Antitumoral effects of 9-cis retinoic acid in adrenocortical cancer

Abstract

The currently available medical treatment options of adrenocortical cancer (ACC) are limited. In our previous meta-analysis of adrenocortical tumor genomics data, ACC was associated with reduced retinoic acid production and retinoid X receptor-mediated signaling. Our objective has been to study the potential antitumoral effects of 9-cis retinoic acid (9-cisRA) on the ACC cell line NCI-H295R and in a xenograft model. Cell proliferation, hormone secretion, and gene expression have been studied in the NCI-H295R cell line. A complex bioinformatics approach involving pathway and network analysis has been performed. Selected genes have been validated by real-time qRT-PCR. Athymic nude mice xenografted with NCI-H295R have been used in a pilot in vivo xenograft model. 9-cisRA significantly decreased cell viability and steroid hormone secretion in a concentration- and time-dependent manner in the NCI-H295R cell line. Four major molecular pathways have been identified by the analysis of gene expression data. Ten genes have been successfully validated involved in: (1) steroid hormone secretion (HSD3B1, HSD3B2), (2) retinoic acid signaling (ABCA1, ABCG1, HMGCR), (3) cell-cycle damage (GADD45A, CCNE2, UHRF1), and the (4) immune response (MAP2K6, IL1R2). 9-cisRA appears to directly regulate the cell cycle by network analysis. 9-cisRA also reduced tumor growth in the in vivo xenograft model. In conclusion, 9-cisRA might represent a promising new candidate in the treatment of hormone-secreting adrenal tumors and adrenocortical cancer.

Fig. 1

Effects of 9-cisRA on cell viability assay (a), on cortisol (b), and DHEA (c) secretion in five different concentrations and three different treatment periods and the relative mRNA expression level of ABCA1 and ABCG1 RXR target genes relative to the housekeeping gene ZNF625 in one concentration and four different treatment times (d) in NCI-H295R cells (n = 3, mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 relative to control)

Fig. 2

Pathways with related gene expression patterns involved in the steroid hormone metabolism after 9-cisRA treatment in NCI-H295R cells (left sides of circular symbols representing gene expression) and investigated microarray studies in ACC (right sides of circular symbols representing gene expression)

Fig. 3

Pathways with related gene expression patterns involved in retinoic acid signaling after 9-cisRA treatment in NCI-H295R cells (left sides of circular symbols representing gene expression) and investigated microarray studies in ACC (right sides of circular symbols representing gene expression)

Fig. 4

Pathways with related gene expression patterns involved in cells cycle regulation (Ataxia Telangiectasia Mutated (ATM) Signaling, Growth arrest and DNA-damage-inducible (GADD45) Signaling) after 9-cisRA treatment in NCI-H295R cells (left sides of circular symbols representing gene expression) and investigated microarray studies in ACC (right sides of circular symbols representing gene expression)

Fig. 5

Results of qRT-PCR validation of eight selected genes relative to the housekeeping gene R3HDM2. Results are represented by ddCT (cycle threshold) compared to the control after three different 9-cisRA treatment in concentrations at 24-h treatment time (mean ± SD, *p < 0.05; **p < 0.01, ***p < 0.001 relative to control n = 6)

Fig. 6

Results of the in vivo xenograft studies. a Growth curve of tumors in control and 9-cisRA-treated mice. b Diagrams showing the normalized tumor volume at the end of treatment (day 28) in control and 9-cis retinoic acid-treated (9-cisRA) animals. Values are expressed as mean ± SE. c Differences in tumor weight on the day 28 of 9-cis retinoic acid treatment between control animals and mice exposed to 9-cisRA. Results are expressed as mean ± SE. d Representative pictures of H&E staining and Ki67 immunoreaction on tumors from control and 9-cis retinoic acid-treated nude mice. Note the alteration in histology of 9-cisRA-treated tumors, where tumor cells are arranged in cords, reminding one of the structure of normal adrenal cortex. Arrows point to dividing cells. Scale bars represent 50 and 25 μm for insets. e Diagrams displaying Ki67 index obtained by counting immunopositive cells on sections from control and 9-cisRA tumors. Results are expressed as mean ± SE, ***p < 0.001