Roles of protein kinase R in cancer: Potential as a therapeutic target.

Double-stranded (ds) RNA-dependent protein kinase (PKR) is a ubiquitously expressed serine/threonine protein kinase. It was initially identified as an innate immune antiviral protein induced by interferon (IFN) and activated by dsRNA. PKR is recognized as a key executor of antiviral host defense. Moreover, it contributes to inflammation and immune regulation through several signaling pathways. In addition to IFN and dsRNA, PKR is activated by multiple stimuli and regulates various signaling pathways including the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells pathways. PKR was initially thought to be a tumor suppressor as a result of its ability to suppress cell growth and interact with major tumor suppressor genes. However, in several types of malignant disease, such as colon and breast cancers, its role remains controversial. In hepatocellular carcinoma, hepatitis C virus (HCV) is the main cause of liver cancer, and PKR inhibits HCV replication, indicating its role as a tumor suppressor. However, PKR is overexpressed in cirrhotic patients, and acts as a tumor promoter through enhancement of cancer cell growth by mediating MAPK or signal transducer and activator of transcription pathways. Moreover, PKR is reportedly required for the activation of inflammasomes and influences metabolic disorders. In the present review, we introduce the multifaceted roles of PKR such as antiviral function, tumor cell growth, regulation of inflammatory immune responses, and maintaining metabolic homeostasis; and discuss future perspectives on PKR biology including its potential as a therapeutic target for liver cancer.

acute lymphoblastic leukemia

chronic lymphocytic leukemia

eukaryotic initiation factor 2 alpha

endoplasmic reticulum

extracellular signal‐regulated kinase 1/2

hepatitis B virus

hepatocellular carcinoma

hepatitis C virus

high mobility group box 1

interferon

inhibitor of B (IkB) kinase

interferon regulatory factor 1

interferon‐stimulated gene

liver cirrhosis

mitogen‐activated protein kinase

non‐alcoholic steatohepatitis

nuclear factor kappa‐light‐chain‐enhancer of activated B cells

platelet‐derived growth factor

PKR‐eIF2α phosphorylation homology domain

PKR‐like endoplasmic reticulum‐resident protein kinase

poly(rI)·poly(rC)

double‐stranded (ds) RNA‐dependent protein kinase

signal transducer and activator of transcription 3

INTRODUCTION

Double‐stranded PKR is a ubiquitously expressed serine/threonine protein kinase that was initially identified as an innate immune antiviral protein induced by IFN.1 It is also recognized as a host IFN‐stimulated gene.2 PKR was discovered after it was observed that cell extracts prepared from IFN‐treated vaccinia virus‐infected cells were sensitive to a translational block after the addition of exogenous mRNA3 and pIC, a synthetic analog of dsRNA.4 These studies led to the identification of a protein with dsRNA‐dependent kinase activity,5, 6 now known as PKR.7

PKR binds to dsRNA, resulting in a number of conformational changes, in which homodimerization appears to be most important according to biochemical and genetic analyses.8 PKR homodimerization leads to rapid autophosphorylation of a stretch of amino acids, namely Ser242, Thr255, Thr258, Ser83, Thr88, Thr89, Thr90, Thr446, and Thr451, termed the activation segment.9 Among others, residues Thr446 and Thr451 are consistently phosphorylated during activation,8, 10, 11 which further stabilizes the PKR dimer and increases its catalytic activity. Then, phosphorylated PKR phosphorylates Ser51 on the alpha subunit of eIF2α,12 and phosphorylated eIF2α inhibits the initiation of translation and decreases the rate of protein synthesis (Figure 1).

Figure 1

Antiviral effects of double‐stranded RNA‐dependent protein kinase (PKR). Double stranded‐RNA produced by RNA viral replication and interferon are potent activators of PKR. Activated PKR induces PKR dimerization and PKR phosphorylation. Then, PKR phosphorylates eukaryotic initiation factor‐2 alpha (eIF2α), which inhibits protein synthesis, including that of virally encoded proteins. HCV, hepatitis C virus

In addition to its established role in antiviral activities, PKR contributes to the regulation of inflammation and immune responses through several signaling pathways. In addition to IFN and dsRNA, PKR is activated by multiple stimuli, including cytokines such as tumor necrosis factor alpha (TNF), interleukin 1 (IL‐1),13, 14 LPS, through the Toll‐interleukin 1 receptor domain‐containing adaptor protein (TIRAP) and Toll‐like receptor 4 (TLR4) signaling pathways,15 PDGF through STAT3 and ERK 1/2 phosphorylation,16 heat shock protein 90,17 and some cellular stressors including arsenite, thapsigargin, and hydrogen peroxide.18, 19

PKR regulates various signal transduction pathways such as the MAPK, STAT,20, 21 and NF‐κB pathways,22 IRF123 or IRF3,24 and activating transcription factors.25 PKR has also been implicated as a general transducer of apoptosis,26, 27 and was shown to trigger autophagy through eIF2α‐mediated activation of microtubule‐associated protein light chain 3 (LC3).28 Thus, it is clear that PKR has multi‐functional roles in the regulation of inflammatory and immune signaling.

