Danielle M. Garshott

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.

Induction of BCL2-interacting killer, BIK, is mediated for anti-cancer activity of curcumin in human head and neck squamous cell carcinoma cells

Naturally occurring diarylheptanoid curcumin (CUR), a principal component of the Asian spice turmeric, has been shown to have anti-cancer effects in many tumor types. However, a detailed mechanism regarding CUR induced tumor cell killing remain to be comprehensively explored. Using two head neck squamous cell carcinoma (HNSCC) cell lines FaDu (hypopharyngeal) and Cal27 (tongue), we demonstrated a novel mechanism by which CUR levies the cytotoxic effect. We found that CUR induced upregulation of pro-apoptotic Bik, down-regulation of survival signaling by AKT and NF-κB prior to the induction of the caspase-cascade reduction of cell proliferation, are primary mechanisms of CUR-induced cell death, thus providing insights into the anti-tumor activity of CUR in HNSCC cells.

Borrelidin Induces the Unfolded Protein Response in Oral Cancer Cells and Chop-Dependent Apoptosis

Oral squamous cell carcinoma (OSCC) is the most common cancer affecting the oral cavity, and US clinics will register about 30,000 new patients in 2015. Current treatment modalities include chemotherapy, surgery, and radiotherapy, which often result in astonishing disfigurement. Cancers of the head and neck display enhanced levels of glucose-regulated proteins and translation initiation factors associated with endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Previous work demonstrated that chemically enforced UPR could overwhelm these adaptive features and selectively kill malignant cells. The threonyl-tRNA synthetase (ThRS) inhibitor borrelidin and two congeners were discovered in a cell-based chemical genomic screen. Borrelidin increased XBP1 splicing and led to accumulation of phosphorylated eIF2α and UPR-associated genes, prior to death in panel of OSCC cells. Murine embryonic fibroblasts (MEFs) null for GCN2 and PERK were less able to accumulate UPR markers and were resistant to borrelidin. This study demonstrates that UPR induction is a feature of ThRS inhibition and adds to a growing body of literature suggesting ThRS inhibitors might selectively target cancer cells.

Overexpression of CHOP in Myelinating Cells Does Not Confer a Significant Phenotype under Normal or Metabolic Stress Conditions.

The PKR-like endoplasmic reticulum kinase (PERK) pathway of the unfolded protein response (UPR) is protective against toxic accumulations of misfolded proteins in the endoplasmic reticulum, but is thought to drive cell death via the transcription factor, CHOP. However, in many cell types, CHOP is an obligate step in the PERK pathway, which frames the conundrum of a prosurvival pathway that kills cells. Our laboratory and others have previously demonstrated the prosurvival activity of the PERK pathway in oligodendrocytes. In the current study, we constitutively overexpress CHOP in myelinating cells during development and into adulthood under normal or UPR conditions. We show that this transcription factor does not drive apoptosis. Indeed, we observe no detriment in mice at multiple levels from single cells to mouse behavior and life span. In light of these data and other studies, we reinterpret PERK pathway function in the context of a stochastic vulnerability model, which governs the likelihood that cells undergo cell death upon cessation of UPR protection and while attempting to restore homeostasis. SIGNIFICANCE STATEMENT Herein, we tackle the biggest controversy in the UPR literature: the function of the transcription factor CHOP as a protective or a prodeath factor. This manuscript is timely in light of the 2014 Lasker award for the UPR. Our in vivo data show that CHOP is not a prodeath protein, and we demonstrate that myelinating glial cells function normally in the presence of high CHOP expression from development to adulthood. Further, we propose a simplified view of UPR-mediated cell death after CHOP induction. We anticipate our work may turn the tide of the dogmatic view of CHOP and cause a reinvestigation of its function in different cell types. Accordingly, we believe our work will be a watershed for the UPR field.

Discovery of sulfonamidebenzamides as selective apoptotic CHOP pathway activators of the unfolded protein response

Cellular proteins that fail to fold properly result in inactive or disfunctional proteins that can have toxic functions. The unfolded protein response (UPR) is a two-tiered cellular mechanism initiated by eukaryotic cells that have accumulated misfolded proteins within the endoplasmic reticulum (ER). An adaptive pathway facilitates the clearance of the undesired proteins; however, if overwhelmed, cells trigger apoptosis by upregulating transcription factors such as C/EBP-homologous protein (CHOP). A high throughput screen was performed directed at identifying compounds that selectively upregulate the apoptotic CHOP pathway while avoiding adaptive signaling cascades, resulting in a sulfonamidebenzamide chemotype that was optimized. These efforts produced a potent and selective CHOP inducer (AC50 = 0.8 μM; XBP1 > 80 μM), which was efficacious in both mouse embryonic fibroblast cells and a human oral squamous cell cancer cell line, and demonstrated antiproliferative effects for multiple cancer cell lines in the NCI-60 panel.

