Our previous gene expression study of human HCC identified GPC3 as the most highly overexpressed membrane-bound protein in HCC compared with nontumor liver and the second most highly overexpressed secretory protein (after AFP) [34
]. Until lately, much of the focus on GPC3 has been on its diagnostic potential. In this study, we validated the therapeutic potential of GPC3 in HCC and reported the involvement of TGF-β2 in GPC3-mediated signaling in HCC cells. Specifically, we showed that suppression of GPC3 in HCC cells enhanced TGF-β2 expression and signaling, which inhibited cell proliferation and cell cycle progression, and induced replicative senescence.
GPC3 is a rational target for the treatment of HCC because it is predominantly expressed in HCC tumors compared with its adjacent nontumor or cirrhotic tissues [5
], implying specificity. In addition, it potentially regulates multiple pathways involved in hepatocarginogensis [20,22–24
], implying a broad spectrum of activity. Several recent studies have investigated different approaches of targeting GPC3, such as the use of anti-GPC3 monoclonal antibody [35
] and HLA-A2- and -A24-restricted GPC3-derived peptide for the immunotherapy for HCC [36
]. These approaches are based on the induction of antibody-dependent cellular cytotoxicity and/or complement-dependent cytotoxicity and on the peptide induction of cytotoxic T lymphocytes, respectively, which, in turn, reduced HCC tumor mass. A mutated, soluble form of GPC3 was also reported to inhibit Wnt signaling in HCC cells, leading to antitumor effects [37
]. Our study demonstrates for the first time that targeting GPC3 at the translational level in GPC3-positive HCC cells activates TGF-β signaling, which, in turn, partially mediates the antitumor effects of GPC3 suppression in HCC cells in vitro
and in vivo
The TGF superfamily, including TGF-β, activin, and BMPs, modulates many cellular responses, such as cell division, differentiation, and cell fate decision [38
]. Three different isoforms of TGF-β, TGF-β1, TGF-β2, and TGF-β3, have similar but not identical biologic activities [39
]. TGF-β signaling is mediated through the binding of TGF-β with TGF-β receptors (TGFBR), which, in turn, recruit and phosphorylate downstream receptor-regulated SMADs (R-SMADs), SMAD2 or SMAD3. Activation of R-SMADs may form signaling complexes with SMAD4 and translocate to the nucleus eliciting tumor suppressive or oncogenic effects [40
]. Whereas TGF-β/SMAD signaling can be both promoting and suppressing, its role in HCC progression is not completely understood [41
]. Recently, TGF-β1 was reported to induce cellular senescence and inhibit tumor growth in HCC cell lines by a p53-independent and p21Cip1
-dependent pathway [33
Our results indicate that the antiproliferative effects of GPC3 in HCC cells are partially mediated by TGF-β signaling. Suppression of GPC3 in HCC cells overexpressing GPC3 inhibited cell proliferation associated with an increase in phosphorylation of SMAD2/3 and also arrested cell cycle progression at the G1 phase, associated with down-regulation of cyclin A and cyclin D1, accumulation of p15Ink4b and p21Cip1, and down-regulation of Rb phosphorylation. GPC3 suppression also caused an accumulation of SA-β-Gal, an indicator of replicative senescence. These cellular changes were recapitulated by the addition of rhTGF-β2 to the HCC cells in culture, confirming the involvement of TGF-β2 in cell cycle arrest and replicative senescence. Moreover, the cotransfection of siRNAs against GPC3 and TGF-β2 partially reversed these effects on cell proliferation, cell cycle progression, replicative senescence, and the associated molecular changes. We further observed an inverse correlation between the expression of GPC3 and TGF-β2, as GPC3 suppression upregulated TGF-β2 at both transcriptional and translational levels. Thus, GPC3 may inversely regulate TGF-β2, which, in turn, partially mediates the subsequent cellular and molecular changes observed in HCC cells on GPC3 suppression.
The two HCC cell lines studied, HepG2 and Huh7, responded to different extents toward GPC3 suppression, probably because of the different genetic contexts between the two cell lines. The poorer response of Huh7 cells may be explained by the following reasons. First, Huh7 cells have shorter cell doubling time (24 hours) than HepG2 cells (33 h) and have a lower percentage of cells in the G1 phase (56.39% ± 2.47%) than HepG2 cells (64.57% ± 2.84%) when cultured in Dulbecco modified Eagle medium supplemented with 10% FBS (P
= .01). The shorter doubling time of Huh7 cells may allow the cells to escape faster from the effect of transient siRNA knockdown. Second, we observed that Wnt signaling was inactivated in HepG2 cells but not in Huh7 cells after GPC3 suppression (Figure W1
). Cooperatively, the regulation of Wnt and TGF-β signaling pathways by GPC3 in HepG2 cells may lead to greater inhibitory effects on cell growth and cell cycle progression in this cell line. Third, in Huh7 cells, the ERK pathway might be the predominant pathway regulating proliferation that is affected by GPC3 suppression (Figure W1D
), and therefore, we observed a negligible effect of TGF-β2 siRNA on reversing cell growth inhibition caused by GPC3 suppression. Fourth, Huh7 cells have endogenous TGF-β2 expression but HepG2 cells do not. The endogenous expression of TGF-β2 may mask the effect contributed by the low and transient increase in TGF-β2 levels caused by GPC3 suppression.
In summary, we have shown that suppression of GPC3 activates the TGF-β signaling pathway in HCC cells, eventually inhibiting cell proliferation, arresting cell cycle progression at the G1
phase, and inducing replicative senescence. The extent and biologic effects of GPC3 suppression vary, depending largely on the genetic context of the cells, which reflects the heterogeneity of HCC tumors. Combined with previous reports that interference with GPC3 expression additionally regulates at least two major signaling pathways (Wnt/β-catenin and ERK/MAPK) in HCC, we suggest that interfering with GPC3 expression and function exert broad-spectrum biologic effects that might be beneficial for the treatment of the heterogeneous subtypes of HCC. Targeting GPC3 in HCC is widely applicable because it is overexpressed in a large percentage (>60%) of HCC patients [4
]. Further studies of the mechanisms by which GPC3 regulates multiple signaling pathways may reveal other points of intervention for this important target.