Abstract
Background/Aim: Despite advances in therapy, lung cancer remains the leading cause of cancer-related mortality. R-spondin (RSPO) 3 and its receptor, leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4), drive tumor progression in a subset of lung adenocarcinomas by potentiating Wnt signaling. In addition to RSPO3, LGR4 is also a receptor for receptor activator of nuclear factor-кB ligand (RANKL). Here, we investigated whether MHP1-AcN, our previously developed RANKL-derived peptide, acts on LGR4 and examined its effects on RSPO3-LGR4 signaling in A549 lung cancer cells.
Materials and Methods: An A549 xenograft model was established by subcutaneous inoculation in BALB/c nude mice, followed by daily intraperitoneal administration of MHP1-AcN. Tumor growth was assessed by volume and weight. In vitro, the effect of MHP1-AcN on A549 cell proliferation, cytotoxicity, migration, and invasion were evaluated. Molecular interactions and signaling pathways were analyzed using immunoprecipitation and immunoblotting.
Results: Intraperitoneal administration of MHP1-AcN significantly reduced tumor volume and weight in the subcutaneous A549 xenograft model. Mechanistically, MHP1-AcN directly interacted with LGR4 and disrupted RSPO3-induced LGR4-IQ motif-containing GTPase activating protein 1 complex formation. MHP1-AcN inhibited A549 cell proliferation without inducing cytotoxicity and suppressed RSPO3-enhaced phosphorylation of LRP6 and accumulation of β-catenin. Furthermore, MHP1-AcN dose-dependently inhibited A549 cell migration and invasion by reducing focal adhesion kinase phosphorylation and disrupting F-actin organization.
Conclusion: These findings demonstrate MHP1-AcN as a novel LGR4 antagonist that inhibits RSPO3-LGR4-Wnt signaling, tumor growth, and metastatic potential in lung adenocarcinoma, highlighting its potential as a therapeutic agent targeting this pathway.
Introduction
Lung cancer is one of the most common cancers worldwide and remains the leading cause of cancer-related mortality. Non-small cell lung cancer (NSCLC), which primarily includes lung adenocarcinoma and lung squamous cell carcinoma, accounts for approximately 70-80% of all lung cancer cases (1, 2). Multiple oncogenic signaling pathways including KRAS, PI3K/AKT/mTOR, and Wnt signaling, have been implicated in lung cancer tumorigenesis and prognosis (3, 4). Aberrant activation of Wnt signaling, driven by genetic mutations and/or epigenetic alterations, plays a critical role in the initiation and progression of NSCLC (5).
Recent studies have identified R-spondins (RSPOs) as key regulators of Wnt signaling. RSPOs are a group of four highly related secreted proteins with diverse roles in embryonic development, organogenesis, and tumorigenesis (6). They exert effects by binding leucine-rich repeat-containing G protein-coupled receptors (LGR) 4-6, which potentiate canonical Wnt signaling (7, 8). Among these receptors, LGR4 is highly expressed in most primary lung adenocarcinomas and is frequently co-expressed with elevated levels of RSPO3 (9). Activation of the RSPO3-LGR4 axis recruits IQ motif-containing GTPase activating protein 1 (IQGAP1) and subsequently increases its affinity for disheveled (DVL), leading to assembly of a Wnt signalosome super-complex. This complex promotes phosphorylation of lipoprotein-receptor-related protein (LRP) 6, activating canonical Wnt signaling and coordinating actin cytoskeleton dynamics through non-canonical Wnt signaling. Consequently, aberrant activation of RSPO3-LGR4-IQGAP1 signaling enhances the proliferation, motility, and metastasis of lung adenocarcinoma cells (9, 10).
