Celastrol induces unfolded protein response-dependent cell death in head and neck cancer.

The survival rate for patients with oral squamous cell carcinoma (OSCC) has not seen marked improvement in recent decades despite enhanced efforts in prevention and the introduction of novel therapies. We have reported that pharmacological exacerbation of the unfolded protein response (UPR) is an effective approach to killing OSCC cells. The UPR is executed via distinct signaling cascades whereby an initial attempt to restore folding homeostasis in the endoplasmic reticulum during stress is complemented by an apoptotic response if the defect cannot be resolved. To identify novel small molecules able to overwhelm the adaptive capacity of the UPR in OSCC cells, we engineered a complementary cell-based assay to screen a broad spectrum of chemical matter. Stably transfected CHO-K1 cells that individually report (luciferase) on the PERK/eIF2α/ATF4/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR pathways, were engineered [1]. The triterpenoids dihydrocelastrol and celastrol were identified as potent inducers of UPR signaling and cell death in a primary screen and confirmed in a panel of OSCC cells and other cancer cell lines. Biochemical and genetic assays using OSCC cells and modified murine embryonic fibroblasts demonstrated that intact PERK-eIF2-ATF4-CHOP signaling is required for pro-apoptotic UPR and OSCC death following celastrol treatment.

Complementary cell-based high-throughput screens identify novel modulators of the unfolded protein response.

Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than 2 decades, indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, the authors hypothesized that high-throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small-molecule activators of the apoptotic arm of the UPR to control or kill OSCC. They have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR subpathways. An 66 K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of prefractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80 µM. A series of citrinin derivatives was isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds, the authors examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, it was found that patulin at 2.5 to 10 µM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34, and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.