Abstract

   Clear cell renal cell carcinoma (ccRCC) exhibits considerable
   heterogeneity, with approximately 25% of localized cases susceptible to
   relapse, highlighting the challenge of the absence of reliable
   predictive biomarkers for personalized treatment. Meanwhile, metastatic
   renal cell carcinoma is characterized by unfavorable survival rates,
   and although Sunitinib offers partial benefits, the clinical advantages
   are often constrained by drug resistance and adverse side effects.
   Here, MAGI3 was associate with ccRCC progression, as identified through
   comprehensive bioinformatics analysis of clinical datasets. A low level
   of MAGI3 emerged as a high-risk factor for ccRCC, indicating its
   potential as a prognostic marker. Individuals with MAGI3 expression in
   middle-to-low levels displayed a significantly poorer survival rate,
   indicating a need for additional treatment even in the early stages of
   ccRCC. Furthermore, patients with MAGI3 expression in middle-to-high
   levels exhibited increased sensitivity to Sunitinib compared to those
   with lower MAGI3 levels, suggesting that individuals with MAGI3
   expression at middle levels may potentially benefit from Sunitinib
   treatment even in the early stages of ccRCC. Through its interaction
   with the MAS receptor, MAGI3 has been identified as a regulator of cell
   proliferation and a determinant of Sunitinib resistance in ccRCC,
   operating via the Ang-(1-7)/MAS/ERK axis. The loss of MAGI3 expression
   in ccRCC patients activated the ERK signaling pathway, contributing to
   both cancer progression and Sunitinib resistance. Therefore, our study
   not only highlight MAGI3’s pivotal role in ccRCC progression and
   Sunitinib resistance, but also reinforces MAGI3’s prospective value as
   a predictive marker.

   graphic file with name 41419_2025_7427_Figa_HTML.jpg

   Subject terms: Tumour-suppressor proteins, Targeted therapies, G
   protein-coupled receptors

Introduction

   Renal cell carcinoma (RCC) is a highly lethal urological malignancy,
   with clear cell renal cell carcinoma (ccRCC) being the most prevalent
   subtype, and its incidence steadily rising in recent years [[54]1,
   [55]2]. The survival rates exhibit significantly difference between
   early-stage or localized RCC and metastatic RCC (mRCC), with localized
   RCC exhibiting a generally favorable rate, while mRCC falls within a
   range of 5–12% in a 5-year period [[56]3, [57]4]. Surgical nephrectomy
   stands as a highly effective treatment for early-stage or localized
   ccRCC. Traditional therapeutic strategies generally do not incorporate
   additional adjuvant therapies, especially for individuals at low risk
   or in early-stage cases, such as stage I. Despite its effectiveness,
   the substantial heterogeneity within ccRCC leads to diverse outcomes,
   even among patients in the same stage, complicating treatment and
   management. Notably, about 25% of patients with localized ccRCC
   experience relapse following local therapy [[58]5, [59]6].
   Nevertheless, the absence of reliable biomarkers presents a challenge
   in accurately identifying high-risk localized ccRCC patients for
   stratification and personalized management.

   mRCC is a life-threatening disease characterized by poor overall
   survival (OS) rates. Despite advancements in diagnostic techniques,
   approximately 20–30% of ccRCC patients present with local or distant
   metastases at the time of diagnosis [[60]7]. The current therapeutic
   approaches demonstrate limited efficacy, mainly attributed to mRCC’s
   intrinsic resistance to chemotherapy or radiotherapy [[61]8]. Cytokine
   treatment benefits only a small subset of patients, yielding a
   discouraging 5-year survival rate of just 9% [[62]9, [63]10].

   Recently, several small molecule tyrosine kinase inhibitors (TKIs) have
   been developed to treat advanced renal cell carcinoma (aRCC) or mRCC
   [[64]11]. Notably, Sunitinib has proven to be an effective first-line
   treatment, markedly enhancing both progression-free survival (PFS) and
   overall survival (OS) in aRCC or mRCC cases [[65]12–[66]14]. Despite
   its effectiveness, Sunitinib has limitations, with an objective
   response rate of approximately 30%, and a significant number of
   patients eventually experiencing relapse due to the development of drug
   resistance [[67]15, [68]16].

   Multiple lines of evidence indicate that resistance to Sunitinib in
   ccRCC is driven by various factors, including interactions with the
   tumor microenvironment, increased tumor invasiveness and metastasis,
   and resistance mediated by noncoding RNAs [[69]17–[70]19]. Tumors also
   have the ability to activate alternative pathways that bypass the
   targeted receptors, promoting cell survival, proliferation, and
   angiogenesis. Overcoming Sunitinib resistance presents a significant
   challenge, primarily attributed to the insufficient inhibition of its
   intended targets. This inadequacy compromises the drug’s efficacy in
   effectively controlling tumor growth [[71]20]. Sunitinib, a
   multi-targeted tyrosine kinase inhibitor (TKI), exerts inhibitory
   effects on VEGFR, PDGFR, and EGFR within tumor cells. Among the
   downstream pathways activated by these surface receptors, the MAPK/ERK
   pathway plays a crucial role [[72]21]. This pathway transmits growth
   and survival signals from receptors to nuclear transcription machinery
   [[73]22, [74]23]. Dysregulation of the MAPK/ERK pathway is a common
   phenomenon in various human cancers, resulting in alterations in gene
   expression, and promoting cancer cell proliferation, progression and
   angiogenesis [[75]24, [76]25]. Recent studies have highlighted the
   efficacy of inhibiting the MAPK/ERK pathway in inducing apoptosis and
   impeding metastasis in RCC cells [[77]26]. The anti-tumor effects of
   Sunitinib primarily result from its ability to inhibit the ERK pathway,
   leading to increased apoptosis [[78]27–[79]29]. However, resistance to
   Sunitinib often develops due to insufficient ERK inhibition or
   excessive activation through alternative pathways that operate
   independently of growth factor receptors in cancer cells [[80]30]. This
   phenomenon has been observed in various cancer types, including RCC
   tumors [[81]31–[82]33]. Nevertheless, the precise molecular mechanisms
   responsible for dysregulated ERK activation in mRCC remain not
   completely understood.

   In this study, an analysis of differentially expressed genes (DEGs)
   between tumor and normal renal tissues revealed the significant
   enrichment of the MAPK/ERK cascade within the DEGs. Among the subset of
   genes related to the MAPK pathway, MAGI3 expression correlates with
   well-prognostic ccRCC, even at an early stage. Through its interaction
   with the MAS receptor, MAGI3 was identified as a regulator of cell
   proliferation and a determinant of Sunitinib resistance in ccRCC,
   operating via the Ang-(1-7)/MAS/ERK axis. Loss of MAGI3 expression in
   ccRCC patients activated the ERK signaling pathway, contributing to
   both cancer progression and Sunitinib resistance. These findings
   highlight MAGI3 as a novel tumor suppressor in ccRCC and indicate the
   importance of the ACE2/Ang-(1-7)/MAS axis in Sunitinib resistance in
   ccRCC. Additionally, this research highlights the potential of MAGI3 as
   a predictive marker, offering promise in tailoring treatments for
   early-stage ccRCC patients and thereby improving clinical
   decision-making.

