MDM4 promotes chemoresistance in bladder cancer by attenuating P53-mediated EMT

Introduction

Bladder cancer is one of the most common malignancies of the genitourinary system, characterized by high rates of recurrence and mortality (1). For muscle-invasive and metastatic bladder cancer, cisplatin-based combination chemotherapy remains the cornerstone of systemic treatment, providing significant survival benefits (2-4). For muscle-invasive bladder cancer (MIBC) and metastatic bladder cancer, cisplatin-based combination chemotherapy remains the cornerstone of systemic treatment. In the neoadjuvant setting (NAC) for localized MIBC, cisplatin-based regimens aim to achieve pathological downstaging and eradicate micrometastases, providing a 5–8% overall survival benefit (5). Conversely, in the metastatic setting, these regimens serve as first-line palliative therapy to control tumor burden and prolong life, although efficacy is frequently limited by chemoresistance (6,7). Despite this, the efficacy of chemotherapy is severely limited by drug resistance, which can be either intrinsic (pre-existing) or acquired (developing after treatment exposure). This resistance is a major clinical obstacle leading to therapeutic failure and dismal patient outcomes (8,9). Acquired resistance, in particular, involves a complex interplay of molecular changes within tumor cells, including increased drug efflux, enhanced DNA damage repair mechanisms, and evasion of apoptosis. Consequently, the 5-year survival rate for metastatic bladder cancer remains poor, largely due to the emergence of chemoresistant cell populations. Therefore, elucidating the specific molecular pathways that drive chemoresistance is urgently needed to identify novel therapeutic targets and develop more effective strategies to overcome treatment failure.

The epithelial-mesenchymal transition (EMT) is a complex cellular program increasingly implicated in cancer progression, metastasis, and the acquisition of therapeutic resistance (10,11). This dynamic process facilitates metastatic dissemination through lymphatic and hematogenous routes, enabling detached tumor cells to intravasate into vessels and migrate to distant sites. Crucially, upon reaching secondary sites like lymph nodes, disseminated cells undergo mesenchymal-epithelial transition (MET) to re-establish epithelial characteristics and form metastatic colonies (10,12). During EMT, cancer cells lose their epithelial characteristics and gain mesenchymal features, which endows them with enhanced motility, invasiveness, and a strong resistance to apoptosis induced by chemotherapeutic agents (13). The tumor suppressor p53, often referred to as the “guardian of the genome”, plays a pivotal role in mediating the cellular response to chemotherapy-induced DNA damage, primarily by inducing cell cycle arrest or apoptosis (14-16). Consequently, the functional inactivation of the p53 pathway is a well-established mechanism of chemoresistance in many human cancers, allowing tumor cells to evade the cytotoxic effects of treatment (17,18).

Murine double minute 4 (MDM4, also known as MDMX) has emerged as a critical negative regulator of p53. It binds directly to the N-terminal transactivation domain of p53, thereby inhibiting its transcriptional activity without promoting its degradation (19-21). Overexpression of MDM4 has been documented in a wide range of human tumors, where it often correlates with a wild-type p53 status and is frequently associated with poor clinical prognosis and resistance to therapy (22-24). By functionally inactivating p53, MDM4 can subvert crucial anti-tumor responses initiated by chemotherapy. However, the specific role of MDM4 in mediating chemoresistance in bladder cancer, particularly through its regulation of the p53-EMT axis, has not been fully elucidated.

In this study, we sought to investigate the role of MDM4 in bladder cancer chemoresistance. Our results demonstrate that MDM4 is highly expressed in chemoresistant bladder cancer cells. We show that knockdown of MDM4 resensitizes resistant cells to chemotherapy by reactivating the p53 pathway and reversing the EMT phenotype, both in vitro and in vivo. These findings identify the MDM4/p53/EMT axis as a key mechanism driving chemoresistance and highlight MDM4 as a potential therapeutic target to overcome treatment failure in bladder cancer. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-907/rc).

Methods

Cell culture

Human urothelial cancer cell lines T24 was preserved in our lab (purchased from ATCC). All cells were cultured in DMEM (GE Healthcare Life Sciences, Connecticut, USA) with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Science, Massachusetts, USA) and 100 mg/mL penicillin/streptomycin (Yeason Biotechnology, Shanghai, China) at 37 ℃ in a 5% CO2 humidified incubator.

