DeltaNp63alpha confers tumor cell resistance to cisplatin through the AKT1 transcriptional regulation

Abstract

Strategies to address resistance to platin drugs are greatly needed in human epithelial cancers (e.g., ovarian, head/neck, and lung) where platins are used widely and resistance occurs commonly. We found that upon ΔNp63α overexpression, AKT1 and phospho-AKT1 levels are upregulated in cancer cells. Investigations using gel-shift, chromatin immunoprecipitation and functional reporter assays implicated ΔNp63α in positive regulation of AKT1 transcription. Importantly, we found that ΔNp63α, AKT1, and phospho-AKT levels are greater in 2008CI3 CDDP-resistant ovarian cancer cells than in 2008 CDDP-sensitive cells. siRNA-mediated knockdown of ΔNp63α expression dramatically decreased AKT1 expression, whereas knockdown of either ΔNp63α or AKT1 decreased cell proliferation and increased death of ovarian and head/neck cancer cells. Conversely, enforced expression of ΔNp63α increased cancer cell proliferation and reduced apoptosis. Together, our findings define a novel ΔNp63α-dependent regulatory mechanism for AKT1 expression and its role in chemotherapeutic resistance of ovarian and head/neck cancer cells.

Figure 1. ΔNp63α and AKT1 mRNA and protein levels in JHU-022 cells after CDDP exposure

Levels of endogenous ΔNp63α, AKT1 and p-AKT1 in JHU-022 cells with (a) increasing dose (0-10μM, 24h) and (b) increasing time (10μM, 0 to 24h) of CDDP treatment. (c). Levels of ΔNp63α, AKT1 and p-AKT1 after transfection with increasing concentrations (0-1.5μg) of ΔNp63α. (d). Levels of ΔNp63α, AKT1 and p-AKT1 in JHU-022 cells after transfection with siRNA against ΔNp63α, TAp63α and control. Left panels are immunoblotting with indicated antibodies. Right panels are semi-qRT-PCR. We used β-actin level as a loading control for immunoblotting, and GAPDH as a loading control for semi-qRT-PCR

Figure 2. Transcriptional regulation of ΔNp63α on AKT1 promoter

(a). JHU-022 cells were transfected with the pGL3B-AKT1-1400 promoter-driven luciferase construct, Renilla luciferase plasmid, and with or without pcCDNA-3.1, pcDNA3.1-HA-TAp63α or pcDNA3.1-HA-ΔNp63α. 48h post transfection of the Firefly luciferase activity was determined. (b). Gel-shift assay with the 6His-tagged ΔNp63α protein along with 4 DNA probes matching the AKT1 promoter sequence. (c). Dose-dependent binding of ΔNp63α to AKT1#3 probe (100 fmol) with increasing concentration of ΔNp63α (1μg, 2μg, 5μg and 10μg). (d). The ChIP assay with JHU-022 and H1299 cells, 72h after transfection with the ΔNp63α or TAp63α expression constructs or control vector. JHU-022 cells were also transfected with the ΔNp63α siRNA or control siRNA.

Figure 3. Effect of ΔNp63α on cell viability

(a) JHU-022 and H1299 cells after concentration-dependent transfection with ΔNp63α. (b). JHU-022 cells after transfection with the ΔNp63α, TAp63α and AKT1 constructs followed by 10μM CDDP treatment for 24h. (c). JHU-022 cells after transfection with the ΔNp63α, TAp63α, AKT1 or control siRNA followed by 10μM CDDP treatment for 24h. (d). JHU-022 cells after transfection with the ΔNp63α, TAp63α, AKT1 and control siRNA. 24h after siRNA transfection cells were transfected with or without 1μg of AKT1 construct. After 24h of AKT1 transfection, cells were treated with 10μM CDDP for 24h. Cell viability was evaluated by the (a-d) MTT, (b) Caspase-3 and TUNEL assays, and immunoblotting analysis of Caspase-3 and PARP1 cleavage. For various experiments, MTT values from control siRNA, empty vector, or untransfected cells were taken as 100%.

Figure 4. ΔNp63α and AKT1 levels affect CDDP resistance in sensitive/resistance ovarian cancer cells

(a). Levels of ΔNp63α, AKT1 and p-AKT1 in OV2008 and OV2008C13 cells tested by immunoblotting (left panel) and semi-qRT-PCR (right panel). Levels of ΔNp63α, AKT1 and p-AKT1 in OV2008 and OV2008C13 cells after transfection with siRNA against ΔNp63α, TAp63α and control (b, upper panels), with increasing concentrations of ΔNp63α (0-1.5μg) overexpression (b, lower panels), and after dose-dependent CDDP (0-5μM) exposure for 24h (c) tested by immunoblotting (left panel) and semi-qRT-PCR (right panel). (d). Binding of ΔNp63α to the AKT promoter in OV2008 and OV2008CI3 cells was analyzed by the ChIP assay. We used β-actin level as a loading control for immunoblotting, and GAPDH as a loading control for semi-qRT-PCR

Figure 5. ΔNp63α down regulation makes OV2008VI3 cell sensitive to CDDP exposure

(a-c). OV2008CI3 cells were transfected with siRNA to ΔNp63α, TAp63α, AKT1 and control for 72h followed by treatment with 10μM CDDP for 24h. (a). MTT assay. (b). Immunoblotting analysis of PARP1 cleavage. (c). Caspase-3 assay. (d). OV2008VCI3 cells were transfected with siRNA to ΔNp63α, TAp63α, AKT1 and control. After 72h cells were subjected to dose-dependent CDDP exposure for 24h followed by MTT assay. (d). Values from cells transfected with an empty vector (data not shown) were taken as 100%.

Figure 6. ΔNp63α overexpression renders OV2008 cell resistant to CDDP exposure

(a-c). OV2008 cells were transfected with the ΔNp63α, TAp63α and AKT1 expression constructs followed by 2.5μM CDDP treatment for 24h and used for (a) MTT assay, (b) immunoblotting analysis for PARP1 cleavage assay, and (c) caspase-3 assay. (d). OV2008 cells were transfected with the ΔNp63α, TAp63α and AKT1 expression constructs followed by dose-dependent CDDP exposure (0-25μM) for 24h and used for MTT assay. Values from cells transfected with an empty vector (data not shown) were taken as 100%.