In addition to binding RSPOs, LGR4 also functions as a decoy receptor for receptor activator of nuclear factor-кB ligand (RANKL), thereby negatively regulating RANKL-induced osteoclast differentiation (11). In addition to its role in bone metabolism, RANKL/RANK signaling regulates toll-like receptor (TLR)-related inflammation in macrophages and microglia (12, 13). Our previous studies demonstrated that a RANKL partial peptide, MHP1-AcN, which contains the DE and part of the EF loop of RANKL, inhibits TLR2-, 3-, 4-, and 9-related inflammation through CD14 (14) and RANK (15). MHP1-AcN exerts anti-inflammatory effects in multiple TLR-related disease models, including ischemic stroke, psoriasis, and acute lung injury (15, 16), However, whether MHP1-AcN interacts with LGR4 signaling remains unexplored. In this study, we investigated the effects of MHP1-AcN on tumor growth in human A549 lung adenocarcinoma xenografts and examined its potential modulation of RSPO3-LGR4-IQGAP1 signaling.
Materials and Methods
Ethics approval and animal model. All experiments were approved by the Institutional Animal Care and Use Committee of The University of Osaka (approved number: 27-020-032) and conducted in accordance with The University of Osaka guidelines, which are based on the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines 2.0. All procedures were performed under isoflurane anesthesia, and all efforts were made to minimize animal suffering. At experimental endpoints, mice were euthanized by CO2 inhalation followed by cervical dislocation. For subcutaneous A549 xenograft model, six-week-old female BALB/cAJcl-nu/nu mice (18-20 g) were purchased from CLEA Japan, Inc. (Tokyo, Japan) and housed under specific pathogen-free conditions. The human lung adenocarcinoma cell line A549 was obtained from the RIKEN BioResource Research Center (BRC; RCB0098, Tsukuba, Japan) and maintained under a 5% carbon dioxide (CO2) atmosphere at 37°C in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin mixed solution (Nacalai). A549 cells (2×106) were resuspended in 50 μl phosphate-buffered saline (PBS) and mixed with Matrigel (Corning Life Science, NY, USA) at a 1:1 ratio. The cell suspension was subcutaneously inoculated into the dorsal flanks of each mouse. When tumors reached ~150 mm3, mice were randomized to receive intraperitoneal injections of MHP1-AcN (600 μg/mouse/day) or vehicle control (PBS) for up to 35 days (n=6 for each group). Tumor volumes (V) were calculated by caliper measurements of the width (W) and length (L) of each tumor using the formula: V=0.5 × (L × W2). Tumors were measured three times per week by an investigator blinded to treatment allocation. Throughout the study, mice were monitored daily for body weight, tumor ulceration, general activity, and grooming behavior. Mice were humanely euthanized when tumor volume reached 1,500 mm3, when body weight loss exceeded 20% of baseline, or when any signs of distress, tumor ulceration, or impaired mobility were observed.
Peptide design and synthesis. MHP1-AcN and biotinylated MHP1-AcN were designed and prepared as described previously (14). Peptides were dissolved in double-distilled water (ddH2O) to prepare 2 mg/ml stock solutions and stored at 4°C.
Cell culture and transfection. HEK293 and HEK293 cells stably expressing TCF/LEF-dependent luciferase reporter plasmid (pGL4.49) were obtained and cultured as previously described (17). HEK293 cells were transfected with pCMV-LGR4-Myc-DDK (OriGene, Rockville, MD, USA), a modified pCMV-LGR4-DDK construct lacking the Myc tag (modified using the KOD Plus Mutagenesis Kit; TOYOBO, Osaka, Japan), or pcDNA3-IQGAP1-Myc (Addgene, Watertown, MA, USA) using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA), according to the manufacturer’s instructions. Cells were harvested 24 h after transfection for subsequent analyses.
Luciferase reporter assay. For the TCF/LEF luciferase reporter assay, HEK293 cells stably expressing the TCF/LEF-dependent firefly luciferase reporter plasmid pGL4.49 were seeded at a density of 1×104 cells per well in 96-well plates. Cells were stimulated with recombinant human Wnt3a (50 ng/ml; R&D Systems, Minneapolis, MN, USA) in combination with recombinant human RSPO1, RSPO2, or RSPO3 (20 ng/ml; PeproTech, Rocky Hill, NJ, USA) in the presence or absence of MHP1-AcN or recombinant human RANKL (PeproTech) for 24 h. Luciferase activity was measured using the Luciferase Assay System (Promega, Madison, WI, USA) on a microplate luminometer (Centro XS3 LB960; Berthold Technologies). Luciferase signals were normalized to total protein concentration, determined using the Bradford Protein Assay Kit (Takara Bio Inc., Kusatsu, Japan).
