Rap2B promotes proliferation, migration, and invasion of human breast cancer through calcium-related ERK1/2 signaling pathway

Abstract

Rap2B, a member of GTP-binding proteins, is widely upregulated in many types of tumors and promotes migration and invasion of human suprarenal epithelioma. However, the function of Rap2B in breast cancer is unknown. Expression of Rap2B was examined in breast cancer cell lines and human normal breast cell line using Western blot analysis. Using the CCK-8 cell proliferation assay, cell cycle analysis, and transwell migration assay, we also elucidated the role of Rap2B in breast cancer cell proliferation, migration, and invasion. Results showed that the expression of Rap2B is higher in tumor cells than in normal cells. Flow cytometry and Western blot analysis revealed that Rap2B elevates the intracellular calcium level and further promotes extracellular signal-related kinase (ERK) 1/2 phosphorylation. By contrast, calcium chelator BAPTM/AM and MEK inhibitor (U0126) can reverse Rap2B-induced ERK1/2 phosphorylation. Furthermore, Rap2B knockdown inhibits cell proliferation, migration, and invasion abilities via calcium related-ERK1/2 signaling. In addition, overexpression of Rap2B promotes cell proliferation, migration and invasion abilities, which could be neutralized by BAPTM/AM and U0126. Taken together, these findings shed light on Rap2B as a therapeutic target for breast cancer.

Figure 1. Protein expression level of Rap2B in breast cancer cell lines.

(A) Western blot analysis of Rap2B expression in MCF-10A, MDA-MB-231, Bcap-37, SK-BR-3 and MCF-7 group with Rap2B antibody. β-actin served as loading control. The intensity of Rap2B was quantified by densitometry (software: Image J, NIH). (B) Western blot analysis of Rap2B in the Rap2B knockdown and control siRNA group for both Bcap-37 and MDA-MB-231 cell lines. The intensity of Rap2B was quantified by densitometry. (C) Western blot analysis to measure the protein levels of Rap2B in breast cancer cells transfected with Rap2B plasmids and pcDNA3.1-Myc3 plasmids. The intensity of Rap2B was quantified by densitometry. All experiments were carried out in triplicate. Data are presented as mean ± SD (n = 3). **P < 0.01 in comparison with respective group.

Figure 2. Effect of Rap2B on cell proliferation of breast cancer cells.

(A) CCK-8 cell proliferation assay after Rap2B knockdown in Bcap-37 and MDA-MB-231 cells. (B) CCK-8 cell proliferation assay after Rap2B overexpressed in breast cancer cells. (C) Influence of Rap2B gene silencing on the cell cycle of breast cancer cells. The percentage of G1 population cells was measured by flow cytometry after Rap2B knockdown in breast cancer cells. (D) Influence of Rap2B gene overexpression on cell cycle analysis of breast cancer cells. The percentage of cells at G1 stage was calculated using ModFit LT 3.0 software. All experiments were carried out in triplicate. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 in comparison with respective group.

Figure 3. Rap2B can increase the intracelluar calcium level and induce ERK1/2 phosphorylation.

(A) Rap2B siRNA could decrease intracelluar calcium level in Bcap-37 and MDA-MB-231 cell lines, as measured by a flow cytometer. (B) Rap2B overexpression increased intracelluar calcium level in breast cancer cells. (C) After breast cancer cells were transfected and starved, some cells were treated with 10% FBS and some were not. Western blot analysis of the protein levels of Rap2B, p-ERK, and ERK in Rap2B knockdown and control siRNA group for both cell lines. (D) After transfection and starvation, cells were incubated in the presence or absence of U0126 for 30 min. Then, some cells were treated with 10% FBS and some were not. Western blot analysis of the protein levels of Rap2B, p-ERK, and ERK in Rap2B overexpression and control group for both cell lines. (E) After transfection and starvation, cells were incubated in the presence or absence of BAPTA/AM for 30 min. Then, some cells were treated with 10% FBS. Western blot analysis of the protein levels of Rap2B, p-ERK, and ERK in Rap2B over-expression and control group for both cell lines. All experiments were carried out in triplicate. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 in comparison to respective group, ^#P < 0.05, ^##P < 0.01 in comparison with respective 10% FBS-untreated group, and ^&P < 0.05, ^&&P < 0.01 in comparison with respective inhibitor-untreated group.

Figure 4. Knockdown of Rap2B inhibits breast cancer cell migration and invasion abilities.

(A) Wound-healing assay was performed after Rap2B knockdown in Bcap-37 and MDA-MB-231 cell lines. (B) Cell migration assay was carried out after Rap2B knockdown in breast cancer cells. (C) A matrigel cell invasion assay was carried out after Rap2B knockdown in breast cancer cells. All experiments were carried out in triplicate. Data are presented as mean ± SD (n = 3). **P < 0.01 in comparison with respective group.

Figure 5. Rap2B overexpression promotes breast cancer cell migration and invasion abilities.

(A)Wound-healing assay was performed after reintroduction of Rap2B in Ctrl, Rap2B, Rap2B + BAPTA/AM and Rap2B + U0126 group in Bcap-37 and MDA-MB-231 cell lines. Starved cells were incubated with 30 μmol/L BAPTA/AM and 10 μmol/L U0126. (B) Cell migration assay was carried out after overexpression of Rap2B in Ctrl, Rap2B, Rap2B+BAPTA/AM and Rap2B+U0126 group in breast cancer cells. Starved cells were incubated with 30 μmol/L BAPTA/AM and 10 μmol/L U0126. (C) A matrigel cell invasion assay was carried out after overexpression of Rap2B in Ctrl, Rap2B, Rap2B + BAPTA/AM and Rap2B + U0126 group in breast cancer cells. Starved cells were incubated with 30 μmol/L BAPTA/AM and 10 μmol/L U0126. All experiments were carried out in triplicate. Data are presented as mean ± SD (n = 3). **P < 0.01 in comparison with respective group, and ^&P < 0.05, ^&&P < 0.05 in comparison with respective inhibitor-untreated group.

Figure 6. A schematic diagram illustrating the proposed Rap2B-induced calcium-related ERK1/2 signaling pathway involved in breast cancer cell proliferation, migration, and invasion.

Rap2B increases the intracellular calcium level, which subsequently phosphorylates the downstream target of ERK1/2, thereby promoting breast cancer cell proliferation, migration, and invasion in vitro. Together, it will support the novel notion that Rap2B promotes tumor cell proliferation, migration, and invasion via calcium-related ERK1/2 signaling pathway.