Stressing the Ubiquitin-Proteasome System without 20S Proteolytic Inhibition Selectively Kills Cervical Cancer Cells Ravi K. Anchoori1,2, Saeed R. Khan2,4, Thanasak Sueblinvong 5 , Alicia Felthauser5, Yoshie Iizuka5, Riccardo Gavioli3, Federica Destro3, Rachel Isaksson Vogel5, Shiwen Peng 2 , Richard B. S. Roden1,2,6, Martina Bazzaro5* 1 Department of Pathology, Johns Hopkins University, Baltimore, Maryland, United States of America, 2 Department of Oncology, Johns Hopkins University, Baltimore, Maryland, United States of America, 3 Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy, 4 FDA-CDER Division of Product Quality Research, Silver Spring, Maryland, United States of America, 5 Masonic Cancer Center and Department of Obstetrics, Gynecology and Women’s Heath, University of Minnesota Twin Cities, Minneapolis, Minnesota, United States of America, 6 Department of Gynecology and Obstetrics, Johns Hopkins University, Baltimore, Maryland, United States of America Abstract Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate, and for specific signaling pathways, notably HPV E6-targeted degradation of p53 and PDZ proteins. Natural compounds with antioxidant properties including flavonoids and triterpenoids hold promise as anticancer agents by interfering with ubiquitin-dependent protein degradation. An increasing body of evidence indicates that their a-b unsaturated carbonyl system is the molecular determinant for inhibition of ubiquitin-mediated protein degradation up- stream of the catalytic sites of the 20S proteasome. Herein we report the identification and characterization of a new class of chalcone-based, potent and cell permeable chemical inhibitors of ubiquitin-dependent protein degradation, and a lead compound RAMB1. RAMB1 inhibits ubiquitin-dependent protein degradation without compromising the catalytic activities of the 20S proteasome, a mechanism distinct from that of Bortezomib. Treatment of cervical cancer cells with RAMB1 triggers unfolded protein responses, including aggresome formation and Hsp90 stabilization, and increases p53 steady state levels. RAMB1 treatment results in activation of lysosomal-dependent degradation pathways as a mechanism to compensate for increasing levels of poly-ubiquitin enriched toxic aggregates. Importantly, RAMB1 synergistically triggers cell death of cervical cancer cells when combined with the lysosome inhibitor Chloroquine. Citation: Anchoori RK, Khan SR, Sueblinvong T, Felthauser A, Iizuka Y, et al. (2011) Stressing the Ubiquitin-Proteasome System without 20S Proteolytic Inhibition Selectively Kills Cervical Cancer Cells. PLoS ONE 6(8): e23888. doi:10.1371/journal.pone.0023888 Editor: Lin Zhang, University of Pennsylvania School of Medicine, United States of America Received June 30, 2011; Accepted July 29, 2011; Published August 31, 2011 Copyright: 2011 Anchoori et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Institutes of Health ATIP and SPORE in Cervical Cancer P50 CA098252 to SRK, RKA and RBSR and HERA foundation (Health, Empowerment, Research, and Awareness) and Department of Defense Ovarian Cancer Research Program (OCRP) OC093424 to MB. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mbazzaro@umn.edu Introduction Ubiquitin-dependent protein degradation via the ubiquitin- proteasome system (UPS) is crucial for the regulation of many cellular processes including cell cycle progression, differentiation and apoptosis in both normal and cancer cells [1]. Aberrant expression of components of the UPS system including ubiquitin- ligases, de-ubiquitinating enzymes and proteasomes has been reported in several cancer settings including cervical cancer [1,2,3], suggesting that in order to sustain their higher levels of metabolic activity cancer cells rely more heavily upon the proper function of the UPS as compared to their normal counterpart [4,5,6,7]. Thus, molecules capable of interfering with ubiquitin- dependent protein degradation, including Bortezomib, show anticancer activity [5]. Human Papillomavirus (HPV) is the primary cause of cervical cancer and responsible for 5% of all cancers worldwide [8]. While HPV vaccines can be an effective preventive measure against cervical cancer, there are currently no virus-specific therapies for it, and the efficacy of standard surgical and chemo/radiotherapies is limited for advanced disease [9]. Expression of two viral oncogenes, E6 and E7, is necessary for the induction and maintenance of the transformed phenotype [10]. The E6 oncoprotein exerts its oncogenic activity by binding to the E3 ubiquitin ligase E6-AP and redirects its activity towards p53 and other tumor suppressor proteins for their rapid ubiquitin- mediated proteasomal degradation [11,12,13]. This reduces the level of this key cellular cell cycle regulator without its mutation. Therefore, we hypothesized that stabilization of p53 via preventing its ubiquitin-mediated degradation will have therapeu- tic potential for cervical cancer and possibly for other cancers wild- type for p53. Natural compounds of the flavonoid and triterpenoids families including curcumin, Celastrol, green tea polyphenols and chalcones have shown promise as antineoplastic agents in a variety of cancer settings including cervical [14], colon [15,16], oesophageal [17], pancreatic [18] and prostate [19,20,21] cancer, linked to pro-apoptotic properties as associated with proteasomal inhibition. We have recently shown that chalcone-derivatives PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23888