The ubiquitin-proteasome system (UPS) is the key intracellular machinery for protein degradation, regulating the localization and stability of thousands of proteins within a cell (1). The UPS encompasses a series of essential components, including ubiquitin, ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), ubiquitin ligases (E3s), deubiquitinating enzymes (DUBs), and the 26S proteasome (2–4). Due to the major role of the UPS in maintaining protein homeostasis, it is a central player in regulating essential cellular functions, including cell differentiation, cell cycle progression, apoptosis, DNA repair, and drug resistance (5–8). The widespread influence of the UPS on cell biology has important implications for human health, as abnormal UPS function is associated with numerous human conditions, including neurodegenerative diseases, autoimmune diseases, and cancer (9, 10). For this reason, a number of small molecule inhibitors targeting the UPS were developed for therapeutic intervention, but unfortunately, drug resistance and toxicity remain significant challenges (11, 12), especially in hematopoietic cells (13–15). Novel therapeutic approaches will be required to effectively target the UPS with less toxicity.

The 26S proteasome is comprised of two main subcomplexes, the 20S core complex and the 19S regulatory complex, which work together to recognize ubiquitylated peptides and promote UPS-dependent protein degradation (Figure 1) (22–25). The 20S core complex contains the protein degradative machinery, whereas the 19S regulatory complex functions by recognizing, binding, unfolding, and translocating ubiquitylated peptides into the 20S core complex for degradation (22, 23, 26). Traditional proteasome inhibitors (e.g., bortezomib, carfilzomib, ixazomib) function by binding to and inhibiting the 20S core complex. Bortezomib (Velcade^®), a reversible inhibitor of chymotrypsin-like proteasome activity, acts by binding to and inhibiting the 20S beta 5 subunit of the proteasome (β5, PSMB5) (27), leading to the accumulation of ubiquitylated proteins and cell death (28). However, proteasome inhibitors like bortezomib are prone to resistance mechanisms, due to mutations in the molecular target of bortezomib, PSMB5, highlighting the need for alternative therapeutic strategies (29–31).

One strategy to overcome proteasome inhibitor resistance is to target the 19S regulatory complex instead of the 20S core complex. Importantly, the 19S complex contains numerous components subdivided into ATP-dependent and ATP-independent subunits (Figure 1) (32). The ATP-dependent subunits include the proteasome 26S subunit, ATPases 1–6 (PSMC1-6, Supplementary Table S1), which make up the base of the 19S complex, whereas the ATP-independent subunits include proteasome 26S subunit, non-ATPases 1–16 (PSMD1-16) (25), which make up the 19S lid (33). The lid is important for interactions with ubiquitin, whereas the base is required for unfolding the protein to allow it to enter the 20S core (22). The ATPase subunits play critical roles in substrate engagement, unfolding, translocation, and opening the gate of the core complex (34–37). Several groups have demonstrated the potential for targeting the 19S adhesion-regulating molecule 1 (ADRM1/hRPN13) protein in bortezomib resistance (38–44), leading to the development of the putative ADRM1/hRPN13 inhibitor, RA190 (45–48). Intriguingly, data from our lab and others demonstrated that certain members of the 19S lid might serve as better molecular targets for proteasome inhibition (24, 25, 38, 49–51). Indeed, our research has shown that knockdown of 26S proteasome non-ATPases 1 (PSMD1/hRPN2) and 3 (PSMD3/hRPN3), two members of the 19S regulatory complex (17), impaired survival and induced apoptosis of myeloid leukemia cells but not normal cord blood CD34⁺ hematopoietic stem and progenitor cells (25, 49), implying they may be good molecular targets for proteasome inhibition in human diseases. However, while the PSMD subunits of the 19S proteasome are non-ATPases that do not contain catalytically active target sites, the PSMC subunits are ATPases that could be targeted more efficiently with small molecule inhibitors. Therefore, we hypothesized that ATPase members of the 19S proteasome would serve as biomarkers and putative therapeutic targets in acute myeloid leukemia and several different solid tumors. Using data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC), expression of the ATPase PSMC subunits of the 19S proteasome was elevated in several cancers compared with normal controls, which often correlated with worse overall survival. Altogether, our data suggest that components of the 19S proteasome could serve as prognostic biomarkers and novel therapeutic targets in multiple human malignancies.

