
==== Front
Cell Commun Signal
Cell Commun Signal
Cell Communication and Signaling : CCS
1478-811X
BioMed Central London

1805
10.1186/s12964-024-01805-5
Research
NDR1 mediates PD-L1 deubiquitination to promote prostate cancer immune escape via USP10
Fu Meiling 1
Li Jinxin 2
Xuan Zuodong 1
Zheng Zeyuan 1
Liu Yankuo 1
Zhang Zeyi 1
Zheng Jianzhong 1
Zhong Min 1
Liu Bin 1
Du Yifan 1
Zhang Lei Drzhanglei@xmu.edu.cn

3
Sun Huimin hmsun@xah.xmu.edu.cn

14
1 https://ror.org/00mcjh785 grid.12955.3a 0000 0001 2264 7233 Department of Urology, Xiang’an Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, 361101 China
2 https://ror.org/02bjs0p66 grid.411525.6 0000 0004 0369 1599 Department of Urology, Changhai Hospital, Naval Medical University (Second Military Medical University), Shanghai, China
3 https://ror.org/00mcjh785 grid.12955.3a 0000 0001 2264 7233 School of Public Health, Xiamen University, Xiamen, 361101 China
4 https://ror.org/00mcjh785 grid.12955.3a 0000 0001 2264 7233 Central Laboratory, School of Medicine, Xiang’an Hospital of Xiamen University, Xiamen University, Xiamen, 361101 China
3 9 2024
3 9 2024
2024
22 42912 6 2024
22 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Prostate cancer (PCa) is one of the most common male genitourinary system malignancies. Despite the significant benefits of anti-PD-L1 immune checkpoint inhibitor therapy in other cancers, the reasons for its poor therapeutic efficacy in prostate cancer (PCa) remain unclear.NDR1 plays an important role in innate immunity, but its role in tumor immunity and immunotherapy has not been investigated. The role of NDR1 in the immune microenvironment of PCa and the related mechanisms are unknown. Here, we found a positive correlation between NDR1 and PD-L1 expression in PCa. NDR1 significantly inhibits CD8 + T cell infiltration and function, thereby promoting immune escape in prostate cancer.More importantly, NDR1 inhibition significantly enhanced CD8 + T cell activation, which enhanced the therapeutic effect of anti-PD-L1. Mechanistic studies revealed that NDR1 inhibits ubiquitination-mediated PD-L1 degradation via the deubiquitinase USP10, upregulates PD-L1, and promotes PCa immune escape. Thus, our study suggests a unique PD-L1 regulatory mechanism underlying PCa immunotherapy failure. The significance of NDR1 in PCa immune escape and its mechanism of action were clarified, and combined NDR1/PD-L1 inhibition was suggested as an approach to boost PCa immunotherapy effectiveness.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12964-024-01805-5.

Keywords

Prostate cancer
NDR1/STK38, PD-L1
Immune escape
Immune checkpoint Blockade
Anti-PD-1 therapy
issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
==== Body
pmcIntroduction

Prostate cancer (PCa) ranks as a frequently occurring malignant tumor within the male genitourinary system, holding the position of the second most prevalent cancer among men [1]. Surgery, radiotherapy and androgen deprivation therapy (ADT) are the mainstays of conventional PCa treatment [2], with ADT serving as the primary therapy for advanced PCa [3]. Prostate cancer patients ultimately failed ADT treatment due to drug resistance and developed metastatic castration resistant prostate cancer (mCRPC) [4]. Moreover, in vivo research suggests that ADT therapy may increase side effects such as neuroendocrine differentiation (NED) and PCa cell invasion [5].

In recent years, immunotherapy, particularly immune checkpoint inhibitors (ICIs) like anti PD-1/PD-L1, has shown promise in treating various solid tumors [6], including lung [7] and kidney cancer [8]. However, using anti PD-1/PD-L1 alone hasn't yielded satisfactory results in prostate cancer treatment [9]. PCa is a "cold tumor" that is mainly characterized by poor T-cell infiltration, low tumor mutation load and immunosuppressive tumor microenvironment, so immunotherapy is not effective in PCa [10]. In addition recent insights in cancer immunotherapy suggest that PD-L1 expression levels in cancer cells may influence the response to anti-PD-L1 therapy [11]. Investigating PD-L1 regulation in prostate cancer may uncover why immunotherapy isn't effective and could be crucial in finding complementary treatments to enhance its response.

NDR1, part of the AGC kinase family, is also known as serine-threonine protein kinase 38 (STK38) [12]. As a part of the Hippo signaling system, NDR1 can work in tandem with NDR2, LATS1, and LATS2 [13] and is primarily engaged in cellular mitosis, embryonic development, and centrosomal replication [14]. The role of NDR1 in cancers is complicated. Previous studies have shown that NDR1 can be used as the proto-oncogene of tumors to promote the occurrence and development of tumors, including breast cancer [15] and in small cell lung cancer [16], etc. Some studies have also shown that NDR1 can be used as the tumor suppressor gene to inhibit the progress of tumors, including gastric cancer [17], skin cancer [18], etc. Especially in prostate cancer, there have been reports of both cancer suppression [19] and cancer promotion [20]. NDR1 is necessary for thymocyte export and T-cell migration, according to certain research, and in vivo tests have revealed that NDR1 exert a vital role in the innate immune response [21]. Liu et al. found through bioinformatics research that NDR1 is closely related to cancer immunity [22]. However, the significance of NDR1 in the immunological microenvironment of PCa and the related underlying mechanisms are unclear.

In our research, we confirmed that NDR1 increases PD-L1 expression and inhibits T cell infiltration, promoting immune escape in prostate cancer. Crucially, inhibiting NDR1 significantly improves the effectiveness of anti-PD-L1 therapy, offering a fresh approach to enhance PCa immunotherapy. This study aimed to unveil NDR1's role in regulating PD-L1 and its contribution to immune evasion in PCa, while investigating combination therapy options to enhance immunotherapy response.

Methods and materials

Cell line and cell culture

All cells, including prostate cancer cells (PC3, 22rv1, and DU145), murine prostate cancer RM-1 cells, and HEK293T, were obtained from the ATCC.

All cells were cultured in complete medium (1640 medium [Gibco, China], then supplemented with 10% heat-inactivated fetal bovine serum [Gibco, China]), 1% penicillin/streptomycin (Macklin, China) were routinely cultured. All cells applied in this study were cultured at 37 °C in a humidified atmosphere of 5% CO2.

Plasmids and transfection

Transfer plasmids or siRNAs to the destination according to the requirements of the transfection reagent.

Plasmids were purchased from Sino Biological: pCMV3-STK38-Myc (HG12319-NM), pCMV3-PD-L1-His (HG10084-CH), pCMV3-USP10-HA (HG17833-CY).

Western blot assay

Place cells in a lysate containing proteasome inhibitors for lysis and obtain a protein solution by denaturing the lysate. After electrophoresis, membrane transfer, room temperature sealing for 1 h, overnight incubation of the corresponding first antibody, and room temperature incubation of the second antibody for 1 h, the obtained protein solution can be imaged through chemiluminescence.

Immunoprecipitation

For Co-IP determination, cell lysis uses IP specific lysis solution (Thermo Fisher Scientific, MA, USA), while others are similar to regular cell lysis. Divide the final obtained protein solution into two parts, one part is directly added to the loading buffer for boiling denaturation as the input group, and the other part is antibodies according to the requirements of the magnetic bead (Bimake, USA) instruction manual. For continuous IP samples, anti HA antibodies and magnetic beads are added to the first Anti Myc IP sample for a second IP.

Protein half-life assay

After applying cycloheximide (CHX, 100ug/ml) to the culture medium, cells were collected at 4.8.12.24 h for Western blot assay.

Ubiquitination analysis

After transfection of Myc-NDR1, HA-Ub, His-PD-L1 or HA-USP10, Myc-Ub, His-PD-L1 into HEK293T cells for 48 h. After 48 h of transfection, the cells were treated with MG132 (40 ng/ul) for 6 h, followed by immunoprecipitation with anti His antibodies.

