Background
Hepatocellular carcinoma (HCC) is the sixth most prevalent human malignancy in the world and the third leading cause of cancer-related mortality [
1]. Relative 5-year survival rate of HCC is only 15 %, which emphasizes the importance of primary prevention of HCC. Chronic infection by hepatitis B and hepatitis C virus, exposure to aflatoxin, alcoholic injury and genetic disorders have proven to play a critical role in the development of HCC [
2], however, the etiology remains unknown in almost 50 % of HCC patients. Recent studies suggest that nonalcoholic fatty liver disease (NAFLD) is associated with an increased risk of HCC [
3,
4], but it remains unclear whether NAFLD is a causative factor for HCC [
5].
Steatosis is the initial stage of NAFLD, which can progress into more pathological stages including nonalcoholic steatohepatitis (NASH), fibrosis and cirrhosis, with the result of the increased risk for HCC development. Previous studies had indicated the significant contribution of NASH to HCC development, where pro-inflammatory cytokine and chemokines favors malignant transformation of hepatocytes by providing a tumor microenvironment [
6,
7]. Inflammatory cascades through interactions of numerous signaling pathways progressively stimulated hepatocyte proliferation and apoptosis [
8]. However, the potential causal relationships among inflammation, steatosis and HCC development need more supporting evidence.
Tumor progression locus 2 (TPL2), a serine-threonine kinase, functions as a critical regulator of inflammatory pathways and mediates oncogenic events by phosphorylating its downstream targets extracellular signal regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs) and P38 [
9] and subsequently up-regulating the production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) [
10]. The role of
Tpl2 in acute and chronic inflammatory disorders and the determination of cellular death/survival ratios in the inflammatory microenvironment had been well-documented [
11]. Our previous work demonstrated that whole-body ablation of
Tpl2 attenuates high fat diet (HFD) induced hepatic inflammatory lesions compared to wild-type control mice, with a concomitant significant reduction in hepatic inflammatory genes expression [
12]. However, the potential role of
Tpl2 in tumorigenesis remains inconsistent [
11,
13]. Several reports supported its oncogenic role in breast cancer [
14], lymphoma [
15] and prostate cancer [
16], while others had suggested an anti-oncogenic activity in lung [
17], colitis-associated tumorigenesis [
18] and skin tumorigenesis [
19]. This inconsistency could be attributed to the complexity of
Tpl2’s role in terms of specific organs, different stages of carcinogenesis or the animal models used. However, there had no reports investigating the potential contribution of
Tpl2 to HCC development by using
Tpl2 knockout mouse model to date.
In the present study, we investigate the role of Tpl2 and its potential mechanisms in the development of hepatic steatosis, inflammation and tumors including HCC. Both wild-type and Tpl2 knockout male mice were initiated with [diethylnitrosamine (DEN)] at 2 weeks of age, and 4 weeks later, both groups mice were given the high carbohydrate diet (HCD) feeding for 24 weeks to induce hepatic steatosis, inflammation and HCC.
Discussion
The role of hepatic inflammation induced by dietary factors, such as HCD and HFD, in promoting DEN-initiated HCC development had been demonstrated in previous studies [
6,
22,
25]. The present study, for the first time, revealed the significantly lower incidence of hepatic tumor with no HCC development in HCD-fed,
Tpl2 knockout mice in contrast to wild-type mice which all developed HCC. The significant difference in tumor pathological types between
Tpl2 knockout mice and wild-type mice supported the critical role of
Tpl2 as a promoter in tumor progression from hepatic hepatocellular adenoma to HCC. Furthermore, we provided a strong evidence that the TPL2 ablation decreased hepatic inflammatory response and hepatic steatosis in
Tpl2 knockout mice. These effects could inhibit the malignant transformation of hepatocytes and the progression of liver tumor by suppressing tumor-promoting microenvironment and alleviating the malignant effects of dys-regulation of lipid metabolism on HCC.
TPL2 mediated inflammatory response by phosphorylating ERK and JNK, two downstream targets of TPL2 signaling pathway. In our study, the significant lower protein levels of hepatic p-JNK and p-ERK in
Tpl2 knock out mice was consistent with the decreased hepatic inflammation, which supported the role of activated ERK (p-ERK) and JNK (p-JNK) in mediating the pro-inflammatory effect of
Tpl2. The decreased hepatic inflammation induced by HCD feeding supported by the fewer hepatic inflammatory foci detected and the lower levels of inflammatory cytokine expression of
Il-1β,
Il-18,
Mcp-1 and
Nalp3 in
Tpl2 knockout mice, as compared with wide type mice. This is also in agreement with our previous study that
Tpl2 knockout mice fed HFD had lower levels of inflammation compared to wild-type mice [
12]. The present work further indicated the role of
Tpl2 in mediating hepatic inflammation and HCC development induced by HCD.
