Conditional RNA interference in vivo to study mutant p53 oncogenic gain of function on tumor malignancy

Abstract

Mutant p53 proteins are thought to have acquired a "gain of function" (GOF) activity that mainly contributes to tumor aggressiveness. Previously we reported that constitutive downregulation of mutant p53 by RNA interference reduces the tumorigenicity of cancer cells in an animal model; however, effects of adaptation to long-term mutant p53 inhibition could not be excluded. To address this point, mimicking more physiological conditions, we now describe the establishment of a lentiviral-based system for conditional interference with mutant p53 expression. In vivo studies assessed the efficacy of conditional RNA interference in inhibiting gain of function activity of mutant p53 proteins by reducing tumor growth ability. Moreover by using this system, microarray data were validated in vitro and in vivo and putative mutant p53 target genes that may contribute to its gain of function effects in cancer were identified. Results are confirmatory that depletion of mutant p53 protein impacts on tumor malignancy and validated the inducible lentiviral-based system as an efficient tool to study the gain of function activity of human tumor derived p53 mutants.

Mutant p53-induced Up-regulation of Mitogen-activated Protein Kinase Kinase 3 Contributes to Gain of Function

“…Furthermore, RNA interference (RNAi) studies demonstrated that depletion of mutp53 renders cancer cells more sensitive to DNA-damaging chemotherapeutic agents in vitro (9,10) and reduces tumor malignancy both in vitro and in vivo (10). In agreement with these results, tumor growth delay studies, performed in the HT29 xenograft model, showed that conditional silencing of mutp53 does not only impact on tumor growth but leads to tumor architecture modifications, with consistent reduction in stromal invasion and tumor angiogenesis (11). At the molecular level, these GOF effects were shown to be linked to the ability of mutp53 to modulate the expression of several genes, such as MDR1 (12), c-MYC (13), CD95 (Fas/APO-1) (14), EGR1 (9), MSP/MST-1 (15), GEF-H1 (16), ID2 (17), GRO1 (18), PPARGC1A, FRMD5 (19), and ID4 (20), supporting the hypothesis that mutp53-specific transcriptional activity is required for at least some of the mutp53 GOF effects.…”

“…Viral Vectors-Lentiviral vectors were produced in HEK293T cells by transient transfection as described previously (11). Lentiviruses were harvested 48 h later, centrifuged for 5 min at 3,000 rpm, aliquoted, and stored at Ϫ80°C.…”

“…All cell cultures were supplemented with 10% fetal bovine serum (GIBCO/Invitrogen, Grand Island, NY), 2 mM L-glutamine (Invitrogen), 100 units/ml penicillin, and 100 g/ml streptomycin (Invitrogen). HEK293T packaging cell lines (11) were maintained in Dulbecco's modified Eagle's medium high glucose (GIBCO/Invitrogen) 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and 1ϫ nonessential amino acids (GIBCO/Invitrogen). All cell lines were maintained at 37°C in a humidified environment of 5% CO 2 .…”

“…Recent studies found that MAP2K3 up-regulation was involved in invasion and progression of gliomas and breast tumors (29). Our recent quantitative PCR validation of microarray data, from cells expressing endogenous mutp53 (HT29 and SKBR3) and xenograft tumors, indicated that MAP2K3 (also known as MKK3) is a mutp53 target gene (11). To unveil a possible role of MAP2K3 up-regulation in mutp53 GOF activity, we studied the molecular mechanisms through which different mutp53 proteins up-regulate MAP2K3 expression and the biologic effects of its up-regulation in different cell lines.…”

Vaccinia Virus-Mediated Expression of Transcription Factor Klf4 Enhances Oncolytic Virotherapy of Colorectal Cancer

“…On the contrary, R273H p53 has been shown to inhibit apoptosis through transcriptional activation of genes like NFKB2 or BCL2L1 [22,23]. In HT-29 cells infected for 24, 48, and 72 hours with GLV-1h68 and GLV-1h291, similar distributions of total live, early-and lateapoptotic cells were observed (Figure 3c).…”