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. 2025 Nov 26;86(8):e70202. doi: 10.1002/ddr.70202

rFIP‐GMI Suppresses IGF‐1–Induced Invasion and Migration in Breast Cancer Cells via PI3K/Akt/β‐Catenin Inhibition

Wen‐Ling Liao 1,2, Yu‐Ying Wu 3,4, Yu‐Fan Liu 5, Pei‐Chi Lan 3, Yu‐Chun Cheng 6, Yueh‐Tzu Hung 3, Hsin‐Wen Liang 3, Huei‐Jane Lee 7, Yi‐Hsien Hsieh 3, Chun‐Wen Cheng 3,8,✉
PMCID: PMC12650121  PMID: 41294378

ABSTRACT

Insulin‐like growth factor‐1 (IGF‐I) promotes breast cancer (BC) progression by activating the phosphatidylinositol 3‐kinase (PI3K)/Akt pathway, which enhances invasion and migration through β‐catenin–mediated epithelial–mesenchymal transition (EMT). Triple‐negative breast cancer (TNBC), an aggressive BC subtype lacking hormone receptors and HER2 expression, exhibits high metastatic potential, poor prognosis, and limited therapeutic options. The recombinant fungal immunomodulatory protein from Ganoderma microsporum (rFIP‐GMI) possesses anti‐inflammatory, anti‐allergic, and anticancer activities; however, its role in suppressing tumor invasion and migration remains unclear. In this study, we investigated the molecular mechanism of rFIP‐GMI in TNBC cell lines, Hs578T and MDA‐MB‐231. Cell invasion and migration were evaluated using Boyden chamber and Transwell migration assays, while Western blot analysis and nuclear/cytoplasmic fractionation were employed to analyze protein expression and β‐catenin localization. rFIP‐GMI significantly inhibited IGF‐1–induced invasion and migration in both TNBC cell lines. Mechanistically, rFIP‐GMI suppressed PI3K and Akt phosphorylation, thereby activating glycogen synthase kinase‐3 beta (GSK3β) and promoting β‐catenin phosphorylation and degradation. This led to reduced nuclear β‐catenin accumulation and downregulation of oncogenic targets, including c‐Myc, cyclin D1, and MMP‐9. Conversely, treatment with the proteasome inhibitor MG132 confirmed that rFIP‐GMI stabilized cytoplasmic β‐catenin phosphorylation and blocked its nuclear translocation. Collectively, these findings demonstrate that rFIP‐GMI inhibits IGF‐1–driven invasion and migration in TNBC by inactivating the PI3K/Akt/β‐catenin axis, highlighting its potential as a therapeutic agent for this aggressive TNBC subtype.

Keywords: breast cancer, EMT, IGF‐1, PI3K/Akt pathway, rFIP‐GMI

1. Introduction

Tumor cell heterogeneity, driven by genetic, mutational, and epigenetic events, gives rise to phenotypic diversity that reflects dynamic and reversible cell state transitions in response to varying intra‐ and extracellular cues between primary and metastatic sites within the same tumor clone. One of the most prominent manifestations of this plasticity across multiple carcinoma types is the epithelial–mesenchymal transition (EMT), a process that confers tumor cells with motility and invasive mesenchymal characteristics, thereby promoting intratumor heterogeneity and metastatic dissemination (Hashemi et al. 2022). Mechanically, EMT is orchestrated by multiple signaling pathways, including transforming growth factor‐β (TGF‐β) (Azmi 2013; Fuxe et al. 2010), Wnt/β‐catenin (Azmi 2013), Notch (Capaccione et al. 2014), and receptor tyrosine kinases (RTKs) (Cervello et al. 2017). Among these, the type 1 insulin‐like growth factor receptor (IGF‐1R), a member of the RTK family, plays a central role in regulating cell proliferation, differentiation, and metabolic homeostasis. Activation of IGF‐1R by its ligands–insulin‐like growth factor 1 (IGF‐1), IGF‐2, or insulin, induces EMT by downregulating epithelial markers such as E‐cadherin and upregulating mesenchymal markers such as N‐cadherin and vimentin, ultimately promoting cancer cell stemness, invasiveness, and metastasis (Cevenini et al. 2018).

