Makorin Ring Finger Protein 1 Inhibits Cell Proliferation in Renal Angiomyolipoma via the ERK/MAPK Signaling Pathway.

Introduction
Renal angiomyolipomas (AMLs) are benign kidney tumors comprising a mix of blood vessels, smooth muscles, and fat cells [1–3]. They are clonal tumors arising from abnormal differentiation of transformed renal progenitor cells [4, 5]. Clinically, AML is divided into sporadic AML (S-AML) and tuberous sclerosis complex (TSC)-associated AML (TSC-AML), with approximately 80% of AML cases being sporadic. Although most renal AMLs are asymptomatic, tumor hemorrhage remains the most serious complication. Tumor size is the primary factor influencing the risk of hemorrhage [6, 7]. A previous study reported a significant difference in median tumor size between hemorrhagic (8 cm) and non-hemorrhagic (4.1 cm) S-AMLs [7]. TSC-AML is associated with autosomal dominant disorders caused by mutations in the TSC1 and TSC2 genes, which are located on chromosomes 9q34.3 and 16p13.3, respectively, tends to present with multiple and bilateral kidney lesions, predominantly affecting younger individuals [8–10]. Epithelioid AML (E-AML) is a histological variant of renal AML with distinct clinical and pathological features. E-AML, a potentially malignant variant, was first described by Martignoni in 1995 and is characterized by epithelial dysplasia [11, 12]. Approximately 22% of E-AML cases exhibit invasive or metastatic behavior [13], distinguishing it from the more indolent nature of classic AML subtypes. E-AML can occur sporadically or in association with TSC, further highlighting its heterogeneous pathogenesis [14, 15].
A few biological pathways are involved in the development of renal AML. Activation of the PI3K/Akt/mTOR pathway was observed in TSC-AML as well as in E-AML and S-AML. E-AML harbors TSC1 or TSC2 mutations and is more sensitive to mTOR inhibitor treatment [13, 16, 17]. TSC1 and TSC2 are integral to the PI3K/AKT/mTOR pathway and influence cell proliferation, differentiation, metabolism, and drug resistance [18–20]. Moreover, our team found that Kruppel-like factor 2 (KLF2) suppresses renal AML cell growth by downregulating the IL-6/JAK/STAT3 signaling pathway [21]. Besides the PI3K/AKT/mTOR and IL-6/JAK/STAT3 pathways, the molecular mechanisms underlying AML development remain unclear. Therefore, further investigation into the regulatory factors of tumor growth could offer valuable insights for developing more effective AML treatment strategies.
Makorin ring finger protein 1 (MKRN1), also known as RING finger protein 61 (RNF61), acts as both a transcriptional coregulator and ubiquitin E3 ligase [22, 23]. It modulates the stability of key regulatory proteins via ubiquitination and proteasome-mediated degradation. Specifically, MKRN1 targets p53 and p21, facilitating their degradation and balancing cellular survival and apoptosis [23]. Additionally, MKRN1 ubiquitinates and degrades human telomerase reverse transcriptase (hTERT), Fas-associated protein with death domain (FADD), and proliferator-activated receptor γ (PPARγ) [24–26]. hTERT is vital for telomere length maintenance and is typically repressed in normal cells but activated in many cancers [27]. FADD is involved in apoptosis and necroptosis via the formation of the death-inducing signaling complex, while PPARγ is crucial for adipocyte differentiation [25, 26]. Emerging evidence indicates that MKRN1 exerts a context-dependent role in cancer, acting either as a tumor suppressor or an oncogene depending on the tumor type and cellular environment. For example, MKRN1 has been shown to function as a tumor suppressor in cervical cancer and clear cell renal cell carcinoma [28, 29], whereas it promotes tumorigenesis in colorectal cancer [30]. Given that MKRN1 is downregulated in renal clear cell carcinoma, we explored its role in benign renal tumors, particularly AML. Therefore, we aimed to investigate the function of MKRN1 in renal AMLs to better understand its specific contributions and underlying mechanisms in this tumor type.
The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway is a pivotal signaling cascade that governs essential cellular functions, including proliferation, differentiation, and survival [31, 32]. This pathway is frequently disrupted in cancer and contributes significantly to tumorigenesis, progression, and metastasis. Hyperactivation of the ERK/MAPK pathway is often associated with poor prognosis in various cancers [33]. Several therapeutic inhibitors targeting the components of this pathway have been developed, including MEK inhibitors (e.g., trametinib) and B-Raf inhibitors (e.g., vemurafenib) [34]. These inhibitors have demonstrated efficacy in treating cancers with specific genetic mutations. However, their clinical effectiveness is often curtailed by the rapid development of resistance mechanisms [35]. In this study, we investigated the role of MKRN1 in renal AML development and the regulatory mechanisms between MKRN1 and the ERK/MAPK pathway.