Generation of cisplatin-resistant T24 cell line (T24-CR)

A cisplatin-resistant T24 subline (designated T24-CR) was generated from parental T24 bladder cancer cells via continuous exposure to increasing concentrations of cisplatin (Sigma-Aldrich, Missouri, USA) over 6 months. Briefly, cells were initially treated with 0.5 µM cisplatin for 72 h, recovered in drug-free medium, and repeatedly re-challenged with incrementally higher cisplatin doses (0.2–0.5 µM increases per cycle). Before experiments, cells were passaged at least twice in drug-free medium. Resistance was validated by CCK-8 assay (Yeason, Shanghai, China), showing significantly higher IC₅₀ in T24-CR compared to parental cells (see Figure 1A).

Figure 1 MDM4 is highly expressed in chemoresistant bladder cancer cells. (A) Establishment of a chemotherapy-resistant T24 cell line and cytotoxicity analysis by CCK-8 assay. (B) RT-qPCR analysis of MDM4 mRNA levels in chemotherapy-resistant cells. The data are presented as the mean ± SD. Significance was assessed using Student’s t-test, **, P<0.01. (C,D) Western blot analysis (C) and corresponding quantification (D) of MDM4 protein levels in chemotherapy-resistant cells. The data are presented as the mean ± SD. Significance was assessed using Student’s t-test, ***, P<0.001. CCK-8, Cell Counting Kit-8; MDM4, murine double minute 4; RT-qPCR, reverse-transcription quantitative real-time polymerase chain reaction; SD, standard deviation.

Cell viability assay

The sensitivity of T24, T24-CR, and MDM4-knockdown T24-CR cells to cisplatin (Sigma-Aldrich, USA) was assessed by CCK-8 Cell Counting Kit (Yeason Biotechnology, China). Briefly, we treated both cells with cisplatin for 48 h at different concentrations to determine cell viabilities. All the working dilutions were prepared in water and replicated three times. Dose response curves to calculate cell inhibition values were plotted using GraphPad Prism Software (version 8.0).

Reverse-transcription quantitative real-time PCR (RT-qPCR)

RNAs were extracted using TRIzol reagent (Yeasen, Shanghai, China) according to the manufacturer’s instructions. The cDNAs were synthesized with Hifair^® III 1^st Strand cDNA Synthesis SuperMix for qPCR (YEASEN), and qPCR were performed in ViiA™ 7 Real-Time PCR Detection System (Thermo Fisher Scientific) using the Hieff^® qPCR SYBR^® Green Master Mix (Yeasen). The primers used are as Table S1.

Western bloting analysis

Cells were lysed directly in RIPA buffer (Beyotime, Shanghai, China). Lysates were separated by SDS-PAGE and electrophoretically transferred onto PVDF membranes (Millipore, Darmstadt, Germany). Membranes were blocked with 5% skim milk for ≥1 hour at room temperature, followed by overnight incubation with primary antibodies at 4 ℃. The next day, membranes were washed three times with 1× TBST buffer and then incubated with HRP-conjugated goat anti-mouse/rabbit secondary antibody for 2 hours at room temperature. Protein bands were finally visualized using the Tanon-5200S chemiluminescent imaging system (Tanon, Shanghai, China). Antibody details are provided in Table S2.

Gene knockdown

To knockdown MDM4 expression in the T24-CR cell line, short hairpin RNA (shRNA)-mediated silencing was performed. Lentiviral particles carrying MDM4-specific shRNA sequences were generated using a pLKO.1 vector (Shanghai Generay Biotech, Shanghai, China). A non-targeting scrambled shRNA was used as negative control. T24-CR cells were seeded in 6-well plates and infected with the lentivirus in the presence of polybrene (8 µg/mL). After 24 hours, the virus-containing medium was replaced with fresh complete medium. To establish stable knockdown cells, transduced cells were selected with puromycin (2 µg/mL) for 7 days. Knockdown efficiency was confirmed at protein levels via Western blotting.

Cell migration and invasion assays

Cell migratory and invasive capacities were evaluated using the wound healing assay and Transwell assay, respectively. For the wound healing assay, 5×10⁵ cells were seeded into 6-well plates. A sterile pipette tip was used to create a uniform scratch in the confluent cell monolayer. After treatment with cisplatin for 48 h, images of cell migration were acquired at 0 and 48 h.