Ganoderma, a genus of polypore fungi in the Ganodermataceae family, has been widely used in traditional Asian medicine for its diverse potential health‐promoting properties. Increasing evidence indicates that several Ganoderma species, including G. lucidum (Lingzhi or Reishi), contain bioactive compounds such as polysaccharides, peptidoglycans, triterpenes, and fungal immunomodulatory proteins (FIPs) with biological activity. These compounds modulate immune responses to maintain immune balance and protect against immunodeficiency, autoimmunity, and tumor progression. Both in vitro and in vivo studies have demonstrated the immunomodulatory and antitumor properties of Ganoderma extracts (Table 1) (Bai et al. 2020; Boh 2013; Cancemi et al. 2024; Cao et al. 2018; Chen et al. 2006; Hsu et al. 2009; Hua et al. 2023; Lin et al. 2010; Lin et al. 2021; Lin and Zhang 2004; Paterson 2008; Xu et al. 2016; Yang et al. 2016; Zhang et al. 2013). Among these, the recombinant fungal immunomodulatory protein derived from Ganoderma microsporum (rFIP‐GMI, NCBI protein ID: AGU04723.1) has attracted increasing attention for its therapeutic potential. rFIP‐GMI has been shown to alleviate oxidative damage (Chao et al. 2022), reduce inflammation, and modulate immune function in metabolic disorders (Su et al. 2024), including cancer (Hsu et al. 2009; Lu et al. 2019). Mechanistic studies further revealed that rFIP‐GMI inhibits the invasion of lung cancer cells by eliminating reactive oxygen species (ROS) and reducing tumor necrosis factor‐alpha (TNF‐α) levels (Lin et al. 2010). Collectively, these findings identify rFIP‐GMI as a bioactive fungal protein with significant therapeutic promise in cancer prevention and treatment.

Table 1.

Summarization of the anticancer effects, cancer types studied, and the mechanisms involved in various studies on Ganoderma immunomodulatory extracts, offering a clear comparison of their broad applications in cancer research.

Ganoderma species Research subject (Cancer type) Anticancer effects Main mechanism Reference
G. atrum Breast cancer Induces cell death Upregulation of a tumor necrosis factor TNFSF8 Xu et al. (2016)
G. atrum Transplantable sarcoma tumor Induces macrophage activation Promotion of TLR4‐mediated NF‐kB and MAPK signaling Zhang et al. (2013)
G. lucidum Colon cancer Induces cell apoptosis Upregulation of Bax and downregulation of Bcl‐2 Bai et al. (2020)
G. lucidum Leukemia Induces cell apoptosis, promotes cell differentiation Suppression of MAPK pathway Yang et al. (2016)
G. lucidum Various cancer cell lines Inhibits tumor growth, increases anticancer activity Modulation of immune response Boh (2013)
G. lucidum Multiple cancer models Inhibits tumor outgrowth Enhancement of immune function Paterson (2008)
G. lucidum Colorectal cancer Enhances immune response, delays tumor progression Modulation of immune cell activity Chen et al. (2006)
G. lucidum Multiple cancer models Inhibits cell growth and promotes cell apoptosis Modulation of immune response Lin and Zhang (2004)
G. lucidum Leukemia Suppresses tumor growth, induces cell apoptosis Activation of macrophages and T cells Cancemi et al. (2024)
G. microsporum EGFR‐positive lung cancer cells Inhibits cell viability Induction of EGFR endocytosis and degradation Hua et al. (2023)
G. microsporum Lung cancer Decreases cancer cell invasiveness Reduction of NF‐κB activation Lin et al. (2010)
G. spp Various cancer cell lines Inhibits tumor cell outgrowth Inhibition of NF‐κB and MAPK signaling to reduce inflammation Cao et al. (2018)
G. sinense Hepatoma Induces cell cycle arrest Activation of the ER stress pathway Lin et al. (2021)
G. tsugae Epidermoid carcinoma cells Decreases cell angiogenesis Inhibition of EGFR‐mediated signaling pathway Hsu et al. (2009)
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In breast cancer (BC), IGF‐1 is a crucial regulator of mammary epithelial cell development (Ruan and Kleinberg 1999), and elevated serum IGF‐1 levels have been associated with increased all‐cause mortality in women with breast malignancies (Duggan et al. 2013). Triple‐negative breast cancer (TNBC), an aggressive BC subtype lacking estrogen receptor, progesterone receptor, and HER2 expression, appears to be particularly influenced by IGF‐1 signaling. Accumulating evidence indicates that the IGF‐1/IGF‐1R axis plays a pivotal role in TNBC biology (Davison et al. 2011). Mechanistically, IGF‐1/IGF‐1R signaling promotes TNBC cell proliferation and survival through activation of the focal adhesion kinase (FAK)–yes‐associated protein (YAP) pathway, underscoring its tumor‐promoting potential (Rigiracciolo et al. 2020). More recently, the multifaceted roles of the IGF‐1/IGF‐1R axis in TNBC pathogenesis and therapeutic targeting have been comprehensively reviewed (Kumar and Chaudhri 2024). Together, these findings emphasized the biological and clinical importance of IGF‐1 signaling in TNBC and provided a strong rationale for exploring its contribution to tumor progression and patient outcomes.

