Hepatocellular carcinoma is typically associated with a poor prognosis.
Most patients are diagnosed with advanced disease which has a 5 year survival of ~ 2%. Therefore prevention of HCC represents the best strategy to reduce mortality and morbidity. This requires the identification of patients at risk for HCC and the development of safe chemopreventive agents. Type II diabetics have significant increased risk for developing HCC (3
). The increased risk represents a growing health concern since diabetes rates are increasing, due in part to the obesity epidemic. Metformin is a first line drug of choice for the treatment of type II diabetes. In addition to its anti-diabetic effects, preclinical studies show that metformin has anticancer properties in vitro
and in vivo
). Epidemiological evidence shows a significant reduction in HCC in diabetic patients taking metformin (14
). Surprisingly, there have been no preclinical studies on the ability of metformin to inhibit HCC despite the liver being the main metformin responsive tissue. The studies we describe here show that metformin significantly protects against HCC formation and tumor growth. In addition, our data shows this is part via downregulation of multiple steps in de novo lipogenesis.
Several potential mechanisms have been proposed for inhibitory action of metformin on tumor growth (19
). Early reports suggested that metformin exerts its effect via activation of the energy sensor AMPK (17
). However in our studies we did not observe an increase in phosphorylated AMPK in the livers of treated mice, although fed and fasting glucose levels as well as gluconeogenic targets were reduced. This was further confirmed by the lack of phosphorylation of AMPK downstream targets, ACC and TSC2. This is in line with several recent studies highlighting AMPK independent effects of metformin on glucose homeostasis and tumor growth in vitro
and in vivo
). Although administration of metformin to mice did not alter AMPK activation in liver, we did observe activation in muscle. Indeed, the original manuscript describing metformin-mediated activation of AMPK in vivo was shown in muscle (18
These studies and others still contradict several studies showing AMPK activation in the liver by metformin. One likely explanation may be that mice were treated for an extended period of time in our experiments where as the other studies used short-term treatment (17
). In addition, it was recently demonstrated by Memmott et al, that AMPK activation by metformin in the liver may be route dependent (34
). They showed that intraperitoneal (IP) but not oral metformin treatment increased the phosphorylation of AMPK in liver. It is believed that IP administration leads to a higher systemic concentration compared to oral administration (34
). It is important to note that metformin is currently approved for orally administration, and therefore IP administered metformin is not clinically appropriate.
This prompted us to investigate other potential mechanisms responsible for the chemopreventive effects of metformin. De novo lipogenesis represents a common feature of many types of cancers and in particular HCC. The expression and activity of the two main fatty acid synthesis enzymes, ACC and FASN are elevated in several different cancer types including HCC. ACC is the rate-limiting step of de novo fatty acid synthesis, which converts acetyl CoA to malonyl CoA. FASN generates palmitate from acetyl CoA and malonyl CoA. Similarly our studies show that metformin reduces FASN expression in the livers of treated mice. In addition, we observe a decrease in ACC expression as well. In support of the potential of metformin to inhibit cancer growth via fatty acid synthesis, recently Algire et al showed that metformin treatment of tumor bearing mice led to a reduction in FASN expression in tumors (37
). Although metformin is reported to reduce fatty acid synthesis by activating AMPK, since we did not observe an increase in AMPK activation, an AMPK independent mechanism is most likely responsible. Regardless, these studies are the first to show the effect of metformin on lipogenic pathways in an autochthonous cancer model.
ACC and FASN utilize acetyl CoA for de novo lipogenesis in the cytoplasm. However acetyl CoA derived from glucose is generated in the mitochondria. In order for acetyl CoA to participate in fatty acid synthesis, it must be made available in the cytoplasm. Cells accomplish this by converting acetyl CoA and oxaloacetate (OAA) in the TCA cycle into citrate, which can then be exported to the cytoplasm. In the cytoplasm ATP-citrate lyase (ACLY) converts citrate back to OAA and acetyl CoA. We show that metformin also reduces ACLY expression. This would further reduce the ability of cells to perform de novo lipogenesis from glucose. The importance of ACLY in cancer is highlighted by studies showing that genetic or chemical inhibition of ACLY has anticancer effects (41
). The ability of metformin to reduce cell growth by inhibiting the gene expression of several genes driving fatty acid synthesis was confirmed using a genetic rescue approach. SREBP1c is a master regulator of lipogenesis in the liver. In addition, ACLY, ACC and FASN are direct transcriptional targets of SREBP1c. Ectopic expression of SREBP1c induced lipogenic gene expression and blocked the growth inhibitory effects of metformin in HCC cell lines. This demonstrates that the ability of metformin to reduce cell growth is in part mediated via inhibition of expression of multiple lipogenic genes. Therefore unlike inhibitors of fatty acid synthesis that target only one step, our data demonstrates that metformin regulates multiple pathways involved in fatty acid synthesis.
The studies we describe here were performed in non-diabetic mice. However, metformin is most likely as effective in diabetic or obese conditions. Indeed, recent studies show that metformin is more effective at reducing tumor growth in mice receiving a diet promoting obesity and diabetes (31
). In addition, the mechanism of action suggests it may be more effective in diabetic models. Diabetes is associated with increased fatty acid synthesis and hepatic steatosis (25
) both of which promote HCC (25
). Future studies will determine the ability of metformin to protect against HCC in diet and genetic rodent models of diabetes and obesity.
Our data also has relevance for cancer prevention with regard to other risk factors associated with HCC. NAFLD and obesity are independent risk factor for HCC. Both of these conditions are associated with increased lipogenesis and hepatic steatosis. However, it should be noted that NAFLD, obesity and diabetes are often co-morbidities, and found in most cases of HCC. These studies also have bearing for patients with HBV and HCV. Many patients with HBV and HCV have hepatic steatosis, and increased fatty acid synthesis (45
). The significance of this is underscored by a 100-fold increased risk of HCC in patients the co-morbidities of diabetes and obesity with hepatitis (49
). Therefore this represents a particularly important candidate group for consideration for metformin as a chemoprevention approach.
One of the advantages of metformin is its relatively safe toxicity profile. In addition, metformin is already approved for diabetes and therefore its introduction into the clinic streamlined. There are currently over 20 clinical trials investigating the role of metformin as an anticancer agent (50
). However, none of these are investigating the ability of metformin to reduce cancer in the liver, its main target organ of action. Prior to these trials it would be valuable to determine at what time point during liver carcinogenesis metformin is effective. In conclusion our data demonstrates that metformin inhibits HCC in part by inhibition of hepatic lipogenesis. This provides a rationale for clinical trials into the efficacy of metformin in HCC in patients that can readily be identified such as diabetics and other pathologies associated with hepatic lipogenesis.