Inhibition of the Proteasome β2 Site Sensitizes Triple-Negative Breast Cancer Cells to β5 Inhibitors and Suppresses Nrf1 Activation

Abstract

The proteasome inhibitors carfilzomib (Cfz) and bortezomib (Btz) are used successfully to treat multiple myeloma, but have not shown clinical efficacy in solid tumors. Here we show that clinically achievable inhibition of the β5 site of the proteasome by Cfz and Btz does not result in loss of viability of triple-negative breast cancer cell lines. We use site-specific inhibitors and CRISPR-mediated genetic inactivation of β1 and β2 to demonstrate that inhibiting a second site of the proteasome, particularly the β2 site, sensitizes cell lines to Btz and Cfz in vitro and in vivo. Inhibiting both β5 and β2 suppresses production of the soluble, active form of the transcription factor Nrf1 and prevents the recovery of proteasome activity through induction of new proteasomes. These findings provide a strong rationale for the development of dual β5 and β2 inhibitors for the treatment of solid tumors.

Keywords: CRISPR; NFE2L1; Nrf1; bortezomib; carfilzomib; proteasome; triple-negative breast cancer; ubiquitin.

Figure 1. The sensitivity of TNBC cells to Btz and Cfz does not correlate with inhibition of the β5 site

a) TNBC and MM cell lines were treated with Btz for 1h, recovered in drug-free media for 48h, then assayed for viable cells with Alamar Blue. Dose-response curves were generated by plotting the averages of 2–5 biological replicates and used to determine the average IC₅₀. See Table 1 for confidence intervals. MM data are from (Shabaneh et al., 2013). TNBC subtype assignments are from (Charafe-Jauffret et al., 2009). b,c) Cells were treated with Btz for 1h, then immediately assayed for proteasome inhibition using site-specific substrates from Proteasome-Glo. In a parallel experiment, viable cells were quantified 48h after treatment as in (a). d,e) Viable cells were plotted against inhibition of the β5 and β1 sites after Btz treatment. f,g) Cells were treated with Cfz for 1h, and analyzed as in (b). h) Viable TNBC cells were plotted against inhibition of the β5 site after Cfz treatment. Values in (b–h) are mean ± S.E.M of 2–3 biological replicates.

Figure 2. Inhibition of the β2 subunit sensitizes cells to β5 inhibitors better than inhibition of the β1 subunit

a,b) SUM149 cells were treated for 1h with LU-102 or NC-021. The active site probes MV-151 and BODIPY-NC-001 (Verdoes et al., 2010) were used to determine site inhibition in cell extracts. Band intensities were quantified by densitometry. The graphs are averages ± S.E.M of two independent experiments. c) SUM149 cells were treated with NC-021 or LU-102 for 1h and viable cells were determined with Alamar Blue after 48h. d,e) Cells were treated with Cfz, Cfz+5μM NC-021, or Cfz+3μM LU-102 for 1h and viable cells were determined after 48h. Combination indexes are listed in Fig S1e. f) SUM149 cells were treated with 100nM Cfz, 3μM LU-102, and 5μM NC-021 for 1h. Cells were stained with Annexin V after 36h. Values are mean ± S.E.M of 2–3 biological replicates. g) Average IC₅₀s were plotted relative to the average IC₅₀ for Btz or Cfz only. h,i) Viable cells after 1h co-treatment with Cfz + 3μM LU-102 (h) or Cfz + 5μM NC-021 (i) were plotted against inhibition of β5 sites by Cfz (as in Fig. 1h). NC-021 and LU-102 do not alter inhibition of the β5 sites by Cfz (not shown).

