Abstract
Growth arrest DNA damage-inducible gene 45 (GADD45) family proteins play a crucial role in regulating cellular stress responses and apoptosis. The present study explored the prognostic and predictive role of GADD45γ in hepatocellular carcinoma (HCC) treatment. GADD45γ expression in HCC cells was examined using quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. The control of GADD45γ transcription was examined using a luciferase reporter assay and chromatin immunoprecipitation. The in vivo induction of GADD45γ was performed using adenoviral transfer. The expression of GADD45γ in HCC tumor tissues from patients who had undergone curative resection was measured using qRT-PCR. Sorafenib induced expression of GADD45γ mRNA and protein, independent of its RAF kinase inhibitor activity. GADD45γ induction was more prominent in sorafenib-sensitive HCC cells (Huh-7 and HepG2, IC50 6-7 μM) than in sorafenib-resistant HCC cells (Hep3B, Huh-7R, and HepG2R, IC50 12-15 μM). Overexpression of GADD45γ reversed sorafenib resistance in vitro and in vivo, whereas GADD45γ expression knockdown by using siRNA partially abrogated the proapoptotic effects of sorafenib on sorafenib-sensitive cells. Overexpression of survivin in HCC cells abolished the antitumor enhancement between GADD45γ overexpression and sorafenib treatment, suggesting that survivin is a crucial mediator of antitumor effects of GADD45γ. GADD45γ expression decreased in tumors from patients with HCC who had undergone curative surgery, and low GADD45γ expression was an independent prognostic factor for poor survival, in addition to old age and vascular invasion. The preceding data indicate that GADD45γ suppression is a poor prognostic factor in patients with HCC and may help predict sorafenib efficacy in HCC.
Highlights
Sorafenib, a multikinase inhibitor, is the current standard systemic therapy for patients with advanced hepatocellular carcinoma (HCC) [1]
GADD45γ induction was more prominent in sorafenibsensitive HCC cells than in sorafenib-resistant HCC cells and was independent of the mitogen-activated protein kinase-extracellular signalregulated kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling in HCC cells because the RAF inhibitor, ZM336372, or the MEK inhibitor, U0126, could not induce GADD45γ expression (Figure 1A and Supplementary Figure S1)
The suppression of GADD45γ expression by using siRNA increased the resistance to sorafenib in sorafenibsensitive HCC cells; this was evident with the increased IC50 and reduced sorafenib-induced apoptosis (Figure 1B and Supplementary Figure S2A)
Summary
A multikinase inhibitor, is the current standard systemic therapy for patients with advanced hepatocellular carcinoma (HCC) [1]. The antitumor mechanisms of sorafenib may involve complex interactions in cellular signaling pathways, which are independent of the inhibitory effects of sorafenib on Raf/MEK/ERK activities [3,4,5,6] Clarification of these “off-target effects” of sorafenib will facilitate identification of predictive biomarkers for treatment efficacy and combination therapy design for HCC [3]. Growth arrest DNA damage-inducible gene 45 (GADD45) family proteins have been reported to play essential roles in cellular stress response, survival, senescence, and apoptosis regulation [7] Both tumorpromoting and tumor-suppressing effects of GADD family proteins have been reported, depending on the cell types tested, the types of oncogenic stresses, and the interaction with other cellular signaling pathways [7].
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