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

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

Early and consistent overexpression of ADRM1 in ovarian high-grade serous carcinoma

Abstract Background Ovarian carcinoma is highly dependent on the ubiquitin proteasome system (UPS), but its clinical response to treatment with the proteasome inhibitor bortezomib has been disappointing. This has driven exploration of alternate approaches to target the UPS in ovarian cancer. Recently, proteasome inhibitors targeting the 19S regulatory particle-associated RPN13 protein have been described, such as RA190. RPN13, which is encoded by ADRM1, facilitates the recognition by the proteasome of its polyubiquinated substrates. Inhibition of RPN13 produces a rapid, toxic accumulation of polyubiquitinated proteins in ovarian and other cancer cells, triggering apoptosis. Here, we sought to determine if RPN13 is available as a target in precursors of ovarian/fallopian tube cancer as well as all advanced cases, and the impact of increased ADRM1 gene copy number on sensitivity of ovarian cancer to RA190. Methods ADRM1 mRNA was quantified by RNAscope in situ hybridization and RPN13 protein detected by immunohistochemistry in high grade serous carcinoma (HGSC) of the ovary and serous tubal intraepithelial carcinoma (STIC). Amplification of ADRM1 and sensitivity to RA190 were determined in ovarian cancer cell lines. Results Here, we demonstrate that expression of ADRM1mRNA is significantly elevated in STIC and HGSC as compared to normal fallopian tube epithelium. ADRM1 mRNA and RPN13 were ubiquitously and robustly expressed in ovarian carcinoma tissue and cell lines. No correlation was found between ADRM1 amplification and sensitivity of ovarian cancer cell lines to RA190, but all were susceptible. Conclusions RPN13 can potentially be targeted by RA190 in both in situ and metastatic ovarian carcinoma. Ovarian cancer cell lines are sensitive to RA190 regardless of whether the ADRM1 gene is amplified

Structure-function analyses of candidate small molecule RPN13 inhibitors with antitumor properties.

We sought to design ubiquitin-proteasome system inhibitors active against solid cancers by targeting ubiquitin receptor RPN13 within the proteasome's 19S regulatory particle. The prototypic bis-benzylidine piperidone-based inhibitor RA190 is a michael acceptor that adducts Cysteine 88 of RPN13. In probing the pharmacophore, we showed the benefit of the central nitrogen-bearing piperidone ring moiety compared to a cyclohexanone, the importance of the span of the aromatic wings from the central enone-piperidone ring, the contribution of both wings, and that substituents with stronger electron withdrawing groups were more cytotoxic. Potency was further enhanced by coupling of a second warhead to the central nitrogen-bearing piperidone as RA375 exhibited ten-fold greater activity against cancer lines than RA190, reflecting its nitro ring substituents and the addition of a chloroacetamide warhead. Treatment with RA375 caused a rapid and profound accumulation of high molecular weight polyubiquitinated proteins and reduced intracellular glutathione levels, which produce endoplasmic reticulum and oxidative stress, and trigger apoptosis. RA375 was highly active against cell lines of multiple myeloma and diverse solid cancers, and demonstrated a wide therapeutic window against normal cells. For cervical and head and neck cancer cell lines, those associated with human papillomavirus were significantly more sensitive to RA375. While ARID1A-deficiency also enhanced sensitivity 4-fold, RA375 was active against all ovarian cancer cell lines tested. RA375 inhibited proteasome function in muscle for >72h after single i.p. administration to mice, and treatment reduced tumor burden and extended survival in mice carrying an orthotopic human xenograft derived from a clear cell ovarian carcinoma

Effects of RAMB treatment on the levels of polyubiquitinated proteins in HeLa cells.

<p><i>Left panel:</i> immunoblot analysis of ubiquitinated proteins in HeLa cells after 6 hours exposure with or without 10 µM RAMBs. Bortezomib was used as positive control. Equal protein loading in each lane was verified by using an antibody against GAPDH. <i>Right panel:</i> Quantification of the Ubiquitin/GAPDH ratios.</p

RAMB1 treatment prevents anchorage-dependent colony formation and synergizes with Chloroquine to kill cervical cancer cells.

<p>A. Equal numbers of SiHa and Caski cells (10³) were seeded into 6-wells plastic dishes and treated with or without RAMB1 at the indicated concentrations over a period of 10 days. Colonies were visualized by crystal violet staining. B. <i>Left panel</i>: Quantification of colony number in mock versus RAMB1 treated HeLa cells. <i>Middle panel</i>: Quantification of colony size in mock versus RAMB1 treated cervical cancer cells. <i>Right panel</i>: Representative experiment of HeLa cells (10²) which were seeded into 6-wells plastic dishes and treated with or without RAMB1 at the indicated concentration over a period of 10 days. Colonies were visualized by crystal violet staining and manually counted using an inverted microscope. <i>Inserts:</i> Representative example of colony size in mock versus RAMB1-treated cells. <i>Middle panel:</i> Quantification of colony number. <i>Right panel</i>: Quantification of colony size, representative of three independent experiments. **, P<0.02. C. CaSki cells were treated with checkerboard dilution series of RAMB1 and Chloroquine over a period of 24 hours. Cell viability was measured by XTT assay and calculated as percent of control untreated cultures. Synergy is expressed as combination index (CI).</p

Effect of RAMB treatment upon cervical cancer cell lines and primary human keratinocytes.

<p>Cultures of HPV-transformed cervical cancer cells (SiHa, CaSki and ME180) or primary human keratinocytes were treated with the indicated concentrations of RAMB1 (<i>left panel</i>) or RAMB4 (<i>right panel</i>) over a period of 48 hours. Cell viability was determined by XTT assay and plotted as a fraction of the untreated control cultures.</p