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PSMD5 Inactivation Promotes 26S Proteasome Assembly During Colorectal Tumor Progression Avi Levin1,2, Adi Minis1, Gadi Lalazar3, Jose Rodriguez1, and Hermann Steller1

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PSMD5 Inactivation Promotes 26S Proteasome Assembly During Colorectal Tumor Progression Avi Levin1,2, Adi Minis1, Gadi Lalazar3, Jose Rodriguez1, and Hermann Steller1 Published OnlineFirst May 1, 2018; DOI: 10.1158/0008-5472.CAN-17-2296 Cancer Molecular Cell Biology Research PSMD5 Inactivation Promotes 26S Proteasome Assembly during Colorectal Tumor Progression Avi Levin1,2, Adi Minis1, Gadi Lalazar3, Jose Rodriguez1, and Hermann Steller1 Abstract Protein degradation by the ubiquitin–proteasome system (UPS) is central to protein homeostasis and cell survival. The active 26S proteasome is a PSMD5 large protease complex consist- Malignant ing of a catalytic 20S subunit transformation and 19S regulatory particles. PSMD5/S5b Cancer cells are exposed to con- down-regulation siderable protein overload due PSMD5 to high metabolic rates, repro- PSMD5 grammed energy metabolism, and aneuploidy. Here we report a mechanism that facilitates the Normal cell Malignant cell assembly of active 26S protea- somes in malignant cells. Upon Downregulation of PSMD5/S5b promotes 26S proteasome assembly, thereby increasing the tumorigenic transformation of proteolytic capacity of colorectal cancer cells, allowing them to maintain protein homeostasis. the gut epithelium, 26S pro- teasome assembly was signifi- © 2018 American Association for Cancer Research cantly enhanced, but levels of individual subunits were not changed. This enhanced assembly of 26S proteasomes increased further with tumor progression and was observed specifically in transformed cells, but not in other rapidly dividing cells. Moreover, expression of PSMD5, an inhibitor of proteasome assembly, was reduced in intestinal tumors and silenced with tumor progression. Reexpression of PSMD5 in tumor cells caused decreased 26S assembly and accumulation of polyubiquitinated proteins. These results suggest that inhibition of cancer-associated proteasome assembly may provide a novel therapeutic strategy to selectively kill cancer cells. Significance: Enhanced cancer-associated proteasome assembly is a major stress response that allows tumors to adapt to and to withstand protein overload. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3458/F1.large.jpg. Cancer Res; 78(13); 3458–68. Ó2018 AACR. Introduction vated protein synthesis can generate significant amounts of super- fluous and damaged proteins, which are potentially toxic. How- Protein synthesis and degradation are coordinated to allow ever, cells can increase their rates of protein degradation to adaptation to a rapidly changing microenvironment (1–3). Ele- maintain protein homeostasis (4, 5). Selective protein degrada- tion plays central roles in multiple biological processes including 1Strang Laboratory of Apoptosis and Cancer Biology, The Rockefeller University, removal of misfolded and potentially toxic proteins, control of New York, New York. 2Division of Gastroenterology, University of Iowa, Iowa cell-cycle progression, regulation of gene expression, and changes City, Iowa. 3Laboratory of Cellular Biophysics, The Rockefeller University, New in cell size and morphology (6, 7). The selective degradation of York, New York. most intracellular proteins is carried out by the ubiquitin-protea- Note: Supplementary data for this article are available at Cancer Research some system (UPS; refs. 8, 9). Proteins tagged with polyubiquitin Online (http://cancerres.aacrjournals.org/). chains are hydrolyzed into small peptides by the 26S proteasome Corresponding Authors: Hermann Steller, The Rockefeller University, 1230 York in an energy-dependent manner. The 26S proteasome is assem- Avenue, New York, NY 10065. Phone: 212-327-7075; Fax: 212-327-7076; E-mail: bled from a cylinder-shaped 20S proteolytic complex and a PA700 [email protected]; and Avi Levin, University of Iowa Hospitals and Clinics. (19S) ATPase-regulatory complex (10). The assembly and activity 200 Hawkins Drive, Iowa City, IA 52242. Phone: 319-356-7368; E-mail: of the 26S proteasome is tightly regulated by a large number of [email protected] cofactors and chaperones (8). Cancer cells are more exposed to doi: 10.1158/0008-5472.CAN-17-2296 proteotoxic stress due to high metabolic rates, reprogrammed Ó2018 American Association for Cancer Research. energy metabolism, and aneuploidy (5, 11–13). Despite this 3458 Cancer Res; 78(13) July 1, 2018 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst May 1, 2018; DOI: 10.1158/0008-5472.CAN-17-2296 Enhanced Proteasome Assembly Mediates Tumor Proteostasis pressure, they successfully proliferate and efficiently withstand this several modifications: large bowels with and without tumors were challenge by fully exploiting the cells' defense mechanisms