TrkA promotes MDM2-mediated AGPS ubiquitination and degradation to trigger prostate cancer progression

Zhang, Yu; Huang, Zhenlin; Li, Keqiang; **e, Guoqing; Feng, Yuankang; Wang, Zihao; Li, Ningyang; Liu, Ruoyang; Ding, Yinghui; Wang, Jun; Yang, **jian; Jia, Zhankui

doi:10.1186/s13046-023-02920-w

TrkA promotes MDM2-mediated AGPS ubiquitination and degradation to trigger prostate cancer progression

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Published: 11 January 2024

Volume 43, article number 16, (2024)
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TrkA promotes MDM2-mediated AGPS ubiquitination and degradation to trigger prostate cancer progression

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Yu Zhang^1,2,4^na1,
Zhenlin Huang¹^na1,
Keqiang Li^1,4^na1,
Guoqing **e^1,4,
Yuankang Feng¹,
Zihao Wang¹,
Ningyang Li¹,
Ruoyang Liu¹,
Yinghui Ding³,
Jun Wang¹,
**jian Yang¹ &
…
Zhankui Jia¹

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Abstract

Background

As a novel necrosis manner, ferroptosis has been increasingly reported to play a role in tumor progression and treatment, however, the specific mechanisms underlying its development in prostate cancer remain unclear. Growing evidence showed that peroxisome plays a key role in ferroptosis. Herein, we identified a novel mechanism for the involvement of ferroptosis in prostate cancer progression, which may provide a new strategy for clinical treatment of prostate cancer.

Methods

Label-Free Mass spectrometry was used to screen and identify candidate proteins after ferroptosis inducer-ML210 treatment. Immunohistochemistry was undertaken to explore the protein expression of AGPS in prostate cancer tissues compared with normal tissues. Co-immunoprecipitation and GST pull-down were used to identify the directly binding of AGPS to MDM2 in vivo and in vitro. CCK8 assay and colony formation assay were used to illustrate the key role of AGPS in the progression of prostate cancer in vitro. The xenograft model was established to verify the key role of AGPS in the progression of prostate cancer in vivo.

Results

AGPS protein expression was downregulated in prostate cancer tissues compared with normal tissues from the first affiliated hospital of Zhengzhou University dataset. Lower expression was correlated with poorer overall survival of patients compared to those with high expression of AGPS. In addition, AGPS can promote ferroptosis by modulating the function of peroxisome-resulting in the lower survival of prostate cancer cells. Furthermore, it was shown that AGPS can be ubiquitinated and degraded by the E3 ligase-MDM2 through the proteasomal pathway. Meanwhile, kinase TrkA can promote the combination of AGPS and MDM2 by phosphorylating AGPS at Y451 site. It was verified that kinase TrkA inhibitor—Larotrectinib can increase the susceptibility of prostate cancer cells to ferroptosis, which leads to the inhibition of prostate cancer proliferation to a great extent in vitro and in vivo.

Conclusion

Based on these findings, we proposed the combination of ferroptosis inducer and TrkA inhibitor to synergistically exert anti-tumor effects, which may provide a new strategy for the clinical treatment of prostate cancer.

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Introduction

Prostate cancer (PCa) is the most common malignancy in men, which has the second highest fatality rate among male tumors and accounts for 20% of annually diagnosed cancer [1]. In recent years, with aging and changes in dietary habits, the incidence of PCa has increased rapidly [2,3,4]. Androgen deprivation therapy (ADT) has become the standard of care for patients with advanced PCa [5, 6]. However, PCa cells gradually become insensitive to ADT treatment and eventually develop into castration-resistant PCa (CRPC) [7], and the current radiotherapy or drug treatment of CRPC is ineffective and non-specific [8]. Therefore, the exploration of new potential therapeutic targets for PCa treatment is particularly important.

