Elsevier

Journal of Hepatology

Volume 55, Issue 5, November 2011, Pages 1041-1048
Journal of Hepatology

RESEARCH ARTICLE
Signal transducer and activator of transcription 3 is a major kinase-independent target of sorafenib in hepatocellular carcinoma

https://doi.org/10.1016/j.jhep.2011.01.047Get rights and content

Background & Aims

Recently, we reported that sorafenib sensitizes hepatocellular carcinoma (HCC) cells to TRAIL through the inhibition of signal transducer and activator of transcription 3 (STAT3). Here, we report that sorafenib inhibits HCC via a kinase-independent mechanism: SHP-1 dependent STAT3 inactivation.

Methods

SC-1 is a sorafenib derivative that closely resembles sorafenib structurally but with no kinase inhibition activity. HCC cell lines (PLC5, Huh-7, Hep3B, and Sk-Hep1) were treated with sorafenib or SC-1 and apoptosis and signal transduction were analyzed. In vivo efficacy was determined in nude mice with Huh-7 xenografts.

Results

SC-1 showed similar effects to sorafenib on growth inhibition and apoptosis in all tested HCC cell lines. SC-1 down-regulated phosphorylation of phospho-STAT3 (p-STAT3) at tyrosine 705 in all tested HCC cells. Expression of STAT3-driven genes, including Cyclin D1 and Survivin, was also repressed by SC-1. Luciferase reporter assay confirmed the inhibition of transcriptional activity of STAT3 in both sorafenib-treated and SC-1-treated cells. Ectopic expression of STAT3 in PLC5 cells abolished apoptosis in SC-1-treated cells. Sorafenib and SC-1 up-regulated SHP-1 activity. Knockdown of SHP-1, but not SHP-2 or PTP-1B, by small interference RNA reduced apoptosis induced by SC-1. Finally, SC-1 reduced Huh-7 tumor growth significantly in vivo, which was associated with down-regulation of p-STAT3 and up-regulation of SHP-1 activity.

Conclusions

STAT3 is a major kinase-independent target of sorafenib in HCC.

Introduction

Human hepatocellular carcinoma (HCC) is the fifth most common cancer and the third most common cause of cancer-related deaths worldwide [1], [2], [3]. Unfortunately, chemotherapy and surgical adjuvant are not effective for patients with advanced HCC. Molecular targeted therapy shows promise for HCC treatment. Sorafenib (Nexavar®) is the first and only targeted therapy approved for HCC. Sorafenib is a multiple kinase inhibitor that represses the activity of Raf-1 and other tyrosine kinases such as VEGFR2, VEGFR3, Flt-3, PDGFR, and FGFR-1 [4], [5]. In phase III studies, sorafenib showed survival benefit in patients with late stage HCC, which led to the clinical approval of sorafenib in HCC [6], [7]. Sorafenib has also been clinically approved for the treatment of renal carcinoma and preclinical studies have shown that sorafenib is also effective in other types of cancer cells, such as non-small cell lung cancer and pancreatic cancer [8], [9], [10].

Recently, we reported that signal transducer and activator of transcription 3 (STAT3) plays a major role in mediating the effects of sorafenib on TRAIL-sensitization through the inhibition of STAT3-related anti-apoptotic genes including Mcl-1 and Survivin [11]. STAT3 is implicated in the transcriptional reciprocation of genes that are associated with cell proliferation and survival. STAT3 is activated by IL-6 cytokine and leptin as well as other growth factors, including epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), and platelet-derived growth factor receptor (PDGFR) through tyrosine phosphorylation [12]. After dimerization, STAT3 translocates into the nucleus where it activates gene transcription by binding to specific DNS sequences. Many STAT3-related genes such as Mcl-1, Cyclin D1, Bcl-2, Bcl-xL, and Survivin play a role in cell proliferation and survival signaling. However, there are many factors that act as negative regulators of STAT3 signaling pathway. For example, SOCS protein family has two domains: a Src homology 2 (SH2) domain and a C terminal SOCS box. The SH2 domain of SOCS-1 mediates the direct interaction with JAK and represses the JAK/STAT pathway [13]. The protein inhibitor of activated STAT (PIAS) family blocks the DNA-binding activity of STAT3 and inactivates STAT3-mediated transcription in the nucleus [14]. In addition, JAK/STAT3 signaling is down-regulated by protein tyrosine phosphatases like SH2-domain-containing cytosolic phosphatases (SHP-1 and SHP-2), and protein tyrosine phosphatase 1B (PTP-1B) [15].

