Anti-Proliferative and Anti-Steroidogenic Effect of TCF7/Beta-Catenin Complex Inhibition in Adrenocortical Tumor Cells

Program: Abstracts - Orals, Poster Previews, and Posters
Session: FRI 330-350-Tumorigenesis, Metastasis, and Therapies for Cancer
Basic/Translational
Friday, March 6, 2015: 1:00 PM-3:00 PM
Hall D-F, Tumor Biology (San Diego Convention Center)

Poster Board FRI-339
Leticia Ferro Leal, MSc1, Ana Carolina Bueno, PhD.1, Debora Cristiane Gomes, MD, PhD2, Rafael Haikal Abduch1, Danila Akemi Arahata1, Rogério Lenotti Zuliani1, Margaret De Castro, MD, PhD1 and Sonir Roberto Rauber Antonini, MD. PhD.1
1Ribeirao Preto Medical School - University of Sao Paulo, Ribeirao Preto, Brazil, 2School of Medicine of Federal University of Uberlandia, Uberlandia MG, Brazil
Background: β-catenin mutations and/or Wnt/β-catenin pathway abnormal activation are common in adrenocortical tumors (ACTs). PNU-74654 is a non-FDA approved drug that can acts as Tcf/β-catenin complex antagonist.

Objective: to investigate the in vitro effects of PNU-74654 on β-catenin-dependent transcription, adrenal steroidogenesis and cell viability in adrenal (NCI-H295) and non-adrenal (HeLa) cell lines.

Methods: NCI-H295 and HeLa cell lines were treated with vehicle (DMSO) and PNU-74654 (5, 10, 50, 100 and 200μM) for 24-96h (NCI-H295) or 48h (HeLa) after treatment to evaluate cell viability (MTS-based proliferation assay), β-catenin expression and localization (western blot and immunofluorescence), mRNA expression of CTNNB1, AXIN2, TCF7, SF1 and CYP21A2(qPCR), and adrenal steroids production (radioimmunoassay).

Results: the inhibition of Tcf/β-catenin complex impaired cell proliferation in a dose-dependent manner. NCI-H295 cell viability decreased by 22%, 27%, 50 and 97% at 10, 50, 100 and 200μM PNU-74654 (p<0.0001), respectively, 96h after treatment. Instead, HeLa cell viability was not affected after treatment by PNU-74654, indicating that this drug-like acts specifically in cells/tissues in which Wnt pathway is activated. Immunofluorescence showed markedly cytotoxic effect as well as reduction of nuclear and cytoplasmic β-catenin expression 48h after treatment of PNU-74654 at 10, 50 and 100μM. β-catenin was also decreased 48 and 96h after PNU-74654 treatment as shown by western blot. In line, PNU-74654 treatment decreased CTNNB1 mRNA expression at 50 and 100μM (p=0.04 and p=0.0034, respectively), but did not affect TCF7 mRNA expression. β-catenin target genes, AXIN2 and CCND1, presented higher mRNA expression at 50μM 48h after treatment (p=0.01 and p=0.0048, respectively). Indeed, PNU-74654 treatment at 10, 50 and 100μM impaired steroidogenesis as shown by decrease in mRNA expression of CYP21A2 (12%, 63% and 88%, respectively; p<0.001) and SF1 (20%, 52% and 66%, respectively; p<0.001). In agreement, cortisol, testosterone, androstenedione and SDHEA secretion was reduced 24 and 48h after treatment with PNU-74654 at 50, 100 and 200μM. Interestingly, adrenal steroidogenesis was impaired already at 24h, before cell viability reduction, showing a potential direct effect on adrenal steroids secretion.

Conclusion: In NCI-H295 adrenal cell line, but not in HeLa cell line, PNU-74654, a TCF7/β-catenin inhibitor, impaired cell proliferation, β-catenin expression and nuclear localization, and adrenal steroids secretion. Thus, Tcf/β-catenin complex inhibitors might become a new target approach for ACTs showing Wnt pathway activation.

Nothing to Disclose: LFL, ACB, DCG, RHA, DAA, RLZ, MD, SRR

*Please take note of The Endocrine Society's News Embargo Policy at https://www.endocrine.org/news-room/endo-annual-meeting

Sources of Research Support: Sao Paulo Research Foundation (FAPESP) grants 2011/13807-4 and 2011/10512-3 and CNPq-Brazil Grant 474273/2011-3