Wnt pathway inhibition via the targeting of Frizzled receptors results in decreased growth and tumorigenicity of human tumors

Abstract

The Wnt/β-catenin pathway, which signals through the Frizzled (Fzd) receptor family and several coreceptors, has long been implicated in cancer. Here we demonstrate a therapeutic approach to targeting the Wnt pathway with a monoclonal antibody, OMP-18R5. This antibody, initially identified by binding to Frizzled 7, interacts with five Fzd receptors through a conserved epitope within the extracellular domain and blocks canonical Wnt signaling induced by multiple Wnt family members. In xenograft studies with minimally passaged human tumors, this antibody inhibits the growth of a range of tumor types, reduces tumor-initiating cell frequency, and exhibits synergistic activity with standard-of-care chemotherapeutic agents.

Fig. 1.

Anti-Fzd antibody OMP-18R5 inhibits Wnt signaling. (A) OMP-18R5 antibody inhibits recombinant Wnt3A signaling as assessed by β-catenin responsive TOP-FLASH luciferase reporter and a FOP-FLASH control reporter. (B) OMP-18R5 antibody (50 nM) inhibits the ability of several Wnts to induce canonical signaling. Shown is relative luciferase signal normalized to control Renilla luciferase and relative to signal in the absence of Wnt (no Wnt). Bars represent the mean ± SD. (C) OMP-18R5 blocks phosphorylation of LRP6 and the accumulation of active β-catenin. HEK-293 cells were treated with purified Wnt3A and OMP-18R5, as indicated. Cells were lysed in the presence of phosphatase inhibitor and Western blot analysis was performed. OMP-18R5 inhibited the induction of phosphorylated (Ser1490) LRP6 by Wnt3A and attenuated the Wnt induced accumulation of β-catenin and unphosphorylated (Ser37, Thr41) β-catenin.

Fig. 2.

Anti-Fzd antibody OMP-18R5 binds to 5 of the 10 human Fzd receptors and inhibits Wnt binding. (A) Binding curves of OMP-18R5 to cells that overexpress the indicated Fzd. Binding was assessed by flow cytometry analysis with cells transfected with cDNA encoding the indicated FZD protein. (B) Epitope mapping of OMP-18R5 to FZD8. Expression vectors comprising N-terminal FLAG-tagged FZD8 variants encoding the indicated amino acid substitution were transiently transfected along with GFP and OMP-18R5 binding was assessed by flow cytometry. (C) Surface rendering of the cysteine-rich domain of FZD8 (18), highlighting important residues involved in OMP-18R5 binding in green and highly conserved residues in pink. (D) Binding of OMP-18R5 to FZD inhibits the interaction of Wnt with FZD. Wnt3A, FZD5-Fc and OMP-18R5 were coincubated as indicated, and then OMP-18R5 and FZD5-Fc were removed by protein A immunoprecipitation. Supernatant was then assayed for Wnt activity in an 8xTCF luciferase reporter assay.

Fig. 3.

Inhibition of canonical Wnt signaling inhibits tumor growth as monotherapy and in combination with chemotherapy. Results of nine tumor models: colon tumor C28 (A), breast tumor T3 (B), lung NSCLC Lu24 (C), pancreatic tumor PN8 (D), breast tumor PE13 (E), teratocarcinoma line PA1 (F), pancreatic tumor PN4 (G), breast tumor PE13 (H), and colon tumor C28 (I). Mean tumor volumes with SEs are presented (n = 10). (G and H) Duration of chemotherapy and antibody treatment is indicated by brackets. Asterisks denote statistical significance P < 0.02 (* vs. control Ab; ** vs. chemotherapy alone).

Fig. 4.

Inhibition of Wnt signaling in pancreas tumor induces mucininous differentiation and decreased nuclear β-catenin and cellular proliferation. Shown is staining of sections of pancreatic tumor PN4 with Alcian blue to highlight mucin (A and B), and with anti–β-catenin (C and D) and with anti-K_i-67 to indicate proliferating cells (E and F). Sections are from tumors following treatment (41 d) with control antibody (A, C, and E) or OMP-18R5 (B, D, and F).

Fig. 5.

Expression changes induced by Wnt pathway inhibition. Shown is the relative mRNA expression (normalized to control antibody and to human control mRNA GAPDH) for the indicated human genes from pancreatic PN4 tumors treated with OMP-18R5, gemcitabine, or the combination. Data shown is average from four tumors in each treatment group analyzed individually. Shown are genes with statistically significant (Student’s t test P < 0.05) expression difference in response to OMP-18R5 or combination treatment relative to either control or gemcitabine treatment arms) with the exception of CDH1 which is included for completeness.

Fig. 6.

Inhibition of Wnt reduces tumorigenicity and induces the presence of nontumorigenic cells. Limiting dilution analysis of the ability of human cells isolated from treated pancreatic PN4 tumor (A) and breast tumor PE13 (B) to initiate tumor growth in the absence of further treatment following secondary transplant to a new cohort of mice. The primary tumor was treated with control antibody or OMP-18R5 or chemotherapy, as indicated. The tumor initiating frequency (TIC) refers to the average number of cells determined to be required to cause tumor growth in the recipient cohort.