Findings suggest that several factors contribute to antiandrogen resistance, including genetic alterations, lineage plasticity,1,2 chromatin remodeling,3 and ectopic mutagenesis.4 However, the most prevalent mechanism is the restoration of androgen receptor (AR) signaling, through AR gene amplification, AR mutations, and AR splice variants. Importantly, dysregulation of ubiquitination-based protein degradation could lead to the accumulation of cancer-promoting proteins and therapy resistance in various cancers. Despite its importance, the molecular function of ubiquitination-driven AR degradation in prostate cancer is not yet fully understood.
The ubiquitin-proteasome system plays a vital role in maintaining cellular health by degrading unnecessary or harmful proteins. Within this system, the coordinated efforts of E1 (activating), E2 (conjugating), and E3 (ligating) enzymes are essential. The E2 enzyme UBE2J1, located in the endoplasmic reticulum (ER), is particularly crucial. UBE2J1’s malfunction plays a key role in cancer, affecting DNA repair, cell growth, and immune response.5 It helps break down proteins, influencing cell cycle control and blood vessel formation. UBE2J1 is also involved in degrading proteins associated with the endoplasmic reticulum and regulates immune responses against RNA virus infections.6 Despite its importance, the specific ways in which UBE2J1 contributes to cancer progression and resistance to treatments, especially in prostate cancer and against antiandrogen therapies, are not fully understood.
Our study identifies UBE2J1 as the primary E2 enzyme responsible for AR ubiquitination in PCa.7 The loss of UBE2J1 in PCa patients leads to impaired AR degradation, resulting in AR protein accumulation and the development of resistance. Utilizing a ubiquitination-based AR degrader can restore AR ubiquitination, thereby decreasing proliferation of antiandrogen-resistant PCa tumors. These valuable findings from this study enlighten the mechanism through which PCa maintains increased AR protein levels, developing resistance to antiandrogen therapies.
Figure 1. UBE2J1 is frequently deleted in prostate cancer patients.
To understand the role of UBE2J1, we knockout (KO) UBE2J1 in two antiandrogens sensitive human PCa cell models. We found that UBE2J1-KO cells had a remarkable growth advantage in the presence of enzalutamide compared to wild type cells. This change in cell growth was diminished in the absence of enzalutamide treatment, indicating that the growth benefit conferred by UBE2J1-KO tends to become of crucial importance when original AR signaling proves to be insufficient for the survival of PCa cells when influenced by antiandrogens. To gain more evidence for this hypothesis, we evaluated the role of UBE2J1 in vivo using an LNCaP/AR xenograft model. The UBE2J1-KO cells exhibited increased growth under both castration and enzalutamide treatments. We also observed increased Ki67 staining in UBE2J1-KO cells, suggesting that loss of this gene in tumors can overcome the proliferation inhibition prompted by antiandrogens, thus supporting the hypothesis that UBE2J1-KO leads to resistance to antiandrogens in AR-dependent PCa.
It was crucial to recognize the molecular mechanism facilitating antiandrogen resistance due to UBE2J1-loss. Therefore, we conducted RNA-Seq analysis on the vehicle-treated condition to identify changes in transcriptomics, rather than those brought by drug-mediated AR suppression. It was found that UBE2J1-loss resulted in an enrichment in AR signaling pathway, which was then confirmed by conducting qPCR on UBE2J1-KO cells under androgen deprivation and enzalutamide treatment. To profoundly understand the mechanism behind the enhanced AR signaling, we showed that AR protein levels were significantly higher in UBE2J1-KO cells, indicating an accumulation of AR proteins. Immunofluorescence staining (IF) also revealed higher levels of AR proteins and AR target genes like NKX3.1 in UBE2J1-KO cells treated with enzalutamide. Equivalent results were seen in xenografted tumors from castrated mice treated with enzalutamide. Further AR ChIP-qPCR analysis revealed that in androgen-deprivation conditions with dihydrotestosterone (DHT) treatment, UBE2J1-KO cells exhibited increased AR binding at AR sites, unlike vehicle-treated cells. This indicates that UBE2J1 loss leads to androgen resistance by elevating AR protein levels and reinstating AR signaling in treated prostate cancer cells.
To explore deeper into the molecular mechanism behind this, we hypothesized that UBE2J1 loss impedes ubiquitination-dependent AR degradation, causing AR accumulation and signaling restoration. Testing this, we treated wildtype and UBE2J1-KO cells with cycloheximide (CHX) to halt protein synthesis and measure protein half-life. Western blot results from various time points showed a prolonged AR half-life in UBE2J1-KO cells, implying slower AR degradation. This was further confirmed by the fact that proteasomal degradation inhibition through epoxomicin treatment eliminated AR level differences between UBE2J1-KO and wild-type cells. Co-immunoprecipitations (co-IP) and western blots demonstrated that AR interacts with UBE2J1 and UBE2J1 loss reducing K48-linked ubiquitination of AR. We also performed co-immunoprecipitations (co-IP) and western blots and found that i) AR and UBE2J1 do interact with each other, ii) UBE2J1 loss diminished the K48-linked ubiquitination of AR.