Ferroptosis is a unique iron-dependent and non-apoptotic form of necrosis due to lipid peroxidation. Several studies have reported that ferroptosis is involved in various degenerative diseases and tumors [9, 10]. Recent studies have displayed that peroxisome plays an important role in the occurrence of ferroptosis via lipid peroxidation by promoting the production of PUFAs and inhibiting the reduction of PUFAs [11, 12]. The discovery of the ferroptosis peroxisome-dependent pathway gradually revealed the importance of ether phospholipids (ePLs) in ferroptosis [Full size image

The TCGA data indicated that AGPS was significantly downregulated in PCa tissues (Supplementary Fig. 1b) and the expression differences were more pronounced at higher T stages (Supplementary Fig. 1c). And we also found the different expression of AGPS in different prostate cancer cell lines from the CCLE database (https://sites.broadinstitute.org/ccle/) (Supplementary Fig. 1d). But the mRNA expression seems to be no significantly different in different Gleason scores (Supplementary Fig. 1e). The comparison of 20 pairs of PCa tumor and normal tissues demonstrated that AGPS mRNA expression was not very significantly different between the tumor tissues and normal tissues (Supplementary Fig. 1f), which coordinated with the results in the cell lines (Fig. 1g). Then, we detected the protein levels of AGPS between prostate tissues and the normal tissues, result suggested that AGPS protein expression decreased in prostate cancer compared with the normal tissues (Fig. 1e). Additionally, we examined the protein expression of AGPS in different prostate cancer cell lines by Western blot (Fig. 1f), which lead to the same results.

Subsequently, we selected 89 pairs of PCa tumor and normal tissues for IHC staining and found that the positive rate of AGPS was significantly lower in PCa tumors than in normal tissues (Fig. 1g and h). We then performed a prognostic analysis based on the expression of AGPS in the 89 pairs of cases collected and found a significant association between AGPS and poor prognosis in PCa patients (Fig. 1i, j and k). These results suggest that AGPS is downregulated in PCa tissues and cells. Aditionally, AGPS is associated with better prognosis in PCa patients.

AGPS inhibits the proliferation of prostate cancer cells by promoting the formation of peroxisome and ferroptosis

In order to investigate whether AGPS acts through the ferroptosis in prostate cancer, we conducted colony formation experiments as an initial approach. Specifically, we employed overexpression of AGPS in 22Rv1 cells, which exhibit relatively low AGPS expression levels. Subsequently, we treated the cells with a combination of cellular autophagy inhibitor (CQ,25 μM), ferroptosis inhibitor (Ferrostatin-1,1 μM), and apoptosis inhibitor (Z-VAD-FMK,40 μM), commencing drug administration on the third day. On the seventh day, we assessed the clonal growth of the cells and found that the addition of the ferroptosis inhibitor alone resulted in a significant restoration of cell growth (Supplementary Fig. 2a and b). To investigate the specific role of AGPS in PCa through ferroptosis, we selected three cell lines (i.e., DU145, 22Rv1 and PC-3) for further investigations. DU145 and PC-3 reported relatively higher AGPS expression, whereas 22Rv1 reported a lower expression (Fig. 1f). We knocked down AGPS expression with three different lentivirus packaging short hairpin RNA (shRNA) in DU145 and PC-3 cell lines and over-expressed AGPS in 22Rv1 cell line (Fig. 2a and b, supplementary Fig. 2e, f). Then we examined the sensitivity of PCa cells to ferroptosis inducers. We found that the IC50 of both DU145 and PC-3 cells to ML210 and RSL3 was remarkably decreased after AGPS knockdown (Fig. 2c, supplementary Fig. 2g) as well as increased significantly after AGPS over-expression in 22Rv1 cells, indicating a correlation between AGPS expression and sensitivity to ferroptosis inducers in PCa. We also noticed the expression of peroxisome membrane protein 70 (PMP70) significantly reduced after AGPS knockdown and increased after AGPS over-expression, which suggested a variation in peroxisome production (Fig. 2d and e, Supplementary Fig. 2c, i,j and k). But there were no significant changes in Fe²⁺ (Supplementary Fig. 2d and h). Our findings reported that when induced with ML210, mitochondria with increased membrane density and atrophy mitochondria reduced after AGPS knockdown through TEM (Transmission electron microscopy) (Fig. 2f, Supplementary Fig. 2l). Similarly, malondialdehyde (MDA), the main product of ferroptosis, was greatly reduced after AGPS knockdown, meanwhile, increased after AGPS over expression (Fig. 2g and Supplementary Fig. 2m). These verified our hypothesis that AGPS could promote the ferroptosis process.

