Soluble glypican 3 inhibits the growth of hepatocellular carcinoma in vitro and in vivo

Abstract

The heterogeneity of the molecular pathology of HCC poses a formidable obstacle to the development of non-cytotoxic therapies. Several pro-tumorigenic signaling pathways can be aberrantly activated in HCC, including those triggered by Wnts. Glypican-3 (GPC3), a membrane-bound heparan sulfate proteoglycan that is overexpressed in most HCCs, promotes the growth of these tumors by stimulating Wnt signaling. Because GPC3 binds with high affinity to Wnts, and its growthpromoting activity requires attachment to the cell membrane, we have hypothesized that a mutated GPC3 lacking the GPI anchoring domain (sGPC3) will block Wnt signaling and inhibit the growth of Wnt-dependent tumors. In addition, because sGPC3 displays heparan sulfate chains, this secreted glypican could also inhibit HCC growth by blocking the activity of other heparin-binding growth factors. To test this hypothesis, HCC cell lines were infected with an sGPC3-expressing lentivirus or virus control, and the effect of sGPC3 on the in vitro and in vivo growth was investigated. In addition, the signaling pathways targeted by sGPC3 were identified. We observed that sGPC3-expressing cells had lower proliferation rate. In addition, sGPC3 significantly inhibited the in vivo growth of the Huh6, HepG2 and Huh7 HCC cell lines. sGPC3 blocked Wnt signaling in Huh6-and Huh7-derived tumors and Erk1/2 and Akt phosphorylation in tumors generated by Huh7 and HepG2 cells, respectively. An anti-angiogenic effect in Huh7 and HepG2-derived tumors was also observed. We conclude that sGPC3 can inhibit HCC tumorigenicity by blocking the activity of several pro-tumorigenic growth factors.

Glypican-3 binds to frizzled and plays a direct role in the stimulation of canonical Wnt signaling

“…Our laboratory has recently reported that a mutated GPC3 that is secreted into the extracellular environment because it cannot be anchored to the cell membrane can inhibit canonical Wnt signaling in various HCC cell lines (Capurro et al, 2005;Zittermann et al, 2010). The results presented here are consistent with our previous report.…”

Stromal heparan sulfate differentiates neuroblasts to suppress neuroblastoma growth

“…Lentiviral vectors consisted of the same construct as those used in adenoviral vectors cloned into a pSMPUW-Neo backbone (TβRIII constructs) or a pLKO.1-puro backbone (TβRIII shRNA construct and nontargeted control). The DNA constructs for GPC3 and sGPC3 (lacking the GPI membrane anchor) were gifts of Jorge Filmus (University of Toronto, Toronto, Ontario, Canada) (74). Transient DNA transfections were performed using Lipofectamine (Invitrogen) according to the manufacturer's instructions.…”

Therapeutic Potential of Targeting Glypican-3 in Hepatocellular Carcinoma

“…GPC3 binds to Wnt through its core protein, and needs to be attached to the cell membrane to be able to display its growth-promoting activity. Based on these findings, it was hypothesized that GPC3 stimulates Wnt signaling by facilitating and/or stabilizing the interaction of Wnt with Frizzled (Fz), its signaling receptor, and that a secreted version of GPC3 would inhibit autocrine and/or paracrine Wnt signaling by removing this growth factor from the cell surface [47]. Furthermore, it was proposed that because sGPC3 displays heparan sulfate chains, this soluble glypican could also block the activity of other heparin-binding growth factors that are produced by HCC tumors.…”