Methods

Collection of Tissue Specimens and Data from Medical Records
This retrospective, observational study was approved by the Human Subject Research Ethics Committee and Internal Review Board of Chang Gung Memorial Hospital at Linkou, Taoyuan, Taiwan (IRB No.: 202202242B0 and 202300883B0). Sixty-one formalin-fixed, paraffin-embedded (FFPE) renal AML samples from 13 men and 48 women, aged 18–81 years, and six frozen tissue samples collected between 2002 and 2017 were analyzed. All patients provided written informed consent prior to undergoing surgery. Clinical and pathological information was retrieved from the medical records.

Hematoxylin and Eosin Staining
Histological evaluation of tumor tissues was performed on 5-μm-thick sections prepared from FFPE samples. The sections were first deparaffinized by heating at 56–60°C for 15 min to overnight, followed by two washes in xylene for 5 min each. Rehydration was carried out through a graded ethanol series (100%, 90%, and 80%, each for 3 min) and rinsed briefly with distilled water for 2 min. The slides were then stained with hematoxylin (Sigma-Aldrich, St. Louis, MO, USA) for 3 min, rinsed in water for 1 min, counterstained with eosin for 45 s, and washed again in tap water for 1 min. Dehydration was achieved by sequential immersion in 90% and 100% ethanol for 1 min each, followed by two changes of xylene for 2 min. The stained slides were finally air dried and coverslipped for microscopic examination.

Immunohistochemistry and Scoring
FFPE tissue sections (5 μm thick) were stained with hematoxylin-eosin (Sigma-Aldrich) to assess histological features and protein expression. Slides were placed in an oven at 56–60°C for 15 min or overnight, then dewaxed in xylene, and rehydrated through a series of ethanol solutions (100%, 90%, 80%). After rinsing the slides in tap water for 30 s, antigen retrieval was performed by boiling them in 0.01 m sodium citrate (pH 6.0) for 20 min, followed by cooling at room temperature and washing with Tris-buffered saline containing 0.1% Tween-20. The tissue sections were stained with an MKRN1 antibody (cat. No. A300-990A, Bethyl Laboratories, Montgomery, TX, USA), HMB-45 (cat. No. 282M-94, Millipore, Burlington, MA, USA), and phospho-ERK (Thr202/Tyr204) (cat. No. 4370, Cell Signaling Technology, Danvers, MA, USA) using an UltraVision Quanto Detection System HRP DAB kit (Lab Vision Corporation, South San Francisco, CA, USA) according to the manufacturer’s instructions. Digital images were captured using an Aperio Digital Pathology Slide Scanner (Leica Biosystems, Nussloch, Germany). Three independent researchers scored the immunohistochemistry (IHC) staining based on the percentage of positive cells and staining intensity and calculated a histoscore (H score) ranging from 0 to 300. The samples were categorized into low- and high-score groups based on mean H scores for further analysis.

Quantitative Reverse Transcription-Polymerase Chain Reaction
RNA was extracted from frozen tissues using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The RNA concentration was measured using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). A PrimeScript 1st Strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan) was used for reverse transcription. The qRT-PCR for MKRN1 was performed using the KAPA SYBR FAST qPCR Kit (KAPA BIOSYSTEMS; Roche, Wilmington, MA, USA) on a StepOnePlusTM Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The following primer sequences were used: MKRN1: forward, 5′-CCA​ATG​GAT​GCT​GCC​CAG​AGA​T-3′; reverse, 5′-GGT​TGG​CTT​TCT​CAT​AGA​CCA​CC-3′ and β-actin: forward, 5′-CAC​CAT​TGG​CAA​TGA​GCG​GTT​C-3′; reverse, 5′-AGG​TCT​TTG​CGG​ATG​TCC​ACG​T-3′. qRT-PCR was conducted in triplicate, with β-actin as the internal control.