We also performed patient data analysis to evaluate the impact of UBE2J1-loss in various clinical settings. Between the two metastatic Castration Resistant Prostate Cancer (mCRPC) patient cohorts: the Stand Up to Cancer (SU2C) and Alumkal 2020 cohorts, we noticed that patients with UBE2J1-loss developed resistance to antiandrogen faster than patients with wildtype UBE2J1. To validate these findings, we used patient-derived explants (PDE) and noticed that UBE2J1 levels tend to decrease upon treatment with enzalutamide. AR degrader was used to diminish AR protein levels, which did not affect the UBE2J1 protein levels. Immunohistochemistry (IHC) staining of malignant and matched benign prostate tumor samples showed a remarkable reduction of UBE2J1 and an increase in AR and AR target NK3.1 protein.
Figure 2. Patients with UBE2J1 deletion and low expression correlated with worse survival.
To see if restoring AR degradation could reverse enzalutamide resistance and decrease proliferation of PCa cells, we treated wildtype and UBE2J1-KO LNCaP/AR cells with AR degraders and evaluated the growth of these cells. Through cell proliferation and FACS-based competition assays, we noticed a complete reversal in growth advantage of UBE2J1-KO cells over wild-type cells, thus providing evidence that restoration of AR degradation could be a strategy to overcome resistance. We also showed a decrease in growth using AR degraders on patient-derived organoids that exhibit low levels of UBE2J1 and are highly resistant to enzalutamide.
Figure 3. UBE2J1 works as the E2 ubiquitin-conjugating enzyme for AR ubiquitination in PCa. Loss of UBE2J1 in PCa leads to antiandrogen resistance by increasing AR accumulation and Inhibition of AR using AR degrader could rescue this resistance.
In conclusion, cumulatively these results show that UBE2J1-loss confers antiandrogen resistance by impairing AR degradation, leading to AR accumulation. By restoring AR degradation, we can overcome enzalutamide resistance in PCa, thereby laying the groundwork for future clinical studies and trials.
Written by: Xiaoling Li,1 Atreyi Mukherji,1 Ping Mu1,2,3
- Department of Molecular Biology, University of Southwestern Medical Center, Dallas, Texas, USA
- Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center, Dallas, USA
- Hamon Center for Regenerative Science and Medicine, UT Southwestern Medical Center, Dallas, USA
- Mu, P.; Zhang, Z.; Benelli, M.; Karthaus, W. R.; Hoover, E.; Chen, C.-C.; Wongvipat, J.; Ku, S.-Y.; Gao, D.; Cao, Z.; et al. SOX2 promotes lineage plasticity and antiandrogen resistance in TP53- and RB1-deficient prostate cancer. Science 2017, 355 (6320), 84-88. DOI: 10.1126/science.aah4307.
- Deng, S.; Wang, C.; Wang, Y.; Xu, Y.; Li, X.; Johnson, N. A.; Mukherji, A.; Lo, U. G.; Xu, L.; Gonzalez, J.; et al. Ectopic JAK-STAT activation enables the transition to a stem-like and multilineage state conferring AR-targeted therapy resistance. Nat Cancer 2022, 3 (9), 1071-1087. DOI: 10.1038/s43018-022-00431-9 From NLM Medline.
- Zhang, Z.; Zhou, C.; Li, X.; Barnes, S. D.; Deng, S.; Hoover, E.; Chen, C. C.; Lee, Y. S.; Zhang, Y.; Wang, C.; et al. Loss of CHD1 Promotes Heterogeneous Mechanisms of Resistance to AR-Targeted Therapy via Chromatin Dysregulation. Cancer Cell 2020, 37 (4), 584-598 e511. DOI: 10.1016/j.ccell.2020.03.001.
- Li, X.; Wang, Y.; Deng, S.; Zhu, G.; Wang, C.; Johnson, N. A.; Zhang, Z.; Tirado, C. R.; Xu, Y.; Metang, L. A.; et al. Loss of SYNCRIP unleashes APOBEC-driven mutagenesis, tumor heterogeneity, and AR-targeted therapy resistance in prostate cancer. Cancer Cell 2023, 41 (8), 1427-1449 e1412. DOI: 10.1016/j.ccell.2023.06.010 From NLM Medline.
- Burr, M. L.; Cano, F.; Svobodova, S.; Boyle, L. H.; Boname, J. M.; Lehner, P. J. HRD1 and UBE2J1 target misfolded MHC class I heavy chains for endoplasmic reticulum-associated degradation. Proc Natl Acad Sci U S A 2011, 108 (5), 2034-2039. DOI: 10.1073/pnas.1016229108 From NLM Medline.
- Feng, T.; Deng, L.; Lu, X.; Pan, W.; Wu, Q.; Dai, J. Ubiquitin-conjugating enzyme UBE2J1 negatively modulates interferon pathway and promotes RNA virus infection. Virol J 2018, 15 (1), 132. DOI: 10.1186/s12985-018-1040-5 From NLM Medline.
- Rodriguez Tirado, C.; Wang, C.; Li, X.; Deng, S.; Gonzalez, J.; Johnson, N. A.; Xu, Y.; Metang, L. A.; Sundar Rajan, M.; Yang, Y.; et al. UBE2J1 is the E2 ubiquitin-conjugating enzyme regulating androgen receptor degradation and antiandrogen resistance. Oncogene 2023. DOI: 10.1038/s41388-023-02890-5.
National Institutes of Health; Department of Defense; Cancer Prevention Research Institute of Texas (CPRIT); Prostate Cancer Foundation; Welch Foundation; UTSW Deborah and W.A. Tex Moncrief, Jr. Scholar in Medical Research Award; UTSW Harold C. Simmons Cancer Center Award.
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