LDH toxicity test and T-cell killing test

Co-culture PC3 cells transfected with plasmids (NDR1 and pCMV) in a 1:10 ratio with human peripheral blood monocytes (activated by co-stimulation with anti-CD3/CD28 magnetic beads T (ThermoFisher, USA)) for 48 h. The supernatants of the acquired cells were assayed for LDH content according to LDH kit (Beyotime Biotechnology, China) to detect the LDH content.

Establishment of mouse tumor model

All animal experiments received approval from the Ethics Committee of Xiamen University School of Medicine. We obtained 6–8-week-old male C57BL/6 and C57BL/6-nu mice from Xiamen University's Experimental Animal Center.

Inject tumor cells(RM-1 control cells and RM-1-shNDR1 or RM-1 NDR1 cells (1 × 106)) subcutaneously into mice and measure the tumor volume every 5 days. After the experiment is completed, tumors were obtained from mice and weighed for subsequent experiments.

For treating mice, we randomly divided them into four groups: control group, 17AAG (NDR1 inhibitor) treatment group, anti PDL1 treatment group and combined treatment group. Every three days, mice were treated with intraperitoneal injection of anti PD-L1 (dose 200ug/animal, Bio X Cell, USA) and 17AAG (dose 75 mg/kg, MedChemExpress, China) to simultaneously measure tumor volume and mouse weight. After five treatments, all mice were euthanized, and subsequent experiments were conducted.

Mouse tumor and human lymphocyte extraction

After digesting mouse tumor tissue with collagenase IV (C8160, Solarbio, China), tumor infiltrating lymphocytes were isolated from percoll cell separation solution(P8370, Solarbio, China).

Normal human peripheral blood was extracted according to Human Peripheral Blood Single Nucleated Cell Isolation Reagent (P8610, Solarbio, China).

RT-qPCR assay

According to the requirements of the kit, extract total RNA from cells (AC0202-A, SparkJake, China) and reverse transcription (RK20400, ABclonal, China) for RT-PCR.

Perform RT-PCR using complementary DNA and SYBRGreen II (RK21203, ABclonal, China). Primers sequences were in Table S1.

Antibodies

NDR1 (A-8) (sc-365555, Santa Cruz), PD-L1 (#13,684,cst), USP10 (#8501,cst), H3 (#4499,cst) His-tag (AE068, ABclonal), HA-tag (AE008, ABclonal), Myc-tag (AE010, ABclonal).CD25 (ab227834, Abcam). PD-L1 (2B11D11, proteintech). anti-human antibody(Biolegend): PE-CD3 (317,307), PE-PD-L1 antibody (329,705);anti-mouse antibody(Biolegend):FITC-CD45 (157,214), perCP- CD45 (103,129), APC-CD8 (126,614), BV421-CD69(124,529).

Statistical analysis

The data was statistically analyzed with GraphPad Prism 8 software from GraphPad Software Inc. in San Diego, CA, USA. Statistically significant results were represented as * (p < 0.05), ** (p < 0.01), or *** (p < 0.001), while 'ns' indicated no significant difference.

Results

NDR1 promotes tumor growth by suppressing the immune response

To investigate the effect of NDR1 on PCa, we first created cell lines with stable NDR1 knockdown and transient NDR1 overexpression with the mouse PCa cell line RM-1 as the parental cell line (Fig. 1A, B). Following that, control and NDR1 overexpression (RM-1-NDR1)/NDR1 knockdown (RM-1-shNDR1) cells were implanted subcutaneously into C57BL/6 mice and T-cell-deficient C57BL/6-nu nude mice to create subcutaneous transplantation tumor models respectively. The procedures used for tumor generation and detection are shown in Fig. 1C. The results showed that in C57BL/6 mice, the tumor weight and volume in the RM-1-NDR1 group were significantly higher than those in the control group (Fig. 1D-F), but the tumor weight and volume in the RM-1-shNDR1 group were significantly lower (Fig.S1A-C). The findings imply that NDR1 promotes the growth of PCa in immunocompetent C57BL/6 mice. However, the tumor weight and volume of the RM-1-NDR1 group in immunodeficient C57BL/6-nu nude mice were significantly lower than those of the control group (Fig. 1G-I). Contrary conclusions were observed in immunocompetent and immunocompromised mice. In immunocompromised mice,NDR1 may exert tumor suppressive effects, while in immunocompromised mice,NDR1 may promote the progression of prostate cancer. The close relationship between NDR1 and immunity may be used to partially explain the complexity of both pro cancer and anti cancer reports of NDR1 in prostate cancer.Fig. 1 NDR1 promotes tumor growth by suppressing the immune. A-B Stable cell lines with overexpression and knockdown of NDR1 was constructed in RM1 cell line. C The diagram illustrates the subcutaneous injection of either control RM-1 cells or RM-1 cells with silenced NDR1 into C57BL/6 mice. D-F Display representative images (D), tumor growth curve (E), and tumor weight (F) of control or RM-1 NDR1 cells injected into C57BL/6 mouse tumors. Measure the tumor at the designated time point and dissect the tumor at 25 days (n = 5). G-I Display representative images (G), tumor growth curve (H), and tumor weight (I) of control or RM-1 NDR1 cells injected into C57BL/6-nu mouse tumors. Measure the tumor at the designated time point and dissect the tumor at 15 days (n=5).

NDR1 suppresses immune response by inhibiting T cell response

To ascertain whether NDR1 suppresses the immune response to promote tumor growth. First, We conducted a pathway enrichment analysis using TCGA data, revealing an association between NDR1 and immunity (Fig. 2A). We then examined immune cell infiltration in PCa cells with altered NDR1 expression and observed a negative correlation between NDR1 and CD8 + T-cell infiltration into tumors (Fig. 2B). Therefore, we proposed that NDR1 inhibits T-cell responses to promote tumor growth. By performing flow analysis on the subcutaneous tumors of the C57BL/6 mice mentioned above (Fig. 1D and S1A), we found that the percentage of CD8 + T cell infiltration in subcutaneous tumors of mice injected with RM-1 NDR1 cells was significantly lower compared to the control group (Fig. 2C), while it was significantly higher in mice that had received injections of RM-1 ShNDR1 cells (Fig.S2A). Moreover, our immunohistochemical staining results were consistent with the flow cytometry results (Fig. 2E). CD69 is a CD8 + T-cell activation marker [23]. These results indicate a substantial decrease in the CD8 + T cell population expressing CD69 within the tumors of mice in the RM-1 NDR1 group as opposed to the control group (Fig. 2D). However, in subcutaneous tumors of mice injected with RM-1 ShNDR1 cells, there was a notable increase in the percentage of CD8 + T cells expressing CD69. (Fig.S2B). The important growth factor for T-cell activation, IL-2, is a crucial part of T cells [24]. Moreover, activated CD8 + T cells can generate IL-2 and TNF-α, which enhance the cytotoxicity of CD8 + T cells [25], so we detected IL-2 and TNF-α in tumor tissues by RT‒qPCR, and compared with the control group, The results showed that the expressions of IL-2 and TNF-α were decreased in the over-expression group(Fig.S2C). At the same time, we demonstrated once again with immunohistochemical staining that the overexpression of NDR1 resulted in a decrease in IL-2 expression (Fig. 2F). The above results indicate that NDR1 may inhibit CD8 + T-cell function and promote tumor immune escape by inhibiting CD8 + T-cell infiltration and activation in vivo.Fig. 2 NDR1 suppresses immune response by inhibiting T cell response. A Gene enrichment analysis of NDR1 in the KEGG database in PRAD. B NDR1 expression is associated with immune cell infiltration based on CIBERSORT. C-D Flow cytometry was used to analyze tumor-infiltrating lymphocytes (n = 5). E-F IHC assessed CD8 and CD25 expression in mouse tumor tissues (× 200 above; × 400 below), with a scale bar of 100 mm or 50 mm. G Schematic diagram showing co-culture of tumor cells and activated T cells. H Co-culture PC3 cells overexpressed with control or NDR1 with activated T cells, and detect the apoptosis of PC3 by flow cytometry. I Representative images and statistical analysis results of T cell killing test. J Statistical analysis results of LDH toxicity test

In addition, we co-cultured human PCa cells with anti-CD3/CD28 magnetic bead-activated T cells (see Fig. 2G for schematic) and the results showed that tumor cells overexpressing NDR1 reduced apoptosis (Fig. 2H), the cytotoxity of T cells on tumor cells was attenuated (Fig. 2I). Lactate dehydrogenase (LDH) is an enzyme that is stable in the cytoplasm, and normally exists only in the cell. When the cell membrane is harmed, it's swiftly released into the culture medium. Through the determination of the content of LDH in cell supernatant on the degree of damage can be judged cells [26]. We found that LDH levels in the supernatant were significantly lower when NDR1 was overexpressed than in the control group, indicating that the cytotoxicity of T cells against these cells was reduced (Fig. 2J). Due to our concern that the killing effect on tumor cells is caused by cytokines secreted by T cells in the supernatant, we co cultured the supernatant with tumor cells (Fig. S2D). The results showed that the cytokines in the supernatant had no killing effect on tumor cells (Fig. S2E-F), further confirming that NDR1 may promote tumor growth and immune escape by inhibiting T cell responses.

