Making sorafenib irresistible: In vivo screening for mechanisms of therapy resistance in hepatocellular carcinoma hits on Mapk14
Authors: Matias Avila, Carmen Berasain
Journal: Hepatology (2015)

Potential conflict of interest: Nothing to report. Rudalska R, Dauch D, Longerich T, McJunkin K, Wuestefeld T, Kang TW, et al. In vivo RNAi screening identifies a mechanism of sorafenib resistance in liver cancer. Nat Med 2014;20:1138‐1146. (Reproduced with permission.) RudalskaR, DauchD, LongerichT, McJunkinK, WuestefeldT, KangTW, et al. In vivo RNAi screening identifies a mechanism of sorafenib resistance in liver cancer. Nat Med2014;20:1138‐1146. (Reproduced with permission.) Abstract In solid tumors, resistance to therapy inevitably develops upon treatment with cytotoxic drugs or molecularly targeted therapies. Here, we describe a system that enables pooled shRNA screening directly in mouse hepatocellular carcinomas (HCC) in vivo to identify genes likely to be involved in therapy resistance. Using a focused shRNA library targeting genes located within focal genomic amplifications of human HCC, we screened for genes whose inhibition increased the therapeutic efficacy of the multikinase inhibitor sorafenib. Both shRNA‐mediated and pharmacological silencing of Mapk14 (p38α) were found to sensitize mouse HCC to sorafenib therapy and prolong survival by abrogating Mapk14‐dependent activation of Mek‐Erk and Atf2 signaling. Elevated Mapk14‐Atf2 signaling predicted poor response to sorafenib therapy in human HCC, and sorafenib resistance of p‐Mapk14‐expressing HCC cells could be reverted by silencing Mapk14. Our results suggest that a combination of sorafenib and Mapk14 blockade is a promising approach to overcoming therapy resistance of human HCC. Comment Despite significant improvements in the management and treatment of patients with hepatocellular carcinoma (HCC), the prognosis of this disease is still very poor, HCC being very resistant to conventional chemotherapy. Most targeted drugs tested have not shown positive results with the exception of the multikinase inhibitor sorafenib, the only approved systemic therapy for advanced HCC, which shows a modest clinical benefit. The reason for this limited effect and for the failure of all other targeted agents against HCC is likely multifactorial, including the molecular complexity of this tumor and the presence of primary and acquired drug resistance mechanisms, as also occurs in other solid malignancies.1 In this scenario improving the efficacy of sorafenib, an inhibitor of RAF (RAF1 and BRAF) and vascular endothelial growth factor receptor‐2/‐3 kinases, is therefore of paramount importance. However, the mechanisms of sorafenib resistance are still not well known. In a recent study published in Nature Medicine, Rudalska et al.2 developed an unbiased screen platform to identify target genes whose inhibition could enhance the therapeutic efficacy of sorafenib in HCC. To this end they took advantage of a well‐controlled transposon‐based mouse model of aggressive and multifocal HCC, in which oncogenic NrasG12V is delivered into the livers of p19Arf‐deficient mice. Albeit Nras and p19 are not frequently mutated in human HCC, tumors histologically similar to moderately to poorly differentiated human HCCs displaying limited clinical response to sorafenib treatment arise in these mice.2 This oncogene‐encoding transposon vector was further engineered to harbor pools of mir‐30–based short hairpin RNAs (shRNAs) that would allow one to screen for and identify genes that could mediate sorafenib resistance (Fig. 1A). One important step was the selection of the shRNAs to be tested. The authors reasoned that critical genes driving hepatocarcinogenesis, and potentially involved in therapy resistance, might be found in focal chromosomal amplifications. From such focal genomic amplifications identified in HCC3 the authors designed a focused shRNA library including 253 shRNAs, which was cloned in the NrasG12V‐coexpressing transposon vector and delivered to p19Arf‐deficient mice that were subsequently treated or not with sorafenib. After 5 weeks, genomic DNA was extracted from tumorous livers and the abundance of each shRNA was determined by deep sequencing. While most shRNAs did not influence sorafenib sensitivity, 19 shRNAs were depleted more than 10‐fold in sorafenib‐treated mice. The target genes of these shRNAs negatively selected upon sorafenib treatment would be good candidates to mediate drug resistance. After applying stringent selection criteria, one candidate gene, the mitogen‐activated protein kinase 14 (Mapk14), also called P38α, was selected for further studies. As the authors mention, previous reports performed shRNA‐based dropout screens to identify novel genes and pathways that could reverse cancer drug resistance. However, the study of Rudalska et al.2 would be the first to apply this strategy to tumors developing in vivo. This is particularly important for a solid tumor like HCC, given the key role played by chronic parenchymal injury in hepatocarcinogenesis.4 Perhaps here lies one of the few limitations of the present study. Most HCCs develop on a chronically inflamed and fibrotic liver, while the oncogene‐defined HCC model used in this screen apparently lacked these important components, which can also influence the response to sorafenib; and therefore, relevant genes may have been missed. Another potentially limiting aspect inherent to the model was the fact that the HCC cells targeted in vivo were of murine origin. Despite significant overlap between the molecular profiles of human and mouse HCCs, there are also important differences. One relevant example here would be the frequent amplification and overexpression of the fibroblast growth factor‐19 gene in human tumors but not in mouse HCCs.3 Nevertheless, in a series of well‐designed genetic and pharmacological studies, the authors cogently demonstrated that knocking down Mapk14 expression or inhibiting its kinase activity