Is 26S proteasome non-ATPase regulatory subunit 6 a potential molecular target for intrahepatic cholangiocarcinoma?

Abstract

In this editorial we comment on the article by Tang et al published in the recent issue of World Journal of Hepatology. Drug therapy of intrahepatic cholangiocarcinoma (iCCA) poses an enormous challenge since only a small proportion of patients demonstrate beneficial responses to therapeutic agents. Thus, there has been a sustained search for novel molecular targets for iCCA. The study by Tang et al evaluated the role of 26S proteasome non-ATPase regulatory subunit 6 (PSMD6), a 19S regulatory subunit of the proteasome, in human iCCA cells and specimens. The authors employed clustered regularly interspaced short palindromic repeat (CRISPR) knockout screening technology integrated with the computational CERES algorithm, and analyzed the human protein atlas (THPA) database and tissue microarrays. The results show that PSMD6 is a gene essential for the proliferation of 17 iCCA cell lines, and PSMD6 protein was overexpressed in iCCA tissues without a significant correlation with the clinicopathological parameters. The authors conclude that PSMD6 may play a promoting role in iCCA. The major limitations and defects of this study are the lack of detailed information of CRISPR knockout screening, in vivo experiments, and a discussion of plausible mechanistic cues, which, therefore, dampen the significance of the results. Further studies are required to verify PSMD6 as a molecular target for developing novel therapeutics for iCCA. In addition, the editorial article summarizes the latest advances in molecular targeted drugs and recently emerging immunotherapy in the clinical management of iCCA, development of proteasome inhibitors for cancer therapy, and advantages of CRISPR screening technology, computational methods, and THPA database as experimental tools for fighting cancer. We hope that these comments may provide some clues for those engaged in the field of basic and clinical research into iCCA.

Key Words: Cholangiocarcinoma; 26S proteasome non-ATPase regulatory subunit 6; Molecular targeted therapies; Proteasome inhibitors; Clustered regularly interspaced short palindromic repeat

Core Tip: Drug therapy of intrahepatic cholangiocarcinoma (iCCA) poses a big challenge and seeking potential molecular targets is being actively pursued. The study published by Tang et al indicated that 26S proteasome non-ATPase regulatory subunit 6 (PSMD6) gene is essential for cell proliferation and PSMD6 protein was overexpressed in iCCA tissues, indicating that PSMD6 may play a promoting role in iCCA. However, this study has several limitations and defects, such as the lack of detailed information of CRISPR knockout screening, in vivo experiments, and exploration of plausible mechanisms, reducing the significance of the results. Further studies are required to verify the role of PSMD6 as a molecular target for iCCA.

INTRODUCTION

The incidence of cholangiocarcinoma (CCA) originating from the epithelial cells lining the bile duct system is rising globally[1]. Distinct from extrahepatic CCA, which has a relatively higher curative opportunity, intrahepatic CCA (iCCA) is characterized by a very low resectable rate and an extremely poor prognosis[2]. This devastating cancer is the second most common primary liver cancer after hepatocellular carcinoma and accounts for about 3% of gastrointestinal malignancies[2]. Even after radical resection, recurrence and metastasis are common in iCCA, which is exceedingly resistant to therapeutic drugs because of its high heterogeneity, heavy mutation burden, and redundancy of noisy signaling pathways[3,4]. Such a dilemma highlights an urgent need to seek novel molecular targets for combating iCCA.

In the observational study by Tang et al[5], the authors employed clustered regularly interspaced short palindromic repeat (CRISPR) knockout screening technology and the computational CERES algorithm to evaluate the role of 26S proteasome subunit non-ATPase 6 (PSMD6) gene in 17 iCCA cell lines, which all had gene effect scores less than -1, indicating that this gene is essential for cell proliferation based on previously established criteria[6,7]. They next utilized the human protein atlas (THPA) database and tissue microarrays containing 42 pairs of iCCA and adjacent non-cancerous specimens to examine PSMD6 protein, which showed marked overexpression in iCCA. However, the expression level of PSMD6 protein was not significantly correlated with clinicopathological parameters including age, gender, pathological grade, and tumor-node-metastasis stage. The authors conclude that PSMD6 may play a promoting role for iCCA.

CURRENT PROGRESS IN CLINICAL DRUG THERAPIES FOR CCA

Despite some therapeutic progress, the proportion of iCCA patients surviving 5 years after diagnosis remains disturbingly low. The standard first-line chemotherapeutic drugs for advanced CCA are gemcitabine and cisplatin, and when the disease becomes refractory, second-line drugs including oxaliplatin, fluorouracil, or capecitabine are used in combination with gemcitabine. However, these regimens exhibit limited survival benefits despite the substantial side effects[8].

