Targeting the Wnt pathway in human cancers: Therapeutic targeting with a focus on OMP-54F28

Abstract

The Wnt signaling pathways are a group of signal transduction pathways that play an important role in cell fate specification, cell proliferation and cell migration. Aberrant signaling in these pathways has been implicated in the development and progression of multiple cancers by allowing increased proliferation, angiogenesis, survival and metastasis. Activation of the Wnt pathway also contributes to the tumorigenicity of cancer stem cells (CSCs). Therefore, inhibiting this pathway has been a recent focus of cancer research with multiple targetable candidates in development. OMP-54F28 is a fusion protein that combines the cysteine-rich domain of frizzled family receptor 8 (Fzd8) with the immunoglobulin Fc domain that competes with the native Fzd8 receptor for its ligands and antagonizes Wnt signaling. Preclinical models with OMP-54F28 have shown reduced tumor growth and decreased CSC frequency as a single agent and in combination with other chemotherapeutic agents. Due to these findings, a phase 1a study is nearing completion with OMP-54F28 in advanced solid tumors and 3 phase 1b studies have been opened with OMP-54F28 in combination with standard-of-care chemotherapy backbones in ovarian, pancreatic and hepatocellular cancers. This article will review the Wnt signaling pathway, preclinical data on OMP-54F28 and other Wnt pathway inhibitors and ongoing clinical trials.

Introduction

Wnt genes, defined for their sequence homology to Integration 1 (INT-1) in mice (Nusse & Varmus, 1982) and its homologue Wingless (Wg) in Drosophila (Cabrera et al., 1987), were shown early on to be imperative for cell fate determination and cell polarity during development. The vital role of Wnt signaling in development was largely elucidated in Drosophila, where loss of Wg led to segment polarity defects in mutant embryos (Nusslein-Volhard & Wieschaus, 1980). Since then, Wnt signaling has been shown to be important in development and axis formation in many organisms, including nematodes, frogs, mice and humans (Bodmer et al., 1987, McMahon and Moon, 1989, Moon et al., 1993, Nusse and Varmus, 1982, Rocheleau et al., 1997, Thorpe et al., 1997).

Three Wnt signaling pathways have been defined, including the canonical, non-canonical planar cell polarity pathway and the non-canonical Wnt/Ca²⁺ pathway. Of the three, the canonical Wnt pathway is the best described. Here, a cysteine-rich Wnt ligand binds the extracellular cysteine-rich domain (CRD) at the amino terminus of a seven pass transmembrane receptor termed Frizzled (FZ/Fzd [Vinson et al., 1989, Bhanot et al., 1996]) and low-density lipoprotein (LDL) receptor-related protein 5/6 (LRP5/6) that acts as a co-receptor (Pinson et al., 2000, Tamai et al., 2000, Wehrli et al., 2000) to start the activation of the canonical Wnt signaling pathway. Nineteen Wnt ligands have been identified along with 10 Fzd receptors (Huang & Klein, 2004). Various Wnt ligands have been shown to bind to particular Fzd receptors, but this interaction is promiscuous wherein one Wnt can bind multiple Fzd receptors (Bhanot et al., 1996).

Wnt glycoproteins are relatively hydrophobic and insoluble possibly due to cysteine palmitoylation by Porcupine (PORC [Willert et al., 2003, Zhai et al., 2004]). However, PORC is required for Wnt signaling, suggesting that palmitoylation is essential in Wnt ligand secretion and pathway activation. Wnt ligands can activate signaling by both autocrine and paracrine signaling (Bafico et al., 2004). Wnt signaling can be inhibited through the binding of soluble Dickkopf (DKK) to LRP5/6 (Glinka et al., 1998) or secreted Frizzled-related protein (SFRP) binding to Wnt ligands due to their sequence homology to the CRD domain of Fzd (Hoang et al., 1996). Wnt inhibitor factor (WIF) proteins, due to their similarity to the extracellular domain of derailed/RYK Wnt transmembrane receptors, can also regulate Wnt signaling by interacting with Wnt ligands (Hsieh et al., 1999a).

