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Targeting polo-like kinase 1, a regulator of p53, in the treatment of adrenocortical carcinoma

Background Adrenocortical carcinoma (ACC) is an aggressive cancer with a 5 year survival rate of 20–30 %. Various factors have been implicated in the pathogenesis of ACC including dysregulation of the G2/M transition and aberrant activity of p53 and MDM2. Polo-like kinase 1 (PLK-1) negatively modulates p53 functioning, promotes MDM2 activity through its phosphorylation, and is involved in the G2/M transition. Gene expression profiling of 44 ACC samples showed that increased expression of PLK-1 in 29 % of ACC. Consequently, we examined PLK-1’s role in the modulation of the p53 signaling pathway in adrenocortical cancer. Methods We used siRNA knock down PLK-1 and pharmacological inhibition of PLK-1 and MDM2 ACC cell lines SW-13 and H295R. We examined viability, protein expression, p53 transactivation, and induction of apoptosis. Results Knocking down expression of PLK-1 with siRNA or inhibition of PLK-1 by a small molecule inhibitor, BI-2536, resulted in a loss of viability of up to 70 % in the ACC cell lines H295R and SW-13. In xenograft models, BI-2536 demonstrated marked inhibition of growth of SW-13 with less inhibition of H295R. BI-2536 treatment resulted in a decrease in mutant p53 protein in SW-13 cells but had no effect on wild-type p53 protein levels in H295R cells. Additionally, inhibition of PLK-1 restored wild-type p53’s transactivation and apoptotic functions in H295R cells, while these functions of mutant p53 were restored only to a smaller extent. Furthermore, inhibition of MDM2 with nutlin-3 reduced the viability of both the ACC cells and also reactivated wild-type p53′s apoptotic function. Inhibition of PLK-1 sensitized the ACC cell lines to MDM2 inhibition and this dual inhibition resulted in an additive apoptotic response in H295R cells with wild-type p53. Conclusions These preclinical studies suggest that targeting p53 through PLK-1 is an attractive chemotherapy strategy warranting further investigation in adrenocortical cancer. Electronic supplementary material The online version of this article (doi:10.1186/s40169-015-0080-3) contains supplementary material, which is available to authorized users.

(GSE10297 and GSE19750). The data were filtered to include only normal adrenal samples without evidence of disease in the contralateral gland and ACC tumor sample from patients over 18 years of age.
Using the Expression File Creator in GenePattern [3], the data were normalized by gcRMA [4] with quantile normalization and background subtraction. Batch effects were minimized using COMBAT [5] with the non-parametric option, floored at 0.00001, and log2 transformed. Normalized expression data was extracted for the PLK1 (202240_at) and TP53 (201746_at) probe sets. MDM2 is represented by multiple probes, so we extracted probe sets 211832_s_at, 205386_s_at, 229711_s_at, and 217542_at. The geometric mean of these probe sets was computed for each sample. The relative fold change to the geometric mean of the corresponding normal samples for each study was computed.

Analysis for p53 mutation
Mutations of the coding regions of TP53 are uncommon in adult sporadic ACC. A total of 31 ACC and two ACC cell lines, SW-13 and H295R were analyzed for TP53 mutations through a combination of methods. We sequenced exons 1-11 of TP53 amplified from DNA extracted from bulk tumor (21 of 31) as well as from the aneuploid nuclei from flow sorted tumors (11 of the 31) [6]. Of the 31 ACC tumors, one failed sequencing. Four mutations were detected in 3 tumors (Supplemental Tables 1   and 2). Of note, none of the mutations detected have been reported in ACC previously. Because mutations in small tumor cell populations can be missed when sequencing from bulk material, we examined the 6 tumors that were sequenced from both bulk material and sorted aneuploid material for evidence of missed mutations. No additional mutations were detected. We detected no mutation of TP53 in H295R. SW-13 carried the previously documented H193Y mutation. These results are in line with the sequencing done by the Sanger Institute as part of the COSMIC Cell Lines Project [7].
The 11 tumors subject to flow-sorting were also examined for loss of heterozygosity (LOH) of p53 using the diploid cell population as the "normal" control. LOH occurred in 3 of 11 (27%) of the ACC tumors studied. No tumor with LOH also harbored a mutation. Although the SNP variant with a proline allele of codon 72 has been associated with an increased risk of ACC [8], we did not observe any difference in the proline allele frequency from that of the expected population frequency in our data.

Analysis of regulator of p53 activity
The low rate of mutations in p53, the lack of overlap of mutations with LOH, and a lack of an increase in the prevalence of a proline allele in codon 72 in our ACC samples suggests that disruption of the p53 pathway may involve regulators of p53. MDM2 is the predominant negative regulator of p53 activity. Previous studies have shown that conversion of T to G at position 309 generates a strong SP1 binding site and increases MDM2 expression [9,10]. Most our tumors did over-express MDM2 at the RNA level (Supplemental Table 3). Therefore, we genotyped SNP 309 in MDM2 in the ACC tumors. We did not observe a change in allele frequency from the expected population frequency, nor did we observe a significant increase MDM2 expression in tumors with a G allele compared to tumors homozygous for the T allele (Tables 1 and 3).