Cell Culture
The study utilized HK2, a human proximal tubular epithelial cell line, and UMB and SV7, which are human TSC-related and sporadic renal AML cell lines, respectively. HK2 cells were cultured in keratinocyte serum-free medium (Invitrogen, Waltham, MA, USA) with 5 ng/mL human recombinant epidermal growth factor (EGF) (Gibco, Waltham, MA, USA) and 0.05 mg/mL bovine pituitary extract (Gibco). UMB and SV7 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (ATCC) supplemented with 10% fetal bovine serum and 4,500 mg/L D-glucose. Cells were maintained at 37°C in a humidified incubator with 5% CO2.

Cell Transfection
Cells were precultured 1 day prior to transfection to ensure optimal confluency (approximately 70–80%) at the time of transfection. Cells were then transfected with either a MKRN1-overexpressing plasmid or a control vector (OriGene, Rockville, MD, USA) using Lipofectamine™ 2000 Reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocol. Transfections were carried out in six-well plates under standard culture conditions (37°C, 5% CO2). After 24 h of incubation, total RNA was extracted using the RNeasy Mini Kit (cat No. 74104, Qiagen) according to the manufacturer’s instructions. In parallel, total protein was isolated using RIPA buffer supplemented with protease and phosphatase inhibitors for subsequent Western blotting analysis.

Cell Proliferation Assay
After 24 h of transfection with either pCMV Entry or pCMV-MKRN1, cells were harvested and seeded at a density of 1 × 104 cells per well in 96-well plates (Corning, Corning, NY, USA). Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8; Sigma-Aldrich) following the manufacturer’s protocol. Absorbance at 450 nm was measured at multiple time points over a 168-h period using a SpectraMax® ABS Plus microplate reader (Molecular Devices, San Jose, CA, USA), with all measurements performed in triplicate.

Western Blotting
Cells were lysed with PRO-PREPTM Protein Extraction Solution (iNtRON Biotechnology, Kirkland, WA, USA) and protein concentration measured using a Qubit® 2.0 Fluorometer (Invitrogen). Proteins were resolved on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) for 1 h at room temperature, followed by incubation with primary antibodies at the appropriate dilution ratios. Subsequently, membranes were incubated with species-specific secondary antibodies under identical conditions. The primary antibodies used in this study included anti-MKRN1 (cat. No. ab72054, Abcam), anti-GFP (cat. No. ab290, Abcam), anti-MEK1/2 (cat. No. 9122, Cell Signaling Technology), anti-phospho-MEK (Ser217/221) (cat. No. 9121, Cell Signaling Technology), anti-ERK1/2 (cat. No. 4695, Cell Signaling Technology), anti-phospho-ERK (Thr202/Tyr204) (cat. No. 4370, Cell Signaling Technology), c-myc (cat. No. 5605, Cell Signaling Technology), cyclin D1 (cat. No. G124-326, BD Biosciences, Franklin Lakes, NJ, USA), anti-β-actin (cat. No. GTX629630, GeneTex, Irvine, CA, USA), and anti-GAPDH (cat. No. MAB374, Millipore). Protein bands were detected using the Immobilon Western Chemiluminescent HRP Substrate (Millipore) and captured with the UVP ChemStudio Plus imaging system, operated via VisionWorks LS 8.22 software (Analytik Jena AG, Jena, Germany).

