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Signaling Crosstalk between the Wnt, BMP and Endolysosomal Pathways in Development and Disease

  • Author(s): Ploper, Diego
  • Advisor(s): De Robertis, Edward M
  • et al.
Abstract

Few processes are as complex as development from a one cell embryo to a multicellular adult with specialized tissues. However, organisms use a surprisingly small number of signaling pathways for such an intricate task. As a result, the integration and coordination of these few pathways must play a central role in development. Throughout evolution, signaling factors may acquire new functions and be recruited to different pathways, generating novel signaling nodes. This thesis describes two of these innovations involving the Wnt signaling pathway. In the first, a secreted Wnt antagonist acquired new functions by also inhibiting an extracellular protease. In the second, a transcription factor involved in organelle biogenesis was brought under regulation by Wnt signaling through Glycogen Synthase Kinase 3 (GSK3) phosphorylation sites identified during this work.

The Bone Morphogenetic Protein (BMP) and Wnt pathways pattern the early Xenopus embryo. At gastrula, dorsal-ventral (DV) polarity is determined by a gradient of BMP signaling, created and reinforced by an extracellular network of interacting proteins secreted by two gastrula centers, the ventral center and the Spemann organizer. Key factors in the maintenance of this gradient are Chordin, a BMP inhibitor, and tolloid, a metalloproteinase that degrades Chordin. Surprisingly, secreted Frizzled Related Protein (sFRP) antagonists of Wnt signaling were found to have adopted novel roles in the BMP pathway. In particular, the sFRP Crescent, while retaining its anti-Wnt properties, was found in this thesis to competitively inhibit tolloids and protect Chordin from proteolytic degradation. Together with Sizzled, its ventrally expressed counterpart, Crescent regulates DV patterning by hindering the proteolytic activity of an extracellular protease and causing an increase in Chordin.

Wnt signaling requires the endolysosomal pathway in order to sequester negative regulators such as GSK3 and Axin1 inside multivesicular bodies (MVBs). Thus, the Wnt and endolysosomal pathways converge at the level of MVBs. Expansion of the MVB compartment, by lysosomal inhibition or mutations in Presenilin (a protein mutated in Alzheimer’s), caused an enhancement of Wnt signaling mediated by increased sequestration of inside MVBs of GSK3, a key negative regulator of Wnt signaling. A similar expansion of the MVB/late endolysosomal compartment is triggered in cells in which the transcriptional regulators of lysosomal biogenesis are dysregulated.

The melanoma oncogene MITF was shown here to drive the biogenesis of endolysosomes, which upon Wnt stimulation lead to increased sequestration of GSK3 and other components of the β-Catenin destruction complex. In this way MITF, an oncogene amplified in 20% of melanomas, enhanced canonical Wnt signaling. By inhibiting GSK3 phosphodegron-mediated ubiquitination and proteasomal degradation, Wnt signaling stabilizes a plethora of cellular proteins. This study identifies MITF as a transcription factor that is regulated by GSK3 and Wnt through particular phosphorylation sites. This finding deepens knowledge about this lineage-addiction oncogene that determines melanoma phenotypes.

The work presented in this dissertation covers a wide range of experimental systems, from Xenopus embryos to melanoma cell lines, and addresses unexpected crosstalks of the Wnt signaling pathway in development and disease. A secreted Wnt inhibitor, Crescent, evolved a role in the BMP pathway by inhibiting tolloid proteases. This thesis also underscores the importance of the endolysosomal pathway for canonical Wnt signaling. The discovery of novel phosphorylations in the oncogene MITF, and its role as a driver of endolysosomal biogenesis in melanoma, suggests that Wnt might regulate other MiT family members in the same manner. In the final part of this dissertation we present a review on how endolysosomal biogenesis and Wnt may intersect with MiT family oncogenic activity.

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