The Wnt signaling pathway plays an essential role in development and tissue homeostasis and is aberrantly activated in many human cancers(1). There are two main Wnt signaling pathways, the canonical Wnt pathway, involving regulation of B-catenin, and the non-canonical Wnt pathway, which mediates signaling through other effector molecules. While canonical Wnt has been extensively researched, non-canonical Wnt remails relatively unexplored. ZNRF3, an E3 ubiquitin ligase, negatively regulates Wnt signaling and targets Frizzled (FZDs) receptors for degradation. Since FZDs can be in complex with co-receptors that mediate canonical (e.g., LRP5/6) or non-canonical Wnt (e.g., ROR1/ROR2, or RYK), ZNRF3 has the potential to regulate both pathways(2,3). To study ZNRF3 and non-canonical Wnt, we are using the adrenal gland as a model. Located above the kidneys, it produces hormones to regulate stress, metabolism, and homeostasis(4). Wnt signaling is known to be critical for adrenal development and homeostasis(5), and ZNRF3 is frequently inactivated in adrenal cancer(6). Prior work in the lab has revealed that mouse adrenals with Znrf3 conditionally knocked out (cKO) display significant hyperplasia at 6 weeks of age(7). While this phenotype is highly dependent on Wnt ligand availability, it remains unclear if this phenotype requires canonical or non-canonical Wnt, or perhaps both. To examine a potential role of non-canonical Wnt in ZNRF3-dependent signaling, the lab measured expression of non-canonical co-receptors in adrenal glands of Znrf3 cKO mice. This revealed that RYK was more highly expressed as compared to the controls, suggesting it may be a ZNRF3 target and key mediator of non-canonical Wnt. To further examine the role of RYK in normal adrenal biology and in relation to ZNRF3, we generated a Ryk cKO mouse model, where the gene has been selectively inactivated in all adrenal cortex cells using Cre/lox technology, and a double knockout (dKO) model where both Znrf3 and Ryk are simultaneously inactivated. We have found a 35% and 27% reduction in adrenal size in male and female mice, respectively, in the Znrf3;Ryk dKOs as compared to Znrf3 cKOs. Using immunohistochemistry (IHC), we have found that while there is no significant difference in proliferation (p=0.55), there is a significant increase in myeloid cells in the dKO as compared to the Znrf3 cKO (p=0.021). Prior work has shown myeloid cells phagocytose damaged adrenal cortex cells(8), suggesting loss of Ryk may enhance immune-mediated cell clearance. Our next step is to perform RNA sequencing to determine differences in the transcriptome of the control adrenal versus the Ryk cKO, and in the Znrf3 cKO versus the Znrf3;Ryk dKO. Based on the signaling pathways that are changed, we will perform follow-up studies to functionally validate these results. We will also conduct further IHC analyses to investigate other possible explanations for the hyperplastic reduction. (1) MacDonald, B. T.; Tamai, K.; He, X. Wnt/β-Catenin Signaling: Components, Mechanisms, and Diseases. Developmental Cell 2009, 17 (1), 9-26. https://doi.org/10.1016/j.devcel.2009.06.016.
(2) Fradkin, L. G.; Dura, J.-M.; Noordermeer, J. N. Ryks: New Partners for Wnts in the Developing and Regenerating Nervous System. Trends in Neurosciences 2010, 33 (2), 84-92. https://doi.org/10.1016/j.tins.2009.11.005.
(3) Green, J.; Nusse, R.; van Amerongen, R. The Role of Ryk and Ror Receptor Tyrosine Kinases in Wnt Signal Transduction. Cold Spring Harb Perspect Biol 2014, 6 (2), a009175. https://doi.org/10.1101/cshperspect.a009175.
(4) Adrenal Glands, 2022.
(5) Berthon, A.; Martinez, A.; Bertherat, J.; Val, P. Wnt/β-Catenin Signalling in Adrenal Physiology and Tumour Development. Molecular and Cellular Endocrinology 2012, 351 (1), 87-95. https://doi.org/10.1016/j.mce.2011.09.009.
(6) Hao, H.-X.; Jiang, X.; Cong, F. Control of Wnt Receptor Turnover by R-Spondin-ZNRF3/RNF43 Signaling Module and Its Dysregulation in Cancer. Cancers 2016, 8 (6), 54. https://doi.org/10.3390/cancers8060054.
(7) Basham, K. J.; Rodriguez, S.; Turcu, A. F.; Lerario, A. M.; Logan, C. Y.; Rysztak, M. R.; Gomez-Sanchez, C. E.; Breault, D. T.; Koo, B.-K.; Clevers, H.; Nusse, R.; Val, P.; Hammer, G. D. A ZNRF3-Dependent Wnt/β-Catenin Signaling Gradient Is Required for Adrenal Homeostasis. Genes Dev 2019, 33 (3-4), 209-220. https://doi.org/10.1101/gad.317412.118.
(8) Warde, K. M.; Liu, L.; Smith, L. J.; Lohman, B. K.; Stubben, C. J.; Ekiz, H. A.; Ammer, J. L.; Converso-Baran, K.; Giordano, T. J.; Hammer, G. D.; Basham, K. J. Senescence-Induced Immune Remodeling Facilitates Metastatic Adrenal Cancer in a Sex-Dimorphic Manner; preprint; Cancer Biology, 2022. https://doi.org/10.1101/2022.04.29.488426.