Activation of the IGF‐1/IGF‐1R pathway triggers downstream signaling cascades that promote tumor cell proliferation and resistance to apoptosis, primarily through the PI3K/Akt pathway (Lee et al. 1999). Recent studies have shown that FIPs can negatively regulate PI3K/Akt signaling and induce autophagy in human lung adenocarcinoma cells (Xie et al. 2018), while rFIP from G. lucidum induces apoptosis by inhibiting the Akt–mTOR pathway in multidrug‐resistant lung cancer cells (Chiu et al. 2015). Although several FIPs have been reported to modulate diverse oncogenic pathways, their role in regulating IGF‐1–induced invasion and migration of highly invasive TNBC cells remains underexplored. We hypothesize that inhibition of IGF‐1–induced PI3K/AKT signaling by rFIP‐GMI may suppress IGF‐1–driven malignant phenotypes, including enhanced migration and invasion. To the best of our knowledge, this study is the first to demonstrate that rFIP‐GMI inhibits the progression of BC cells by modulating the PI3K/Akt pathway and suppressing β‐catenin activation in IGF‐1–stimulated TNBC cells. By targeting this signaling cascade, our findings provide novel mechanistic insight and establish rFIP‐GMI as a promising therapeutic candidate for the management of highly aggressive TNBC.

2. Materials and Methods

2.1. Reagents

rFIP‐GMI is a fungal immunomodulatory protein (GMITM) provided by MycoMagic Biotechnology Company Ltd. (New Taipei, Taiwan). It was generated and enhanced from a recombinant protein derived from Ganoderma microsporum (NCBI protein ID AGU04723.1). The protocol for purification rFIP‐GMI and assay of the endotoxin levels was detailed in previous report accordingly (Lu et al. 2018).

2.2. Cells and Cell Culture

Human breast cancer cell line, Hs578T and MDA‐MB‐231 were obtained from the American Type Culture Collection (Manassas, VA, USA). These cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc., Grand Island, NY, USA) containing 0.1 mM sodium pyruvate, 10% FBS, 2 mM l‐glutamine, 100 IU/mL penicillin, and 100 µg/mL streptomycin (BioSource, Rockville, MD, USA), and supplemented with 10% fetal bovine serum and then cultured in a humidified incubator at 37°C that was supplied with 5% CO2.

2.3. In Vitro Cell Invasion and Migration Assays

To estimate the capacity for cell motility and invasiveness towards a chemo‐attractant, the Boyden chamber assay with a membrane with 8 µm‐pore‐size or the Transwell migration assay (IBIDI™ Culture Inserts, IBIDI, Martinsried, Germany) was exactly performed as described previously (Liao et al. 2022). A total of 5 × 104 serum‐free cells (200 μL) were seeded in triplicate in culture medium onto the apical surface of each hanging‐insert and placed into wells that held culture medium (500 μL) supplemented with 10% FBS. Cell migration and invasion were assessed over a 16 h period, with migration performed without Matrigel and invasion performed with Matrigel. Later, the lower surface of the insert was fixed with 100% methanol and stained with Giemsa solution (Sigma‐Aldrich, St. Louis, MO, USA) or 0.4% crystal violet for 15 min where appropriate. Non‐migrating cells were removed from the upper surface using a cotton stick and the migrated cells were counted. The invaded cells were quantified by counting five random high‐power visions using an Olympus Ckx41 light microscope (Tokyo, Japan).

2.4. Nuclear and Cytoplasmic Protein Fractionation

Cell components were fractionated into nuclear and cytosolic fractions using a previously documented methodology (Liu and Fagotto 2011) with a minor modification. Initially, cell pellets were obtained by centrifugation after scraping and washed twice with phosphate buffered saline (PBS). The resulting cell suspension was centrifuged at 1800 × g for 5 min at 4°C. The resulting pellet was then suspended in cytoplasmic lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.5 mM DTT, 20% glycerol, and 0.2 mM EDTA) supplemented with cocktail protease inhibitor (Sigma‐Aldrich, St. Louis, MO, USA) and kept on ice for 15 min. Subsequently, the cell lysate was subjected to aspiration five times using a syringe with a narrow‐gauge (No. 27) needle. Further centrifugation at 8000 × g for 5 min at 4°C allowed the collection of the supernatant containing the cytoplasmic protein extract. The remaining cellular pellet was resuspended in nuclear extract buffer (20 mM HEPES pH 7.9, 0.5 M KCl, 25% glycerol, 0.5 mM DTT, and 0.2 mM EDTA) supplemented with cocktail protease inhibitor. The resulting supernatant was incubated at 4°C for 30 min, followed by recovery of the supernatant containing nuclear protein extract through centrifugation at 12,000 × g for 10 min at 4°C. Protein quantification was carried out using Pierce™ Bradford Plus Protein Assay kits (Thermo Fisher Scientific, Waltham, MA, USA) and protein extracts were subsequently stored at −80°C.

