WNT4 and WNT3A activate cell autonomous Wnt signaling independent of PORCN and secretion

The enzyme PORCN is considered essential for Wnt secretion and signaling. However, PORCN inhibition did not phenocopy the effects of WNT4 knockdown in WNT4-dependent breast cancer cells, suggesting an atypical role for PORCN in WNT4 signaling. WNT4 or WNT3A were over-expressed in cell lines (breast and ovarian cancer, and fibrosarcoma). Conditioned medium from these cell lines, and co-culture systems, were used to assess Wnt secretion and activity. The dependence of Wnt secretion on PORCN and WLS was also tested. We observed that WLS was universally required for Wnt secretion and paracrine signaling. In contrast, the dependence of WNT3A secretion and activity on PORCN varied across cell lines, and WNT4 secretion was PORCN-independent in all models. Surprisingly, WNT4 did not present paracrine activity in any tested context. Absent the expected paracrine activity of secreted WNT4, we identified cell autonomous Wnt signaling activation by WNT4 and WNT3A, independent of PORCN and secretion. Direct transfection of Wnt protein activated the Wnt second messenger proteins DVL2 and DVL3, independent of activation of membrane receptors. The PORCN-independent, cell-autonomous Wnt signaling demonstrated herein may be critical in WNT4-driven cellular contexts, or those which are otherwise considered to have dysfunctional Wnt signaling. Summary Statement Wnt proteins can mediate an atypical mode of cell-autonomous signaling, distinct from paracrine signaling, that is independent of both palmitoylation by PORCN and Wnt secretion.


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Wnt signaling is an ancestrally conserved pathway that plays fundamental roles in embryonic 51 development and adult tissue homeostasis. Dysregulation of Wnt signaling is a causative factor for a 52 range of human pathologies, including several forms of cancer (reviewed in (1)). As a result, inhibition of 53 Wnt signaling has become an attractive therapeutic target in ongoing clinical trials, with some strategies 54 targeting the upstream activation of signaling by Wnt proteins (1-3). Wnt proteins comprise a family of 55 secreted glycoproteins that act as intercellular ligands, which stimulate a myriad of signal transduction 56 cascades regulating cellular proliferation, stem cell renewal, cell motility, angiogenesis, and apoptosis (1, 57 4-6). Wnt proteins are post-translationally modified by the O-acyltransferase Porcupine (PORCN), which 58 palmitoylates Wnt proteins at single serine residues (2,(7)(8)(9). This lipidation forms a binding motif for 59 interaction with Wntless (WLS), which chaperones Wnt proteins to the plasma membrane for secretion 60 (8,10,11). Once secreted, Wnt proteins signal in a paracrine manner, binding nearby receptor complexes.

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The Wnt protein WNT4 is critical in organogenesis of endocrine organs and regulation of bone mass, and 72 underlies steroid hormone-related phenotypes in humans (15)(16)(17)(18)(19)(20)(21)(22). WNT4 dysregulation via loss-of-73 function mutation results in developmental female to male sex reversal (23)(24)(25)(26). Similarly, WNT4 74 polymorphisms are associated with endocrine dysfunction, gynecological malignancies, reduced bone 75 density with premature skeletal aging, and related phenotypes (27)(28)(29)(30)(31)(32)(33). WNT4 is also critical in mammary 76 gland development, as Wnt4 knockout in mouse mammary gland prevents progesterone-driven ductal 77 elongation and branching during pregnancy (34,35). In this context, activated progesterone receptor 78 drives expression of Wnt4 in mammary gland luminal cells resulting in paracrine signaling that supports 79 maintenance of the mammary stem cell niche (6,(36)(37)(38). Despite these observed critical roles of WNT4 80 in both normal and malignant tissues, WNT4 signaling is crudely understood due to varied context-81 dependent functions. In a cell type-and tissue-specific manner, WNT4 (human or murine) has been 82 shown to regulate either canonical or non-canonical Wnt signaling, and has been shown to either activate 83 or suppress signaling (described in references herein). Further, conflicting reports exist as to whether 84 Wnt4 can or cannot activate canonical Wnt signaling in the murine mammary gland (36,39). As such, 85 WNT4 has been described as a "problem child" among Wnt proteins. It is also unclear which FZD 86 receptor complexes are utilized by WNT4, as WNT4 is often required for distinct, non-redundant 87 functions versus other Wnt proteins (reviewed in (35)). Since WNT4 has myriad downstream signaling 88 effects, inhibition of WNT4 upstream of Wnt effector pathways (e.g. with PORCN inhibitors) is an 89 attractive approach to block WNT4 signaling in a "pathway indifferent" manner to treat WNT4-related 90 pathologies.