Quantitative real-time reverse transcription PCR (RT-qPCR). A549 cells were seeded at a density of 1×105 cells per well in 12-well plates and treated with MHP1-AcN for 24 h. mRNA isolation and cDNA synthesis were performed as previously described (14). Gene-specific TaqMan primers were purchased and designated according to the following identifications: MYC, Hs00153408_m1; AXIN2, Hs00610344_m1; CCDN1, Hs00765553_m1; GAPDH, Hs02786624_g1; LGR4, Hs00173908_m1; LGR5, Hs00969422_m1; LGR6, Hs00663887_m1; RSPO1, Hs00543475_m1; RSPO2, Hs04400416_m1; RSPO3, Hs00262176_m1 (Thermo Fisher Scientific). 5′ nuclease assays were performed in MicroAmp Optical 384-well reaction plates using the QuantStudio 6 Pro Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The expression of specific genes was normalized to that of GAPDH and quantified using the ΔΔCt method (18).
Immunoprecipitation, western blotting, and immunostaining. Immunoprecipitation (IP), western blotting, and immunostaining were performed as previously described (14). For LGR4 competitive binding assays, HEK293 cells were transfected with FLAG-tagged LGR4 and incubated for 24 h, followed by stimulation with biotinylated recombinant human RSPO1 (50 ng/ml; R&D Systems) in the presence or absence of MHP1-AcN for 2 h. Protein complexes were immunoprecipitated using high-capacity streptavidin agarose (Thermo Fisher Scientific). For co-immunoprecipitation experiments, HEK293 cells were cultured in 6-cm dishes to approximately 70% confluence and co-transfected with pcDNA3-IQGAP1-Myc and pCMV-LGR4-DDK for 24 h. Cells were then stimulated with recombinant human Wnt3a (50 ng/ml) and RSPO3 (20 ng/ml), with or without MHP1-AcN for 4 h. Protein complexes were eluted using 30 μl of 2 × Laemmli sample buffer, and 20 μl of the eluate was subjected to SDS–PAGE. Immunoprecipitation was performed using high-capacity streptavidin agarose (Thermo Fisher Scientific) or anti-c-Myc agarose affinity gel (Sigma-Aldrich, St. Louis, MO, USA), as indicated. The following primary antibodies were used for western blotting: anti-LGR4 (PA5-67868, Invitrogen), anti-FLAG (F1804, Sigma-Aldrich), anti-c-Myc (sc-40, Santa Cruz Biotechnology, Dallas, TX, USA), anti-IQGAP1 (610611, BD Bioscience, Franklin Lakes, NJ, USA), anti-paxillin (610051, BD Bioscience), anti-GAPDH (MAB374, Sigma-Aldrich), anti-mouse IgG-HRP (NA931V, GE Healthcare, Boston, MA, USA), anti-rabbit IgG-HRP (NA934V, GE Healthcare), phalloidin-TRITC (P1951, Sigma-Aldrich), HRP-streptavidin (Sigma-Aldrich), anti-pLRP6, anti-LRP6, anti-β-catenin, anti-pFAK, anti-FAK, and anti-DVL2 from Cell Signaling Technology (Danvers, MA, USA). For immunofluorescence staining, Alexa Fluor 488– or 546–conjugated secondary antibodies (Invitrogen) and TRITC-conjugated phalloidin (P1951, Sigma-Aldrich) were used as indicated. Images were acquired using a confocal microscope (BZ-X800, Keyence, Osaka, Japan).