PKR has been implicated as an ER stress‐regulated kinase, as well as a PERK. Both PKR and PERK are activated by autophosphorylation, after which they phosphorylate eIF2α.29 Several studies have reported the probable role of PKR in ER stress‐induced neural cell death in Alzheimer's disease and Huntington's disease.30, 31 Moreover, Lee et al32 found that PKR plays a significant role in ER stress‐induced apoptosis mediated by protein activator of interferon‐induced protein kinase (PACT).

PKR AND HUMAN HEPATITIS C VIRUS

Hepatitis C virus infection is a major public health concern. About 150 million individuals are infected worldwide, and each year, 3‐4 million individuals become infected with the virus.33, 34 HCV is the leading cause of chronic liver disease and the most common indication for liver transplantation. HCV establishes persistent infection and induces chronic hepatitis, which leads to LC and frequently to HCC. However, the detailed mechanisms underlying the progression of LC to HCC remain unknown. Both dsRNA produced by HCV replication and the HCV core protein can activate PKR.35 It is likely that PKR activation by the core protein is associated with the similar ability of the latter to bind dsRNA, thereby providing the PKR activator substrate and possible mechanism of PKR activation during HCV infection.36 In the liver tissue of patients with chronic HCV, PKR mRNA is significantly increased compared with that in patients with other etiologies.37, 38 In contrast, consistent with the important role of PKR in the control of HCV infection, HCV has several PKR‐inactivation strategies (Figure 2). For example, the IFN sensitivity‐determining region in the HCV‐1b NS5A region within the PKR‐binding domain inhibits IFN‐induced PKR, thereby influencing responses to IFN‐based therapies.39, 40 In addition, the PKR‐eIF2α phosphorylation homology domain of the HCV E2 gene also inhibits IFN‐induced PKR.41 However, it remains unclear whether these proteins contribute to persistent HCV or are resistant to the antiviral activities of PKR.9, 42 Tokumoto et al43 showed that HCV protein expression is directly dependent on PKR expression. In this report, HCV core protein levels significantly increased upon knockdown of PKR; conversely, overexpression of PKR significantly suppressed HCV core levels in cell lines transfected with full‐length HCV constructs. Moreover, PKR expression was responsible for the antiviral effects of IFN against HCV. Hence, in contrasting actions, PKR suppressed HCV replication, and HCV suppressed PKR function. In chronic HCV infection, the functions of HCV and PKR are balanced. In patients with chronic HCV infection, we speculate that HCV function against PKR is greater than the anti‐HCV function of PKR, so active inflammation is maintained. However, in liver cancer, PKR is overexpressed and its functions dominate, resulting in cancer progression (Figure 3).

Figure 2

Anti‐double‐stranded RNA‐dependent protein kinase (PKR) effects as a result of hepatitis C virus (HCV). HCV has developed PKR inhibitory strategies for their persistence. The NS5A of HCV‐1b with a wild‐type interferon sensitivity determining region (ISDR) sequence within the PKR‐binding domain has the potential to block the interferon (IFN)‐induced PKR that mediates various aspects of the antiviral effects. The E2 gene of HCV interacts with the PKR‐eukaryotic initiation factor‐2 alpha phosphorylation homology domain and inhibits PKR activation by IFN

Figure 3

Relationship between double‐stranded RNA‐dependent protein kinase (PKR) and hepatitis C virus (HCV). In patients with chronic HCV infection, HCV function against PKR is greater than the anti‐HCV function of PKR, so active inflammation is maintained. However, in liver cancer, PKR is overexpressed, and its functions dominate, resulting in cancer progression

