Identification of the Genes Chemosensitizing Hepatocellular Carcinoma Cells to Interferon-α/5-Fluorouracil and Their Clinical Significance

Tomohiko Sakabe,Kohei Shomori,Kazue Gonda,Daisaku Yamada,Hiroaki Nagano,Goshi Shiota,Junya Azumi,Hiroshi Wada,Keita Kanki,Hiroyuki Tsuchiya,Yusuke Mizuta

doi:10.1371/journal.pone.0056197

Tomohiko Sakabe, Kohei Shomori + Show 9 more

Open Access

https://doi.org/10.1371/journal.pone.0056197

Copy DOI

Abstract

The incidence of advanced hepatocellular carcinoma (HCC) is increasing worldwide, and its prognosis is extremely poor. Interferon-alpha (IFN-α)/5-fluorouracil (5-FU) therapy is reportedly effective in some HCC patients. In the present study, to improve HCC prognosis, we identified the genes that are sensitizing to these agents. The screening strategy was dependent on the concentration of ribozymes that rendered HepG2 cells resistant to 5-FU by the repeated transfection of ribozymes into the cells. After 10 cycles of transfection, which was initiated by 5,902,875 sequences of a ribozyme library, three genes including protein kinase, adenosine monophosphate (AMP)-activated, gamma 2 non-catalytic subunit (PRKAG2); transforming growth factor-beta receptor II (TGFBR2); and exostosin 1 (EXT1) were identified as 5-FU-sensitizing genes. Adenovirus-mediated transfer of TGFBR2 and EXT1 enhanced IFN-α/5-FU-induced cytotoxicity as well as 5-FU, although the overexpression of these genes in the absence of IFN-α/5-FU did not induce cell death. This effect was also observed in a tumor xenograft model. The mechanisms of TGFBR2 and EXT1 include activation of the TGF-β signal and induction of endoplasmic reticulum stress, resulting in apoptosis. In HCC patients treated with IFN-α/5-FU therapy, the PRKAG2 mRNA level in HCC tissues was positively correlated with survival period, suggesting that PRKAG2 enhances the effect of IFN-α/5-FU and serves as a prognostic marker for IFN-α/5-FU therapy. In conclusion, we identified three genes that chemosensitize the effects of 5-FU and IFN-α/5-FU on HCC cells and demonstrated that PRKAG2 mRNA can serve as a prognostic marker for IFN-α/5-FU therapy.

Highlights

Hepatocellular carcinoma (HCC) is the third most common cancer, the incidence of which is reportedly increasing worldwide [1]
The viability of cells transfected with the recovered plasmid DNA (pDNA) significantly increased during the progression from 0 to 10 cycles of screening at 1–10 mg/mL of 5-FU, suggesting that ribozymes become densely concentrated by the screening (Figure 1C)
Since selective knockdown of the genes by specific small interfering RNA induced resistance to 5-FU, we subsequently focused on PRKAG2, TGFBR2, and exostosin 1 (EXT1) (Figure 1E and 1F)

Summary

Introduction

Hepatocellular carcinoma (HCC) is the third most common cancer, the incidence of which is reportedly increasing worldwide [1]. Sorafenib, an oral multikinase inhibitor, was reported to improve the median overall survival rate in advanced HCC patients [2,3]. It did not improve overall survival and prognosis in advanced HCC patients with portal vein tumor thrombosis (PVTT) [4]. Interferon-alpha (IFN-a)/5-fluorouracil (5FU) combination therapy showed favorable effects in advanced HCC patients [5], especially compared to those with PVTT [6,7,8], its maximum efficacy was only 25%–50% in some countries including Japan and USA, suggesting that chemoresistance limits the therapeutic potential of IFN-a and 5-FU. Limited information is currently available on the genes involved in enhancing chemosensitivity to this therapy

Methods

Results

Conclusion