Immunofluorescence Staining and Quantification of Ki-67
SV7 and UMB cells (1 × 105) were seeded onto sterilized glass coverslips in 6-well plates and incubated overnight. Cells were then fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at 4°C and permeabilized with 0.1% Triton X-100 in PBS for 7 min at room temperature. To block nonspecific binding, cells were incubated with 1% bovine serum albumin in PBS for 1 h at room temperature. After blocking, cells were incubated overnight at 4°C with mouse anti-Ki-67 monoclonal antibody (cat. No. ab279653, BD Biosciences; 1:500 dilution). After washing three times with PBS, Alexa Fluor 488-conjugated goat anti-mouse IgG (cat. No. A11001, Invitrogen) was applied for 1 h at room temperature in the dark. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich; 1 μg/mL) for 15 min. Slides were mounted using CitiFluor™ AF1 (Agar Scientific, UK), a glycerol-based antifade reagent that reduces photobleaching of fluorescent dyes, and visualized using a Zeiss Vert.A1 inverted fluorescence microscope (Carl Zeiss Microscopy GmbH, Germany).
For each experimental condition, immunofluorescent images were acquired at 10× magnification. Five randomly selected non-overlapping fields were imaged per sample. In each field, the total number of DAPI-stained nuclei and the number of Ki-67-positive cells (green nuclear staining) were manually counted using ImageJ software. The Ki-67 positive rate was calculated as the ratio of Ki-67-positive cells to the total number of cells per field. Each experiment was independently performed in biological triplicates (n = 3). Data were analyzed using unpaired two-tailed Student’s t test, and results were expressed as mean ± standard deviation. A p value <0.05 was considered statistically significant.

Gene Set Enrichment Analysis
RNA sequencing (RNA-Seq) was conducted to identify differentially expressed genes regulated by MKRN1. Total RNA was extracted from cells transfected with MKRN1 or control plasmids, and high-throughput sequencing was carried out. The resulting data were analyzed to determine gene expression profiles. To explore the biological pathways affected by MKRN1, gene set enrichment analysis (GSEA) was performed using the hallmark gene sets from the Molecular Signatures Database (MSigDB, version 7.5.1). Enriched pathways were identified based on normalized enrichment scores (NESs), nominal p values, and false discovery rate, providing insights into the functional impact of MKRN1 on cellular processes.

Statistical Analysis
All experiments were performed in triplicate. Data are presented as the mean ± standard deviation from the indicated number of independent experiments. Statistical analysis was conducted using Student’s t test with SPSS software (version 22.0; IBM Corp., Armonk, NY, USA). A p value of <0.05 was considered statistically significant.

Results

MKRN1 Expression Is Downregulated in Renal AML Tissues
To evaluate MKRN1 expression in renal AMLs, we performed IHC, quantitative RT-PCR, and Western blotting analysis using tumor and adjacent normal tissues. Tissue sections from 61 renal AML cases, which included 33 S-AML, 6 TSC-AML, and 22 E-AML cases, and 51 adjacent normal renal tissues were examined. The clinicopathologic features of all 61 AML patients included in this study, such as age, sex, tumor subtype, and hemorrhagic presentation, are summarized in online supplementary Table S1.
IHC staining revealed markedly lower MKRN1 expression in AML tumor tissues compared to normal renal tissues. HMB-45, a melanocytic marker, showed robust expression in the tumor components, validating the AML diagnosis. Hematoxylin and eosin staining confirmed the histological classification (shown in Fig. 1a). AML tumor sections encompassing various histological components, such as pericytes, myocytes, and epithelioid cells, demonstrated low or undetectable MKRN1 levels. In contrast, normal kidney tissues from the healthy margins of these tumors exhibited strong nuclear and cytoplasmic staining in renal tubules and endothelial cells. Quantification of MKRN1 IHC staining using H-score analysis demonstrated a significant reduction in MKRN1 expression in tumor tissues compared to normal tissues (p < 0.0001, shown in Fig. 1b). Among AML subtypes, TSC-AML displayed significantly higher MKRN1 levels than both S-AML (p = 0.0016) and E-AML (p = 0.0013), whereas no significant difference was observed between S-AML and E-AML (shown in Fig. 1c). Consistent with these results, MKRN1 mRNA expression was significantly reduced in AML tumor tissues compared to matched normal tissues from the same patients (n = 6, p = 0.003, paired t test, shown in Fig. 1d).
To further validate these findings at the protein level, we conducted Western blotting analysis on six matched S-AML tissue pairs. MKRN1 protein was markedly downregulated in tumor samples relative to adjacent normal tissues (shown in Fig. 1e), and densitometric quantification confirmed a statistically significant decrease (p < 0.001, shown in Fig. 1f). Consistent with the tissue-based observations, MKRN1 expression was also significantly reduced in AML-derived cell lines (SV7 and UMB) compared with normal renal epithelial cells (HK2) at both the protein and mRNA levels (shown in Fig. 1g–i). These results provide direct evidence that endogenous MKRN1 downregulation occurs in both AML tissues and AML cell lines.

