Tight control of cell-cell communication is essential for the generation of a normally patterned embryo. A critical mediator of key cell-cell signaling events during embryogenesis is the highly conserved Wnt family of secreted proteins. Recent biochemical and genetic analyses have greatly enriched our understanding of how Wnts signal, and the list of canonical Wnt signaling components has exploded. The data reveal that multiple extracellular, cytoplasmic, and nuclear regulators intricately modulate Wnt signaling levels. In addition, receptor-ligand specificity and feedback loops help to determine Wnt signaling outputs. Wnts are required for adult tissue maintenance, and perturbations in Wnt signaling promote both human degenerative diseases and cancer. The next few years are likely to see novel therapeutic reagents aimed at controlling Wnt signaling in order to alleviate these conditions.

/pdf/the-wnt-signaling-pathway-in-development-and-disease-z4j8s29ao8.pdf

The Wnt signaling pathway in development and disease.

https://www.cell.com/cell/pdf/S0092-8674(06)01344-4.pdf

Wnt/beta-catenin signaling in development and disease.

Wnt/β-catenin signaling: components, mechanisms, and diseases

https://web.stanford.edu/group/nusselab/cgi-bin/wnt/sites/default/files/reviews/Cell%202012%20Clevers.pdf

Wnt/β-catenin signaling and disease.

The canonical Wnt cascade has emerged as a critical regulator of stem cells. In many tissues, activation of Wnt signalling has also been associated with cancer. This has raised the possibility that the tightly regulated self-renewal mediated by Wnt signalling in stem and progenitor cells is subverted in cancer cells to allow malignant proliferation. Insights gained from understanding how the Wnt pathway is integrally involved in both stem cell and cancer cell maintenance and growth in the intestinal, epidermal and haematopoietic systems may serve as a paradigm for understanding the dual nature of self-renewal signals.

Wnt signalling in stem cells and cancer

Wnt signalling is involved in numerous events in animal development, including the proliferation of stem cells and the specification of the neural crest. Wnt proteins are potentially important reagents in expanding specific cell types, but in contrast to other developmental signalling molecules such as hedgehog proteins and the bone morphogenetic proteins, Wnt proteins have never been isolated in an active form. Although Wnt proteins are secreted from cells, secretion is usually inefficient and previous attempts to characterize Wnt proteins have been hampered by their high degree of insolubility. Here we have isolated active Wnt molecules, including the product of the mouse Wnt3a gene. By mass spectrometry, we found the proteins to be palmitoylated on a conserved cysteine. Enzymatic removal of the palmitate or site-directed and natural mutations of the modified cysteine result in loss of activity, and indicate that the lipid is important for signalling. The purified Wnt3a protein induces self-renewal of haematopoietic stem cells, signifying its potential use in tissue engineering.

/pdf/wnt-proteins-are-lipid-modified-and-can-act-as-stem-cell-1qt4flvafx.pdf

Wnt proteins are lipid-modified and can act as stem cell growth factors

Wnt proteins, regulators of development in many organisms, bind to seven transmembrane-spanning (7TMS) receptors called frizzleds, thereby recruiting the cytoplasmic molecule dishevelled (Dvl) to the plasma membraneFrizzled-mediated endocytosis of Wg (a Drosophila Wnt protein) and lysosomal degradation may regulate the formation of morphogen gradients Endocytosis of Frizzled 4 (Fz4) in human embryonic kidney 293 cells was dependent on added Wnt5A protein and was accomplished by the multifunctional adaptor protein beta-arrestin 2 (betaarr2), which was recruited to Fz4 by binding to phosphorylated Dvl2 These findings provide a previously unrecognized mechanism for receptor recruitment of beta-arrestin and demonstrate that Dvl plays an important role in the endocytosis of frizzled, as well as in promoting signaling

https://science.sciencemag.org/content/sci/301/5638/1391.full.pdf

Dishevelled 2 Recruits ß-Arrestin 2 to Mediate Wnt5A-Stimulated Endocytosis of Frizzled 4