Elevated hepatic DNL could promote hepatic steatosis [
26]. Interestingly, in our present study,
Tpl2 ablation resulted in a significant decrease of hepatic steatosis, and the down-regulated protein expression of genes related to DNL, such as ACC and SCD1, which was associated the decreased protein expression of AKT phosphorylation and SREBP-1C.
Previous studies have suggested that AKT activation is essential and sufficient to stimulate DNL and lipid accumulation through the induction of SREBP-1C [
27,
28]. Furthermore, the promoting role of dysregulated lipid metabolism [
29] and lipogenesis induced by activated AKT in HCC development had been documented [
30]. Thus, the decrease in steatosis and AKT activation could further explain the decreased HCC in
Tpl2 knockout mice.
However, the exact role of
Tpl2 in regulating the genes related to DNL is unclear. It has been reported that the regulation of lipid metabolism and hepatic steatosis mediated by the activation of JNK, the downstream target of TPL2, is associated with activated ER stress, especially in the PERK-eIF2a pathway [
31]. Our observation of the down-regulated expression of p-JNK, PERK and p-eIF2a in
Tpl2 knockout mice with the decreased hepatic steatosis supported the involvement of ER stress in TPL2/JNK mediated steatosis. Recent reports have demonstrated that ER stress is closely associated with hepatic lipogenesis with elevated DNL [
26,
32,
33], and population-based studies also support the positive regulation of ER stress in hepatic lipogenesis [
34,
35]. Since newly synthesized unfolded proteins in the ER is a major cause of activated ER stress and activated ER stress could further induce lipogenesis, the vicious cycle could result in the progression of steatosis [
21]. In our present study, the deceased expression of the lipogenic enzymes ACC, SCD1 and SREBP-1C could decrease the impact of protein folding in the ER, and alleviate ER stress in
Tpl2 knockout mice. PERK-mediated signaling can also promote apoptosis through inducing pro-apoptotic CHOP expression, the no significant difference of the protein level of CHOP suggested that HCD induced expression of PERK predominantly mediated lipogenesis, but not pro-apoptotic effects in our present study. Furthermore, the expression of GRP78, a chaperone protein that attenuates ER stress [
36], were not different between the
Tpl2 knockout and wild-type mice, combining with our observation that
Tpl2 knockout mice had relatively lower levels of PERK and p-eIF2a, the downstream molecular of GRP78, than wild-type mice, we therefore concluded that
Tpl2 mediated hepatic lipogenesis by targeting the axis of TPL2/JNK/ER stress/p-eIF2a, the downstream of GRP78.
Our previous study had shown that HCD feeding promoted DEN-initiated HCC development accompanying with the induction of the hepatic ER stress-mediated PERK activation, which subsequently induced the elevated expression of pro-survival markers AKT and ERK1/2 [
22]. Our present study demonstrated that
Tpl2 ablation decreased ER stress mediated PERK expression and eIF-2a activation, which might account for the decreased tumor incidence. Since ER-dependent cell fate is associated with the activation of JNK/ERK [
37], and the pro-survival role of activated ERK can be mediated by AKT phosphorylation and the involvement of PERK/eIF2a signaling [
38‐
40], thus, in the present study, the down-regulated activation of ERK, AKT and PERK/eIF2a provided further explanation for the decreased incidence of HCC in
Tpl2 knockout mice. The exact relationship between TPL2 and ER stress requires further investigation.
In summary, our present study demonstrated that
Tpl2 played significant role in DEN-initiated, NAFLD associated HCC development by using
Tpl2 knockout mouse model. Both TPL2/ERK/JNK axis mediated hepatic inflammation and TPL2/JNK/ERS/p-eIF2a axis mediated hepatic lipogenesis synergistically promoted HCC development. These data provided strong molecular evidence supporting
Tpl2 as a promoter in HCC development. Interestingly, it has been shown that luteolin, one of the common phytonutrients present in celery, parsley, broccoli and herbal spices, could target
Tpl2 and inhibit its activity in vitro [
41]. Recent in vivo studies also supported the preventive effects of luteolin on DEN-initiated alcohol-promoted hepatic carcinogenesis in mice [
42], and DEN-initiated HCC development in rats [
43]. Taking all into consideration, dietary or pharmacologic interventions targeting
Tpl2 could be a potential direction for HCC prevention in the future.
Competing interests
The authors declare no conflicts of interest.
Authors’ contributions
XLL and KQH conducted RT-PCR and Western blot. CL participated in the histo-pathological analysis. DES and ASG participated in the animal breeding. BCI and XDW participated in the design of the study. XLL performed the statistical analysis. XLL and XDW drafted the manuscript. All authors read and approved the final manuscript.