PKR AND MALIGNANT DISEASES

Initially, PKR was thought to be a tumor suppressor as its overexpression in mammalian, insect, and yeast cells led to the suppression of cell growth,44, 45 indicating its role in inhibiting cell proliferation. In addition, the expression of several PKR dominant‐negative mutants led to the malignant transformation of NIH 3T3 cells, causing tumorigenesis in nude mice.46, 47 These results suggested that PKR was able to activate some apoptotic signals, thereby supporting the notion that it may be a tumor suppressor.27, 48 Moreover, PKR interacts with major tumor suppressor genes such as p53 and phosphatase and tensin homolog, and plays essential roles in their tumor suppressor functions.49, 50 However, the role of PKR in cancer biology remains a subject of debate. In Jurkat T cells, the reduced expression of PKR also corresponded with reduced PKR activity,51 and the resultant effects were characterized in the promonocytic leukemia cell line U937.52 Furthermore in 21 of 28 chronic lymphoid leukemia cells, PKR was expressed but not active.53 However, several reports have indicated that PKR is overexpressed and activated in several hematopoietic malignancies. For example, PKR is generally overexpressed in AML and ALL.54 In addition, the status of PKR activity was recently examined in AML and ALL cell lines, and patient samples and both cell types had significantly higher levels of phosphorylated PKR/PKR activation compared with normal controls.55 In thyroid carcinoma, there was a reverse correlation between PKR expression and Ki‐67 labeling, suggesting that tumor cells with low PKR expression had much higher proliferative activity than those with high PKR expression in thyroid carcinoma.56 The reverse correlation between PKR expression and tumor cell proliferation has been reported in head and neck squamous cell carcinoma.57 Furthermore, He et al58 reported that in patients with non‐small‐cell lung carcinoma, those with high levels of phosphorylated PKR or phosphorylated eIF2α had significantly longer median survival than those with little or no expression. By contrast, Kim et al59 showed by immunohistochemical analysis that primary melanomas had minimal PKR immunoreactivity, but lymph node metastases expressed high levels of PKR protein. In the same report, analyses of colon cancer specimens showed that malignant transformation from normal mucosa to adenoma and adenocarcinoma was coincident with an increase in PKR expression. Kim et al60 also reported that PKR autophosphorylation and phosphorylation were much higher in human breast cancer cell lines than in non‐transformed mammary epithelial cell lines. Moreover, Roh et al61 demonstrated that patients with small‐sized peripheral lung cancer and high‐grade PKR expression had significantly shorter survival than those with low‐grade expression. These conflicting data suggest that the precise role of PKR in cancer may differ depending upon the pathological type of tumor, stage of tumor development, and tumor microenvironment.

PKR AND LIVER CANCER

Chronic HCV infection is specific to liver cancer (not to other types of cancer); hence, among the many PKR upstream triggers previously described, HCV infection and IFN signaling might be liver cancer‐specific triggers. Moreover, in HCC related to NASH, nutrition or energy excess might contribute to liver cancer. The first report on the relationship between PKR and HCC was by Shimada et al62 in 1998. In that report, the authors showed that PKR expression in HCV‐related HCC was higher in moderate‐ to well‐differentiated carcinomas compared with poorly differentiated HCC or LC. Because this study was not carried out using HCC/non‐HCC tissues pairs, it was not determined whether PKR levels in the surrounding tissue were also variable. To clarify the role of PKR in HCV‐related hepatocarcinogenesis, expression of PKR protein in paired malignant and surrounding non‐malignant tissues was examined from patients with HCV‐related HCC.63 The results showed that PKR protein levels were consistently increased in HCC‐related HCC tissues compared with surrounding non‐HCC tissue, and a similar increase was seen in eIF2α expression. In addition, HCV copy number was reduced in HCC compared with LC tissue, indicating that overexpressed PKR in HCC tissues retained its antiviral function against HCV. Importantly, sequence data indicated that increased PKR expression was not only functional but was also wild type, showing that mutant PKR had not accumulated. We studied the molecular mechanisms of PKR in HCV‐related HCC using two permissive cell lines for HCV replication (JFH1 and H77s), as well as human HCC specimens with HCV infection.64 In HCV‐related HCC cell lines, PKR upregulated c‐Fos and c‐Jun activities through activation of ERK1/2 and JNK, respectively, subsequently increasing HCC cell proliferation. Moreover, coordinated upregulation of c‐Fos and c‐Jun signaling was confirmed in human HCC specimens. c‐Fos and c‐Jun transcription factors have been implicated in carcinogenesis, and they regulate genes including important regulators of invasion and metastasis, proliferation, differentiation, and survival in several types of cancer.65, 66 However c‐Fos and c‐Jun activation through PKR signaling has been reported in HCV‐related liver cancer only.63, 64 c‐Fos and c‐Jun activation by PKR signal may be as a result of HCV chronic infection, so this signaling in liver cancer associated with other etiologies is not yet clear.

Recently, Wang et al67 reported that PKR plays a key role in increasing the proliferation and migration of HepG2 human HCC cells, and mouse xenograft models also confirmed the tumorigenic role of PKR in HepG2 cells. In that report, the tumor‐promoting function of PKR was mediated by the STAT3 transcription factor. The authors used human primary tumor samples and the surrounding tissue to show that the expression of total and phosphorylated PKR was upregulated in tumor tissues compared with the surrounding tissues. Importantly, patients did not have HBV or HCV infection, indicating that PKR is also activated in non‐HCV‐related liver cancer. These data do not support the role of PKR as a classic tumor suppressor, but suggest that PKR may have a positive regulatory role in controlling tumor growth and progression in liver cancer (Figure 4).