The findings derived from the CCK8 and clone formation assays further substantiate that the downregulation of AGPS elicits a stimulatory effect on the proliferation of PCa cells, while the overexpression of AGPS exerts an inhibitory influence on their proliferation (Fig. 2h-j, Supplementary Fig. 2n-p). To further verify the role of AGPS in PCa, we performed a subcutaneous transplantation tumor model using severely immunodeficient mice and found that the tumor progressed by AGPS knockdown (Fig. 2k-m).

Overall, the above results suggested that AGPS can contribute to the development of ferroptosis by promoting peroxisome formation and inhibiting the proliferation of PCa cells.

MDM2 ubiquitinates and modifies AGPS and promotes its degradation

To understand the specific mechanisms involved in the lower expression of AGPS in PCa, we treated PCa cells with the proteasome inhibitor MG132 and the lysosomal inhibitor hydroxychloroquine. We found that AGPS expression was significantly upregulated after MG132 treatment, but no significant changes were observed in hydroxychloroquine-treated cells (Supplementary Fig. 3a). Thus, we speculated that the degradation of AGPS protein might be related to the proteasome pathway. We established the interaction between E3 ligases and AGPS using the Ubibrowser (http://ubibrowser.ncpsb.org/) to predict the possible E3 ligases of AGPS and used the Biogird database (https://thebiogrid.org/) to identify the potentially interacting proteins with AGPS. The combined analysis indicated that MDM2 proteins may interact with and regulate AGPS proteins (Supplementary Fig. 3b). Meanwhile, we transfected different E3 ligases and DUBs in PCa cells, and the level of AGPS protein was significantly decreased in the MDM2 ectopically transfected cells (Fig. 3a), which was consistent with our predictions from the analysis. We then ectopically transfect MDM2 but found no remarkable changes in AGPS at the transcriptional level (Fig. 3b). Subsequently, we found that MDM2 knockdown significantly increased the AGPS expression in a dose-dependent manner (Fig. 3c and d), and the process could be hindered by MG132 treatment (Fig. 3e). This result suggested that MDM2 affects AGPS stability via the ubiquitin–proteasome pathway. Meanwhile, the half-life of AGPS significantly decreased after ectopic expression of MDM2, which confirmed that MDM2 could degrade AGPS (Fig. 3f and g).

Next, we examined the ubiquitination level of AGPS after ectopic transfection with MDM2 and found that MDM2 overexpression increased the ubiquitination level of AGPS, indicating that MDM2 could alter the ubiquitination level of AGPS (Fig. 3h). On a molecular level, we examined the possible interactions between MDM2 and AGPS. The co-immunoprecipitation (co-IP) assay confirmed that both ectopically expressed and endogenous AGPS and MDM2 interacted with each other (Fig. 3i and j). To investigate the type of ubiquitin linkage on AGPS, we mutated the lysine (K) and arginine (R) residues on ubiquitin and evaluated their effects on AGPS ubiquitination after MDM2 transfection. Results demonstrated that K6R-, K11R-, K27R-, K29R-, K33R- and K63R-ubiquitin could trigger MDM2-mediated AGPS ubiquitination, but the lysine 48 (K48) R-ubiquitin largely inhibited the ubiquitination of AGPS (Fig. 3k). These results also support the notion that MDM2 could mediate AGPS ubiquitination and degradation. To further verify our conclusion, we overexpressed AGPS with or without MDM2 overexpression (Supplementary Fig. 3c). As expected, we observed that mitochondria with increased membrane density and atrophy mitochondria increased significantly, and the phenomenon was reversed by MDM2 overexpression (Supplementary Fig. 3d and e). Similarly, the level of MDA was reversed by the overexpression of MDM2 (Supplementary Fig. 3f). On the contrary, the CCK8 and clone formation assays indicated that AGPS overexpression inhibited tumor cell growth, and concomitant overexpression of MDM2 resulted in significantly higher PCa cell activity as compared to the experimental group overexpressing AGPS alone (Supplementary Fig. 3g-i).

