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Title: The Stress-Regulated Transcription Factor CHOP Promotes Hepatic Inflammatory Gene Expression, Fibrosis, and Oncogenesis}
Article:
Abstract:
Viral hepatitis, obesity, and alcoholism all represent major risk factors for hepatocellular carcinoma( HCC). Although these conditions also lead to integrated stress response( ISR) or unfolded protein response( UPR) activation, the extent to which these stress pathways influence the pathogenesis of HCC has not been tested. Here we provide multiple lines of evidence demonstrating that the ISR-regulated transcription factor CHOP promotes liver cancer. We show that CHOP expression is up-regulated in liver tumors in human HCC and two mouse models thereof. Chop-null mice are resistant to chemical hepatocarcinogenesis, and these mice exhibit attenuation of both apoptosis and cellular proliferation. Chop-null mice are also resistant to fibrosis, which is a key risk factor for HCC. Global gene expression profiling suggests that deletion of CHOP reduces the levels of basal inflammatory signaling in the liver. Our results are consistent with a model whereby CHOP contributes to hepatic carcinogenesis by promoting inflammation, fibrosis, cell death, and compensatory proliferation. They implicate CHOP as a common contributing factor in the development of HCC in a variety of chronic liver diseases.
Introduction:
HCC constitutes nearly 90 percent of all liver cancers in the United States, and liver cancer is the fifth most common cancer as well as the third most common cause of cancer related death worldwide[1],[2]. Risk factors for HCC include obesity, alcoholism, and viral hepatitis, due to the hepatic inflammation, fibrosis, and cirrhosis associated with these conditions[3]–[5]. In contrast to most other cancers, HCC prevalence has more than doubled in the U. S. over the past 20 years[2]. Stimuli that predispose to HCC show a striking commonality in their association with stress in the endoplasmic reticulum( ER), as well as signaling through the related unfolded protein response( UPR) and integrated stress response( ISR) pathways[6]–[10]. However, it is not known whether either the UPR or ISR contributes to HCC pathogenesis. The ER participates in diverse physiological processes including membrane protein folding and trafficking, calcium storage, drug detoxification, lipid and sterol synthesis, and various steps of lipid and sugar catabolism. ER stress is caused by disruption of the organelle's protein folding capacity, and can be induced by varied physiological and pathological stimuli[6]. ER stress is sensed by the UPR, which is initiated by three ER-resident transmembrane proteins and which culminates in alterations to gene expression that improve ER function. One of these initiating proteins is the ER-resident kinase PERK, which when activated phosphorylates the translation initiation factor eIF2α to transiently inhibit protein synthesis and to stimulate gene expression via the eIF2α-regulated transcription factor ATF4[7],[8]. eIF2α can also be phosphorylated by other kinases in response to diverse stresses( classically: viral infection, amino acid deprivation, or heme deficiency). This pathway, termed the integrated stress response( ISR), thus shares many transcriptional targets with the UPR[9]. IRE1α and ATF6α initiate the other two branches of the UPR, but are not thought to be activated by stimuli that do not cause ER stress; thus, the ISR is characterized by eIF2α-dependent signaling in the absence of IRE1α and ATF6α signaling. The UPR and/or ISR are implicated in the pathogenesis of multiple types of tumors including breast, colon, prostate, brain and lung[10]–[15]. Evidence for UPR activation in human HCC tumors also exists[16]. UPR- and ISR-mediated apoptosis appears to require the ATF4-dependent transcription factor CHOP( C/EBP Homologous Protein)[17]. Thus CHOP induction has been proposed as a strategy for ameliorating cancer, and pharmacological ER stresses that induce CHOP can kill cancer cells, including hepatomas, in vitro[18],[19]. However, in at least one case CHOP appears to promote oncogenesis, when it is fused by genomic rearrangement with either the FUS/TLS protein or the EWS protein[20]–[22]. Although the common association of conditions that predispose to HCC with activation of the UPR and ISR points to a possible role for these pathways in HCC pathogenesis, their contribution to the development and progression of liver cancer has not been tested.
