María Salazar

Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells

Autophagy can promote cell survival or cell death, but the molecular basis underlying its dual role in cancer remains obscure. Here we demonstrate that delta(9)-tetrahydrocannabinol (THC), the main active component of marijuana, induces human glioma cell death through stimulation of autophagy. Our data indicate that THC induced ceramide accumulation and eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation and thereby activated an ER stress response that promoted autophagy via tribbles homolog 3-dependent (TRB3-dependent) inhibition of the Akt/mammalian target of rapamycin complex 1 (mTORC1) axis. We also showed that autophagy is upstream of apoptosis in cannabinoid-induced human and mouse cancer cell death and that activation of this pathway was necessary for the antitumor action of cannabinoids in vivo. These findings describe a mechanism by which THC can promote the autophagic death of human and mouse cancer cells and provide evidence that cannabinoid administration may be an effective therapeutic strategy for targeting human cancers.

Linking ER Stress to Autophagy: Potential Implications for Cancer Therapy

Different physiological and pathological conditions can perturb protein folding in the endoplasmic reticulum, leading to a condition known as ER stress. ER stress activates a complex intracellular signal transduction pathway, called unfolded protein response (UPR). The UPR is tailored essentially to reestablish ER homeostasis also through adaptive mechanisms involving the stimulation of autophagy. However, when persistent, ER stress can switch the cytoprotective functions of UPR and autophagy into cell death promoting mechanisms. Recently, a variety of anticancer therapies have been linked to the induction of ER stress in cancer cells, suggesting that strategies devised to stimulate its prodeath function or block its prosurvival function, could be envisaged to improve their tumoricidial action. A better understanding of the molecular mechanisms that determine the final outcome of UPR and autophagy activation by chemotherapeutic agents, will offer new opportunities to improve existing cancer therapies as well as unravel novel targets for cancer treatment.

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.

Loss of striatal type 1 cannabinoid receptors is a key pathogenic factor in Huntington’s disease

Endocannabinoids act as neuromodulatory and neuroprotective cues by engaging type 1 cannabinoid receptors. These receptors are highly abundant in the basal ganglia and play a pivotal role in the control of motor behaviour. An early downregulation of type 1 cannabinoid receptors has been documented in the basal ganglia of patients with Huntingtons disease and animal models. However, the pathophysiological impact of this loss of receptors in Huntingtons disease is as yet unknown. Here, we generated a double-mutant mouse model that expresses human mutant huntingtin exon 1 in a type 1 cannabinoid receptor-null background, and found that receptor deletion aggravates the symptoms, neuropathology and molecular pathology of the disease. Moreover, pharmacological administration of the cannabinoid Δ(9)-tetrahydrocannabinol to mice expressing human mutant huntingtin exon 1 exerted a therapeutic effect and ameliorated those parameters. Experiments conducted in striatal cells show that the mutant huntingtin-dependent downregulation of the receptors involves the control of the type 1 cannabinoid receptor gene promoter by repressor element 1 silencing transcription factor and sensitizes cells to excitotoxic damage. We also provide in vitro and in vivo evidence that supports type 1 cannabinoid receptor control of striatal brain-derived neurotrophic factor expression and the decrease in brain-derived neurotrophic factor levels concomitant with type 1 cannabinoid receptor loss, which may contribute significantly to striatal damage in Huntingtons disease. Altogether, these results support the notion that downregulation of type 1 cannabinoid receptors is a key pathogenic event in Huntingtons disease, and suggest that activation of these receptors in patients with Huntingtons disease may attenuate disease progression.

A combined preclinical therapy of cannabinoids and temozolomide against glioma.

Glioblastoma multiforme (GBM) is highly resistant to current anticancer treatments, which makes it crucial to find new therapeutic strategies aimed at improving the poor prognosis of patients suffering from this disease. Δ9-Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoid receptor agonists inhibit tumor growth in animal models of cancer, including glioma, an effect that relies, at least in part, on the stimulation of autophagy-mediated apoptosis in tumor cells. Here, we show that the combined administration of THC and temozolomide (TMZ; the benchmark agent for the management of GBM) exerts a strong antitumoral action in glioma xenografts, an effect that is also observed in tumors that are resistant to TMZ treatment. Combined administration of THC and TMZ enhanced autophagy, whereas pharmacologic or genetic inhibition of this process prevented TMZ + THC-induced cell death, supporting that activation of autophagy plays a crucial role on the mechanism of action of this drug combination. Administration of submaximal doses of THC and cannabidiol (CBD; another plant-derived cannabinoid that also induces glioma cell death through a mechanism of action different from that of THC) remarkably reduces the growth of glioma xenografts. Moreover, treatment with TMZ and submaximal doses of THC and CBD produced a strong antitumoral action in both TMZ-sensitive and TMZ-resistant tumors. Altogether, our findings support that the combined administration of TMZ and cannabinoids could be therapeutically exploited for the management of GBM. Mol Cancer Ther; 10(1); 90–103. ©2011 AACR.