For the Transwell invasion assay, 1×10⁴ cells suspended in 200 µL serum-free DMEM were added to the upper compartment of Matrigel-coated Transwell inserts (Corning, New York, USA), while 400 µL complete DMEM was placed in the lower chamber. Following 24 h of cisplatin treatment, the cells were fixed, stained with 0.5% crystal violet, and imaged under an inverted microscope (Olympus, Hamburg, Germany).

Apoptosis analysis

To determine the apoptotic rate and cell cycle profile, flow cytometric analysis was carried out in this study. Three cell groups, including T24, T24-CR, and MDM4-silenced T24-CR cells, were incubated with cisplatin for 24 hours. After incubation, cells were detached with EDTA-free trypsin (Yeason Biotechnology, China) and washed with PBS buffer. Apoptotic cells were quantified by staining with Annexin V-FITC and PI, using a commercial apoptosis detection kit (Yeason Biotechnology, China), and analyzed on a flow cytometer. Meanwhile, cell cycle distribution was examined using a dedicated cell cycle staining kit obtained from the same manufacturer (Yeason Biotechnology, China).

Murine model and study design

Experiments were performed under a project license (No. 2025SYXK8) granted by the Ethics Committee board of Second Affiliated Hospital of Naval Medical University, in compliance with institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration. Male wild-type BALB/c-nu/nu mice (Phenotek, China) between 6 and 8 weeks of age were used and maintained in specific pathogen-free conditions in animal facilities. Mice were randomly divided into three groups (n=6): the T24-WT group, the T24-CR group, and the T24-CR-shMDM4 group. A subcutaneous xenograft model was established by inoculating 3×10⁵ cells of either T24-WT, T24-CR, or MDM4-knockdown T24-CR into each mouse. Tumor size and volume were monitored throughout the study. Kaplan-Meier survival curves were generated to analyze the experimental outcomes. The experimental endpoint was defined as death or a tumor volume ≥2,000 mm³. Tumor Volume using the following formula: Tumor Volume = 0.5 × length × width².

Immunohistochemical (IHC) staining

IHC staining was performed as follows: Dewaxed tissue sections were first rehydrated through a graded ethanol series. For antigen retrieval, sections were heated in 10 mmol/L citrate buffer (pH 6.0) using a heat-induced epitope retrieval method. Endogenous peroxidase activity was quenched by incubating sections in 3% hydrogen peroxide (H₂O₂) diluted in methanol. To block nonspecific antibody binding, sections were treated with 10% horse serum for 30 minutes at room temperature. Primary antibodies were then added to the sections, which were incubated overnight (16 hours) at 4 ℃ in a humidified environment. After three washes with PBS to eliminate unbound primary antibodies, sections were incubated with a biotin-conjugated goat anti-mouse/rabbit IgG secondary antibody. Immunoreactivity was visualized using a 3,3'-diaminobenzidine (DAB) substrate kit. Finally, stained slides were digitally imaged using the Pannoramic SCAN II system. Detailed information regarding the primary and secondary antibodies used is provided in Table S2.

Statistical analysis

All experimental data were presented as means ± standard deviation (SD). Differences were determined by Student’s t-test with in two groups, or the one-way analysis of variance (ANOVA) if more than two groups with the significance level set at P<0.05 (GraphPad Prism8, Bethesda, MD).

Results

MDM4 is highly expressed in chemoresistant bladder cancer cells

To investigate the mechanisms underlying chemoresistance in tumor cells, we first successfully established a T24-CR. The resistance phenotype was validated through a CCK-8 cytotoxicity assay, which confirmed the reduced sensitivity of T24-CR cells to chemotherapeutic treatment (Figure 1A). As MDM4 is a key negative regulator of the p53 tumor suppressor and its overexpression has been linked to therapeutic resistance in several cancers, we next sought to determine whether its expression was altered in these resistant cells. Using RT-qPCR, we found that the mRNA levels of MDM4 were significantly elevated in the T24-CR cells compared to the T24-WT cells (Figure 1B). To confirm this finding at the protein level, we performed a Western blot analysis. Consistent with the transcriptomic data, the expression of MDM4 protein was also markedly increased in the T24-CR cells (Figure 1C,1D). Collectively, these results demonstrate that MDM4 is highly expressed at both the mRNA and protein levels in chemoresistant bladder cancer cells, suggesting its potential involvement in the acquisition of drug resistance.

