bioRxiv preprint doi: https://doi.org/10.1101/333906; this version posted September 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Running Title: Secretion-independent Wnt signaling Research article WNT4 and WNT3A activate cell autonomous Wnt signaling independent of secretion Deviyani M. Rao1, Rebecca L. Ferguson1, Tomomi M. Yamamoto2, Benjamin G. Bitler2, Matthew J. Sikora1 Affiliation: 1Dept. of Pathology, 2Dept. of Obstetrics and Gynecology, University of Colorado Anschutz Medical Campus Corresponding author: Matthew J. Sikora, PhD.; Mail Stop 8104, Research Complex 1 South, Room 5117, 12801 E. 17th Ave.; Aurora, CO 80045. Tel: (303)724-4301; Fax: (303)724-3712; email: [email protected]. Twitter: @mjsikora Funding This work was supported by R00 CA193734 (MJS) and R00 CA194318 (BGB) from the National Institutes of Health, and by a grant from the Cancer League of Colorado, Inc (MJS). Authors' contributions DMR and MJS conceived of the project and experiments. DMR, RLF, and MJS designed and performed experiments. RLF, DMR, and TMY developed models for the project. DMR, RLF, BGB, and MJS contributed to data analysis and interpretation. DMR wrote the draft manuscript; all authors read and revised the manuscript, and have read and approved of this version of the manuscript. bioRxiv preprint doi: https://doi.org/10.1101/333906; this version posted September 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Competing interests The authors have nothing to disclose. bioRxiv preprint doi: https://doi.org/10.1101/333906; this version posted September 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Abstract Background: Wnt protein secretion and signaling canonically require the enzyme PORCN, suggesting PORCN inhibitors can effectively treat Wnt-dependent forms of cancer. We tested the ability of PORCN inhibitors to inhibit the growth of WNT4-dependent breast cancer cell lines, but found PORCN inhibitors did not phenocopy WNT4 knockdown. This led us to investigate the role of PORCN in WNT4 signaling. Methods: Breast cancer cell line MDA MB 134VI, fibrosarcoma cell line HT1080, and PORCN-knockout HT1080 were used as model systems for WNT4 signaling (i.e. derived from WNT4-responsive tissues; breast and bone, respectively), along with additional breast and ovarian cancer cell lines. Using these models, WNT3A or WNT4 were constitutively over-expressed. Conditioned medium was used to assess Wnt secretion and paracrine activity, and dependence on the Wnt secretion proteins PORCN and Wntless (WLS). Secreted Wnt protein activity was compared to recombinant Wnt protein. Results: Whereas WLS was universally required for Wnt secretion and paracrine signaling, the role of PORCN varied across contexts. PORCN was not required for WNT3A secretion in all cell lines, but PORCN was universally required for paracrine activity of WNT3A. In contrast, WNT4 secretion was PORCN-independent in all models tested. However, while recombinant WNT4 activated Wnt signaling in treated cells, secreted WNT4 did not present paracrine activity in any tested context. Absent the expected paracrine activity of secreted WNT4, we examined Wnt signaling directly in cells over-expressing Wnt proteins. This revealed cell autonomous activation of Wnt signaling, independent of secretion, by both WNT4 and WNT3A. Direct transfection of Wnt protein activated non-canonical Wnt signaling independent of membrane receptor activation. Conclusions: PORCN is differentially required for secretion and paracrine activity of WNT4 and WNT3A, specific to the cell type expressing Wnt proteins and the paracrine recipient of Wnt signals. Wnt proteins can activate cell autonomous signaling, independent of PORCN and of secretion. This PORCN- independent mode of Wnt signaling may be critical in cell contexts that are otherwise considered to have dysfunctional Wnt signaling. 