2.5. Western Blot Analysis

Protein expression levels were analyzed using Western blot, as described previously (Taylor and Posch 2014). Briefly, cells were lysed in a buffer containing 50 mM Tris‐HCl mM (pH 7.4), 100 mM NaCl, 5 mM EDTA, 50 mM NaF, 10 mM NaPP, 1% Triton X‐100, 1 mM DTT, and a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). Total protein concentration was determined using the Bradford assay to ensure consistent lysate quantities. For electrophoresis, 20 μg of protein per sample was loaded onto SDS–polyacrylamide gels. Proteins were transferred onto an Immobilon–PSQ polyvinylidene difluoride (PVDF) membrane. Primary antibodies used included phosphor‐Akt (Ser473; no. 9271), GSK3β (no. 9315), and phospho‐β‐catenin (Ser675; no. 9567) from Cell Signaling Technology (Danvers, MA, USA); phospho‐PI3K p85 (Tyr467/Tyr199; #bs‐3332R) from Bioss, Inc. (China); Lamin A (133A2) from Abcam (Cambridge, UK); c‐Myc (no. GTX103436) and MMP‐9 (no. GTX100458) were purchased from GeneTex International Co. (Hsinchu City 300 Taiwan); β‐actin (NB600‐501) from Novus Biologicals LLC (Littleton, CO, USA); and Akt (sc‐8312), cyclin D1 (sc‐8396), and β‐catenin (sc‐7963) from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase–conjugated secondary antibodies were used for detection. Protein signals were visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore Corporation, Temecula, CA, USA). Chemiluminescence signals were captured using the LAS–4000 system with Fujifilm Image Read LAS–4000 software version 2.0. Quantification of immunoblot band intensities was performed with ImageJ software (version 1.52; National Institute of Health, USA).

2.6. Statistical Analysis

All experiments were performed in triplicate and statistical analyzes were performed with GraphPad Pro Prism 8.01 (GraphPad, CA, USA). Data are expressed as mean ± standard deviation (SD). The expression levels of the Western blots of interest from exposures to rFIP‐GMI were compared with the results between two groups using an unpaired Student's t‐test and one‐way ANOVA was used for multigroup comparisons. p < 0.05 was considered statistically significant.

3. Results

3.1. rFIP‐GMI Significantly Reduces Invasion and Migration in Highly Invasive BC Cells

Metastasis accounts for more than 60% of deaths related to solid cancer (Dillekås et al. 2019). To address this, we investigated the effect of rFIP‐GMI on BC cell motility and invasiveness. Consistent with our hypothesis, rFIP‐GMI treatment significantly reduced the percentages of migratory and invasive cells in the highly invasive Hs578T and MDA‐MB‐231 BC cell lines, as assessed by the Boyden chamber assay (Figure 1A,B). Specifically, after treatment with 0.8 μM rFIP‐GMI, migration capacity decreased by more than 60% and invasion capacity by more than 80% in Hs578T cells treated with rFIP‐GMI compared to the control group (p < 0.001). Similarly, MDA‐MB‐231 cells treated with 0.4 μM rFIP‐GMI exhibited a reduction in migration and invasion capacities of more than 60% compared to the control group (Figure 1C,D). Recent studies have shown that FIPs negatively regulate the PI3K/Akt pathway to suppress the invasiveness of lung adenocarcinoma cells (Xie et al. 2018). Consistent with these findings, the Western blot results (Figure 1E) showed that the administration of rFIP‐GMI significantly decreased the levels of phosphorylated levels of both PI3K [p‐PI3K‐p85)] (Figure 1F) and Akt (p‐Akt) (Figure 1G), suggesting that rFIP‐GMI is effective against activation of the PI3K/Akt pathway in inhibiting TNBC cell invasion and migration.

Figure 1.

Figure 1

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rFIP‐GMI reduces invasion and migration in triple‐negative breast cancer (TNBC) cell lines. (A) Hs578T and (B) MDA‐MB‐231 cells were treated with rFIP‐GMI for 24 h and cell migration and invasion were evaluated using the Boyden chamber assay. Representative micrographs show Giemsa‐stained cells on the membrane after 16 h of incubation, captured at ×400 magnification. Quantitative analyses of (C) migration and (D) invasion are presented as mean ± SD from three independent experiments. **p < 0.01; p ***< 0.001 versus control. (E) Representative Western blot images and (F, G) densitometric analyses of phosphorylated PI3K (p‐pI3K) and phosphorylated Akt (p‐Akt), with β‐actin used as the loading control. Data are presented as mean ± SD from three independent experiments. ns, not significant; *p < 0.05, **p < 0.01.

