Mechanisms by which obesity accelerates hepatocarcinogenesis: molecular studies in foz/foz mice
Hepatocellular carcinoma (HCC) is the second most common fatal cancer worldwide and the fastest increasing cause of cancer death in Australia. Epidemiological data indicate that obesity and diabetes are independent risk factors. This may be driven by the development of non-alcoholic fatty liver disease (NAFLD), but the molecular mechanisms driving such promotion of hepatocarcinogenesis are unclear. This thesis focuses on the potential role of obesity-related...[Show more] metabolic signalling pathways to promote hepatocarcinogenesis in a murine carcinogen-induced model. In addition, the cellular responses to DNA damage that could be relevant to obesity-enhanced hepatocarcinogenesis are addressed. Experiments were performed using foz/foz mice, a novel obese mouse model, and the carcinogen diethylnitrosamine (DEN). foz/foz mice have a spontaneous mutation of the Alström gene (Alms1). This impairs appetite regulation, so causing metabolic obesity, with secondary physical inactivity, hyperinsulinemia, diabetes, dyslipidemia, hyperleptinemia, and hypoadiponectinemia. Atherogenic (Ath) diet-fed foz/foz mice exhibit severe non-alcoholic steatohepatitis (NASH) with liver fibrosis. DEN-injected obese, diabetic foz/foz mice develop HCC at 6 months, whereas lean wildtype (Wt) mice do not develop liver tumours until 9 months. At 3 months, livers of DEN-injected foz/foz mice showed increased liver injury with apoptosis and compensatory hepatocellular proliferation compared to Wt littermates, and this was associated with more dysplastic hepatocytes. Livers of foz/foz mice (vs Wt) exhibited up-regulation of DNA-damage sensors (ATM and ATR). Despite this, there was inadequate cell cycle checkpoint controls by CHK1 and CHK2, at least in part affecting the ability of p53 to inhibit proliferation of damaged and altered hepatocytes during the progression of dysplastic hepatocytes to HCC. On the other hand, these studies found no evidence to support the concept that obesity enhances hepatocarcinogenesis by activation of inflammatory signalling (IL-6/STAT3) or via NF-κB. The hyperleptinemia exhibited by these obese mice did not induce STAT3 activation. One of the design strengths of the present study was the opportunity to examine the pre-malignant phase of hepatocarcinogenesis. Consistent with hyperinsulinemia and resultant increased bioavailability of IGF-1, Akt and nutrient-sensing mTORC1 were activated in fatty livers from obese diabetic mice at 3 months. The possibility that this may be functionally relevant for control of hepatocyte growth was indicated by enhanced phosphorylation of both S6 and eIF4B, the key downstream targets of mTORC1 which regulate protein synthesis, cell growth and proliferation. Further, AMPK activation was reduced in livers from obese mice; since AMPK supresses mTORC1 signalling, this lack of restraint could contribute to sustained mTORC1 signalling in foz/foz mice. In order to test whether mTORC1 activation was responsible for facilitating growth and survival of altered hepatocytes in obese mice, we administered rapamycin over 3 months to inhibit mTORC1. This strategy failed to prevent growth of dysplastic hepatocytes; further, consistent with a study conducted contemporaneously (Umemura et al, 2014), rapamycin tended to increase liver injury. Another attractive explanation to the link between obesity and enhanced HCC is oxidant stress, as observed here by increased hepatic GSSG levels and a decreased GSH to GSSG ratio. Further, the redox-responsive NRF2 pathway was up-regulated in livers from obese mice. By promoting nucleotide synthesis, sustained up-regulation of NRF2 signalling, in combination with increased PI3K/Akt signalling, could contribute to growth promotion of preneoplastic and/or cancer cells. In addition to NRF2, we also observed activation of JNK in livers from obese foz/foz vs lean Wt mice, as well as in HCCs derived from obese mice. JNK has been implicated in both NASH pathogenesis and hepatocarcinogenesis. In order to establish whether JNK1 is an essential mediator of enhanced hepatocarcinogenesis by obesity, we examined whether JNK1 deletion would alter hepatocarcinogenesis in atherogenic diet-fed mice (other work in the host lab had shown that this diet, like obesity, accelerated hepatocarcinogenesis). Atherogenic diet-fed Jnk1-/- and Wt mice were similarly overweight. JNK1 deletion did not alter adipose mass, but slightly impaired glucose tolerance. Atherogenic dietary feeding increased liver tumorigenesis in both DEN-treated Jnk1-/- and Wt compared with chow-fed. However, Atherogenic diet-fed Jnk1-/- mice