Mutant p53-R273H mediates cancer cell survival and anoikis resistance through AKT-dependent suppression of BCL2-modifying factor (BMF).

p53 is the most frequently mutated tumor-suppressor gene in human cancers. Unlike other tumor-suppressor genes, p53 mutations mainly occur as missense mutations within the DNA-binding domain, leading to the expression of full-length mutant p53 protein. Mutant p53 proteins not only lose their tumor-suppressor function, but may also gain new oncogenic functions and promote tumorigenesis. Here, we showed that silencing of endogenous p53-R273H contact mutant, but not p53-R175H conformational mutant, reduced AKT phosphorylation, induced BCL2-modifying factor (BMF) expression, sensitized BIM dissociation from BCL-XL and induced mitochondria-dependent apoptosis in cancer cells. Importantly, cancer cells harboring endogenous p53-R273H mutant were also found to be inherently resistant to anoikis and lack BMF induction following culture in suspension. Underlying these activities is the ability of p53-R273H mutant to suppress BMF expression that is dependent on constitutively active PI3K/AKT signaling. Collectively, these findings suggest that p53-R273H can specifically drive AKT signaling and suppress BMF expression, resulting in enhanced cell survivability and anoikis resistance. These findings open the possibility that blocking of PI3K/AKT will have therapeutic benefit in mutant p53-R273H expressing cancers.

Mutant p53 mediates survival of breast cancer cells.

BACKGROUND: p53 is the most commonly mutated tumour-suppressor gene in human cancers. Unlike other tumour-suppressor genes, most p53 cancer mutations are missense mutations within the core domain, leading to the expression of a full-length mutant p53 protein. Accumulating evidence has indicated that p53 cancer mutants not only lose tumour suppression activity but also gain new oncogenic activities to promote tumourigenesis. METHODS: The endogenous mutant p53 function in human breast cancer cells was studied using RNA interference (RNAi). Gene knockdown was confirmed by quantitative PCR and western blotting. Apoptosis was evaluated by morphological changes of cells, their PARP cleavage and annexin V staining. RESULTS: We show that cancer-associated p53 missense mutants are required for the survival of breast cancer cells. Inhibition of endogenous mutant p53 by RNAi led to massive apoptosis in two mutant p53-expressing cell lines, T47D and MDA-MB-468, but not in the wild-type p53-expressing cells, MCF-7 and MCF-10A. Reconstitution of an RNAi-insensitive mutant p53 in MDA-MB-468 cells completely abolished the apoptotic effects after silencing of endogenous mutant p53, suggesting the specific survival effects of mutant p53. The apoptotic effect induced by mutant p53 ablation, however, is independent of p63 or p73 function. CONCLUSION: These findings provide clear evidence of a pro-survival 'gain-of-function' property of a subset of p53 cancer mutants in breast cancer cells.

Preclinical toxicokinetic evaluation of phortress [2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole lysylamide dihydrochloride] in two rodent species.

BACKGROUND AND AIMS: The 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole prodrug Phortress exerts potent and selective antitumour activity in vitro and in vivo. Preclinical toxicokinetic studies in 2 rodent species were undertaken to determine Phortress' maximum tolerated dose and advise a safe starting dose for clinical evaluation. METHODS: Plasma pharmacokinetic parameters were determined by high-performance liquid chromatography and fluorescence detection following Phortress administration to mice (10 mg/kg, intravenously on days 1 and 8). Phortress (20 mg/kg, on days 1 and 8) was administered to CYP1A1/betaGAL reporter mice; tissues were examined macro- and microscopically. Toxicological and pharmacodynamic endpoints were examined in organs of rodents receiving Phortress (10 mg/kg or 20 mg/kg, on days 1 and 8). CYP1A1 expression and Phortress-derived DNA adducts were determined in lungs and livers (on days 11 and 36). RESULTS: No accumulation of Phortress was detected in murine plasma. beta-Galactosidase activity inferred Phortress-derived induction of cyp1a1 transcription in the livers of transgenic mice; no total body weight loss was encountered in these animals. However, a fall in lung:body weight and kidney:body weight ratios, raised serum alkaline phosphatase levels and hepatic histopathological disturbances in animals receiving 20 mg/kg Phortress indicate organ sites of potential toxicity. CYP1A1 protein was induced transiently in the lungs of both species and in the livers of rats. Elimination of hepatic DNA adducts and rat pulmonary adducts was evident; however, murine pulmonary adducts persisted. CONCLUSION: Rodent preclinical toxicology established that mice represent the more sensitive rodent species, resolving a maximum tolerated dose of 10 mg/kg Phortress.

