Original Article/Liver
Torin2 overcomes sorafenib resistance via suppressing mTORC2-AKT-BAD pathway in hepatocellular carcinoma cells

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Abstract

Background

Sorafenib is an oral multi-kinase inhibitor that was approved by the US Food and Drug Administration for the treatment of patients with advanced hepatocellular carcinoma (HCC). However, resistance to sorafenib is an urgent problem to be resolved to improve the therapeutic efficacy of sorafenib. As the activation of AKT/mTOR played a pivotal role in sorafenib resistance, we evaluated the effect of a dual mTOR complex 1/2 inhibitor Torin2 on overcoming the sorafenib resistance in HCC cells.

Methods

The sorafenib-resistant Huh7 and Hep3B cell lines were established from their parental cell lines. The synergistic effect of sorafenib and Torin2 on these cells was measured by cell viability assay and quantified using the Chou-Talalay method. Apoptosis induced by the combination of sorafenib and Torin2 and the alteration in the specific signaling pathways of interest were detected by Western blotting.

Results

Sorafenib treatment inversely inhibited AKT in parental but activated AKT in sorafenib-resistant Huh7 and Hep3B HCC cells, which underscores the significance of AKT activation. Torin2 and sorafenib synergistically suppressed the viability of sorafenib-resistant cells via apoptosis induction. Torin2 successfully suppressed the sorafenib-activated mTORC2-AKT axis, leading to the dephosphorylation of Ser136 in BAD protein, and increased the expression of total BAD, which contributed to the apoptosis in sorafenib-resistant HCC cells.

Conclusions

In this study, Torin2 and sorafenib showed synergistic cytostatic capacity in sorafenib-resistant HCC cells, via the suppression of mTORC2-AKT-BAD pathway. Our results suggest a novel strategy of drug combination for overcoming sorafenib resistance in HCC.

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.

References (31)

  • W. Tang et al.

    The mechanisms of sorafenib resistance in hepatocellular carcinoma: theoretical basis and therapeutic aspects

    Signal Transduct Target Ther

    (2020)
  • S.M. Wilhelm et al.

    BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis

    Cancer Res

    (2004)
  • Y. Ito et al.

    Activation of mitogen-activated protein kinases/extracellular signal-regulated kinases in human hepatocellular carcinoma

    Hepatology

    (1998)
  • L. Liu et al.

    Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5

    Cancer Res

    (2006)
  • K.F. Chen et al.

    Activation of phosphatidylinositol 3-kinase/Akt signaling pathway mediates acquired resistance to sorafenib in hepatocellular carcinoma cells

    J Pharmacol Exp Ther

    (2011)
  • Cited by (0)

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