To summarize, these findings indicate that NDR1 inhibits cytotoxic T cells in both in vivo and in vitro settings, thereby significantly promoting PCa cell growth and immune evasion.

NDR1 is positively correlated with PD-L1 expression in PCa

To explore the relationship between NDR1 and PD-L1, we overexpressed NDR1 in PC3 cells, resulting in a corresponding increase in PD-L1 expression (Fig. 3A). Knocking down NDR1 in DU145 cells led to downregulation of PD-L1 (Fig. 3B). We used flow cytometry to detect PD-L1 expression on the cell membrane, and the results were consistent (Fig. 3C). Next, we utilized the TIMER2.0 database, the analysis demonstrated a positive correlation between NDR1 and PD-L1(Fig. 3D). Moreover, WB and IHC of tumor tissues from mice described above (Fig. 1D) showed that PD-L1 increased with the increase of NDR1 (Fig. 3E, F). Finally, we used samples from prostate cancer patients for further validation. The immunohistochemical staining results showed that as the tumor progressed, the expression level of NDR1 increased, and the expression level of PD-L1 also increased (Fig. 3G, H). In summary, our research findings further confirm the positive correlation between the expression of NDR1 and PD-L1 in prostate cancer.Fig. 3 NDR1 is positively correlated with PD-L1 expression in PCa. A In NDR1-overexpressing PC3 cells, we assessed NDR1 and PD-L1 expression using WB. B In NDR1 knockdown DU145 cells, we examined NDR1 and PD-L1 expression via WB. C Evaluation PD-L1 expression on the cell surface. D Analyzing the correlation between NDR1 and PD-L1 using TIMER2.0 database. E-F Detection the levels of NDR1 and PD-L1 in mouse tumor tissues via WB and IHC. G-H IHC staining and statistical results of NDR1 and PD-L1 in patients (n = 23)

NDR1 binds to PD-L1 and enhances PD-L1 stability

Next, we explored the mechanism of action of NDR1 and PD-L1. First, we investigated the interaction between NDR1 and PD-L1. The Co-IP results showed that NDR1 coprecipitated with PD-L1, and the same results were found for endogenous IP(Fig. 4A, B). Immunofluorescence experiments showed colocalization of NDR1 and PD-L1 in the spatial structure (Fig. 4C). The above results indicate that NDR1 can bind to PD-L1 in PCa cell lines. Since knockdown of NDR1 resulted in an opposite trend of PD-L1 at the mRNA level and protein level (Fig. 3A, B), we considered that it might be due to the effect of NDR1 on the stability of PD-L1. To test whether NDR1 regulates the stability of PD-L1 protein, we examined the expression level of PD-L1 in PCa cells treated with the protein synthesis inhibitor CHX. Overexpression of NDR1 in PC3 significantly increased the stability and half-life of PD-L1, whereas knockdown of NDR1 in DU145 cells significantly destabilized the PD-L1 protein (Fig. 4D, E). This indicates that NDR1 inhibited PD-L1 degradation and increased PD-L1 stability. The autophagy-lysosomal pathway and the ubiquitin‒proteasome pathway are the major pathways of protein degradation [27]. We treated DU145 cells with the ubiquitin‒proteasome pathway inhibitor MG132 and the autophagy‒lysosome pathway inhibitor chloroquine diphosphate (CQ) and found that MG132 could reverse PD-L1 regulation by NDR1 to some extent, but not after CQ treatment (Fig. 4F). Moreover, we found that overexpression of NDR1 can inhibit PD-L1 ubiquitination (Fig. 4G). In summary, the above results indicate that NDR1 binds to PD-L1 and enhances its stability by inhibiting the ubiquitination pathway.Fig. 4 NDR1 binds to PD-L1 and enhances PD-L1 stability. A Exogenous PD-L1 coimmunoprecipitated with NDR1 in 293 T cells. B NDR1 was coimmunoprecipitated with PD-L1 in PC3. C IF experiment to determine the spatial localization of NDR1 and PD-L1. D PC3/pCMV and PC3/NDR1 cells were subjected with CHX (100 μg/mL) or MG132 at specified intervals to detect PD-L1-degrading protein levels. E DU145/shmock and DU145/shNDR1 cells were subjected with CHX (100 μg/mL) or MG132 at specified intervals to detect PD-L1-degrading protein levels. F PD-L1 level in NDR1 knockdown cells subjected with MG132 and CQ by WB. G The impact of NDR1 on PD-L1 ubiquitination in 293 T cells was detected

USP10 binds to PD-L1 and mediates its deubiquitination

Promoting protein stability can be achieved by removing the ubiquitin pathway from proteins, which is achieved through deubiquitinases (DUBs) [28], namely deubiquitination. Similar to ubiquitination, deubiquitination mediated regulation requires specific DUB.We speculate that NDR1 affects the stability of PD-L1 by affecting its deubiquitinase. To identify the potential deubiquitinase of PD-L1, we intersected the mass spectrometry results of NDR1 with those of PD-L1. The highest-scoring deubiquitinase identified was USP10 (Fig. 5A). First, we checked whether USP10 and PD-L1 could bind. The Co-IP showed that USP10 coprecipitated with PD-L1, the same results were found for the endogenous IP experiment (Fig. 5B, C). Immunofluorescence experiments showed colocalization of USP10 and PD-L1 in the spatial structure (Fig. 5D). These results suggest that USP10 and PD-L1 bind in PCa.Fig. 5 USP10 binds to PD-L1 and mediates its deubiquitination. A Identification of the number of candidate proteins interacting with NDR1 and PD-L1 in PC3 cells through CO-IP-MS analysis. B Exogenous PD-L1 coimmunoprecipitated with USP10 in 293 T cells. C USP10 was coimmunoprecipitated with PD-L1 in PC3 cells. D IF experiment was conducted to determine the spatial localization of USP10 and PD-L1. E USP10 expression in prostate cancer cells. F Detection of PD-L1 expression level in PC3 cells after concentration gradient increase of USP10 by WB. G Detection of PD-L1 expression in DU145 cells with siUSP10 gene knockdown by WB. H PD-L1 expression levels in DU145 cells were assessed using a concentration gradient of the USP10 inhibitor (Spautin-1) via immunoblot analysis. I PC3/pCMV and PC3/USP10 cells were subjected with CHX (100 μg/mL) at specified intervals to detect PD-L1-degrading protein levels. J The impact of USP10 on PD-L1 ubiquitination in 293 T cells was detected

Then, we detect the expression of USP10 in several PCa cell line. As shown (Fig. 5E), USP10 was highly expressed in DU145 cells but expressed at low levels in PC3 and 22rv1 cells. Thus, we overexpressed USP10 in PC3 and 22rv1 cell lines, the expression level of PD-L1 increased with the increase of USP10 (Fig. 5F and S3A). Knockdown of USP10 using siRNA technology (Figure.S3B) or USP10 inhibitor (Spautin-1 [29]) in DU145 cells resulted in a decrease in PD-L1 expression level and a dose-dependent decrease (Fig. 5G, H and S3C). We examined the expression level of PD-L1 in PCa cells treated with the protein synthesis inhibitor CHX, and overexpression of USP10 prolonged the half-life of PD-L1 (Fig. 5I). A ubiquitination assay showed that USP10 in PC3 cells remarkably increased the stability and half-life of PD-L1(Fig. 5J). In brief, these findings indicate that USP10 binds to PD-L1, promoting its deubiquitination.