Figure 3. Mutant CRISPR cell lines with inactive β2 subunits are more effectively sensitized to β5 inhibition than cells with inactive β1 subunits

a) Activity of each active site was determined with activity-based probes. “i” designates subunits of lymphoid-tissue specific immunoproteasomes. The faint band between β1 and β1i in the β1ΔT1 mutant is due to BODIPY-NC-001 cross-reacting with the β5/5i subunit (see Fig. S2a). b) Sequences of mutant alleles. T₁ is the active site threonine. Lowercase letters indicate CRISPR-introduced T1A and synonymous mutations. subs.= substitution, del.=deletion. c) Western blot confirming expression of mutant subunits. d) Cells were treated with Cfz for 1h and viable cells were determined at 48h. Data are averages ± S.E.M. of 3–7 biological replicates. e) Cells were injected into the mammary fat pads of female NSG mice. Day 0 indicates the day that tumors were deemed palpable, defined as 14mm³. Mice were treated intravenously with 1.5 mg/kg Cfz twice-weekly on consecutive days. ***P<0.001, two-way ANOVA, compared to vehicle control, n=8–14/group (Table S1).

Figure 4. β2 inhibition increases accumulation of ubiquitinated protein and inhibits proteasome activity recovery in Cfz-treated cells

a,c) Cells were treated for 1h with 100 nM Cfz, 3 μM LU-102, and 5 μM NC-021, harvested at the times indicated, and analyzed by western blot. b) Cells were treated as in (a) and harvested. Caspase activity was determined in cell extracts using the caspase substrate Ac-DEVD-amc. d) CRISPR cell lines were treated for 1h with 100nM Cfz and harvested at 9h, then analyzed by western blot. e) Cells were treated for 1h with the same concentrations of inhibitors as in (a) and (b), and β5 occupancy was determined with activity-based probes as in Fig. 2ab. See Fig. S3 for β1 and β2 occupancies. Data are presented as mean ± S.E.M of 2–3 biological replicates.

Figure 5. Co-inhibition of β5 and β2 suppresses soluble, active Nrf1 and prevents upregulation of proteasome genes

a–c) Cells were treated for 1h with 100nM Cfz, 3 μM LU-102, and/or 5 μM NC-021 (a,b), or 100 nM Cfz (c), harvested at times indicated, lysed with CHAPS buffer, and analyzed by western blot. Cfz+NC-021 18h is loaded on both gels to allow comparison. Representative results of 2 experiments are shown. FL=full-length, cl=cleaved. d) SUM149 cells were treated for 1h and harvested at 16h. The soluble fraction was isolated with CHAPS buffer, the pellet was treated with DNase and high-salt to isolate the chromatin-bound fraction, and aggregates were subsequently solubilized by sonication in 2% SDS. Fractions were analyzed by western blot. Origin Recognition Complex subunit 2 (ORC2) is a marker for chromatin-bound proteins. e) MDA-MB-231 cells were treated as in (a), harvested at 10h, and mRNA was measured by RT-PCR. mRNA levels are relative to the control gene PGK1. f) Cells were treated as in (c), harvested at 12h, and mRNA was measured by RT-PCR. (g,h) Cells were treated with 60nM control, Nrf1, or DDI2 siRNA, then treated with indicated compounds for 1h. Samples for westerns were harvested at 16h. Remaining cells were stained with Annexin V after 36h. i) Model for the effect of proteasome inhibition on Nrf1 regulation. ***P<0.001, **P<0.01, *P<0.05, unpaired t-test. Data in (e–h) are presented as mean ± S.E.M of 2–5 biological replicates.

Figure 6. LU-102 sensitizes cell lines derived from other solid tumors to Cfz and Btz

a,b) Percentage of viable cells were plotted against inhibition of the β5 or β1 sites after 1h treatment with Cfz. Cells lines are derived from lung (A549, H23, Hop-62, H226), renal (TK-10), or ovarian (OVCAR4) cancer. c) Average IC₅₀s were plotted relative to the average IC₅₀ for Btz or Cfz only. d) Percentage of viable cells were plotted against inhibition of the β5 site after 1h treatment with Cfz + 3 μM LU-102. LU-102 did not reduce viability when used as a single agent and did not alter inhibition of the β5 site by Cfz (not shown).