The orphan G protein-coupled receptor GPR55 promotes cancer cell proliferation via ERK

GPR55 is an orphan G protein-coupled receptor that may be engaged by some lipid ligands such as lysophosphatidylinositol and cannabinoid-type compounds. Very little is known about its expression pattern and physio-pathological relevance, and its pharmacology and signaling are still rather controversial. Here we analyzed the expression and function of GPR55 in cancer cells. Our data show that GPR55 expression in human tumors from different origins correlates with their aggressiveness. Moreover, GPR55 promotes cancer cell proliferation, both in cell cultures and in xenografted mice, through the overactivation of the extracellular signal-regulated kinase cascade. These findings reveal the importance of GPR55 in human cancer, and suggest that it could constitute a new biomarker and therapeutic target in oncology.

Cannabinoids Inhibit Glioma Cell Invasion by Down-regulating Matrix Metalloproteinase-2 Expression

Cannabinoids, the active components of Cannabis sativa L. and their derivatives, inhibit tumor growth in laboratory animals by inducing apoptosis of tumor cells and impairing tumor angiogenesis. It has also been reported that these compounds inhibit tumor cell spreading, but the molecular targets of this cannabinoid action remain elusive. Here, we evaluated the effect of cannabinoids on matrix metalloproteinase (MMP) expression and its effect on tumor cell invasion. Local administration of Delta(9)-tetrahydrocannabinol (THC), the major active ingredient of cannabis, down-regulated MMP-2 expression in gliomas generated in mice, as determined by Western blot, immunofluorescence, and real-time quantitative PCR analyses. This cannabinoid-induced inhibition of MMP-2 expression in gliomas (a) was MMP-2-selective, as levels of other MMP family members were unaffected; (b) was mimicked by JWH-133, a CB(2) cannabinoid receptor-selective agonist that is devoid of psychoactive side effects; (c) was abrogated by fumonisin B1, a selective inhibitor of ceramide biosynthesis; and (d) was also evident in two patients with recurrent glioblastoma multiforme. THC inhibited MMP-2 expression and cell invasion in cultured glioma cells. Manipulation of MMP-2 expression by RNA interference and cDNA overexpression experiments proved that down-regulation of this MMP plays a critical role in THC-mediated inhibition of cell invasion. Cannabinoid-induced inhibition of MMP-2 expression and cell invasion was prevented by blocking ceramide biosynthesis and by knocking-down the expression of the stress protein p8. As MMP-2 up-regulation is associated with high progression and poor prognosis of gliomas and many other tumors, MMP-2 down-regulation constitutes a new hallmark of cannabinoid antitumoral activity.

AMBRA1 links autophagy to cell proliferation and tumorigenesis by promoting c-Myc dephosphorylation and degradation

Inhibition of a main regulator of cell metabolism, the protein kinase mTOR, induces autophagy and inhibits cell proliferation. However, the molecular pathways involved in the cross-talk between these two mTOR-dependent cell processes are largely unknown. Here we show that the scaffold protein AMBRA1, a member of the autophagy signalling network and a downstream target of mTOR, regulates cell proliferation by facilitating the dephosphorylation and degradation of the proto-oncogene c-Myc. We found that AMBRA1 favours the interaction between c-Myc and its phosphatase PP2A and that, when mTOR is inhibited, it enhances PP2A activity on this specific target, thereby reducing the cell division rate. As expected, such a de-regulation of c-Myc correlates with increased tumorigenesis in AMBRA1-defective systems, thus supporting a role for AMBRA1 as a haploinsufficient tumour suppressor gene.

Stimulation of the midkine/ALK axis renders glioma cells resistant to cannabinoid antitumoral action

Identifying the molecular mechanisms responsible for the resistance of gliomas to anticancer treatments is an issue of great therapeutic interest. Δ9-Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoids inhibit tumor growth in animal models of cancer, including glioma, an effect that relies, at least in part, on the stimulation of autophagy-mediated apoptosis in tumor cells. Here, by analyzing the gene expression profile of a large series of human glioma cells with different sensitivity to cannabinoid action, we have identified a subset of genes specifically associated to THC resistance. One of these genes, namely that encoding the growth factor midkine (Mdk), is directly involved in the resistance of glioma cells to cannabinoid treatment. We also show that Mdk mediates its protective effect via the anaplastic lymphoma kinase (ALK) receptor and that Mdk signaling through ALK interferes with cannabinoid-induced autophagic cell death. Furthermore, in vivo Mdk silencing or ALK pharmacological inhibition sensitizes cannabinod-resistant tumors to THC antitumoral action. Altogether, our findings identify Mdk as a pivotal factor involved in the resistance of glioma cells to THC pro-autophagic and antitumoral action, and suggest that selective targeting of the Mdk/ALK axis could help to improve the efficacy of antitumoral therapies for gliomas.

TRB3 links ER stress to autophagy in cannabinoid anti-tumoral action

Δ9-tetrahydrocannabinol (THC), the main active component of marijuana, is being investigated as a potential anti-tumoral agent. We find that THC stimulates an endoplasmic reticulum (ER) stress-related signaling pathway, which activates autophagy via inhibition of the Akt/mTORC1 axis. We also show that autophagy is upstream of apoptosis in cannabinoid-induced cancer cell death and that activation of this pathway is necessary for the anti-tumoral action of cannabinoids in vivo.