Abstract
Transforming growth factor-beta (TGF-β) signaling has gained extensive interest in hepatocellular carcinoma (HCC). The small molecule kinase inhibitor galunisertib, targeting the TGF-β receptor I (TGF-βRI), blocks HCC progression in preclinical models and shows promising effects in ongoing clinical trials. As the drug is not similarly effective in all patients, this study was aimed at identifying new companion diagnostics biomarkers for patient stratification. Next-generation sequencing-based massive analysis of cDNA ends was used to investigate the transcriptome of an invasive HCC cell line responses to TGF-β1 and galunisertib. These identified mRNA were validated in 78 frozen HCC samples and in 26 ex-vivo HCC tissues treated in culture with galunisertib. Respective protein levels in patients blood were measured by enzyme-linked immunosorbent assay. SKIL, PMEPA1 ANGPTL4, SNAI1, Il11 and c4orf26 were strongly upregulated by TGF-β1 and downregulated by galunisertib in different HCC cell lines. In the 78 HCC samples, only SKIL and PMEPA1 (P<0.001) were correlated with endogenous TGF-β1. In ex-vivo samples, SKIL and PMEPA1 were strongly downregulated (P<0.001), and correlated (P<0.001) with endogenous TGF-β1. SKIL and PMEPA1 mRNA expression in tumor tissues was significantly increased compared with controls and not correlated with protein levels in the blood of paired HCC patients. SKIL and PMEPA1 mRNA levels were positively correlated with TGF-β1 mRNA concentrations in HCC tissues and strongly downregulated by galunisertib. The target genes identified here may serve as biomarkers for the stratification of HCC patients undergoing treatment with galunisertib.
Highlights
Standard therapy for patients with advanced hepatocellular carcinoma (HCC) is currently restricted to the use of sorafenib
As a first step to the discovery of biomarkers for companion diagnostics of galunisertib, genome-wide massive analysis of cDNA ends (MACE) transcription profiles of approximately 152 million mRNAs from the HLF cell line were obtained from three independent samples, each from either untreated controls or cells treated with TGF-β1, galunisertib and TGF-β1 plus galunisertib after short (2 h) and longer (48 h) incubation, respectively
To better resolve the time frame of TGF-β1 induction and galunisertib inhibition, respectively, we investigated the abundance of the mRNAs of the candidate genes over a wider time frame ranging from 15 min up to 48 h after treatment in three independent experiments per time point (Figure 2a)
Summary
Standard therapy for patients with advanced hepatocellular carcinoma (HCC) is currently restricted to the use of sorafenib. Smad-2 inhibition increases the E-cadherin level, reducing the migratory and invasive capabilities of HCC cells.[10,11] In addition, in different preclinical models galunisertib inhibited HCC progression, decreased tumor growth and metastasis and abolished the cross-talk between the surrounding stroma and the tumor (reviewed in Giannelli et al.[12]). Based on these data, galunisertib was tested in a phase 2 clinical trial in patients who failed to react to prior sorafenib treatment (NCT01246986). Here we aim to discover and validate new biomarkers in preclinical models of HCC for companion diagnostics of galunisertib
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