Identification of WEE1 as a target to make AKT inhibition more effective in melanoma

Abstract

AKT3 is one of the major therapeutic targets in melanoma but clinically targeting AKT3 alone seems to be an ineffective therapeutic approach. To identify unique strategies to enhance the efficacy of targeting AKT3, a screen was undertaken where AKT3 was co-targeted with a panel of kinases important in melanoma development. The screen identified WEE1 as the most potent target that when inhibited along with AKT3 would enhance the efficacy of targeting AKT3 in melanoma. RNAi mediated inhibition of AKT3 and WEE1 synergistically inhibited the viability of melanoma cells leading to a 65-75% decrease in tumor development. This approach was effective by mechanistically modulating pathways associated with the transcription factors p53 and FOXM1. Simultaneously regulating the activity of these two transcriptionally driven pathways, cooperatively deregulated cell cycle control and DNA damage repair to synergistically kill melanoma cells. This study uniquely identifies a potential approach to improve the efficacy of targeting AKT3 in melanoma.

Keywords: AKT3; Melanoma; WEE1; synergy.

Figure 1.

Targeting AKT3 and WEE1 synergistically inhibited melanoma cell growth. (A) and B, Dose-response curves of siRNA targeting AKT3 or WEE1 were undertaken by nucleofecting increasing amounts of siRNA targeting WEE1 and AKT3 followed by measurement of cellular viability after 3 d of growth in serum-free medium. For combination analysis, 50 or 100 picomoles of AKT3 were combined with increasing amounts of WEE1 siRNA. (C) and (D), The combination index (CI) analysis showed synergism between AKT3 and WEE1 when targeted together. Columns; mean (n = 6–8); error bars, SEM. (E) and (F), siRNAs targeting AKT3 alone or in combination with increasing amounts of WEE1 siRNA were introduced into melanoma cells via nucleofection and protein levels were measured 2 d later. ERK2 served as a control for equal protein loading. Western blot analysis was reproduced at least twice.

Figure 2.

Targeting of AKT3 and WEE1 inhibited melanoma tumor growth. Targeting AKT3 and WEE1 synergistically inhibited melanoma tumor growth in vivo. UACC 903 (2A) and 1205 Lu (2B) cells were nucleofected with AKT3 and WEE1 siRNA alone or in combination and 48 hours later, viable cells were s.c. injected into left and right flanks of nude mice. Cells transfected with scrambled siRNA were used as a control. Developing tumors were measured on alternate days for 3 weeks. Significance was measured by one-way analysis of variance, followed by the post hoc test, *P < 0.05, **P < 0.01, ***P < 0.01. Error bars show SEM. Data were obtained from duplicate experiments with 3 mice per group, containing 2 tumors per mouse. C. Western blot analysis showing knockdown of AKT3 and WEE1 in tumor lysates of UACC 903 xenografts harvested at day 9. ERK2 served as a control for equal protein loading. (D) and E, Analysis of size and time matched tumors from animals injected with UACC 903 melanoma cells transfected with scrambled siRNA controls or siRNA to AKT3, WEE1 or AKT3, and WEE1. Tumor sections were immunostained for Ki-67 (2D) or TUNEL (2E) to measure the proliferation and apoptosis, respectively. Bar graphs show the fold change in Ki-67 or TUNEL-positive cells compared with the scrambled siRNA control. Data were obtained from 3 to 4 tumors with 4 to 5 fields averaged per tumor. Significance was measured by one-way analysis of variance, followed by the post hoc test, **P < 0.01, ***P < 0.001, NS; Non-significant. Columns, mean; error bars, SEM.

Figure 3.

Alteration in gene expression and protein levels through genome-wide RNA sequence and RPPA analysis respectively. A, Venn diagram showing significantly altered genes identified following RNA-Seq analysis of UACC 903 cells transfected with siRNAs targeting AKT3, WEE1 or both. B, Enrichment analysis of 40 genes significantly altered by the combination. C, RPPA array analysis showing some of the altered proteins in UACC 903 melanoma cells transfected with siAKT3, siWEE1 or the combination. In RNA-Seq experiments, significant genes were determined using the intensity difference test with a p-value threshold of 0.05 and Benjamini and Hochberg multiple testing correction.

Figure 4.

Targeting AKT3 and WEE1 increased p53 while reducing FOXM1 and CDK1 signaling. (A) and (B). Knockdown of WEE1 kinase led to a dose-dependent decrease in the phosphorylation of its substrate CDK1. Dose-dependent increase in the phosphorylation of H2AX was observed. Consequently, this resulted in increased p53, p21 as well as p27 levels, which are known to be inhibitory to cell proliferation. ERK2 served as a control for equal protein loading.

Figure 5.

Diagram showing the mechanism of synergism for co-targeting AKT and WEE1 signaling pathways. Inhibition of siWEE1 (1) suppresses inhibitory phosphorylation of CDK1 leading to early-G2/M progression. This leads DNA damage (2) and activates p53 signaling. p53 inhibits cell cycle progression by induction of p21, allowing DNA damage repair. If the DNA damage is not repairable, p53 induces apoptosis. However, in many cancer cells, apoptotic cascades are suppressed by oncogenic alterations. Over-activated AKT inhibits pro-apoptotic factors while inducing antiapoptotic factors (3). AKT signaling also enhances cell cycle progression by CyclinD1 mediated phosphorylation of RB (4) and inhibition of p27 (5). Furthermore, AKT phosphorylates and induces Polo-like kinase 1 (PLK1) (6), which in turn inhibits pro-apoptotic functions of p53 and its family members, p63 and p73 (7). In addition, PLK1 also induces FOXM1 activity and M-phase progression (8). Proteins that were validated by Western blotting are shown in bold.