Overview

Today is well-established that cannabinoid administration curbs the growth of different types of tumor xenografts in rats and mice. These actions are based on the ability of cannabinoids to inhibit tumor angiogenesis and activate apoptosis of cancer cells.

Results obtained in our group during the last few years have led us to characterize (at least in part) the signalling routes responsible for these antineoplasic actions of cannabinoids.

Thus, binding of cannabinoids to their corresponding receptors in the plasma membrane of tumor cells triggers an early accumulation of de novo-synthesized ceramide (an event that takes place in the ER), which leads in turn to ER dilation and increased eukaryotic initiation translation factor 2α (eIF2α) phosphorylation, two hallmarks of the ER stress response. Activation of this response (an adaptive cellular reaction to ER alterations) induces the up-regulation of several genes, including the stress-regulated protein p8 and its downstream targets, the transcription factors ATF-4 and CHOP and the pseudokinase Tribbles homologue 3 (TRB3). Our investigations have led us to identify the important role played by these proteins in the regulation of autophagy (another cellular process activated in response to certain stress situations). Stimulation of autophagy by cannabinoids activates apoptosis and promotes cancer cell death.

Mechanism of cannabinoid antitumoral action

One of the objectives of our research is dissecting the molecular mechanisms responsible for cannabinabinoid antitumoral action. This objective is aimed not only at optimizing the killing actions of these agents in cancer cells but also at identifying molecular targets which may help to design new pharmacological strategies directed to reduce tumour growth. In the context of these objectives our team is developing several interrelated projects:

(i) Role of sphingolipids in the stimulation of ER stress and autophagy. De novo synthesis of ceramide takes place in the ER and therefore changes in the levels of this sphingolipid (or of some of the intermediaries of its synthesis) may affect the functionality of this organelle. As de novo-synthesized ceramide is required for cannabinoid-induced ER stress, autophagy and apoptosis, we are investigating the mechanism through which local accumulation of different species of this sphingolipid (or changes on its subcellular location) may regulate these processes in the context of cancer cell death.

(ii) Role of p8 and TRB3 on the regulation of the Akt/mTORC1 axis and autophagy. Our investigations showed that a key step in the induction of autophagy by cannabinoids relies on the inhibition of the Akt/mammalian target of rapamycin complex 1 (mTORC1) axis by the pseudokinase TRB3. We are currently investigating the molecular mechanisms by which p8 and TRB3 regulate the Akt/mTORC1 axis promoting thus autophagy and cancer cell death through the regulation of Atg1 (an autophagy related gene which encodes for a protein kinase which might pay a crucial role in the stimulation of autophagy).

Dual role of autophagy in cancer

Autophagy is a complex cellular process by which cytoplasmic components including organelles are targeted for degradation to the lysosomes. Intriguingly, the final outcome of the activation of the autophagy program is highly dependent on the cellular context and the strength and duration of the stress-inducing signals. Thus, besides its role in cellular homeostasis, autophagy can be a form of programmed cell death, designated type II programmed cell death, or play a cytoprotective role, for example in situations of nutrient starvation. Accordingly, autophagy has been proposed to play an important role in both tumor progression and promotion of cancer cell death, although the molecular mechanisms responsible for this dual action of autophagy in cancer have not been elucidated. Thus, one of the ongoing projects in our laboratory is trying to understand the differential regulatory mechanisms underlying the actions of cannabinoids and other pro-autophagic drugs.

Optimization of cannabinoid antitumoral action in gliomas

One of the most striking features of gliomas is their high resistance to conventional anti-tumoral therapies. Thus, it is widely believed that strategies aimed at reducing the mortality caused by these tumors should consist of targeted therapies capable of providing the most efficacious treatment for each individual cell subpopulation, tumor and patient. This new therapeutic approach would require not only the utilization of new cocktails of chemotherapeutic drugs but, the identification of the markers associated with the resistance of tumor cells to these new therapies. Accordingly, an additional aim of our laboratory is trying to improve the efficacy of the cannabinoid antitumoral action by understanding the mechanisms of resistance to the treatment with these agents and finding the optimal drug combination which could help to enhance their antineoplasic effects.

(i) Identification of the factors for resistance to cannabinoid antitumoral action. In order to identify the molecular features associated with the resistance of glioma cells to cannabinoid treatment, we analyzed the gene expression profile of several human astrocytoma cell lines as well as primary cultures obtained from human glioma biopsies. By using this procedure, we identified a series of genes whose expression is associated with a higher resistance to cannabinoid treatment. We are at the moment testing the potential use of these genes as predictors of the responsiveness of a particular brain tumor to cannabinoid administration.

(ii) Molecular mechanisms mediating the resistance to cannabinoid antitumoral action in gliomas. In addition, we also identified several individual genes [including the EGFR-ligand Amphiregulin  (Lorente et al., Glia, in press)] as direct mediators of the resistance to cannabinoid antitumoral action. One of our aims is therefore investigating the signal transduction pathways by which these proteins mediate the resistance of gliomas to cannabinoid treatment. In addition we are testing whether pharmacological or genetic inhibition of these genes (or of the pathways that they activate) could be a therapeutic strategy to enhance the response of these tumours to anticancer therapies.

(iii) Combinational therapies enhancing cannabinoid anti-tumoral action. In order to enhance the response of tumor cells to cannabinoid anti-tumoral action (particularly in those cells that are resistant to the cell-killing effect of these agents), we are investigating the synergic action of the combined administration of different anti-tumoral agents with cannabinoids. Our results indicate for example that administration of cannabinoids together with temozolomide (the drug of reference for the management of malignant gliomas) produces a very remarkable synergic inhibition of tumor growth. We are therefore testing whether other drugs that induce autophagy or can activate ER stress may also enhance the anti-tumoral action of cannabinoids.