NDR1 promotes binding of USP10 and PD-L1 and inhibits PD-L1 ubiquitination degradation

Following this, we wanted to further explore whether NDR1 regulates PD-L1 through USP10. The Co-IP results showed that NDR1 coprecipitated with USP10, and the same results were found with endogenous IP experiments (Fig. 6A-B and S4A). Immunofluorescence experiments showed colocalization of USP10 and NDR1 in the spatial structure (Fig. 6C). The above results suggest that USP10 and NDR1 bind in PCa.Fig. 6 NDR1 promotes binding of USP10 and PD-L1 and inhibits PD-L1 ubiquitination degradation. A Exogenous NDR1 coimmunoprecipitated with USP10 in 293 T cells. B USP10 was coimmunoprecipitated with NDR1 in PC3 cells. C IF experiment revealed the spatial localization of USP10 and NDR1. D Sequential IP assay for NDR1-USP10-PD-L1 interaction. E-F NDR1 knockdown in DU145 cells was assessed for its impact on the protein and mRNA expression of NDR1 and USP10. G-H co-IP assays and IF analysis to explore the relationship between USP10 and PD-L1 in 293T cells, with or without NDR1 overexpression. I: Intranuclear USP10 and PD-L1 expression levels in PC3 cells following a concentration gradient increase of NDR1. J The ubiquitination of intranuclear PD-L1 was examined in 293T cell by WB. K The expression of USP10,NDR1and PD-L1 was detected in NDR1 knockdown or overexpressed DU145 cells with or without Transfected USP10

Since both USP10 and PD-L1 can interact with NDR1, Continuous IP analysis confirmed that NDR1, PDL1 and USP10 can form trimers (Fig. 6D). Subsequently, we wanted to explore whether NDR1 regulates PD-L1 by affecting USP10. We discovered that although NDR1 did not affect the RNA and protein levels of USP10 (Fig. 6E, F and S4B), immunoprecipitation experiments and immunofluorescence experiments showed that overexpression of NDR1 enhanced the interaction between USP10 and PD-L1 (Fig. 6G, H). Interestingly, we found that overexpression of NDR1 resulted in an increase in USP10 in the nucleus and an increase in PD-L1 (Fig. 6I), while an intranuclear ubiquitination assay showed that overexpression of USP10 inhibited the ubiquitination of PD-L1 (Fig. 6J). Research has indicated that nuclear PD-L1 can activate diverse immune response pathways, facilitating immune evasion [30, 31]. Knocking out NDR1 in DU145 cells resulted in a decrease in PD-L1 expression, but overexpressing USP10 resulted in an upregulation of PD-L1 expression. These results indicate that USP10 plays an important role in the regulation of PD-L1 by NDR1(Fig. 6K). Moreover, We examined PD-L1 expression in PCa cells treated with CHX. In NDR1 knockdown DU145 cells, PD-L1 degraded rapidly, but its degradation was slowed down when USP10 was overexpressed. (Fig. S6C). These results showed that NDR1 promotes the binding of USP10 and PD-L1, inhibits the ubiquitination degradation of PD-L1 and upregulated PD-L1.

Inhibiting NDR1 significantly enhances the effectiveness of anti-PD-L1 therapy for PCa

The above findings demonstrate that NDR1 increases PD-L1 expression in prostate cancer and suppresses T cell infiltration in the tumor microenvironment. To further confirm whether combining NDR1 inhibition with an anti-PD-L1 antibody can inhibit tumor growth and enhance T cell infiltration, we subcutaneously injected RM1 cells into mice and randomly divided them into four groups: control, NDR1 inhibitor (17AAG [32]), anti-PD-L1, and combination treatment (Fig. 7A). The experimental results revealed that the combination treatment group reduced tumor burden compared to the monotherapy groups (Fig. 7B-D). Moreover, there were no significant changes in the mice's body weight (Fig. 7E) and HE staining indicated no noticeable tissue damage or lesions in the liver and kidneys (Fig. 7F). This suggests that 17AAG and anti-PD-L1 are relatively safe in in vivo tests. Additionally, the combined treatment group exhibited increased infiltration of CD69 + CD8 + T cells (Fig. 7G). These findings imply that NDR1 inhibitors enhance the effectiveness of anti-PD-L1 therapy in prostate cancer by boosting CD8 + T cell activity.Fig. 7 Inhibiting NDR1 significantly enhances the effectiveness of anti-PD-L1 therapy for PCa. A The process of treatment strategies. B-D tumor weight (B),Tumor representation images (C) and tumor growth curves (D) (n = 3) in four groups of mice. E Weight of four groups of mice. F HE staining of liver and kidney tissues of four groups of mice. G The tumor infiltrated lymphocytes were analyzed by flow cytometry (n = 3)

Discussion

Tumor immune escape refers to the evasion of tumor cells from the recognition and attack of the immune system through various mechanisms. This promotes tumor growth, metastasis, and is a vital strategy for tumor survival and development [33]. Tumor cells evade immune recognition, in part due to the development of an immunosuppressive tumor microenvironment [34]. The PD-L1/PD-1 signaling pathway is a vital element of tumor immunosuppression [35]. Inhibiting T cell cytotoxicity allows tumors to escape, which is a major contributor to tumor immune evasion. Therefore, understanding the mechanism of PD-L1 expression regulation is of great significance for further improving the effectiveness of PD-1/PD-L1 targeted therapy.

However, PD-L1 expression has complex regulatory mechanisms, such as gene transcription [36] and posttranscriptional and posttranslational modifications [37]. Evidence suggests that PD-L1 protein expression is generally regulated by proteasomal degradation pathway [37–39]. In non-small cell lung cancer, OTUB1 acts as a ubiquitin enzyme for PD-L1, which boosts PD-L1 expression and facilitates immune evasion [40]. In OSCC, USP9x was discovered as a deubiquitinating enzyme for PD-L1 [41]. Moreover, CSN5 exerts a role as a deubiquitinating enzyme of PD-L1 in a variety of tumors, including hepatocellular carcinoma [42], colorectal cancer [43], and breast cancer [37]. In PCa, USP22 was shown to act as a deubiquitinating enzyme for PD-L1 [44]. However, it's essential to conduct further screening of deubiquitinating enzymes in PCa to delve deeper into the regulation of PD-L1 expression and advance tumor immunotherapy.

Ubiquitination and deubiquitination are reversible processes regulated by E3 ligases and deubiquitinating enzymes, and abnormalities in deubiquitinating enzymes are associated with poor disease prognosis [45]. USP10 is abundantly expressed in PCa and significantly correlates with a negative patient prognosis [46]. It has been shown that USP10 promotes AR signaling and stabilizes G3BP2 protein, inhibits p53 activity, and promotes PCa growth [46]. Significantly, Spautin-1, an inhibitor of USP10, hinders EGFR phosphorylation and its downstream signaling. In vivo experiments have demonstrated that Spautin-1, whether used alone or combined with enzalutamide, markedly enhances the anticancer effects in PCa [47]. A recent study illustrated that USP10 may promote tumor growth by affecting immunity, but the exact mechanism is still unclear [48].

Immune checkpoint inhibitors, a common form of tumor immunotherapy, combat tumors by inhibiting immune checkpoints and reactivating T cell responses against them [49]. For "cold tumors" such as PCa, T cell infiltration within the tumor is small, and immune checkpoint inhibitors have limited effects [50]. Transforming 'cold tumors' into 'hot tumors' is crucial to enhancing immunotherapy effectiveness for PCa. Studies indicate this can be achieved by boosting T-cell responses, like CAR-T therapy, or blocking inhibitory signals, such as MDSC depletion. However, potential side effects must not be overlooked [51]. Enhancing immune checkpoint expression and fostering effector T cell infiltration can also shift 'cold tumors' towards becoming 'hot tumors' [52]. In this study, we observed that NDR1 upregulated PD-L1 expression, while suppressing CD8 + T cell infiltration and activation. Inhibition of NDR1, conversely, enhanced effector T cell infiltration, potentially transforming PCa from a 'cold tumor' to a 'hot tumor' and improving anti-PD-L1 therapy efficacy.