The increasing recognition of targetable genetic alterations has ushered a new era of molecular targeted therapies for CCA. However, the use of molecularly driven agents is limited to a small subgroup of CCA patients. Among this class of drugs, pemigatinib was the first approved agent for previously treated, unresectable locally advanced or metastatic CCA with gene fusion or rearrangement of fibroblast growth factor receptor (FGFR) 2 reported in 2020[9], followed by infigratinib (a specific FGFR1-3 kinase inhibitor)[10] and ivosidenib for CCA with mutant isocitrate dehydrogenase 1[11] in 2021. However, the United States Food and Drug Administration (FDA) announced the withdrawal of the approval of infigratinib for treating CCA in May of 2024. The cruel reality indicates the twists and turns for developing effective drugs in the campaign to fight CCA. In addition, zanidatamab, a human epidermal growth factor receptor 2 (HER2)-targeted bispecific antibody, and futibatinib, an oral highly selective irreversible small-molecule inhibitor of FGFR1-4, have received a breakthrough therapy designation for the treatment of CCA, although they have not yet been formally approved by the FDA[10].

The recently emerging immunotherapy, mainly immune checkpoint inhibitors, has also been applied in the clinical management of CCA. The FDA approved durvalumab targeting programmed death-ligand 1 in September of 2022, and pembrolizumab blocking programmed death-1 in October of 2023, in combination with gemcitabine and cisplatin, for the treatment of locally advanced or metastatic CCA[12,13]. However, the long-term efficacy remains to be demonstrated notwithstanding the expensive costs.

DEVELOPMENT OF PROTEASOME INHIBITORS FOR CANCER THERAPY AND POTENTIAL ROLE OF PSMD6 IN CCA

The 26S proteasome is a pivotal component of the ubiquitin-proteasome system since it catalyzes over 80% of protein degradation in growing mammalian cells, thus playing a crucial role in cellular proteostasis[14]. The 26S proteasome is composed of the 20S catalytic core particle and one or two 19S regulatory particles. The 19S regulatory particles are formed by a base, which contains six ATPases associated with diverse cellular activities subunits (PSMC1-PSMC6) and three non-ATPases (PSMD1, PSMD2, and ADRM1), and a lid, which consists of nine subunits [PSMD3, 6, 7, 8, 11, 12, 13, 14, and SHFM1 (split hand/foot malformation type 1)][15]. A compromised complex assembly or a dysfunctional proteasome is associated with the underlying pathophysiology of diseases including cancer and has been exploited as drug targets for therapeutic interventions[16]. Proteasome inhibition mainly focuses on the 20S core, which can act alone to cause the degradation of ubiquitin-independent proteins[17]. Several proteasome inhibitors targeting the 20S core, such as bortezomib, carfilzomib, and ixazomib, have been developed and used for clinical treatment of multiple myeloma and mantle cell lymphoma[18]. By inhibiting proteasome function, these drugs display anti-cancer activities by suppressing the NF-κB pathway, activating the MAPK pathway, stabilizing p53, reducing degradation of pro-apoptotic proteins, inducing proteotoxic crisis, and triggering endoplasmic reticulum stress[18].

PSMD6 (also known as Rpn7) is one of the lid subunits in the 19S regulatory particles of the 26S proteasome. The role of PSMD6 in human diseases has been investigated in a limited number of studies, which found that PSMD6 is associated with Parkinson’s disease[19,20], Alzheimer’s disease[21], and type 2 diabetes[22,23]. PSMD6 was demonstrated to be one of six genes in a cuproptosis prognostic signature for esophageal squamous cell carcinoma by using bioinformatics analysis[24]. In another study, PSMD6 was shown to serve as a potential prognostic and diagnostic biomarker for early-stage pancreatic ductal adenocarcinoma (PDCA), and the gene set enrichment analysis further revealed that PSMD6 was involved in various biological processes and signaling pathways, such as p53, cyclin-dependent kinase inhibitor 2A (CDKN2A), Myc, and KRAS (Kristen rat sarcoma viral oncogene homolog)[25]. However, the role of PSMD6 has not been confirmed in either clinical analysis or cell assays or animal experiments in the two studies[24,25]. Depletion of a single subunit of the 19S regulatory particles, including PSMD1, PSMD6, and PSMD11, impaired the viability of human triple-negative breast cancer MDA-MB-231 cells, and human colon cancer Colo321 and HCT116 cells in vitro, but the results have not been validated in vivo[26]. PSMD1 depletion inhibited the growth of MDA-MB-231 and OVCAR8 (from human high-grade ovary adenocarcinoma) cells in cell culture and animal models, and suppressed patient-derived ovarian cancer xenografts[27]. This study by Tang et al[5] may be, to our knowledge, the first that demonstrates the significance of PSMD6 in CCA. Although the underlying mechanisms have not been explored in this study, it is conjectured that PSMD6 may participate in regulating cell proliferation, apoptosis, metabolism, and invasiveness since PSMD6 is involved in the signaling pathways of p53, CDKN2A, Myc, and KRAS in PDCA[25]. In addition, it has been demonstrated that suppression of 26S proteasomes in cancer cells activates the unfolded protein response and caspase-3[26], and modulates the cell cycle[28] and metabolic regulation[29].

