Developmental biologists have postulated for years that many of the molecules that are essential for regulating embryonic development are also active in adults, where they participate in tissue homeostasis, in regenerative responses to acute injury, and in disease processes. Supporting this idea, Wnts belong to a family of secreted proteins that activate multiple receptor-mediated signal transduction pathways in both embryos and adults. For example, as a very junior faculty member I made the serendipitous observation that over-expressing Wnt1 in early Xenopus embryos stimulates the formation of a new Spemann gastrula organizer center, resulting in two-headed tadpoles. Investigating the biochemical and cellular processes that explain this dramatic phenotype enabled us to identify many of the core components of the evolutionarily conserved Wnt/ß-catenin pathway, and to identify their mechanisms of action.
As described below, the arc of research in my lab has evolved from the early days of examining Wnt signaling in development to a new focus on the roles of Wnt signaling in stem and progenitor cells in regenerative processes, the involvement of Wnt signaling in diseases, and the identification of new therapies based on modulating Wnt signaling. This change in emphasis has already yielded insights into disease mechanisms and identified new therapeutic avenues.
Wnt signaling, regeneration, and stem and progenitor cells
Through experiments in Xenopus, zebrafish, and mice we and others previously established that there is a striking requirement for Wnt/β-catenin signaling in regenerating tissues, and that enhancing signaling accelerates regeneration. Based on these initial studies we have elected to pursue a dual track approach to understanding the roles and control of stem and progenitor cells in regenerative processes. On the one hand, we have been focusing on human embryonic stem cells (hESCs and induced pluripotent stem cells (iPSCs). Our findings to date include the observation that human embryonic stem cells can only undergo self-renewal if Wnt signaling is qiescent, while activating signaling promotes differentiation.
The other approach we have taken is to study regenerative processes and progenitor cell populations in model organisms. For example, working with Chuck Murry (University of Washington) we have identified Wnt pathway modulators that play key roles in cardiac progenitor cells in vertebrates. In other studies, we have been working with Irwin Bernstein (Fred Hutchinson Cancer Research Center) and Gordon Keller (University of Toronto) on the roles of Wnt signaling in hematopoietic progenitor cells. Collectively these and other ongoing studies suggest that Wnt signaling plays important roles in stem and progenitor cells, and that it should be possible to leverage these insights to develop regenerative therapies.
Leveraging advances in technology to identify the mechanisms of Wnt signaling
As noted above, our laboratory is increasingly focused on identifying how Wnt signaling is involved in diseases, and on identifying Wnt-based therapies. This change in emphasis is driven in part by maturation of the Wnt field, but also by the emergence of new technologies that enable us to answer questions that were previously intractable. For example, we have established facilities for high throughput screening with siRNAs and small molecules, and used this technology to accelerate the discovery of both endogenous Wnt pathway components as well as small molecule modulators. We have also used these facilities to successfully conduct both genome-wide siRNA screens (with Rosetta/Merck), as well as kinome screens to identify kinases that regulate ß-catenin signaling in melanomas.
It is significant that using these advanced screening tools has time and again enabled us to identify new endogenous regulators of Wnt/ß-catenin signaling that were not previously detected by genetic screens. For example, our discovery that the FDA approved drug Riluzole regulates ß-catenin signaling led us to determine that the indirect cellular target of Riluzole, GRM1, is unexpectedly involved in repressing ß-catenin signaling. High throughput screening also led us to identify Bruton's tyrosine kinase (BTK) as a negative regulator of Wnt/β-catenin signaling and to identify WIKI4 as a novel small-molecule inhibitor of Wnt/ß-catenin signaling.
The next key technology we have adopted is mass spectrometry, which we have used to identify protein interaction networks in cells, including complexes of SCRIB, NOS1AP, and VANGL, and complexes of MINDBOMB1 and the Wnt receptor, RYK. In addition, we have pioneered the integration of proteomic data (which define complexes of proteins but do not reveal protein function) with siRNA data (which identify functional genes but not how the encoded proteins interact to form a signaling network). By integrating two different screens, which compensate for each other’s weaknesses, we have developed a general approach for accelerating the study of any signaling network (Figure 2). Finally, in unpublished work we have used phospho-proteomic approaches to determine whether there are changes in the phosphorylation of discrete polypeptides following stimulation of Wnt signaling.
Therapeutic modulation of Wnt signaling
We have a long-standing interest in Wnt signaling in cancer, notably melanoma. This interest arose when we found that elevated β-catenin signaling unexpectedly correlates with improved patient survival in melanoma, likely by decreasing cell proliferation and promoting differentiation. These results are in stark contrast to colorectal cancer, where elevated β-catenin correlates with a poorer prognosis. We have built on these initial investigations by identifying Riluzole as an FDA-approved drug that modulates ß-catenin signaling, and which is in clinical trials for melanoma by an independent lab at Rutgers. We have also demonstrated that targeted BRAF inhibitors, recently approved as a therapy for melanoma, require endogenous ß-catenin in order to be therapeutic, and that elevating ß-catenin signaling enhances the efficacy of the BRAF inhibitor. Strikingly, we have also shown that melanoma cells that are resistant to targeted bRAF inhibitors can be sensitized to undergo apoptosis by reducing levels of AXIN1, a negative regulator of ß-catenin.
Wnt signaling may also be a useful therapeutic target to enhance neurogenesis in the adult brain. We have previously shown that Traumatic Brain Injury (TBI) leads to the transient activation of Wnt/ß-catenin signaling in neural progenitor populations in the mouse brain. More recently, we have identified a widely prescribed drug as being able to activate Wnt/ß-catenin signaling in the brain, which has implications for a number of therapies.
In summary, our laboratory is focused on identifying the functions and mechanisms of action of Wnt signaling networks in vertebrates. We are leveraging these insights to better understand the roles of Wnt signaling in human diseases and to contribute to the discovery and understanding of new therapies.