DNA damage and cell cycle arrest induced by 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) is attenuated in aryl hydrocarbon receptor deficient MCF-7 cells.

The fluorinated benzothiazole analogue 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) is a novel agent with potent and selective antitumour properties and, in the form of its L-lysylamide prodrug Phortress (NSC 710305), is a current candidate for early phase clinical studies. Previous findings have indicated that cytochrome P450 1A1 (CYP1A1) may play a role in the antitumour activity of molecules in the benzothiazole series including the nonfluorinated parent compound 2-(4-amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495) (Kashiyama et al, 1999; Chua et al, 2000; Loaiza-Pérez et al, 2002). In this study, we assessed and verified that a fully functional aryl hydrocarbon receptor (AhR) signalling pathway is a necessary requisite for the induction of efficient cytotoxicity by 5F 203 in MCF-7 wild-type sensitive cells. Drug exposure caused MCF-7 sensitive cells to arrest in G(1) and S phase, and induced DNA adduct formation, in contrast to AhR-deficient AH(R100) variant MCF-7 cells. In sensitive MCF-7 cells, induction of CYP1A1 and CYP1B1 transcription (measured by luciferase reporter assay and real-time reverse transcriptase-polymerase chain reaction (RT-PCR)), and 7-ethoxyresorufin-O-deethylase (EROD) activity was demonstrated, following treatment with 5F 203. In contrast, in resistant AH(R100) cells, drug treatment did not affect CYP1A1 and CYP1B1 transcription and EROD activity. Furthermore, AH(R100) cells failed to produce either protein/DNA complexes on the xenobiotic responsive element (XRE) sequence of CYP1A1 promoter (measured by electrophoretic mobility shift assay) or DNA adducts. The data confirm that activation of the AhR signalling pathway is an important feature of the antitumour activity of 5F 203.

Antitumour 2-(4-aminophenyl)benzothiazoles generate DNA adducts in sensitive tumour cells in vitro and in vivo.

2-(4-Aminophenyl)benzothiazoles represent a potent and highly selective class of antitumour agent. In vitro, sensitive carcinoma cells deplete 2-(4-aminophenyl)benzothiazoles from nutrient media; cytochrome P450 1A1 activity, critical for execution of antitumour activity, and protein expression are powerfully induced. 2-(4-Amino-3-methylphenyl)benzothiazole-derived covalent binding to cytochrome P450 1A1 is reduced by glutathione, suggesting 1A1-dependent production of a reactive electrophilic species. In vitro, 2-(4-aminophenyl)benzothiazole-generated DNA adducts form in sensitive tumour cells only. At concentrations >100 nM, adducts were detected in DNA of MCF-7 cells treated with 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203). 5F 203 (1 microM) led to the formation of one major and a number of minor adducts. However, treatment of cells with 10 microM 5F 203 resulted in the emergence of a new dominant adduct. Adducts accumulated steadily within DNA of MCF-7 cells exposed to 1 microM 5F 203 between 2 and 24 h. Concentrations of the lysylamide prodrug of 5F 203 (Phortress) > or = 100 nM generated adducts in the DNA of sensitive MCF-7 and IGROV-1 ovarian cells. At 1 microM, one major Phortress-derived DNA adduct was detected in these two sensitive phenotypes; 10 microM Phortress led to the emergence of an additional major adduct detected in the DNA of MCF-7 cells. Inherently resistant MDA-MB-435 breast carcinoma cells incurred no DNA damage upon exposure to Phortress (< or = 10 microM, 24 h). In vivo, DNA adducts accumulated within sensitive ovarian IGROV-1 and breast MCF-7 xenografts 24 h after treatment of mice with Phortress (20 mg kg(-1)). Moreover, Phortress-derived DNA adduct generation distinguished sensitive MCF-7 tumours from inherently resistant MDA-MB-435 xenografts implanted in opposite flanks of the same mouse.