-246 combination to inhibit the growth of cell line xenografts and patient-derived xenografts in vivo.

Overall, this thesis demonstrates an essential role for MDM4 in the establishment of oncogenesis in BC cells expressing wt-p53 and mt-p53 and defines MDM4 as an attractive therapeutic target in both single and combinatorial therapy to treat mt-p53 expressing BC. The efficacy of MDM4 targeting in mt-p53 cancers challenges current dogma and opens the window for a novel therapeutic approach in these difficult to treat BCs.