MDM4 is a critical regulator of chemoresistance and malignancy in bladder cancer

Given the high expression of MDM4 in chemoresistant cells, we next investigated its functional role in bladder cancer. We knocked down MDM4 in the T24-CR cell line, and the knockdown efficiency was confirmed by a significant reduction in MDM4 protein levels (Figure 2A,2B). To determine whether MDM4 knockdown could reverse chemoresistance, we evaluated the cellular response to chemotherapeutic drugs. Silencing MDM4 significantly increased the sensitivity of T24-CR cells to this treatment (Figure 2C). We then evaluated the impact of MDM4 on malignant phenotypes. Knockdown of MDM4 markedly suppressed the proliferative capacity of T24-CR cells (Figure 2D). Furthermore, the migration and invasion capabilities of the resistant cells were also significantly inhibited upon MDM4 knockdown (Figure 2E,2F). To explore a potential mechanism for the increased chemosensitivity, we examined apoptosis. T24-CR cells with MDM4 knockdown exhibited a significantly higher rate of apoptosis following chemotherapy treatment (Figure 2G). Taken together, these findings suggest that MDM4 is a critical regulator of both chemoresistance and malignant phenotypes, including proliferation, migration, and invasion, in bladder cancer cells.

Figure 2 Knockdown of MDM4 suppresses chemoresistance and malignant phenotypes in bladder cancer cells. (A,B) The efficacy of MDM4 knockdown was evaluated by WB (A) and quantified (B). The data are presented as the mean ± SD. **, P<0.01; ****, P<0.0001. (C) The cytotoxicity of T24, T24-CR, and MDM4-knockdown T24-CR cells was determined by CCK-8 assays. (D) Colony formation assay of T24, T24-CR, and MDM4-knockdown T24-CR cells. All images represent data from three independent experiments. (E) Wound healing assays were used to assess the migration of T24, T24-CR, and MDM4-knockdown T24-CR cells. (F) Transwell assays were used to evaluate the invasion of T24, T24-CR, and MDM4-knockdown T24-CR cells. (G) The apoptosis of T24, T24-CR, and MDM4-knockdown T24-CR cells was assessed by FITC-conjugated Annexin V-FITC and PI staining. CCK-8, Cell Counting Kit-8; IC₅₀, half-maximal inhibitory concentration; MDM4, murine double minute 4; SD, standard deviation; T24-CR, cisplatin-resistant T24 cell line.

MDM4 depletion suppresses EMT through reactivation of the P53 signaling pathway

To elucidate the molecular mechanism by which MDM4 regulates chemoresistance and malignancy, we investigated its impact on the EMT and the p53 signaling pathway. Western blot analysis revealed that, compared to parental T24 cells, chemoresistant T24-CR cells exhibited classic hallmarks of EMT, including decreased expression of the epithelial marker E-cadherin and increased expression of the mesenchymal marker Vimentin. We then examined the p53 pathway, as MDM4 is a primary negative regulator of p53 function. While the overall expression level of p53 protein remained unchanged between T24-CR and MDM4 knockdowm T24-CR cells, its activity was clearly suppressed in the resistant cells. Remarkably, the knockdown of MDM4 in T24-CR cells reversed the EMT phenotype, leading to a significant increase in E-cadherin expression and a marked reduction in Vimentin expression (Figure 3A,3B). This reversal is attributed to the restoration of p53’s transcriptional activity, as MDM4’s regulatory role is primarily at the functional, rather than expressional, level. These results suggest that the elevated MDM4 in chemoresistant cells promotes EMT by functionally suppressing the p53 signaling pathway.

Figure 3 Knockdown of MDM4 can restore the activity of the P53 signaling pathway and thereby suppress EMT. (A) Western blot assays showing alterations in EMT and P53 signaling pathway -related proteins between T24, T24-CR, and MDM4-knockdown T24-CR cells. (B) Relative protein expression levels are quantified via gray value analysis. The data are presented as the mean ± SD. *, P<0.05； ****, P<0.0001. EMT, epithelial-mesenchymal transition; MDM4, murine double minute 4; SD, standard deviation; T24-CR, cisplatin-resistant T24 cell line.