against flushed with cold PBS and opened longitudinally, washed with proteotoxic stress. For instance, constitutive activation of the heat 70% ethanol. Large-bowel tumors were dissected from normal shock factor protein 1 (HSF1)-regulated heat shock response large bowel. Normal large bowels and tumors were agitated in a (HSR) pathway is among the recurring features of malignant cells, solution of 2 mmol/L EDTA in PBS for 30 minutes at 4C. Tumors and inhibition of molecular chaperones represents a promising were dissociated using 70-mm strainer. therapeutic strategy for cancer treatment (14). In addition, Crypts were collected as described and embedded onto Matrigel increased proteasome activity can suppress aneuploidy-associated (BD Biosciences) with 50 mL/well in 24-well plates. DMEM/F12 proteotoxic stress (12). Activation of proteasomal degradation by culture medium (Gibco), containing Glutamax (Gibco) and pen- deletion of the proteasome-associated deubiquitylating enzyme icillin/streptomycin was supplemented with B27 (Gibco, 1:50), UBP6 attenuates the aneuploidy-induced changes in cellular pro- 100 ng/mL murine Noggin (Peprotech), 20 ng/mL murine EGF tein composition and improves cell fitness (15). The mechanisms (Peprotech), 10 ng/mL human basic-FGF (Peprotech) and 250 ng/ by which cancer cells overcome proteotoxic stress are of consid- mL human R-Spondin1 (Peprotech), 100 ng/mL mouse Wnt-3a erable scientific and clinical interest as advances in this area may (Peprotech) and 10 mmol/L nicotinamide (Sigma) for large-bowel identify new therapeutic targets. Notably, proteasome inhibitors organoids. Tumor organoids were cultured in medium containing have antitumor activity and are clinically used for the treatment of only B27 (Gibco, 1:50), 100 ng/mL murine Noggin (Peprotech) multiple myeloma and mantle cell lymphoma (16, 17). and 20 ng/mL murine EGF (Peprotech) supplements. The rate-limiting step in proteolysis by the UPS is considered to Lentiviral infection was used to transduce genes into advanced be substrate ubiquitination. However, recent findings indicate that tumor organoids. We generated recombinant lentiviral particles proteolysis rate is significantly changed by regulatory pathways expressing mouse PSMD5 in HEK293T. Because lentivirus parti- that affect the activity of the 26S proteasome itself, for example, by cles cannot penetrate the Matrigel layer, we incubated dissociated posttranslational modifications of proteasome subunits, or by organoids with viral particles in liquid media for 6 hours 37C facilitating the assembly of the 26S proteasome from the core under 5% CO2 (v/v). Next, cells were embedded in Matrigel and proteasome and the regulatory complexes (18, 19). It has been grown into organoids. Two to 3 days after plating of cells, previously shown that colon cancer cells increase expression of puromycin was added (5 mg/mL) to the medium for selection of proteasome subunits in a concerted manner (20). However, upre- successfully transduced cells. gulating the levels of proteasome subunits, while important, is not sufficient to significantly increase proteasome activity. Indeed, the Protein translation rate levels of functional 26S proteasomes depend not only on the The rate of protein translation was measured using the nonra- expression of its subunits, but also on their precise assembly. Work dioactive surface Sensing of translation (SUnSET) labeling method in yeast clearly demonstrates that assembly of 26S can vary (25). Briefly, puromycin (4 mmol/L) was added to culture medium significantly to meet the changing proteolytic needs of a cell (21). of organoids for 30 minutes, then organoids were harvested and Here we investigated a possible role of 26S proteasome assem- puromycin incorporation was detected by Western blot analysis bly for protein homeostasis during tumorigenesis of the mouse using an anti-puromycin antibody, clone 12D10 (1:5,000, Sigma). gut epithelium. We show that 26S proteasome assembly is highly To label new synthesized proteins in vivo, we injected into peri- increased upon tumorigenic transformation of the gut epitheli- toneum puromycin (0.04 mmol/gr of body weight of mice). We um. Importantly, this increase in 26S assembly is cell-autono- euthanized animals 30 minutes after puromycin injection and mous and occurs exclusively in transformed cells, but not in other analyzed tissues by Western blot analysis using an anti-puromycin rapidly dividing cells. In addition, levels of the proteasome antibody, clone 12D10 (1:5,000, Sigma). assembly inhibitor PSMD5/S5B are significantly decreased in tumors, and reexpression of PSMD5 blocks 26S proteasome Cell culture and DNA transfection assembly. Collectively, these results suggest that intestinal cancer HEK293T and HT-29 cells (purchased from ATCC) were grown cells escape proteotoxic stress by reducing PSMD5 to stimulate in DMEM containing 10% FBS and penicillin/streptomycin
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