Anti-tumoral action of cannabinoids on hepatocellular carcinoma: role of AMPK-dependent activation of autophagy

Hepatocellular carcinoma (HCC) is the third cause of cancer-related death worldwide. When these tumors are in advanced stages, few therapeutic options are available. Therefore, it is essential to search for new treatments to fight this disease. In this study, we investigated the effects of cannabinoids – a novel family of potential anticancer agents – on the growth of HCC. We found that Δ9-tetrahydrocannabinol (Δ9-THC, the main active component of Cannabis sativa) and JWH-015 (a cannabinoid receptor 2 (CB2) cannabinoid receptor-selective agonist) reduced the viability of the human HCC cell lines HepG2 (human hepatocellular liver carcinoma cell line) and HuH-7 (hepatocellular carcinoma cells), an effect that relied on the stimulation of CB2 receptor. We also found that Δ9-THC- and JWH-015-induced autophagy relies on tribbles homolog 3 (TRB3) upregulation, and subsequent inhibition of the serine–threonine kinase Akt/mammalian target of rapamycin C1 axis and adenosine monophosphate-activated kinase (AMPK) stimulation. Pharmacological and genetic inhibition of AMPK upstream kinases supported that calmodulin-activated kinase kinase β was responsible for cannabinoid-induced AMPK activation and autophagy. In vivo studies revealed that Δ9-THC and JWH-015 reduced the growth of HCC subcutaneous xenografts, an effect that was not evident when autophagy was genetically of pharmacologically inhibited in those tumors. Moreover, cannabinoids were also able to inhibit tumor growth and ascites in an orthotopic model of HCC xenograft. Our findings may contribute to the design of new therapeutic strategies for the management of HCC.


Introduction
Hepatocellular carcinoma (HCC) is one of the most common solid tumors and the third leading cause of cancer-related death worldwide 1 . Its prognosis remains reserved, with a 5-year survival rate of <5% 2 . It is the most common cause of death in patients with cirrhosis [3][4] and, according to the World Health Organization, the incidence of HCC is expected to increase until 2030. The overall survival of patients with HCC has not significantly improved in the last two decades. Current treatments are only applicable at early stages of tumor development and include tumor resection, liver transplantation, chemoembolization, and sorafenib administration [5][6] . However, approximately half of the patients suffer tumor recurrence. The most important mechanism of liver cancer progression is cell proliferation. Although in recent years several clinical trials have tested the efficacy of agents that selectively target key signaling pathways involved in the control of this process, no relevant improvement in the prognostic/survival of patients with HCC has been achieved so far 7 and, therefore, it is necessary to identify novel therapeutic strategies for the management of HCC.

Cannabinoids are lipid mediators originally isolated from the hemp plant
Cannabis sativa that produce their effects by activating primarily two G-protein coupled receptors: CB 1 , which is highly abundant in the brain, and CB 2 , which is mainly expressed in non-neural tissues. Recently, numerous studies have evidenced the role of cannabinoids as potential anti-tumoral drugs owing to their ability to reduce tumor in different animal models, including glioma 8 , breast cancer 9 10 and prostate cancer 11-12 . Recent research has also reported that the synthetic cannabinoid WIN-55,212-2 inhibits HCC growth [13][14] .
It has been described that Δ 9 -tetrahydrocannabinol (Δ 9 -THC), the main active constituent of marijuana, triggers human glioma cell death through stimulation of an ER stress pathway that activates autophagy and promotes apoptosis [15][16] . Autophagy is a cellular self-digestive process whereby bulk cytoplasmic components and intra-cellular organelles are sequestered into double membrane vesicles named autophagosomes and delivered for degradation to the lysosomes 10,[17][18][19] . In the liver, autophagy may play an important role in the regulation of energy balance for basic cell functions 20 . While functional autophagy acts as a metabolic stress buffer, many lines of evidence supports a role for autophagy in antagonizing cell survival and in promoting cell death and apoptosis 19,[21][22][23][24] . Autophagy plays an important role in cancer, and inhibition of this cellular process has been proposed to contribute to HCC progression [25][26] and, therefore, it is a potentially very important target for liver cancer prevention and treatment.
This study was therefore undertaken to evaluate the potential anti-tumoral activity of cannabinoids in HCC and the mechanisms responsible for cannabinoid action in that devastating disease. We found that, both in cell cultures and in xenografted mice, Δ 9 -THC and the synthetic CB 2 receptor-selective agonist JWH-015 promote human HCC death via autophagy stimulation. We also provide a molecular mechanism underlying CB 2 receptor-mediated anti-tumoral signaling. These observations may pave the way to the design of novel therapeutic strategies for the treatment of hepatocellular carcinoma.