When there is no Wnt ligand present, β-catenin levels are limited by the destruction complex that includes Adenomatous Polyposis Coli (APC) and AXIN. With Wnt signaling “off,” AXIN facilitates the phosphorylation of β-catenin by casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK-3 [Peifer et al., 1994, Yost et al., 1996, Sakanaka et al., 1999, Liu et al., 2002]). These phosphorylated ser/thr sites are recognized by an E3 ubiquitin ligase complex, and β-catenin is subsequently targeted for proteasomal degradation (Aberle et al., 1997). Therefore, β-catenin is maintained at low cytoplasmic and nuclear levels. In the “on” state, Wnt ligand binds the extracellular CRD of the amino terminus of Fzd and the LRP5/6 co-receptor (Dann et al., 2001, Pinson et al., 2000, Tamai et al., 2000). Dishevelled (Dsh/Dvl) is activated and recruited along with the destruction complex to the plasma membrane (Lee et al., 1999, Rothbacher et al., 2000). AXIN also interacts with the plasma membrane, possibly by binding the cytoplasmic tail of LRP5/6 (Mao et al., 2001). This binding is promoted by phosphorylation of LRP5/6 by GSK-3 and CK1 (Davidson et al., 2005, Zeng et al., 2005). AXIN is degraded, and GSK-3 is thus prevented from phosphorylating β-catenin. This leads to the accumulation of β-catenin in the nucleus and its interaction with T-cell factor (TCF) and lymphoid enhancer-binding protein (LEF) transcription factors to activated downstream targets (Behrens et al., 1996, Huber et al., 1996).

Aberrant Wnt signaling was first implicated in cancer in mouse studies, where mouse mammary tumor virus (MMTV) was found to be virally inserted into the promoter region of Int-1, promoting mammary tumors (Nusse and Varmus, 1982, Tsukamoto et al., 1988). It was later found that Int-1 was a homologue to Wg, and thus renamed Wnt (Nusse et al., 1991, Rijsewijk et al., 1987). Since this time, the Wnt pathway has been shown to be aberrantly regulated in many cancers. Abnormal β-catenin activation has been well characterized in colon cancer, where mutations in APC, or less frequently in β-catenin, results in constitutively active β-catenin and consequently active downstream effectors (Morin et al., 1997). While APC and β-catenin mutations are rare in lung cancer, overexpression of Dvl, Wnt-1 and Wnt-2 have all been correlated with non-small cell lung cancer (NSCLC) (He et al., 2004, Pongracz and Stockley, 2006, Ueda et al., 2001, Uematsu et al., 2003, You et al., 2004c). Moreover, increased tumor relapse was associated with a TCF4 Wnt gene signature in lung adenocarcinomas (Nguyen et al., 2009b). Together, these data provide strong evidence for the role of Wnt signaling in lung cancers. Wnt-5a has also been shown to be increased in breast cancer (Lejeune et al., 1995). Several of Fzds that have been shown to be overexpressed in cancers and/or cancer cell migration include Fzd4, Fzd7, Fzd8 and Fzd10 (Fukukawa et al., 2009, Jin et al., 2011, Ueno et al., 2009, Wang et al., 2012b, Yang et al., 2011). These have been shown to activate the canonical and/or non-canonical Wnt pathway. However, these are just a few of the studies linking Fzd overexpression with cancer, and an extensive list was previously covered by Ueno et al. (2013).

Wnt expression has also been associated with metastasis and tumor microenvironment. Inhibition of Wnt signaling by RNAi targeting LEF1 and HOXB9 reduced brain and bone metastasis using a mouse model of lung adenocarcinoma (Nguyen et al., 2009b). The mechanism of LEF1 and HOXB9 metastasis promotion was not elucidated in this study although Wnt signaling, specifically Wnt-1 and Wnt-5a, has been shown to increase proliferation and survival of endothelial cells (Masckauchan et al., 2005, Masckauchan et al., 2006). β-catenin was also shown to correlate with VEGF expression in colon cancer (Easwaran et al., 2003, Zhang et al., 2001), suggesting a role for Wnt signaling in angiogenesis. Moreover, Wnt-5a expression has recently been shown to be increased in NSCLC; its expression in patient tissue was correlated with expression of angiogenesis related proteins such as vascular endothelial cadherin and matrix metalloprotease 2, microvessel density and vasculogenic mimicry, all of which suggest a role for Wnt-5a in promoting angiogenesis (Yao et al., 2014).

Decreased expression of Wnt pathway inhibitors (WIF-1, DKKs, and SFRPs) “allows” for the activation of Wnt signaling and has also been observed in various cancers. For example, the down-regulation of associated Wnt antagonist, WIF-1, has been implicated in the breast, prostate, lung and bladder cancer (Wissmann et al., 2003). Furthermore, WIF-1 has been shown to be epigenetically silenced in lung and bladder cancer (Mazieres et al., 2004, Urakami et al., 2006). Epigenetic silencing of DKK-1 has been shown in colorectal cancer (Aguilera et al., 2006) and SFRP in NSCLC, hepatocellular carcinoma and colorectal cancer (Fukui et al., 2005, Shih et al., 2006, Suzuki et al., 2004). Recent studies suggest that Wnt inhibitors may also play a pro-apoptotic role, where reduced apoptosis and p53 expression were observed in mammary glands isolated from SFRP^-/- mice following induction of DNA damage by ɣ-irradiation (Gauger & Schneider, 2014). In addition, another study suggests that WIF-1 may inhibit angiogenesis. DKK-1 and WIF-1 directly interact and together may act as co-regulators in promoting apoptosis in the human umbilical vein endothelial cell (HUVEC) system (Ko et al., 2014).

