Ubiquitin proteasome system stress underlies synergistic killing of ovarian cancer cells by bortezomib and a novel HDAC6 inhibitor

Abstract

Purpose: Elevated metabolic activity of ovarian cancer cells causes increased ubiquitin-proteasome-system (UPS) stress, resulting in their greater sensitivity to the toxic effects of proteasomal inhibition. The proteasomes and a potentially compensatory histone deacetylase 6 (HDAC6)-dependent lysosomal pathway mediate eukaryotic protein turnover. We hypothesized that up-regulation of the HDAC6-dependent lysosomal pathway occurs in response to UPS stress and proteasomal inhibition, and thus, ovarian cancer cell death can be triggered most effectively by coinhibition of both the proteasome- and HDAC6-dependent protein degradation pathways.

Experimental design: To address this hypothesis, we examined HDAC6 expression patterns in normal and cancerous ovarian tissues and used a novel HDAC6-specific inhibitor, NK84, to address HDAC6 function in ovarian cancer.

Results: Abnormally high levels of HDAC6 are expressed by ovarian cancer cells in situ and in culture relative to benign epithelium and immortalized ovarian surface epithelium, respectively. Specific HDAC6 inhibition acts in synergy with the proteasome inhibitor Bortezomib (PS-341) to cause selective apoptotic cell death of ovarian cancer cells at doses that do not cause significant toxicity when used individually. Levels of UPS stress regulate the sensitivity of ovarian cancer cells to proteasome/HDAC6 inhibition. Pharmacologic inhibition of HDAC6 also reduces ovarian cancer cell spreading and migration consistent with its known function in regulating microtubule polymerization via deacetylation of alpha-tubulin.

Conclusion: Our results suggest the elevation of both the proteasomal and alternate HDAC6-dependent proteolytic pathways in ovarian cancer and the potential of combined inhibition of proteasome and HDAC6 as a therapy for ovarian cancer.

Figure 1. HDAC6 over-expression in ovarian carcinoma and cell lines

A, left panel,immunohistochemical staining of HDAC6 in ovarian tumors. Representative examples of intense HDAC6 staining of high-grade and low-grade ovarian serous carcinomas and weaker staining in serous adenofibroma and serous cystadenoma (imaging with X40 objective), right panel, staining intensity for each case was graded as 0 (no staining), 1 (weak staining), 2 (moderate staining) and 3 (intense staining), and the statistical significance of differences in staining intensities among indicated groups was calculated using the Mann-Whitney U test. Error bars indicate ± SD. B, Western blot analysis of HDAC6 in clinical specimens of serous cystadenoma (lanes 1–4) and serous carcinoma (lanes 5–10). Equal loading was verified by using an antibody directed against β-actin. C, Western blot analysis of HDAC6 immortalized ovarian surface epithelial cells (IOSE-29 and IOSE-397) and ovarian cancer cell lines (SKOV-3, ES-2, TOV-21G). Equal protein loading in each lane was verified by using an antibody directed against β-actin.

Figure 2. NK84 treatment reduces the viability ovarian cancer cells while sparing IOSEs

A, Ovarian cancer cell lines (SKOV-3, ES-2, TOV-21G) and immortalized ovarian surface epithelial cells (IOSE-29 and IOSE-397) were treated with the HDAC6 inhibitor NK84 (A,B) at the concentrations indicated. Cell viability was measured by XTT assay after culturing the cells for 24 hours (A) or 48 hours (B) in presence of NK84 inhibitor. Error bars indicate ± SD.

Figure 3. Simultaneous inhibition of HDAC6 and proteasome activity induces synergistic killing of ovarian cancer cells

Dose-dependent inhibition of the cell viability of ES-2 (A) and TOV-21G (B) in the absence (−) or in the presence (+) of 5μM NK84 HDAC6 inhibitor and PS-341 at the indicated concentration. Cell viability was measured after a 24 hours incubation by XTT assay and the percentage of viable cells is presented relative to mock-treated controls. ***P < 0.001. Error bars indicate ± SD. Lysate of ES-2 cell line treated with NK84 (5 μm) and/or PS-341 at the indicated concentrations was immunoblotted with an antibody recognizing the full-length and cleaved forms of PARP. Equal protein loading was verified by using an antibody directed against β-actin.

Figure 4. Synergistic activity of PS-341 and NK84 on ovarian cancer cells is dependent upon level of metabolic activity

A, Immunoblot analysis of the levels of poly-ubiquitinated proteins in cyclohexamide-exposed (24h) ES-2 ovarian cancer cells. Equal loading was verified by using an antibody directed against β-actin. B, ES-2 ovarian cancer cells pre-exposed to 0, 1 or 5 mg/ml CHX (24 hours) subsequently received treatment with of PS-341 (1nM) and NK84 (5 μM) or mock treatment. Cell viability was as evaluated by XTT assay after 24 hours of treatment. Percentage of viable cells is relative to mock-treated control cells is presented. ** P < 0.02. Error bars indicate ± SD. C, proliferation rate of confluent (+) or sub-confluent (−) ES-2 and TOV-21G ovarian cancer cell lines was measured by XTT assay. Each assay was performed in triplicate. Shown are bars ± SD of proliferative activity measured in terms of optical density at 450 nm on each given day. D, cell viability of ES-2 and TOV-21G ovarian cancer cell lines was evaluated by XTT assay in sub-confluent (−) versus confluent (+) cultures in the presence of PS-341 1nM and NK84 5 μM. Percentage of viable cells is relative to mock-treated control cells. ***P < 0.001. Error bars indicate ± SD.

Figure 5. HDAC6 inhibition prevents aggresome formation in UPS stressed ovarian cancer cells

A, ES-2 ovarian cancer cells were incubated with 5nM PS-341, 10 μM NK84 or the combination of both for 24 hours before fixation and immuno-fluorescent staining of DNA (blue), ubiquitin (green) and vimentin (red) and imaging (X60 objective). B, IOSE-29 cells were incubated with 20nM PS-341 for 24 hours before fixation and immuno-fluorescent staining of DNA (blue), ubiquitin (green) and vimentin (red) and imaging (X60 objective). C, Immunoblot analysis of ubiquitinated protein in ES-2 cells (left panel) or IOSE-29 cells (right panel) 24h after treatment with or without 10 μM NK84 and the indicated concentrations of PS-341. Equal protein loading in each lane was verified by using an antibody directed against β-actin.

Figure 6. Pharmacologic inhibition of HDAC6 impedes cell motility and migration of ovarian cancer cells

A Plates of confluent SKOV-3 cells either mock or NK84 treated (10 μM) were examined by phase-contrast microscopy at the time of removal by scratching (0 hours) and 5 hours later. B, Analysis of the number of SKOV-3 cells spreading across the wound; results are means ± SD of three independent experiments. C, an equivalent number (2.5 × 10⁴) of SKOV-3 or ES-3 cells mock-treated or treated with 10 μM of NK84 HDAC6 inhibitor were seeded in migration chambers and migrating cells were counted per each condition 8 hours thereafter. Three different fields of cells for each condition were counted at 40X. Each assay was performed in triplicate. Shown are means ± SD of migrating cells per microscopic field. D, representative example of migration assay conducted on mock or NK84-treated SKOV-3 cells.