Caspase-2 deficiency accelerates chemically induced liver cancer in mice

Aberrant cell death/survival has a critical role in the development of hepatocellular carcinoma (HCC). Caspase-2, a cell death protease, limits oxidative stress and chromosomal instability. To study its role in reactive oxygen species (ROS) and DNA damage-induced liver cancer, we assessed diethylnitrosamine (DEN)-mediated tumour development in caspase-2-deficient (Casp2−/−) mice. Following DEN injection in young animals, tumour development was monitored for 10 months. We found that DEN-treated Casp2−/− mice have dramatically elevated tumour burden and accelerated tumour progression with increased incidence of HCC, accompanied by higher oxidative damage and inflammation. Furthermore, following acute DEN injection, liver injury, DNA damage, inflammatory cytokine release and hepatocyte proliferation were enhanced in mice lacking caspase-2. Our study demonstrates for the first time that caspase-2 limits the progression of tumourigenesis induced by an ROS producing and DNA damaging reagent. Our findings suggest that after initial DEN-induced DNA damage, caspase-2 may remove aberrant cells to limit liver damage and disease progression. We propose that Casp2−/− mice, which are more susceptible to genomic instability, are limited in their ability to respond to DNA damage and thus carry more damaged cells resulting in accelerated tumourigenesis.

The initiator caspase, caspase-2, has recently emerged as a tumour suppressor with loss of this protein being associated with increased lymphomagenesis in ATM-deficient mice and Eμ-Myc transgenic mice, and MMTV/c-neu-driven mammary carcinoma. [1][2][3][4] Caspase-2 has been shown to protect cells from oxidative stress, DNA damage and genomic instability, and caspase-2 deficiency is linked to chromosomal instability and aneuploidy. 1,5,6 However, currently little is known about the possible role of caspase-2 in carcinogenesis induced by ROS and DNA damage.
Hepatocellular carcinoma (HCC) is among the most common cancers and is the second leading cause of cancer-related deaths worldwide. 7 Development of HCC is multifaceted progressing from DNA damage to dysplasia, adenoma development and malignant transformation of hepatocytes. 7,8 The major risk factors of HCC include viral hepatitis, excessive alcohol consumption, carcinogens and metabolic diseases. 7,8 Despite this, the molecular causes of the disease are relatively less understood. Diethylnitrosamine (DEN) is a DNA alkylating and ROS inducing carcinogen that is widely used as a model to study the progression of HCC in rodents. 9,10 DEN treatment mimics the human disease by inducing DNA damage in proliferating hepatocytes of infant mice. 9 Increases in the generation of ROS and accumulation of DNA damage can stimulate an inflammatory response and hyperproliferation that helps drive tumour progression. [7][8][9][10] It has increasingly been recognised that apoptosis and compensatory proliferation are crucial for progression of certain cancers including HCC. [11][12][13] While loss of apoptosis would indirectly favour proliferation, this is not universally true, for example loss of PUMA, or hepatocyte-specific deletion of Bid, impedes HCC by preventing compensatory proliferation. [14][15][16][17][18][19][20] Metabolic state is another important regulator of HCC progression and there is increasing evidence that caspase-2 has a subtle role in regulating this process. 21,22 Caspase-2 deficiency in mice results in metabolic perturbations and aged caspase-2 mice show increased oxidative stress and DNA damage. 5,21,23 DEN-induced tumour formation involves the generation of DNA adducts, 10 and human HCC is commonly associated with genomic instability. 24 These observations, combined with the known tumour suppressor role in lymphoma and MMTV-driven mammary carcinoma, suggest a possible role of caspase-2 in HCC. Thus, we sought to explore whether caspase-2 deficiency would promote chemically induced hepatic carcinogenesis in liver. We observed that DEN-induced liver tumours in Casp2 − / − mice were consistently larger and more advanced than those in WT mice, providing the first evidence that caspase-2 deficiency has a role in ROS and DNA damaged mediated HCC.

