• Drug Resistance
• Gene Targeting
• Hepatocellular Carcinoma
• Liver
• Molecular Carcinogenesis

Hepatocellular carcinoma (HCC), the most common tumour of the liver, develops in more than 80% of cases on patients with chronically damaged livers owing to excessive alcohol consumption, hepatitis B or C virus (HBV or HCV) infection or obesity. Despite positive results of HBV vaccination programmes and the promising data from the new anti-HCV treatments,1 the incidence of HCC is increasing significantly in Western countries because of the progression of old HCV infections and the almost epidemic prevalence of obesity and metabolic syndrome-associated non-alcoholic fatty liver disease.2

The prognosis of patients with HCC is generally very poor. HCC tumours are resistant to chemotherapy and are usually diagnosed at a late stage when the curative strategies of surgical resection and orthotopic liver transplantation are not applicable. Targeted treatments against specific oncogenes have been shown to be effective in the treatment of leukaemias and solid tumours such as breast, colon and lung carcinomas.3 In 2008, the SHARP (Sorafenib HCC Assessment Randomised Protocol) trials showed an improved overall survival in Child–Pugh class A patients with advanced HCC upon treatment with the antiangiogenic and antiproliferative agent sorafenib.4 This multikinase inhibitor was established as the standard of care for patients with advanced HCC. However, the promising systemic treatment has demonstrated limited survival benefits with very low rates of tumour response, suggesting the existence of primary and acquired drug resistance mechanisms.5

In this situation HCC appears to be a moving target, and a rapid intervention in at least four different aspects is required to improve the outcome of patients with HCC: (1) an understanding of the underlying mechanisms of resistance to sorafenib, the standard first-line treatment for patients with advanced HCC, (2) identification of molecular markers to classify the highly heterogeneous patients with HCC in order to determine their suitability for a specific (targeted or not) treatment, (3) better characterisation of the mechanisms of hepatocarcinogenesis and identification of more reliable prognosis and therapeutic targets from the molecular landscape and (4) establishment and evaluation of synergistic clinical trials to test the combination of new agents targeting different pathways and also use of some of these drugs with chemotherapy and/or other therapeutic interventions.

Most studies of the mechanisms of drug resistance focus on the role of tumour cells. However, as discussed later a growing body of evidence has uncovered the importance of tumour stroma and host genetics in drug delivery and chemoresistance.6 ,7 Both, the molecular heterogeneity and genetic instability of HCC cells are at the origin of primary and acquired resistance to sorafenib. It has been shown recently that patients with overexpression of the small heat shock protein αB-crystallin, which induces epithelial–mesenchymal transition through activation of extracellular signal-regulated kinases (ERKs), fail to respond to sorafenib.8 Many molecular pathways are altered in HCC. The activation of epidermal growth factor receptor (EGFR) and subsequently of ERK and Akt has been implicated both in the inherent and acquired resistance to sorafenib.9 The EGFR ligand amphiregulin (AR) is induced in HCC cells upon sorafenib treatment and its silencing sensitises HCC cells to sorafenib. AR levels are increased in the serum of patients treated with sorafenib9 and AR expression is induced in a subset of HCCs.10 Together these results suggest that AR expression could represent a marker of primary resistance to sorafenib and emphasise the potential of EGFR inhibitors as coadjuvants in the treatment of HCC. Cancer cell metabolism appears to be a new target for intervention and a correlation between the Warburg effect and sorafenib resistance has been recently shown in HCC cells.11 Shen et al11 have shown that the activation of oxphos by pyruvate dehydrogenase kinase inhibitors overcomes both inherent and acquired resistance of HCC cells to sorafenib. Autophagy is a catabolic process induced under metabolic stress conditions, and its role in HCC is controversial. In a recent report Shimizu et al12 showed that sorafenib induces adaptive autophagy in HCC cells, conferring sorafenib resistance on these cells, which is overcome using autophagy inhibitors.

In tumours the survival and growth of HCC cells depends on interactions with multiple factors and cell types present in the tumour microenvironment. Targeting and modulation of the non-tumoral cells present in the stroma is an opportunity for intervention in the treatment and also in the prevention of HCC.6 In fact, the therapeutic effect of sorafenib is based on its antiproliferative and proapoptotic activity on tumour cells, through the inhibition of Raf kinases and the antiapoptotic protein Mcl-1, and also on the antiangiogenic activity on tumour-associated endothelial cells inhibiting vascular endothelial growth factor receptor and platelet-derived growth factor receptor signalling. Liang et al13 have recently shown that this antiangiogenic activity can, however, be responsible for the activation of resistance mechanisms. They show that sustained sorafenib treatment leads to increased intratumour hypoxia and induction of HIF-1α expression that mediates cell survival. They also show that the use of HIF-1α inhibitors abolishes drug resistance.

Cancer stem cells (CSC) are present in the HCC microenvironment and, although their contribution to tumour development and biology is not clear, it has been proposed that they are responsible for therapeutic failure.14 The remarkable study of Xin et al15 provides the first evidence for the potential role of label-retaining cancer cells (LRCC), a new subpopulation of CSC, in sorafenib resistance. They demonstrate that LRCC are relatively resistant to sorafenib, and can be selected from different HCC cell lines upon treatment with the drug. As few as 10 LRCC can generate tumours, leading the authors to postulate that these cells might be responsible for HCC recurrence. In their attempt to characterise the molecular mechanisms implicated in the resistance of LRCC they found that depending on the origin of the LRCC different pathways, including sustained ERK or Akt activation, could be involved.15 Nevertheless, further efforts are needed in order to understand the mechanistic details of sorafenib resistance. Demonstrating the presence of LRCC in HCC biopsy samples, the clinical relevance of CSC in drug resistance and the contribution of their targeting to treatment efficacy are also important issues that merit attention.

All these data show that the establishment of effective treatments for HCC has to take into account the complex biology of the tumour and its microenvironment, in order to overcome the numerous mechanisms of drug resistance that can be put into motion.

In addition, to make things more complicated, and as it has been recently shown for chronic myeloid leukaemia and lung cancer, it will be necessary to pay attention to the genetic profile of the patient, since the resistance mechanisms may be already present in the individual's genome. Ng et al7 have demonstrated that germline deletion polymorphisms in the B cell lymphoma 2 interacting mediator of cell death (BIM) gene, common in East Asian populations, confer intrinsic resistance to cell death induced by tyrosine kinase inhibitors.7

In summary, new perspectives for effective therapeutic strategies in selected patients, based on the combined use of inhibitors of angiogenesis, hypoxia, autophagy, EGFR and growth factors, activators of oxphos and so on, are on the horizon and need further consideration. The work of Xin et al15 adds CSC to this repertoire of promising targets.