Dataset Information

Silencing mutated ?-catenin inhibits cell proliferation and stimulates apoptosis in the adrenocortical cancer cell line H295R.

ABSTRACT:

Context

Adrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine neoplasm, with limited therapeutic options. Activating ?-catenin somatic mutations are found in ACC and have been associated with a poor clinical outcome. In fact, activation of the Wnt/?-catenin signaling pathway seems to play a major role in ACC aggressiveness, and might, thus, represent a promising therapeutic target.

Objective

Similar to patient tumor specimen the H295 cell line derived from an ACC harbors a natural activating ?-catenin mutation. We herein assess the in vitro and in vivo effect of ?-catenin inactivation using a doxycyclin (dox) inducible shRNA plasmid in H295R adrenocortical cancer cells line (clone named sh?).

Results

Following dox treatment a profound reduction in ?-catenin expression was detectable in sh? clones in comparison to control clones (Ctr). Accordingly, we observed a decrease in Wnt/?catenin-dependent luciferase reporter activity as well as a decreased expression of AXIN2 representing an endogenous ?-catenin target gene. Concomitantly, ?-catenin silencing resulted in a decreased cell proliferation, cell cycle alterations with cell accumulation in the G1 phase and increased apoptosis in vitro. In vivo, on established tumor xenografts in athymic nude mice, 9 days of ?-catenin silencing resulted in a significant reduction of CTNNB1 and AXIN2 expression. Moreover, continous ?-catenin silencing, starting 3 days after tumor cell inoculation, was associated with a complete absence of tumor growth in the sh? group while tumors were present in all animals of the control group.

Conclusion

In summary, these experiments provide evidences that Wnt/?-catenin pathway inhibition in ACC is a promising therapeutic target.

SUBMITTER: Gaujoux S

PROVIDER: S-EPMC3567123 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Silencing mutated β-catenin inhibits cell proliferation and stimulates apoptosis in the adrenocortical cancer cell line H295R.

Gaujoux Sébastien S Hantel Constanze C Launay Pierre P Bonnet Stéphane S Perlemoine Karine K Lefèvre Lucile L Guillaud-Bataille Marine M Beuschlein Felix F Tissier Frédérique F Bertherat Jérôme J Rizk-Rabin Marthe M Ragazzon Bruno B

PloS one 20130207 2

<h4>Context</h4>Adrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine neoplasm, with limited therapeutic options. Activating β-catenin somatic mutations are found in ACC and have been associated with a poor clinical outcome. In fact, activation of the Wnt/β-catenin signaling pathway seems to play a major role in ACC aggressiveness, and might, thus, represent a promising therapeutic target.<h4>Objective</h4>Similar to patient tumor specimen the H295 cell line derived from an AC ...[more]

PMID: 23409032

Similar Datasets

Project description:Adropin is a multifunctional peptide hormone encoded by the ENHO (energy homeostasis associated) gene. It plays a role in mechanisms related to increased adiposity, insulin resistance, as well as glucose, and lipid metabolism. The low adropin levels are strongly associated with obesity independent insulin resistance. On the other hand, overexpression or exogenous administration of adropin improves glucose homeostasis. The multidirectional, adropin-related effects associated with the regulation of metabolism in humans also appear to be attributable to the effects of this peptide on the activity of various elements of the endocrine system including adrenal cortex. Therefore, the main purpose of the present study was to investigate the effect of adropin on proliferation and secretory activity in the human HAC15 adrenal carcinoma cell line. In this study, we obtained several highly interesting findings. First, GPR19, the main candidate sensitizer of adrenocortical cells to adropin, was expressed in HAC15 cells. Moreover, GPR19 expression was relatively stable and not regulated by ACTH, forskolin, or adropin itself. Our findings also suggest that adropin has the capacity to decrease expression levels of steroidogenic genes such as steroidogenic acute regulatory protein (StAR) and CYP11A1, which then led to a statistically significant inhibition in cortisol and aldosterone biosynthesis and secretion. Based on whole transcriptome study and research involving transforming growth factor (TGF)-β type I receptor kinase inhibitor we demonstrated that attenuation of steroidogenesis caused by adropin is mediated by the TGF-β signaling pathway likely to act through transactivation mechanism. We found that HAC15 cells treated with adropin presented significantly higher proliferation levels than untreated cells. Using specific intracellular inhibitors, we showed that adropin stimulate proliferation via ERK1/2 and AKT dependent signaling pathways. We have also demonstrated that expression of GPR19 is elevated in adrenocortical carcinoma in relation to normal adrenal glands. High level of GPR19 expression in adrenocortical carcinoma may constitute a negative prognostic factor of disease progression.

Project description:In order to investigate genes regulated by Wnt/Beta-catenin-signaling in immortalized mouse adrenocortical cells, we treated a pair of ATCL7 cell cultures, one with BIO, a small molecule mimicking Wnt/Beta-catenin-signaling, the other with a control treatment. We repeated this 3 additional times resulting in 4 pairs of samples. The Wnt/beta-catenin pathway is not basally active in ATCL7 cells, nor do these cells appear to contain any mutations in the Wnt/Beta-catenin pathway. ATCL7 cells were grown under standard conditions at 37°C in a humidified incubator containing 5% CO2. 250,000 ATCL7 cells per sample were treated with 0.5uM BIO (6-Bromoindirubin-3'-oxime) or 0.01% DMSO (v/v) for 24 hours, in DMEM:F12 growth media containing 100U/mL pencillin/streptomycin, 1X insulin-transferrin-selenium-X, 0.025% fetal bovine serum and 0.025% horse serum. Cells were harvested and RNA was extracted using an RNeasy Plus Mini Kit (Qiagen). Biotinylated cDNA were prepared according to the Ambion WT kit protocol from 250 ng total RNA (GeneAtlas™ WT Expression Kit User Manual P/N 702935 Rev. 3). We assayed the targets with Affymetrix Mouse Gene ST 1.1 strip arrays. We modeled the data using paired T-tests for each probe-set. We also supply a supplementary file holding the data and some statistical analysis, as well as probe-set annotation that we used at that time (users may wish to obtain new annotation though). We analyzed only 28944 probe-sets with category "main", "---", and "flmrna->unmapped" according to Affymetrix annotation. ATCL7 cells were grown under standard growth conditions at 37°C in a humidified incubator containing 5% CO2. 250,000 ATCL7 cells per sample were treated for 24 hours with 0.5uM BIO (6-Bromoindirubin-3'-oxime) or 0.01% DMSO (v/v) in growth media containing 100U/mL pencillin/streptomycin, 1X insulin-transferrin-selenium-X, 0.025% fetal bovine serum and 0.025% horse serum. Cells were harvested and RNA was extracted using an RNeasy Plus Mini Kit (Qiagen). Biotinylated cDNA were prepared according to the Ambion WT kit protocol from 250 ng total RNA (GeneAtlas™ WT Expression Kit User Manual P/N 702935 Rev. 3). We assayed the targets with Affymetrix Mouse Gene ST 1.1 strip arrays.