Hedgehog Signal Inhibitor GANT61 Inhibits the Malignant Behavior of Undifferentiated Hepatocellular Carcinoma Cells by Targeting Non-Canonical GLI Signaling

Abstract

The Hedgehog (HH)–GLI pathway plays an important role in cell dedifferentiation and is therefore pivotally involved in the malignant transformation of cancer cells. GANT61, a selective inhibitor of GLI1 and GLI2, was reported as a promising treatment for cancer in various tissues; however, the biological impact of GANT61 in hepatocellular carcinoma (HCC), especially in undifferentiated HCC cells, remains unclear. In this study, we investigated the antitumor effect of GANT61 using two undifferentiated hepatoma cell lines: HLE and HLF. Quantitative PCR and RT-PCR analyses revealed that these cells express GLI transcripts, showing mesenchymal phenotypes characterized by the loss of epithelial and hepatic markers and specific expression of epithelial–mesenchymal transition (EMT)-related genes. GANT61 significantly reduced the proliferation and cell viability after drug treatment using 5-FU and Mitomycin C. We showed that GLI transcript levels were down-regulated by the MEK inhibitor U0126 and the Raf inhibitor sorafenib, suggesting that non-canonical signaling including the Ras–Raf–MEK–ERK pathway is involved. Sphere formation and migration were significantly decreased by GANT61 treatment, and it is suggested that the underlying molecular mechanisms are the down-regulation of stemness-related genes (Oct4, Bmi1, CD44, and ALDH) and the EMT-related gene Snail1. The data presented here showed that direct inhibition of GLI might be beneficial for the treatment of dedifferentiated HCC.

Hedgehog/GLI1 Transcriptionally Regulates FANCD2 in Ovarian Tumor Cells: Its Inhibition Induces HR-Deficiency and Synergistic Lethality with PARP Inhibition.

“…Recent studies identified novel non-canonical regulation of GLI in various cancers. Especially, nuclear myocardin-related transcription factor in basal cell carcinoma [ 57 ], deregulated GSK3β in colon cancer cells [ 58 ], Ras–Raf–MEK ERK pathway in hepatocellular carcinoma [ 59 ], fibroblast-derived insulin-like growth factor-1 in oral squamous cell carcinoma [ 60 ], MAPK/ERK signaling in lung adenocarcinoma [ 61 ], transcriptionally-active androgen receptors in pancreatic cancer [ 62 ] and SOX2-BRD4 transcriptional complex in melanoma [ 63 ]. Additionally, studies in breast cancer models, EMT cells induce increased metastasis of weakly metastatic, non-EMT tumor cells in a paracrine manner, in part by non-cell autonomous activation of the GLI transcription factor.…”

Mechanism of Targeting the Hedgehog Signaling Pathway against Chemotherapeutic Resistance in Multiple Myeloma

“…Currently, known inhibitors of the Hedgehog signaling pathway include GANT61, cyclopamine, and GANT58 [ 26 ]. GANT61 specifically blocks GLI1 in the Hedgehog signaling pathway and inhibits cell transcription and expression [ 27 ]. Multiple myeloma cells were treated with GANT61 as a Hedgehog signaling pathway blocker.…”

HDAC9 Is Preferentially Expressed in Dedifferentiated Hepatocellular Carcinoma Cells and Is Involved in an Anchorage-Independent Growth

“…We observed a minimum expression of HDAC9 in the normal hepatic cell line HC and well-differentiated HepG2 and HuH1 hepatoma cells that retain a hepatic phenotype, such as hepatic gene expression and drug-metabolizing activities. In contrast, HDAC9 mRNA was positively detected in HLE and HLF cells, typical undifferentiated HCC cell lines that show a mesenchymal phenotype [ 14 , 15 , 16 ]. Consistent with previous reports, class I HDACs were ubiquitously expressed among the cell lines tested, suggesting that HDAC9, but not class I HDACs, may play a specific role in undifferentiated HCC cells [ 17 ].…”

“…CSCs are a sub-population of tumor cells responsible for their initiation, recurrence, and metastasis. Previously, we observed a 0.1–1.0% sphere formation rate in HLE and HLF cells seeded in a low-attachment dish [ 16 ]. In this study, HDAC9 suppression significantly reduced the growth of these spheres in both cell lines.…”