MDM4 targeting confirms efficacy in reversing chemoresistance in vivo

To validate our in vitro findings, we next assessed the effect of MDM4 knockdown on tumor growth and chemoresistance in a xenograft mouse model. As expected, mice bearing tumors from T24-CR cells showed significantly enhanced tumor growth and poorer overall survival compared to the T24-WT group. However, targeting MDM4 in T24-CR cells resulted in a significant reduction in tumor volume and prolonged the overall survival of the mice (Figure 4A,4B). Immunohistochemical analysis of the excised tumor tissues corroborated our in vitro data. Tumors derived from MDM4-knockdown cells displayed lower levels of MDM4 and Vimentin, alongside elevated expression of E-cadherin, when compared to tumors from the T24-CR control group (Figure 4C,4D). Collectively, these in vivo results confirm that targeting MDM4 can effectively reverse chemoresistance and suppress the malignant phenotype of bladder cancer.

Figure 4 Knockdown of MDM4 reverses chemoresistance in bladder cancer in vivo. (A) Tumors injected by T24, T24-CR, and MDM4-knockdown T24-CR cells were removed and measured. (B) Kaplan-Meier curves showing the overall survival of mice. (C) Immunohistochemical staining of MDM4, E-cadherin, Vimentin and P53 in murine tumors. (D) Relative expression levels are quantified via gray value analysis. The data are presented as the mean ± SD. **, P<0.01; ***, P<0.001; ns, not significant. MDM4, murine double minute 4; SD, standard deviation; T24-CR, cisplatin-resistant T24 cell line.

Discussion

Chemotherapeutic resistance remains a formidable challenge in the clinical management of bladder cancer, contributing significantly to treatment failure and poor patient survival (8,25). The molecular mechanisms driving this resistance are complex and not yet fully understood. In this study, we provide compelling evidence for a novel mechanism in which MDM4, a key negative regulator of p53, fuels chemoresistance by suppressing p53-mediated EMT. Our findings collectively identify the MDM4/p53/EMT axis as a critical driver of the resistant phenotype and highlight MDM4 as a potential therapeutic target to overcome this clinical obstacle.

Our initial investigation successfully established a chemoresistant bladder cancer cell line, which exhibited significantly elevated expression of MDM4 at both the mRNA and protein levels. This observation is consistent with a growing body of literature implicating MDM4 as an oncogene whose overexpression is associated with poor prognosis and therapeutic resistance in various malignancies (19,20,22,26,27). While MDM4’s role in tumorigenesis is well-documented, its specific contribution to acquired chemoresistance in bladder cancer has been less clear. Our data firmly establish a strong correlation between high MDM4 expression and the chemoresistant phenotype in bladder cancer cells, providing a solid foundation for exploring its functional role.

The functional significance of this upregulation was demonstrated through our loss-of-function experiments. Knockdown of MDM4 not only resensitized resistant bladder cancer cells to chemotherapy but also broadly suppressed their malignant phenotypes, including proliferation, migration, and invasion, while promoting apoptosis. This dual effect is crucial, as it suggests that targeting MDM4 could simultaneously combat drug resistance and inhibit tumor progression. We further elucidated the underlying molecular mechanism, showing that the aggressive, chemoresistant cells had undergone EMT, a process strongly linked to drug resistance (11,28,29), characterized by a loss of E-cadherin and a gain of Vimentin. Crucially, MDM4 knockdown reversed this EMT phenotype. This MET-like transition may have dual therapeutic implications: beyond overcoming chemoresistance, it could potentially inhibit metastatic dissemination. Since EMT enables physical movement through lymphatic vessels while MET facilitates lymph node colonization, targeting the MDM4/p53 axis might disrupt both processes in bladder cancer progression (30). Given that the status of the p53 pathway is a critical determinant of bladder cancer prognosis (15,16,18), this finding is highly significant. We noted that p53 protein levels remained unchanged, underscoring that MDM4’s primary role is to functionally inhibit p53’s transcriptional activity rather than regulate its expression. By knocking down MDM4, p53 is liberated to resume its tumor-suppressive functions, which include the well-established role of inhibiting EMT (19,31).

To confirm the clinical relevance of our findings, we extended our investigation to an in vivo xenograft model. The results from this model strongly corroborated our in vitro data. Targeting MDM4 significantly attenuated tumor growth and prolonged the survival of mice bearing chemoresistant tumors. Immunohistochemical analysis of these tumors confirmed the molecular changes observed in vitro, with MDM4 knockdown leading to increased E-cadherin and decreased Vimentin expression. This preclinical evidence reinforces the therapeutic potential of targeting the MDM4/p53/EMT axis to overcome chemoresistance in a physiological context.