Figure 4

Cancer suppression and progression effects of double‐stranded (ds) RNA‐dependent protein kinase (PKR). PKR influences liver cancer through mechanisms that function either against viral infection or through the growth of cancer cells. PKR during viral infection in hepatocytes or apoptotic effect shows its function of tumor suppression. In contrast, PKR acts as a tumor promoter through enhancement of cancer cell growth. Thus, PKR works as a molecular Jekyll and Hyde in liver cancer. eIF2α, eukaryotic initiation factor 2 alpha; HCV, hepatitis C virus; HSP, heat shock protein; IL, interleukin; IRF, interferon regulatory factor; MAPK, mitogen‐activated protein kinase; NF‐κB, nuclear factor kappa‐light‐chain‐enhancer of activated B cells; PACT, protein activator of interferon‐induced protein kinase; PDGF, platelet‐derived growth factor; STAT, signal transducer and activator of transcription; TLR4, Toll‐like receptor 4; TNFα, tumor necrosis factor alpha

POSSIBILITY OF PKR AS A THERAPEUTIC TARGET AGAINST LIVER CANCER

Lu et al68 reported that PKR is required for the activation of inflammasomes and the subsequent release of HMGB1 protein, a pro‐inflammatory cytokine. The functions of inflammasomes were recently reported in various inflammation‐induced cancers.69 With the prevalence of HCV expected to decline, the proportion of HCC related to NASH is anticipated to significantly increase as a result of the growing epidemic of obesity and diabetes.70 Nakamura et al71 reported that PKR can respond to nutrient signals, as well as to ER stress, and coordinates the activity of other critical inflammatory kinases, such as JNK, to regulate insulin signaling and metabolism. Dietary and genetic obesity features indicated the activation of PKR in liver tissue, and deletion of PKR led to decrease metabolic disorder as a result of nutrition or energy excess in mice. This report suggests that PKR may be the key molecule in HCC associated with NASH or metabolic disorders. Taken together with the aforementioned reports on the relationship between PKR and liver cancer, it appears that PKR has multifaceted roles in liver cancer, such as antiviral function, promotion of tumor cell growth, regulation of the inflammatory immune response, and maintenance of metabolic homeostasis. Thus, PKR might be an effective therapeutic target in human liver cancer (Figure 5). We consider that inhibition of HCV replication by PKR could indirectly affect tumor suppression. However, once liver cancer has developed, the effects of PKR on decreasing HCV are not sufficient for tumor suppression, whereas overexpressed PKR contributes to tumor progression. It has been proposed that PKR might activate some apoptotic signals or interact with several tumor suppressor genes.26, 72, 73 However, in liver cancer, these tumor suppressor effects have not yet been confirmed. Considering other functions underlying cancer progression in liver cancer, we believe that a strategy for inhibiting PKR might be effective against liver cancer caused by several etiologies. The negative effects against liver cancer by inhibiting PKR, for example, by increasing HCV replication, need to be verified in future studies.

Figure 5

Multi‐functional roles of double‐stranded RNA‐dependent protein kinase (PKR) against liver cancer. PKR has multifaceted roles in liver cancer including antiviral functions, promotion of tumor cell growth, regulation of the inflammatory immune response, induction of autophagy, and maintenance of metabolic homeostasis. PKR may be an attractive therapeutic target for human liver cancer. HMGB1, high mobility group box 1; LC3, light chain 3; MAPK, mitogen‐activated protein kinase; NF‐κB, nuclear factor kappa‐light‐chain‐enhancer of activated B cells; STAT, signal transducer and activator of transcription

CONCLUSIONS AND PERSPECTIVES

Recent progress on the roles of PKR in liver cancer has shown that PKR influences liver cancer through mechanisms via its antiviral properties or by negatively regulating the growth of cancer cells. Studies on PKR during the virus infection of hepatocytes showed its tumor suppression function. However, it can also act as a tumor promoter through enhancement of cancer cell growth. Thus, PKR functions as a molecular Jekyll and Hyde against liver cancer. The complex nature of the relationship between PKR and virus infection in liver cells and liver cell growth presents a unique challenge and opportunity to develop therapeutic intervention strategies for targeting liver cancer. Further studies examining the role of additional molecules in regulating PKR signaling in liver cancer will provide additional insights into the development of small‐molecule or peptide‐based inhibitors for future therapeutic treatments.

CONFLICT OF INTEREST

Research finding: Takeshi Imamura from SONY Corporation and Chugai Corporation.