ZCCHC10 suppresses lung cancer progression and cisplatin resistance by attenuating MDM2-mediated p53 ubiquitination and degradation.

The activation of p53 tumor suppressor is essential for preventing abnormal cell proliferation and carcinogenesis. ZCCHC10 was previously identified as a potential p53-interacting partner in a yeast two-hybrid screen, but the interaction in cells and its subsequent influence on p53 activity and cancer development have not been investigated. In this paper, we demonstrate that ZCCHC10 expression levels are statistically lower in lung adenocarcinoma tissues than the corresponding adjacent noncancerous tissues, and decreased expression of ZCCHC10 mRNA predicts poorer survival of the patients. Ectopic expression of ZCCHC10 in lung cancer cells harboring wild-type p53 dramatically suppresses cell proliferation, colony formation, migration, invasion and cisplatin resistance in vitro, as well as tumor growth and metastasis in vivo. Conversely, knockdown of ZCCHC10 exerts opposite effects in the normal lung cell Beas-2b. However, ZCCHC10 has no influence on the biological behaviors of p53-null (H358) or p53-mutant (H1437) lung cancer cells. Mechanistically, ZCCHC10 binds and stabilizes p53 by disrupting the interaction between p53 and MDM2. The p53 inhibitor pifithrin-α attenuated the influences of ZCCHC10 overexpression on p53 pathway, cell cycle, apoptosis, and epithelial-mesenchymal transition, whereas the p53 activator Nutlin3 could reverse the effects of ZCCHC10 knockdown. Collectively, our results indicate that ZCCHC10 exerts its tumor-suppressive effects by stabilizing the p53 protein and can be used a potential prognostic marker and therapeutic target in lung adenocarcinoma.

The proteasome activator PA28γ, a negative regulator of p53, is transcriptionally up-regulated by p53.

PA28γ (also called REGγ, 11Sγ or PSME3) negatively regulates p53 activity by promoting its nuclear export and/or degradation. Here, using the RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) method, we identified the transcription start site of the PA28γ gene. Assessment with the luciferase assay demonstrated that the sequence -193 to +16 is the basal promoter. Three p53 binding sites were found within the PA28γ promoter utilizing a bioinformatics approach and were confirmed by chromatin immunoprecipitation and biotinylated DNA affinity precipitation experiments. The p53 protein promotes PA28γ transcription, and p53-stimulated transcription of PA28γ can be inhibited by PA28γ itself. Our results suggest that PA28γ and p53 form a negative feedback loop, which maintains the balance of p53 and PA28γ in cells.

AP-2ß enhances p53-mediated transcription of the αB-crystallin gene through stabilizing p53.

The αB-crystallin (CRYAB) is a member of the small heat shock protein family that can be induced by various stresses and pathological conditions. Aberrant expression of CRYAB has been shown to be associated with several neurological diseases and malignant neoplasms. To identify transcriptional regulators of CRYAB expression, we examined its promoter for binding sites of transcription factors and identified four potential AP-2 binding sites in addition to a p53 binding site reported previously. Although the CRYAB promoter contains four consensus binding sequences of AP-2 and can be activated by AP-2α either in the presence or absence of p53, the luciferase assay showed that AP-2ß alone does not regulate the activity of the CRYAB promoter in the absence of p53. However, in the presence of p53, AP-2ß can significantly increase the luciferase activities of both the CRYAB promoter and reporter vector pp53-TA-luc, which contains a p53-responsive element, but no AP-2 binding sites. These data suggest that AP-2ß enhances transactivation of p53 and regulates CRYAB transcription via p53. Further study demonstrated that AP-2ß interacts with p53 and augments its protein stability. Taken together, our results indicate that AP-2ß up-regulates the transcription of the CRYAB gene through stabilizing p53.

A miR-200b/200c/429-binding site polymorphism in the 3' untranslated region of the AP-2α gene is associated with cisplatin resistance.

The transcription factor AP-2α functions as a tumor suppressor by regulating various genes that are involved in cell proliferation and apoptosis. Chemotherapeutic drugs including cisplatin induce post-transcriptionally endogenous AP-2α, which contributes to chemosensitivity by enhancing therapy-induced apoptosis. microRNAs (miRNAs) miR-200b, miR-200c and miR-429 (miR-200b/200c/429) are up-regulated in endometrial and esophageal cancers, and their overexpression correlates with resistance to cisplatin treatment. Using computational programs, we predicted that the 3' untranslated region (UTR) of AP-2α gene contains a potential miRNA response element (MRE) for the miR-200b/200c/429 family, and the single nucleotide polymorphism (SNP) site rs1045385 (A or C allele) resided within the predicted MRE. Luciferase assays and Western blot analysis demonstrated that the miR-200b/200c/429 family recognized the MRE in the 3' UTR of AP-2α gene and negatively regulated the expression of endogenous AP-2α proteins. SNP rs1045385 A>C variation enhanced AP-2α expression by disrupting the binding of the miR-200b/200c/429 family to the 3' UTR of AP-2α. The effects of the two polymorphic variants on cisplatin sensitivity were determined by clonogenic assay. The overexpression of AP-2α with mutant 3' UTR (C allele) in the endometrial cancer cell line HEC-1A, which has high levels of endogenous miR-200b/200c/429 and low levels of AP-2α protein, significantly increased cisplatin sensitivity, but overexpression of A allele of AP-2α has no significant effects, compared with mock transfection. We concluded that miR-200b/200c/429 induced cisplatin resistance by repressing AP-2α expression in endometrial cancer cells. The SNP (rs1045385) A>C variation decreased the binding of miR-200b/200c/429 to the 3' UTR of AP-2α, which upregulated AP-2α protein expression and increased cisplatin sensitivity. Our results suggest that SNP (rs1045385) may be a potential prognostic marker for cisplatin treatment.