Mechanistically, NDR1 increases the expression level of PD-L1 in PCa (Fig. 3) and promotes immune escape from the tumor.These findings fill a gap in the immunomodulatory role of NDR1 in prostate cancer. Next, we used mass spectrometry to screen and analyze USP10, the deubiquitinating enzyme of NDR1 that stabilizes PD-L1 (Fig. 5). Subsequent experiments demonstrated that NDR1 didn't impact USP10 expression but instead hindered PD-L1 degradation by enhancing the interaction between USP10 and PD-L1 (Fig. 6G). Simultaneously, we observed that NDR1 facilitates the nuclear translocation of USP10 (Fig. 6I), enhancing its nuclear presence. Nuclear USP10 acts as a deubiquitinating enzyme, preventing ubiquitination-mediated PD-L1 degradation (Fig. 6J) and thus elevating PD-L1 stability. These findings reveal the mechanism of a novel signaling axis, NDR1/USP10/PD-L1, in immune escape and provide new insights into how NDR1 promotes immune escape in PCa.

Conclusions

In summary, this study identified that NDR1 promotes PCa growth and immune escape, which aids in clarifying the mechanism of PCa development and filling the gap in the related research field. Deeper exploration of its regulatory mechanisms unveiled the significance of the NDR1/USP10/PD-L1 signaling axis in PCa immune evasion. Moreover, we observed that inhibiting NDR1 can enhance the in vivo effectiveness of anti-PD-L1 treatment.Thus, NDR1 inhibitors may become candidates in PCa immunotherapy in the future. These findings will guide the development of new strategies for PCa treatment and provide more effective treatments for patients.

Limitations of the study

However, our research on NDR1 in the immune microenvironment is only the tip of the iceberg, more research is needed to explore, such as using single-cell analysis to explore the entire immune microenvironment and exploring the mechanisms of other immune checkpoints (such as CTLA4). Although we found that NDR1 can stabilize PD-L1 through deubiquitination, its transcriptional level and specific action sites still need further investigation. Overall,our research indicates that NDR1 serves as a potential molecular target for prostate cancer.

Supplementary Information

Additional file 1: Fig. S1. NDR1 promotes tumor growth by suppressing the immune. C: Display representative images (A), tumor growth curve (B), and tumor weight (C) of C57BL/6 mouse tumors injected with control or RM-1 shNDR1 cells. Measure the tumor at the designated time point and dissect the tumor at 25 days (n=5).

Additional file 2: Fig. S2. NDR1 suppresses immune response by inhibiting T cell response. A-B: Flow cytometry was used to analyze tumor-infiltrating lymphocytes (n=5).C: Detection of IL-2 and TNF- α expression in tumor tissue through RT-Qpcr. D: Schematic diagram showing co-culture of tumor cells and incubate T cell supernatant. E: Representative images and statistical analysis results of T cell killing test. F: Statistical analysis results of LDH toxicity test.

Additional file 3: Fig. S3. USP10 binds to PD-L1 and mediates its deubiquitination. A: PD-L1 expression in 22rv1 cells with USP10 gene overexpressing by WB. B: The knockdown efficiency of siUSP10 in DU145 cells through WB. C: PD-L1 expression levels in DU145 cells using USP10 inhibitor(Spautin-1) by WB.

Additional file 4: Fig. S4. NDR1 promotes binding of USP10 and PD-L1 and inhibits PD-L1 ubiquitination degradation. A: Exogenous NDR1 coimmunoprecipitated with USP10 in 293T cells. B: USP10 expression were detected in NDR1 overexpressing PC3 cells by WB.C: Overexpressing USP10 was observed to mitigate the degradation of PD-L1 protein induced by NDR1 knockdown.

Acknowledgements

This work was supported by the Natural Science Foundation of China (No. 81972373) and Natural Science Foundation of Fujian Province of China (No. 2023D008). We also thank Figdraw (www.figdraw.com) for the assistance in creating Graphical abstract. The author thanks Professor Shao Chen from the Department of Urology, Xiang'an Hospital, Xiamen University for providing project guidance.

Authors’ contributions

H.S. and L.Z. initiated the project. M.F. designed, supervised all experiments, and edited the manuscript. J.L. and Z.X. assisted with animal experiments, while Y.B. and Z.Z. supported the WB experiments. Y.L., and M.Z. contributed to the bioinformatics analysis. B.L., Y.D., and S.X. participated in data interpretation and analysis. All authors have reviewed and approved the submitted manuscript.

Funding

This work was supported by the Natural Science Foundation of China (No. 81972373) and Natural Science Foundation of Fujian Province of China (No. 2023D008). We also thank Figdraw (www.figdraw.com) for the assistance in creating Graphical abstract.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All animal experiments were conducted in accordance with the protocol approved by the Experimental Animal Welfare and Ethics Committee of Xiamen University (No. XMULAC20200039).

Consent for publication

All authors agree for publication.

Competing interests

The authors declare no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Meiling Fu, Jinxin Li and Zuodong Xuan contributed equally to this work.
==== Refs
References

