MMP-9 increases HER2/neu expression and alters apoptosis levels in human mammary epithelial cells (HMEC).

HER2/neu overexpression leads to poorer prognosis and higher risk of disease reoccurrence in breast cancer patients. The causative factors responsible for increasing HER2/neu expression levels on mammary cells are not known. We investigated whether factors associated with inflammation or metastasis could induce HER2/neu expression on human mammary epithelial cells (HMECs). Human mammary epithelial cells and several human breast cancer cell lines used in our studies were treated with several agents, including estrogen and matrix metalloproteinase-9 (MMP-9), either alone or in various combinations. Relative expression of HER2/neu on the surface of target cells was assessed using fluorochrome-tagged antibodies and a fluorescence cytometer. HER2/neu gene expression was also determined by Western blot analysis and PCR. Apoptosis levels were also determined. MMP-9, administered either alone or in combination with interleukin-7 and estrogen, caused a significant rise in HER2/neu expression on the surface of HMECs. The induction in HER2/neu protein expression was suppressed using a MMP-9 inhibitor. Similar results were obtained for breast cancer cells treated with the estrogen in combination with MMP-9. MMP-9 treatment significantly decreased apoptotic levels in HMECs. Our results indicate that MMP-9 is a regulator of HER2/neu expression on human mammary epithelial cells and suggest that upregulation of HER2/neu by MMP-9 may be relevant to altering the characteristics of normal mammary cells toward a transformed phenotype.

Estrogen and tamoxifen up-regulate FXYD3 on breast cancer cells: assessing the differential roles of ER α and ZEB1.

Increased expression of the FXYD3 family of proteins has been associated with lung, colorectal, bladder and pancreatic cancers, and recent evidence suggests that elevated FXYD3 may promote tumor cell proliferation in breast cancer as well. However, factors involved in up-regulating the expression of FXYD3 in breast cancer have not been identified. We evaluated whether estrogen and the selective estrogen receptor modulator tamoxifen could regulate the expression of FXYD3 on breast cancer cells. Estrogen receptor (ER) α-positive MCF-7 and ER α-negative MDA-MB-231 human breast cancer cells used in our studies were treated with estrogen, tamoxifen or the combination of these agents. Relative expression of FXYD3 was assessed using fluorochrome-tagged antibodies and a fluorescence cytometer. We found that estrogen and tamoxifen, used alone or in combination, significantly increased FXYD3 on MCF-7 cells. FXYD3 levels did not increase compared to the control samples when ER α-negative 231 cells were treated with estrogen or tamoxifen, alone or in combination, indicating that ER α was required for the increased FXYD3 response. We showed that ER α associates with the transcription factor ZEB1 in MCF-7 cells, and that decreasing ZEB1 protein expression using siRNA disrupts the ability of estrogen, but not tamoxifen, to increase FXYD3 in MCF-7 cells. Our results indicate that there may be two mechanisms, both involving ER α and one requiring ZEB1, through which FXYD3 may be increased by estrogen and tamoxifen in breast cancer cells. Ongoing research endeavors are focusing on identifying cellular components through which estrogen and tamoxifen, alone or in combination, differentially regulate FXYD3 expression in human breast cancer cells.

Antitumor/antiestrogenic effect of the chemokine interferon inducible protein 10 (IP-10) involves suppression of VEGF expression in mammary tissue.

Vascular endothelial growth factor (VEGF) promotes angiogenesis in a number of tumor model systems. We reported previously that estrogen supports the growth of CCL-51 cell-based mammary tumors in mice, which could be blocked with specific chemokines. We investigated whether promotion of tumor growth by estrogen, and suppression of tumor growth by chemokines, was associated with VEGF protein expression. Female C3H mice were treated with vehicle, estradiol, or with one of several chemokines for 72 h. The presence of VEGF in mammary tissue samples was detected and quantified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting using antimurine VEGF antibodies. Estrogen significantly increased mammary VEGF expression. Cotreatment with tamoxifen or the chemokine interferon-inducible protein-10 (IP-10) suppressed the action estrogen on VEGF expression. CCL-51 tumor cells were placed into mammary tissue of C3H mice. Mice were treated every 72-h with either vehicle or estradiol, in the presence or absence of IP-10 for 21 days. Estrogen supported CCL-51 tumor growth, with an average of 2.3 tumors present/animal. Cotreatment of mice with estrogen and IP-10 resulted in significantly lower numbers of tumors in mammary tissue in comparison to animals treated with estrogen alone. VEGF levels in mammary tissue and tumors of IP-10 and estrogen cotreated mice were 40-50% less than those detected in mammary tissue of estrogen-treated mice. Our results suggest that estrogenic support of CCL-51 mammary tumor growth is related to increased VEGF expression, and that the inhibitory action of IP-10 may be related to suppressing VEGF levels in mammary tissue.

Estrogen decreases expression of chemokine receptors, and suppresses chemokine bioactivity in murine monocytes.

PROBLEM: We propose that the ability of estrogen exposure to increase the probability of a woman developing breast cancer may be related to decreased chemokine activity and suppression of immune surveillance in mammary tissue. The present study was conducted to determine whether estrogen could decrease monocyte bioactivity through alteration of chemokine receptor expression. METHOD OF STUDY: We examined the effect of estrogen and tamoxifen on the expression of the chemokine receptors CCR2 and CXCR3 on murine monocytes treated in culture and in vivo. Effects of estrogen on chemokine activation of monocytes were also evaluated. RESULTS: Estrogen and tamoxifen significantly decreased expression of CCR2 and, to a lesser extent, CXCR3 on murine monocytes. Estrogen decreased chemotaxis of monocytes towards MCP-1/JE. The chemokines MCP-1/JE and MIP-1alpha were unable to evoke increases in intracellular calcium in murine monocytes treated with estrogen, alone or in combination with tamoxifen. CONCLUSIONS: Our results show that estrogen suppresses the ability of monocytes to respond to certain chemokines, suggesting that estrogen exposure might decrease immune surveillance in tissues where the action of specific chemokines is involved.

Estrogen decreases chemokine levels in murine mammary tissue: implications for the regulatory role of MIP-1 alpha and MCP-1/JE in mammary tumor formation.

Estrogen contributes to the development of breast cancer through mechanisms that are not completely understood. Estrogen influences the function of immune effector cells, primarily through alterations in cytokine expression. Chemokines are proinflammatory cytokines that attract various immune cells to the site of tissue injury or inflammation, and activate many cell types, including T lymphocytes and monocytes. As an initial step toward ultimately determining whether regulation of chemokine expression and/or biological activity by estrogen could potentially be a contributing factor to the development and progression of mammary tumors, we evaluated the effect of estrogen on the expression of specific chemokines in murine mammary tissue. We also evaluated whether exposure of female mice to various chemokines could alter the growth of mammary tumors in the presence of estrogen. We report here that estrogen significantly decreases levels of the chemokines MIP-1alpha and MCP-1/JE in murine mammary tissue. Co-treatment with 4-hydroxytamoxifen partially reverses the suppressive effect of estrogen on MIP-1alpha levels. Estrogen increases the growth of CCL- 51 cell-based tumors in the mammary glands of female mice. Co-treatment with the chemokine MIP-1alpha or MCP- 1/JE substantially decreases the ability of estrogen to stimulate the formation of CCL-51 cell-based tumors. Our results show that estrogen might influence the bioactivity of specific chemokines through alteration of chemokine expression in mammary tissue, and further suggest that decreases in murine chemokines evoked by estrogen exposure could contribute to the promotion of mammary tumor growth.