Wnt Inactivation for Liver Cancer Therapy
Treatment with the new human monoclonal antibody, HS20, blocks the Wnt/β-catenin signaling pathway and inhibits hepatocellular carcinoma tumor growth in mice.
Hepatocellular carcinoma (HCC) is the fifth most common and third most deadly type of cancer in the world. The majority of cases occur in Asia and Africa, resulting in most cases being diagnosed only at advanced stages of the disease when drug resistance is high. HCC typically follows damage to the liver such as cirrhosis, making radiation and chemotherapy a more challenging prospect. Surgery is also not a very viable option because less than one in four carcinomas can be completely removed. The limitations in these treatment modalities create the need for alternative therapeutic approaches.
One potential avenue of research in HCC treatment has been the Wnt signaling pathway. More than 95 percent of patients with HCC have either upregulation in the expression of the signaling protein Wnt or its receptor Frizzled, or down-regulation of Frizzled receptor inhibitors, making the Wnt pathway a potentially important therapeutic target. Current strategies for disrupting Wnt signaling include monoclonal antibodies against the Wnt protein or the Frizzled receptor and using soluble receptors for Frizzled to neutralize Wnt.
Another approach to attacking Wnt is focusing on molecular partners of the signaling pathway. One candidate molecule is GPC3—one of many heparan sulfate proteoglycans (HSPGs) that act as Wnt co-receptors or modulators and which participate in tumor proliferation and invasion, angiogenesis, and metastasis. GPC3, which is comprised of a core protein and two heparan sulfate (HS) chains, is known to attract Wnt to the cell surface and promote cell proliferation. GPC3 is also expressed in 70 to 100 percent of HCCs, but not in adult normal tissue. A soluble form of the molecule is also known to inhibit tumor growth.
Antibodies against GPC3 have been created for liver cancer therapies, however none has been shown to disrupt Wnt signaling. In search of a novel therapeutic approach to HCC, Mitchell Ho, Ph.D., Head of the Antibody Therapy Section in CCR’s Laboratory of Molecular Biology, and his colleagues developed antibodies to target GPC3’s HS chains, which have been shown to bind a wide variety of growth factors and proteins including Wnt.
Ho’s team first demonstrated the expression of Wnt3a and its receptors, as well as its ability to promote cell proliferation, in a variety of HCC cell lines. They then used mRNA and protein analysis to determine that five of the six HCC cell lines they tested expressed GPC3. Overexpression of GPC3 in one of those lines led to increased Wnt activity in a dose-dependent manner. In contrast, knockdown of GPC3 decreased the Wnt response.
Next, the researchers attempted to isolate an antibody to neutralize GPC3 using phage display, in which a virus is coaxed to display a human antibody on its surface in order to attract binding partners. They found that GPC3 bonded most strongly to a human monoclonal antibody called HS20. Furthermore, they observed that HS20 did not bind to the isolated core protein of GPC3, indicating that the HS chains contain the binding site for the antibody. The team then treated HCC cell lines with HS20 and observed a significant, dose-dependent reduction in Wnt activity. They also observed a significant disruption in Wnt3a-dependent cell proliferation.
HS20 showed significant antitumor activity in nude mice subcutaneously injected with HCC cells. In vivo toxicology studies indicated no toxicity related to HS20. HS20-treated tumors also had less staining for β-catenin, a molecule whose involvement in the canonical Wnt pathway sees it translocated to the nucleus to act as a transcription factor co-activator. Knocking down GPC3 made tumors grow more slowly. Treating these knockdown mice with HS20 eliminated the inhibition of tumor growth, suggesting that inhibition of HCC tumor growth by HS20 is dependent on the presence of GPC3.
Taken together, the study by Ho and colleagues demonstrates the development of a monoclonal antibody, HS20, against a molecule involved in the Wnt signaling pathway, providing a potential therapy for HCC.Summary Posted: Wed, 01/01/2014
Gao W, Kim H, Feng M, Phung Y, Xavier CP, Rubin JS, Ho M. Inactivation of Wnt signaling by a human antibody that recognizes the heparan sulfate chains of glypican-3 for liver cancer therapy. Hepatology, January 6, 2014 PubMed Link