SUMMARY Tetraspanins are commonly believed to act only as “molecular facilitators”, with no direct role in signal transduction. We herein demonstrate that upon ligation, CD37, a tetraspanin molecule expressed on mature normal and transformed B-cells, becomes tyrosine phosphorylated, associates with proximal signaling molecules, and initiates a cascade of events leading to apoptosis. Moreover, we have identified two tyrosine residues with opposing regulatory functions, one lies in the N-terminal domain of CD37 in a predicted “ITIM-like” motif and mediates SHP1-dependent death whereas the second lies in a predicted “ITAM motif” in the C-terminal domain of CD37 and counteracts death signals by mediating phosphatidylinositol 3-kinase-dependent survival.

In cancer cells, the epithelial-mesenchymal transition (EMT) confers the ability to invade basement membranes and metastasize to distant sites, establishing it as an appealing target for therapeutic intervention. Here, we report a novel function of the master metabolic kinase AMPK in suppressing EMT by modulating the Akt-MDM2-Foxo3 signaling axis. This mechanistic link was supported by the effects of siRNA-mediated knockdown and pharmacological activation of AMPK on epithelial and mesenchymal markers in established breast and prostate cancer cells. Exposure of cells to OSU-53, a novel allosteric AMPK activator, as well as metformin and AICAR, was sufficient to reverse their mesenchymal phenotype. These effects were abrogated by AMPK silencing. Phenotypic changes were mediated by Foxo3a activation, insofar as silencing or overexpressing Foxo3a mimicked the effects of AMPK silencing or OSU-53 treatment on EMT, respectively. Mechanistically, Foxo3a activation led to the transactivation of the E-cadherin gene and repression of genes encoding EMT-inducing transcription factors. OSU-53 activated Foxo3a through two Akt-dependent pathways, one at the level of nuclear localization by blocking Akt- and IKKβ-mediated phosphorylation, and a second at the level of protein stabilization via cytoplasmic sequestration of MDM2, an E3 ligase responsible for Foxo3a degradation. The suppressive effects of OSU-53 on EMT had therapeutic implications illustrated by its ability to block invasive phenotypes in vitro and metastatic properties in vivo. Overall, our work illuminates a mechanism of EMT regulation in cancer cells mediated by AMPK, along with preclinical evidence supporting a tractable therapeutic strategy to reverse mesenchymal phenotypes associated with invasion and metastasis.

This study was aimed at elucidating the mechanism by which FTY720, a synthetic sphingosine immunosuppressant, mediated antitumor effects in hepatocellular carcinoma (HCC) cells. The three HCC cell lines examined, Hep3B, Huh7, and PLC5, exhibited differential susceptibility to FTY720-mediated suppression of cell viability, with IC 50 values of 4.5, 6.3, and 11 Mmol/L, respectively. Although FTY720 altered the phosphorylation state of protein kinase B and p38, our data refuted the role of these two signaling kinases in FTY720-mediated apoptosis. Evidence indicates that the antitumor effect of FTY720 was attributable to its ability to stimulate reactive oxygen species (ROS) production, which culminated in protein kinase C (PKC)D activation and subsequent caspase-3-dependent apoptosis. We showed that FTY720 activated PKCD through two distinct mechanisms: phosphorylation and caspase-3-dependent cleavage. Cotreatment with the caspase-3 inhibitor Z-VAD-FMK abrogated the effect of FTY720 on facilitating PKCD proteolysis. Equally important, pharmacologic inhibition or shRNA-mediated knockdown of PKCD protected FTY720-treated Huh7 cells from caspase-3 activation. Moreover, FTY720 induced ROS production to different extents among the three cell lines, in the order of Hep3B > Huh7 >> PLC5, which inversely correlated with the respective glutathione S-transferase P expression levels. The low level of ROS generation might underlie the resistant phenotype of PLC5 cells to the apoptotic effects of FTY720. Blockade of ROS production by an NADPH oxidase inhibitor protected Huh7 cells from FTY720-induced PKCD activation and caspase-3-dependent apoptosis. Together, this study provides a rationale to use FTY720 as a scaffold to develop potent PKCD-activating agents for HCC therapy.

We performed proteomic studies using the GRP78 chaperone-inhibitor drug AR-12 (OSU-03012) as bait. Multiple additional chaperone and chaperone-associated proteins were shown to interact with AR-12, including: GRP75, HSP75, BAG2; HSP27; ULK-1; and thioredoxin. AR-12 down-regulated in situ immuno-fluorescence detection of ATP binding chaperones using antibodies directed against the NH2-termini of the proteins but only weakly reduced detection using antibodies directed against the central and COOH portions of the proteins. Traditional SDS-PAGE and western blotting assessment methods did not exhibit any alterations in chaperone detection. AR-12 altered the sub-cellular distribution of chaperone proteins, abolishing their punctate speckled patterning concomitant with changes in protein co-localization. AR-12 inhibited chaperone ATPase activity, which was enhanced by sildenafil; inhibited chaperone – chaperone and chaperone – client interactions; and docked in silico with the ATPase domains of HSP90 and of HSP70. AR-12 combined with sildenafil in a GRP78 plus HSP27 –dependent fashion to profoundly activate an eIF2α/ATF4/CHOP/Beclin1 pathway in parallel with inactivating mTOR and increasing ATG13 phosphorylation, collectively resulting in formation of punctate toxic autophagosomes. Over-expression of [GRP78 and HSP27] prevented: AR-12 –induced activation of ER stress signaling and maintained mTOR activity; AR-12 –mediated down-regulation of thioredoxin, MCL-1 and c-FLIP-s; and preserved tumor cell viability. Thus the inhibition of chaperone protein functions by AR-12 and by multi-kinase inhibitors very likely explains why these agents have anti-tumor effects in multiple genetically diverse tumor cell types.

Background: Adenosine monophosphate-activated protein kinase (AMPK) modulates cancer cell metabolism and survival. Results: The novel compound OSU-53 directly activates AMPK, inhibits multiple metabolic and oncogenic pathways, and induces apoptosis in triple-negative breast cancer cells. Conclusion: OSU-53 acts through a broad spectrum of antitumor activities downstream of AMPK activation. Significance: OSU-53 is a potent small molecule AMPK activator with translational potential for breast cancer therapy.