3 bioRxiv preprint doi: https://doi.org/10.1101/333906; this version posted September 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Introduction Wnt signaling is an ancestrally conserved pathway that plays fundamental roles in numerous facets of embryonic development and adult tissue homeostasis. Dysregulation of Wnt signaling is a causative factor for a range of human pathologies, including several forms of cancer (reviewed in [1]). As a result, inhibition of Wnt signaling has become an attractive therapeutic target with ongoing clinical trials, with some strategies focused upstream by inhibiting the signaling actions of Wnt proteins [1–3]. Wnt proteins comprise a family of secreted glycoproteins that act as inter-cellular ligands, which stimulate a myriad of signal transduction cascades regulating cellular proliferation, stem cell renewal, cell motility, angiogenesis, and apoptosis [1, 4–6]. Wnt proteins are post-translationally modified by the O- acyltransferase Porcupine (PORCN), which palmitoylates Wnt proteins at conserved serine residues [2, 7, 8]. This lipidation forms a binding motif for interaction with Wntless (WLS), which chaperones Wnt proteins to the plasma membrane for secretion [8–10]. Once secreted, Wnt proteins signal in a paracrine manner, binding nearby receptor complexes. Wnts typically bind a Frizzled (FZD) receptor in conjunction with the LRP5 or LRP6 co-receptor, resulting in second messenger activation (Dishevelled proteins; DVL1/2/3 in humans) and initiation of either canonical (β-catenin-dependent) or non-canonical (β- catenin-independent) signaling [1, 4]. The essential initiating step in Wnt processing is palmitoylation by PORCN, which has prompted the development of PORCN inhibitors, including the IWP class of compounds [10], WNT974 (a.k.a. LGK974) [3], and others [2, 11]. These compounds have been shown to block Wnt secretion, inhibit downstream Wnt signaling, and suppress Wnt-driven tumor growth in animal models [3, 12, 13]. WNT974 is currently in Phase I/II clinical trials for cancer treatment (NCT01351103, NCT02278133). Based on these observations, PORCN inhibitors are an attractive strategy to target Wnt- driven pathologies. The Wnt protein WNT4 is critical in organogenesis of endocrine organs, regulation of bone mass, and in several steroid hormone-related phenotypes in humans [14–21]. WNT4 dysregulation via loss-of-function mutation results in developmental female to male sex reversal [22–25], and WNT4 polymorphisms are 4 bioRxiv preprint doi: https://doi.org/10.1101/333906; this version posted September 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. associated with endocrine dysfunction, gynecological malignancies, reduced bone density with premature skeletal aging, and related phenotypes [26–32]. WNT4 is also critical in mammary gland development, as Wnt4 knockout in mice prevents progesterone-driven ductal elongation and branching of the mammary gland during pregnancy [33, 34]. In this context, activated progesterone receptor drives expression of Wnt4 in mammary gland luminal cells resulting in paracrine signaling that supports maintenance of the mammary stem cell niche [6, 35–37]. Despite these observed critical roles of WNT4 in both normal and malignant tissues, WNT4 signaling is crudely understood due to varied context-dependent functions. Both human and murine WNT4 are reported to activate or suppress either canonical or non-canonical Wnt signaling in a context and tissue-specific manner (described in references herein). Further, conflicting reports indicate Wnt4 can or cannot activate canonical Wnt signaling in the murine mammary gland [35, 38]. It is also unclear which FZD receptor complexes are utilized by WNT4, as WNT4 is often required for distinct, non-redundant functions versus other Wnt proteins, leading to the description of WNT4 as a “problem child” among Wnts (reviewed in [34]). Based on these observations, inhibition of WNT4 signaling upstream of Wnt effector pathways, such as with PORCN inhibitors, is an attractive approach to block WNT4 signaling in a “pathway indifferent” manner and treat WNT4-related pathologies. We recently reported that regulation of WNT4 expression is co-opted by the estrogen receptor in a subtype of breast cancer, invasive lobular carcinoma (ILC) [39, 40]. Estrogen-driven WNT4 is required in ILC cells for estrogen-induced proliferation and survival, as well as anti-estrogen resistance [40]. Though WNT4-driven signaling in ILC is yet to be fully elucidated, ILC cells lack the capacity to engage canonical Wnt signaling, as the characteristic genetic loss of E-cadherin in ILC leads to loss of β-catenin protein [40, 41]. This suggests that ILC should