Materials and methods

Clear cell renal cell carcinoma specimens

   This study retrospectively collected surgical specimens and
   corresponding data from metastatic ccRCC patients at the First Hospital
   of Shanxi Medical University (2016–2021). The inclusion criteria were
   as follows: 1) Patients underwent primary tumor resection, confirming
   ccRCC with at least one measurable metastatic lesion; 2) Patients
   received oral Sunitinib (50 mg/day for 4 weeks, followed by a 2-week
   break) for a 6-week cycle. Computed Tomography scans assessed
   therapeutic response every two cycles; 3) Complete follow-up
   information on Sunitinib responsiveness and prognosis. The exclusion
   criteria were: 1) Other combination therapies; 2) Discontinuation of
   Sunitinib before one cycle completion due to adverse reactions or
   compliance issues. The study included 47 patients (33 males, 14
   females, average age: 54.2 ± 9.6 years). Tissue microarrays, containing
   90 matched pairs of ccRCC and adjacent normal tissues (51 males, 39
   females, average age: 59.0 ± 11.5 years) were sourced from Shanghai
   Outdo Biotech. The research protocol adhered to the Declaration of
   Helsinki, approved by the Ethics Committee of Capital Medical
   University and the First Hospital of Shanxi Medical University. All
   participating patients provided written informed consent for sample use
   and publication of related data.

Animal study

   To establish a xenograft tumor model, 5 ×10^6 control vector or
   MAGI3-overexpressing 786-O cells were subcutaneously injected into the
   dorsal flank of BALB/c nude mice (male, 6 weeks old, weight 16–18 g,
   n = 18). Tumor growth was monitored, and size was calculated using the
   formula: 1/2 × length × width^2. For Sunitinib treatment, 1 ×10^7 cells
   were injected, and mice (male, 6 weeks old, weight 16-18 g, n = 10)
   received Sunitinib (i.g. 50 mg/kg/day) and Ang-(1-7) (s.c. 20
   ug/kg/day) when tumors reached 80–100 mm^3.

   Experiments concluded when tumors did not exceed 1000 mm^3; mice were
   humanely euthanized, tumors dissected, and subjected to fixation,
   paraffin embedding, and IHC staining.

   Magi3 knockout mice (Magi3^−/−) and their wild-type counterparts
   (Magi3^+/+) were generated by Shanghai Model Organisms Center, Inc.,
   and genotyped using specific primers. Ang-(1-7) or A779 (dissolved in
   0.9% saline) was administered intraperitoneally, with doses of 60 ug/kg
   for Ang-(1-7) and 0.5 ug/kg for A779, given 30 minutes prior. Kidney
   tissue samples underwent IHC and western blotting, with each
   experimental group comprising 2 male mice aged 6 weeks and weighing
   between 16 and 20 g.

   Mice were randomly allocated to either the control or experimental
   groups using a random number method. Grouping was conducted without
   utilizing a blind method, and sample sizes were determined based on
   previous experimental experience to ensure that statistical differences
   could be achieved.

   All animal experiments were conducted in compliance with the National
   Institutes of Health guidelines for the Care and Use of Laboratory
   Animals and approved by the Animal Use and Care Committee of Capital
   Medical University (approval numbers AEEI-2020-133 and AEEI-2018-16).
   Our study adhered to the guidelines set forth by our ethics committee,
   which approved a maximal tumor size/burden of 1000 mm^3. We confirm
   that throughout the study, the maximal tumor size/burden limit was
   strictly adhered to and not exceeded.

Statistical analysis

   Statistical analysis utilized GraphPad Prism 7 software. Data, unless
   specified otherwise, is presented as mean ± SEM. Measurement data were
   assessed for normal distribution and homogeneity of variance using
   appropriate tests (homogeneity of variance was considered present when
   P > 0.1). Paired or unpaired t-tests were employed for continuous
   variables, with nonparametric tests used for data not assuming a
   Gaussian distribution. Chi-square or Fisher exact tests were utilized
   to compare distribution differences between two groups. Spearman
   correlation assessed variable correlations. Time-to-event outcomes were
   evaluated using Kaplan–Meier curves with log-rank tests. The
   determination of sample size was based on statistical power analysis to
   ensure sufficient ability to detect a pre-specified effect size. We
   assumed a moderate effect size, with an alpha level set at 0.05, and a
   power (1-β) set at 0.80. A significance level of P < 0.05 (two-tailed)
   was considered statistically significant.

   The remaining materials and methods can be found in the supplementary
   materials.

Results

MAGI3 is identified as a novel prognostic indicator in ccRCC and is involved
in MAPK signaling

   Through differential gene expression analysis across multiple ccRCC
   datasets, we identified 984 differentially expressed genes (384
   upregulated and 600 downregulated), with pathway enrichment
   highlighting significant involvement in cancer-related signaling
   pathways (Supplementary Fig. [83]1A). Notably, six of the top 20
   enriched pathways were associated with the MAPK cascade (Supplementary
   Fig. [84]1B), underscoring its significance in ccRCC development.
   Within the subset of MAPK-related genes, 21 genes, including MAGI3,
   exhibited a notable correlation with the overall survival of ccRCC
   patients (Supplementary Fig. [85]1C). Despite MAGI3 not having been
   previously associated with ccRCC, its role in regulating ERK
   activation, as reported in our earlier publications [[86]34–[87]37],
   prompted a focused exploration into how MAGI3 expression influences
   ccRCC initiation and progression through ERK signaling.

   To validate MAGI3 downregulation in ccRCC, we examined protein levels
   in 110 ccRCC samples from a Chinese cohort, revealing a substantial
   decrease compared to normal renal tissues (Fig. [88]1A, B). mRNA
   analysis also showed significantly reduced MAGI3 levels in ccRCC
   tissues, consistent with TCGA data (Fig. [89]1C) and GEO datasets
   ([90]GSE16449, [91]GSE53757, and [92]GSE66271) (Supplementary Fig.
   [93]1D–F).

Fig. 1. Dysregulated low levels of MAGI3 correlate with poor prognosis in
ccRCC.

