LMO4 mediates trastuzumab resistance in HER2 positive breast cancer cells

Abstract

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Trastuzumab (Herceptin) is an effective antibody drug for HER2 positive breast cancer; de novo or acquired trastuzumab resistance retarded the use of trastuzumab for at least 70% of HER2 positive breast cancers. In this study, we reported LMO4 (a member of LIM-only proteins) promoted trastuzumab resistance in human breast cancer cells. Over-expression of LMO4 was observed in acquired trastuzumab resistance breast cancer cells SKBR3 HR and BT474 HR. Depletion of LMO4 partly abolished the trastuzumab resistance of SKBR3 HR and BT474 HR cells. Forced expression of LMO4 significantly increased trastuzumab resistance of HER2 positive breast cancer cells both in vitro and in vivo. BCL-2 was regulated by LMO4 and mediated the promoting role of LMO4 in trastuzumab resistance of HER2 positive breast cancer cells. High level of LMO4 was associated with worse clinicopathological parameters (including tumor size and histological grade) and lower survival rate in HER2 positive breast cancer patients. LMO4 therefore could be used as a target to develop diagnostic and therapeutic methods for human HER2 positive breast cancer.

Keywords: BCL-2; HER2; LMO4; breast cancer; trastuzumab resistance.

Figure 1

Expression of LMO4 in acquired trastuzumab resistant breast cancer cells. A. SKBR3, BT474 parental cells and trastuzumab resistant cells SKBR3 HR (10 µg/ml), BT474 HR (10 µg/ml) were treated with the indicated concentrations of trastuzumab for 6 days, MTT assay was performed to detected cell viability. B. mRNA levels of LMO4 in SKBR3/SKBR3 HR and BT474/BT474 HR cells were examined by RT-qPCR. GAPDH was used as an endogenous control. C. Protein levels of LMO4 in SKBR3/SKBR3 HR and BT474/BT474 HR cells were examined by western blot. β-actin was used as an endogenous control. *P<0.05. **P<0.01.

Figure 2

Depletion of LMO4 reduced trastuzumab resistance of SKBR3 HR and BT474 HR cells. A. Protein levels of LMO4 in SKBR3 HR and BT474 HR cells after transfection with siLMO4 or siNC were examined by western blot. β-actin was used as an endogenous control. B. Cell viability of SKBR3 HR and BT474 HR cells after transfection with siLMO4 or siNC and treated with trastuzumab (10 µg/ml) or vehicle for 6 days were examined by MTT assay. C. SKBR3 HR and BT474 HR cells were transfected with siLMO4 or siNC and treated with trastuzumab (10 µg/ml) or vehicle for 6 days, cell colony formation in soft agar was examined. *P<0.05. **P<0.01.

Figure 3

Forced expression of LMO4 reduced trastuzumab sensitivity of SKBR3 and BT474 cells in vitro. A. Protein levels of LMO4 in SKBR3 and BT474 cells after transfection with LMO4 plasmid or Vec control were examined by western blot. β-actin was used as an endogenous control. B. Cell viability of SKBR3 and BT474 cells after transfection with LMO4 plasmid or Vec control and treated with trastuzumab (1.25 µg/ml) or vehicle for 6 days were examined by MTT assay. C. SKBR3 and BT474 cells were transfected with LMO4 plasmid or Vec control and treated with trastuzumab (1.25 µg/ml) or vehicle for 6 days, cell colony formation in soft agar were examined. *P<0.05. **P<0.01.

Figure 4

Forced expression of LMO4 reduced trastuzumab sensitivity of breast cancer cells in vivo. A. SKBR3 LMO4 and SKBR3 Vec cells were injected subcutaneously into the dorsal flank of 4-week-old female mice. At 15 days after cell inoculation, these tumors reached about 100 mm³ and mice were randomized to receive trastuzumab (10 mg/kg) or vehicle as control once a week. Mice were sacrificed and tumors were harvested after 39 days. Growth curves of tumors were calculated. B. Ki-67 staining of tumor sections derived from mice injected with SKBR3 LMO4 or SKBR3 Vec cells and received trastuzumab or vehicle control. *P<0.05. **P<0.01.

Figure 5

BCL-2 was regulated by LMO4 in parental and trastuzumab resistant breast cancer cells. A. mRNA levels of BCL-2 in SKBR3 and BT474 cells after transfection with LMO4 plasmid or Vec control were examined by RT-qPCR. GAPDH was used as an endogenous control. B. Protein levels of BCL-2 in SKBR3 and BT474 cells after transfection with LMO4 plasmid or Vec control were examined by western blot. β-actin was used as an endogenous control. C. mRNA levels of BCL-2 in SKBR3 HR and BT474 HR cells after transfection with siLMO4 or siNC were examined by RT-qPCR. D. Protein levels of BCL-2 in SKBR3 HR and BT474 HR cells after transfection with siLMO4 or siNC were examined by western blot. *P<0.05. **P<0.01.

Figure 6

Depletion of BCL-2 reduced trastuzumab resistance of SKBR3 HR and BT474 HR cells. A. Protein levels of BCL-2 in SKBR3 HR and BT474 HR cells after transfection with siBCL-2 or siNC were examined by western blot. β-actin was used as an endogenous control. B. Cell viability of SKBR3 HR and BT474 HR cells after transfection with siBCL-2 or siNC and treated with trastuzumab (10 µg/ml) or vehicle for 6 days were examined by MTT assay. C. SKBR3 HR and BT474 HR cells were transfected with siBCL-2 or siNC and treated with trastuzumab (10 µg/ml) or vehicle for 6 days, cell colony formation in soft agar were examined. *P<0.05. **P<0.01.

Figure 7

LMO4 enhanced trastuzumab resistance of human breast cancer cells through BCL-2. A. SKBR3 and BT474 cells with forced expression of LMO4 were co-transfected with siBCL-2 or siNC. Protein levels of LMO4 and BCL-2 were examined by western blot. β-actin was used as an endogenous control. B. MTT assay; C. Soft agar colony formation assay were performed in SKBR3 and BT474 cells after co-transfected with LMO4 plasmid or Vec control and siBCL-2 or siNC and treated with trastuzumab (1.25 µg/ml) or vehicle for 6 days. *P<0.05. **P<0.01.

Figure 8

Expression of HER2 and LMO4 in human breast cancer tissues and patient survival analysis. A. Protein levels of HER2 in breast cancer tissues were examined using immunohistochemistry. B. Protein levels of LMO4 in HER2++/+++ breast cancer tissues were examined using immunohistochemistry. The magnifications of the images were 200. C. The RFS and OS rates of HER2 positive breast cancer patients with different LMO4 expression levels were analyzed by Kaplan-Meier curves.