Collectively, our experiments revealed that AGPS, a new substrate for MDM2, can be ubiquitinated and degraded by E3 ligase MDM2.

AGPS binds to MDM2 at the C-terminal amino acid sequence of the F443-F455 segment

To verify the specific binding motifs of AGPS and MDM2, we generated GST recombinant proteins for AGPS and MDM2 (Fig. 4a and b). Except for the full length, the two truncated MDM2 recombinant proteins contained the domains of p53 binding domain or not (Fig. 4a). GST pull-down assay reported that the p53 binding domain could bind to AGPS (Fig. 4c), while the C-terminus of AGPS could bind to the C-terminus of MDM2 (Fig. 4d). To verify this, we simulated the spatial structures of AGPS and MDM2 proteins using the Z-DOCK software and subsequently performed molecular docking for the two proteins. The high Z-score suggests a strong binding of the two proteins (Fig. 4e). Moreover, the result of the molecular docking revealed the amino acids in the C-terminus of AGPS that could be specific binding sites for MDM2. This included F443, L447, K448, F450, Y451, I452, K454, and F455.

To validate the binding sites, we constructed the AGPS delete mutant of AGPS F443-F455 region and transfected the mutant AGPS into PCa cells. We found that the protein expression and half-life of mutated AGPS were significantly higher or longer than wild-type AGPS (Fig. 4f–h). In contrast, there seems less binding of mutated AGPS with MDM2and the level of ubiquitination of AGPS by MDM2 also significantly reduced after AGPS mutation (Fig. 4i and j). These results suggest that the binding of mutated AGPS and MDM2 was greatly reduced, which resulted in a decline in ubiquitination and degradation of AGPS. In summary, these experimental results identified the specific binding sites of AGPS and MDM2 and revealed the regulatory relationship between them.

Phosphorylation modification of the Y451 site leads to the accumulation of AGPS in prostate cancer cells

The expression of MDM2 did not significantly change in PCa (Supplementary Fig. 4a), but its substrate, AGPS, was significantly downregulated. Interestingly, a study previously reported that the ubiquitination modification and degradation function of MDM2 for AR was affected by phosphorylation modification [21]. The research proposed the function of MDM2 ubiquitination modification in a phosphorylation-dependent manner. To validate that the phosphorylation of AGPS could affect the binding of AGPS and MDM2 and lead to a change in AGPS expression, we first treated the cells with lambda protein phosphatase (λpp) to eliminate the function of phosphatase. We found that the expression of AGPS increased after λpp treatment, and this effect was removed after combination treatment with MG132 (Fig. 5a). Subsequently, we transfected both AGPS and MDM2 and found MDM2 can no longer degrade AGPS after λpp treatment (Fig. 5b). Likewise, the level of ubiquitination modification of AGPS by MDM2 significantly decreased after λpp treatment (Fig. 5c). These results suggested that the ubiquitination regulation of AGPS might be influenced by the phosphorylation of AGPS.