MKRN1 Suppresses Cell Proliferation in Renal AML Cell Lines
To explore MKRN1’s role in AML cell proliferation, we overexpressed MKRN1 in SV7 and UMB cells. Cell proliferation was monitored for up to 168 h using the CCK-8 assay. MKRN1 overexpression significantly reduced cell proliferation in both SV7 and UMB cells compared with the control group (p < 0.001, shown in Fig. 2a, b). To further confirm these findings, Ki-67 immunofluorescence staining was performed. The proportion of Ki-67-positive cells was markedly decreased in MKRN1-overexpressing SV7 and UMB cells compared with control cells (p = 0.004 and p = 0.001, respectively, shown in Fig. 2c, d). These results demonstrate that MKRN1 overexpression inhibits AML cell proliferation, suggesting that MKRN1 possesses anti-proliferative properties in renal AMLs.

MKRN1 Suppresses Renal AML Cell Proliferation via Inhibition of the ERK/MAPK Pathway
To investigate the mechanisms by which MKRN1 influences AML cells, we performed RNA-seq on UMB and SV7 cells that overexpressed MKRN1 for 48 h. GSEA identified several significantly altered signaling pathways in MKRN1-overexpressing AML cells. Specifically, MKRN1 activation affected the ERK/MAPK signaling pathway in both SV7 (shown in Fig. 3a) and UMB cells (shown in Fig. 3b). Enrichment score (ES) plots for the HALLMARK_KRΑS_SIGNALING_DN pathway were upregulated by MKRN1 activation, whereas the GO_REGULATION_OF_MAPK_CASCADE pathway was downregulated (shown in Fig. 3c, d). These findings suggest that MKRN1 serves as an upstream regulator of the ERK/MAPK signaling pathway, mediating its anti-tumorigenic effects in AMLs.
Further Western blotting analysis revealed that AML cell lines (SV7 and UMB) exhibited markedly reduced MKRN1 expression accompanied by elevated basal levels of phosphorylated ERK (p-ERK) compared to the normal renal epithelial cell line HK2 (shown in Fig. 4a, b), providing direct evidence of MKRN1 downregulation and ERK activation in AML cells. Importantly, ectopic expression of MKRN1 in SV7 and UMB cells significantly decreased phosphorylated MEK and p-ERK levels, while total MEK and ERK expression remained unchanged (shown in Fig. 4c, d). These findings indicate that endogenous MKRN1 downregulation contributes to ERK/MAPK pathway activation in AML cells and that restoration of MKRN1 suppresses ERK/MAPK signaling, supporting its role as a negative regulator of oncogenic signaling and cell proliferation in renal AMLs.

MKRN1 Negatively Regulates ERK Signaling and Suppresses AML Cell Proliferation
IHC analysis revealed that p-ERK expression was markedly elevated in S-AML tumor tissues compared to adjacent normal kidney tissues, where minimal or no p-ERK staining was observed in the renal tubules and endothelial cells (shown in Fig. 5a). Quantification of H scores from 23 normal and 37 tumor samples confirmed that p-ERK levels were significantly higher in AML tissues (p < 0.0001, shown in Fig. 5b). Furthermore, correlation analysis of 19 paired S-AML tissues demonstrated a significant inverse relationship between MKRN1 and p-ERK expression (Pearson correlation coefficient = −0.512, p = 0.021, shown in Fig. 5c). Western blotting of three paired S-AML tissues also confirmed that p-ERK expression was increased in tumor tissues compared to normal counterparts, whereas MKRN1 levels were reduced (shown in Fig. 5d, e).
To functionally test whether MKRN1 inhibits proliferation via ERK suppression, we performed a rescue experiment using EGF, a known activator of the ERK/MAPK pathway. In both SV7 and UMB cells, EGF treatment reversed the anti-proliferative effects of MKRN1 overexpression, as evidenced by restored cell viability and increased Ki-67 positivity (shown in Fig. 6a–d). At the molecular level, EGF also reactivated ERK signaling, restoring p-ERK and cyclin D1 expression that had been downregulated by MKRN1 (shown in Fig. 6e, f). These results demonstrate that MKRN1 inhibits AML cell proliferation, at least in part, through suppression of ERK/MAPK-dependent mitogenic signaling.