Δ 9 -THC and JWH-015-induced autophagy and apoptosis relies on CB 2 receptor activation
To investigate the activity of cannabinoids on HCC cells we first analyzed the effect of Δ 9 -THC (a CB 1 /CB 2 receptor-mixed agonist that constitutes the main psychoactive ingredient of Canabis sativa) and JWH-015 (a CB 2 receptor-selective agonist) on HepG2 and HUH-7 cells, two HCC lines that express CB 1 and CB 2 cannabinoid receptors (Suppl. Fig. 1A). Treatment with Δ 9 -THC reduced the viability of HepG2 and HuH-7 cells, an event that was prevented by co-incubation with SR144528 (SR2, a CB 2 receptor-selective antagonist) but not with SR141716A (SR1, a CB 1 receptor-selective antagonist) (Supp. Fig. 1B). Likewise, JWH-015 decreased the viability of HCC cells, and co-incubation with SR2 abrogated this effect (Suppl. Fig. 1B). These observations support that stimulation of CB 2 receptors is responsible for the decrease of cell viability triggered by cannabinoids on HCC cells.

HuH-7 via autophagy stimulation
It has been recently shown that cannabinoids induce human glioma cell death via autophagy stimulation in vitro and in vivo 15,27 . We therefore examined whether Δ 9 -THC and JWH-015 activate a similar mechanism in HCC cells. Upon autophagy stimulation, the autophagy protein LC3 becomes conjugated to phosphatidylethanolamine (PE), which targets this protein to the membrane of the autophagosomes. The lipidated autophagosome-associated form of LC3 (LC3-II) can be monitored by immunofluorescence (autophagic cells exhibit a characteristic pattern of LC3 puncta) or Western blot (LC3-II has higher electrophoretic mobility than nonlipidated LC3). Immunofluorescence analysis revealed that LC3 exhibited a punctuated distribution, consistent with its translocation to the autophagosome, in cells that had been treated with Δ 9 -THC or JWH-015 (Fig. 1A). Likewise, incubation of HepG2 and HuH-7 cells with Δ 9 -THC or JWH-015 increased the levels of the lipidated form of LC3 ( Fig. 1B). Furthermore, pharmacological inhibition with E64d and pepstatine A of lysosomal proteases (the enzymes responsible for the degradation of the autophagosome content after fusion with the lysosome) enhanced the accumulation of LC3-II (as well as of the autophagosome cargo p62) in cells that had been treated with THC or JWH-015, thus supporting that cannabinoid treatment leads to dynamic autophagy in HCC cells ( Fig. 1B).
Next, we investigated whether autophagy was directly involved in the mechanism of cannabinoid-induced cell death. As shown in Fig. 1C Likewise, knock-down of Atg5 [an essential autophagy gene that is part of one of the two protein conjugation systems required for autophagosome elongation 29-30 ] impaired THC or JWH-015-induced cell death (Fig. 1D). Taken together, these observations strongly support that autophagy is required for cannabinoid-induced HCC cell death.
Many lines of evidence indicate that there is a cross talk between autophagy and apoptosis 31 . To investigate whether cannabinoid-induced autophagy was involved in apoptosis induction, HepG2 and HuH-7 cells were incubated with either Δ 9 -THC or JWH-015 in the presence of the 3-MA, and levels of procaspase 3 were detected by immunoblot. As shown in Fig. 1E, pre-incubation with 3-MA prevented the cleavage of procaspase 3, suggesting that autophagy induction by cannabinoids was previous to and necessary for apoptosis.
AMPK activation and TRB3 upregulation are involved in Δ 9 -THC and JWH-015induced autophagy and apoptosis of HCC cells The mechanisms of autophagy stimulation by cannabinoids in glioma and other types of cancer cells relies on the stimulation of an ER stress-related pathway, which leads to the up-regulation of the pseudokinase TRB3. This latter protein interacts with Akt and promotes the inhibition of the mTORC1 complex, which leads to autophagy stimulation 27 . As shown in Fig. 2, THC and JWH-015 increased the phosphorylation of the alpha subunit of the eukaryotic translation initiation factor 2 (eIF2α, a hallmark of the ER stress response; Fig. 2A), increased TRB3 levels (Fig. 2B) and decreased the phosphorylation of Akt, p70S6 kinase (a well established substrate of mTORC1) and the ribosomal protein S6 (a target of p70S6 kinase) in HepG2 and HUH-7 cells (Fig.   2C). Furthermore, selective knock-down of TRB3 abrogated cannabinoid-induced inhibition of the Akt/mTOR pathway, autophagy and cell death (Suppl. Fig 2), thus supporting that the mechanism by which cannabinoids promote glioma cell death also operates in HCC cells.
Of note, we observed that treatment of HepG2 and HUH-7 cells with THC or JWH-015 increased the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), a key intracellular nutrient status sensor that has been proposed to play a critical role in the regulation of autophagy as induced by hypoxia or nutrient deprivation (Fig. 2B). In addition, pharmacological blockade of CB 2 receptors with SR2 abrogated the effect of Δ 9 -THC and JWH-015 on Akt, S6 and AMPK phosphorylation as well as autophagy ( Fig. 2D and Suppl 1C). We therefore asked whether AMPK may also play a role on the regulation of the antiproliferative effect evoked by cannabinoids in HCC cells. In line with this notion, when AMPK was pharmacologically blocked with dorsomorphin ( Fig. 3A) or genetically inhibited with siRNA ( Fig. 3B), HepG2 and HUH-7 cells were more resistant to cannabinoid-induced cell death. Likewise, AMPK knock-down prevented LC3 lipidation ( Fig. 3C and Suppl. Fig. 2C). These observations support that activation of AMPK is necessary for the stimulation of autophagy-mediated cell death by cannabinoids in HCC cells.

