Frizzled-7-targeting antibody (SHH002-hu1) potently suppresses non–small-cell lung cancer via Wnt/β-catenin signalling

Non-small-cell lung cancer (NSCLC) makes up for ~85% of lung cancer patients, which is the most frequent cause of cancer-related death worldwide. Although, new targeted therapies and personalised medicine have improved outcome, most patients eventually develop progressive disease. Furthermore, acquired resistance to such treatments, limits their long-term efficacy.

The Wnt/β-catenin pathway has been broadly implicated in human cancers, including NSCLC. The aberrant activation of Wnt/β-catenin signaling is tightly linked to the increased prevalence, advancement of malignant progression, and the development of poor prognostics. Metastasis is considered one of the leading causes of treatment failure and death in NSCLC patients, and the Wnt/β-catenin pathway is demonstrated to be correlated strongly with the epithelial-to-mesenchymal transition (EMT).

The epithelial-to-mesenchymal transition (EMT) occurs during normal embryonic development, tissue regeneration, organ fibrosis, and wound healing. It is a highly dynamic process, by which epithelial cells can convert into a mesenchymal phenotype. However, EMT is also involved in tumor progression with metastatic expansion, and the generation of tumor cells with stem cell properties that play a major role in resistance to cancer treatment.

Frizzled-7 (Fzd7) is a key protein expressed on the surface of endothelial cells that controls vascular permeability through the Wnt-canonical pathway. Fzd7 is upregulated along the Wnt/ β-catenin signaling pathway in NSCLC. Given the important role that Fzd7 plays in tumorigenesis and progression, to date, several methods have been designed to antagonize Wnt/β-catenin signaling by targeting Fzd7.

In a previous study, Xie et al generated a humanized antibody targeting Fzd7 with extremely high affinity (SHH002-hu1). In the study presented here, the same group focused on the anti-tumor effects of SHH002-hu1 against NSCLC and the underlying mechanism.

First, they established that the antibody specifically and selectively bound to Fzd7 expressed on the cell surface of the NSCLC cell lines A549 and H1975 in vitro using immunofluorescence and in vivo on xenograft tumours established from the same cell lines using near-infrared imaging. The selectively of SHH002-hu1 was confirmed using siRNA knockdown where A549/H1975 cells showed little binding with SHH002-hu1 when the Fzd7 expression was suppressed by siFzd7.

They continued with performing wound healing, trans-well invasion and proliferation assays, to investigate whether SHH002-hu1 could inhibit the migration and the invasion of A549/H1975 cells and if antibody treatment had any effect on proliferation. They showed that SHH002-hu1 inhibited the migration and invasion of NSCLC cells in an Fzd7-dependent manner, whilst exhibiting little effect on A549/H1975 cell proliferation under serum starvation at the antibody concentration range tested. Interestingly, a colony forming assay conducted in medium containing 10% serum revealed that 100nM SHH002-hu1 or siRNA knockdown of Fzd7 markedly decreased colony formation and growth.

In subsequent experiments, Li et al demonstrated that:

• SHH002-hu1, as well as siFzd7 markedly decreased the transcriptional activity stimulated by Wnt3a, by suppressing the nuclear β-catenin accumulation and translocation from the cytoplasm induced by Wnt3a, which is required for the transcriptional activity of β-catenin. Furthermore, SHH002-hu1 or siFzd7 downregulated the total β-catenin protein expression.
• Additional assays concluded that SHH002-hu1 strongly attenuated the Wnt/β-catenin signaling pathway induced by Wnt3a by competing with Wnt3a for Fzd7 binding.
• Treatment with SHH002-hu1 at 5mg/kg resulted in a significant inhibition of NSCLC xenograft tumor growth relative to PBS control.
• Immunohistochemistry (IHC) and IF staining of SHH002-hu1 treated tumours showed an increased expression of the epithelial marker E-cadherin, and a decreased expression of the proliferation marker Ki-67 as well as the mesenchymal marker N-cadherin suggesting the potential of SHH002-hu1 to impair EMT of NSCLC in vivo.
• In addition, CD31 staining was decreased indicated that SHH002-hu1 significantly inhibited NSCLC angiogenesis.
• A popliteal lymph node metastasis model, where A549/H1975 cells were implanted in the footpad of nude mice, showed decreased popliteal lymph node engagement after treatment with SHH002-hu1 compared to control. Further analysis revealed downregulated expression of nuclear β-catenin as well as total β-catenin in lymph nodes from SHH002-hu1 treated mice.

In this study, the authors show that the antibody SHH002-hu1 exerts excellent targeting toward Fzd7+ NSCLC cells and NSCLC tumor tissue, enabling specific Wnt/β-catenin signaling inhibition, resulting in a significant attenuation of the EMT. In addition, SHH002-hu1 exhibited superior anti-tumor activities by inhibiting the growth of NSCLC xenografts and suppressing the LN metastasis of NSCLC. Furthermore, the authors show that the SHH002-hu1 antibody exhibited antiangiogenic activities against NSCLC in vivo. Taken together, SHH001-hu1 is a potential candidate antibody drug for targeted NSCLC therapy