ERK Pathway Modulates Proliferation in Renal AML Cells
To confirm the functional role of ERK signaling in AML cell proliferation, SV7 and UMB cells were treated with either EGF (20 ng/mL; Sigma-Aldrich) or SCH772984 (1 μm; MedChemexpress, Monmouth Junction, NJ, USA), a selective ERK inhibitor. EGF treatment significantly enhanced proliferation in both cell lines compared to control, while SCH772984 nearly abolished proliferation throughout the time course (p < 0.001, shown in Fig. 7a, b). Immunofluorescence staining further supported these findings, demonstrating that EGF treatment increased the proportion of Ki-67-positive cells, whereas exposure to SCH772984 markedly suppressed Ki-67 expression (shown in Fig. 7c, d).
At the molecular level, Western blotting analysis demonstrated that EGF robustly induced p-ERK and upregulated downstream targets c-Myc and cyclin D1. In contrast, ERK inhibition by SCH772984 suppressed p-ERK and reduced the expression of c-Myc and cyclin D1 in both SV7 and UMB cells (shown in Fig. 7e, f). These findings indicate that ERK pathway activation promotes AML cell proliferation by driving key regulators of cell cycle progression.

Discussion
Renal AMLs are clonal tumors that originate from abnormal differentiation of transformed renal progenitor cells [4, 5]. Although most renal AMLs, including sporadic and TSC-associated AMLs, are benign, E-AML are rare variants with malignant potential. Despite being typically asymptomatic, larger tumors may lead to hemorrhage, resulting in poor clinical outcomes. Understanding the regulatory mechanisms that drive AML development is essential for identifying potential therapeutic targets. The role of TSC in renal AML development is well established; mutations in the tumor suppressor genes TSC1 and TSC2 lead to upregulation of vascular endothelial growth factor, promoting angiogenesis and tumor growth [36, 37]. However, the cytogenetic mechanisms and malignant transformations of renal AMLs remain largely unknown. In our previous studies, we identified a frameshift mutation in TSC2 along with downregulation of TSC1/TSC2, which subsequently increased mTOR expression and activated the PI3K/Akt/mTOR signaling cascade [13]. GSEA and Western blotting revealed that the transcription factor KLF2 downregulates the IL-6/JAK/STAT3 signaling pathway [21]. KLF2 acts as a tumor suppressor, with reduced expression significantly correlated with increased tumor size and a higher frequency of tumor-associated hemorrhage.
Recent studies have demonstrated that MKRN1 acts as a tumor suppressor in renal clear-cell carcinoma and cervical cancer [28, 29], while it functions as an oncogene in colorectal cancer by activating the TGF-β signaling pathway via SNIP1 protein degradation [30]. In our study, MKRN1 was significantly downregulated in renal AML tissues compared to adjacent normal tissues, supporting its tumor-suppressive role in this context (shown in Fig. 1, 2). Consistently, AML-derived cell lines (SV7 and UMB) also exhibited markedly reduced MKRN1 expression compared with normal renal epithelial cells, further confirming endogenous MKRN1 downregulation in AML (shown in Fig. 1g–i). Moreover, MKRN1 expression was higher in TSC-associated AMLs than in sporadic and epithelioid subtypes (shown in Fig. 1c), indicating a context-specific function influenced by genetic background. These findings not only align with prior studies in other tumor types but also underscore MKRN1’s potential as a diagnostic biomarker and therapeutic target in renal AML.
