Compromising the 19S proteasome complex protects cells from reduced flux through the proteasome
- Whitehead Institute for Biomedical Research, United States
- Howard Hughes Medical Institute, Massachusetts Institute of Technology, United States
- Department of Cell Biology, Harvard Medical School, United States
- Stanford University School of Medicine, United States
- Harvard Medical School, United States
- Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Austria
- Netherlands Cancer Institute, Netherlands
- Brigham and Women's Hospital, United States
Figures
Figure 1 with 1 supplement
The 19S regulatory subunits of the proteasome are the most significant mediators of resistance to proteasome inhibitor toxicity.
(A) Schematic representation of the screen. One hundred million KBM7 cells subjected to random gene deletion using retroviral gene-trap insertions were exposed to either MG132 (700 nM) or bortezomib …
Figure 1—figure supplement 1
(A) Schematic representation of the knockout strategy to generate the mutant PSMD12 and PSMC2 ES cells.
Genetraps are in antisense (top), and sense (bottom) orientation. (B) Brightfield (top panels) and fluorescence (lower panels) microscopic imaging of FACS sorted PSMD12 and PSMC2 clones, stably …
Figure 2 with 1 supplement
Reducing expression of 19S subunits increases the levels of active 20S proteasomes and protects cancer cells from proteasome inhibition.
(A) HepG2 cells were infected with 80 shRNAs targeting 20 different subunits of the proteasome and 10 control shRNAs. Infected cells were then exposed to 12 nM bortezomib and cell number was …
Figure 2—figure supplement 1
(A–D) Examining the effect of proteasome subunit knockdown in different cell lines.
80 shRNAs targeting 20 different proteasome subunits and control hairpins were expressed in HepG2 (A), H838 (B), T47D (C), and H1792 (D) cells by viral transduction. Each subunit was targeted by 4 …
Figure 3 with 1 supplement
Inhibition of bortezomib-mediated transcriptional responses in PSMD2 knockdown cells.
RNA-seq gene expression profiling was conducted on HepG2 cells that harbor two different shRNAs targeting PSMD2 (PSMD2-1, PSMD2-2; same cells used in Figure 2F) and on control cells (shLacZ). The …
Figure 3—figure supplement 1
(A) Gene set enrichment analysis using the set of genes that are bound by HSF1 in MCF7 cancer cells under 37° basal conditions (Mendillo et al., 2012) was performed on genes negatively regulated in PSMD2 knockdown cells (siPSMD2) vs control cells (LacZ).
(B, C) Gene set enrichment analysis using the set of genes that are induced following heat shock (B), or genes that when knocked down confer resistance to bortezomib (C) was performed on genes …
Figure 4 with 1 supplement
Transient induction of PSMD2 shRNA is sufficient to promote resistance to proteasome inhibition.
(A) Schematic representation of the experimental design. (B) T47D cells harboring a doxycycline-inducible PSMD2 shRNA were grown in the presence or absence of 1 μg/ml doxycycline for 48 hr. Cells …
Figure 4—figure supplement 1
(A–F) PSMD2 shRNA was induced for 48 hr with 1 μg/ml doxycycline.
Cells were then collected, washed, and plated in the absence of doxycycline 24 hr prior to exposure to increasing concentration of bortezomib (A), MG132 (B), cyclohexamide (C), withaferin A (D), …
Figure 5 with 1 supplement
Reduced expression of 19S subunits correlates with resistance to proteasome inhibitors.
(A, B) Analysis of expression data from 315 cell lines in the Genomics of Drug Sensitivity in Cancer (GDSC) database (Garnett et al., 2012). The levels of 20S proteasome subunit (PSMAs and PSMBs) …
Figure 5—figure supplement 1
The relative expression level of each 19S complex subunit was analyzed in the MG132 resistant and sensitive groups.
Expression levels with deviation of more than twofold from the average were color-coded (red-up, green-down).
Figure 6
Transient 19S subunit reduction confers a competitive survival advantage in the presence proteasome inhibitors.
(A, B) T47D cells that harbor a doxycycline-inducible control shRNA (GFP) or a doxycycline-inducible PSMD2 shRNA (TurboRFP) were incubated with doxycycline for 48 hr. Cells were collected, counted, …
Figure 7 with 1 supplement
Reducing the levels of 19S subunits is an evolutionarily conserved mechanism to acquire resistance to proteasome inhibition.
Proteasome subunit DAmP strains and the BY4741 control strain were grown in the presence or absence of 50 μM MG132 for 48 hr. The relative change in OD induced by MG132 is plotted. Five proteasome …
Figure 7—figure supplement 1
Proteasome subunit DAmP strains and the BY4741 control strain were grown in YPD media and OD600 was measured after 48 hr.
https://doi.org/10.7554/eLife.08467.015Additional files
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Supplementary file 1
KBM7 screening hits for MG132 and bortezomib, insertions and p-values.
- https://doi.org/10.7554/eLife.08467.016
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Supplementary file 2
Validation reagents: lentivirus clones, selected shRNAs, antibodies.
- https://doi.org/10.7554/eLife.08467.017
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Supplementary file 3
RNA-seq analysis.
- https://doi.org/10.7554/eLife.08467.018
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Supplementary file 4
Genomics of drug sensitivity in cancer analysis.
- https://doi.org/10.7554/eLife.08467.019