The ubiquitin-proteasome system (UPS) is a multi-protein complex that plays a critical role in regulating protein homeostasis, apoptosis, and a number of other cellular processes. Cancers can have abnormally increased proteasome activity due to the high metabolic demands for cancer growth that require rapid protein turnover. Therefore, the UPS has become a spotlight in research as a therapeutic target for various cancers. Standard proteasome inhibitors bind to and inhibit the 20S core complex of the UPS (27). However, proteasome inhibitors like bortezomib are prone to resistance mechanisms, highlighting the need for alternative therapeutic strategies (29–31). One strategy to overcome proteasome inhibitor resistance is to target the 19S regulatory complex instead of the 20S core complex. Indeed, knockdown of the 19S PSMD3 subunit significantly impaired the stability of human epidermal growth factor 2 (HER2) in breast cancer, which induced apoptosis and inhibited the growth, proliferation, and colony formation of tumor cells. Furthermore, a high level of PSMD3 mRNA expression was associated with shorter overall survival in breast cancer, especially in HER2+ patients (51). Similarly, our data demonstrated that PSMD3 expression is highly upregulated in chronic myeloid leukemia (CML) patients who have progressed from the chronic to the rapidly fatal blast phase of the disease (49). In marked contrast, however, patients with AML demonstrated reduced expression of PSMD3 mRNA compared with normal specimens (25). Despite this observation, AML patients with higher than average levels of PSMD3 expression had worse outcomes, especially for patients with FLT3 mutations (25). Importantly, PSMD3 knockdown impaired survival and induced apoptosis of CML and AML cells, but not normal cord blood progenitors (25, 49), indicating it may be a good potential target for cancer therapy.

The PSMD subunits of the 19S proteasome are non-ATPases that harbor no catalytically active target sites, which may make them poor cancer drug targets. On the other hand, the PSMC subunits of the 19S proteasome are ATPases that harbor catalytically active target sites. Indeed, proton pump inhibitors, also known as H+/K+ ATPase modulators, have attracted much attention for their clinical implication in gastric acid-related diseases (62, 63), whereas vacuolar ATPase inhibitors are being studied as a potential treatment for therapy-resistant cancers (64). Therefore, we hypothesized that ATPase members of the 19S proteasome would serve as biomarkers of disease progression and possible therapeutic targets in AML and multiple solid tumors, and this was indeed what we observed. Similar to our observations for PSMD3 (25), mRNA encoding PSMC2, PSMC3, and PSMC4 were surprisingly downregulated in AML versus normal mononuclear cells (Figure 2). We speculate that low proteasome subunit expression may explain the suboptimal response of AML patients to proteasome inhibition in certain clinical trials (65). Despite the reduced subunit expression, AML patients with high levels of PSMC2, PSMC3, PSMC4, and PSMC5 expression had a worse overall survival compared with patients demonstrating low-to-medium levels of expression (Figure 2). Interestingly, PSMC1, PSMC2, and PSMC5 mRNA expression were significantly higher in AML patients with mutated versus wild-type FLT3 (Figure 3), which correlated with worse outcomes in the case of PSMC2 and PSMC5 (Figure 4). In the case of solid tumors, all six PSMC subunits were upregulated at the mRNA and/or protein level in multiple different cancers, which in some cases correlated with worse outcomes (Figures 5–7). DNA mutations, deep deletions, or amplifications may explain the differences in PSMC gene expression observed in certain types of cancers.

