To identify additional cell lines that display autocrine Wnt signaling, we tested cell lines with relatively high expression of Axin2 mRNA or phospho-LRP5/6 protein for inhibition of Wnt signaling by Fzd8CRD-Fc protein in Wnt luciferase reporter gene assays. Nine cell lines exhibited autocrine Wnt signaling that was inhibited by Fzd8CRD-Fc protein (Figure 2A), including NSCLC cells NCI-H23 and NCI-H2030 and soft tissue sarcoma cells SW872 and HT-1080 that have previously been reported to have endogenous Wnt signaling based on assays with other Wnt antagonists [23–25]. When signaling was further induced in all nine cell lines by exogenous Wnt3a protein, YW211.31.57 antibody inhibited this response to Wnt3a (Figure 2A, 2B, 2E, and 2G). Surprisingly, YW211.31.57 antibody potentiated auto- crine Wnt signaling in all nine of these cell lines, whereas YW210.09 potentiated autocrine Wnt signaling in five lines and inhibited in three lines (Figure 2A–2D, 2F, and 2G). This reciprocal activity of YW211.31.57 antibody on autocrine and Wnt3a-induced signaling was observed not only using the luciferase reporter, but also for expression of endogenous Wnt target genes such as Axin2 in the six cell lines tested (Figure 2B, 2C, and 2D). In EKVX (Figure 2H) and breast carcinoma Hs578T (data not shown) cell lines, we confirmed that the increase in Wnt signaling by YW211.31 antibody was dependent on autocrine Wnt(s) by demonstrating that this increase is blocked by Fzd8CRD-Fc protein. Potentiation of autocrine Wnt signaling in EKVX and Hs578T cells is also observed with the other five antibody antagonists of Wnt3a-induced signaling identified in the screen (data not shown). Reciprocal Activities of LRP6 Antibodies on Different Wnt Isoforms Our results show that YW211.31 antibody inhibits signaling induced by exogenous Wnt3a protein in all cell lines, but can either inhibit or potentiate autocrine Wnt signaling in a cell line- dependent manner. This suggests that the specific Wnt isoform driving the autocrine signal dictates the activity of the antibody. Therefore, we tested the activity of the antibody on signaling induced by exogenous expression of Wnt3a and other Wnt isoforms. Wnt signaling induced by transfection of Wnt3a in either HEK293 (Figure 3A) or Hs578T (data not shown) cells is inhibited by YW211.31.57 antibody with similar potency to inhibition of signaling induced by Wnt3a protein treatment. Importantly, we found that signaling induced by Wnt1 expression in both cell lines was potentiated by YW211.31.57 antibody, whereas both Wnt1 and Wnt3a signaling are inhibited by Fzd8CRD-Fc protein as expected. Potentiation of Wnt1 signaling was also observed with the other Wnt3a antagonist antibodies identified in the screen (data not shown). YW210.09 antibody also displayed opposing activities against Wnt3a- and Wnt1-induced signaling, which were the reciprocal of YW211.31.57 activities; i.e., potentiation of Wnt3a and inhibition of Wnt1 signaling (Figure 3A). YW210.09 antibody also inhibits endogenous Wnt1 signaling in cultured tumor cells grown from MMTV-Wnt1 tumors, as observed by reduced expression of Wnt target genes Axin2 and Mmp7 to a similar extent as Fzd8CRD-Fc protein treatment (Figure 3B). In MMTV-Wnt1 cells, YW211.31.57 antibody failed to potentiate Wnt1 signaling, possibly because Wnt1 signaling is already maximal in these cells. Having demonstrated that YW211.31.57 and YW210.09 antibodies have reciprocal activities on Wnt signaling initiated by Wnt3a and Wnt1, we extended this analysis to the additional 11 of 19 Wnt genes that induce the luciferase reporter greater than two-fold in HEK293 cells (Figure 3C). Only Wnt3a and Wnt3 activities are inhibited by YW211.31.57 antibody, and both are Wnts Specify LRP6 mAb Activity potentiated by YW210.09. Seven