To address whether down-regulation of p-STAT3 is associated with kinase inhibition of sorafenib, we designed a sorafenib analog, SC-1. Without an amide functional group or a pyridine ring, SC-1 exhibits low binding affinity with the ATP binding sites of tyrosine kinases such as Raf-1 and VEGFR. Our data show that sorafenib and SC-1 inhibit HCC through SHP-1-dependent STAT3 inactivation.

Section snippets

Reagents and antibodies

Sorafenib (Nexavar®) was kindly provided by Bayer Pharmaceuticals (West Haven, CT). Sodium vanadate and SHP-1 inhibitor were purchased from Cayman Chemical (Ann Arbor, MI). Antibodies for immunoblotting such as Raf-1, Cyclin D1, and PARP were purchased from Santa Cruz Biotechnology (San Diego, CA). Other antibodies such as anti-pVEGFR2 (Y1175), VEGFR2, survivin, phospho-STAT3 (Tyr705), and STAT3 were from Cell Signaling (Danvers, MA).

Cell culture

The Huh-7 HCC cell line was obtained from the Health Science

SC-1, a sorafenib derivative lacking the inhibitory function of Raf-1, showed similar cell death effect to sorafenib in HCC cell lines

To investigate the molecular mechanism by which sorafenib inhibits STAT3, we developed a series of sorafenib analogs. SC-1 is a compound modified from sorafenib but without the functional amide group or pyridine ring that are critical to the hydrogen bond interactions between sorafenib and the ATP binding pocket of b-Raf (Fig. 1A, left panel). First, we examined the effects of sorafenib and SC-1 on Raf-1 activity. Raf-1 immunoprecipitated from PLC5 or Hep3B cell extracts was incubated with MEK

Discussion

In this study, we designed a sorafenib derivative, SC-1, without hydrogen donor ability by replacing the pyridine ring and amide functional group with phenyl cyanide. We then explored the ability of sorafenib and SC-1 to inhibit Raf-1 kinase activity in PLC5 cells (Fig. 1A). According to the X-ray structure of b-Raf and sorafenib, the amide group connected to the pyridine ring of sorafenib provides a hydrogen donor and forms a hydrogen bond with the ATP binding pocket of b-Raf. Surprisingly,

Conflict of interest

Dr. Ann-Lii Cheng serves as a consultant and a member of the speaker’s bureau for Bayer-Schering. The other authors have nothing relevant to this manuscript to disclose.

Financial support

Supported by NTUH 100P04 from National Taiwan University Hospital, and NSC98-2314-B-002-067-MY3, NSC98-3112-B-002-037, and NSC99-2314-B-002-017-MY2 from National Science Council, Taiwan.

References (30)

  • E. Fransvea et al.

    HCC heterogeneity: molecular pathogenesis and clinical implications

    Cell Oncol

    (2009)
  • P. Bioulac-Sage et al.

    Hepatocellular adenoma management and phenotypic classification: the Bordeaux experience

    Hepatology

    (2009)
  • M. Di Maio et al.

    Targeted therapies: Role of sorafenib in HCC patients with compromised liver function

    Nat Rev Clin Oncol

    (2009)
  • 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)
  • J.M. Llovet et al.

    Sorafenib in advanced hepatocellular carcinoma

    N Engl J Med

    (2008)
  • Cited by (0)

    These authors contributed equally to this work.

    View full text