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Abstract

Background: Hepatocellular carcinoma (HCC) is an aggressive, largely chemo-resistant cancer with a poor prognosis and is the leading cause of cancer-related deaths worldwide. Currently, there is no effective systemic chemotherapy for HCC. Sorafenib is a multikinase inhibitor commonly used as the first-line treatment for advanced HCC. However, acquired drug resistance significantly limits its clinical effectiveness. The Y-box binding protein (YBX1) is an oncogenic RNA-binding protein found to be upregulated in sorafenib-resistant HCC cells and tissues.

Methods: HCC model SK Hep1, YBX1 overexpression, and knockdown cells were prepared by transient transfection with puromycin selection, and stable cell lines were established through Fluorescence activated cell sorting (FACS) with GFP selection. We then validated cholesterol precursor genes and downstream regulatory genes using qRTPCR and assessed protein levels through western blot and immunofluorescence assays. HepG2 and Huh7 sorafenib-resistant cell lines were generated and maintained at a 10µM sorafenib concentration. Additionally, qRT-PCR and western blot analyses were performed to measure the RNA and protein levels. To identify kinase pathways affected by YBX1, a phosphoproteome array was utilized.

Results: The nuclear translocation of YBX1 is associated with worse outcomes, and cholesterol metabolism pathways appear to be significantly impacted by YBX1. Transforming growth factor beta 1 (TGF-β1) signaling promotes the nuclear translocation of YBX1 and boosts its transcriptional activity. Nuclear YBX1 increases the expression of the sterol regulatory element-binding protein (SREBP2), a key transcription factor in cholesterol biosynthesis, and contributes to metabolic reprogramming and resistance to sorafenib. Overexpression of YBX1 raises SREBP2 levels, while knockdown reduces them in HCC cell line models. Additionally, YBX1 activation supports the PI3K/AKT/mTOR and RAS/RAF pathways, reinforcing cellular proliferation and drug tolerance. Kinases play a critical role in drug resistance, and activation of Akt/mTOR-associated genes was observed in YBX1 overexpression and reversed in YBX1 knockdown cell line model.

Conclusions: Collectively, these findings suggest that YBX1 contributes to sorafenib resistance in HCC through dysregulation of cholesterol metabolism and oncogenic signaling. Targeting the YBX1/TGF-β1/SREBP2 signaling axis offers a promising strategy for overcoming resistance and improving treatment outcomes in HCC.

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Poster

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YBX1-Mediated Activation of SREBP2 Drives Sorafenib Resistance via Cholesterol Biosynthesis in Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) is an aggressive, largely chemo-resistant cancer with a poor prognosis and is the leading cause of cancer-related deaths worldwide. Currently, there is no effective systemic chemotherapy for HCC. Sorafenib is a multikinase inhibitor commonly used as the first-line treatment for advanced HCC. However, acquired drug resistance significantly limits its clinical effectiveness. The Y-box binding protein (YBX1) is an oncogenic RNA-binding protein found to be upregulated in sorafenib-resistant HCC cells and tissues.

Methods: HCC model SK Hep1, YBX1 overexpression, and knockdown cells were prepared by transient transfection with puromycin selection, and stable cell lines were established through Fluorescence activated cell sorting (FACS) with GFP selection. We then validated cholesterol precursor genes and downstream regulatory genes using qRTPCR and assessed protein levels through western blot and immunofluorescence assays. HepG2 and Huh7 sorafenib-resistant cell lines were generated and maintained at a 10µM sorafenib concentration. Additionally, qRT-PCR and western blot analyses were performed to measure the RNA and protein levels. To identify kinase pathways affected by YBX1, a phosphoproteome array was utilized.

Results: The nuclear translocation of YBX1 is associated with worse outcomes, and cholesterol metabolism pathways appear to be significantly impacted by YBX1. Transforming growth factor beta 1 (TGF-β1) signaling promotes the nuclear translocation of YBX1 and boosts its transcriptional activity. Nuclear YBX1 increases the expression of the sterol regulatory element-binding protein (SREBP2), a key transcription factor in cholesterol biosynthesis, and contributes to metabolic reprogramming and resistance to sorafenib. Overexpression of YBX1 raises SREBP2 levels, while knockdown reduces them in HCC cell line models. Additionally, YBX1 activation supports the PI3K/AKT/mTOR and RAS/RAF pathways, reinforcing cellular proliferation and drug tolerance. Kinases play a critical role in drug resistance, and activation of Akt/mTOR-associated genes was observed in YBX1 overexpression and reversed in YBX1 knockdown cell line model.

Conclusions: Collectively, these findings suggest that YBX1 contributes to sorafenib resistance in HCC through dysregulation of cholesterol metabolism and oncogenic signaling. Targeting the YBX1/TGF-β1/SREBP2 signaling axis offers a promising strategy for overcoming resistance and improving treatment outcomes in HCC.

 

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