1. Sung H Ferlay J Siegel RL Laversanne M Soerjomataram I Jemal A Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries CA Cancer J Clin 2021 71 209 249 10.3322/caac.21660 33538338
Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71:209–49.33538338 10.3322/caac.21660
2. Hamdy FC Donovan JL Lane JA Metcalfe C Davis M Turner EL Fifteen-year outcomes after monitoring, surgery, or radiotherapy for prostate cancer N Engl J Med 2023 388 1547 1558 10.1056/NEJMoa2214122 36912538
Hamdy FC, Donovan JL, Lane JA, Metcalfe C, Davis M, Turner EL, et al. Fifteen-year outcomes after monitoring, surgery, or radiotherapy for prostate cancer. N Engl J Med. 2023;388:1547–58.36912538 10.1056/NEJMoa2214122
3. Chen K O'Brien J McVey A Jenjitranant P Kelly BD Kasivisvanathan V Combination treatment in metastatic prostate cancer: is the bar too high or have we fallen short? Nat Rev Urol 2023 20 116 123 10.1038/s41585-022-00669-z 36509970
Chen K, O’Brien J, McVey A, Jenjitranant P, Kelly BD, Kasivisvanathan V, et al. Combination treatment in metastatic prostate cancer: is the bar too high or have we fallen short? Nat Rev Urol. 2023;20:116–23.36509970 10.1038/s41585-022-00669-z
4. Bhoir S De Benedetti A Targeting prostate cancer, the 'Tousled Way' Int J Mol Sci 2023 24 11100 10.3390/ijms241311100 37446279
Bhoir S, De Benedetti A. Targeting prostate cancer, the “Tousled Way.” Int J Mol Sci. 2023;24:11100.37446279 10.3390/ijms241311100
5. Niu Y Guo C Wen S Tian J Luo J Wang K ADT with antiandrogens in prostate cancer induces adverse effect of increasing resistance, neuroendocrine differentiation and tumor metastasis Cancer Lett 2018 439 47 55 10.1016/j.canlet.2018.09.020 30227222
Niu Y, Guo C, Wen S, Tian J, Luo J, Wang K, et al. ADT with antiandrogens in prostate cancer induces adverse effect of increasing resistance, neuroendocrine differentiation and tumor metastasis. Cancer Lett. 2018;439:47–55.30227222 10.1016/j.canlet.2018.09.020
6. Liu Q Li J Zheng H Yang S Hua Y Huang N Adoptive cellular immunotherapy for solid neoplasms beyond CAR-T Mol Cancer 2023 22 28 10.1186/s12943-023-01735-9 36750830
Liu Q, Li J, Zheng H, Yang S, Hua Y, Huang N, et al. Adoptive cellular immunotherapy for solid neoplasms beyond CAR-T. Mol Cancer. 2023;22:28.36750830 10.1186/s12943-023-01735-9
7. Lahiri A Maji A Potdar PD Singh N Parikh P Bisht B Lung cancer immunotherapy: progress, pitfalls, and promises Mol Cancer 2023 22 40 10.1186/s12943-023-01740-y 36810079
Lahiri A, Maji A, Potdar PD, Singh N, Parikh P, Bisht B, et al. Lung cancer immunotherapy: progress, pitfalls, and promises. Mol Cancer. 2023;22:40.36810079 10.1186/s12943-023-01740-y
8. Bedke J Albiges L Capitanio U Giles RH Hora M Ljungberg B The 2022 updated european association of urology guidelines on the use of adjuvant immune checkpoint inhibitor therapy for renal cell carcinoma Eur Urol 2023 83 10 14 10.1016/j.eururo.2022.10.010 36511268
Bedke J, Albiges L, Capitanio U, Giles RH, Hora M, Ljungberg B, et al. The 2022 updated european association of urology guidelines on the use of adjuvant immune checkpoint inhibitor therapy for renal cell carcinoma. Eur Urol. 2023;83:10–4.36511268 10.1016/j.eururo.2022.10.010
9. Rebuzzi SE Rescigno P Catalano F Mollica V Vogl UM Marandino L Immune checkpoint inhibitors in advanced prostate cancer: current data and future perspectives Cancers (Basel) 2022 14 1245 10.3390/cancers14051245 35267553
Rebuzzi SE, Rescigno P, Catalano F, Mollica V, Vogl UM, Marandino L, et al. Immune checkpoint inhibitors in advanced prostate cancer: current data and future perspectives. Cancers (Basel). 2022;14:1245.35267553 10.3390/cancers14051245
10. Galon J Bruni D Approaches to treat immune hot, altered and cold tumours with combination immunotherapies Nat Rev Drug Discov 2019 18 197 218 10.1038/s41573-018-0007-y 30610226
Galon J, Bruni D. Approaches to treat immune hot, altered and cold tumours with combination immunotherapies. Nat Rev Drug Discov. 2019;18:197–218.30610226 10.1038/s41573-018-0007-y
11. Sun C Mezzadra R Schumacher TN Regulation and function of the PD-L1 heckpoint Immunity 2018 48 434 452 10.1016/j.immuni.2018.03.014 29562194
Sun C, Mezzadra R, Schumacher TN. Regulation and function of the PD-L1 heckpoint. Immunity. 2018;48:434–52.29562194 10.1016/j.immuni.2018.03.014
12. Santos PF Fazendeiro B Luca FC Ambrósio AF Léger H The NDR/LATS protein kinases in neurobiology: key regulators of cell proliferation, differentiation and migration in the ocular and central nervous system Eur J Cell Biol 2023 102 151333 10.1016/j.ejcb.2023.151333 37327741
Santos PF, Fazendeiro B, Luca FC, Ambrósio AF, Léger H. The NDR/LATS protein kinases in neurobiology: key regulators of cell proliferation, differentiation and migration in the ocular and central nervous system. Eur J Cell Biol. 2023;102:151333.37327741 10.1016/j.ejcb.2023.151333
13. Irie K Nagai T Mizuno K Furry protein suppresses nuclear localization of yes-associated protein (YAP) by activating NDR kinase and binding to YAP J Biol Chem 2020 295 3017 3028 10.1074/jbc.RA119.010783 31996378
Irie K, Nagai T, Mizuno K. Furry protein suppresses nuclear localization of yes-associated protein (YAP) by activating NDR kinase and binding to YAP. J Biol Chem. 2020;295:3017–28.31996378 10.1074/jbc.RA119.010783
14. Yan M Chu L Qin B Wang Z Liu X Jin C Regulation of NDR1 activity by PLK1 ensures proper spindle orientation in mitosis Sci Rep 2015 5 10449 10.1038/srep10449 26057687
Yan M, Chu L, Qin B, Wang Z, Liu X, Jin C, et al. Regulation of NDR1 activity by PLK1 ensures proper spindle orientation in mitosis. Sci Rep. 2015;5: 10449.26057687 10.1038/srep10449
15. Wang LL Wan XY Liu CQ Zheng FM NDR1 increases NOTCH1 signaling activity by impairing Fbw7 mediated NICD degradation to enhance breast cancer stem cell properties Mol Med 2022 28 49 10.1186/s10020-022-00480-x 35508987
Wang LL, Wan XY, Liu CQ, Zheng FM. NDR1 increases NOTCH1 signaling activity by impairing Fbw7 mediated NICD degradation to enhance breast cancer stem cell properties. Mol Med. 2022;28:49.35508987 10.1186/s10020-022-00480-x
16. Wang LL Wan XY Wang TL Liu CQ Zheng FM NDR1 activates CD47 transcription by increasing protein stability and nuclear location of ASCL1 to enhance cancer stem cell properties and evasion of phagocytosis in small cell lung cancer Med Oncol 2022 39 254 10.1007/s12032-022-01859-w 36224405
Wang LL, Wan XY, Wang TL, Liu CQ, Zheng FM. NDR1 activates CD47 transcription by increasing protein stability and nuclear location of ASCL1 to enhance cancer stem cell properties and evasion of phagocytosis in small cell lung cancer. Med Oncol. 2022;39:254.36224405 10.1007/s12032-022-01859-w
17. Cui DX Zhang L Yan XJ Zhang LX Xu JR Guo YH A microarray-based gastric carcinoma prewarning system World J Gastroenterol 2005 11 1273 1282 10.3748/wjg.v11.i9.1273 15761963
Cui DX, Zhang L, Yan XJ, Zhang LX, Xu JR, Guo YH, et al. A microarray-based gastric carcinoma prewarning system. World J Gastroenterol. 2005;11:1273–82.15761963 10.3748/wjg.v11.i9.1273
18. Hummerich L Müller R Hess J Kokocinski F Hahn M Fürstenberger G Identification of novel tumour-associated genes differentially expressed in the process of squamous cell cancer development Oncogene 2006 25 111 121 10.1038/sj.onc.1209016 16247483