However, the potential of PSMD6 as a molecular target for developing effective therapeutics is still weak. In particular, the limitations and defects of this study as discussed below reduce the significance of the results. At present, no inhibitors directed towards the 19S regulatory subunits have been reported. Besides, opposite results have been reported, where reducing expression of individual 19S regulatory subunits including PSMD6 increased the level of active 20S proteasome and protected cancer cells from proteasome inhibitor-induced toxicity[30], devaluing the potential of PSMD6 inhibition as a therapeutic strategy for cancer treatment. Thus, further studies are required to verify the role of PSMD6 in iCCA.

SCREENING AND VALIDATING MOLECULAR TARGETS BY USING CRISPR TECHNOLOGY, COMPUTATIONAL METHODS, AND THPA DATABASE

The study by Tang et al[5] utilized CRISPR screening technology integrated with the computational CERES algorithm to elucidate the role of the PSMD6 gene in iCCA cells. Over the past decades, the CRISPR technology has emerged as a powerful tool for performing large-scale and loss-of-function screens to identify novel molecular targets because of its simplicity, flexibility, and high efficiency[31]. This system has revolutionized gene editing both at single genes and in multiplexed loss-of-function screens, thus enabling precise genome-scale identification of genes essential for the proliferation and survival of cancer cells[32]. CERES is a computational method for deducing gene essentiality from CRISPR screens in cancer cells to minimize false positive results by correcting the copy number effect because the estimated sgRNA activity as a gene-independent anti-proliferative effect of Cas9-mediated DNA cleavage confounds the measurement of genetic dependency and leads to false positive results in copy number of amplified regions[32]. THPA is a public database aiming to map all the human proteins using an integration of various “omics” technologies and constitutes a valuable tool for researchers studying protein localization and expression in human tissues[33]. The authors adopted the images and data from the Tissue Section on the protein expression profiles in human tissues[34] and the Pathology Section containing information on protein expression from 17 forms of human cancer[35] in the THPA database, enabling a precise comparison of the expression levels of PSMD6 protein between iCCA and normal bile duct epithelial tissues. The exploitation of THPA public resources and CRISPR screening technologies are the strengths of this study.

LIMITATIONS AND DEFECTS OF THIS STUDY

However, this study[5] has obvious limitations and flaws, thus reducing its impact. First, the authors have not provided detailed information of the CRISPR knockout screening technology, such as the resource of 17 iCCA cell lines, specifics of cell culture media and condition, gene delivery system, CRISPR knockout pooled library, sequences of the primers, and cell proliferation assay. All the above procedures largely determine experimental outcomes. For example, even the choice of culture media can influence the physiological relevance of findings from cell culture experiments due to the nutrient compositions and concentrations[36]. Second, the promoting function of PSMD6 in the proliferation of iCCA cells in vitro has not been validated in in vivo experiments. Third, this study has not explored any mechanisms underlying the role of PSMD6. Finally, it incorrectly cites a published report, in which silencing PSMC6 but not PSMD6 by small interference RNAs suppressed the growth of lung adenocarcinoma cells[37].

CONCLUSION

Due to a lack of effective therapeutic drugs for treating iCCA, over the past decades efforts to seek potential molecular targets to fight this notorious cancer have never stopped. This study has identified PSMD6 as a potential gene for iCCA by using CRISPR knockout screening technology, and validated it at the protein level by analyzing the THPA database and tissue microarrays[5]. The authors conclude that PSMD6 may be a promoting factor for iCCA though the expression levels of PSMD6 protein were not significantly correlated with clinicopathological parameters. This study has exploited public resources and modern technologies in attempts to evaluate PSMD6 as a potential molecular target for iCCA, but the significance of this study is not strong because of weaknesses mentioned above. As a 19S regulatory subunit, the role of PSMD6 in the proteasome system is not as decisive as the 20S core, which can act alone to cause ubiquitin-independent protein degradation[16,17]. However, the authors’ attempt is worthy of encouragement as they harnessed public resources and modern technologies in identifying precise molecular targets in this cancer type. Clues on PSMD6 in iCCA may provide an alternative avenue to enhance the effects of proteasome inhibition in fighting cancer since agents targeting the 19S regulatory subunit bind to an alternative site on the proteasome and may be useful in overcoming resistance to proteasome inhibitors directed towards the 20S core[18]. Therefore, it is worthwhile validating the role of PSMD6 in iCCA by employing appropriate animal models, exploring the mechanisms for its action, and developing specific inhibitors in future studies. On the other hand, this study examined the expression of PSMD6 in tissue microarrays containing only 42 specimens. Increasing the number of specimens and identifying subpopulation of iCCA patients with higher PSMD6 expression may provide a clearer understanding of its correlation with clinicopathological parameters.

ACKNOWLEDGEMENTS

We thank Dr. Shiva Reddy (University of Auckland, New Zealand) for revising the manuscript.