   [94]Fig. 1
   [95]Open in a new tab

   A, B Immunohistochemical (IHC) analysis of MAGI3 protein in ccRCC
   specimens. Representative images of MAGI3 staining in adjacent tissues
   or tumors (A). Scale bars: 200 μm. Magnified images of dashed areas are
   displayed in the right panels. Scale bars: 50 μm. Dot plot showing the
   corresponding quantification of the MAGI3 H-score (B). Data presented
   as mean ± SEM. Statistical significance was calculated by the
   Mann–Whitney test. ***P < 0.001. C Dot plot comparing the mRNA
   expression level of MAGI3 in TCGA ccRCC specimens with paired normal
   tissues. Statistical significance is determined using the paired t-test
   (****P < 0.0001). D–F TCGA ccRCC patients were stratified into two
   groups based on ccRCC specimens MAGI3 expression cutoff at 261.5 (reads
   per kilobase of transcript, per million mapped reads, RPKM): high and
   middle-to-low (M/L). Kaplan–Meier survival plots for overall survival
   (OS) (D), progression-free survival (PFS) (E) and disease-specific
   survival (DSS) (F) of the two groups (log-rank test). G Dot plot
   showing the mRNA expression level of MAGI3 in tumor tissues and
   adjacent tissues of TCGA ccRCC patients with stage I. Statistical
   significance was calculated using the unpaired t-tests
   (****P < 0.0001). Kaplan–Meier survival plots for OS (H), PFS (I) and
   DSS (J) according to MAGI3 mRNA level in tumor tissue of TCGA ccRCC
   patients with stage I (log-rank test). K, L Prognostic analysis of OS
   according to MAGI3 histochemistry score (H-score) in tumor tissue of
   ccRCC patients with stage I from Chinese cohort. Representative images
   with different MAGI3 H-scores (K). Scale bars: 200 μm. The magnified
   images are displayed in the right panels. Scale bars: 50 μm.
   Kaplan–Meier survival plots for OS of patients with MAGI3 at high
   levels (H-score ≥ 8) and MAGI3 at middle-to-low levels (M/L,
   H-score ≤ 6) (log-rank test) (L).

   To assess MAGI3’s clinical relevance, the TCGA database excluded
   patients undergoing adjuvant therapy. The remaining cohort was divided
   into high (H, >261.5 RPKM) and middle-to-low (M/L, <261.5 RPKM) MAGI3
   expression groups. Kaplan–Meier analysis revealed a substantial
   divergence, particularly within the M/L group, associated with lower
   10-year overall survival (OS) (32.7% vs. 58.5%), progression-free
   survival (PFS) (37.9% vs. 48.3%), and disease-specific survival (DSS)
   (51.7% vs. 78.6%) rates (Fig. [96]1D–F, Supplementary Table [97]1).
   Notably, in stage I ccRCC patients, M/L MAGI3 expression (<261.5 RPKM)
   correlated with markedly reduced 10-year OS (45.5% vs. 74.6%), PFS (55%
   vs. 69.8%), and DSS (77.9% vs. 98%) compared to high MAGI3 expression
   (Fig. [98]1G–J, Supplementary Table [99]2). Consistent findings were
   validated across independent ccRCC cohorts, confirming the correlation
   between M/L MAGI3 levels [Histochemistry score (H-score) ≤6)] and an
   unfavorable prognosis, particularly at the protein level in stage I
   cases (Fig. [100]1K, L). Cox univariate and multivariate analysis
   identified MAGI3 protein level as an independent prognostic factor for
   stage I ccRCC patients (Supplementary Table [101]3). These results
   establish a significant association between reduced MAGI3 levels (M/L)
   and an unfavorable prognosis in ccRCC patients, revealing MAGI3 as a
   promising novel prognostic marker and providing insights into its
   pivotal role in ccRCC initiation and progression.

Interaction between MAGI3 and MAS receptor

   MAGI3, recognized for its scaffolding role with five PDZ domains, forms
   multi-protein complexes. To gain a deeper understanding of MAGI3’s role
   in ccRCC, it is essential to identify its binding partners within renal
   cells. A previous proteomic analysis suggested a potential interaction
   between MAGI3 and the MAS receptor in human kidney cells, although
   without verification [[102]38]. The potential interaction with the MAS
   receptor in renal cells was identified through GSEA analysis of the
   MAGI3 M/L group in ccRCC, indicating MAGI3’s involvement in MAS
   activation (Fig. [103]2A). Our previous investigation revealed a
   PDZ-binding motif at the carboxyl terminus (CT) of MAS [[104]34],
   implying a direct association between MAGI3 and MAS signaling in ccRCC.

Fig. 2. MAGI3 specifically interacts with MAS through its carboxyl terminus.

   [105]Fig. 2
   [106]Open in a new tab

   A GSEA enrichment plots showing significant activation of MAS receptor
   in ccRCC specimens with low MAGI3 expression from the TCGA dataset. B
   The carboxyl terminus of MAS (MAS-CT) specifically binds to MAGI3 PDZ1
   domains in an overlay assay. PDZ proteins (His tag) are separated
   through SDS-PAGE, and the isolated proteins are overlaid with GST-MAS
   followed by Western blot analysis. C The binding between MAS-CT and
   MAGI3 PDZ1 is disrupted when the ETVV motif in MAS-CT is mutated. GST
   fusion proteins of wild-type and mutant MAS-CT were employed for
   pull-down assays, and immunoblotting utilized an anti-His antibody. D
   The interaction between MAS-CT and MAGI3 is dependent on the ETVV motif
   at the C-terminal. In the pull-down assay, COS-7 cells were transfected
   with Flag-tagged MAGI3, the cell lysates were pulldown with either
   wild-type GST-MAS-CT or the ETVA mutant, followed by Western blot
   analysis. E Co-immunoprecipitation assay demonstrates the interaction
   between full-length MAS and MAGI3 through the ETVV motif at the MAS
   C-terminal. COS-7 cells were transfected with GFP-MAS WT or GFP-MAS
   V325A, both in the presence or absence of co-transfection with
   Flag-MAGI3, followed by Western blot analysis. F Co-immunoprecipitation
   of endogenous MAGI3 and MAS proteins in mouse kidney tissue. G, H
   Immunofluorescence co-localization of MAS and MAGI-3. BHK cells were
   transfected with GFP-MAS WT or GFP-MAS V325A, both in the presence or
   absence of co-transfection with Flag-MAGI3, and the co-localization was
   visualized through merging of individual images, Scale bars: 10 μm.
   Impaired colocalization was observed when the MAS V325A mutation was
   present.

   Through overlay assays, we confirmed a robust interaction between MAS’s
   CT and MAGI3’s first PDZ domain (PDZ1) (Fig. [107]2B). Furthermore, by
   generating mutants in MAS-CT’s PDZ-binding motif (PBM) and conducting
   GST pull-down assays, we determined that mutations in any PBM residue
   abolished the interaction with MAGI3 PDZ1 (Fig. [108]2C). This specific
   binding of MAS-CT to full-length MAGI3 was confirmed by GST pull-down
   assays (Fig. [109]2D). Co-transfection of Flag-MAGI3 and wild-type
   GFP-MAS in cellular experiments confirmed their interaction via
   co-immunoprecipitation assays; however, this interaction was disrupted
   by the MAS point mutant V325A (Fig. [110]2E), consistent with prior
   findings (Fig. [111]2C, D). Importantly, the presence of the endogenous
   MAS/MAGI3 complex was detected in mouse kidney tissue (Fig. [112]2F).