Cell proliferation, cytotoxicity, migration, and invasion assays. Cell proliferation was assessed using the Cell Titer 96 Non-radioactive Cell Proliferation Assay (Promega), and cytotoxicity was assessed using the CytoTox 96 Non-radioactive Cytotoxicity Assay (Promega), according to the manufacturer’s instructions. Briefly, A549 cells were cultured in 96-well plates at a density of 5×103 cells per well for proliferation assays or 5×104 cells per well for cytotoxicity assays. Cells were treated with ddH2O or MHP1-AcN at the indicated concentrations. After 48 h of treatment, cell proliferation was measured, whereas cytotoxicity was assessed after 6 h of treatment. In Boyden chamber migration assays, A549 cells (2.5×104 cells per well) were suspended in serum-free DMEM (50 μl per well) with or without MHP1-AcN and seeded into the upper chambers of a 48-well Boyden chamber system (Neuro Probe, Gaithersburg, MD, USA) fitted with an 8-μm pore-size polycarbonate membrane filter. The lower chambers were filled with DMEM containing 10% FBS (30 μl per well) as a chemoattractant. Cells were allowed to migrate for 15 h at 37°C. For invasion assays, A549 cells (2×105 cells per well) were suspended in serum-free DMEM (500 μl per well) with or without MHP1-AcN and seeded into Matrigel-coated invasion chambers (Corning Life Sciences, Corning, NY, USA). The lower chambers were filled with DMEM containing 10% FBS (750 μl per well) and incubated for 24 h at 37°C. After incubation, non-migrated or non-invaded cells on the upper surface of the membranes were gently removed using cotton swabs. Cells that had migrated or invaded to the lower surface were fixed and stained with Diff-Quik solution (Sysmex, Kobe, Japan). Stained cells were imaged and automatically quantified using a digital microscope (BZ-X800, Keyence). Data are expressed as the mean number of migrated or invaded cells per field, calculated from three randomly selected fields per membrane at 20× magnification.
Statistical analysis. All values are expressed as mean±standard deviation. The significance of tumor volumes was determined using two-way ANOVA with Sidak’s correction for multiple comparisons. Multiple comparisons were evaluated using ANOVA, followed by Dunnett’s multiple comparison test. The groups were compared using unpaired two-tailed Student’s t-test. Differences were considered statistically significant at p<0.05. Statistical analyses were performed using Prism 8.0 (GraphPad Inc., San Diego, CA, USA).
Results
MHP1-AcN inhibited A549 xenograft tumor growth. First, we examined whether MHP1-AcN suppresses tumor growth in a subcutaneous A549 xenograft model using BALB/c nude mice (n=6 for each group) (Figure 1A). Intraperitoneal administration of MHP1-AcN (30 mg/kg) significantly reduced tumor growth and tumor weight compared with vehicle-treated controls (Figure 1B and C). No significant changes in body weight were observed during the treatment period (Figure 1D). These results demonstrate that MHP1-AcN inhibits tumor growth in an A549 lung adenocarcinoma xenograft model.
MHP1-AcN inhibited A549 xenograft tumor growth. (A) Schematic overview of the animal study design. (B) Tumor growth curves showing tumor volume over time following daily intraperitoneal administration of MHP1-AcN or PBS. (C) Quantification of tumor weight at the experimental endpoint. (D) Body weight changes during the treatment period. s.c.: Subcutaneously; i.p.: intraperitoneal. **p<0.01 vs. A549 + PBS.
MHP1-AcN bound LGR4 and disrupted RSPO3-LGR4-IQGAP1-mediated Wnt signaling. Direct interaction between MHP1-AcN and LGR4 was examined using streptavidin pull-down assays in HEK293 cells overexpressing FLAG-tagged LGR4 (Figure 2A). MHP1-AcN competitively inhibited the binding of biotinylated RSPO1 to LGR4 in a dose-dependent manner (Figure 2B). Consistent with this effect, MHP1-AcN significantly suppressed RSPO1-enhanced TCF/LEF luciferase activity (Figure 2C). Because MHP1-AcN was designed based on RANKL, we also tested full-length recombinant RANKL; however, RANKL did not affect RSPO1-enhanced Wnt activity (Supplementary Figure 1). In addition to RSPO1, MHP1-AcN inhibited RSPO2-enhanced (Supplementary Figure 2) and RSPO3-enhanced Wnt signaling (Figure 2D). The A549 lung adenocarcinoma cell line is characterized by high LGR4 and RSPO3 expression, with little or no expression of LGR5, 6, RSPO1 or RSPO2 (Supplementary Figure 3), consistent with previous reports (10, 19), Therefore, subsequent analyses focused on RSPO3-dependent signaling. Co-immunoprecipitation experiments demonstrated that MHP1-AcN impaired the association between LGR4 and IQGAP1 and reduced the subsequent interaction of IQGAP1 with phosphorylated LRP6 and DVL2 following Wnt3a and RSPO3 stimulation (Figure 2E). Together, these findings indicate that MHP1-AcN functions as an LGR4 antagonist, disrupting RSPO3–LGR4–IQGAP1 complex formation and thereby suppressing RSPO-enhanced Wnt signaling.
