No abstract

Although the global incidence of cutaneous melanoma is increasing, survival rates for patients with metastatic disease remain <10%. Novel treatment strategies are therefore urgently required, particularly for patients bearing BRAF/NRAS wild-type tumors. Targeting autophagy is a means to promote cancer cell death in chemotherapy-resistant tumors, and the aim of this study was to test the hypothesis that cannabinoids promote autophagy-dependent apoptosis in melanoma. Treatment with Δ(9)-Tetrahydrocannabinol (THC) resulted in the activation of autophagy, loss of cell viability, and activation of apoptosis, whereas cotreatment with chloroquine or knockdown of Atg7, but not Beclin-1 or Ambra1, prevented THC-induced autophagy and cell death in vitro. Administration of Sativex-like (a laboratory preparation comprising equal amounts of THC and cannabidiol (CBD)) to mice bearing BRAF wild-type melanoma xenografts substantially inhibited melanoma viability, proliferation, and tumor growth paralleled by an increase in autophagy and apoptosis compared with standard single-agent temozolomide. Collectively, our findings suggest that THC activates noncanonical autophagy-mediated apoptosis of melanoma cells, suggesting that cytotoxic autophagy induction with Sativex warrants clinical evaluation for metastatic disease.

Autophagy is considered primarily a cell survival process, although it can also lead to cell death. However, the factors that dictate the shift between these 2 opposite outcomes remain largely unknown. In this work, we used Δ9-tetrahydrocannabinol (THC, the main active component of marijuana, a compound that triggers autophagy-mediated cancer cell death) and nutrient deprivation (an autophagic stimulus that triggers cytoprotective autophagy) to investigate the precise molecular mechanisms responsible for the activation of cytotoxic autophagy in cancer cells. By using a wide array of experimental approaches we show that THC (but not nutrient deprivation) increases the dihydroceramide:ceramide ratio in the endoplasmic reticulum of glioma cells, and this alteration is directed to autophagosomes and autolysosomes to promote lysosomal membrane permeabilization, cathepsin release and the subsequent activation of apoptotic cell death. These findings pave the way to clarify the regulatory mechanisms that determine the selective activation of autophagy-mediated cancer cell death.

Tribbles pseudokinase-3 (TRIB3) has been proposed to act as an inhibitor of AKT although the precise molecular basis of this activity and whether the loss of TRIB3 contributes to cancer initiation and progression remain to be clarified. In this study, by using a wide array of in vitro and in vivo approaches, including a Trib3 knockout mouse, we demonstrate that TRIB3 has a tumorsuppressing role. We also find that the mechanism by which TRIB3 loss enhances tumorigenesis relies on the dysregulation of the phosphorylation of AKT by the mTORC2 complex, which leads to an enhanced phosphorylation of AKT on Ser473 and the subsequent hyperphosphorylation and inactivation of the transcription factor FOXO3. These observations support the notion that loss of TRIB3 is associated with a more aggressive phenotype in various types of tumors by enhancing the activity of the mTORC2/AKT/FOXO axis. Pseudokinases constitute a group of proteins that have a kinase-like domain that lacks at least one of the conserved catalytic residues. 1,2 Different studies have shown that some pseudokinases can exhibit low levels of kinase activity, while others have critical roles as activators of their specific targets. 1,2 Moreover, aberrant regulation of pseudokinases has been implicated in the etiology and progression of a variety of diseases, including cancer. 3 The Tribbles family of pseudokinases was first described in Drosophila as a negative regulator of cell division in early embryogenesis. [4][5][6][7] There are three mammalian Tribbles isoforms (Trib1, Trib2 and Trib3), homologs to the Drosophila tribbles proteins, and they all share a highly conserved central kinase-like domain, which lacks catalytic residues, and a 'tribbles specific' C-terminal domain, which has been proposed to participate in the binding to different Tribbles partners. 8 Tribbles pseudokinase-3 (TRIB3; also named TRB3, NIPK and SKIP3) has been proposed to interact with several proteins, including the transcription factors activating transcription factor 4 (ATF-4) and CHOP 9 as well as with several MAPKs. 10 TRIB3 has also been shown to interact and inhibit AKT, 11 which has been suggested to suppress insulin signaling. 11,12 In addition, administration of different anticancer agents promotes cancer cell death via TRIB3 upregulation and the subsequent inhibition of Akt. [13][14][15][16][17][18][19] However, the precise molecular basis of the regulation of Akt by TRIB3 and whether loss of this pseudokinase may contribute to cancer initiation and progression remains to be clarified.In this study, we investigated the effect of the genetic inactivation of TRIB3 in several cellular and animal models of cancer. Our findings indicate that genetic inhibition of TRIB3 enhances tumorigenesis and that this effect is due -at least primarily -to a selective inactivation of the transcription factor FOXO by the mammalian target of rapamycin complex 2 (mTORC2)/AKT axis. ResultsGenetic inhibition of TRIB3 facilitates oncogene transformation and enhances the tumorigenicity of cancer c...