The retinoblastoma (Rb) protein regulates ferroptosis induced by sorafenib in human hepatocellular carcinoma cells

Highlights

• The retinoblastoma (Rb) protein modulates the response of human liver cancer cells to sorafenib.

• Sorafenib is more cytotoxic in cells with reduced levels of Rb.

• Rb knock-down cells exposed to sorafenib encounter ferroptosis, a new form of regulated necrosis.

• Rb-negative cancer cells encounter increased levels of oxidative stress.

Abstract

Sorafenib is the treatment of reference for advanced hepatocellular carcinoma (HCC), the most frequent form of primary liver tumour. The loss of function of the retinoblastoma (Rb) protein is an important event during liver carcinogenesis, but it is unclear whether the Rb status modulates the response of HCC cells to sorafenib. Here, we examined this question in HCC cells with reduced levels of Rb achieved through stable RNA interference. We show that HCC cells with reduced levels of Rb exhibit a two- to threefold increase in cell death induction upon exposure to sorafenib compared with controls. Sorafenib treatment of Balb/c nude mice that received tumour xenografts derived from HCC cells with reduced Rb levels resulted in complete tumour regression in 50% of the animals treated, compared with tumour stabilization in mice that received control cells. We show that, upon exposure to sorafenib, the Rb-negative status of HCC cells promotes the occurrence of ferroptosis, a form of oxidative necrosis. The findings highlight the role of Rb in the response of HCC cells to sorafenib and the regulation of ferroptosis.

Introduction

Hepatocellular carcinoma (HCC) is the most frequent form of primary liver cancer, and constitutes a major health concern around the world, due to its high mortality rate [1]. Sorafenib, a multi-kinase inhibitor, is currently the only systemic treatment with a proven efficacy in terms of overall survival for advanced forms of HCC [2]. However, sorafenib confers only a modest gain in survival, of a few months, in patients with HCC. Although HCC presents great heterogeneity in individual sensitivity to sorafenib [3], [4], [5], there are currently no biological assays that can predict its efficacy in individual patients. An important aim of biomedical research is to identify the biological alterations that are associated with an optimal efficacy of sorafenib, in order to personalize its use in HCC [5].

Induction of cytotoxicity in cancer cells is one of the main goals of anticancer treatments. Apoptosis was the first form of cell death reported to be induced in HCC cells exposed to sorafenib in vitro and in preclinical models of HCC [6]. However, sorafenib is only weakly pro-apoptotic when it is applied on HCC cells as a single-agent [7]. Recently, we and others reported that sorafenib is able to induce a new form of cell death, distinct from apoptosis, and called ferroptosis [8], [9], [10]. Ferroptosis is a form of regulated necrosis characterized by the occurrence of oxidative stress and membrane lipid peroxidation [10], [11]. Ferroptosis was originally reported in cells exposed to the chemical compound erastin, identified during a search for compounds displaying synthetic lethality in cells with a constitutively active, mutated version of H-RAS [12]. The possibility that ferroptosis might be selectively induced in cancer cells over normal cells has recently fostered interest in this new form of regulated cell death. To date, it is currently unclear which genomic alterations could account for the susceptibility of cancer cells to ferroptosis induced by sorafenib.

The retinoblastoma (Rb) protein is the founding member of a family of proteins that exert a potent regulatory function on the transcription of several genes in eukaryotes [13]. Rb is best known for its regulatory role in cell proliferation and its key role at the G1/S checkpoint, essentially via its ability to regulate the activity of the transcription factors of the E2F family [13]. Loss of function of Rb is a common event in HCC, yet the mechanisms involved are complex. Loss of heterozygosity involving the RB1 locus is frequently observed in HCC [14]. Epigenetic alterations also contribute to the loss of expression of Rb in HCC cells, through the regulation of the methylation of RB1 [15]. The common activation of the cyclin-dependent kinases (CDK) also leads to functional inactivation of Rb in HCC. This activation is typically accounted for by reduced expression of the p16 protein, encoded by the CDKN2A gene, and somatic amplification of the gene encoding Cyclin D1 [14], [16]. Increased levels of ubiquitinylation and protein turn-over of Rb are also reported in HCC, for example as a consequence of Hepatitis C virus (HCV) infection [17]. In mice, the invalidation of Rb was shown to directly contribute to liver carcinogenesis [18], [19]. The loss of function of Rb is therefore clearly a motor event during liver carcinogenesis, but it is not known whether Rb has an impact on the response of HCC to sorafenib.