WNTing no RASt for hepatocellular carcinoma
Authors: Chad Walesky, Wolfram Goessling
Journal: Hepatology (2017)

Potential conflict of interest: Nothing to report. See Article On Page 1581 Hepatocellular carcinoma (HCC) is one of the deadliest malignancies worldwide, and its incidence in the United States is rising. Currently, the most effective and curative treatment strategies for HCC are liver transplantation or surgical resection. The only drug approved by the US Food and Drug Administration, sorafenib, has limited clinical success and survival benefits, illustrating the urgent need to better elucidate the molecular mechanisms of HCC pathogenesis and identify new therapeutic targets. Previous work from the Monga laboratory and other investigators has emphasized the importance of the Wnt/β‐catenin pathway in HCC pathogenesis. A study published in this issue of Hepatology focuses on targeting β‐catenin in HCC induced by overexpression of both K‐RAS and mutant β‐catenin, highlighting the use of lipid nanoparticles (LNPs) as a more targeted approach for the inhibition of select pathways, such as Wnt/β‐catenin, that are identified as important for tumor pathogenesis.1 The importance of the Wnt/β‐catenin pathway for HCC pathogenesis has been demonstrated by multiple groups. Through their previous studies, the Monga laboratory has provided evidence for cross‐talk between Wnt/β‐catenin and other signaling pathways, including nuclear factor kappa B, Hippo/yes‐associated protein, Hedgehog, and epidermal growth factor receptor. In the current study, the authors provide further evidence for the importance of β‐catenin in the pathogenesis of HCC, and particularly HCC that has been induced by coexpression of K‐Ras, an important effector in the RAS/RAF/MEK/ERK signaling pathway. Mutations in K‐RAS are rare in human HCC (∼7%); however, the current study sheds light on an interaction that could provide insight into a subset of HCCs. In this study, mice were delivered mutant, constitutively active G12D‐K‐Ras in combination with mutant β‐catenin (S45Y‐β‐catenin and S33Y‐β‐catenin). Coexpression of mutant K‐Ras and β‐catenin resulted in a profound increase in tumor burden within the liver, characterized as well‐differentiated HCC. It is important to note that K‐Ras mutant tumors had similar characteristics to Met mutant/β‐catenin–induced tumors reported previously.2 RNA microarray analysis comparing gene expression data between the Met/β‐catenin and K‐Ras/β‐catenin models confirms the hypothesis that hMet mutations cause activation of Ras, which then cooperates with mutant β‐catenin in the pathogenesis of HCC. To further solidify this signaling relationship, the authors provide evidence that silencing of β‐catenin gene activation could significantly reduce tumor burden, employing EnCore LNPs for tumor‐selective delivery of a Dicer‐substrate small interfering RNA (siRNA) against β‐catenin.3 This provides an interesting avenue for targeting the Wnt/β‐catenin pathway, because previous strategies have focused mainly on Wnt receptors.4 Mice injected with LNPs targeting β‐catenin showed reduced liver‐to‐body weight ratios and reduction in visible tumors. These mice also demonstrated almost complete absence of nodules tagged for mutant K‐Ras/β‐catenin. However, select mice did not respond to β‐catenin knockdown. The authors concluded that this may be due to tumor heterogeneity: the tumors escaping β‐catenin knockdown were typically negative for glutamine synthetase (GS), an established marker for β‐catenin activation, suggesting that β‐catenin knockdown may be an effective strategy only in GS‐positive HCCs. A cumulative analysis of published studies highlights the importance of an hMet–β‐catenin mechanistic relationship in the context of HCC pathogenesis (Fig. 1). The hMet signaling cascade can be broken into multiple branches. Two major branches of this cascade involve RAS/RAF/MEK/ERK signaling along with phosphoinositide 3‐kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR). It has been reported that hMet signaling in the context of hyperactivated β‐catenin results in increased incidence and progression of HCC. Therefore, it is plausible that a combination of mutant RAS (downstream of hMet signaling) and mutant β‐catenin would also result in this observed increase. This approach does highlight the potential importance of the RAS/RAF/MEK/ERK branch in this mechanism. In both studies, HCC was inhibited by targeting β‐catenin. β‐catenin knockdown led to a significant decrease in ERK activation, but only a small effect on mTOR, and no effect on phosphorylated AKT. These data indicate that the PI3K/AKT/mTOR branch of hMet signaling may