Discussion:
This is the first report to our knowledge that links CHOP to hepatocellular oncogenesis. Our data lead us to propose a working model for the role of CHOP in HCC( Figure 7): Hepatocyte injury, be it in the form of lipotoxicity associated with obesity, the toxicity of ethanol metabolism, viral hepatitis, or genotoxic challenge, leads to activation of the ISR and production of CHOP either directly or after the initial formation of preneoplastic lesions such as the foci of cellular alteration that constitute some of the nodules produced by DEN. CHOP is strongly functionally associated with cell death in multiple contexts[17]. Consistent with that function, there is less cell death in the nodules of DEN challenged Chop−/− mice than wild-type animals( Figure 3). CHOP might promote cell death by enhancing production of reactive oxygen species[8], although we as yet have found no evidence for altered ROS production in Chop−/− mice. Alternatively, CHOP-dependent cell death might also be provoked directly as a consequence of regulation of BCL-dependent pathways[45],[46],[47]. The mechanism by which CHOP promotes hepatocyte cell death will be the subject of future investigation. We propose that initiation of cell death by CHOP provokes an inflammatory response, accounting for why Chop−/− mice show lower basal levels of inflammatory gene expression( Figure 5). Although classically thought to be “silent, ” more recently apoptosis has been shown to stimulate inflammation through several mechanisms including failure to clear apoptotic bodies, activation of pro-inflammatory signals in Kupffer cells that engulf apoptotic bodies, and secretion of pro-inflammatory cytokines by the apoptotic cells themselves[48]. We propose that inflammation stimulates hepatic stellate cell activation and fibrotic deposition, which would account for attenuated fibrosis in DEN-challenged Chop−/− animals( Figure 4). Inflammation also promotes compensatory hepatocyte proliferation through the effects of mitogenic cytokines[38], the diffusible nature of which would explain why Chop−/− animals show fewer Ki-67- and PCNA-positive cells than wild-type animals, and why these cells, unlike caspase-3-positive cells, are more-or-less uniformly distributed throughout the liver( Figure 3). The enhanced fibrosis and proliferation are then more likely to stimulate tumor formation. It is also possible that inflammation and fibrosis might further stimulate CHOP production, amplifying the effects of CHOP on tumorigenesis. This work establishes a functional role for CHOP in hepatic oncogenesis. While previous microarray-based approaches have not suggested Chop as a gene of interest([49] and references therein), our starting focus on UPR- and ISR-regulated genes allowed us to identify and subsequently experimentally validate a potential role for CHOP and provide a plausible pathway by which this role might be realized. The next step will be to test the key causal relationships with higher temporal resolution. Our data in Chop−/− mice allow us to place CHOP upstream of both fibrosis and tumorigenesis, since these animals are partially protected from both. Thus, we favor a model whereby the progression from CHOP expression to liver tumors occurs in a stepwise fashion, because this view is consistent with the prevalent notions of the etiology of HCC. Yet the observation that tumors in wild-type versus Chop−/− animals can be distinguished on at least one basis—the expression of ribosomal genes—and that CHOP expression seems most predominantly associated with nodules and tumors rather than surrounding non-transformed tissue in both mice and humans suggests that CHOP exerts an ongoing influence on tumor biochemistry. The fact that numerous genes encoding ribosomal subunits are upregulated in tumors from Chop−/− mice would suggest that CHOP ultimately inhibits protein synthesis. However, during the canonical ER stress response, CHOP is known to promote protein synthesis through upregulation of the regulatory GADD34 subunit of the PP1 phosphatase that dephosphorylates eIF2α[26]. Thus, it will be necessary to determine whether protein synthesis is actually altered in tumors from Chop−/− mice, and whether such alteration contributes to the apparent retardation of tumor growth seen in these animals. Our findings do not exclude the possibility that CHOP only assumes an aggravating role in fibrosis and tumorigenesis after the process has already begun. Or, alterntively, CHOP might promote the steps of inflammation, fibrosis, and tumorigenesis independently of each other. Further experiments will be needed to determine when CHOP is expressed during the tumorigenic process, and more particularly, whether its expression precedes the formation of neoplastic lesions and fibrotic deposition, and whether it is expressed in less- or more-advanced tumors. These—along with an inducible expression system for CHOP in the liver, which has not to our knowledge been reported—would enable us to determine