Results
Caspase-2 deficiency causes increased incidence of HCC. Casp2 −/− mice injected with DEN at 15 days old consistently showed enlarged livers with increased tumour burden compared with WT mice at 10 months post injection (Figures 1a and c). While all Casp2 −/− mice injected with DEN formed tumours at 10 months, 2/11 DEN-injected WT mice were tumour free (Figure 1b). Histological analysis of livers from young (6-9 weeks) non-DEN-injected tumour free Casp2 −/− mice had no abnormal liver morphology or perturbed hepatocyte nuclei. However, consistent with previous findings 23 by 10 months of age all PBS-injected Casp2 −/− mice displayed an increase in nuclear volume (karyomegaly) and binucleation ( Supplementary Figure 1a and b).
In DEN-injected WT mice, livers were usually macroscopically normal with occasional protrusion of tumour nodules ( Figure 1a). Microscopically, altered hepatic foci were often present (Supplementary Figure 1c). Foci were composed of hepatocytes generally resembling those found in adjacent unaffected hepatic parenchyma, but with a cytoplasmic tinctorial appearance ranging from eosinophilic, basophilic, clear or vacuolated (fatty change). Hepatocytes in tumour masses were generally well differentiated and resembled the tinctorial span found in hepatic foci. Mitoses were rare to absent. Tumour masses were usually well circumscribed, but frequently compressed the surrounding parenchyma, and were designated hepatocellular adenomas (HCAs) (Figure 1d and Supplementary Figure 1c).
Macroscopicaly, livers of Casp2 −/− mice often showed multiple tumour masses of varying size and colour ( Figure 1a). Microscopically, these contained many hepatocytes with enlarged, hyperchromatic, atypical nuclei of round to ovoid, more elliptical, or sometimes lobulated, appearance, sometimes bi-or multinucleated, and often showed prominent nucleoli (Figure 1d and Supplementary Figure 1d  Liver damage and inflammation is often accompanied by increases in hepatic cell death and proliferation. 13 We used immunoblot analysis and TUNEL to determine levels of apoptosis in DEN-injected mice. Slightly increased cleavage of caspase-3 was observed by immunoblotting in DENinjected WT, but not in Casp2 −/− mice ( Figure 3a). However, there was no difference in PARP cleavage or levels of PUMA ( Figure 3a) or in the frequency of TUNEL-positive cells ( Figure 3b). In addition, no difference in cleaved caspase-3 was detected by immunohistochemical analysis of liver sections (Supplementary Figure 3). Proliferation of hepatocytes as assessed by PCNA staining of DEN-injected livers was high in both WTand Casp2 −/− mice, but no difference was observed between genotypes ( Figure 3c).
Altered metabolism and stress-response pathways in advanced stage Casp2 −/− tumours. We next investigated the contribution of altered metabolism and stress/DNA damage response pathways to the increased susceptibility of DEN-induced HCC in Casp2 −/− mice. Activation of stressresponse pathways was analysed by immunoblotting with an increase in phospho-JNK being observed in Casp2 −/− mice along with a trend towards increased ERK activation ( Figure 4 and Supplementary Figure 4).
