Poster Presentation 35th Lorne Cancer Conference 2023

Understanding the relationship between FOXL2C134W and SMADs in driving adult granulosa cell tumour pathogenesis (#158)

Teharn Hegarty 1 2 , Yizhou Yao 1 , Ying Wang 3 , Daniel J. Bernard 3 4 , Hari Sivaraman 5 , Wilson Wong 5 , Peter J. Fuller 1 , Simon Chu 1
  1. Hormone Cancer Therapeutics Laboratory , Hudson Institute of Medical Research, Melbourne, VIC, Australia
  2. Department of Molecular and Translational Science, Monash University, Melbourne, Victoria, Australia
  3. Department of Anatomy and Cell Biology, McGill University, Montreal, Québec, Canada
  4. Department of Pharmacology and Therapeutics, McGill University, Montreal, Québec, Canada
  5. Structural Biology of Inflammation and Cancer Laboratory, Hudson Institute of Medical Research, Melbourne, VIC, Australia

Introduction: Granulosa cell tumours (GCT) are a rare ovarian cancer, accounting for approximately 5% of ovarian cancer diagnoses.  There are two subtypes of GCT, adult GCT (95%) and juvenile GCT (5%). Approximately 97% of adult GCT (aGCT) carry a somatic mutation in the FOXL2 gene (C134W). The FOXL2 gene encodes for a winged helix/forkhead transcription factor, which regulates genes essential for the maintenance of ovarian identity.

The exact role of the FOXL2C134W mutation remains unresolved. Studies have implicated the TGF-beta/activin/SMAD signalling pathway in increasing aGCT cell proliferation. Weis-Banke et al1 showed that the FOXL2C134W mutation results in enhanced binding of FOXL2 to SMAD2/3 and SMAD4, with the FOXL2C134W/SMAD complex binding to novel DNA motifs associated with oncogenesis. We hypothesise that SMADs drive FOXL2C134W-mediated transactivation to promote expression of TGF-beta signalling pathway in aGCT pathogenesis.

Methods & Results: To assess functional consequences of the FOXL2C134W mutation, we co-transfected FOXL2WT or FOXL2C134W into HEK293 cells. We showed that SMAD3 increased FOXL2wt and FOXL2C134W expression as determined by Western blot analysis compared to when SMAD3 was absent. The murine FSHβ promoter-reporter is known to be synergistically activated by SMAD3 and FOXL2wt. In both HEK293 and the aGCT-derived KGN cell line, we observed that increased FSHβ promoter-reporter transactivation occurred with FOXL2C134W with SMAD3 co-expression compared to FOXL2wt. This was further increased when co-expressed with an activated form of the activin type I receptor, ALK4, which phosphorylates SMAD3.

Conclusion: The FOXL2C134W mutation not only displays increased expression when co-transfected with SMAD3, but also results in increased TGF-beta/activin/SMAD signalling, supporting an oncogenic (gain of function) role for this mutation to the pathogenesis of aGCT. The uniqueness of the FOXL2C134W mutation makes it an attractive therapeutic target for GCT. We are currently determining the nature of the SMAD/FOXL2C134W interaction in the GCT context, with the next steps to elucidate the three-dimensional structure of the FOXL2C134W/SMAD complex to identify potential “druggable” regions.

  1. Banke, S. E., Lerdrup, M., Kleine-Kohlbrecher, D., Mohammad, F., Sidoli, S., Jensen, O.N., Yanase, T., Nakamura, T., Iwase, A., Stylianou, A., Abu-Rustum, N.R., Aghajanian, C., Soslow, R., Paula, A.D.C., Koche, R.P., Weigelt, B., & Christensen, J. (2020). Mutant FOXL2C134W Hijacks SMAD4 and SMAD2/3 to Drive Adult Granulosa Cell Tumors. Genome and Epigenome 80(17), 3466-3479.