Hummerich L, Müller R, Hess J, Kokocinski F, Hahn M, Fürstenberger G, et al. Identification of novel tumour-associated genes differentially expressed in the process of squamous cell cancer development. Oncogene. 2006;25:111–21.16247483 10.1038/sj.onc.1209016
19. Yue J Sun H Liu S Yu F Wang S Wang F Downregulation of NDR1 contributes to metastasis of prostate cancer cells via activating epithelial-mesenchymal transition Cancer Med 2018 7 3200 3212 10.1002/cam4.1532 29733518
Yue J, Sun H, Liu S, Yu F, Wang S, Wang F, et al. Downregulation of NDR1 contributes to metastasis of prostate cancer cells via activating epithelial-mesenchymal transition. Cancer Med. 2018;7:3200–12.29733518 10.1002/cam4.1532
20. Paul I Batth TS Iglesias-Gato D Al-Araimi A Al-Haddabi I Alkharusi A The ubiquitin ligase Cullin5(SOCS2) regulates NDR1/STK38 stability and NF-κB transactivation Sci Rep 2017 7 42800 10.1038/srep42800 28216640
Paul I, Batth TS, Iglesias-Gato D, Al-Araimi A, Al-Haddabi I, Alkharusi A, et al. The ubiquitin ligase Cullin5(SOCS2) regulates NDR1/STK38 stability and NF-κB transactivation. Sci Rep. 2017;7:42800.28216640 10.1038/srep42800
21. Tang F Gill J Ficht X Barthlott T Cornils H Schmitz-Rohmer D The kinases NDR1/2 act downstream of the Hippo homolog MST1 to mediate both egress of thymocytes from the thymus and lymphocyte motility Sci Signal 2015 8 ra100 10.1126/scisignal.aab2425 26443704
Tang F, Gill J, Ficht X, Barthlott T, Cornils H, Schmitz-Rohmer D, et al. The kinases NDR1/2 act downstream of the Hippo homolog MST1 to mediate both egress of thymocytes from the thymus and lymphocyte motility. Sci Signal. 2015;8:ra100.26443704 10.1126/scisignal.aab2425
22. Liu Y Shi Z Zheng Z Li J Yang K Xu C Prognostic and immunological role of STK38 across cancers: friend or foe? Int J Mol Sci 2022 23 11590 10.3390/ijms231911590 36232893
Liu Y, Shi Z, Zheng Z, Li J, Yang K, Xu C, et al. Prognostic and immunological role of STK38 across cancers: friend or foe? Int J Mol Sci. 2022;23:11590.36232893 10.3390/ijms231911590
23. Koyama-Nasu R Kimura MY Kiuchi M Aoki A Wang Y Mita Y CD69 imposes tumor-Specific CD8+ T-cell fate in tumor-draining lymph nodes Cancer Immunol Res 2023 11 1085 1099 10.1158/2326-6066.CIR-22-0406 37216576
Koyama-Nasu R, Kimura MY, Kiuchi M, Aoki A, Wang Y, Mita Y, et al. CD69 imposes tumor-Specific CD8+ T-cell fate in tumor-draining lymph nodes. Cancer Immunol Res. 2023;11:1085–99.37216576 10.1158/2326-6066.CIR-22-0406
24. Huang Y Jia A Wang Y Liu G CD8(+) T cell exhaustion in anti-tumour immunity: the new insights for cancer immunotherapy Immunology 2023 168 30 48 10.1111/imm.13588 36190809
Huang Y, Jia A, Wang Y, Liu G. CD8(+) T cell exhaustion in anti-tumour immunity: the new insights for cancer immunotherapy. Immunology. 2023;168:30–48.36190809 10.1111/imm.13588
25. Malik A Sayed AA Han P Tan MMH Watt E Constantinescu-Bercu A The role of CD8+ T-cell clones in immune thrombocytopenia Blood 2023 141 2417 2429 36749920
Malik A, Sayed AA, Han P, Tan MMH, Watt E, Constantinescu-Bercu A, et al. The role of CD8+ T-cell clones in immune thrombocytopenia. Blood. 2023;141:2417–29.36749920
26. Fotakis G Timbrell JA In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride Toxicol Lett 2006 160 171 177 10.1016/j.toxlet.2005.07.001 16111842
Fotakis G, Timbrell JA. In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicol Lett. 2006;160:171–7.16111842 10.1016/j.toxlet.2005.07.001
27. Rusilowicz-Jones EV Urbé S Clague MJ Protein degradation on the global scale Mol Cell 2022 82 1414 1423 10.1016/j.molcel.2022.02.027 35305310
Rusilowicz-Jones EV, Urbé S, Clague MJ. Protein degradation on the global scale. Mol Cell. 2022;82:1414–23.35305310 10.1016/j.molcel.2022.02.027
28. Chan WC Liu X Magin RS Girardi NM Ficarro SB Hu W Accelerating inhibitor discovery for deubiquitinating enzymes Nat Commun 2023 14 686 10.1038/s41467-023-36246-0 36754960
Chan WC, Liu X, Magin RS, Girardi NM, Ficarro SB, Hu W, et al. Accelerating inhibitor discovery for deubiquitinating enzymes. Nat Commun. 2023;14:686.36754960 10.1038/s41467-023-36246-0
29. Qiu W Xiao Z Yang Y Jiang L Song S Qi X USP10 deubiquitinates RUNX1 and promotes proneural-to-mesenchymal transition in glioblastoma Cell Death Dis 2023 14 207 10.1038/s41419-023-05734-y 36949071
Qiu W, Xiao Z, Yang Y, Jiang L, Song S, Qi X, et al. USP10 deubiquitinates RUNX1 and promotes proneural-to-mesenchymal transition in glioblastoma. Cell Death Dis. 2023;14:207.36949071 10.1038/s41419-023-05734-y
30. Gao Y Nihira NT Bu X Chu C Zhang J Kolodziejczyk A Acetylation-dependent regulation of PD-L1 nuclear translocation dictates the efficacy of anti-PD-1 immunotherapy Nat Cell Biol 2020 22 1064 1075 10.1038/s41556-020-0562-4 32839551
Gao Y, Nihira NT, Bu X, Chu C, Zhang J, Kolodziejczyk A, et al. Acetylation-dependent regulation of PD-L1 nuclear translocation dictates the efficacy of anti-PD-1 immunotherapy. Nat Cell Biol. 2020;22:1064–75.32839551 10.1038/s41556-020-0562-4
31. Xiong W Gao Y Wei W Zhang J Extracellular and nuclear PD-L1 in modulating cancer immunotherapy Trends Cancer 2021 7 837 846 10.1016/j.trecan.2021.03.003 33903073
Xiong W, Gao Y, Wei W, Zhang J. Extracellular and nuclear PD-L1 in modulating cancer immunotherapy. Trends Cancer. 2021;7:837–46.33903073 10.1016/j.trecan.2021.03.003
32. Enomoto A Fukasawa T Takamatsu N Ito M Morita A Hosoi Y The HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin modulates radiosensitivity by downregulating serine/threonine kinase 38 via Sp1 inhibition Eur J Cancer 2013 49 3547 3558 10.1016/j.ejca.2013.06.034 23886587
Enomoto A, Fukasawa T, Takamatsu N, Ito M, Morita A, Hosoi Y, et al. The HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin modulates radiosensitivity by downregulating serine/threonine kinase 38 via Sp1 inhibition. Eur J Cancer. 2013;49:3547–58.23886587 10.1016/j.ejca.2013.06.034
33. Damiris K Abbad H Pyrsopoulos N Cellular based treatment modalities for unresectable hepatocellular carcinoma World J Clin Oncol 2021 12 290 308 10.5306/wjco.v12.i5.290 34131562
Damiris K, Abbad H, Pyrsopoulos N. Cellular based treatment modalities for unresectable hepatocellular carcinoma. World J Clin Oncol. 2021;12:290–308.34131562 10.5306/wjco.v12.i5.290
34. Marin-Acevedo JA Kimbrough EO Lou Y Next generation of immune checkpoint inhibitors and beyond J Hematol Oncol 2021 14 45 10.1186/s13045-021-01056-8 33741032
Marin-Acevedo JA, Kimbrough EO, Lou Y. Next generation of immune checkpoint inhibitors and beyond. J Hematol Oncol. 2021;14:45.33741032 10.1186/s13045-021-01056-8
35. Jiang X Wang J Deng X Xiong F Ge J Xiang B Role of the tumor microenvironment in PD-L1/PD-1-mediated tumor immune escape Mol Cancer 2019 18 10 10.1186/s12943-018-0928-4 30646912
Jiang X, Wang J, Deng X, Xiong F, Ge J, Xiang B, et al. Role of the tumor microenvironment in PD-L1/PD-1-mediated tumor immune escape. Mol Cancer. 2019;18:10.30646912 10.1186/s12943-018-0928-4
36. Han C Fu YX β-Catenin regulates tumor-derived PD-L1 J Exp Med 2020 217 e20200684 10.1084/jem.20200684 32860048
Han C, Fu YX. β-Catenin regulates tumor-derived PD-L1. J Exp Med. 2020;217:e20200684.32860048 10.1084/jem.20200684
