TP53 transcription factor for the NEDD9/HEF1/Cas-L gene: potential targets in Non-Small Cell Lung Cancer treatment

Abstract

Lung cancer is a serious public health problem. Although there has been significant progress in chemotherapy, non-small cell lung cancer is still resistant to current treatments, primarily because of the slow rate of cell development. It is thus important to find new molecules directed against targets other than proliferation agents. Considering the high proportion of mutant proteins in tumor cells, and the high rate of mutation of the TP53 gene in all cancers, and in NSCLC in particular, this gene is a perfect target. Certain new molecules have been shown to restore the activity of mutated p53 protein, for example PRIMA-1, which reactivates the His273 mutant p53. In a previous study, we presented triazine A190, a molecule with a cytostatic activity that blocks cells in the G1 phase and induces apoptosis. Here, we show that A190 not only restores mutant p53 activity, but also induces an overexpression of the NEDD9 gene, leading to apoptotic death. These findings might offer hope for the development of new targeted therapies, specific to tumor cells, which spare healthy cells.

Figure 1

(A) Expression of NEDD9 tested by qRT-PCR on synchronized NSCLC-N6-L16 cells treated or not with A190 at IC50 at 10, 15, 30 and 45 hours. (B) Expression of NEDD9 (a and c) or TP53 (b and d) tested by qRT-PCR on synchronized NSCLC-N6-L16 cells (a and b) or synchronized A549 cells (c and d) treated or not with A190 at IC50 from 15 to 17 hours. The results are expressed as a ratio of mRNA quantity of the genes tested and of β-actin (control gene). The values are mean ± S.D. (n = 8 for each group). (* p < 0.002 - ** p < 0.001 - ***p < 0.0002).

Figure 2

Flow cytometry analysis of the comparative effects of different cytostatic molecules on synchronized NSCLC-N6-L16 cells treated at IC50 for 16 hours (a: total cells b: non-apoptotic cells c: apoptotic cells).

Figure 3

Flow cytometry analysis of the comparative effects of different cytostatic molecules on synchronized A549 cells treated at IC50 for 16 hours (a: total cells b: non-apoptotic cells c: apoptotic cells).

Figure 4

Observation of the degree of fixation of p53 to DNA in NSCLC-N6-L16 or A549 cells treated or not with PRIMA-1 or A190 at IC50. The values are mean ± S.D. from triplicate samples. (* p < 0.006 - ** p < 0.0001).

Figure 5

(a) ChIP assay control on synchronized NSCLC-N6-L16 with Anti-RNA Polymerase II Antibody and ChIP Positive Control Primers (GAPDH promoter) (b) ChIP assays on synchronized NSCLC-N6-L16 treated or not with A190 with p53 antibody and NEDD9 specific primers.

Figure 6

Chemical structure of the triazine A190.