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We recently reported that regulation of WNT4 expression is co-opted by the estrogen receptor in a subtype 93 of breast cancer, invasive lobular carcinoma (ILC) (40,41). Estrogen-driven WNT4 is required in ILC 94 cells for estrogen-induced proliferation and survival, as well as anti-estrogen resistance (41). Though 95 WNT4-driven signaling in ILC is yet to be fully elucidated, ILC cells lack the capacity to engage 96 canonical Wnt signaling, as the characteristic genetic loss of E-cadherin in ILC leads to loss of β-catenin 97 protein (41,42). This suggests WNT4 drives non-canonical Wnt signaling in ILC cells. Though the 98 specific non-canonical pathway activated by WNT4 is unknown, PORCN inhibition should be an 99 effective strategy to block WNT4 upstream and treat this subtype of breast cancer. However, treatment of 100 ILC cells with PORCN inhibitors did not suppress growth or survival. These unexpected results initiated 101 further studies into the mechanisms of WNT4 secretion and signaling. In this report, we show WNT4 102 secretion is mediated by atypical mechanisms. Our observations challenge the paradigm that PORCN-103 mediated secretion is required for Wnt signaling, and suggest a novel process by which Wnt proteins,

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LGK974. Proliferation was compared to untreated cells, and cells treated with the anti-estrogen 114 fulvestrant (Fulv) or transfected with siRNA targeting WNT4 (siWNT4), both of which strongly suppress 115 growth. Cell death was monitored by SyTOX green fluorescence, and proliferation results were 116 confirmed at the experimental endpoint by dsDNA quantification. As we previously reported, siRNA-117 mediated WNT4 knockdown or Fulv halt proliferation, and WNT4 knockdown induces cell death ( Fig.   118 1A). However, neither genetic nor chemical PORCN inhibition had any effect on cell proliferation or 119 survival of MM134 cells (Fig. 1A,B). Similar results were obtained in ILC cell line SUM44PE, as 120 PORCN inhibitor at concentrations up to 1μM did not affect proliferation (Supplemental Fig. 1)

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Consistent with the lack of effect of cell proliferation, PORCNi treatment had no effect on WNT4 138 secretion, and WNT3A secretion was also unaffected by PORCNi (Fig. 2B, top). Similarly, siPORCN 139 had no effect on secretion of either WNT4 or WNT3A (Fig. 2B, bottom). However, WLS was required 140 for Wnt secretion, as siWLS suppressed secretion of both WNT3A and WNT4 from MM134 (Fig. 2B,   141 bottom). These data suggest that Wnt processing and secretion may be atypical in ILC cells, but the 142 PORCN-independent secretion of WNT4 is a potential mechanism of PORCNi resistance (Figure 1).

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To further examine the function of recombinant and secreted WNT4, we used MC3T3-E1 as an additional 223 "receiver" cell line (Supplemental Fig. 2). MC3T3-E1 cells are another bone-like model that are highly 224 responsive to exogenous Wnt protein and induce alkaline phosphatase production upon Wnt signaling 225 activation, which can be measured by colorimetric assay (see Materials and Methods; (48)). Conditioned 226 medium from Wnt-expressing cells as above was used to treat "receiver" 3T3-E1 cells, and alkaline 227 phosphatase (AP) activity was used as the readout for activation of paracrine Wnt signaling. As expected, 228 increasing concentrations of either rWNT3A or rWNT4 increased AP activity (Fig. 4A), and both 229 rWNT3A and rWNT4 induced DVL and LRP6 phosphorylation in 3T3-E1 cells (Supplemental Fig. 4A).

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This confirmed that 3T3-E1 respond to paracrine WNT4, and could be used to assess the activity of   LGK974. 'Ctrl' represents TOP-FLASH transfected HT1080-PKO without co-culture. WNT signaling activity, as measured by luminescence, was performed using a dual luciferase assay. Statistics obtained using Student's unpaired t-test compared to the no co-culture control (ctrl). * represents p<0.005. Points represent mean of 3 technical replicates ±SD. Results are representative of two independent experiments. (F-G) HT1080-PKO control cells were treated for 24 hours with recombinant WNT protein (at concentrations of 10ng/ml, 50ng/ml, 100ng/ml, 250ng/ml, or 500ng/ml). (F) Immunoblots of whole cell lysates were run and (G) mRNA extracted for qPCR were performed as above (A-D). Statistics obtained using ANOVA with Dunnett's multiple correction. Points represent mean of 2 technical replicates ±SD. LGK974: bone, ovarian cancer is a relevant context as WNT4 mediates Müllerian tissue and ovary development 254 (23,25). Wnt expression and secretion were assessed as above, and paracrine activity of secreted Wnt 255 proteins was tested using the 3T3-E1 receiver model.

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Despite strong WNT3A over-expression in cell lysate, secreted WNT3A from SUM44PE in conditioned 260 medium was at detection limits. Over-expression of neither WNT3A nor WNT4 induced AP activity 261 versus parental SUM44PE cells (Fig. 5C).
LGK974 and siWLS modestly decreased basal AP activity 262 versus respective controls, which may be due to secretion inhibition of other Wnt proteins in these cells.

