Original Article/LiverTorin2 overcomes sorafenib resistance via suppressing mTORC2-AKT-BAD pathway in hepatocellular carcinoma cells
Introduction
Globally, primary liver cancer is the seventh most common cancer and the second leading cause of cancer-related death. Among the primary liver malignancy, hepatocellular carcinoma (HCC) accounts for approximately 75% of the total cases [1]. HCC is natively resistant to traditional systemic chemotherapy [2]. Sorafenib is approved by US Food and Drug Administration in 2007 as the first-line drug for treatment of advanced HCC. However, the clinical efficacy of sorafenib was not satisfactory, as the median overall survival of patients that received sorafenib was only prolonged by 2–3 months compared with that of the placebo group in large-scale clinical trials [3,4]. Accumulating evidence showed that some patients with HCC responded to sorafenib in the initial stage but the tumor eventually progressed during sorafenib therapy [4], indicating that the development of acquired resistance is a huge obstacle in sorafenib treatment.
The mechanisms involved in the sorafenib resistance in HCC are diverse. An increasing number of studies indicated that the compensatory effect and cross-talk between the signaling pathways, the generation of cancer stem cells, as well as the tumor microenvironment contributed to sorafenib resistance [5,6]. Since the therapeutic effect of sorafenib monotherapy is limited, the development of novel drug combination strategies is urgent to overcome the resistance to sorafenib.
Sorafenib is a multi-target kinase inhibitor, whose targets include receptor tyrosine kinases such as VEGFR2–3, PDGFR, FGFR-1 and c-Kit, as well as B-RAF and RAF-1 [7]. PI3K-AKT-mTOR and MAPK/ERK (RAS-RAF-MEK-ERK) signaling pathways are two important tumor-promoting downstream cascades of the above receptor tyrosine kinases. The MAPK/ERK pathway is frequently over-activated in HCC tissue [8], which can be blocked directly by sorafenib through B-RAF and RAF-1 inhibition [9]. However, sorafenib does not directly suppress and even activates the PI3K/AKT/mTOR pathway, which confers resistance capacity of the HCC cells [10]. In a variety of pre-clinical studies, the efficiency of sorafenib combined with PI3K/AKT inhibitors for sorafenib-resistant HCC cells has been confirmed [11,12]. Nonetheless, the combination of dual mTOR complex 1/2 (mTORC1/2) inhibitor plus sorafenib still needs to be evaluated.
Torin2 is a novel second-generation ATP-competitive dual mTORC1/2 inhibitor [13], which has shown the capacity of suppressing the proliferation in breast cancer, HCC, and ovarian cancer cells [14], [15], [16]. However, it remains unclear whether Torin2 may overcome the sorafenib resistance in HCC cell lines. Therefore, this study aimed to assess the cytostatic effect of Torin2 in combination with sorafenib for the treatment of sorafenib-resistant HCC cells and to explore the underlying mechanism.
Section snippets
Cell culture, chemicals and antibodies
Parental Hep3B human HCC cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Parental Huh7 human HCC cell line was purchased from Chinese Academy of Sciences Committee Type Culture Collection cell bank (Shanghai, China). HCC cell lines were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) (ATCC) plus 10% fetal bovine serum (HyClone, Marlborough, MA, USA). Cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C. The growth medium
Confirmation of sorafenib resistance in Hep3B-SR and Huh7-SR cell lines
A cell viability assay of parental and sorafenib-resistant Huh7 and Hep3B cell lines was carried out after the treatment of various concentrations of sorafenib (Fig. 1). The IC50 of sorafenib was 4.39-fold potent in the Huh7-SR (IC50 = 13.42 μmol/L) than that in the parental Huh7 (Huh7-P) (IC50 = 3.05 μmol/L) cells. Similarly, IC50 of sorafenib was 3.66-fold potent in the Hep3B-SR (IC50 = 13.52 μmol/L) than that in the parental Hep3B (Hep3B-P) (IC50 = 3.69 μmol/L) cells, confirming that the
Discussion
Our study found that compared with monotherapy of each drug, the combined therapy of sorafenib and Torin2 suppressed the Huh7-SR and Hep3B-SR cell viability and the capacity of colony formation more effectively. Similarly, the pro-apoptotic effect in the sorafenib-resistant HCC cells was greater when the two drugs were used in combination.
AKT has been identified as a key factor that promotes sorafenib resistance in HCC cells and the phosphorylation of Ser473 is significant for its full
Acknowledgments
None.
CRediT authorship contribution statement
Yi-Ting Hu: Investigation, Methodology, Writing - original draft. Zhe-Yue Shu: Methodology, Writing - original draft. Jing-Hua Jiang: Methodology, Software. Qin-Fen Xie: Data curation, Funding acquisition. Shu-Sen Zheng: Conceptualization, Supervision, Writing - review & editing.
Funding
This study was supported by a grant from Medical and Health Science and Technology Program of Zhejiang Province (2019RC076).
Ethical approval
Not needed.
Competing interest
No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
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