While our study provides strong evidence for this pathway, we acknowledge its limitations. Our findings are based on a single bladder cancer cell line, and further validation in a broader range of cell lines, patient-derived xenografts, and clinical samples is necessary to establish the generalizability of this mechanism. Furthermore, the xenograft model used lacks a competent immune system, which can influence tumor biology and response to therapy.

Looking forward, our findings open several avenues for future research and clinical application. The challenge of directly restoring the function of a mutated or lost tumor suppressor like p53 is well-known in oncology. Therefore, targeting its negative regulators presents a more viable and attractive therapeutic strategy. In this context, our identification of MDM4 as a key driver of resistance highlights it as an exceptionally important therapeutic target in bladder cancer. Its elevated expression in resistant tumors suggests a potential therapeutic window. Firstly, MDM4 expression levels could serve as a potential predictive biomarker to identify bladder cancer patients at high risk of developing chemoresistance. Secondly, and more importantly, the development of small-molecule inhibitors designed to specifically disrupt the MDM4-p53 interaction is an active and promising area of cancer research (32,33). Such inhibitors could effectively “release the brakes” on p53, reactivating its tumor-suppressive functions. Our study provides a strong rationale for prioritizing the clinical investigation of these inhibitors, potentially in combination with conventional chemotherapy, as a novel strategy to resensitize resistant tumors and improve treatment outcomes for patients with advanced bladder cancer.