AMPK and TRB3 regulate cannabinoid-induced autophagy of HCC cells through different mechanisms
AMPK has been shown to inhibit mTORC1 32 . Unlike Akt, AMPK activates TSC2, a GTPase-activating protein responsible for the blockade of mTORC1 32 . Therefore we next studied whether this was the mechanism by which AMPK stimulated autophagy in was not affected by LKB1 silencing (Fig. 4A

Autophagy is required for Δ 9 -THC and JWH-015 anti-tumoral action in human HCC xenografts
To investigate the ability of Δ 9 -THC and JWH-015 to inhibit HCC growth in vivo we Taken together, these observations robustly support that cannabinoid anti-tumoral action in HCC relies on AMPK stimulation, Akt inhibition and activation of autophagy in HCC cells both in vitro and in vivo.

Discussion
In this study we show that the natural cannabinoid Δ 9 -THC and the CB 2 receptorselective agonist JWH-015 inhibit HCC cell growth via stimulation of autophagy.
Importantly, although the human hepatocellular carcinoma cell lines used (HepG2 and HuH-7) expressed both CB 1 and CB 2 receptors, only CB 2 activation was involved in the pro-autophagic and anti-proliferative effect induced by cannabinoids on these cells. This is in line with the recent observation that the synthetic cannabinoid WIN-55,212-2 induced apoptosis in HepG2 cells in a process that was partially inhibited by the CB 2 receptor-selective antagonist AM630 13 . Moreover, it has been previously shown that CB 2 receptors are overexpressed in HCC and correlate with good prognosis 35 . Those findings, together with ours, support that stimulation of CB 2 receptors could be a new therapeutic strategy to promote HCC death.
Our study shows that the mechanism of cannabinoid anti-tumoral action in HCC relies on the stimulation of autophagy and the subsequent activation of apoptosis.
Depending on the physiopathological setting, autophagy has been proposed to protect from apoptosis, act as an apoptosis-alternative pathway to induce cell death, or act together with apoptosis as a combined mechanism for cell death 36-37 . However, very little is known on the role that the interchange between these two cellular processes plays in the control of tumor growth in response to anticancer agents. Our observations are in line with previous results obtained in human glioma cells 15 and support that stimulation of autophagy in response to cannabinoid treatment leads to apoptosis.
Nevertheless, further research is still necessary to clarify the precise mechanisms linking both cellular processes upon cannabinoid treatment.
Stimulation of autophagy in many cellular settings relies on the inhibition of the mTORC1 complex, which plays a central role in the control of protein synthesis, cell growth and cell proliferation through the regulation of several downstream targets. As a result of its central position in the control of cellular homeostasis, mTORC1 integrates signals from different inputs. One of the most important upstream regulators of mTORC1 is the pro-survival kinase Akt, which phosphorylates and inactivates TSC2 (an inhibitor of the mTORC1 activator Rheb) and PRAS-40. Thus, Akt activation stimulates mTORC1 and inhibits autophagy. In this work we found that cannabinoid treatment of HCC cells leads to Akt and mTORC1 inhibition, which is in agreement with our recent studies in glioma cells 15 . Thus, it had been previously shown that inhibition of Akt/mTORC1 pathway by cannabinoids relies on the stimulation of an ER stress-related pathway which leads to the up-regulation of the pseudokinase TRB3, the inhibition of the Akt/mTORC1 axis and the induction of autophagy 15,27 . In the present study, Δ 9 -THC and JWH-015 promoted ER stress and increased TRB3 expression. In addition, Akt/mTORC1 inhibition and autophagy were abolished when ceramide biosynthesis was inhibited or when TRB3 expression was silenced, thus suggesting that this could be a general mechanism of cannabinoid anti-tumoral action. Of importance, we also found that Δ 9 -THC and JWH-015 activate AMPK in HCC cells and that pharmacological or genetic inhibition of this kinase has a similar inhibitory effect on cannabinoid-induced cell death and autophagy. AMPK has been shown to negatively regulate mTORC1 via TSC2 activation, which also leads to autophagy stimulation 38 , and therefore we asked whether cannabinoids also inhibit mTORC1 through this mechanism in HCC cells. In disagreement with this possibility, our data show that -

Materials and Methods
Reagents. One day prior to the experiments, medium was changed to 0.5 % FBS medium.
Experiments were done when cell monolayers were 80% confluent. After blocking with 5% skim dried milk, immunoblot analysis was performed followed by enhanced chemoluminescence detection.