whether CHOP facilitates fibrosis and initial tumor formation, or instead promotes further tumorigenesis once tumors have already formed; either possibility would be consistent with the data presented here. It is not currently clear whether the CHOP-independent ISR pathways activated during transformation also promote tumor growth or act against it, nor is the identity of the ISR kinase activated during the process. The balance of data suggest that ISR activation is generally anti-tumorigenic[50], yet ISR activation can clearly promote tumor growth in at least certain instances[15],[51],[52]. Liver-specific manipulation of the eIF2α kinases( PERK, PKR, HRI, and GCN2) will allow their roles to be tested. Also of potential use in this respect is a recently described inhibitor of PERK[53],[54], which, by blunting CHOP upregulation, might either protect against or reverse HCC in mouse models. The approximately 1000-fold selectivity of this molecule for PERK over other eIF2α kinases[53] will also enable determination of whether PERK is required for CHOP upregulation in these models. Another question to be addressed in future work is the cell autonomy of CHOP's pro-oncogenic effects. CHOP expression is associated with hepatocytes rather than macrophages, bile ducts, or stellate cells in human HCC( Figure 6), and appears to be so in mouse tumors as well( unpublished data). However, the immune and inflammatory genes that are differentially expressed in Chop−/− livers( Figure 5) are largely expressed in immune cells rather than hepatocytes. It thus seems most likely that CHOP acts cell-non-autonomously by promoting inflammation and compensatory proliferation. Or, CHOP dependence might arise entirely from CHOP function in a non-hepatocyte cell type( such as Kupffer cells), with tumor-associated CHOP expression being merely coincidental. Alternatively, the fact that TLS-CHOP and EWS-CHOP fusions show direct transformation potential leaves open the possibility that CHOP is directly transforming in hepatocytes as well. The transforming activity of fused CHOP in myxoid liposarcoma has been attributed to its ability to interfere with the activity of other transcription factors of the C/EBP family, thereby interfering with the adipogenic differentiation program, including expression of the inducing factor PPARγ2[55]–[57]. Given the association of dysregulated lipid metabolism with HCC—particularly via the activity of PPAR family members[58]–CHOP could promote HCC through altered lipid metabolism in parallel to an inflammatory mechanism, or such alterations could be the upstream stimulus that induces inflammation. Consistent with this idea, we recently demonstrated that CHOP alters expression of lipid metabolic genes including Ppara through interactions with C/EBP family members[59]. Or, CHOP might act cell-autonomously to de-differentiate cells and thereby loosen the constraints on their proliferative capacity. Ultimately, tissue-specific manipulation of CHOP expression will be required to discriminate among these hypotheses. CHOP expression is correlated with poor prognosis in both colorectal cancer and mesothelioma[60],[61] and elevated CHOP expression was seen in the central hypoxic regions of human cervical tumors[51]. Thus, understanding the mechanisms, both direct and indirect, by which CHOP promotes oncogenesis will likely have implications for other cancers beyond HCC. Our results have revealed in CHOP an unexpected molecular link between hepatocarcinogenesis and the stimuli that predispose to it. Understanding how CHOP is induced by these stimuli and how it ultimately promotes tumorigenesis will suggest additional points for potential therapeutic intervention, which are sorely needed for HCC. Note added in proof: It has come to our attention that, consistent with the results shown here, another group has found that Chop−/− mice are resistant to chemical hepatocarcinogenesis[62].
Liver cancer is the third most common cause of cancer death worldwide . It is most commonly caused by viral hepatitis , alcoholism , or obesity , all of which activate cellular stress responses in the liver . However , the contribution of these responses to disease pathogenesis was unknown . We found that expression of the stress-regulated transcription factor CHOP—widely thought to be anti-oncogenic because of its cell death-promoting properties—was associated with both human liver cancer and two mouse models thereof . In response to challenge with a tumor-causing agent , mice lacking CHOP developed fewer tumors , exhibited less cell death , compensatory cellular proliferation , and liver scarring ( fibrosis ) , and showed lower expression of immune and inflammatory genes . These findings establish CHOP as a biomarker for liver cancer and demonstrate its importance in promoting liver tumor formation . They raise the possibility that promotion of tumorigenesis by CHOP is a common feature of liver cancer caused by viral infection , alcoholism , and obesity .