Additionally, Co-IP results confirm that the binding of AGPS and MDM2 was affected by λpp (Fig. 5d). We transfected AGPS△F443-F455 and wild-type AGPS in PCa cells and treated the cells with λpp. The result indicated λpp only affected the wild-type AGPS and not the mutated AGPS (Fig. 5e), which verified that AGPS might be modified by phosphorylation at the binding site with MDM2, thus affecting the binding of the two proteins. Among these binding sites, we found that Y451 could be modified by phosphorylation and is highly conserved in evolution (Fig. 5f). Consequently, we speculated that the phosphorylation modification of Y451 was the primary cause. To confirm this speculation, we used the Z-DOCK software to simulate the spatial conformation of AGPS after the phosphorylation of the Y451 site and used this model to dock again with MDM2. Result showed that the binding of the two proteins was greatly increased after docking, and the Z-score significantly increased (Fig. 5g), which indicated that the phosphorylation of Y451 promoted the binding between AGPS and MDM2. To verify this, we constructed an Y451-mutated AGPS and found that the expression and half-life of AGPS significantly increased (Fig. 5h-j). Similarly, the level of AGPS ubiquitination was significantly reduced after simultaneous overexpression of MDM2 after the Y451 mutation (Fig. 5l). Co-IP experiments revealed that the binding of AGPS to MDM2 was significantly weakened after mutation at Y451 (Fig. 5k), which explained the reduced ubiquitination level and protein accumulation of AGPS. Our findings confirmed that AGPS can be phosphorylated at Y451, thus enhancing its binding to MDM2 and AGPS accumulation.

TrkA promotes the ubiquitinated degradation of AGPS by MDM2 by modifying the phosphorylation of AGPS

To future illustrate the regulation mechanism, we screened several common kinases from both databases that might modify the phosphorylation of AGPS at the Y451 position (The sequence of AGPS used was obtained from two separate databases: https://services.healthtech.dtu.dk/service.php?NetPhos-3.1 and www.phosphonet.ca), including EGFR/ERBB4, Ack/TNK2, Met/MSTIR, PDGFR/CFFIP, and Trk/NTRK1. Only the TrkA inhibitor, Larotrectinib, but not any other inhibitor could significantly increase the expression of endogenous AGPS (Fig. 6a). Interestingly, we conducted an intersection analysis between these kinases and AGPS-related proteins in the Biogrid database, revealing that solely TrkA exhibited a connection with AGPS (Supplementary Fig. 4b). This finding further substantiates our observed phenomenon. Furthermore, we discovered that TrkA is overexpressed and phosphorylated in prostate cancer, suggesting a potential association with disease progression. Consequently, we selected the TrkA inhibitor Larotrectinib for further investigation. Also, Co-IP results suggested that Larotrectinib could inhibit the phosphorylation of AGPS tyrosine (Fig. 6b), which was consistent with our previous results that TrkA can modify the phosphorylation of AGPS.

Interestingly, it has been reported that TrkA is aberrantly activated in PCa and the phosphorylation level is positively correlated with the Gleason score [22, 23]. We examined the phosphorylation level of TrkA in PCa cells and also found that p-TrkA was significantly higher in PCa cells than in RWPE-1 cells (Fig. 6c). Immunohistochemistry in prostate tissues also indicated the expression of p-TrkA gradually increased with increasing Gleason score. On the contrary, AGPS expression is a negative correlation with the Gleason score and p-TrkA level (Fig. 6d, Supplementary Fig. 4c). Meanwhile, we found that the expression of AGPS was significantly elevated with p-TrkA inhibitor Larotrectinib (Fig. 6e and f). Also, the ubiquitination level of AGPS was significantly reduced and AGPS protein half-life significantly increased after Larotrectinib treatment which was consistent with our previous findings (Fig. 6g-i). To further corroborate our findings, we knocked down NTRK1 (Supplementary Fig. 4d) and found that the ubiquitination level of AGPS and the binding of AGPS and MDM2 were significantly reduced (Fig. 6j, k). This indicated that TrkA phosphorylation modification of AGPS promoted the binding of AGPS and MDM2, leading to the degradation of AGPS. Likewise, the protein half-life of AGPS was increased (Supplementary Fig. 4e and f).

