Molecular roles of ZNRF3 on WNT signaling in adrenal cortex maintenance and cancer

The adrenal cortex is a critical mediator of the mammalian stress response through production of corticosteroid hormones and coordination of hypothalamic-pituitary-adrenal signaling responses.  Comprised of many layers of structurally and functionally differentiated cellular zones, discrete differences in the ability to synthesize, secrete, and respond to peptide hormone stimulation exist in different cellular compartments.  While the biochemistry and zonal distribution of steroid hormone production are well characterized, very little progress has been made in defining the contributions of particular cellular groups and the mechanisms underlying adrenal cell turnover and repopulation. The highly conserved Wnt signaling pathway regulates the critical processes of stem/progenitor cell renewal in multiple organ systems and several studies in the Hammer laboratory have implicated the Wnt/β-catenin pathway in adrenal development and homeostasis.  My initial project in the Hammer lab investigates the contribution of the newly identified Wnt signaling regulator, ZNRF3, to critical aspects of the adrenal progenitor cell biology, adrenal cortex maintenance, and cancer.
ZNRF3 and negative regulation of Wnt signaling.
Activation of the Wnt pathway is temporally and spatially controlled through tightly regulated negative and positive feedback loops. We and others have shown that disruption of the regulatory mechanisms resulting in excessive Wnt activation in adrenal tissue leads to the expansion of the progenitor cell compartment and ultimately to tumor formation (3). Conversely, age-dependent depletion of adrenocortical progenitor cells and organ failure is observed in aged mice harboring loss of function mutations (1). Thus, regulation of Wnt signaling level is an important aspect of adrenal homeostasis and cellular maintenance.
Ultimately, the dynamics of pathway activation or repression depend on the interplay between the regulatory components. One of such components is the control of the availability of the Fzd receptors at the cell membrane. Recent studies have shown that a delicate interplay between ZNRF3 and the LGR4/5 receptors is decisive for terminating Wnt pathway activation. In progenitor cell types, the ubiquitin-protein ligase activity of ZNRF3 actively attenuates Wnt signaling though ubiquitination and degradation of Wnt receptor complex components Fzd and LRP6. However, in the presence of R-spondins, the LGR4/5 ligands, ZNFR3 interacts with LRG4/5 and is removed from the membrane, keeping the Wnt pathway in an activated state (5, 6).   Thus, ZNRF3 has a critical role in limiting Wnt signaling in the stem cell compartment. Consistent with this model, conditional mouse knock-out of the ubiquitin ligases ZNRF3 and its homologous Wnt target, RNF43 in the gut epithelium, results in high levels of β-catenin activation and strong proliferative expansion of the intestinal stem cell niche, which ultimately leads to tumor formation (7). While ZNRF3 and R-spondins can interact with multiple frizzled receptors and LGRs, in the human adrenal, Wnt4, Wnt2b and Wnt5a serve as the most abundant ligands for the adrenal-expressed FRZ1, FRZ7, FRZ3, FRZ8 and associated LRP6 in the subcapsular cortex.  Enriched expression of RSPO3 and LGR4 complete the components of the Wnt receptor complex in the subcapsular adrenal cortex.  These initial findings support the study of ZNRF3 and its coregulators in adrenal progenitor homeostasis.

Wnt signaling over-activation in adrenocortical tumorigenesis
Gain of function (GOF) mutations of critical components of the Wnt signaling pathway have been implicated in many types of cancers including familial and sporadic adrenocortical carcinoma (ACC). The observation of activating CTNNB1 mutations in both benign and malignant tumors suggests that Wnt disruption is an early event in the tumorigenesis (4). The University of Michigan Endocrine Oncology Program has been the coordinating center for the NCI-sponsored adrenocortical carcinoma TCGA (The Cancer Genome Atlas) project. Recently, our preliminary data demonstrated that mutations affecting components of the Wnt pathway (CTNNB1, APC and the recently described ZNRF3) are present in ~40% of the ACC samples, being the most commonly affected pathway. A surprising ~20% of adrenocortical carcinoma samples examined presented with (LOF) mutation in ZNRF3. An additional ~15% of tumors contained gain of function (GOF) mutations in β-catenin and additionally highlighted the importance of Wnt pathway alterations for the pathogenesis of adrenal cancer. Therefore, ZRNF3 represents a new Wnt signaling pathway constituent with a significant role in adrenal cell regulation and therapeutic target in cancer.
Despite the evident importance of ZNRF3 for tumorigenesis, it’s the mechanistic role in the regulation of normal adrenal cortex homeostasis is unknown. The goals of my research are to define the role of ZNRF3 in adrenal homeostasis and tumorigeneis. Given that Wnt signaling preserves adrenal progenitor cells in an undifferentiated, and steroidogenically inactive state (2), we hypothesize:

 

1. ZNRF3 is a critical contributor to maintaining adrenocortical homeostasis.
2. Tumors that harbor ZNRF3 mutations are derived from progenitor cell populations. 
3. ZNRF3 LOF tumors will be exquisitely sensitive to pharmaceutical intervention targeting Wnt ligand – receptor interactions.

 

To test our hypothesis we will to assess ZNRF3 function in adrenal progenitor cells both in vivo and in vitro. The Hammer lab has recently characterized the Wnt-responsive adrenocortical cells as a heterogeous mix of SHH-producing progenitor cells (ShhLacZ reporter mice) and differentiated cells of the aldosterone-producing zG . We will utilize Tcf/Lef:H2B-GFP reporter mice to monitor and characterize the Wnt-responsive cells of the adrenal cortex under a variety of physiologic perturbations and pathologic conditions.  Additionally, we routinely use FACS sorting of these isolated cells to define novel Wnt-target genes that regulate the pluripotency of this progenitor population (2).  Furthermore, by reproducing ZNRF3 LOF mutations in these cell populations, we hope to assess tumorigenesis using mouse xenograft models.  Additional studies will include determining the adrenal-specific mechanism of ZNRF3 action in terms of interaction with LGR5 and RSPO3 and using small molecule inhibitors of this interaction to explore tumor sensitivity to Wnt ligand – receptor antagonists. 

It is clear that there is biological and therapeutic significance in further understanding regulatory modules controlling β-catenin activity and ZNRF3 is a promising new target. Together the proposed experiments will provide insights into the normal function of ZNRF3 and hopefully lead to improved understanding of tumorigenic transformation in adrenocortical carcinoma to aid in developing therapeutic interventions for ACC patients.

References

1. Kim, A.C., Reuter, A.L., Zubair, M., Else, T., Serecky, K., Bingham, N.C., Lavery, G.G., Parker, K.L. and Hammer, G.D. (2008) Targeted disruption of beta-catenin in Sf1-expressing cells impairs development and maintenance of the adrenal cortex. Development, 135, 2593-2602.
2. Walczak, E.M., Kuick, R., Finco, I., Bohin, N., Hrycaj, S.M., Wellik, D.M. and Hammer, G.D. (2014) Wnt signaling inhibits adrenal steroidogenesis by cell-autonomous and non-cell-autonomous mechanisms. Mol Endocrinol, 28, 1471-1486.
3. Heaton, J.H., Wood, M.A., Kim, A.C., Lima, L.O., Barlaskar, F.M., Almeida, M.Q., Fragoso, M.C., Kuick, R., Lerario, A.M., Simon, D.P. et al. (2012) Progression to adrenocortical tumorigenesis in mice and humans through insulin-like growth factor 2 and β-catenin. Am J Pathol, 181, 1017-1033.
4. Berthon, A., Sahut-Barnola, I., Lambert-Langlais, S., de Joussineau, C., Damon-Soubeyrand, C., Louiset, E., Taketo, M.M., Tissier, F., Bertherat, J., Lefrançois-Martinez, A.M. et al. (2010) Constitutive beta-catenin activation induces adrenal hyperplasia and promotes adrenal cancer development. Hum Mol Genet, 19, 1561-1576.
5. Peng, W.C., de Lau, W., Madoori, P.K., Forneris, F., Granneman, J.C., Clevers, H. and Gros, P. (2013) Structures of Wnt-antagonist ZNRF3 and its complex with R-spondin 1 and implications for signaling. PLoS One, 8, e83110.
6. de Lau, W., Peng, W.C., Gros, P. and Clevers, H. (2014) The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength. Genes Dev, 28, 305-316.
7. Koo, B.K., Spit, M., Jordens, I., Low, T.Y., Stange, D.E., van de Wetering, M., van Es, J.H., Mohammed, S., Heck, A.J., Maurice, M.M. et al. (2012) Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors. Nature, 488, 665-669.

 

Curriculum Vitae

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