escape the influence of β‐catenin inhibition and contribute to the observed tumor heterogeneity. It is possible that stimulation of β‐catenin has led to hyperactivation of the hMet signaling pathway through a positive‐feedback mechanism.5 Once β‐catenin is inhibited, the GS‐negative tumors may be stimulated by the residual hyperactivated PI3K/AKT/mTOR branch of hMet signaling.Figure 1: Working model for Met‐Ras‐β‐catenin cross‐talk in the pathogenesis of HCC. Past studies have elucidated multiple branches by which Met can stimulate hepatic proliferation and survival during liver regeneration and cancer pathogenesis, with β‐catenin emerging as a potent target for HCC therapies. Expression of mutant β‐catenin along with stimulation of Met or expression of mutant RAS leads to a profound increase in tumor burden. Inhibition of β‐catenin leads to potent inhibition of GS‐positive tumors with direct effects on β‐catenin targets, but also leads to inhibition of the RAS/RAF/MEK/ERK branch of Met signaling through inhibition of ERK. Select tumors (GS‐negative) can escape β‐catenin inhibition, suggesting that PI3K/AKT/mTOR signaling may function independently of β‐catenin to promote HCC growth.Although this study has further illustrated the importance of the Wnt/β‐catenin signaling pathway in the pathogenesis of liver cancer, there are no clinically relevant therapies available that target Wnt/β‐catenin in liver disease. This is most likely due to the ubiquitous requirement for intact Wnt/β‐catenin signaling for organ homeostasis, particularly in the intestine and bone marrow.6 In a recent review by Lu et al., as of late 2016, 34 clinical trials were listed to be targeting Wnt/β‐catenin signaling.4 Furthermore, there are 242 articles within PubMed that have focused on screens for inhibitors of Wnt/β‐catenin signaling, revealing 76 different chemical entities. Of the inhibitors being studied, 59% (45/76) have been designed to inhibit activation of the receptors, whereas 28% (21/76) are being designed to target β‐catenin directly. Popular targets for inhibition have been Wnt, Frizzled, and LRP6, with a recent target of interest being the Wnt secretion protein Porcupine, which is currently undergoing a phase 1 clinical trial in five independent studies. There are currently 19 ongoing clinical trials focusing on the inhibition of Wnt signaling, five of which are targeting β‐catenin directly. A concerning detail is that only five of these trials are recruiting patients with a condition of solid tumors, and of these five trials, only one has listed HCC as an indication directly. Furthermore, none of the current clinical trials focusing directly on targeting β‐catenin are recruiting HCC patients. Because Wnt/β‐catenin signaling is extremely important for tissue homeostasis, there are two issues to address when considering it as a drug target: 1) At which signaling level in the cell can Wnt/β‐catenin signaling be disrupted? At the level of Wnt ligand processing, at the receptor level, by targeting β‐catenin directly, or at its transcriptional interface? 2 How do we ensure tumor specificity? In the current study, the authors used EnCore LPNs to deliver siRNA against β‐catenin. EnCore LNPs are currently in clinical development for tumor‐directed delivery of siRNA and have been used for delivery of β‐catenin–targeting siRNA for various tumor types in mice.7 In the current study, the investigators demonstrate tumor‐specific uptake of LNPs. Furthermore, treatment with LNPs targeting β‐catenin resulted in decreased cell proliferation and increased cell death. However, as mentioned previously, a subset of tumors escaped β‐catenin knockdown and continued to proliferate. This subset of tumors was found to be positive for mutant β‐catenin, but negative for the β‐catenin target GS. These observations suggest that inhibition of β‐catenin alone may only be effective in a subset of GS‐positive HCCs. Regardless, this work highlights a successful strategy for specifically targeting select pathways in which global pharmacologic approaches have seen little to no success. In conclusion, studies such as the one published in this issue of Hepatology highlight the need for further definitive elucidation of Wnt/β‐catenin as a target for HCC therapies and continue to shed light on mechanisms surrounding this pathway in the context of HCC development. Furthermore, the current study demonstrates a novel strategy for targeting signaling pathways that play an important role in cancer pathogenesis but also have an essential function in organ homeostasis. Hopefully, these approaches can enhance our ability to treat HCC and other Wnt‐dependent cancers in the future.