Overall, TrkA is aberrantly activated in PCa and modifies the phosphorylation of AGPS at Y451, which promotes the binding of AGPS and MDM2 and significantly downregulates AGPS expression in PCa.

Larotrectinib promotes the sensitivity of prostate cancer cells to ferroptosis by inhibiting the phosphorylation of TrkA

In recent years, the application of ferroptosis attracted a lot of attention in tumor therapy, some studies reported the use of ferroptosis inducers RSL3 and Erastin in the treatment of PCa [24]. Therefore, we speculate Larotrectinib could promote the accumulation of AGPS and exert a positive effect on ferroptosis inducers in tumor therapy. To validate our claim, we treated the cells with ML210 and Larotrectinib alone or in combination. We noticed different degrees of elevation of AGPS with the combination application of both drugs (Fig. 7a, Supplementary Fig. 5b). Also, we found that the prostate cancer cells were much more sensitive to ferroptosis inducer when combination administration of Larotrectinib (Fig. 7b, Supplementary Fig. 5c). In addition, PMP70 level and membrane density, and atrophy mitochondria reduced were greatly increased in the combination application of ML210 and Larotrectinib (Fig. 7c-f, Supplementary Fig. 5e). Also, MDA level also increased when combination application of ML210 and Larotrectinib (Fig. 7g, Supplementary Fig. 5f). Thus, we conclude that Larotrectinib-induced accumulation of AGPS could significantly increase the sensitivity of PCa cells to ferroptosis, and this was more pronounced when ML210 was used concomitantly.

Next, we examined the effect of both drugs on cell proliferation. The CCK8 assay and colony formation results suggested that the inhibition of cell proliferation by the combination of both drugs was significantly higher than the individual drug treatments (Fig. 7h and i, Supplementary Fig. 5a, h and i). To further verify the therapeutic efficacy of the two drugs in vivo, we established organoid culture from prostate cancer tissues, when cultured with different drugs, the result showed that the sphere area of organoids generated through the synergistic application of two drugs exhibits a significantly reduced magnitude compared to the individual drug effects, aligning with the outcomes obtained from our in vitro experimental investigations (Fig. 7j, Supplementary Fig. 5j).Finally, we performed a subcutaneous transplantation tumor model using severely immune-deficient mice and found that the tumor suppression effect of Larotrectinib in combination with ML210 was much greater than that of the individual drug treatments (Fig. 7k-m).

In conclusion, Larotrectinib can increase the sensitivity of PCa cells to ferroptosis by promoting the accumulation of AGPS, thereby enhancing the therapeutic effect of ferroptosis inducers on tumors. This provides new ideas for the clinical treatment of PCa.

Discussion

Previous studies focused on the role of AGPS in the synthesis of ether lipids, which affects the progression of various diseases by regulating lipid synthesis. It has been reported that AGPS can inhibit the progression of hepatocellular carcinoma and glioma by regulating the cell cycle and the activity of signal transduction pathways such as MAPK [25]. In contrast, there are very few reports related to the role of AGPS in peroxisome production and tumor progression. In the synthesis of peroxisomes, AGPS promotes peroxisome production via the activation of GNPAT [26]. Our study revealed that AGPS inhibited the proliferation of PCa cells by promoting peroxisome production, thereby leading to lipid peroxidation in PCa cells and enhancing the process of ferroptosis (Fig. 8).

