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.

Adrenocortical carcinoma (ACC) is an aggressive cancer with a 5-year survival rate of 20-30%. Mitotane is the only approved drug for the treatment of patients with ACC. It often carries significant toxicities which result in the discontinuation of treatment. There are no approved second line therapies. The increased incidence of ACC in the cancer predisposition syndrome, Li-Fraumeni, suggests the involvement of the p53 pathway in ACC pathogenesis. Our analysis of the gene expression profiles of 19 ACC samples identified dysregulation of the G2/M transition and the activity of the p53 modulator, MDM2 as important in ACC pathogenesis. Polo-like kinase-1 (PLK1) is involved in the G2/M transition and acts to promote MDM2 activity through its phosphorylation. We observed that PLK1 inhibition by siRNA results in up to a 70% reduction in viability in the ACC cell lines SW-13 and H295R. Therefore, we studied PLK1 inhibition as a potential therapeutic strategy. We used the small molecule inhibitor, BI-2536, to inhibit PLK1 function in SW-13 and H295R. Drug-dose response curves demonstrated that both cell lines are sensitive to pharmacological inhibition of PLK1 (IC50 doses of 0.0094815 μM and 0.062805 μM for SW-13 and H295R, respectively.) Murine xenograft studies demonstrated that BI-2536 resulted in a statistically significant reduction in tumor growth in SW-13 but not H295R. Examination of p53 protein levels in the presence of the drug showed a dose-dependent reduction in p53 levels in SW-13, which carries a homozygous p53 mutation. The same was not true in H295R, which is p53 wild-type. To test the hypothesis that BI-2536 was decreasing mutant p53 levels by promoting its proteasomal degradation, both cell lines were treated with previously determined inhibitory concentrations of BI-2536, either alone or in combination with the proteasome inhibitor, MG132. Western blot analysis showed recovery of p53 protein when cells were concomitantly treated with MG132, supporting a role for BI-2536 as a regulator of proteasomal degradation. Both ACC cell lines are relatively insensitive to MDM2 inhibition by nutlin-3 (NCI60 GI50 values range from ∼4 uM to ∼2 uM versus ACC cell lines @13-15uM), possibly because PLK1 stimulates MDM2 and renders nutlin-3 ineffective. We therefore assessed the ability of BI-2536 to sensitize ACC cell lines to the effects of the MDM2 inhibitor. BI-2636 did show synergy with nutlin-3, shifting the IC50 in SW-13 from 19.78 μM to 6.45 μM and from 12.75 μM to 2.84 μM in H295R. These results demonstrate that inhibition of PLK1 alone or in combination with an MDM2 inhibitor warrants further investigation as a treatment for patients with ACC. Cells with mutant p53 are more sensitive to the growth inhibitory effects of BI-2536. In the context of wild-type p53, the combined inhibition of both PLK1 and MDM2 results in loss of viability. Further clinical development of PLK1 as a target should take p53 mutation status into account. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 978. doi:1538-7445.AM2012-978

Adrenocortical carcinoma (ACC) is a lethal tumor of the adrenal cortex with limited treatment options. The only possibility for cure is complete surgical resection, but 50-70% of patients present with metastatic disease, precluding curative surgery. The outcome for patients with ACC has remained unchanged in the past 25 years, with the overall 5-year survival of patients undergoing surgical resection being ∼40%. Patients that present with advanced disease fare considerably worse with 5-year survival rates of 10-20%. The standard chemotherapy regimen of mitotane (the only approved agent), etoposide, doxorubicin, and cisplatin, has a response rate of 23.2%. A better understanding of pathobiology is needed to improve the treatment results for this disease. Previous genomic studies combined with expression microarray studies have implicated lack of H19 expression coupled with IGF2 over-expression, beta-catenin activation, and loss of p53 pathway function as important events in ACC, but no single alteration has been seen in all ACC. We used a combination of whole genome sequencing, exome sequencing, and RNA-sequencing on three tumor/normal pairs from patients. Using data from spectral karyotyping of the two ACC cell lines, SW-13 and NCI-H295R, we mined the sequencing data for structural rearrangements in regions of common breakpoints. We identified a novel recurrent translocation, t(4;8)(p16.2;p23.1) in all three tumors that was not present in the normal sequence. Using fluorescent in situ hybridization (FISH) we validated the translocation in the three tumors and both SW-13 and H295R. An additional five of five ACC scored positive for the translocation, as did three malignant non-ACC tumors (malignant pheochromocytoma, pancreatic neuroendocrine tumor, and carcinoid) suggesting that this finding has implications beyond ACC. The recurrent translocation t(4;8) (p16.2; p23.1) involves two genes of unknown function, BC042823 (uc003gho.2, IMAGE:5275587) on chromosome 4 and BC030294 (ucoo3wrb.1, IMAGE:5396854) on chromosome 8. RNA- sequencing data and RT-PCR with primers designed to both derivative chromosomes revealed transcription from both derivative chromosomes. The cDNA products were sequenced by Sanger sequencing and show partial homology to a non-coding RNA, LOC100506990, at chr8:12,338,893-12,452,929. RNA-sequencing data revealed this locus is highly transcribed in normal adrenal gland but reduced in expression in the three ACCs. In contrast to other recurrent translocations previously found in other cancers, this translocation is only present in 18-30% of the tumor nuclei scored by FISH, suggesting that it is a necessary alteration in malignant progression, but underscoring the question of how it might cooperate with events in other sub-clones to initiate tumor formation and progression. Citation Format: Michael J. Demeure, Kaiti Schwartz, Aditi Bapat, Claire Linnehan, Hollie Benson, Rebecca Reiman, Lori Phillips, Christophe Legendre, Raghu Metpally, Ahmet Kurdoglu, David Craig, John Carpten, Winnie S. Liang, Waibhav Tembe, Kimberly J. Bussey. Whole-genome and RNA sequencing identify a novel recurrent translocation in adrenocortical carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1781. doi:10.1158/1538-7445.AM2013-1781