Chlorotoxin targets ERα/VASP signaling pathway to combat breast cancer

Abstract

Breast cancer is one of the most common malignant tumors among women worldwide. About 70-75% of primary breast cancers belong to estrogen receptor (ER)-positive breast cancer. In the development of ER-positive breast cancer, abnormal activation of the ERα pathway plays an important role and is also a key point leading to the failure of clinical endocrine therapy. In this study, we found that the small molecule peptide chlorotoxin (CTX) can significantly inhibit the proliferation, migration and invasion of breast cancer cells. In in vitro study, CTX inhibits the expression of ERα in breast cancer cells. Further studies showed that CTX can directly bind to ERα and change the protein secondary structure of its LBD domain, thereby inhibiting the ERα signaling pathway. In addition, we also found that vasodilator stimulated phosphoprotein (VASP) is a target gene of ERα signaling pathway, and CTX can inhibit breast cancer cell proliferation, migration, and invasion through ERα/VASP signaling pathway. In in vivo study, CTX significantly inhibits growth of ER overexpressing breast tumor and, more importantly, based on the mechanism of CTX interacting with ERα, we found that CTX can target ER overexpressing breast tumors in vivo. Our study reveals a new mechanism of CTX anti-ER-positive breast cancer, which also provides an important reference for the study of CTX anti-ER-related tumors.

Keywords: ERα; VASP; breast cancer; chlorotoxin; treatment.

Figure 1

Preparation and purification of chlorotoxin. (A) The CTX recombination protein was isolated and identified by Tris‐Tricine‐SDS‐PAGE electrophoresis. Lane 1: Low Range Protein ladder marker, Lane 2: pGEX‐6p‐1‐CTX‐induced whole‐cell protein without IPTG, Lane 3: whole‐cell protein after induction of IPTG by pGEX‐6p‐1‐CTX, Lane 4: wash with PBS heteroprotein, Lane 5: concentrated GST‐CTX fusion protein after ultrafiltration, Lane 6: GST protein and CTX protein after digestion, Lane 7: HPLC purification of the obtained CTX protein. (B) The isolated CTX protein was purified by RP‐HPLC. (C) The molecular weight of the purified protein was determined by the MALDI‐TOF‐MS method. CTX, chlorotoxin; RP‐HPLC, reversed‐phase high‐performance liquid chromatography

Figure 2

Chlorotoxin can inhibit the proliferation, migration, and invasion of breast cancer cells in a concentration‐ and time‐dependent manner. MCF‐7 (A) and MDA‐MB‐231 (B) cells were treated with CTX at 0, 0.05, 0.5, and 5 μmol/L for 0, 12, 24, 48, and 72 hours, respectively. Cell proliferation was measured by a CCK‐8 assay. MCF‐7 (C) and MDA‐MB‐231 (D) breast cancer cells were treated with 0, 0.05, 0.5, and 5 μmol/L CTX for 24 hours. Cell migration was tested by a wound healing assay. (E) MCF‐7 and MDA‐MB‐231 breast cancer cells were treated with 0, 0.05, 0.5, and 5 μmol/L CTX for 24 hours. Cell migration was tested by a transwell assay. (F) MCF‐7 and MDA‐MB‐231 breast cancer cells were treated with 0, 0.05, 0.5, and 5 μmol/L CTX for 24 hours. Cell invasion was tested by a transwell assay. *P < 0.05, **P < 0.01, ***P < 0.001. CTX, chlorotoxin

