Stressing the Ubiquitin-Proteasome System without 20S Proteolytic Inhibition Selectively Kills Cervical Cancer Cells

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 α-β 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.

Small-Molecule RA-9 Inhibits Proteasome-Associated DUBs and Ovarian Cancer In Vitro and In Vivo via Exacerbating Unfolded Protein Responses

“…The deubiquitinating enzyme inhibitor RA-9 was synthesized and purified as we have previously described (15). The proteasome inhibitor Bortezomib was purchased from Cayman Chemical.…”

“…Cell viability was determined by WST-1 or CellTiter96® AQ ueous One Solution Cell Proliferation assays as previously described (15-17). Briefly, cells were seeded at the concentration of 1,000 or 10,000 per well in 100 μL medium in 96-well plate and treated with the indicated concentrations of drugs.…”

“…Previously, we showed that ovarian cancer cells are under higher levels of ubiquitin-proteasome stress as compared to normal cells, rendering them selectively sensitive to inhibition of ubiquitin-dependent protein degradation (11, 12). Recent work from our laboratory suggests that the presence of an α-β carbonyl system constitutes a key molecular determinant for a new series of small-molecule inhibitors of cysteine-based DUBs, thus paving the way to the further development of inhibitors of this class of proteases for cancer treatment (15). …”

HPV16 activates the promoter of Oct4 gene by sequestering HDAC1 from repressor complex to target it to proteasomal degradation

“…For example, the E6 oncoprotein targets cellular proteins with PDZ domain, such as p53, to proteasomal degradation via E6 associated protein (E6AP) mediated ubiquitination [16]. These reduced proteins can be rescued by proteasome inhibitors which were also confirmed to execute a selective toxicity against HPV positive cervical cancer cells [17,18]. On the other hand, although sumoylation of HDAC1 protein can strength the stability of it [19], E6 oncoprotein destabilizes HDAC1 protein by reducing the level of the sole sumo conjugation enzyme, Ubc9 [20].…”

Inhibition of proteasome deubiquitinase activity: a strategy to overcome resistance to conventional proteasome inhibitors?

“…Interestingly the specificities of α, β-unsaturated ketone-containing compounds towards various DUBs have been reported to vary considerably. For example whereas b-AP15 was reported to be specific to proteasomal deubiquitinases (D'Arcy et al, 2011), the structurally related compound RA-9 was reported to display more broad range activity and shown to inhibit UCHL1, UCHL3, USP2 and USP8 in addition to the proteasomal DUBs (Anchoori et al, 2011;Coughlin et al, 2014;Issaenko and Amerik, 2012). The chalcone-derivative G5 was also reported to display a broad spectrum of inhibition of DUB activity (Aleo et al, 2006;Nicholson et al, 2008).…”