Cantharidins Induce ER Stress and a Terminal Unfolded Protein Response in OSCC

Mortality and morbidity associated with oral squamous cell carcinoma (OSCC) remain unacceptably high with disfiguring treatment options and a death rate of 1 per hour in the United States. The approval of cituximab for advanced OSCC has been the only new treatment for these patients since the 1970s, although it has not significantly increased overall survival. To address the paucity of effective new therapies, we undertook a high-throughput screen to discover small molecules and natural products that could induce endoplasmic reticulum (ER) stress and enforce a terminal unfolded protein response (UPR) in OSCC. The terpenoid cantharidin (CNT), previously used to treat various malignancies in culture-specific medical practices for over 2,000 y, emerged as a hit. CNT and its analog, cantharidic acid, potently induced protein and gene expression profiles consistent with the activation of ER stress, the UPR, and apoptosis in OSCC cells. Murine embryonic fibroblasts null for the UPR-associated transcription factors Atf4 or Chop were significantly protected from CNT, implicating a key role for the UPR in the death response. These data validate that our high-throughput screen can identify novel modulators of UPR signaling and that such compounds might provide a new therapeutic approach to treating patients with OSCC.

Recent Trends in Oral Cavity Cancer Research Support in the United States.

The objectives were to characterize oral cavity cancer (OCC) funding from the National Institutes of Health (NIH) with a secondary aim of comparing NIH support provided to OCC and other malignancies. NIH awards supporting OCC inquiry from 2000 to 2014 were accessed from the NIH RePORTER database. These data were used to evaluate temporal trends and the role of human papilloma virus and to determine the academic training and professional profiles of the principal investigators. Comparison of 2014 funding levels with other malignancies was also performed, controlling for incidence. Overall funding totals decreased considerably after 2009. Funding administered through the National Institute of Dental and Craniofacial Research (NIDCR) was 6.5 times greater than dollars awarded by the National Cancer Institute in 2000. During the period evaluated, NIDCR support decreased in most years, while National Cancer Institute support increased and approached NIDCR funding levels. Funding for human papilloma virus–related projects gradually rose, from 3.4% of dollars in 2000 to 2004 to 6.2% from 2010 to 2014 (P < 0.05). A majority of principal investigators had a PhD omnia solus (57%), and 13% possessed dual PhD/clinical degrees. Among clinicians with specialty training, otolaryngologists and oral/maxillofacial pathologists garnered the most funding. OCC had a 2014 funding:incidence ratio of

Mucosal Therapy for Potentially Malignant Diseases and OSCC

785, much lower than for other malignancies. There has been increased volatility in funding support in recent years possibly due to budget cuts and sequestration. The National Cancer Institute has played an increasingly important role in supporting OCC research, concomitant with decreasing NIDCR support. Our findings suggest that OCC is underfunded relative to other non–oral cavity malignancies, indicating a need to increase the focus on rectifying the disparity.

The Unfolded Protein Response as a Therapeutic Target for Head and Neck Squamous Cell Carcinoma

Despite remarkable advances in our ability to manage and treat a number of hematologic and solid malignancies, there has been a paucity of new chemotherapeutic agents to significantly improve the outcomes and survival of oral cavity squamous cell carcinoma (OSCC) patients (Furness et al., Cochrane Database Syst Rev (4):CD006386, 2011). Surgical resection and radiation and systemic chemotherapy are first-line options used in the management of most patients. The cure rate for early-stage OCSS patients remains very promising; however, these lifesaving surgeries are often accompanied by tremendous sacrifices. Many patients are left with cosmetic and functional disfigurement and require adjunctive support for basic functions such as chewing, swallowing, and, in some cases, even breathing. The exploration of local delivery methods, such as mucosal therapy, holds promise due to decreased systemic toxicity, consequently decreased morbidity, and improved patient quality of life (Saba et al., Oral Oncol 51:112–118, 2015). Furthermore, topical therapies offer an alternative in the treatment of malignant precursor lesions, such as leukoplakia and erythroplakia, so that patients may be treated earlier and remain in the purview of nonsurgical oral healthcare providers.

Dimethyl Fumarate Ameliorates Myoclonus Stemming From Protein Misfolding In Oligodendrocytes

The epidemiology of head and neck malignancies and the survival rates for oral cancer patients are discussed in a cogent and thorough fashion throughout Targeting Oral Cancer. It is clear that a wide range of challenges, including disparity in health-care resources and research funding opportunities, and low prioritization by foundations and other organizations have left head and neck squamous cell carcinoma (HNSCC) patients in a remote corner of a huge outcome gap. Despite progress in multimodal therapies, responses to treatment and survival rates experienced by HNSCC patients lag egregiously behind the improvements that have been realized in other cancers. We should not lose focus of the fact that the 5-year survival rate for HNSCC is less than 50 % and very often accompanied by challenging and painful disfigurement. These dire statistics highlight the urgency of the problem. Also, comprehensive data indicating that ~70 % of HNSCC patients are still diagnosed at stages III or IV (Ferrari et al., Expert Opin Pharmacother 10(16):2625–32, 2009; Specenier and Vermorken, Expert Rev Anticancer Ther 8(3):375–91, 2008) underscores our need to diagnose patients sooner. Our continued inability to treat patients with chemotherapy whose metastatic and recurrent disease have outpaced the limitations of surgery and radiation therapy sentences them to an average median progression-free survival of ~6 months (Ferrari et al., Expert Opin Pharmacother 10(16):2625–32, 2009; Specenier and Vermorken, Expert Rev Anticancer Ther 8(3):375–91, 2008). Novel therapeutic targets and innovative strategies are urgently needed for a cancer that has seen little improvement over the last three decades. In this chapter we will discuss targeting the unfolded protein response (UPR) with small molecules and natural products as a novel anti-cancer approach in HNSCC models.