Although Wnt signaling is not as well correlated with head and neck squamous cell carcinoma (HNSCC) as with other cancers, such as colon cancer, recent studies provide evidence that Wnt signaling is an attractive target in HNSCC. Wnt pathway activation has been shown in HPV positive HNSCC, possibly driven by E6 and E7 (Rampias et al., 2010). β-catenin nuclear accumulation was also observed in the majority of patient HNSCC tumor samples (Wend et al., 2013). Up-regulation of several Fzd receptors was observed in HNSCC, including Fzd1, Fzd7a, Fzd10b, Fzd2 and Fzd13 (Rhee et al., 2002). Furthermore, Wnt expression may affect radiosensitivity in HNSCC cell lines, where β-catenin nuclear accumulation was correlated with radiation-resistance (Chang et al., 2008). Similarly, radiation-resistant mouse mammary progenitor cells were associated with active Wnt signaling (Chen et al., 2007, Woodward et al., 2007). Wnt expression has been correlated with therapy resistance in prostate cancer, where Wnt16B increased following therapy and lessened DNA damage following treatment with a topoisomerase inhibitor (Sun et al., 2012). In this study, Wnt16B increased growth and proliferation. Taken together, these studies suggest that Wnt expression not only promotes cancer cell proliferation, but may also affect treatment efficacy. Furthermore, the up-regulation of Wnt16B originating specifically in the stroma compartment, and through tumor-stroma interactions promoting therapy resistance in the tumor compartment, suggests that the stroma is a favorable target for therapy. Consistent with this, human ovarian fibroblasts released Wnt16B into the stroma compartment following DNA damage by radiation or chemotherapy (Shen et al., 2014). Interestingly stromal Wnt16B activated the Wnt signaling pathway in dendritic cells (DCs), causing the release of interleukin-10 (IL-10) and tumor growth factor-β (TGF-β) and regulatory T-cell differentiation. Thus, Wnt16B may not only confer therapy resistance, but also alter the tumor microenvironment and as the authors suggest, possibly promote immune evasion.

Wnt signaling is important in stem cell homeostasis. In the intestinal villi Wnt signaling is particularly important in stem cell maintenance as well as in determining stem cell fate (Batlle et al., 2002, Korinek et al., 1998). Wnt signaling has also been shown to be essential in stem cell proliferation and hair follicle development and may function to activate stem cells in the bulge to more proliferative progenitor cells, as well as determining cell fate (Andl et al., 2002, Choi et al., 2013, Lien et al., 2014, Lowry et al., 2005). Similarly, Wnt overexpression in hematopoietic stem cells leads to the expansion of progenitor cells, suggesting that Wnt signaling is also important in hematopoiesis (Austin et al., 1997).

Aberrant Wnt signaling in the stem cell compartment has been shown to contribute to tumorigenesis. Here, it is important to note that while some authors appropriately choose conservative terminology in the definition of CSCs, for the purpose of coherency in this review, we loosely combine tumor-initiating, tumor propagating and CSCs into one term, as CSCs. Loss of APC, consequently leading to the accumulation of β-catenin, in colorectal cells resulted in cells maintaining a phenotype similar to progenitor cells of the crypt (Sansom et al., 2004). In another approach, high levels of Wnt expression were observed in CSCs from colon cancer grown as spheroids (Vermeulen et al., 2010). Similarly, Wnt-1, -3 and -5a all promoted murine mammosphere growth, a method that enriches for stem cells, and results suggested both canonical and non-canonical Wnt signaling could promote growth (Many & Brown, 2014). Furthermore, hair follicle tumors were observed to have stable expression of β-catenin in mice (Gat et al., 1998). A recent study found hair follicle stem cells (HFSC) treated with dimethylbenzanthracene (DMBA) and 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced sebaceous neoplasms in C57BL/6 mice, as well as increased Wnt10b expression in basal cells via immunostaining (Qiu et al., 2014). Here the authors propose a model wherein increased Wnt10b results in proliferation and differentiation of HFSCs and thus promoting sebaceous neoplasms. High levels of Wnt expression were also observed in granulocyte-macrophage progenitors isolated from chronic myeloid leukemia (CML) patients and correlated with increased self-renewal (Jamieson et al., 2004). Fzd4 was suggested to regulate “stemness” of cancer cells and promote invasiveness in glioma cells (Jin et al., 2011). In HNSCC cell lines, side populations sorted by Hoechst efflux, a functional assay for enriching stem cells, were more invasive and tumorigenic in nude mice, and importantly these populations exhibited higher Wnt signaling (Song et al., 2010). Together, the data suggest that the same Wnt signaling mechanisms that regulate stem cells, when abnormal, may contribute to the tumorigenic potential of CSCs.