Previous studies have reported altered metabolic profile in Casp2 −/− mice. 19 During the development of HCC and HCA, the activity of several enzymes involved in glycogen metabolism, oxidative pentose phosphate pathway and glycolysis is different. 25 It is known that adenomas largely contain basophilic cells with high fat or glycogen content. 25 In addition, G-6-Pase and ALPase activity increases as adenomas develop into carcinomas while G6PDH activity declines in carcinomas. 25 DEN injection did not alter the levels of glycogen or G-6-P in WT or Casp2 −/− mice (Figures 5a and b). However, compared with WT, glycogen levels were lower in both PBS-and DEN-injected Casp2 −/− mice and G-6-P was lower in DEN-injected Casp2 −/− mice (Figures 5a and b). In addition, G6PDH activity was reduced in DEN-injected compared with PBS-injected Casp2 −/− mice (Figure 5c). Another group of proteins that regulate metabolism and have been linked with DEN-induced HCC is Sirtuins. [26][27][28] We therefore measured Sirt1 and 3 expression and activity. Sirt1 activity was reduced in DEN-treated Casp2 −/− mice, but not in WT (Figure 5d), whereas Sirt3 activity was significantly increased by DEN in WT mice but not in Casp2 −/− mice (Figure 5e). Surprisingly, an increase in total Sirt1 protein levels was detected in DEN-treated Casp2 −/− mice (Figure 5f). This may be a compensatory response from the decrease in Sirt1 activity. No differences in total Sirt3 protein levels were observed (Figure 5f). P53 is frequently inactivated (mutated) in HCC, impairing the DNA damage response. 24 Therefore, we investigated p53 and p21 levels by immunoblotting. Total p53 protein levels were somewhat increased in DEN-treated WT Increased cellular damage in the absence of cell death is responsible for proliferative advantage in Casp2 −/− mice. To further investigate the mechanisms that lead to increased tumour incidence and burden in Casp2 −/− mice, we carried out short-term DEN exposure. After 24 h DEN, little to no TUNEL-positive cells were observed in Casp2 −/− mice liver compared with WT but at 48 h both WT and Casp2 −/− mice displayed TUNEL-positive hepatocytes with no difference between genotypes (Figure 6a). Damage was localised to the central canal regions. γH2Ax is a well-known marker for dsDNA breaks and 48 h after DEN injection, γH2Ax was increased in both WT and Casp2 −/− mice, with Casp2 −/− mice having significantly higher number of γH2Ax-positive cells compared with WT ( Figure 6b). Notably, although reduced TUNEL reactivity was observed at 24 h in Casp2 −/− mice, pale necrotic centrolobular region was evident in both WT and Casp2 −/− mice (Supplementary Figure 5). PCNA staining was then performed on the same sections. At 48 h post DEN injection, there were significantly more PCNA-positive cells in the Casp2 −/− mice compared with WT mice (Figure 6c). To assess the extent of liver damage, we measured serum ALT and observed increased activity in Casp2 −/− mice at 48 h (Figure 6d). Compared with WT, IL6 levels were increased in Casp2 −/− mice at 24 and 48 h and IL1α levels at 48 h after DEN injection (Figures 6g and e). Furthermore, IL1α and IL1β were further increased in DEN-injected Casp2 −/− mice at 48 h compared with 24 h (Figures 6e and f).  Figure 7d).

Discussion
In this study, we provide a direct evidence for caspase-2 in delaying DEN initiated liver tumours. We find higher DNA damage in Casp2 −/− mice when exposed to DEN compared with WT mice. Casp2 − / − mice also had increased liver damage and formed aggressive HCCs. Hepatocarcinogenesis is a multistep process that can be caused by a number of agents that ultimately lead to malignant transformation of hepatocytes. [7][8][9][10]29 HCC induction by DEN is a commonly utilised mouse model. 9,10 DEN is an alkylating agent that induces DNA damage by reacting with nucleophiles such as DNA bases. 9,10 Furthermore, DEN bioactivation involves the activity of P450 enzymes which produces ROS and results in oxidative stress. 9,30 ROS-induced DNA, protein and lipid damage is known to contribute to hepatocarcinogenesis. [30][31][32] Single DEN dose causes HCC in 80-100% rodents in about 45-105 weeks 33 with the sequential emergence of preneoplastic hepatic foci, HCAs and then HCC. 25  WT mice were classified as adenomas, suggesting that tumours in Casp2 − / − mice were more advanced.