MHP1-AcN directly interacted with LGR4 and inhibited RSPO-enhanced Wnt signaling. (A) HEK293 cells overexpressing FLAG-tagged LGR4 were incubated with ddH2O (NC), MHP1-AcN, or biotinylated MHP1 (MHP-B). Streptavidin agarose pull-down assays showed MHP1-AcN directly bound LGR4. (B) HEK293 cells overexpressing FLAG-tagged LGR4 were incubated with biotinylated RSPO1 (50 ng/ml) and MHP1-AcN at indicated concentrations. RSPO1 binding to LGR4 was assessed by pull-down assay followed by detection with HRP-conjugated streptavidin. Normal HEK293 cell lysates served as NC. (C, D) HEK293 cells stably expressing a TCF/LEF-dependent luciferase reporter were stimulated with Wnt3a (50 ng/ml), RSPO1 (20 ng/ml) or RSPO3 (20 ng/ml) in the presence or absence of MHP1-AcN for 24 h. Luciferase activity was normalized to total protein content in each well. *p<0.05, **p<0.01 vs. Wnt3a + RSPO. (E) HEK293 cells Myc-tagged IQGAP1 and FLAG-tagged LGR4 were stimulated with Wnt3a and RSPO3 in the presence or absence of MHP1-AcN (100 μg/ml). MHP1-AcN disrupted the association between LGR4 and IQGAP1 and reduced the subsequent interaction of IQGAP1 with phosphorylated LRP6 and DVL2. NC: Negative control.
MHP1-AcN inhibited A549 cell proliferation by suppressing Wnt/β-catenin signaling. An MTT assay showed that MHP1-AcN dose-dependently inhibited A549 cell proliferation, with an IC50 value of 3.70 μg/ml (Figure 3A). The lactate dehydrogenase (LDH)-based cytotoxicity assays showed that MHP1-AcN did not induce cytotoxicity even at the highest concentration tested (Figure 3B), indicating that the reduction in cell viability was not due to nonspecific cytotoxic effects. Next, we assessed the effect of MHP1-AcN on Wnt/β-catenin signaling in A549 cells. Upon stimulation with Wnt3a and RSPO3, MHP1-AcN suppressed phosphorylation of LRP6 and reduced total β-catenin levels (Figure 3C). Consistently, the expression of β-catenin target genes MYC and AXIN2 was downregulated by MHP1-AcN (Figure 3D), whereas LGR4 or RSPO3 expression showed only a modest reduction (Supplementary Figure 4). Collectively, these results indicate that MHP1-AcN selectively suppresses RSPO3-enhanced Wnt/β-catenin signaling and inhibits the proliferation of lung cancer cells.
MHP1-AcN inhibited A549 cell proliferation by suppressing Wnt/β-catenin signaling. (A) A549 cells were treated with MHP1-AcN at the indicated concentrations for 48 h and cell viability was analyzed by the MTT assay. (B) MHP1-AcN induced cytotoxicity was evaluated by LDH release assay. (C) Stimulation by Wnt3a (50 ng/ml) and RSPO3 (20 ng/ml) induced phosphorylation of LRP6 and total β-catenin accumulation, which were reduced by MHP1-AcN. (D) mRNA expression of Wnt target genes MYC, AXIN2 and CCND1 in A549 cells after incubation with MHP1-AcN for 24 h. *p<0.01 vs. normal A549 cells.