3.2. rFIP‐GMI Can Attenuate IGF‐1‐induced BC Cell Invasion and Migration

High levels of IGF‐1 in serum are associated with increased all‐cause mortality in women with breast malignancy (Duggan et al. 2013), and IGF‐1/IGF‐1R signaling triggers downstream activation of the PI3K/Akt pathway, leading to proliferation, survival, and metastasis of epithelial tumors (Cevenini et al. 2018). To explore the molecular mechanism by which rFIP‐GMI inhibits BC cell progression, we evaluated its effect on IGF‐1‐induced activation of this pathway. In this study, initial results demonstrated that rFIP‐GMI significantly reduced Hs578T and MDA‐MB‐231 cell invasion and migration by more than 80% after treatment at concentrations of 1.6 µM and 0.8 µM, respectively (Figure 1). Based on these findings, we next performed rFIP‐GMI at 0.8 µM and 0.4 µM on Hs578T and MDA‐MB‐231 cells that were stimulated by IGF‐1 (100 ng/mL) to assess the inhibitory effects of cell invasiveness and explore the difference in protein expression levels. To our expectations, the micrograph results revealed that rFIP‐GMI treatment significantly inhibited IGF‐1–induced invasion and migration in these BC cell lines (Figure 2A). Quantitative analysis corroborated these observations, showing that IGF‐1 stimulated Hs578T and MDA‐MB‐231 cell invasion and migration capabilities were reduced by more than 60% after administration of rFIP‐GMI (Figure 2B,C).

Figure 2.

rFIP‐GMI inhibits IGF‐1—induced invasion and migration by suppressing the PI3K/Akt/β‐catenin pathway. (A) Representative images of Hs578T and MDA‐MB‐231 cells treated with rFIP‐GMI in the Transwell migration assay. Cells were exposed to rFIP‐GMI for 24 h, and white arrows indicate invaded cells. Quantitative analyses of (B) migration and (C) invasion are shown as mean ± SD from three independent experiments. **p < 0.01; ***p < 0.001 versus IGF‐1 (100 ng/mL) alone. (D) Representative Western blot images of cells co‐treated with IGF‐1 and rFIP‐GMI compared with IGF‐1 alone. (E–H) Densitometric analyses of p‐pI3K, p‐Akt/Akt, GSK3β, and phosphorylated β‐catenin (p‐β‐catenin/β‐catenin), with β‐actin as the loading control. Data are presented as mean ± SD from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

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3.3. rFIP‐GMI Suppresses IGF‐1‐induced BC Cell Progression by Inhibiting the PI3K/Akt/β‐catenin Pathway

Furthermore, the results of the Western blot analysis demonstrated that rFIP‐GMI suppresses the PI3K‐Akt signaling pathway (Figure 2D), thus reducing the levels of the phosphorylated form of the p‐PI3K‐p85 and p‐Akt proteins by more than 40% (Figure 2E,F). Specifically, rFIP‐GMI did not significantly affect GSK3β expression levels (Figure 2G). Therefore, sustained activity of GSK3β can lead to an increase in β‐catenin phosphorylation (p‐β‐catenin) (Figure 2H) in both TNBC cell lines stimulated with IGF‐1 after treatment with rFIP‐GMI compared to those treated with IGF‐1 alone (p < 0.05). This finding is consistent with previous reports (Aberle et al. 1997), which indicate that phosphorylated β‐catenin is targeted for ubiquitination, resulting in its degradation via the proteasome. Consequently, this degradation prevents β‐catenin nuclear translocation and downregulates the transcription of β‐catenin‐dependent oncogenes (Karim et al. 2004). Collectively, these findings highlight the therapeutic potential of rFIP‐GMI in inhibiting IGF‐1–induced BC cell invasion and migration, which is attributed to modulation of the PI3K/Akt/β‐catenin pathway.

3.4. rFIP‐GMI Enhances β‐Catenin Phosphorylation by Inactivating Akt in IGF‐1–Stimulated BC Cells

To investigate the inhibitory effect of rFIP‐GMI on temporal dynamics of phosphorylated Akt and β‐catenin protein levels in IGF‐1–induced BC cells, we performed quantitative Western blots on protein lysates collected over a 6 h time course from two IGF‐1–stimulated cell lines treated with rFIP‐GMI (Figure 3A,D). Quantification of the phosphorylated–to–native protein ratio revealed that Akt phosphorylation was significantly suppressed after 2 h of rFIP‐GMI treatment. Specifically, p‐Akt levels were reduced by more than 40% and 75% in IGF‐1–treated Hs578T and MDA‐MB‐231 cells, respectively (Figure 3B,E). Moreover, extending rFIP‐GMI treatment beyond 2 h, up to 6 h, did not result in further significant reduction in p‐Akt levels.

