Dysregulation of HER2/HER3 signaling axis in Epstein-Barr virus-infected breast carcinoma cells

Abstract

The role of Epstein-Barr virus (EBV) in the pathogenesis of breast cancer has been of long-standing interest to the field. Breast epithelial cells can be infected by EBV through direct contact with EBV-bearing lymphoblastoid cells, and EBV infection has recently been shown to confer breast cancer cells an increased resistance to chemotherapeutic drugs. In this study, we established EBV-infected breast cancer MCF7 and BT474 cells and demonstrated that EBV infection promotes tumorigenic activity of breast cancer cells. Firstly, we showed that the EBV-infected MCF7-A and BT474-A cells exhibited increased anchorage-independent growth in soft agar. The increased colony formation capacity in soft agar was associated with increased expression and activation of HER2/HER3 signaling cascades, as evidenced by the findings that the treatment of HER2 antibody trastuzumab (Herceptin), phosphatidylinositol 3-kinase inhibitor, or MEK inhibitor completely abolished the tumorigenic capacity. In the EBV-infected breast cancer cells, the expression of EBV latency genes including EBNA1, EBER1, and BARF0 was detected. We next showed that BARF0 alone was sufficient to efficiently up-regulate HER2/HER3 expression and promoted tumorigenic activity in MCF7 and BT474 cells by the use of both overexpression and small interfering RNA knock-down. Collectively, we demonstrated that EBV-encoded BARF0 promotes the tumorigenic activity of breast cancer cells through activation of HER2/HER3 signaling cascades.

FIG. 1.

EBV-infected MCF7 and BT474 cells display type I latency viral gene expression profile. (A) RT-PCR analyses of EBV gene expression profiles in the parental (MCF7 and BT474), vector control (MCF7-Neo^r and BT474-Neo^r), or EBV-infected (MCF7-A and BT474-A) cells using primers specific for the designated transcript. MCF7-A and BT474-A cells are positive for EBNA1, EBER1, and BARF0 but negative for RK-BARF0, EBNA2, LMP1, LMP2A, and Zta transcripts. RNA from B95.8 virus-transformed lymphoblastoid cells (LCL) was included as a positive control. S26 was detected as an internal control. (B) Western blot analysis of EBV-encoded proteins in MCF7-A and BT474-A cells. Fifty micrograms of protein extracts was detected by 8% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane for immunoblotting with specific antibodies. Both cells are positive for EBNA1 but negative for EBNA2 and LMP1. Cell lysate from LCL was included as a positive control. α-Tubulin was detected as an internal control.

FIG. 2.

EBV infection enhances anchorage-independent growth of breast cancer cells. MCF7, MCF7-A, and MCF7-Neo^r cells, as well as BT474, BT474-A, and BT474-Neo^r cells, were seeded in 6-cm dish in culture medium containing 0.33% low-melting-temperature agarose over a 0.5% agarose layer and culture for 2 weeks. Colonies were then stained with 0.05% crystal violet, and colonies larger than 60 μm were counted. (A) Photomicrographs show a representative size distribution of MCF7, MCF7-A, MCF7-Neo^r, BT474, BT474-A, and BT474-Neo^r colonies. Bar, 100 μm. (B) The number of colonies is expressed as a percentage of the number of colonies in parental controls. Results from three independent experiments were averaged and are presented as means ± standard error as shown. The Student's t test was used to determine the significance of enhancement ★, P < 0.05.

FIG. 3.

EBV infection upregulates the expression of HER2 and HER3. (A) HER2 and HER3 mRNA expression levels in parental (MCF7 and BT474), EBV-infected (MCF7-A and BT474-A), and Neo^r (MCF7-Neo^r and BT474-Neo^r) cell lines were determined by RT-PCR. S26 was detected as an internal control. Signal changes relative to the parental control are shown. (B) Protein expression of HER1, HER2, HER3, and HER4 was detected in immunoblotting with specific antibodies. α-Tubulin was detected as an internal control. Increases in protein expression (n-fold) relative to the parental control are shown. (C) After being washed in phosphate-buffered saline, MCF7, MCF7-A, and MCF7-Neo^r cells were harvested in lysis buffer, and 500 μg of each lysate was immunoprecipitated with a HER2 antibody. The immune complexes were divided into equal parts for detecting HER2, HER3, p-Tyr, and p85 with specific antibodies in immunoblotting. α-Tubulin detected from 10% of cell lysates used for immunoprecipitation served as an internal control.