As mentioned previously, high expression of all six ATPase subunits of the 19S proteasome correlated with worse overall survival in multiple human malignancies. However, the expression of PSMC4 appeared to have the greatest effect, correlating with reduced overall survival in 8/24 TCGA tumors (Figure 6). The first evidence for an oncogenic role of PSMC4 was in prostate cancer. In that study, expression of the genes encoding PSMC4, PSMB5, and the E3 ubiquitin ligase NEDD4L, were significantly upregulated in prostate cancer cells compared with the corresponding adjacent normal prostate tissue (66). PSMC4 was also shown to facilitate interactions between the proteasome and the endoplasmic reticulum, allowing for cellular ubiquitination of specific mitochondrial proteins (67). Interestingly, this association was shown to favor the resistance of breast cancer cells to anthracycline treatments (68). Additionally, the proteasome is dynamically phosphorylated during the cell cycle at threonine 25 of the 19S PSMC4 subunit, which contributes to cell proliferation and tumorigenesis (37). Through a kinome-wide screen, they identified dual-specificity tyrosine-regulated kinase 2 (DYRK2) as the primary kinase that phosphorylates PSMC4, leading to enhanced peptide translocation and degradation (37). Genome editing or small-molecule mediated inhibition of DYRK2 significantly bypassed bortezomib resistance in both multiple myeloma and breast cancer cells (69). PSMC4 is also considered a biomarker in endometrial cancer (70–72), breast cancer (60), hepatocellular carcinoma (73), oral squamous cell carcinoma (74), and laryngeal carcinoma (75). Combined with our data in the present study, PSMC4 may be a novel biomarker and therapeutic target for drug-resistant cancer patients demonstrating high levels of PSMC4 expression.

Consistent with our findings, higher proteasome activity has been detected in colon cancer tissue compared with the surrounding normal tissue (76). Furthermore, PSMC2-6 were shown to be more highly expressed at the mRNA and protein level in breast cancer compared with normal breast tissue, which again correlated with worse outcomes (60). Proteasome inhibitors like bortezomib, carfilzomib, or ixazomib have been very effective in the treatment of multiple myeloma, but resistance to therapy is still a major obstacle (13–15, 27, 30). These drugs target the PSMB5 subunit of the 20S proteasome, and mutations in PSMB5 can confer drug resistance in in vitro models (31). However, an analysis of 1,500 multiple myeloma patients revealed that these mutations were rare in the clinic (31). A recent meta-analysis study investigated the potential role of PSMC subunits in multiple myeloma and the correlation of mRNA expression with proteasome inhibitor resistance. This analysis included approximately 2000 newly diagnosed and advanced multiple myeloma patients, demonstrating 36 single nucleotide variants in the ATP/ADP binding pockets, which affected the conformation of individual subunits and the structure of the proteasome as a whole. For instance, an acquired mutation of PSMC2 at Y429S after bortezomib therapy correlated with relapse, thereby confirming an association of PSMC2 with proteasome inhibitor resistance (61). Another study identified 19S proteasome subunits as key determinants of resistance to proteasome inhibitors. This study performed a bortezomib screening on the KBM7 human myeloid leukemia cell line, revealing that PSMC2-6 served as key players in proteasome inhibitor resistance, and that downregulation of PSMC5 increased proteasome inhibitor resistance (77). Combined with our data, the PSMC subunits of the 19S proteasome could be novel prognostic biomarkers and putative therapeutic targets in AML and multiple types of solid tumors. These novel cancer drug targets are worthy of future investigation in the fields of cancer therapy and drug resistance.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

BT: data curation, investigation, methodology, writing-original draft, writing-review and editing, visualization, and formal analysis. JA: data curation, investigation, methodology, writing-original draft, writing-review and editing, visualization, and formal analysis. MG and IO: data curation, investigation, visualization, supervision, writing-original draft, and writing-review and editing. JS, MK, VV, EM, DT, AR, CJ, and DE: writing-review and editing. AE: conceptualization, methodology, formal analysis, data curation, supervision, validation, writing-original draft, and writing-review and editing. All authors contributed to the article and approved the submitted version.

This work was funded by the Elsa U. Pardee Foundation (AE), the Scholarly Activity and Research Program (SARP) at Paul L. Foster School of Medicine, and the Department of Molecular and Translational Medicine at Texas Tech University Health Sciences Center at El Paso.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.