developed fewer and smaller tumours than similarly fed Wt mice, and this culminated in a substantially reduced tumour burden. The attenuation of hepatocarcinogenesis by JNK1 deletion was associated with reversal of liver injury, hepatocyte apoptosis and hepatocyte proliferation. To establish whether the effects of JNK1 could be targeted therapeutically, we tested the efficacy of a selective JNK1 inhibitor CC930 (Celgene) against DEN-induced hepatocarcinogenesis in obese foz/foz NOD.B10 mice. Daily gavage of CC930 (200 mg/kg body weight) for 18 wks did not reduce liver injury or hepatocyte cell death, and accordingly failed to prevent the progression of DEN-induced liver tumorigenesis in foz/foz mice. Moreover, despite initial reduction of liver injury after 6 weeks of gavaging, longer term CC930 treatment appeared to increase serum ALT in obese mice, confounding interpretation of any effects on hepatocarcinogenesis. While obesity and diabetes are independent risk factors for HCC, the relative contribution of each metabolic condition to hepatocarcinogenesis has not been clarified. Insulin resistance and glucose intolerance are common in obese subjects, but some individuals do not develop these metabolic complications; such persons are referred to as “metabolically healthy obesity (MHO)”. In order to establish whether obesity per se (MHO) or diabetes (“metabolic obesity”) is more relevant to the acceleration of hepatocarcinogenesis, we compared DEN-induced HCC in equally obese but non-diabetic foz/foz BALB/c mice with the obese diabetic foz/foz NOD.B10 mice used earlier. At 6 months, the number of liver tumours was significantly higher in foz/foz NOD.B10 than foz/foz BALB/c mice (100% vs 40%), and liver nodules were more numerous and larger. Liver injury and hepatocyte apoptosis were also increased in foz/foz NOD.B10 mice compared with foz/foz BALB/c counterparts, but hepatocyte proliferation (cyclin D1 and PCNA) was similar. Akt/mTORC1 activation did not differ between the strains, but JNK was activated in foz/foz NOD.B10 mice (vs Wt) but not in foz/foz BALB/c mice. The activation of JNK was associated with enhanced oxidative stress, as indicated by increased NRF2 activation in foz/foz NOD.B10 mice but not in foz/foz BALB/c mice. Such oxidative stress may contribute to an enhanced DNA damage response in foz/foz NOD.B10 mice compared with foz/foz BALB/c mice, and/or activation of JNK and/or NRF2 which could act as signals for promotion of hepatocarcinogenesis, as discussed earlier. Physical activity is important for prevention and correction of obesity and its metabolic complications (it improves insulin sensitivity), and has a protective effect against development of some cancers. To test whether voluntary exercise could be sufficient to slow HCC development in obese mice, we provided them with an in-cage exercise wheel from weaning until 12 or 24 weeks of age. foz/foz mice spent as much time running as Wt mice; on average they travelled 4 km/day. With such physical activity, foz/foz mice demonstrated a slowing of weight gain compared with their sedentary counterparts, although at the end of experiments they still weighed considerably more than Wt mice. Exercise prevented the accelerated growth of dysplastic hepatocytes at 3 months and eventually reduced tumour burden at 6 months in foz/foz mice. Despite improved insulin sensitivity in exercising foz/foz mice, there was no evidence to support inhibition of mTORC1 activation as a protective mechanism. Instead, beneficial effects on the pathological severity of fatty liver disease, on JNK signalling, and on the p53 response to DNA injury occurred, each of which individually or severally could play an important role. In summary, the results of experiments described in this thesis indicate that accelerated DEN-induced HCC in obese diabetic mice cannot readily be attributed to IL6/STAT3 or NF-κB activation, or to direct effects of Akt/mTORC1 activation. Instead, the critical mechanism for obesity-enhanced hepatocarcinogenesis lies in the disconnection between hepatocellular injury and DNA damage, and the resultant unrestrained proliferative response of altered hepatocytes. Pharmacological inhibition of mTORC1 and JNK may not be effective to counter the effects of obesity in promoting hepatocarcinogenesis, though further work is required to study the combination of such agents with NRF2 inhibitor and/or AMPK activators. Meanwhile, the impressive protective effect of exercise for reducing the growth of dysplastic hepatocytes and delaying eventual HCC in obese mice should lead to similar studies in humans with NASH, who are at risk of HCC.
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