Human Cytomegalovirus Interleukin-10 Promotes Proliferation and Migration of MCF-7 Breast Cancer Cells

Breast cancer is the most common malignancy affecting women worldwide. While a small fraction of breast cancers have a hereditary component, environmental and behavioral factors also impact the development of cancer. Human cytomegalovirus (HCMV) is a member of the Herpesviridae family that is widespread in the general population and has been linked to several forms of cancer. While HCMV DNA has been found in some breast cancer tissue specimens, we wanted to investigate whether a secreted viral cytokine might have an effect on cancerous or even pre-cancerous cells. HCMV encodes an ortholog of the human cellular cytokine interleukin-10 (IL-10). The HCMV UL111A gene product is cmvIL-10, which has 27% sequence identity to IL-10 and binds the cellular IL-10 receptor (IL-10R) to induce downstream cell signaling. We found that MCF-7 human breast cancer cells express IL-10R and that exposure to cmvIL-10 results in enhanced proliferation and increased chemotaxis of MCF-7 cells. PCR arrays revealed that treatment with cmvIL-10 alters expression of cell adhesion molecules and increases MMP gene expression. In particular, MMP-10 gene expression was found to be significantly up-regulated and this correlated with an increase in cell-associated MMP-10 protein produced by MCF-7 cells exposed to cmvIL-10. These results suggest that the presence of cmvIL-10 in the tumor microenvironment could contribute to the development of more invasive tumors

Human breast cancer cells express the IL-10 receptor.

<p>A) MDA-MB-231 cells were stained with anti-IL-10R-PE antibody (black line) or isotype control (gray line) and analyzed by flow cytometry. B) Cells were untreated or treated with 100 ng/ml cmvIL-10 for 15 min and stained for IL-10R followed by TRITC-conjugated secondary antibody, then visualized by fluorescence microscopy. Red corresponds to IL-10R, blue corresponds to DAPI staining of the nucleus. C) RNA was harvested from MDA-MB-231 cells and mock- or HCMV-infected HFF cells (MOI = 1, 72 hrs post-infection), reverse-transcribed and IE1 or β-actin gene specific primers were used for PCR. D) MDA-MB-231 and mock- or HCMV-infected HFFs were cultured on glass coverslips, fixed and stained for IE1 followed by FITC-conjugated secondary (green). These results are representative of three independent experiments.</p

Human breast cancer cells are protected from apoptosis by cmvIL-10.

<p>A) MDA-MB-231 cells were treated with 100 µM etoposide in the presence or absence of 100 ng/ml cmvIL-10 for 48 hrs, then stained for Annexin V and analyzed by flow cytometry. B) MDA-MB-231 cells were grown in 96-well dishes and treated with the indicated doses of etoposide in the presence or absence of 100 ng/ml cmvIL-10 for 48 hours, then cell viability evaluated via the addition of Cell Titer Glo and detection of resulting chemiluminescence. C) Cells were cultivated in the presence of 10 µM etoposide with or without 100 ng/ml cmvIL-10. Cell viability was evaluated at the indicated time points via Cell Titer Glo. Error bars represent standard error of three replicates per condition. * indicates p<0.01, Student’s <i>t</i>-test. These results are representative of two independent experiments.</p

cmvIL-10 stimulates proliferation and increases DNA synthesis in human breast cancer cells.

<p>A) MDA-MB-231 cells were grown in 96-well dishes and treated with the indicated doses of cmvIL-10. Cell Titer Glo was added at the indicated time points to measure cell viability, represented as relative light units (RLUs) based on the resulting chemiluminescence. B) Cell growth in the presence or absence of 100 ng/ml cmvIL-10 via Cell Titer Glo. C) BrdU incorporation in the presence of 100 ng/ml cmvIL-10 or hIL-10. D) Standard cell counts of cultures from 6-well dishes containing 100 ng/ml cmvIL-10 or hIL-10 using a hemacytometer. E) BrdU incorporation was assessed at 72 hrs in cells cultured in the presence of 10 ng/ml of each indicated cytokine. F) Cells were treated with 10 µM of each indicated inhibitor or an equivalent volume of DMSO in the presence of absence of 10 ng/ml cmvIL-10 and BrdU incorporation measured after 72 hrs. Error bars represent standard error among three replicates for each condition. * indicates p<0.05, Student’s <i>t</i>-test. These results are representative of three independent experiments.</p

cmvIL-10 induces Stat3 phosphorylation in human breast cancer cells.

<p>A) MDA-MB-231 cells were treated with 100 ng/ml cmvIL-10, hIL-10, or IFNγ for 15 min, then lysed and Western blotted with the indicated antibodies. B) Cells were grown in 96-well dishes and treated with the indicated doses of cmvIL-10 for 15 min before lysis in the well followed by quantification of total vs. pStat3 levels. Results are represented as the normalized ratio of pStat3 to total Stat3 in relative fluorescence units (RFUs). *  =  p < 0.01, Student’s <i>t</i>-test. Error bars represent standard error for three replicates of each condition. C) MDA-MB-231 cells were cultured and supernatants collected at the indicated time points were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. Control indicates purified recombinant protein (cmvIL-10, hIL-10, or serpin E1/PAI) was loaded as a positive control for each respective antibody. Results are representative of three independent experiments.</p

Human breast cancer cells exhibit enhanced chemotaxis when exposed to cmvIL-10.

<p>MDA-MB-231 cells were seeded at a density of 2×10⁵ cells in a total volume of 0.1 ml in the upper chamber of an 8 µm trans-well filter. A) Complete media containing the indicated concentrations of EGF in the presence or absence of 100 ng/ml cmvIL-10 or hIL-10 was placed in the lower chamber. After 5 hrs, cells in the lower chamber were harvested and quantified by the addition of Cell Titer Glo to measureme luminescence. B) Complete medium containing the indicated concentrations of cmvIL-10 in the presence or absence of 1 ng/ml EGF. Cells traversing the filter after 5 hrs were quantified as described. Error bars represent standard error. * indicates p < 0.05, Student’s <i>t</i>-test. Results are representative of three independent experiments.</p

cmvIL-10 Stimulates the Invasive Potential of MDA-MB-231 Breast Cancer Cells

Cancer is the result of unregulated cell growth that leads to tumor formation, and in many cases, metastases. Although there are several risk factors associated with cancer, one area that remains poorly understood is the impact of infectious disease. Human cytomegalovirus (HCMV) is a member of the herpesvirus family that is highly prevalent in the population. HCMV usually causes clinical disease only in immune compromised individuals, but recent evidence suggests that HCMV may be strongly associated with some forms of cancer, particularly glioblastoma and breast cancer. We investigated the possibility that cmvIL-10, a viral cytokine with homology to human IL-10 that is secreted from infected cells, could act in a paracrine manner to alter the tumor microenvironment, induce cell signaling, and increase the invasive potential of cancer cells. We found that human MDA-MB-231 breast cancer cells express the IL-10 receptor and that exposure to cmvIL-10 results in activation of Stat3, a transcription factor strongly associated with enhanced metastatic potential and chemo-resistance. In addition, cmvIL-10 stimulated an increase in DNA synthesis and cell proliferation, protected MDA-MB-231 cells from etoposide-induced apoptosis, and also greatly enhanced chemotaxis toward epidermal growth factor (EGF). These results suggest a significant and wide-ranging role for cmvIL-10 in the progression of breast cancer and could have broad implications for the diagnosis and treatment of cancer in HCMV-positive patients