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

Highlights

• Celastrol activates the Unfolded Protein Response (UPR).

• Intact Perk-eIF2〈-Atf4-Chop signaling is required for efficient celastrol-mediated cell death.

• Celastrol induces Bak, Puma and Trb3.

• Celastrol enhances protein ubiquitination prior to death.

• Oral squamous cell carcinoma cell lines with deficient SMAD4/TGF®R signaling are more resistant to celastrol.

Abstract

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.

Introduction

A remarkable incidence of oral squamous cell carcinoma (OSCC) persists in the United States. Progress in durable patient responses has been only incremental since the introduction of cisplatin in 1978, and approximately 40,000 Americans will be newly diagnosed with oral cavity and oropharyngeal cancer this year [2]. In an attempt to improve the outcomes of conventional chemotherapy we have utilized a cell-based high throughput screening technique to identify novel small molecules that intensify the unfolded protein response (UPR) and selectively kill malignant cells [1].

The unfolded protein response (UPR) is a cell׳s rejoinder to the accumulation of misfolded protein in the lumen of the endoplasmic reticulum (ER). Innositol-requiring enzyme 1 alpha (IRE1α), activating transcription factor 6 (ATF6), and protein kinase RNA-like endoplasmic reticulum kinase (PERK) are three ER transmembrane sensors that continuously monitor the status of luminal protein folding. Broadly, the UPR consists of genetically distinct pathways that are simultaneously activated to either adapt to a folding challenge or initiate cell death when a stress is particularly robust or protracted. The adaptive response is directed primarily through the un-conventional splicing of a 26 base intron from X-box binding protein (XBP1) mRNA by IRE1αSpliced XBP1 yields a potent transcription factor that increases the expression of foldases, chaperones and other heat shock factors and glucose-related proteins that return to the ER in an attempt to remedy the folding defect. Although the precise mechanism governing the switch from UPR-mediated adaptation toward cell death remains an incomplete story, it is clear, when the adaptive response becomes overwhelmed, that the activation of the PERK-eIF2α-axis induces ATF4 and CHOP expression prior to apoptosis [3].

Given the rapid growth rate and highly secretory nature of many solid and hematological tumors it is not surprising that many human cancers are characterized by increased expression of translation factors and high basal levels of UPR signaling and stress. Recent studies have revealed increased expression of eukaryotic initiation factors (eIFs) and UPR-related chaperones in breast [4], [5], bladder [6], lung [7], thyroid [8], lymphoma [9], colorectal [10], leukemia [11], larynx [12], [13] and OSCC [11], [14], [15]. As malignant cell populations begin to grow and invade host tissue the extracellular tumor milieu becomes increasingly starved of oxygen, glucose and other nutrients as the rate of expansion outpaces the capacity of its vasculature. The cellular stress caused by these harsh conditions leads to IRE1α- and PERK (ATF4) -mediated angiogenesis and cell survival [16]. Importantly, Xbp1-/- fibroblasts and Xbp1 knockdown cells failed to form tumors in mice [17], and PERK -/- cells and xenografts are unable to tolerate hypoxia [18]. When considered together, the increased expression of translation factors and the demand for UPR-driven angiogenesis in the tumor stroma strongly support the hypothesis that targeting the UPR with small molecules will be a productive therapeutic approach.

A cell-based high throughput screen was engineered using CHO cells transfected with luciferase reporters to specifically monitor XBP1 splicing or CHOP promoter activation [1]. A library of approximately 66,000 compounds was screened at the University of Michigan Center for Chemical Genomics and celastrol, a triterpenoid compound isolated from the plant family Celastraceae [19], emerged as a hit that could activate both UPR reporters.

Although most studies have focused on the anti-inflammatory properties of celastrol, there are a growing number of reports highlighting its use as a promising anti-cancer compound in breast leukemia, melanoma, myeloma, pancreatic and prostate cell culture and xenograft models [20], [21], [22], [23], [24]. The anti-proliferative effects of celastrol have been attributed to mechanisms involving diverse signaling networks that include the inhibition of pro-survival NF-B signaling, proteasome inhibition and the up-regulation of pro-apoptotic Bcl-2 family members and down regulation of anti-apoptotic genes such as Bcl-2 and XIAP. Since celastrol has been shown to potently induce the expression of heat shock proteins [25] and was able to activate UPR luciferase reporters in our screen, we hypothesized that its ability to induce cell death and reduce xenograft tumor burden in several models might be dependent on its ability to activate the UPR.