   Immunofluorescence co-localization studies provided additional evidence
   of the MAGI3 and MAS interaction. Co-transfection of wild-type MAS and
   MAGI3 showed substantial co-localization (Fig. [113]2H a-c), while the
   MAS V325A mutant exhibited minimal co-localization with MAGI3 (Fig.
   [114]2H d-f).

   Collectively, these findings provide evidence of a physical association
   between MAS and MAGI3 in renal cells, facilitated by the PDZ1 domain of
   MAGI3 and the C-terminal of MAS.

MAGI3 suppresses MAS-mediated cell proliferation in ccRCC

   To elucidate the biological significance of MAGI3, we analyzed the TCGA
   ccRCC dataset, stratifying data into MAGI3 expression high and
   middle/low groups. GSEA revealed significant enrichment of the cell
   proliferation gene signature in the MAGI3 M/L group (Supplementary Fig.
   [115]2A). Reduced MAGI3 expression showed a strong association with
   larger tumor size (Supplementary Fig. [116]2B), revealing the link
   between MAGI3 and cell proliferation in ccRCC. Considering that cell
   proliferation is a critical hallmark often driven by MAPK/ERK
   activation, and the MAS receptor is associated with promoting cell
   growth [[117]39–[118]41], we explored the impact of the MAS/MAGI3 axis
   on ccRCC cell proliferation.

   Stable transfections of 786-O and 769-P ccRCC cells were established
   with control or MAGI3 constructs (Fig. [119]3A). Cell growth, assessed
   through CCK8 colorimetric and colony-formation assays under low serum
   conditions in the presence of Ang-(1-7), revealed that MAGI3
   overexpression significantly reduced Ang-(1-7)-induced cell
   proliferation (Fig. [120]3B) and colony formation (Fig. [121]3C, D) in
   ccRCC cells. Conversely, depleting MAGI3 expression in 786-O and 769-P
   ccRCC cells (Fig. [122]3E) significantly increased cell proliferation
   (Fig. [123]3F) and clonogenicity (Fig. [124]3G, H). Additionally, MAGI3
   overexpression induced cell cycle arrest in the G0/G1 phase in ccRCC
   cells (Supplementary Fig. [125]2C).

Fig. 3. MAGI3 inhibits proliferation of ccRCC cells.

   [126]Fig. 3
   [127]Open in a new tab

   A MAGI3 overexpression was confirmed via Western blot analysis in 786-O
   and 769-P cells following transfection with lentivirus carrying MAGI3
   coding sequence or control vectors. B Increased MAGI3 expression
   reduced cell viability in 786-O and 769-P cells, as assessed over a
   5-day period using a CCK8 assay. C, D Upregulated MAGI3 expression
   decreased colony formation ability, evident in crystal violet staining
   and photographs of ccRCC cell colonies formed over a 10-day culture
   period (C). Quantitative analysis shows a reduced number of colonies
   (D). E MAGI3 knockdown was confirmed in 786-O and 769-P cells using
   lentivirus-mediated delivery of shRNA, as evidenced by Western blot
   analysis. F Decreased MAGI3 expression enhanced cell viability,
   observed over a 5-day period through CCK8 assays in 786-O and 769-P
   cells. G, H Reduced MAGI3 expression increased colony formation in
   ccRCC cells, as observed through crystal violet staining and colony
   photography (G). Quantitative analysis indicated an increased number of
   colonies (H). I, J The inhibitory effect of MAGI3 on cell proliferation
   was abolished by blocking the MAS receptor with A779, as demonstrated
   in CCK8 (I) and cell colony formation (J) assays in
   MAGI3-overexpressing 786-O cells and control cells treated with A779.
   Statistical significance is indicated by ***(P < 0.001); NS
   non-significant.

   Importantly, the inhibitory impact of MAGI3 on ccRCC cell proliferation
   (Fig. [128]3I) and colony formation (Fig. [129]3J) was nullified with
   the administration of the MAS receptor antagonist A779, indicating that
   MAGI3 constrains cell proliferation via the MAS receptor.

MAGI3 suppresses MAS-mediated ERK signaling pathway in ccRCC cell

   The MAPK/ERK pathway, a critical regulator of cellular processes like
   proliferation, has been identified to associate with MAGI3
   (Supplementary Fig. [130]1B). GSEA analysis further reveals a
   significant enrichment of the gene signature related to ERK activation
   in the MAGI3 M/L group of ccRCC, establishing an association between
   MAGI3 and the activation of MAPK/ERK signaling in ccRCC cells
   (Supplementary Fig. [131]3A).

   To explore the connection between the MAPK/ERK pathway and
   Ang-(1-7)/MAS signaling, 786-O cells transfected with the MAS receptor
   were stimulated with varying Ang-(1-7) concentrations for different
   durations. ERK phosphorylation peaked at 10 minutes (Fig. [132]4A and
   Supplementary Fig. [133]3B), exhibiting a concentration-dependent
   increase, with maximum activation observed at an Ang-(1-7)
   concentration of 10^-6 M (Fig. [134]4B and Supplementary Fig. [135]3C).

Fig. 4. MAGI3 inhibits MAS-mediated ERK activation.

   [136]Fig. 4
   [137]Open in a new tab

   A Time-dependent analysis of ERK activation in 786-O cells stimulated
   with 10 μM Ang-(1-7). Phosphorylated ERK1/2 was assessed at different
   time points following Ang-(1-7) treatment in serum-starved 786-O cells.
   B Dose-response evaluation of Ang-(1-7)-induced ERK activation in 786-O
   cells. Various concentrations of Ang-(1-7) were administered to
   serum-starved COS-7 cells for 5 minutes, and ERK phosphorylation was
   measured. C MAGI3 inhibits Ang-(1-7)-induced ERK activation in 786-O
   cells. 786-O cells, transfected with Flag or Flag-MAGI3 underwent serum
   starvation and were stimulated with 10 μM Ang-(1-7) for varying
   durations. MAGI3 exhibited substantial inhibition of ERK
   phosphorylation, with the most pronounced effect observed at
   10 minutes. D MAGI3 knockdown promotes MAS-mediated ERK activation in
   786-O cells. 786-O cells with reduced MAGI3 expression and control
   cells were serum-starved and stimulated with 10 μM Ang-(1-7) for 5 min.
   ERK phosphorylation was measured via Western blot. E Specific
   inhibition of Ang-(1-7)-induced ERK activation in 786-O cells by
   blocking MAS activation with A779. Serum-starved 786-O cells
   transiently overexpressing Flag or Flag-MAGI3 were treated with the MAS
   antagonist A779 (1 μM) and 10 μM Ang-(1-7) for 5 min. Both MAGI3
   overexpression and A779 treatment effectively suppressed MAS-mediated
   ERK phosphorylation. F MAGI3’s interaction with MAS contributes to the
   inhibition of MAS-induced ERK activation. 786-O cells, transfected with
   MAS or MAS V325A mutant, and MAGI3 were serum-starved and stimulated
   with 10 μM Ang-(1-7) for 5 min. Western blot analysis revealed that
   MAGI3 significantly inhibited MAS-mediated ERK activation but had
   little effect on MAS V325A. Ang-(1-7)-induced ERK activation in the
   absence of MAGI3 was used as a control. G, H MAGI3’s inhibitory effect
   on cell proliferation is abolished when the ERK pathway is blocked with
   SCH772984 in MAGI3-overexpressing 786-O cells, as demonstrated by CCK8
   (G) and cell colony formation assays (H). Statistical significance is
   denoted by ***(P < 0.001); NS indicates non-significance.