markedly increased the therapeutic activity of sorafenib in their in vivo model of hepatocarcinogenesis. Interestingly, Mapk14 knockdown also potentiated sorafenib responses in an orthotopic model of advanced tumors derived from NrasG12V‐ and p19Arf‐deficient HCC cells. Most importantly, pharmacological inhibition of MAPK14 also enhanced sorafenib therapeutic performance in murine HCC cell lines harboring other oncogenic drivers (i.e., AKT‐1 and c‐MYC) and in human HCC cells. Given the molecular heterogeneity of HCC, this was a relevant observation, suggesting that MAPK14 could also mediate sorafenib resistance across the wide spectrum of HCC genetic subclasses.Figure 1: In vivo RNA interference screening for sorafenib resistance genes in HCC. (A) Outline of the transposon‐based mouse model of HCC. Transposable elements encoding oncogenic NrasG12V and a miR30‐based shRNA library (Amplicon set) are delivered into the livers of p19Arf‐deficient mice by hydrodynamic tail vein injection. One week later, half of the mice were treated with sorafenib and the other half with vehicle (carrier). Five weeks later genomic DNA was isolated from tumoral livers, and the abundance of each shRNA in sorafenib‐treated and control mice was determined by deep sequencing. Those HCC cells that received shRNAs sensitized to the effects of the drug were depleted. Two independent shRNAs targeting MAPK14 were depleted more than 10‐fold, and thus, this gene was selected for further studies. (B) Sorafenib targets the RAF‐MEK‐ERK cascade, but in sorafenib‐resistant HCC cells this pathway is overactivated. As demonstrated by Rudalska et al.,2 sorafenib also triggers the activity of MAPK14 (p38α), which is a stress‐activated protein kinase. Through at least two pathways MAPK14 mediates resistance to sorafenib: by feeding into the MEK‐ERK cascade downstream of RAF and by activation of the pro‐oncogenic transcription factor ATF‐2. Abbreviations: HCC, hepatocellular carcinoma; MAPK14, mitogen‐activated protein kinase 14; sh, short hairpin.It should be considered that MAPK14 (P38α) is the major isoform of the stress‐activated kinases and that its activity controls the expression of inflammatory mediators, cytokines, as well as survival and antioxidant genes. In fact, hepatocyte‐specific deletion of Mapk14 results in enhanced oxidative stress, liver injury, and increased HCC development in an experimental model of chronic liver injury and carcinogenesis.6 Therefore, MAPK14 also plays a hepatoprotective role; and thus, its untimely inhibition might result in increased liver damage and even in enhanced tumorigenesis. Consequently, the safety of long‐term administration of MAPK14 inhibitors must be carefully evaluated. Rudalska et al.2 also provide valuable insights into the mechanism of HCC sensitization to sorafenib upon Mapk14 inhibition. The RAF kinases and their downstream mitogen‐activated protein kinase kinase (MEK)/extracellular signal–regulated kinase (ERK) kinase cascade are regarded as central therapeutic targets of sorafenib in HCC cells and key drivers in hepatocarcinogenesis, being upregulated in a great proportion of HCCs.7 Indeed, the expression of negative regulators of this pathway is frequently impaired in HCC, and growth factor–mediated autocrine/paracrine loops activating the RAF‐MEK‐ERK cascade are present in HCC cells and have been implicated in sorafenib resistance.1 Interestingly, sorafenib‐mediated attenuation of RAF‐MEK‐ERK–dependent signaling was significantly potentiated upon Mapk14 silencing in murine HCC cells, suggesting the existence of a crosstalk between the RAF‐MEK‐ERK cascade and MAPK14 signaling. Paradoxically, activation of the RAF‐MEK‐ERK cascade by sorafenib has been recently related with drug resistance,8 and Rudalska et al.2 also found increased levels of phosphorylated MAPK14 in sorafenib‐treated cells that were no longer responsive to the drug. Interestingly, MAPK14 knockdown in these cells resulted in reduced p‐MEK and p‐ERK levels and down‐regulation of cell cycle–associated markers, indicating that MAPK14 was indeed driving a resistance mechanism by feeding into the MEK‐ERK cascade. Additionally, the authors noticed that MAPK14 silencing resulted in a pronounced down‐regulation of the phosphorylation of the transcription factor ATF2 and of ATF2‐dependent expression of cancer‐associated genes. Bearing this in mind, Rudalska et al. went on to demonstrate that ATF2 silencing also restored sorafenib sensitivity in HCC cells, indicating that besides MAPK14 crosstalk into the MEK‐ERK pathway the MAPK14‐ATF2 axis also limited the therapeutic efficacy of sorafenib (Fig. 1B). Together, these findings strongly supported the involvement of MAPK14 in primary and acquired sorafenib resistance in HCC cells. This notion was further strengthened by the finding of increased pretherapeutic staining levels of p‐MAPK14 and p‐ATF2 in liver biopsies of patients with shorter survival on sorafenib therapy. In view of all this, the authors propose a therapeutic strategy whereby maximum efficacy would be obtained by the combination of RAF inhibition (sorafenib) plus MAPK14 blockade (l‐skepinone). This strategy could be clinically tested after patients have undergone standard sorafenib treatment or ab initio if high levels of p‐ATF2 are detected in a pretherapeutic biopsy. Nevertheless, and as pointed out before, preclinical safety evaluations of the simultaneous long‐term administration of sorafenib and MAPK14 inhibitors should be conducted in models of liver injury–associated hepatocarcinogenesis. The work of Rudalska et al.2 is certainly a pioneer study paving the way for more comprehensive in vivo screens in liver pathophysiology. This technology can be applied not only for the identification of drug sensitizer genes, including those that eventually will participate in l‐skepinone resistance, but also for the elucidation of suppressor and driver genes in hepatocarcinogenesis. Author names in bold designate shared co‐first authorship.