Figure 3

CTX can significantly inhibit the expression levels of ERα and VASP in breast cancer cells. (A) MCF‐7 and MDA‐MB‐231 cells were treated with 0, 0.05, 0.5, 5, and 10 μmol/L CTX for 24 hours and harvested for detecting the mRNA expression levels of ERα and VASP by RT‐qPCR. (B) MCF‐7 and MDA‐MB‐231 cells were treated with 5 μmol/L CTX for 0, 12, 24, and 48 hours, respectively, and harvested for detecting the mRNA expression levels of ERα and VASP by RT‐qPCR. (C) MCF‐7 and MDA‐MB‐231 cells were treated with 0, 0.05, 0.5, and 5 μmol/L CTX for 24 hours, and harvested for detecting the protein expression levels of ERα and VASP by western blotting. (D) MCF‐7 and MDA‐MB‐231 cells were treated with 5 μmol/L CTX for 0, 12, 24, and 48 hours, respectively, and harvested for detecting the protein expression levels of ERα and VASP by western blotting. (E) The effect of CTX, E2, Tam, and ERα overexpression on the activity of ERα promoter reporter gene was detected by a luciferase reporter gene assay in MCF‐7 cells. MCF‐7 (F‐H) and MDA‐MB‐231 (I) cells were treated with 5 μmol/L CTX for 24 hours. The effect of CTX treatment on the expression and distribution of ERα, VASP, MMP2, and actin was detected by immunofluorescence. *P < 0.05, **P < 0.01, ***P < 0.001. CTX, chlorotoxin; ERα, estrogen receptor α; RT‐qPCR, quantitative reverse transcription polymerase chain reaction

Figure 4

CTX can directly interact with ERα and affect the protein secondary structure of ERα‐LBD. The binding of CTX and ERα was detected by a pull‐down assay (A) and internal fluorescence emission spectroscopy assay (B). The effect of CTX on the secondary structure of ERα‐LBD was detected by a circular dichroism assay (C). CTX, chlorotoxin; ERα, estrogen receptor α

Figure 5

VASP was a target gene of ERα signaling pathway. (A) E2 or Tam was used to activate or inhibit the ERα signaling pathway, respectively. In addition, the mRNA expression of ERα and VASP was detected by RT‐qPCR. (B) After transfection of ERα overexpression plasmid, VASP mRNA expression was detected by RT‐qPCR. (C) MCF‐7 cells were treated with 0, 0.01, 0.1, and 1 μmol/L E2 for 24 hours. The protein expression levels of ERα and VASP were detected by western blotting. (D) MCF‐7 cells were treated with 0, 0.01, 0.1, and 1 μmol/L Tam for 24 hours, the protein expression levels of ERα and VASP were detected by western blotting. (E) ERα can bind to the promoter of VASP, which was detected by ChIP assay. (F) The wild‐type and mutated ERα binding site, and the truncated of VASP promoter reporter plasmids were constructed. (G) The effect of ERα overexpression on the activity of wild‐type or truncated VASP promoter reporter, which was detected by luciferase reporter gene assay. (H) The effect of ERα overexpression on the activity of wild‐type and mutated ERα binding site of VASP promoter reporter, which was detected by luciferase reporter gene assay. *P < 0.05, **P < 0.01, ***P < 0.001. ChIP, chromatin immunoprecipitation; ERα, estrogen receptor α; RT‐qPCR, quantitative reverse transcription polymerase chain reaction

Figure 6

CTX can target ER‐positive breast tumors in vivo. (A) The CTX: Cy5.5 recombination protein was purified by RP‐HPLC. (B) The molecular weight of the purified CTX: Cy5.5 recombination protein was determined by MALDI‐TOF‐MS method. MCF‐7 (C) and MDA‐MB‐231 (D) breast cancer cells were transplanted into nude mice, respectively, and the distribution of CTX in mice was observed by small animal in vivo imaging technique. (The arrow and circle indicated the location of tumors in vivo, and the square indicated the tumors ex vivo.) The effect of CTX on MCF‐7 tumor (E) and the growth curve and weight (F) of tumors were present. (G) The effect of CTX on the growth curve and weight of MDA‐MB‐231 tumors were present. (H) The liver and kidney tissues of nude mice were collected and detected by H&E staining. ***P < 0.001. CTX, chlorotoxin; ER, estrogen receptor; RP‐HPLC, reversed‐phase high‐performance liquid chromatography; H&E, hematoxylin and eosin

Figure 7

Working model for the regulation of proliferation and migration of breast cancer cells by CTX. CTX can directly interact with ERα to inhibit the expression of ERα, which inhibits the ERα/VASP signaling pathway, leading to suppression of cell growth and migration in breast cancer. CTX, chlorotoxin; ER, estrogen receptor