MHP1-AcN inhibited A549 migration, invasion and focal adhesion signaling. MHP1-AcN dose-dependently inhibited A549 cell migration and invasion, as assessed by Boyden chamber-based cell migration assays and Matrigel-coated invasion assays (Figure 4A and B). To elucidate the underlying mechanisms, we focused on RSPO-LGR4-IQGAP1 signaling, which recruits actin assembly proteins to couple receptor activation to focal adhesion formation. In this pathway, focal adhesion kinase (FAK) and paxillin are key components of the RSPO-LGR4-IQGAP1 complex and regulate F-actin-mediated cytoskeletal organization, thereby promoting cell migration and invasion (10). A549 cells treated with MHP1-AcN exhibited lower levels of paxillin expression and actin stress fibers formation, with increased actin accumulation at the cell cortex (Figure 4C). In addition, MHP1-AcN inhibited Wnt3a and RSPO3-induced phosphorylation of FAK and paxillin expression (Figure 4D). These findings suggest that MHP1-AcN suppress lung cancer metastatic potential through focal adhesion signaling and actin cytoskeletal dynamics.
MHP1-AcN inhibited A549 migration, invasion and focal adhesion signaling. (A) A549 cells were treated with MHP1-AcN at the indicated concentrations. Transwell assays showed reduced migration and invasion, with representative images of stained cells. (B) Quantitative analysis confirmed dose-dependent decreases in migrated and invaded cells. **p<0.01 vs. MHP1-AcN untreated cells. (C) After 24 h with MHP1-AcN (100 μg/ml), confocal microscopy revealed disrupted paxillin (green), F-actin (red, phalloidin), and DAPI-stained nuclei (blue). Scale bar=10 μm. (D) Western blotting analysis showed the MHP1-AcN reduced Wnt3a and RSPO3-induced phosphorylation of FAK and paxillin.
Discussion
In the present study, we demonstrated that the novel peptide MHP1-AcN directly interacts with LGR4 and disrupted RSPO3-induced LGR4-IQGAP1 complex formation, thereby inhibiting RSPO3-enhanced Wnt signaling. Systemic administration of MHP1-AcN suppressed tumor growth in vivo and reduced the proliferation, migration, and invasion of A549 lung adenocarcinoma cells.
Although this study primarily focused on RSPO3-LGR4 signaling, MHP1-AcN also inhibited RSPO1- and RSPO2-enhanced Wnt signaling. Previous structural studies have shown that leucine-rich repeat domains (LRRs) 1-9 of LGR4 are important for RSPO binding (20-22), whereas LRR domains 3-12 constitute the predicted interaction interface with RANKL (11). Based on these findings, we speculate that the RANKL partial peptide MHP1-AcN interacts with key LGR4 regions that overlap with RSPO-binding sites, thereby competitively impairing RSPO–LGR4 interactions. However, definitive confirmation will require comprehensive competitive binding assays and high-resolution structural analyses.
MHP1-AcN markedly reduced total β-catenin protein levels in Wnt3a and RSPO3-stimulated A549 cells, yet induced only modest downregulation of canonical Wnt target genes MYC, AXIN2, and CCND1 at the mRNA level. This discrepancy likely reflects post-transcriptional regulation of β-catenin, including enhanced proteasomal degradation via the destruction complex (upon LRP6 phosphorylation inhibition) or reduced translation efficiency, rather than primary transcriptional suppression (23). Such mechanisms are common in Wnt pathway modulation, where protein stability drives signaling output more than target gene transcription
In a phase Ia/b clinical trial, the monoclonal anti-RSPO3 antibody OMP-131R10 (Rosmantuzumab) showed limited clinical benefit in patients with advanced colorectal and ovarian cancers (24). In contrast, preclinical studies have demonstrated that RSPO3 blockade suppresses tumor proliferation in lung cancer, colorectal cancer and acute myeloid leukemia (25) using patient-derived xenograft models. One possible explanation for this discrepancy is that the clinical trial did not stratify patients based on RSPO3-driven tumor dependency (26). Moreover, RSPO3 is not the only gain-of-function alteration among the RSPO family. RSPO2 fusions also promote colorectal tumorigenesis (27-29). RSPO1 overexpression has been reported in ovarian cancer (30) in addition to RSPO3. From this perspective, the ability of MHP1-AcN to suppress RSPO1-, RSPO2-, and RSPO3-enhanced Wnt signaling may offer broader clinical utility than monoclonal antibodies targeting RSPO3 alone.