37. Lim SO Li CW Xia W Cha JH Chan LC Wu Y Deubiquitination and Stabilization of PD-L1 by CSN5 Cancer Cell 2016 30 925 939 10.1016/j.ccell.2016.10.010 27866850
Lim SO, Li CW, Xia W, Cha JH, Chan LC, Wu Y, et al. Deubiquitination and Stabilization of PD-L1 by CSN5. Cancer Cell. 2016;30:925–39.27866850 10.1016/j.ccell.2016.10.010
38. Mezzadra R Sun C Jae LT Gomez-Eerland R de Vries E Wu W Identification of CMTM6 and CMTM4 as PD-L1 protein regulators Nature 2017 549 106 110 10.1038/nature23669 28813410
Mezzadra R, Sun C, Jae LT, Gomez-Eerland R, de Vries E, Wu W, et al. Identification of CMTM6 and CMTM4 as PD-L1 protein regulators. Nature. 2017;549:106–10.28813410 10.1038/nature23669
39. Liu Y Liu X Zhang N Yin M Dong J Zeng Q Berberine diminishes cancer cell PD-L1 expression and facilitates antitumor immunity via inhibiting the deubiquitination activity of CSN5 Acta Pharm Sin B 2020 10 2299 2312 10.1016/j.apsb.2020.06.014 33354502
Liu Y, Liu X, Zhang N, Yin M, Dong J, Zeng Q, et al. Berberine diminishes cancer cell PD-L1 expression and facilitates antitumor immunity via inhibiting the deubiquitination activity of CSN5. Acta Pharm Sin B. 2020;10:2299–312.33354502 10.1016/j.apsb.2020.06.014
40. Liu Z Wang T She Y Wu K Gu S Li L N(6)-methyladenosine-modified circIGF2BP3 inhibits CD8(+) T-cell responses to facilitate tumor immune evasion by promoting the deubiquitination of PD-L1 in non-small cell lung cancer Mol Cancer 2021 20 105 10.1186/s12943-021-01398-4 34416901
Liu Z, Wang T, She Y, Wu K, Gu S, Li L, et al. N(6)-methyladenosine-modified circIGF2BP3 inhibits CD8(+) T-cell responses to facilitate tumor immune evasion by promoting the deubiquitination of PD-L1 in non-small cell lung cancer. Mol Cancer. 2021;20:105.34416901 10.1186/s12943-021-01398-4
41. Jingjing W Wenzheng G Donghua W Guangyu H Aiping Z Wenjuan W Deubiquitination and stabilization of programmed cell death ligand 1 by ubiquitin-specific peptidase 9, X-linked in oral squamous cell carcinoma Cancer Med 2018 7 4004 4011 10.1002/cam4.1675 29992764
Jingjing W, Wenzheng G, Donghua W, Guangyu H, Aiping Z, Wenjuan W. Deubiquitination and stabilization of programmed cell death ligand 1 by ubiquitin-specific peptidase 9, X-linked in oral squamous cell carcinoma. Cancer Med. 2018;7:4004–11.29992764 10.1002/cam4.1675
42. Chen J Lin Z Liu L Zhang R Geng Y Fan M GOLM1 exacerbates CD8(+) T cell suppression in hepatocellular carcinoma by promoting exosomal PD-L1 transport into tumor-associated macrophages Signal Transduct Target Ther 2021 6 397 10.1038/s41392-021-00784-0 34795203
Chen J, Lin Z, Liu L, Zhang R, Geng Y, Fan M, et al. GOLM1 exacerbates CD8(+) T cell suppression in hepatocellular carcinoma by promoting exosomal PD-L1 transport into tumor-associated macrophages. Signal Transduct Target Ther. 2021;6:397.34795203 10.1038/s41392-021-00784-0
43. Liu C Yao Z Wang J Zhang W Yang Y Zhang Y Macrophage-derived CCL5 facilitates immune escape of colorectal cancer cells via the p65/STAT3-CSN5-PD-L1 pathway Cell Death Differ 2020 27 1765 1781 10.1038/s41418-019-0460-0 31802034
Liu C, Yao Z, Wang J, Zhang W, Yang Y, Zhang Y, et al. Macrophage-derived CCL5 facilitates immune escape of colorectal cancer cells via the p65/STAT3-CSN5-PD-L1 pathway. Cell Death Differ. 2020;27:1765–81.31802034 10.1038/s41418-019-0460-0
44. McCann JJ Vasilevskaya IA Poudel Neupane N Shafi AA McNair C Dylgjeri E USP22 Functions as an oncogenic driver in prostate cancer by regulating cell proliferation and DNA epair Cancer Res 2020 80 430 443 10.1158/0008-5472.CAN-19-1033 31740444
McCann JJ, Vasilevskaya IA, Poudel Neupane N, Shafi AA, McNair C, Dylgjeri E, et al. USP22 Functions as an oncogenic driver in prostate cancer by regulating cell proliferation and DNA epair. Cancer Res. 2020;80:430–43.31740444 10.1158/0008-5472.CAN-19-1033
45. Antao AM Tyagi A Kim KS Ramakrishna S Advances in Deubiquitinating enzyme inhibition and applications in cancer therapeutics Cancers (Basel) 2020 12 1579 10.3390/cancers12061579 32549302
Antao AM, Tyagi A, Kim KS, Ramakrishna S. Advances in Deubiquitinating enzyme inhibition and applications in cancer therapeutics. Cancers (Basel). 2020;12:1579.32549302 10.3390/cancers12061579
46. Takayama KI Suzuki T Fujimura T Takahashi S Inoue S Association of USP10 with G3BP2 inhibits p53 signaling and contributes to poor outcome in prostate cancer Mol Cancer Res 2018 16 846 856 10.1158/1541-7786.MCR-17-0471 29378906
Takayama KI, Suzuki T, Fujimura T, Takahashi S, Inoue S. Association of USP10 with G3BP2 inhibits p53 signaling and contributes to poor outcome in prostate cancer. Mol Cancer Res. 2018;16:846–56.29378906 10.1158/1541-7786.MCR-17-0471
47. Liao Y Guo Z Xia X Liu Y Huang C Jiang L Inhibition of EGFR signaling with Spautin-1 represents a novel therapeutics for prostate cancer J Exp Clin Cancer Res 2019 38 157 10.1186/s13046-019-1165-4 30975171
Liao Y, Guo Z, Xia X, Liu Y, Huang C, Jiang L, et al. Inhibition of EGFR signaling with Spautin-1 represents a novel therapeutics for prostate cancer. J Exp Clin Cancer Res. 2019;38:157.30975171 10.1186/s13046-019-1165-4
48. Gao D Zhang Z Xu R He Z Li F Hu Y The prognostic value and immune infiltration of USP10 in pan-cancer: a potential therapeutic target Front Oncol 2022 12 829705 10.3389/fonc.2022.829705 35433424
Gao D, Zhang Z, Xu R, He Z, Li F, Hu Y, et al. The prognostic value and immune infiltration of USP10 in pan-cancer: a potential therapeutic target. Front Oncol. 2022;12:829705.35433424 10.3389/fonc.2022.829705
49. Sharma P Goswami S Raychaudhuri D Siddiqui BA Singh P Nagarajan A Immune checkpoint therapy-current perspectives and future directions Cell 2023 186 1652 1669 10.1016/j.cell.2023.03.006 37059068
Sharma P, Goswami S, Raychaudhuri D, Siddiqui BA, Singh P, Nagarajan A, et al. Immune checkpoint therapy-current perspectives and future directions. Cell. 2023;186:1652–69.37059068 10.1016/j.cell.2023.03.006
50. Benoit A Vogin G Duhem C Berchem G Janji B Lighting up the fire in the microenvironment of cold tumors: a major challenge to improve cancer immunotherapy Cells 2023 12 1787 10.3390/cells12131787 37443821
Benoit A, Vogin G, Duhem C, Berchem G, Janji B. Lighting up the fire in the microenvironment of cold tumors: a major challenge to improve cancer immunotherapy. Cells. 2023;12:1787.37443821 10.3390/cells12131787
51. Whiteside TL Demaria S Rodriguez-Ruiz ME Zarour HM Melero I Emerging opportunities and challenges in cancer immunotherapy Clin Cancer Res 2016 22 1845 1855 10.1158/1078-0432.CCR-16-0049 27084738
Whiteside TL, Demaria S, Rodriguez-Ruiz ME, Zarour HM, Melero I. Emerging opportunities and challenges in cancer immunotherapy. Clin Cancer Res. 2016;22:1845–55.27084738 10.1158/1078-0432.CCR-16-0049
52. Xiong W Gao X Zhang T Jiang B Hu MM Bu X USP8 inhibition reshapes an inflamed tumor microenvironment that potentiates the immunotherapy Nat Commun 2022 13 1700 10.1038/s41467-022-29401-6 35361799
Xiong W, Gao X, Zhang T, Jiang B, Hu MM, Bu X, et al. USP8 inhibition reshapes an inflamed tumor microenvironment that potentiates the immunotherapy. Nat Commun. 2022;13:1700.35361799 10.1038/s41467-022-29401-6
