Gradient Rotating Magnetic Fields Impairing F-Actin-Related Gene CCDC150 to Inhibit Triple-Negative Breast Cancer Metastasis by Inactivating TGF-β1/SMAD3 Signaling Pathway

Abstract

Triple-negative breast cancer (TNBC) is the most aggressive and lethal malignancy in women, with a lack of effective targeted drugs and treatment techniques. Gradient rotating magnetic field (RMF) is a new technology used in oncology physiotherapy, showing promising clinical applications due to its satisfactory biosafety and the abundant mechanical force stimuli it provides. However, its antitumor effects and underlying molecular mechanisms are not yet clear. We designed two sets of gradient RMF devices for cell culture and animal handling. Gradient RMF exposure had a notable impact on the F-actin arrangement of MDA-MB-231, BT-549, and MDA-MB-468 cells, inhibiting cell migration and invasion. A potential cytoskeleton F-actin-associated gene, CCDC150, was found to be enriched in clinical TNBC tumors and cells. CCDC150 negatively correlated with the overall survival rate of TNBC patients. CCDC150 promoted TNBC migration and invasion via activation of the transforming growth factor β1 (TGF-β1)/SMAD3 signaling pathway in vitro and in vivo. CCDC150 was also identified as a magnetic field response gene, and it was marked down-regulated after gradient RMF exposure. CCDC150 silencing and gradient RMF exposure both suppressed TNBC tumor growth and liver metastasis. Therefore, gradient RMF exposure may be an effective TNBC treatment, and CCDC150 may emerge as a potential target for TNBC therapy.

Fig. 1.

Gradient RMF cell culture device and its effect on cytoskeleton and motility of MDA-MB-231 cell at 5 Hz, 0.41 T. (A) Schematic diagram of gradient RMF cell culture platform. (B) Partial enlargement of gradient RMF device. Y-component of the magnetic flux density distribution in the vertical (C) and horizontal (D) directions in N–N mode. Effect of gradient RMF exposure on morphology (E), cytoskeleton (F), polarization coefficient (G), spreading area (H), migration (I), invasion (J), and wound-healing capacity (K) of MDA-MB-231 cells. n = 3. Statistical analysis was conducted using t test and one-way ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001 versus NC group.

Fig. 2.

Correlation between CCDC150 expression and TNBC. (A) Screening of key gradient RMF response cytoskeleton-related gene CCDC150 in TNBC. (B) CCDC150 expression in TNBC tumor and adjacent tissue in TCGA database. (C) Correlation between CCDC150 expression and stage of tumor development according to TCGA database. (D) Effect of CCDC150 expression on overall survival rate in TNBC. (E) Expression of CCDC150 in different subtypes of TNBC. (F) Sankey diagram of CCDC150 expression in TNBC. Biological process (G) and cellular component categories (H) of CCDC150 in TNBC. CCDC150 expression in TNBC tumor tissues of clinical patients (I) and TNBC cells (J). (K) Immunohistochemical staining results of CCDC150 expression in TNBC tumor tissues. n = 3. Statistical analysis was conducted using t test, one-way ANOVA, or two-way ANOVA, and post hoc tests were carried out. *P < 0.05, **P < 0.01, and ***P < 0.001 versus NC group.

Fig. 3.

Effects of CCDC150 on migration, invasion, and cytoskeleton F-actin rearrangement in MDA-MB-231 cells. (A) Transfection efficiency after treatment with CCDC150-specific siRNA. Effect of CCDC150 knockdown on cell viability (B), cycle (C), apoptosis (D), migration capacity (E), invasion capacity (F), and wound-healing capacity (G) of MDA-MB-231 cells. Fluorescent staining of cytoskeleton F-actin in MDA-MB-231 cells (H), and cell polarization coefficient (I) and spreading area (J) after CCDC150 silencing. (K) Expression level of EMT biomarkers after CCDC150 silencing. GAPDH was used as a reference gene. n = 3. Statistical analyses were conducted using t test and one-way ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001 versus NC group.

Fig. 4.

Effect of gradient RMF exposure or CCDC150 knockdown on TNBC tumor growth in situ. Schematic diagram (A) and profile display (B) of gradient RMF animal handling device. (C) Y-component of the magnetic flux density distribution with different spacing alignments in N–N mode. (D) Flow chart of experiments with gradient RMF exposure or CCDC150 knockdown of TNBC xenografts in nude mice. Body weight (E), tumor volume (F), tumor image (G), and tumor weight (H) in different groups (NC siRNA treatment, si-CCDC150 treatment, gradient RMF exposure). H&E (I) and Ki67 (J) staining of tumor tissue. (K) Major organ weights of mice in each group. (L) Expression level of CCDC150 and EMT biomarkers in different treatment groups. GAPDH was used as a reference gene. n = 7. Statistical analyses were conducted using t test, one-way ANOVA, or two-way ANOVA, and post hoc tests were carried out. *P < 0.05, **P < 0.01, and ***P < 0.001 versus NC group.

Fig. 5.

Gradient RMF exposure or CCDC150 silencing inhibits liver colonization and metastasis of MDA-MB-231 cells. (A) H&E staining images of liver tissues of nude mice. (B) ALT and AST level in nude mice. (C) ALT/AST ratio in nude mice. (D) Experiment flowchart of MDA-MB-231 cell coculture with LX-2 cells. (E) α-SMA levels in LX-2 cells after coculture with CM-1 collected from MDA-MB-231 cells (group A, normal medium; group B, CM-1 collected from MDA-MB-231 cells; group C, CM-1 collected from MDA-MB-231 cells after transfection with CCDC150 siRNA; group D, CM-1 collected from MDA-MB-231 cells after gradient RMF exposure). Effect of CM-2 from LX-2 cells on MDA-MB-231 cell migratory capacity (F), invasive capacity (G), colony formation capacity (H), wound-healing capacity (I), and EMT biomarker level (J). GAPDH was used as the reference gene. n = 3. Statistical analyses were conducted using t test, one-way ANOVA, or two-way ANOVA, and post hoc tests were carried out. *P < 0.05, **P < 0.01, and ***P < 0.001 versus NC group.

Fig. 6.

Gradient RMF exposure inactivates the TGF-β1/SMAD3 signaling pathway by down-regulating CCDC150 in MDA-MB-231 cells. (A) Expression of biomarkers of TGF-β1/SMAD3 signaling pathway after CCDC150 knockdown. (B) Expressions of TGF-β1/SMAD3 signaling pathway and EMT biomarkers after treatment with gradient RMF and TGF-β1 agonist and its inhibitor. Effect of gradient RMF exposure and TGF-β1/SMAD3 signaling pathway on migration (C), invasion (D), wound healing (E), and colony formation capacity (F) of MDA-MB-231 cells. (G) Potential mechanisms by which gradient RMF exposure suppresses TNBC progression by regulating CCDC150. GAPDH was used as the reference gene. n = 3. Statistical analyses were conducted using t test, one-way ANOVA, or two-way ANOVA, and post hoc tests were carried out. *P < 0.05, **P < 0.01, and ***P < 0.001 versus NC group.