We further explored the mechanistic role of MKRN1 using transcriptomic profiling. Transcriptomic profiling showed that MKRN1 overexpression significantly downregulated KRAS and ERK-MAPK gene sets in SV7 and UMB cells (shown in Fig. 3). Heatmaps revealed reduced expression of MAPK activators including FGFR1, FGFR3, TLR3, SPHK1, and MAPK3 and increased DUSP6, an ERK-specific phosphatase, suggesting that MKRN1 dampens ERK signaling by suppressing upstream drivers and enhancing dephosphorylation. Functional assays demonstrated an inverse relationship between MKRN1 and p-ERK (shown in Fig. 4, 5, 6). MKRN1 overexpression decreased p-ERK, cyclin D1, and c-Myc and inhibited proliferation measured by CCK-8 and Ki-67. ERK inhibition with SCH772984 produced similar suppression and further reduced growth (shown in Fig. 7). Causality was supported by a rescue experiment in which EGF restored p-ERK and cyclin D1 and partially rescued proliferation in MKRN1-overexpressing cells. SV7 and UMB display high basal ERK activity, which limits the magnitude of EGF responses, especially in UMB, yet the strong growth inhibition by ERK blockade in both lines confirms that AML proliferation depends on ERK signaling. Together these findings indicate that MKRN1 indirectly restrains AML cell growth by attenuating upstream activators of the MEK-ERK pathway rather than directly targeting ERK itself.
Prior studies in AML xenografts have shown phosphorylation of AKT and ERK1/2, implicating concomitant activation of the PI3K/AKT and MAPK pathways in tumor growth and angiogenesis [38]. Consistent with this, our ongoing data indicate PTEN loss and PI3K/AKT/mTOR activation in E-AML, while estrogen can rapidly trigger MAPK signaling via the GPR30-MMP-EGFR axis [39]. Together with our findings that MKRN1 downregulates ERK/MAPK signaling (shown in Fig. 3–6) and inversely correlates with p-ERK in tissues, these observations support a model in which ERK/MAPK contributes to AML pathogenesis and intersects with oncogenic and hormonal cues. Given evidence that ERK can be co-activated in TSC-deficient tumors alongside mTORC1 [40–42], MKRN1 downregulation may facilitate a “dual-pathway activation” state (ERK + mTORC1). This provides a mechanistic rationale to explore ERK pathway inhibition or MKRN1 reactivation in combination with mTOR-targeted therapy, particularly in tumors with high ERK activity [43].
In summary, this study identifies MKRN1 as a negative regulator of the ERK/MAPK pathway in renal AML. Its downregulation is associated with increased p-ERK signaling and tumor cell proliferation, particularly in aggressive subtypes. These findings suggest that MKRN1 may serve as both a prognostic marker and a potential therapeutic target. Targeting the ERK/MAPK pathway through MKRN1 modulation could offer a novel strategy for managing renal AML, especially in cases with TSC deficiency or malignant potential. Future studies incorporating larger clinical cohorts and in vivo models will be necessary to confirm MKRN1’s clinical utility and explore dual-inhibition therapies tailored to AML molecular subtypes.

Statement of Ethics
The study protocol was reviewed and approved by the Human Subject Research Ethics Committee and the Institutional Review Board of Chang Gung Memorial Hospital at Linkou (Approval No. 202202242B0 and 202300883B0). All patients provided written informed consent prior to undergoing surgery. For this retrospective study using de-identified human specimens and clinical data, the requirement for additional written informed consent was waived by the Institutional Review Board. All procedures were conducted in accordance with the principles of the Declaration of Helsinki.

Conflict of Interest Statement
The authors have no conflicts of interest to declare.

Funding Sources
This work was supported by the Chang Gung Memorial Hospital of Taiwan (Grant No. CIRPG3K0011-2) and the National Science and Technology Council (NSTC) of Taiwan (Grant No. MOST 111-2314-B-182A-112-, 2022; NSTC 113-2314-B-182A-015-, 2024). The funder played no role in the design, data collection, data analysis, or reporting of this study.

Author Contributions
T.-H.C., J.-S.T.P., and C.-K.C. contributed to study conception, design, data collection, data analysis, statistical analysis, and manuscript writing. Y.-H.C., C.-Y.L., and T.-K.W. performed IHC analysis and scoring. Y.-H.C. and C.-Y.L. performed sample collection and pathology data analysis.