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However, these data with SUM44PE are consistent with our observations of PORCN-independent Wnt 264 secretion and inactive paracrine activity of secreted Wnt proteins in ILC. As observed in HT1080,

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As WNT4 is required for bone regeneration and cell proliferation (22), we examined if WNT4 is similarly 302 essential for proliferation and/or viability of HT1080 or HT1080-PKO. We hypothesized that WNT4 303 might be dispensable in HT1080, due to redundant Wnt signaling. However, without functional PORCN 304 for secretion and paracrine signaling of Wnt family members, HT1080-PKO may become reliant on cell-305 autonomous PORCN-independent WNT4 signaling. Knockdown of WNT4 induced ~21% cell death at 306 48h post-knockdown in HT1080 (Fig. 6C), leading to a modest suppression of proliferation (Fig. 6D). In 307 contrast, WNT4 knockdown in HT1080-PKO strongly suppressed growth, and cell death was accelerated 308 (~70% cell death at 48h post-knockdown). Importantly, minimal cell death was induced by WLS

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To determine whether cell autonomous Wnt signaling could be activated directly by intra-cellular Wnt 316 protein, we transfected recombinant Wnt protein directly in to HT1080-PKO cells and examined 317 activation of Wnt signaling as above, 4 hours post-treatment. Of note, these experiments used higher Wnt 318 protein concentrations (~4000ng/mL) than in above paracrine signaling studies (Fig. 3); rWnt protein is 319 also not identical to Wnt proteins produced endogenously (Supplemental Fig. 4). Compared to control 320 transfection with FITC-labeled antibody, transfection of WNT3A or WNT4 activated DVL2/3 321 phosphorylation in HT1080-PKO (Fig. 6E). Though paracrine treatment with this high concentration of 322 Wnt protein was sufficient to activate DVL2/3 with either WNT3A or WNT4, LRP6 was only 323 phosphorylated with paracrine WNT3A. This indicates that transfected Wnt protein activates DVL2/3 324 independent of extracellular activity, as observed in HT1080-PKO Wnt-overexpressing cells (Fig. 6A,B).

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The Wnt modifying enzyme PORCN is commonly described as a gatekeeper for the secretion of Wnt 328 proteins, and thus PORCN inhibition is an approach to broadly block Wnt signaling without targeting cell 329 type-or tissue-specific downstream Wnt pathways. WNT4 signaling is required for survival and 330 proliferation of ILC cells, but we show that PORCN is dispensable, calling into question the role of 331 PORCN in WNT4 signaling. PORCN was not required for WNT4 secretion from a panel of cell lines, as 332 genetic or chemical PORCN blockade had no effect on WNT4 section. However, WNT4 was not capable 333 of activating paracrine Wnt signaling in any model tested, despite the ability of recombinant human 334 WNT4 to do so in a context-dependent manner. These data together suggest that secreted WNT4 may not 335 be responsible for driving signaling in WNT4-expressing cells. Instead, we determined that WNT4 and 336 WNT3A can activate cell autonomous, intra-cellular signaling independent of secretion. This unique 337 mode of Wnt signaling (Fig. 7) is likely essential for the survival and proliferation of WNT4-dependent   Wnt4 had no effect on insulin secretion (53). These data parallel our findings and support that WNT4 has 372 a novel function in signaling independent of PORCN, secretion, and paracrine signaling.

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Our data, together with the above reports on PORCN-independent Wnt signaling, highlight that the roles 375 of PORCN and WLS in Wnt modification, secretion, and signaling are context-dependent across 376 individual Wnt proteins in a cell-type specific manner. This observed context-dependence includes not 377 only the specific "receiver" cells in question (perhaps best understood in the context of differentially 378 expressed FZD receptors), but also includes the source of the Wnt protein (Fig. 7, Supplemental Fig .7).  lysates. This non-specific target largely precludes detection of WNT4, but cutting immunoblot 580 membranes immediately below a 50kD ladder marker prevents this issue. This non-specific band was not 581 detected in WNT4 immunoblots from conditioned medium. Similarly, in immunoblots of conditioned 582 medium WNT3A MAB13242 detects a prominent non-specific band at ~60kD that precludes detection of 583 secreted WNT3A; cutting membranes above a 50kD ladder marker prevents this issue. This non-specific 584 band was not detected in WNT3A immunoblots from cell lysates.  MM134 and SUM44PE were plated and 24hrs later started treatment with either anti-estrogen fulvestrant (Fulv) or increasing concentrations of WntC59. At the timecourse completion of either 5 or 7 days, total ATP was measured using Promega Cell-Titer Glo. Points represent the mean of 6 biological replicates + SD.

Fulv
Cells +/-WNT over-expression are allowed to condition medium for up to 7 days (as described in the Methods) after siRNA transfection or PORCNi treatment as indicated.

Cells collected
Cells lysed for Western Blot, mRNA analysis