   In investigating MAGI3’s molecular mechanism, we examined
   Ang-(1-7)-induced ERK activation in 786-O cells with MAGI3
   overexpression and knockdown. MAGI3 overexpression significantly
   reduced Ang-(1-7)-induced ERK phosphorylation (Fig. [138]4C), while
   MAGI3 depletion amplified ERK activity (Fig. [139]4D). A779, a MAS
   receptor antagonist, almost completely inhibited Ang-(1-7)-induced ERK
   activation (Fig. [140]4E). These findings suggest that Ang-(1-7)
   primarily signals through MAS to activate the ERK pathway, and MAGI3
   can restrain this activation. The inhibitory effect of MAGI3 on ERK
   activation is dependent on the MAS/MAGI3 interaction, as demonstrated
   by reduced phosphorylated ERK levels in cells with wild-type MAS but
   not in those with the MAS-V325A mutant, disrupting the MAS/MAGI3
   interaction (Fig. [141]4F and Supplementary Fig. [142]3D).

   Examining the impact of blocking the ERK signaling pathway on MAGI3’s
   effects on MAS-mediated cell functions, we found that the ERK inhibitor
   SCH772984 effectively reduced Ang-(1-7)-induced proliferation and
   colony formation in 786-O cells, confirming the crucial role of the ERK
   signaling pathway in MAS-mediated cell growth. Notably, SCH treatment
   showed no additional inhibition of cell growth in MAGI3-overexpressing
   cells, indicating that MAGI3 hampers MAS-mediated renal cell
   proliferation by suppressing the ERK signaling pathway (Fig. [143]4G,
   H). These results highlight the significant contribution of the
   MAS/MAGI3/ERK axis to ccRCC cell proliferation.

   To delve into the mechanisms of MAGI3 disrupting the ERK pathway via
   interaction with MAS, we utilized AlphaFold for structural prediction.
   Using protein files from the PDB database, we discerned detailed
   protein surfaces. Structural analysis highlighted a hydrophobic pocket
   in MAGI3-PDZ1 and an alignment of MAS receptor’s C-terminal with the
   class I PDZ-binding motif (PBM), forming the basis for their reciprocal
   binding (Fig. [144]5A–C).

Fig. 5. Molecular dynamics simulation of the carboxyl terminus of MAS bound
with MAGI3 to block the coupling of G protein with the intracellular loop 2
(ICL2) of MAS.

   [145]Fig. 5
   [146]Open in a new tab

   A Predicted AlphaFold models of MAGI3-PDZ1, with the carboxylate
   binding loop (Gly-419, Phe-420, Gly-421, Phe-422, Thr-423, Ile-424)
   highlighted in blue, and other residues in green. MAS-PBM is colored in
   oranges (Cys-316, Asn-317, Thr-318, Val-319, Thr-320, Val-321) and
   magentas (Glu-322, Thr-323, Val-324, Val-325). B, C Molecular
   simulation of MAGI3-PDZ1/MAS-PBM complex. Front (B) and side (C) views
   of the interaction between MAGI3-PDZ1/MAS-PBM. D. Ribbon diagram
   representation of MAGI3-PDZ1 and MAS-PBM structure. Colors correspond
   to those in Fig. 5A. The “ETVV” motif of MAS is labeled. E Stereoview
   of the detailed interface of MAGI3-PDZ1/MAS-PBM complex. Dotted lines
   represent chemical bonds. Right panels show magnifications of the
   wireframe areas on the left. The “GFGFTI” motif of MAGI3-PDZ1 and
   “ETVV” motif of MAS are labeled. F Crystal structure of MRGPRX2-Gq
   signaling complex (PDB: 7S8N). The ICL2 that interacts with Gαq protein
   on MRGPRX2 is colored in pink. G Structural comparison of MAS AlphaFold
   model with MRGPRX2. Lower panel show magnifications of the wireframe
   area on the upper. H Molecular dynamics simulation of MAS/MAGI3-PDZ1
   complex, indicating the interaction of MAGI3-PDZ1 with MAS-PBM
   obscuring the ICL2 on MAS that interacts with G protein.

   Further analysis revealed the chemical bonds between MAS’s C-terminal
   motif “ETVV” and the hydrophobic loop sequence “GFGFTI” of MAGI3’s
   PDZ1, emphasizing the crucial role of the “ETVV” motif in MAGI3-MAS
   binding (Fig. [147]5D, E, Supplementary Table [148]4), consistent with
   our previous overlay and GST pulldown experiments (Fig. [149]2B, C).
   Subsequently, we investigated the impact of MAGI3-PDZ1 and MAS
   interaction on MAS’s coupling with G-proteins. Literature and
   structural studies suggested the intracellular loop 2 (ICL2) as the
   potential G-protein coupling site for MAS, similar to MRGPR2 (Fig.
   [150]5F, G, Supplementary Table [151]5) [[152]42–[153]44]. Notably, the
   interaction orchestrated by MAS’s “ETVV” motif with MAGI3-PDZ1
   significantly obstructed MAS’s ICL2 coupling with G-proteins (Fig.
   [154]5H). These findings lead us to propose that MAGI3’s interaction
   with MAS impedes G-protein coupling with the MAS receptor, ultimately
   inhibiting the activation of the ERK pathway.

MAGI3 regulates cell proliferation through the MAS/ERK axis in vivo

   In tumor-bearing experiments with BLAB/c nude mice injected with
   MAGI3-overexpressing 786-O cells and treated with Ang-(1-7), MAGI3
   overexpression significantly inhibited xenograft tumor growth. These
   tumors exhibited reduced weight and volume, along with decreased
   expression of Ki67 and phosphorylated ERK, emphasizing MAGI3’s
   regulatory role (Fig. [155]6A–E).

Fig. 6. MAGI3 inhibits MAS-induced ccRCC tumor proliferation in vivo.