Ferroptosis is iron-dependent cell death caused by lipid peroxidation due to excessive accumulation of ROS [10]. In this process, GPX4 is mainly responsible for degrading lipid peroxides and removing toxic intermediates. The inhibition of GPX4 initiates ferroptosis. The development of small molecule covalent inhibitors of GPX4 to effectively target cancer cells is appealing in modern drug development [18]. The currently available GPX4 inhibitors, such as RSL3 and ML162, exert their inhibitory effects by covalently linking the selenocysteine residue at position 46 on the active site of GPX4 through a reactive alkyl chloride moiety. ML210, a molecule with a nitro isoxazole structure, is widely used for inducing ferroptosis [27]. Researchers have explored the antitumor molecular mechanism of ML210 to inhibit GPX4 and reported a tumor-suppressive effect in a variety of tumors [28]. Our study also verified that ML210 could achieve the therapeutic effect of inhibiting PCa progression by promoting ferroptosis. However, its therapeutic effect is limited because of the complex ferroptosis process, thus new therapeutic modalities need to be further explored (Fig. 8).

TrkA is a tyrosine kinase receptor and is closely related to the progression of many diseases. Its activation can activate pathways such as AKT for disease development [23]. TrkA is highly phosphorylated in a variety of tumors, and its inhibitors are effective in tumor suppression. Among them, Larotrectinib has been clinically used as an inhibitor of TrkA in various tumors such as liver cancer and breast cancer [29]. Our study revealed that in PCa, abnormally activated TrkA promoted the degradation of AGPS via MDM2. Therefore, the inhibition of TrkA could inhibit the degradation of AGPS via MDM2 and enhance the sensitivity of PCa cells to ML210. The combination of the TrkA inhibitors Larotrectinib and ML210 increased the lethality of ML210 and Larotrectinib towards PCa cells (Fig. 8). Our study provided a theoretical basis for the use of Larotrectinib in the treatment of PCa in the clinical setting.

MDM2 often appears as a complex with p53 in vivo and could suppress ferroptosis by degrading p53 [30,31,32,33]. Since tumor Suppressor p53 and its mutants have widely reported involved in ferroptosis development and progression [34, 35]. This will undoubtedly lead to the consideration of whether MDM2 affects ferroptosis through p53 and whether AGPS are involved in this process. In order to eliminate the influence of p53 as far as possible, many experiments were carried out in both p53 positive (22Rv1, DU145) and p53 negative cell lines (PC-3), which including the detection of peroxisome level, ferroptosis level and mitochondrial membrane potential. These findings suggested that under the condition of p53 deletion, MDM2-AGPS can still affect the occurrence of ferroptosis by regulating the formation of peroxisome and ferroptosis inducer drug sensitivity in a p53 independent manner. Since ferroptosis involves in different processes and molecular pathways, further research is needed to better understand the mechanism of MDM2 and AGPS in ferroptosis.

Conclusion

Over all these findings, our study identified AGPS as a novel substrate of MDM2. Their interaction could be regulated by TrkA and its inhibitor, Larotrectinib. Larotrectinib could enhance the sensitivity of PCa cells to ML210, and the combined application of Larotrectinib and ML210 could achieve a better anti-tumor effect on PCa. Our study is important for exploring new clinical treatment modalities for PCa.

Availability of data and materials

The raw data generated in this study are available upon request, from the corresponding author.

Abbreviations

PCa:: Prostate cancer
AGPS:: Alkylglycerone phosphate synthase Gene
MDM2:: Mousedouble minute 2
TrkA:: Tyrosine kinase receptor A
IHC:: Immunohistochemistry
RT-qPCR:: Real-time quantitative polymerase chain reaction
TEM:: Transmission electron microscope

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Acknowledgements

The authors thank public data of TCGA. We thank Dr. Gu(guluosha@163.com) from Peking Union Medical College Hospital for drawing the hypothetic model. We thank the BioRender (www.biorender.com) website for the hypothetic model.

Funding

This work was supported by National Natural Science Foundation of China (grant number:882172564), National Natural Science Foundation of China (grant number:82303032) and the Henan Science and Technology Research Program (grant number: 2018020142).

Author information

Yu Zhang, Zhenlin Huang and Keqiang Li contributed equally to this work.

