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Multidrug resistance is one of the reasons for low survival of advanced hepatocellular carcinoma (HCC). Our previous studies indicate that the hedgehog signalling is involved in hepatic carcinogenesis, metastasis and chemo‐resistance. The present study aims to uncover molecular mechanisms underlying hepatoma chemo‐resistance. TAP1 and GLI1/2 gene expression was assessed in both poorly differentiated hepatoma cells and HCC specimens. Potential GLI‐binding site in the TAP1 promoter sequence was validated by molecular assays. Approximately 75% HCC specimens exhibited an elevated expression of hedgehog GLI1 transcription factor compared with adjacent liver tissue. Both GLI1/2 and TAP1 protein levels were significantly elevated in poorly differentiated hepatoma cells. Both Huh‐7‐trans and Huh‐7‐DN displayed more karyotypic abnormalities and differential gene expression profiles than their native Huh‐7 cells. Sensitivity to Sorafenib, doxorubicin and cisplatin was remarkably improved after either GLI1 or TAP1 gene was inhibited by an RNAi approach or by a specific GLI1/2 inhibitor, GANT61. Further experiments confirmed that hedgehog transcription factor GLI1/2 binds to the TAP1 promoter, indicating that TAP1 is one of GLI1/2 target genes. In conclusion, TAP1 is under direct transcriptional control of the hedgehog signalling. Targeting hedgehog signalling confers a novel insight into alleviating drug resistance in the treatment of refractory HCC. [ABSTRACT FROM AUTHOR]

The high expression of sonic hedgehog ligand (SHh) is closely correlated to the metastasis, drug resistance and poor prognosis of hepatocellular carcinoma (HCC). Therefore, sensitive, specific and efficient detection methods for SHh are needed for the early diagnosis and assessment of prognosis. Herein, an aptamer, AP32 that specifically binds to SHh (K D = 25.7 ± 4.1 nM) was obtained by SELEX technology with further optimization. In vivo experiments confirmed that AP32 has the potential to be an imaging probe for Huh-7 cell-derived xenograft. The interaction mode in 3-dimensional configuration between the aptamer and SHh was established by molecular simulation and confirmed by mutations at key sites of the aptamer. An aptasensor-based assay was successfully developed by conjugating Texas-Red-labeled AP32 to microbeads, and was used to analyze SHh content in hepatoma cell lysates, serum and HCC specimens. The method exhibited a broad detection range from 0.07 to 62.5 nM with a low detection limit of 69 pM, and a recovery rate of 104.6 ± 3.9% in serum. When the assay was used to measure SHh content in tissue lysates, the results demonstrated that it possessed 57.1% positivity, 100% specificity in distinguishing 28 HCC specimens from normal tissues, and was compensatory for detection of HCC in AFP-negative cases. Moreover, elevated SHh levels are indicative of portal vein invasion at 77.8% positive rate. This novel aptasensor-based SHh assay may offer a reliable means in predicting early metastasis and poor prognosis in hepatocellular carcinoma. Image 1 • A specific aptamer was identified to detect sonic ligand of the hedgehog signaling with an aptasensor-based assay. • The assay detects SHh content at pM level in solid or liquid phases of tissue. • Increased SHh in HCC was distinguishable from normal tissue, and is compensative for negative AFP value. • Elevated SHh levels in HCC specimens are indicative of portal vein invasion. • This assay represents a robust means in the prediction of early metastasis and poor prognosis for HCC. [ABSTRACT FROM AUTHOR]

Hedgehog (Hh) signaling is a classic morphogen in controlling embryonic development and tissue repairing. Aberrant activation of Hh signaling has been well documented in liver cancer, including hepatoblastoma, hepatocellular carcinoma (HCC) and cholangiocarcinoma. The present review aims to update the current understanding on how abnormal Hh signaling molecules modulate initiation, progression, drug resistance and metastasis of HCC. The latest relevant literature was reviewed with our recent findings to provide an overview regarding the molecular interplay and clinical relevance of the Hh signaling in HCC management. Hh signaling molecules are involved in the transformation of pre-carcinogenic lesions to malignant features in chronic liver injury, such as nonalcoholic steatohepatitis. Activation of GLI target genes, such as ABCC1 and TAP1, is responsible for drug resistance in hepatoma cells, with a CD133−/EpCAM− surface molecular profile, and GLI1 and truncated GLI1 account for the metastatic feature of the hepatoma cells, with upregulation of matrix metalloproteinases. A novel bioassay for the Sonic Hh ligand in tissue specimens may assist HCC diagnosis with negative α-fetoprotein and predict early microvascular invasion. In-depth exploration of the Hh signaling deepens our understanding of its molecular modulation in HCC initiation, drug sensitivity and metastasis, and guides precise management of HCC on an individual basis. [ABSTRACT FROM AUTHOR]

A couple of molecular-targeting medications, such as Lenvatinib, are available for the treatment of hepatocellular carcinoma (HCC) in addition to Sorafenib in an advanced stage. Approval for the use of immune check-point inhibitors, such as Nivolumab and Pembrolizumab has shifted the paradigm of current HCC treatment, and the monotherapy or in combination with Lenvatinib or Sorafenib has significantly extended overall survival or progression-free survival in a large portion of patients. A combination of programmed cell death ligand-1 (PD-L1) inhibitor Atezolizumab with a vascular endothelial growth factor (VEGF) inhibitor, Bevacizumab, has recently achieved promising outcome in unresectable HCC patients. Other immunotherapy, such as chimeric antigen receptor T (CAR-T) cell therapy has achieved an evolutional success in hematologic malignancies, and has extended its use in deadly solid tumors, such as HCC. Although there exist various barriers, novel approaches are developed to move potential adoptive T cell therapy strategies, including cytokine-induced killer (CIK) cells, tumor-infiltrating lymphocytes (TIL), T cell receptor (TCR) T cells, CAR-T cells, to clinical application. Unlabelled Image [ABSTRACT FROM AUTHOR]

Hedgehog signaling is activated in response to liver injury, and modulates organogenesis. However, the role of non-canonical hedgehog activation via TGF-β1/SMAD3 in hepatic carcinogenesis is poorly understood. TGF-β1/SMAD3-mediated non-canonical activation was found in approximately half of GLI2-positive hepatocellular carcinoma (HCC), and two new GLI2 isoforms with transactivating activity were identified. Phospho-SMAD3 interacted with active GLI2 isoforms to transactivate downstream genes in modulation of stemness, epithelial-mesenchymal transition, chemo-resistance and metastasis in poorly-differentiated hepatoma cells. Non-canonical activation of hedgehog signaling was confirmed in a transgenic HBV-associated HCC mouse model. Inhibition of TGF-β/SMAD3 signaling reduced lung metastasis in a mouse in situ hepatic xenograft model. In another cohort of 55 HCC patients, subjects with high GLI2 expression had a shorter disease-free survival than those with low expression. Moreover, co-positivity of GLI2 with SMAD3 was observed in 87.5% of relapsed HCC patients with high GLI2 expression, indicating an increased risk of post-resection recurrence of HCC. The findings underscore that suppressing the non-canonical hedgehog signaling pathway may confer a potential strategy in the treatment of HCC. [Display omitted] • Hedgehog signaling transcription factor GLI2 levels correlate with HCC recurrence. • Non-canonical activation by TGF-β/SMAD3 was found in 50% of GLI2-positive HCC samples. • SMAD3 interacted with active GLI2 isoforms to drive HCC progression. • Two newly identified GLI2 isoforms possess transactivating activity. [ABSTRACT FROM AUTHOR]