The Wnt signaling pathway plays a critical role in embryogenesis and tumorigenesis. However, biological roles of Wnt growth factors have not been fully characterized in prostate development and the pathogenesis of prostate cancer. In this study, we used Wnt3a-conditioned medium (Wnt3a-CM) and purified Wnt3a proteins to investigate whether there is a direct effect of Wnt3a on androgen receptor (AR)-mediated transcription and to determine its role in the growth of prostate cancer cells. We demonstrated that Wnt3a-CM either induces AR activity in the absence of androgens or enhances AR activity in the presence of low concentrations of androgens, whereas purified Wnt3a showed a pronounced effect in the presence of low concentrations of ligands. We also showed that Wnt3a-CM and the purified Wnt3a enhance the level of cytosolic and nuclear β-catenin, suggesting an involvement of β-catenin in this regulation. Moreover, treatment of LNCaP cells with Wnt3a-CM and purified Wnt3a significantly enhances cell growth in the absence of androgens. Our findings demonstrate that Wnt3a plays an important role in androgen-mediated transcription and cell growth. These results suggest a novel mechanism for the progression of prostate cancer.

https://cancerres.aacrjournals.org/content/canres/64/24/8860.full.pdf

Wnt3a Growth Factor Induces Androgen Receptor-Mediated Transcription and Enhances Cell Growth in Human Prostate Cancer Cells

Cancer therapy has been revolutionized by the recent developments of immune-checkpoint inhibitors (ICI) to harness the power of the immune system in fighting cancer. However, most patients fail to have durable responses or become resistant to ICI, highlighting the need to identify new mechanisms of immune evasion in cancer and develop novel therapeutic modalities. Recently, the glyco-immune checkpoint axis (sialoglycan/Siglec pathway) has emerged as a new mechanism of immune regulation involving both innate and adaptive immunity and an important mechanism of cancer immune escape. Upon ligation of sialoglycan to ITIM-containing Siglecs on immune cells, this pathway suppresses multiple facets of anti-cancer immunity, including cancer antigen release, cancer antigen presentation, and priming and activation of anti-cancer T cells. However, a therapeutic intervention of this axis remains a great challenge due to the overlapping and promiscuous receptor-ligand interactions between 15 Siglecs and dense array of various sialoglycans in humans. To overcome this hurdle and block the glyco-immune checkpoint axis, we describe here a new therapeutic modality named EAGLE (Enzyme-Antibody Glyco-Ligand Editing), which is antibody-like, multi-functional, and comprised of a tumor-associated antigen-binding moiety and a sialidase moiety, allowing selectively removing terminal sialic acids, the critical binding carbohydrate of Siglecs, from sialoglycans on tumor cells. We demonstrated that EAGLE decreased sialic acid levels of tumor cells and enhanced anti-tumor immune responses using multiple human system models mimicking immunosuppressive tumor microenvironment and immunocompetent syngeneic mouse tumor models. EAGLE treatment released cancer cell-mediated immunosuppression, restored dendritic cell functions, enhanced CD8+ T-cell proliferation/activation, and induced proinflammatory cytokines IFNγ, IL-17A, IL-2, IL-6, and TNFα in human coculture assays of cancer cells with dendritic cells or PBMC in the presence or absence of primary endothelial cells. Systematic administration of EAGLE increased tumor-infiltrating immune cells and led to significant anti-tumor activities with complete regressions as monotherapy in syngeneic mouse tumor models. Re-challenge experiments in cured mice from the EAGLE treatment resulted in a complete rejection of tumor cells, demonstrating that EAGLE induced anti-tumor immunological memory. We further revealed that the mechanism of action of EAGLE involved both innate and adaptive immunity because depleting macrophages or CD8+ T-cells decreased or abolished its efficacy. Moreover, EAGLE in combination with anti-PD1 mAb treatment achieved ~100% cures in syngeneic EMT6-Her2 models. In summary, EAGLE is a novel and promising immunomodulatory therapeutic modality inhibiting the glyco-immune checkpoints and has the potential to overcome resistance to current immunotherapies. Citation Format: Lizhi Cao, Adam Petrone, Wayne Gatlin, Jenny Che, Abhishek Das, Robert LeBlanc, Zakir Siddiquee, Sujata Nerle, Michal Stanczak, Michele Mayo, Lihui Xu, Karl Normington, Jeff Brown, Wei Yao, Carolyn Bertozzi, James Broderick, Heinz Laubli, Li Peng. A novel therapeutic modality of inhibiting the glyco-immune checkpoint axis to treat cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-109.

Jeffrey Brown

Papers

Wnt proteins are lipid-modified and can act as stem cell growth factors

Dishevelled 2 Recruits ß-Arrestin 2 to Mediate Wnt5A-Stimulated Endocytosis of Frizzled 4

Wnt3a Growth Factor Induces Androgen Receptor-Mediated Transcription and Enhances Cell Growth in Human Prostate Cancer Cells

Abstract LB-109: A novel therapeutic modality of inhibiting the glyco-immune checkpoint axis to treat cancer