Productive Infection of Human Breast Cancer Cell Lines with Human Cytomegalovirus (HCMV)

Abstract

Breast cancer is the leading cause of cancer deaths among women worldwide. There are many known risk factors for breast cancer, but the role of infectious disease remains unclear. Human cytomegalovirus (HCMV) is a widespread herpesvirus that usually causes little disease. Because HCMV has been detected in breast tumor biopsy samples and is frequently transmitted via human breast milk, we investigated HCMV replication in breast tumor cells. Four human breast cancer cell lines with different expression profiles for the key diagnostic markers of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), were infected with a bacterial artificial chromosome-derived HCMV clinical strain TB40/E tagged with green fluorescent protein (GFP). Fluorescence microscopy confirmed that all four breast cancer cell lines supported virus entry. RNA was isolated from infected cells and the expression of immediate early (UL123), early (UL54), and late (UL111A) genes was confirmed using PCR. Viral proteins were detected by immunoblotting, and viral progeny were produced during the infection of breast tumor cells, as evidenced by subsequent infection of fibroblasts with culture supernatants. These results demonstrate that breast tumor cells support productive HCMV infection and could indicate that HCMV replication may play a role in breast cancer progression.

Keywords: HCMV; breast cancer; cancer; cytomegalovirus.

Figure 1

HCMV infection of breast cancer cells. Monolayer cultures of the indicated cell types were infected with HCMV strain TB40/E-GFP for 72 h (MOI = 1). Images were captured using bright field and fluorescence microscopy. Scale bar = 100 µm.

Figure 2

Breast cancer cells support viral gene expression. RNA was harvested from mock- or HCMV-infected cells at 72 hpi, reverse-transcribed to cDNA, and PCR was performed using gene-specific primers. Resulting bands were visualized by agarose gel electrophoresis.

Figure 3

Viral proteins are produced in breast cancer cells. Cells were mock-infected or infected with TB40/E-GFP (MOI = 1). After 96 h, cell lysates were prepared, separated via SDS-PAGE, and immunoblotted using (A) antibodies directed against IE1/2 proteins, or (B) serum from a HCMV-seropositive donor. Representative viral proteins are indicated.

Figure 4

Breast cancer cells produce infectious progeny. Monolayer cultures of NuFF cells were treated with supernatants (SN) that had been harvested from mock- or HCMV-infected NuFF, MCF-7, or MDA-MB-231 cultures at 96 hpi. Cultures were examined by bright field and fluorescence microscopy 96 h after treatment with SN. Scale bar = 100 µm.