Wnt isoforms in addition to Wnt1 are potentiated by YW211.31.57 and inhibited by YW210.09. A third class of Wnt isoforms (Wnt7a, 7b, and 10a) exhibit signaling activity that is not inhibited by either antibody but is potentiated by at least YW211.31.57. In Hs578T cells transfected with different Wnt isoforms, the antibodies display most of these same activities. In particular, YW211.31.57 inhibits Wnt3a and Wnt3 and potenitates all 11 of the other Wnt isoforms that induce the luciferase reporter at least two-fold. YW210.09 also potentiates Wnt3 and Wnt3a in Hs578T cells, as well as inhibits 5 of the 7 Wnt isoforms that are antagonized in HEK293 cells and can be tested in Hs578T cells. The other two Wnt isofoms in this class, Wnt8a and Wnt9b, are not significantly affected by YW210.09 antibody in Hs578T cells. Since RNAi experiments indicate that Wnt3a signaling is transduced by both LRP6 and LRP5 in Hs578T cells, but only by LPR6 in HEK293 cells (data not shown), Wnt8a and Wnt9b might signal primarily through LRP5 in Hs578T cells. Similar to HEK293 cells, Wnt isoforms in the third class are not inhibited by either antibody in Hs587T cells, and we can add to this class Wnt4, which did not induce signaling in HEK293 cells. Only the activity of Wnt7b in this class behaves differently in that YW210.09 antibody potentiates its signaling in Hs578T but not HEK293 cells. In contrast to the Wnt isoform-specific activities of YW211.31.57 and YW210.09 antibodies, Fzd8CRD-Fc protein can potently inhibit the activity of all Wnts tested except Wnt6 and Wnt9b in HEK293 cells. In Hs578T cells, YW211.31.57 and YW210.09 antibodies potentiate both autocrine Wnt signaling and signaling induced by expression of Wnt4, Wnt7a, and Wnt7b. Thus these three Wnt isoforms are candidates for those that drive autocrine signaling in Hs578T cells. Multiple siRNAs against Wnt7b, but not the other Wnt isoforms, inhibit autocrine signaling in Hs578T cells, identifying the specific Wnt protein mediating signaling (Figure S2C). Since autocrine Wnt signaling in PA-1 cells is inhibited by YW211.31.57 antibody and potentiated by YW210.09, Wnt3 or Wnt3a likely activate endogenous signaling in these cells. Indeed, siRNAs against Wnt3 but not Wnt3a inhibit autocrine Wnt signaling in PA-1 cells (Figure S2A). In NCI-H23 NSCLC cells and in M14 melanoma cells, potentiation of autocrine Wnt signaling by YW211.31 antibody and antagonism by YW210.09 are consistent with Wnt2 RNAi inhibiting endogenous signaling in NCI-H23 cells [23], and with endogenous Wnt1 expression in M14 cells [35]. Using multiple siRNAs, we confirm that Wnt2 expression in NCI-H23 cells (Figure S2D) and Wnt1 in M14 cells (Figure S2B) are required for autocrine Wnt signaling. Wnt Isoforms Specify Different Activities of LRP6 Antibodies Different Wnt isoforms may preferentially bind different FZD isoforms expressed endogenously in the various cell lines, and could conceivably account for the differential activities of our LRP6 antibodies. To examine this possibility, we expressed chimeric proteins covalently linking different Wnt-FZD pairs to test whether the specific Wnt or FZD isoform determines the activity of the LRP6 antibody. Wnt3a or Wnt1 fused to either FZD4 or FZD5 potently activates Wnt signaling in HEK293 cells in the presumed absence of endogenous Wnt expression, whereas overexpression of FZD4 or FZD5 does not induce Wnt signaling (data not shown). YW211.31.57 antibody inhibits the signaling activity of Wnt3a fused to either FZD4 or FZD5, and potentiates the activity of Wnt1 fused to either FZD4 or FZD5 (Figure 3D). YW210.09 antibody shows the reciprocal activity against the Wnt1 chimeras, inhibiting both. Thus the activity of the antibody PLoS ONE | www.plosone.org 4 September 2010 | Volume 5 | Issue 9 | e12682