Figure 3.

Figure 3

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rFIP‐GMI decreases phosphorylated Akt and increases phosphorylated β‐catenin levels in BC cells. (A, D) Representative Western blot images showing p‐Akt and p‐β‐catenin expression in Hs578T and MDA‐MB‐231 cells. Cells were stimulated with IGF‐1 for 0.5 h, followed by rFIP‐GMI treatment at the indicated time points. (B, E) Densitometric analyses of the p‐Akt/Akt ratio, and (C, F) densitometric analyses of the p‐β‐catenin/β‐catenin ratio. β‐actin was used as the loading control. Data are presented as mean ± SD from three independent experiments. Statistical annotations: a, versus untreated control; b, versus IGF‐1 alone; c, d, and e versus IGF‐1 plus rFIP‐GMI for 0.5, 1.0, and 2.0 h, respectively. p < 0.05 was considered statistically significant. Fisher's exact two‐tailed test was applied; ptrend < 0.001.

In the canonical signaling pathway, p‐Akt phosphorylates and inactivates GSK3β, thereby preventing GSK3β from phosphorylating β‐catenin. This inhibition allows β‐catenin to accumulate in the nucleus, where it activates oncogenic transcriptional programs that support cancer cell survival and progression (Shah and Kazi 2022). Conversely, reduced levels of p‐Akt reactivate GSK3β, leading to enhanced β‐catenin phosphorylation, which triggers its ubiquitination and subsequent proteasomal degradation. Consistent with this mechanism, we observed a time‐dependent increase in p‐β‐catenin during the 6 h rFIP‐GMI treatment period in both IGF‐1–stimulated Hs578T and MDA‐MB‐231 cells, compared to IGF‐1–treated controls or other time points (Figure 3C,F). Together, these findings suggest that rFIP‐GMI inhibits IGF‐1–induced BC progression by suppressing Akt phosphorylation, thereby promoting β‐catenin phosphorylation and degradation, ultimately acting as a molecular switch to prevent EMT activation.

3.5. rFIP‐GMI Blocks β‐Catenin Nuclear Localization via Phosphorylation

As previously reported, nuclear localization of β‐catenin is a critical event driving oncogenic gene transcription during cancer progression. To determine whether rFIP‐GMI promotes β‐catenin phosphorylation leading to proteasomal degradation, IGF‐1–stimulated BC cells treated with the proteasome inhibitor MG132. As shown in Figure 4A, MG132 treatment markedly increased p‐β‐catenin levels in cells co‐treated with IGF‐1 and rFIP‐GMI. Specifically, p‐β‐catenin expression was approximately twofold higher in MG132–treated group compared with cells without MG132 (Figure 4B,C). confirming that rFIP‐GMI–induced β‐catenin phosphorylation targets the protein for proteasomal degradation. To further assess the effect of rFIP‐GMI on nuclear translocation, we performed nuclear and cytoplasmic fractionation assays (Figure 4D). The results showed a significant (> 50%) reduction in nuclear β‐catenin levels in rFIP‐GMI–treated cells compared with controls (Figure 4E,F). These findings indicate that rFIP‐GMI inhibits the nuclear accumulation of β‐catenin by promoting its phosphorylation and subsequent degradation. Collectively, these results suggest that rFIP‐GMI modulates the GSK3β/β‐catenin axis to suppress oncogenic transcription, underscoring its potential as a therapeutic agent in BC.

Figure 4.

Figure 4

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rFIP‐GMI reduces nuclear β‐catenin levels in IGF‐1‐treated BC cells. (A) Representative Western blot images showing reduced p‐β‐catenin expression in Hs578T and MDA‐MB‐231 cells after rFIP‐GMI treatment; this effect was reversed by MG132, a proteasome inhibitor. (B, C) Densitometric analyses of β‐catenin and p‐β‐catenin protein levels. Data are presented as mean ± SD from three independent experiments. *p < 0.05; **p < 0.01 versus control. (D) Western blot analysis of cytoplasmic and nuclear fractions showing decreased nuclear β‐catenin levels following rFIP‐GMI treatment. β‐actin and lamin were used as cytoplasmic and nuclear loading controls, respectively. (E, F) Quantification of nuclear β‐catenin levels in Hs578T and MDA‐MB‐231 cells, presented as mean ± SD from three independent experiments.