FIG. 4.

EBV infection stimulates ERK and Akt activation through HER2/HER3 signaling pathway. (A) Western blot analysis of the activation of ERK and Akt downstream of HER2/HER3. Fifty micrograms of protein extracts was detected by 8% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes for immunoblotting with antibodies specific to phospho-ERK1/2, ERK1/2, phospho-Akt, or Akt kinases. α-Tubulin was detected as an internal control. (B) Trastuzumab inhibits phosphorylation of ERK and Akt in MCF7-A cells. MCF7-A cells were treated with trastuzumab (10 μg/ml). At the indicated time points, the cells were washed and harvested in lysis buffer, and 50 μg of protein lysates was analyzed in immunoblotting with antibodies as indicated.

FIG. 5.

The anchorage-independent growth of MCF7-A cells was attenuated by trastuzumab, PI3K inhibitor LY294002 or ERK inhibitor U0126. MCF7, MCF7-A, and MCF7-Neo^r cells (1 × 10⁴ cells/well) were seeded in 6-cm dishes in culture medium containing 0.33% low-melting-temperature agarose over a 0.5% agarose layer and were cultured for 2 weeks in the presence of DMSO or trastuzumab (10 μg/ml), U0126 (20 μM), or LY294002 (20 μM). Colonies were then stained with 0.05% crystal violet.

FIG. 6.

Expression of HER2 and HER3 were upregulated in MCF7-HA-BARF0 cells. (A) MCF7 was transfected with EBNA1, BARF0, or EBER expression plasmids, and transfectants were subjected to RT-PCR analysis of HER2, HER3, EBNA1, BARF0, and EBER1. EBV-infected MCF7 (MCF7-A) was included as a positive control. MCF7-Hyg^r and MCF7-Neo^r served as vector-harboring controls. S26 was detected as an internal control. (B) Total protein extracts from cells in panel A were detected by 8% or 12% (for BARF0) SDS-PAGE and transferred onto nitrocellulose membrane for immunoblotting with specific antibodies against HER2, HER3, EBNA1, or HA. α-Tubulin was detected as an internal controls. According to the protein marker (indicated at right), the approximately 21-kDa molecular masses of HA-BARF0 can be detected in BARF0 transfectants. (C) MCF7, MCF7-A-pSuper (vector-harboring cells), and MCF7-A-siBARF0 cells lysates were examined by RT-PCR for HER2, HER3, EBNA1, BARF0, and EBER1 expression (upper panel). Total protein extracts from cells were separated by 8% SDS-PAGE and transferred onto nitrocellulose membrane for blotting with antibodies specific to the HER2 and HER3. α-Tubulin was detected as an internal control (lower panel). (D) The pNulit or pGL3-erb3 plasmid was transfected into MCF7-HA-BARF0 cells or MCF7-Neo^r cells. The Renilla reporter plasmid (1:10 ratio with target reporter) was also included in all experiments to normalize variation in transfection efficiency. Lysates were harvested 48 h posttransfection, and luciferase activity was determined with the Dual-Glo Luciferase assay system (Promega). Results are expressed in relative light units as the ratio of the Renilla luciferase activity to the increase in induction (n-fold) relative to the activity observed in MCF7-Neo^r cells. These results represent the means and standard deviations of three independent experiments.

FIG. 7.

Trastuzumab inhibits activation of PI3K/Akt kinase and mitogen-activated protein kinase kinase in MCF7-HA-BARF0 cells. (A) MCF7-Neo^r or MCF7-HA-BARF0 cells were cultured in the presence of 10 μg/ml trastuzumab, 20 μM U0126, or 20 μM LY294002 for 48 h. Cell lysates were separated by 12% SDS-PAGE and transferred onto nitrocellulose membrane for immunoblotting with phospho-ERK1/2, ERK1/2, phospho-Akt, Akt kinases, or HA antibodies. α-Tubulin was detected as an internal control. (B) MCF7-Neo^r or MCF7-HA-BARF0 cells (1 × 10⁴ cells/well) were seeded in 6-cm dishes in culture medium containing 0.33% low-melting-temperature agarose over a 0.5% agarose layer and cultured in the presence of DMSO, trastuzumab (10 μg/ml), U0126 (20 μM), or LY294002(20 μM) for 2 weeks. Colonies were then stained with 0.05% crystal violet.