   [156]Fig. 6
   [157]Open in a new tab

   A–C Upregulation of MAGI3 inhibits MAS-induced tumor growth in
   xenografted ccRCC cells in BLAB/c nude mice. After subcutaneously
   implanting 786-O cells overexpressing MAGI3 or control vector cells,
   subcutaneous injections of 0.5 mg/kg Ang-(1-7) were administered every
   24 h upon tumor formation. Tumor volumes were monitored at specified
   intervals, with the largest tumors allowed to reach 1000 mm^3. Upon
   sacrifice, tumor weights were recorded. Displayed are representative
   images of xenograft tumors (A) tumor weight (B) and tumor growth curves
   (C). D, E Downregulation of pERK and Ki-67 levels in transplanted
   tumors with MAGI3 overexpression. Paraffin sections from the 786-O
   ccRCC tumors in nude mice were subjected to immunohistochemistry
   staining, utilizing MAGI3, pERK, and Ki-67 antibodies. Scale bars: 50
   μm. Data are presented as mean ± SEM. Significance levels are indicated
   as *, **, *** (P < 0.05, P < 0.01, P < 0.001). F Representative
   immunohistochemistry staining of MAGI3 and pERK in kidney tissues from
   Magi3^−/− or Magi3^+/+ mice. Scale bars: 200 μm. Right panels exhibit
   magnifications of the dashed areas on the left. Scale bars: 50 μm. G
   Magi3 knockdown fails to trigger ERK phosphorylation by blocking MAS
   activation with A779. Western blot analysis of pERK in kidney tissues
   from Magi3^−/− or Magi3^+/+ mice, with or without A779 pretreatment. H
   MAGI3’s impact on ccRCC cell proliferation mediated by the MAS/ERK
   axis. Enrichment plots from gene set enrichment analysis (GSEA) reveal
   significant activation of MAS, ERK, cell cycle, and proliferation
   pathways in ccRCC specimens with middle-low (M/L, <261.5 RPKM) MAGI3
   expression from the TCGA ccRCC patients with stage I.

   To confirm MAGI3’s impact on ERK activation in renal cell in vivo,
   Magi3-knockout (KO) mice were generated. Immunohistochemical results
   consistently showed a significant accumulation of phosphorylated ERK in
   the kidneys of Magi3-deficient mice (Fig. [158]6F). Importantly,
   Western blot analysis demonstrated that Magi3 knockdown did not induce
   ERK pathway activation in mice treated with A779 (Fig. [159]6G).

   The in vivo mouse experiments, along with clinical data analysis
   (Supplementary Figs. [160]2A, [161]3A, and Fig. [162]2A), unequivocally
   demonstrate a substantial rise in MAS-mediated ERK pathway activation
   and its pro-proliferative effects in the absence of MAGI3, even in the
   early stages of ccRCC (Fig. [163]6H). In summary, these findings
   collectively confirm the pivotal role of MAGI3 in inhibiting renal cell
   proliferation by modulating the MAS/ERK axis in both in vitro and in
   vivo settings.

MAGI3 modulates Sunitinib sensitivity in ccRCC cells

   Given that the inhibition of the ERK signaling pathway is a major
   mechanism through which Sunitinib suppresses tumor cell proliferation,
   we examined the impact of MAGI3 expression on Sunitinib sensitivity in
   ccRCC cells. Firstly, we analyzed the expression changes of MAIG3 in
   Sunitinib resistant renal cancer cells in the [164]GSE64052 database,
   and the results showed that the mRNA level of MAGI3 was significantly
   downregulated in Sunitinib-resistant cells (Supplementary Fig.
   [165]4A). Subsequently, our cell experiments showed that overexpression
   of MAGI3 significantly reduced the IC50 of Sunitinib in 786-O cells
   (from 28.19 to 10.12 μM) and 769-P cells (from 79.71 to 16.60 μM) under
   Ang1-7 stimulation. Interestingly, MAGI3-induced cellular sensitivity
   to Sunitinib was not further enhanced with pre-treatment using A779 to
   block MAS activation (Fig. [166]7A, B). Conversely, depletion of MAGI3
   expression increased the IC50 of Sunitinib in 786-O cells (from 19.96
   to 50.60 μM) and 769-P cells (from 99.29 to 273.5 μM) under Ang1-7
   stimulation. However, pre-treatment with A779 significantly mitigated
   the Sunitinib resistance induced by MAGI3 knockdown (Fig. [167]7C, D).
   Colony formation assays supported these findings, demonstrating that
   MAGI3 overexpression increased sensitivity in 786-O cells, while MAGI3
   knockdown reduced sensitivity in 769-P cells. Importantly,
   pre-treatment with A779 nullified the changes in Sunitinib sensitivity
   resulting from MAGI3 manipulation (Fig. [168]7E–H).

Fig. 7. MAGI3 improves Sunitinib sensitivity by suppressing the MAS/ERK
signaling pathway.

   [169]Fig. 7
   [170]Open in a new tab

   A, B MAGI3 overexpression reduces Sunitinib IC50 in 786-O and 769-P
   cells with MAS activation. Survival curves of MAGI3-overexpressing
   786-O (A) and 769-P (B) cells treated with increasing sunitinib
   concentrations for 48 h, with or without A779 pretreatment. Mean ± SEM;
   n = 3; two-way ANOVA. C, D With MAS activation, MAGI3 knockdown
   increases sunitinib IC50 in 786-O and 769-P cells. Survival curves of
   MAGI3 knockdown 786-O (C) and 769-P (D) cells treated with increasing
   sunitinib concentrations for 48 h, with or without A779 pretreatment.
   Mean ± SEM; n = 3; two-way ANOVA. E, F MAGI3 overexpression
   significantly inhibits colony formation in sunitinib-treated 786-O
   cells with MAS activation. Quantification analysis of colony formation
   assays in MAGI3-overexpressing 786-O cells treated with increasing
   concentrations of sunitinib for 10 days, with or without A779
   pretreatment. Mean ± SEM; n = 3; one-way ANOVA. G, H MAGI3 knockdown
   significantly promotes colony formation in sunitinib-treated 769-P
   cells in the presence of MAS activation. Quantification analysis of
   colony formation assays in MAGI3 knockdown 769-P cells treated with
   increasing concentrations of sunitinib for 10 days, with or without
   A779 pretreatment. Mean ± SEM; n = 3; one-way ANOVA. I–L MAGI3 enhances
   Sunitinib sensitivity in renal cell carcinoma xenografts. Nude mice
   transplanted with MAGI3-overexpressing 786-O cells or control cells
   were treated with Ang-(1-7) and Sunitinib. Representative images of
   xenograft tumors (I) tumor growth curves, two-way ANOVA (J) and tumor
   weight (K) are shown. Representative IHC staining of pERK and ki67 in
   xenografted tumors are shown in L. Data are presented as mean ± SEM.
   *P < 0.05, **P < 0.01, ***P < 0.001.

   In the Sunitinib-treated subcutaneous xenograft tumor model, MAGI3
   overexpression led to significantly slowed tumor growth, reduced
   weight, and diminished levels of phosphorylated ERK and ki67, affirming
   the pivotal role of the MAS/MAGI3 axis in regulating Sunitinib
   sensitivity in ccRCC cells (Fig. [171]7I–L).