3.6. rFIP‐GMI Suppresses β‐catenin Downstream Oncogene Expression

Nuclear accumulation of β‐catenin activates Wnt/β‐catenin signaling and is strongly associated with cancer progression, cellular differentiation, and lymph node metastasis (Lecarpentier et al. 2019). To further confirm the inhibitory effect of rFIP‐GMI on this pathway, we examined the expression of key β‐catenin downstream oncogenes–c‐Myc, cyclin D1, and MMP‐9–which are critical regulators of proliferation, invasion, and migration. Western blot analysis revealed that rFIP‐GMI markedly reduced the protein levels of these targets compared with control cells (Figure 5A), and densitometric analyses confirmed a significant decrease (Figure 5B–D). These findings provide direct evidence that rFIP‐GMI suppresses β‐catenin signaling by downregulating its oncogenic effectors, thereby contributing to its antimetastatic activity in BC cells.

Figure 5.

Figure 5

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rFIP‐GMI suppresses β‐catenin downstream oncogene expression. (A) Representative Western blot images of c‐Myc, cyclin D1, and MMP‐9, with β‐actin as the loading control. (B–D) Densitometric analyses of c‐Myc, cyclin D1, and MMP‐9 expression. Data are presented as mean ± SD from three independent experiments. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.

4. Discussion

This study provides a comprehensive evaluation of the anticancer effects of rFIP‐GMI, highlighting its potent inhibitory activity against the highly invasive TNBC cell lines Hs578T and MDA‐MB‐231. Our results demonstrate that rFIP‐GMI significantly reduces the phosphorylation of PI3K‐p85 and Akt, key regulators of IGF‐1–induced cancer cell progression. By inactivating the PI3K/Akt pathway, rFIP‐GMI promotes β‐catenin phosphorylation and proteasome–mediated degradation, thereby suppressing invasion and migration of IGF‐1–stimulated BC cells. These findings are consistent with previous reports showing that IGF‐1 signaling promotes TNBC progression through activation of FAK–YAP signaling (Rigiracciolo et al. 2020) and elevated Akt and mitogen‐activated protein kinase in TNBC models (Davison et al. 2011). In agreement with these observations, rFIP‐GMI treatment markedly attenuated IGF‐1–induced PI3K/Akt activation and reduced tumor cell invasion and migration, further supporting the role of rFIP‐GMI as a potential inhibitor of the IGF‐1–driven signaling in this highly invasive BC subtype.

The anticancer properties of Ganoderma spp. extracts are well established, particularly their ability to inhibit cancer cell survival and progression. For instance, polysaccharides derived from Ganoderma lucidum modulate EMT markers by downregulating N‐cadherin and vimentin while upregulating E‐cadherin expression, thus suppressing cervical cancer metastasis and promoting apoptosis (Jin et al. 2020). Similarly, the immunomodulatory protein from Ganoderma microsporum has been reported to inhibit lung tumor growth by attenuating epidermal growth factor receptor (EGFR)–mediated tyrosine kinase activation (Hua et al. 2023). Furthermore, fungal immunomodulatory proteins have been shown to enhance β‐catenin degradation through a GSK3β‐dependent proteasomal pathway, leading to downregulation of oncogene transcription factors, such as survivin and cyclin D1, ultimately inducing apoptosis in lung cancer cells (Hsin et al. 2018). In the present study, Western blot analysis and cytosol/nuclear protein fractionation revealed a marked reduction in nuclear β‐catenin levels, accompanied by increased cytosolic phosphorylated β‐catenin in IGF‐1–stimulated BC cells treated with rFIP‐GMI. These effects were further validated using the proteasome inhibitor MG132, confirming that rFIP‐GMI promotes β‐catenin degradation via a proteasome–dependent mechanism.

Tumor metastasis remains the primary cause of cancer‐related mortality, underscoring the urgent need for effective antimetastatic interventions. EMT is a central process in this progression, with the Wnt/β‐catenin signaling known to drive cancer cell invasiveness, migration, and acquisition of stem‐like properties. Numerous natural compounds and plant‐derived extracts with anticancer activity have been reported to inhibit metastasis by modulating EMT, often through regulation of matrix metalloproteinases (MMPs) and extracellular matrix (ECM) remodeling (Avila‐Carrasco et al. 2019; Gajos‐Michniewicz and Czyz 2024; Li et al. 2019). Fungal bioactive proteins, however, represent a relatively underexplored class of therapeutic agents with significant promise. In this context, our findings demonstrate that rFIP‐GMI suppresses EMT in TNBC cell lines by inactivating the PI3K/Akt pathway, resulting in decreased nuclear β‐catenin accumulation. This suppression subsequently downregulated β‐catenin downstream oncogenic targets, including c‐Myc, cyclin D1, and MMP‐9. These findings are consistent with previous evidence showing that enhanced ubiquitin–proteasome activity facilitates the degradation of β‐catenin and associated oncogenic proteins (Aberle et al. 1997; Easwaran et al. 1999; Hideshima et al. 2001). Collectively, these findings establish rFIP‐GMI as a novel inhibitor of Wnt/β‐catenin signaling and provide strong support for its therapeutic potential in controlling TNBC metastasis.

