Triple-negative human breast cancers (TNBC) lack three proteins commonly targeted by chemotherapy; estrogen and progesterone receptors, and her-2-neu. Most current protocols used to treat TNBC are largely ineffective and aggressive tumors frequently re-emerge, leading to metastasis and patient death. Thus, new therapies for TNBC are needed. Recent studies show that around 80% of TNBC express mutant p53 (mtp53), a functionally defective form of the p53 tumor suppressor protein. If mtp53 is converted into the active wild-type protein (wtp53), tumor suppressor functions are recovered. Most p53 mutations occur in the DNA-binding domain, causing normal regulation of p53 target genes involved in apoptosis, cell-cycle arrest, and angiogenesis to be blocked. This promotes metastasis and renders tumors resistant to chemotherapy. APR-246 is a small-molecule drug that re-activates mtp53 by covalent modification of the DNA-binding core domain of the mutant protein through alkylation of thiol groups and has been shown to reactivate mtp53 and restore p53 function. We examined whether APR-246 could inhibit TNBC growth, both in vitro and in vivo. Cell viability assays and FACS were used to measure in vitro TNBC cell growth and apoptosis respectively, in MDA-MB-231 and MDA-MB-468 cells, with MCF-7 cells (which express wtp53) as controls. Analysis of TNBC growth in vivo was assessed in a mouse model of MDA-MB-231 derived xenografts. Nuclear extracts of APR-246-treated TNBC cells exhibited significantly increased p53 DNA binding compared with untreated cells, indicating that APR-246 converts mtp53 to wtp53 in these cells. APR-246 significantly reduced TNBC cell viability in vitro, but had no effect on normal mammary cells or wtp53-expressing MCF-7 cells. Pro-apoptotic proteins, Bax, p21 and caspase-3 were elevated in APR-246 treated cells, while the cell survival protein Bcl-2 was suppressed. In the xenograft model, animals were given an intravenous (iv) tail vein injection of APR-246 alone (100 mg/kg/day) once tumors reached 100 mm3. A second group received an intraperitoneal (ip) injection of 2aG4 antibody (100 µg/mouse/day), which targets phosphatidylserine and disrupts tumor blood vessel formation. A third group was given both APR-246 and 2aG4 using the same doses above. A control group received antibody C44 (100 µg/mouse/day, ip) and/or PBS (0.1 mL/day, iv). A total of 18 treatments were used. Administration of APR-246 alone or in combination with 2aG4, significantly reduced TNBC tumor growth, as well as two markers of angiogenesis (vascular endothelial growth factor expression and blood-vessel density). APR-246 in combination with 2aG4 completely eradicated almost 20% of the TNBC tumors. We conclude that TNBC is inhibited by APR-246 and 2aG4. Such treatment could represent an effective and innovative means of combating these particularly aggressive and deadly types of cancer.
Read full abstract