One reason for the advanced tumorigenic state may be higher ROS levels in Casp2 − / − mice leading to increased oxidative stress-induced DNA damage. We have shown earlier that Casp2 −/− mice experience excessive oxidative stress with ageing and oxidative stress response 5,23 and here again show increased susceptibility to more oxidative DNA and protein damage after DEN injection. Sirtuins contribute to the response to oxidative stress and the lower Sirt1 and Sirt3 activities in DEN-injected Casp2 − / − mice may in part contribute to increased susceptibility to DEN-induced damage. Sirt3 has several substrates involved in oxidative stress response such as MnSOD. 34 Consistent with the lack of increased Sirt3 activity as observed in WT, SOD activity was lower in DEN-injected Casp2 −/− mice, which is likely to contribute to higher oxidative damage in this group. Further, higher serum ALT levels and ALP levels indicate increased liver injury in this group of mice.
A number of signalling pathways have been implicated in DEN-induced liver carcinogenesis, and stress activated JNK expression was higher in Casp2 −/− mice. JNK activation leads to upregulation of several proapoptotic genes such as TNFα. 35 A dual role for JNK in development of HCC has been proposed. 36 In hepatocytes, higher JNK activity prevents HCC development while JNK activation in non-parenchymal liver cells promotes HCC development. 36 p53 is a well-known tumour suppressor that also regulates the DNA damage response. 37 DEN-injected WT mice showed a significant upregulation of total p53 protein and p53 expression (Supplementary Figure 6b). On the other hand, mdm2 was reduced in DEN-injected Casp2 −/− mice (Supplementary Figure 6c). As mdm2 is a target of p53, 38 reduced gene expression in Casp2 −/− mice may be a consequence of failure of p53 activation in this group. Our immunoblot data indicated increased p53 and p21 protein levels in DEN-injected Casp2 −/− mice. While no change in protein levels of PUMA, another p53 target that mediates DNA damage was observed, following DEN treatment control Casp2 −/− mice had elevated puma gene expression (Supplementary Figure 6d), which correlates with the observations that DNA damage is already higher in ageing Casp2 −/− mice. 22 Levels of noxa, another proapoptotic BH3-only protein, remained unchanged (Supplementary Figure 6e).
Interestingly, we also observed that IL1α, IL1β and IL6 levels were higher in DEN-injected Casp2 − / − mice. Inflammation has a key role in various types of cancers. 39,40 The classical liver inflammatory cytokines described in a number of HCC-linked liver inflammation models include IL6, TNF-α, IL1α and IL1β. 40,41 Several studies have reported higher IL6 levels in cases of HCC. 42,43 DEN exposure promotes production of IL6 in Kupffer cells and stimulates tumour growth. Male mice have higher IL6 levels compared with females due to oestrogen-mediated inhibition reflecting the lower incidence of DEN-induced HCC in females 44 and its importance in HCC progression can be realised from the fact that after ablation of IL6 male mice do not produce any more HCC than female mice. 43,45 Previous studies have demonstrated that in two different models of DNA damage-driven mouse tumours, deficiency of caspase-2 had no significant effect. The authors found that lymphomagenesis in mice induced by repeated exposure to low-dose γ-irradiation, or formation of fibrosarcoma by injection of 3-methylcholanthrene (3-MC), a carcinogen forming bulky adducts with DNA, were similar in WT and Casp2 −/− mice. 46 These data suggested that tumour suppression by caspase-2 is not involved in DNA damage-induced tumourigenesis. Our data on the other hand suggest that caspase-2 is a strong suppressor of DEN-induced HCC. Although it remains unclear why suppressor effects of caspase-2 are so vastly different in these models of tumourigenesis, it is possible that different mechanisms of tumourigenesis (HCC versus lymphomagenesis and fibrosarcoma) contribute to differences in DNA damage response. For example, in addition to DNA damage, increased inflammatory and compensatory proliferation responses also have important roles in HCC, but may have limited roles in γ-irradiationinduced lymphomagenesis and 3-MC-dependent fibrosarcoma formation. Further studies will be required to fully understand the tumour suppressive effects of caspase-2 in