Beyond its demonstrated antimetastatic potential in vitro, FIP‐GMI has also shown robust in vivo efficacy in suppressing tumor growth, invasion, and metastasis across multiple cancer models, including A549 lung cancer, PANC‐1 pancreatic cancer, and MCF‐7 BC xenografts (Lin et al. 2010; Qu et al. 2018; Wu et al. 2021). These studies suggest that the antitumor effects of FIP‐GMI are not restricted to a single cancer type but may involve a broader mechanism of action. In our current study, the most pronounced reduction in p‐Akt levels occurred within 2 h of rFIP‐GMI treatment, after which no further suppression was observed despite prolonged exposure. This kinetic pattern implies that rFIP‐GMI exerts a rapid yet time‐limited inhibitory effect on PI3K/Akt signaling, thereby defining a therapeutic window for its optimal activity. Such temporal regulation may be particularly relevant for dosing strategy design; as excessive or prolonged treatment might not yield additional benefits and could potentially activate compensatory signaling pathways. Taken together, these results reinforce the therapeutic promise of rFIP‐GMI as a Wnt/β‐catenin pathway modulator and highlight the importance of optimizing treatment timing to maximize its antimetastatic efficacy.

5. Conclusions

Our study demonstrates that rFIP‐GMI suppresses BC invasion and metastasis by inhibiting the phosphorylation of PI3K‐p85 and Akt, thereby preventing nuclear β‐catenin accumulation and blocking its downstream oncogenic transcriptional activity (Figure 6). While these findings provide compelling mechanistic evidence, they are primarily derived from in vitro experiments. Therefore, additional validation using BC xenograft models is required to substantiate its therapeutic efficacy in vivo. Future investigations should also explore the potential crosstalk of rFIP‐GMI with other oncogenic signaling pathways, particularly in TNBC, where effective therapeutic options remain limited. Collectively, these results identify rFIP‐GMI as a promising antimetastatic candidate and underscore the need for comprehensive in vivo and translational studies to fully elucidate its molecular mechanisms and clinical potential.

Figure 6.

Figure 6

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Proposed mechanism by which rFIP‐GMI inhibits TNBC cell invasion and migration via modulation of the PI3K/Akt signaling pathway. (A) IGF‐1 stimulation activates the PI3K/Akt pathway, leading to nuclear accumulation of β‐catenin and upregulation of genes associated with invasion and migration. (B) rFIP‐GMI disrupts this cascade by reducing Akt phosphorylation, thereby promoting β‐catenin ubiquitination and downregulating oncogenic targets involved in cancer progression. Collectively, these effects suppress tumor growth and metastasis. The schematic was created using BioRender (https://www.biorender.com/; agreement number: IN27UAZUU3).

Authorship Contributions

Wen‐Ling Liao designed the experiments, analyzed the data and drafted the manuscript. Yu‐Ying Wu and Yu‐Fan Liu performed the experiments, data curation, investigation and methodology. Pei‐Chi Lan, Yu‐Chun Cheng, Yueh‐Tzu Hung, Hsin‐Wen Liang performed data curation and methodology. Huei‐Jane Lee and Yi‐Hsien Hsieh provided assistance for the conduct of the experiment; Chun‐Wen Cheng contributed to the conceptualization, project administration, supervision, writing−review and revised the manuscript. Wen‐Ling Liao and Chun‐Wen Cheng performed funding acquisition. All authors reviewed and approved the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

This study was financially supported by the Grants of China Medical University Taichung, Taiwan (R.O.C) (CMU‐112‐SR‐49 to Wen‐Ling Liao) and Chung Shan Medical University, Taichung, Taiwan (R.O.C) (CSMU‐INT‐113‐10 to Chun‐Wen Cheng).

Liao, W.‐L. , Wu Y.‐Y., Liu Y.‐F., et al. 2025. “rFIP‐GMI Suppresses IGF‐1–Induced Invasion and Migration in Breast Cancer Cells via PI3K/Akt/β‐Catenin Inhibition.” Drug Development Research 86, 10.1002/ddr.70202.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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