   In a clinical study using IHC staining on ccRCC patients undergoing
   Sunitinib treatment, those with higher MAGI3 levels and lower
   phosphorylated ERK levels displayed a favorable response, while poor
   responders exhibited the opposite (Fig. [172]8A–C). A significant
   negative correlation between MAGI3 protein levels and phosphorylated
   ERK levels was identified (r = −0.6, Fig. [173]8D). Patients with MAGI3
   in middle-to-high levels (H/M, H-score ≥4) demonstrated a substantially
   higher response rate to Sunitinib compared to those with lower levels
   (H-score ≤3) (47.1% vs. 15.8%, Fig. [174]8E), highlighting the
   association between MAGI3 expression and Sunitinib response in a
   clinical context. Kaplan–Meier survival analysis revealed that patients
   with lower MAGI3 H-scores had worse OS [Hazard Ratio (HR) = 2.64, 95%
   Confidence Interval (CI): 1.043–6.680] and PFS (HR = 2.619, 95% CI:
   1.123–6.105) compared to those with MAGI3 H/M in H-scores (Fig.
   [175]8F, G).

Fig. 8. High MAIG3 levels associated with increased sensitivity to Sunitinib,
and the reduction of ERK signaling in ccRCC specimens.

   [176]Fig. 8
   [177]Open in a new tab

   A IHC analysis of MAGI3 and p-ERK in ccRCC specimens from 47
   Sunitinib-treated patients. Representative images of MAGI3 and p-ERK
   staining in Sunitinib responders (R) and non-responders (NR). Scale
   bars: 200 μm. The right panels display magnified views of the dashed
   areas on the left. Scale bars: 50 μm. Quantification of MAGI3 H-score
   (B) and p-ERK H-score (C) in ccRCC specimens. Data is presented as
   mean ± SEM. Statistical significance was determined using the
   Mann–Whitney test. *P < 0.05, **P < 0.01. D Correlation analysis
   (Spearman) between MAGI3 H-score and p-ERK H-score in ccRCC specimens.
   E Evaluation of the response ratio to Sunitinib in patients with MAGI3
   high-to-middle (H/M) levels (H-score ≥4) (chi-square test).
   Kaplan–Meier (KM) survival plots for overall survival (OS) (F) and
   progression-free survival (PFS) (G) based on MAGI3 H-score in ccRCC
   specimens (log-rank test). H Comparison of MAGI3 mRNA expression levels
   in ccRCC specimens between patients with favorable responses (Living)
   and those with poor responses (Death) to Sunitinib in the TCGA dataset
   of 48 patients. Data is presented as mean ± SEM, with statistical
   significance determined using the Mann–Whitney test. * indicates
   significance at P < 0.05. I ccRCC patients with MAGI3 mRNA levels above
   200 RPKM (high-middle, H/M) had a notably higher response ratio to
   Sunitinib, as indicated by chi-square test results. J Kaplan–Meier
   survival plots demonstrate the association between MAGI3 mRNA
   expression levels and overall survival based on the log-rank test. K
   GSEA shows significant activation of MAS, proliferation, and ERK
   pathways in ccRCC specimens with low MAGI3 expression among patients
   treated with Sunitinib, derived from the TCGA dataset.

   This observation was validated by the TCGA dataset. Responders to
   Sunitinib treatment exhibited higher MAGI3 levels than non-responders
   (Fig. [178]8H). Patients with high-middle (H/M, MAGI3 mRNA levels >200
   RPKM) levels showed a significantly higher response rate to Sunitinib
   treatment compared to those with low (L, MAGI3 levels <200 RPKM) levels
   (72.7% vs. 24.3%) (Fig. [179]8I). Kaplan–Meier analysis indicated that
   patients in the L group had worse OS compared to those in the H/M
   group, with an HR of 3.126 (95% CI: 1.388–7.04) (Fig. [180]8J,
   Supplementary Table [181]6). Additionally, the low-MAGI3 group
   exhibited notable enrichment of the gene signature associated with cell
   proliferation, MAS receptor activation, and ERK1/2 activation (Fig.
   [182]8K).

   Taken together, these findings strongly suggest that the downregulation
   of MAGI3 expression in ccRCC patients initiates MAS/ERK signaling
   activation, contributing to Sunitinib resistance.

Discussion

   Surgical nephrectomy is a highly effective treatment for localized
   ccRCC patients. Traditional approaches generally do not incorporate
   additional adjuvant therapies, especially for low-risk or early-stage
   cases like stage I. However, due to the substantial heterogeneity of
   ccRCC, approximately 25% of localized patients face a risk of relapse
   post-surgery [[183]45]. Thus, stratifying localized ccRCC is essential
   to optimize treatment outcomes.

   Prognosis prediction in ccRCC has traditionally relied on tumor
   staging, histological grading, or lymphatic infiltration, offering
   insights into tumor anatomy and histology [[184]46]. Yet, ccRCC’s
   remarkable heterogeneity leads to varying outcomes, even among patients
   with similar clinical characteristics [[185]47]. This diversity arises
   from differences in molecular pathogenesis, including distinct gene and
   protein expressions, even within the same stage or grade
   [[186]48–[187]50]. Therefore, molecular markers, particularly specific
   gene expressions, hold the potential to significantly improve the
   precision of outcome predictions and enable more tailored treatment
   strategies for ccRCC patients [[188]51].

   This study establishes a significant correlation between reduced MAGI3
   expression and an unfavorable prognosis in ccRCC, revealed through
   comprehensive analysis across multiple patient cohorts (Supplementary
   Fig. [189]1). In order to reflect the predictive effect of MAGI3 on the
   natural prognosis of ccRCC patients who did not receive postoperative
   adjuvant therapy, we removed patients who received adjuvant therapy
   after surgery from the TCGA database and conducted survival analysis on
   patients who only received surgical treatment. In addition, the ccRCC
   tissue we tested were also from patients who did not receive
   postoperative adjuvant therapy. The results show that reduced MAGI3
   levels emerge as a substantial risk factor and prognostic indicator for
   adverse clinical outcomes, particularly in early-stage ccRCC. Among
   Stage I ccRCC patients, those with middle-to-low MAGI3 expression
   (<261.5 RPKM or H-score ≤ 6) experience a notable decrease in 10-year
   OS (45.5% vs. 74.6%), PFS (55% vs. 69.8%), and DSS (77.9% vs. 98%)
   compared to those with high MAGI3 expression (>261.5 RPKM or
   H-score ≥ 8) (Fig. [190]1). These findings position MAGI3 as a
   promising prognostic marker, enhancing outcome prediction accuracy and
   enabling effective patient stratification.