Authors and Affiliations

Department of Urology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China
Yu Zhang, Zhenlin Huang, Keqiang Li, Guoqing **e, Yuankang Feng, Zihao Wang, Ningyang Li, Ruoyang Liu, Jun Wang, **jian Yang & Zhankui Jia
Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, 55905, USA
Yu Zhang
Department of Otorhinolaryngology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China
Yinghui Ding
Academy of Medical Science, Zhengzhou University, Zhengzhou, 450052, China
Yu Zhang, Keqiang Li & Guoqing **e

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Yu Zhang
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Zhenlin Huang
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Keqiang Li
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Guoqing **e
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Yuankang Feng
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Zihao Wang
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Ningyang Li
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Ruoyang Liu
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Yinghui Ding
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Jun Wang
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**jian Yang
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Zhankui Jia
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Contributions

YZ and ZLH conceived and designed the study. YZ, KQL and ZLH performed experiments and drafted the manuscript. GQX and RYL performed experiments and analyzed the data. ZHW and JW collected the patient samples and analyzed clinical data. KQL, YKF and YHD performed the statistical analysis. ZKJ and JJY provided technical and methodological support. ZKJ supervised the study. ZKJ and ZLH reviewed and revised the manuscript. All authors read and approved the final version.

Corresponding authors

Correspondence to Jun Wang, **jian Yang or Zhankui Jia.

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Ethics approval and consent to participate

Approval was obtained for all animal studies under the guidelines of the First Affiliated Hospital of Zhengzhou University. All clinical study was approved by Ethics Committee of the First Affiliated Hospital of Zhengzhou University and written informed consent was obtained from all subjects. Ethics approval number: 2022-KY-0976.

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The authors declare no conflict of interest.

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Additional file 1:

Supplementary Fig 1. (Supplemental to Fig. 1) AGPS is down-regulated in prostate cancer and negatively correlated with prognosis. Supplementary Fig 2. (Supplemental to Fig. 2) AGPS inhibits the proliferation of PC-3 cells. Supplementary Fig 3. (Supplemental to Fig. 3) MDM2 inhibits ferroptosis through regulating AGPS- a p53-independent pathway. Supplementary Fig 4. (Supplemental to Fig. 6) TrkA has a significant effect on the stability of the protein of AGPS. Supplementary Fig 5. (Supplemental to Fig. 7) Effectiveness of the combination of ML210 and Larotrectinib, in the PC3 cell line. Supplementary Table 1.Oligonucleotides used for relative gene expression by qRT-PCR. Supplementary Table 2. The oligonucleotides of si- or sh- RNAs.

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Zhang, Y., Huang, Z., Li, K. et al. TrkA promotes MDM2-mediated AGPS ubiquitination and degradation to trigger prostate cancer progression. J Exp Clin Cancer Res 43, 16 (2024). https://doi.org/10.1186/s13046-023-02920-w

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Received: 14 September 2023
Accepted: 30 November 2023
Published: 11 January 2024
DOI: https://doi.org/10.1186/s13046-023-02920-w

TrkA promotes MDM2-mediated AGPS ubiquitination and degradation to trigger prostate cancer progression

Abstract

Background

Methods

Results

Conclusion

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Introduction

AGPS inhibits the proliferation of prostate cancer cells by promoting the formation of peroxisome and ferroptosis

MDM2 ubiquitinates and modifies AGPS and promotes its degradation

AGPS binds to MDM2 at the C-terminal amino acid sequence of the F443-F455 segment

Phosphorylation modification of the Y451 site leads to the accumulation of AGPS in prostate cancer cells

TrkA promotes the ubiquitinated degradation of AGPS by MDM2 by modifying the phosphorylation of AGPS

Larotrectinib promotes the sensitivity of prostate cancer cells to ferroptosis by inhibiting the phosphorylation of TrkA

Discussion

Conclusion

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