hMAGEA2 promotes progression of breast cancer by regulating Akt and Erk1/2 pathways

Abstract

Breast cancer is the most abundant cancer worldwide and a severe problem for women. Notably, breast cancer has a high mortality rate, mainly because of tumor progression and metastasis. Triple-negative breast cancer (TNBC) is highly progressive and lacks the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Therefore, there are no established therapeutic targets against TNBC. In this study, we investigated whether the expression of human melanoma-associated antigen A2 (MAGEA2) is associated with TNBC. We found that hMAGEA2 is significantly overexpressed in human TNBC tissues; we also observed oncogenic properties using TNBC cell lines (MDA-MB-231 and MDA-MB-468). The overexpression of hMAGEA2 in MDA-MB-231 cell line showed dramatically increased cellular proliferation, colony formation, invasion, and xenograft tumor formation and growth. Conversely, knockdown of hMAEGA2 in MDA-MB-468 cell line suppressed cellular proliferation, colony formation, and xenograft tumor formation. Additionally, we showed that hMAGEA2 regulated the activation of Akt and Erk1/2 signaling pathways. These data indicate that hMAGEA2 is important for progression of TNBC and may serve as a novel molecular therapeutic target.

Keywords: Akt; breast cancer; hMAGEA2; metastasis; triple-negative breast cancer.

Figure 1. hMAGEA2 is overexpressed in human triple-negative breast cancer (TNBC) tissues and breast cancer tissues with TNBC

(A) Immunostaining of hMAGEA2 in human TNBC tissues (upper panel) and breast cancer tissues with TNBC (lower panel). (B) Relative density of expression of hMAGEA2 in human TNBC tissues and breast cancer tissues with TNBC. hMAGEA2 expression was higher in human TNBC tissues and breast cancer with TNBC than that in precancer tissue. (Means ± SD, *p < 0.05, compared with control).

Figure 2. Establishment of stable overexpression of hMAGEA2 in TNBC cell lines

(A) The level of endogenous expression of hMAGEA2 in MDA-MB-231 and MDA-MB-468 cell lines assessed using RT-PCR. (B) Relative mRNA expression of stably overexpressed hMAGEA2 in MDA-MB-231 and MDA-MB-468 cell lines; β-actin was used as normalization control. (C) Protein expression of stable hMAGEA2 overexpression in MDA-MB-231 and MDA-MB-468 cell lines; β-actin was used as loading control. (Means ± SD, *p < 0.05, **p < 0.01, compared with control).

Figure 3. hMAGEA2 enhances proliferation capacity and anchorage-independent colony formation in TNBC cell lines

(A) Relative optical density (O.D.) values of MDA-MB-231 cell line overexpressing hMAGEA2 were compared with those not overexpressing hMAGEA2 using the CCK-8 assay. O.D. was measured after 0, 24, 48, and 72 h. (B) Relative O.D. values of MDA-MB-468 cell line overexpressing hMAGEA2 were compared with those not overexpressing hMAGEA2. O.D. values were measured after 0, 24, 48, and 72 h. (C) Representative images of colonies were acquired using microscopy (x50 magnification, scale bar = 250 μm) (left panel) Colony numbers were counted in MDA-MB-231 and MDA-MB-468 cells lines overexpressing hMAGEA2 and those not overexpressing hMAGEA2. Colony diameters were compared in MDA-MB-231 and MDA-MB-468 cell lines stably overexpressing hMAGEA2 and those not overexpressing hMAGEA2. (right panel) (Means ± SD, *p < 0.05, compared with control) (D) Relative expression of Ki-67 and survivin mRNA was compared between MDA-MB-231 and MDA-MB-468 cell lines overexpressing hMAGEA2 and those not overexpressing hMAGEA2. (Means ± SD, *p < 0.05, **p < 0.01, compared with control).

Figure 4. hMAGEA2 enhances metastasis ability in TNBC cell line

(A) Representative microscopy images of invaded MDA-MB-231 and MDA-MB-468 cell lines overexpressing hMAGEA2 compared with those not overexpressing hMAGEA2 (x100 magnification, scale bar = 250 μm) (upper panel). Percentage of invaded cells (low panel) (Means ± SD, ***p < 0.001, compared with control). (B) Protein expression of epithelial-mesenchymal transition (EMT) markers E-cadherin and vimentin. β-actin was used as loading control.

Figure 5. hMAGEA2 activates p-Akt and p-Erk1/2 in TNBC cell line

(A) Protein expression of phospho-Akt, total-Akt, phospho-Erk1/2, total-Erk1/2, phospho-JNK, total-JNK, phospho-p38, and total-p38, measured in MDA-MB-231 cell line overexpressing hMAGEA2 and those not overexpressing hMAGEA2. β-actin was used as loading control. (B) Relative protein expression level of p-Akt and p-Erk1/2 in MDA-MB-231 cell line overexpressing hMAGEA2, and those not overexpressing hMAGEA2. (Means ± SD, *p < 0.05, **p < 0.01, compared with control).

Figure 6. hMAGEA2 enhances xenograft tumor formation via activation of p-Akt and p-Erk1/2

(A) Growth curve of xenograft tumor formation after the injection of MDA-MB-231 cell line overexpressing hMAGEA2, and those not overexpressing hMAGEA2, in Balb/c nude mice. (Means ± SD, *p < 0.05) (B) The amount of hMAGEA2 was confirmed using hematoxylin and eosin (H&E) staining and immunohistochemistry in sectioned xenograft tumor tissues generated using MDA-MB-231 cell line stably overexpressing hMAGEA2 (x50 magnification, scale bar = 250 μm) (upper panel). The expression of phospho-Akt and phospho-Erk1/2 in xenograft tumor tissues was detected using immunofluorescence (x200 magnification, scale bar = 250 μm) (lower panel).

Figure 7. Knockdown of hMAGEA2 inhibits progression of TNBC

(A) Establishment of hMAGEA2 knockdown in MDA-MB-468 cell line. Decrease in the expression of hMAGEA2 mRNA (left panel) and protein (right panel) compared with those of scrambled control shRNA and shMAGEA2. (B) Relative OD values were measured at 0, 12, 24, 36, and 48 h in scrambled control shRNA and shMAGEA2 using the CCK-8 assay (upper panel). Colony formation was compared between scrambled control shRNA and shMAGEA2 (x50 magnification, scale bar = 250 μm) (lower panel). (C) Protein expression of phospho-Akt, total-Akt, phospho-Erk1/2, total-Erk1/2, phospho-p38, and total-p38 was assessed in scrambled control shRNA and shMAGEA2. (D) Growth curve of xenograft tumor formation after injection of cell lines expressing shMAGEA2 and scrambled control shRNA in Balb/c nude mice (left panel). Using sectioned xenograft tumor tissues, H&E staining and immunohistochemistry were performed. (x50 magnification, scale bar = 250 μm) (right panel). (Means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 compared with control).