Consistent with previous reports showing that IQGAP1 regulates both canonical and non-canonical Wnt pathways downstream of RSPO–LGR4 signaling (10), MHP1-AcN inhibited both β-catenin–dependent signaling, as assessed by TCF/LEF reporter activity, and β-catenin–independent signaling pathways involved in focal adhesion assembly and actin cytoskeletal dynamics. Nonetheless, alternative mechanisms cannot be excluded. LGR4 is known to promote the clearance of the membrane-bound E3 ligases RNF43 and ZNRF3 upon RSPO binding, thereby preventing Wnt receptor degradation and enhancing Wnt signaling independently of heterotrimeric G proteins or β-arrestin (7, 8, 31, 32). MHP1-AcN may therefore modulate RNF43/ZNRF3-mediated receptor turnover to suppress Wnt signaling.
In addition, LGR4 has been reported to activate the Gαs-cAMP-PKA-CREB pathway and promote pro-inflammatory responses in macrophages during myocardial infarction (33). Tumor-associated macrophage infiltration is closely associated with tumor proliferation and metastatic potential in non–small cell lung cancer (34-36). Thus, modulation of LGR4-mediated G-protein signaling by MHP1-AcN may represent an additional mechanism contributing to tumor suppression. Future studies should evaluate MHP1-AcN’s effects on RNF43/ZNRF3 turnover and Gαs-cAMP signaling in relevant models.
RANKL/RANK signaling promotes an immunosuppressive tumor microenvironment in various cancer, including tumor of apocrine origin, where denosumab targeting tumor-associated macrophages has shown therapeutic potential (37). Notably, our RANKL-derived peptide MHP1 does not activate but instead inhibits RANKL-indued osteoclastogenesis. Whether MHP1 also exerts effects on RANKL-associated tumors required further investigations.
Conclusion
MHP1-AcN, a RANKL-derived partial peptide, binds to LGR4 and effectively inhibits RSPO3-potentiated Wnt/β-catenin signaling by disrupting the RSPO3-LGR4-IQGAP1 signaling complex. These findings identify MHP1-AcN as a novel LGR4 antagonist that exerts antitumor and antimetastatic effects in lung adenocarcinoma models, highlighting its therapeutic potential by targeting the RSPO-LGR4 pathway.
Acknowledgements
We appreciate the helpful support and constructive discussions provided by all members of the Department of Health Development and Medicine. All individuals acknowledged have agreed to be listed.
Footnotes
Authors’ Contributions
N.J., H.H., M.S. and H.N. designed the experiments; N.J. and H.H. performed the experiments; N.J., H.H., M.S., C.C., H.N. and R.M. analyzed the data; N.J. and M.S. wrote the manuscript and edited the figures. Authors approved the submitted version of the manuscript.
Supplementary Material
The supplementary file includes supplementary Figure 1 to Figure 4, and can be found here: https://doi.org/10.6084/m9.figshare.32020281.
Conflicts of Interest
The Department of Gene & Stem Cell Regenerative Therapy is an endowed department supported by the AS medical support. The Department of Health Development and Medicine is an endowed department supported by AnGes, Daicel, and FunPep and collaborates with these companies. The Department of Clinical Gene Therapy is financially supported by Novartis, AnGes, Shionogi, Boehringer, Fancl, Rohto, and FunPep. R.M. is a stockholder of AnGes and Funpep and had received an honorarium at AnGes. H.N. is a scientific advisor to FunPep.
Funding
This work was supported by the Translational Research Program, and the Strategic Promotion for Practical Application of Innovative Medical Technology (TR-SPRINT) from the Japan Agency for Medical Research and Development, AMED.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
- Received February 12, 2026.
- Revision received March 16, 2026.
- Accepted April 3, 2026.
- Copyright © 2026 The Author(s). Published by the International Institute of Anticancer Research.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
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