   This study unveiled MAGI3’s anti-tumor role in ccRCC progression by
   inhibiting ERK activation induced by the Ang-(1-7)/MAS axis. Building
   upon our prior work, which linked the Ang-(1-7)/MAS axis to ccRCC cell
   migration [[191]34], our current findings illustrate MAGI3’s ability to
   suppress cell proliferation mediated by Ang-(1-7)/MAS through ERK
   pathway inhibition in renal cells (Figs. [192]3 and [193]4,
   Supplementary Fig. [194]2). Further validation through subcutaneous
   xenograft models, Magi3-knockout mice, and clinical analyses
   underscored the crucial involvement of the MAGI3/MAS/ERK axis in RCC
   development (Fig. [195]6, Supplementary Figs. [196]2, [197]3). As the
   ERK pathway plays a pivotal role in cell survival, cycle progression,
   and proliferation [[198]33], recent studies emphasizing Ang-(1-7)/MAS
   axis-induced ERK activation in ccRCC gain significance [[199]52]. Our
   study revealed that MAGI3 significantly reduces Ang-(1-7)-induced ERK
   phosphorylation in a dose- and time-dependent manner through its
   interaction with the MAS receptor, shedding light on the critical role
   of the MAGI3/MAS/ERK axis in RCC development and progression.

   Our study reveals a direct physical interaction between MAGI3 and the
   MAS receptor in renal cells, supported by multiple protein interaction
   assays and immunofluorescence co-localization studies (Fig. [200]2).
   This association is mediated through the PDZ1 domain of MAGI3 and the
   C-terminal of MAS. Structural analysis and dynamic simulation using
   AlphaFold suggest that MAS’s intracellular loop 2 (ICL2) serves as a
   potential G-protein coupling site (Fig. [201]5F, G). The interaction of
   MAS’s C-terminal motif “ETVV” and the hydrophobic groove of MAGI3-PDZ1,
   hinders MAS’s ICL2 from coupling with G proteins (Fig. [202]5D–H). This
   interference by MAGI3 inhibits the activation of the ERK pathway,
   providing insights into the modulation of MAS/ERK signaling.

   This study highlights MAGI3’s substantial impact on modulating
   Sunitinib sensitivity in ccRCC, suggesting its potential as a predictor
   of Sunitinib response. MAGI3 overexpression enhances sensitivity, while
   knockdown reduces it in ccRCC (Fig. [203]7). Notably, manipulating
   MAGI3 doesn’t affect Sunitinib IC50 in ccRCC, post A779 treatment.
   Among patients receiving Sunitinib treatment, those with MAGI3 protein
   at H/M levels (H-score ≥4) show significantly improved survival (47.1%
   vs. 15.8%) (Fig. [204]8A–G), confirmed by independent datasets (Fig.
   [205]8H–J). GSEA indicates MAS/ERK signaling enrichment in ccRCC
   patients with low MAGI3 expression (Fig. [206]8K). These collective
   evidences emphasize the significant role of MAGI3 in regulating
   Sunitinib responsiveness in ccRCC cells through the MAS/ERK pathway.

   As mentioned earlier, patients with MAGI3 levels in the middle to low
   range (H-score ≤ 6 or <261.5 RPKM) in stage I ccRCC demonstrated
   significantly lower survival rates compared to those with MAGI3-high
   levels (H-score ≥ 8 or >261.5 RPKM) (54.4% vs. 86.7% with a 5-year OS,
   Fig. [207]1K, L), suggesting the critical necessity for additional
   treatment interventions. Among patients with MAGI3 M/L levels,
   identified as a subset at high risk of recurrence, roughly 20% of
   individuals (5 out of 22 patients from Chinese cohort and 40 out of 245
   patients from TCGA, respectively) exhibited MAGI3 expression at a
   moderate level (protein H-score between 4 ~ 6 or mRNA between
   200 ~ 261.5 RPKM). Notably, our study suggests that ccRCC patients with
   MAGI3 levels in the middle to high range (protein H-score ≥ 4 or mRNA
   level >200 RPKM) may derive substantial benefits from Sunitinib
   treatment. Therefore, it is proposed that about 20% of ccRCC patients
   in stage I with MAGI3 exhibited in middle levels (H-score = 4 ~ 6 or
   mRNA between 200 ~ 261.5 RPKM) may benefit from Sunitinib adjuvant
   therapy. This collective evidence underscores the potential of MAGI3 as
   a predictive marker for tailoring treatment in early-stage ccRCC
   patients. However, further verification is necessary.

   Based on the primary findings of this study, we have also preliminarily
   explored the potential of MAGI3 as a prognostic biomarker in other
   types of renal cancer, such as kidney renal papillary cell carcinoma
   and chromophobe renal cell carcinoma. Prognostic analysis indicated
   that there were no significant differences in overall survival between
   patients with high and low MAGI3 expression in these types of renal
   cancers (Supplementary Fig. [208]4B). This highlights the specificity
   of MAGI3 in ccRCC. Furthermore, we are considering targeting MAGI3 to
   improve resistance to Sunitinib in ccRCC patients. An initial approach
   could involve screening for drugs that mimic the function of
   MAGI3-PDZ1, thereby achieving negative regulation of the MAS-ERK
   pathway to address Sunitinib resistance in ccRCC patients. This
   strategy underscores the therapeutic potential of targeting specific
   molecular interactions within the signaling pathways that contribute to
   drug resistance.

   This study acknowledges certain limitations that should be considered
   when interpreting its findings. One significant limitation is the
   relatively small cohort of ccRCC patients analyzed, which might
   restrict our ability to definitively establish MAGI3 as a reliable
   predictor for Sunitinib sensitivity. The power of our findings is
   constrained not only by the size of the sample but also by the
   retrospective nature of the data, emphasizing the necessity for future
   prospective studies.

   In summary, this study reveals that reduced MAGI3 expression in ccRCC
   patients activates the ERK signaling pathway, promoting cell
   proliferation and Sunitinib resistance. MAGI3 functions as a novel
   tumor suppressor in ccRCC, emphasizing the critical role of the
   MAGI3/MAS/ERK axis in Sunitinib resistance. Diminished MAGI3 levels are
   associated with an unfavorable prognosis, particularly in early-stage
   ccRCC, suggesting its potential as a valuable prognostic biomarker.
   This underscores the prospective use of MAGI3 as a predictive marker
   for personalized treatment in early-stage ccRCC, enhancing clinical
   decision-making.

Supplementary information

   [209]supplementary figures^ (442.2KB, pdf)
   [210]supplementary table 1^ (37KB, doc)
   [211]supplementary table 2^ (36.5KB, doc)
   [212]supplementary table 3^ (16KB, docx)
   [213]supplementary table 4^ (14.6KB, docx)
   [214]supplementary table 5^ (14.7KB, docx)
   [215]supplementary table 6^ (36.5KB, doc)
   [216]Supplementary figure and table legends^ (14.3KB, docx)
   [217]supplementary materials and methods^ (19.9KB, docx)
   [218]original data^ (550.1KB, pdf)

Acknowledgements