Conclusions

In conclusion, our study identifies MDM4 as a critical driver of chemoresistance in bladder cancer, noting its significant overexpression in resistant cells. We demonstrate that targeting MDM4 reverses this resistance by reactivating the p53 pathway and suppressing EMT. These findings, validated both in vitro and in vivo, establish the MDM4/p53/EMT axis as a key mechanism and highlight MDM4 as a promising biomarker and therapeutic target for drug-resistant bladder cancer.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-907/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-907/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-907/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-907/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Experiments were performed under a project license (No. 2025SYXK8) granted by the Ethics Committee board of Second Affiliated Hospital of Naval Medical University, in compliance with institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Sung H, Ferlay J, Siegel RL, 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. [Crossref] [PubMed]
2. Adjuvant Chemotherapy for Muscle-invasive Bladder Cancer. A Systematic Review and Meta-analysis of Individual Participant Data from Randomised Controlled Trials. Eur Urol 2022;81:50-61. [Crossref] [PubMed]
3. Ghodoussipour S, Bivalacqua T, Bryan RT, et al. A Systematic Review of Novel Intravesical Approaches for the Treatment of Patients with Non-muscle-invasive Bladder Cancer. Eur Urol 2025;88:33-55. [Crossref] [PubMed]
4. Liu W, Tian J, Zhang S, et al. The utilization status of neoadjuvant chemotherapy in muscle-invasive bladder cancer: a systematic review and meta-analysis. Minerva Urol Nephrol 2021;73:144-53. [Crossref] [PubMed]
5. Grossman HB, Natale RB, Tangen CM, et al. Neoadjuvant chemotherapy plus cystectomy compared with cystectomy alone for locally advanced bladder cancer. N Engl J Med 2003;349:859-66. [Crossref] [PubMed]
6. Sherif A, Rintala E, Mestad O, et al. Neoadjuvant cisplatin-methotrexate chemotherapy for invasive bladder cancer -- Nordic cystectomy trial 2. Scand J Urol Nephrol 2002;36:419-25. [Crossref] [PubMed]
7. Sherif A, Holmberg L, Rintala E, et al. Neoadjuvant cisplatinum based combination chemotherapy in patients with invasive bladder cancer: a combined analysis of two Nordic studies. Eur Urol 2004;45:297-303. [Crossref] [PubMed]
8. Li F, Zheng Z, Chen W, et al. Regulation of cisplatin resistance in bladder cancer by epigenetic mechanisms. Drug Resist Updat 2023;68:100938. [Crossref] [PubMed]
9. Galluzzi L, Senovilla L, Vitale I, et al. Molecular mechanisms of cisplatin resistance. Oncogene 2012;31:1869-83. [Crossref] [PubMed]
10. Singh A, Settleman J. EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene 2010;29:4741-51. [Crossref] [PubMed]
11. Dongre A, Weinberg RA. New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer. Nat Rev Mol Cell Biol 2019;20:69-84. [Crossref] [PubMed]
12. Lambert AW, Pattabiraman DR, Weinberg RA. Emerging Biological Principles of Metastasis. Cell 2017;168:670-91. [Crossref] [PubMed]
13. Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol 2014;15:178-96. [Crossref] [PubMed]
14. Simpson KL, Rothwell DG, Blackhall F, et al. Challenges of small cell lung cancer heterogeneity and phenotypic plasticity. Nat Rev Cancer 2025;25:447-62. [Crossref] [PubMed]
15. Li WW, Li VW, Tsakayannis D. p53 and bladder cancer. N Engl J Med 1995;332:957-author reply 958. [Crossref] [PubMed]
16. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell 1997;88:323-31. [Crossref] [PubMed]
17. Jing X, Yang F, Shao C, et al. Role of hypoxia in cancer therapy by regulating the tumor microenvironment. Mol Cancer 2019;18:157. [Crossref] [PubMed]
18. Duffy MJ, Synnott NC, O’Grady S, et al. Targeting p53 for the treatment of cancer. Semin Cancer Biol 2022;79:58-67. [Crossref] [PubMed]
19. Marine JC, Jochemsen AG. MDMX (MDM4), a Promising Target for p53 Reactivation Therapy and Beyond. Cold Spring Harb Perspect Med 2016;6:a026237. [Crossref] [PubMed]
20. Wade M, Li YC, Wahl GM. MDM2, MDMX and p53 in oncogenesis and cancer therapy. Nat Rev Cancer 2013;13:83-96. [Crossref] [PubMed]
21. Hu K, Yin F, Yu M, et al. In-Tether Chiral Center Induced Helical Peptide Modulators Target p53-MDM2/MDMX and Inhibit Tumor Growth in Stem-Like Cancer Cell. Theranostics 2017;7:4566-76. [Crossref] [PubMed]
22. Ellison V, Polotskaia A, Xiao G, et al. A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin. Nucleic Acids Res 2025;53:gkaf627. [Crossref] [PubMed]
23. Karni-Schmidt O, Lokshin M, Prives C. The Roles of MDM2 and MDMX in Cancer. Annu Rev Pathol 2016;11:617-44. [Crossref] [PubMed]
24. Li Q, Lozano G. Molecular pathways: targeting Mdm2 and Mdm4 in cancer therapy. Clin Cancer Res 2013;19:34-41. [Crossref] [PubMed]
25. Huang P, Wang J, Yu Z, et al. Redefining bladder cancer treatment: innovations in overcoming drug resistance and immune evasion. Front Immunol 2025;16:1537808. [Crossref] [PubMed]
26. Böttger V, Böttger A, Garcia-Echeverria C, et al. Comparative study of the p53-mdm2 and p53-MDMX interfaces. Oncogene 1999;18:189-99. [Crossref] [PubMed]
27. Espadinha M, Lopes EA, Marques V, et al. Discovery of MDM2-p53 and MDM4-p53 protein-protein interactions small molecule dual inhibitors. Eur J Med Chem 2022;241:114637. [Crossref] [PubMed]
28. Cheng Y, Mo F, Li Q, et al. Targeting CXCR2 inhibits the progression of lung cancer and promotes therapeutic effect of cisplatin. Mol Cancer 2021;20:62. [Crossref] [PubMed]
29. Yuan J, Liu M, Yang L, et al. Acquisition of epithelial-mesenchymal transition phenotype in the tamoxifen-resistant breast cancer cell: a new role for G protein-coupled estrogen receptor in mediating tamoxifen resistance through cancer-associated fibroblast-derived fibronectin and β1-integrin signaling pathway in tumor cells. Breast Cancer Res 2015;17:69. [Crossref] [PubMed]
30. Liu Y, He S, Wang XL, et al. Tumour heterogeneity and intercellular networks of nasopharyngeal carcinoma at single cell resolution. Nat Commun 2021;12:741. [Crossref] [PubMed]
31. Liu Y, Wang X, Wang G, et al. The past, present and future of potential small-molecule drugs targeting p53-MDM2/MDMX for cancer therapy. Eur J Med Chem 2019;176:92-104. [Crossref] [PubMed]
32. Wang C, Wan S, Li K, et al. TPI1 promotes p53 ubiquitination in bladder cancer by recruiting AKT to enhance MDM2 phosphorylation. Pharmacol Res 2025;215:107695. [Crossref] [PubMed]
33. Saha G, Ghosh MK. The key vulnerabilities and therapeutic opportunities in the USP7-p53/MDM2 axis in cancer. Biochim Biophys Acta Mol Cell Res 2025;1872:119908. [Crossref] [PubMed]