WISP-2 mRNA and protein was overexpressed in preneoplastic and cancerous cells of human breast. Statistical analyses show a significant association between WISP-2 expression and estrogen receptor (ER) positivity. In normal breast, the expression was virtually undetected. The studies showed that WISP-2 is an estrogen-induced early response gene in MCF-7 cells and the expression was continuously increased to reach a maximum level at 24 h. The estrogen effect was inhibited by a pure antiestrogen (ICI 182,780). Human mammary epithelial cells, in which WISP-2 expression was undetected or minimally detected, responded to 17beta-estradiol by upregulating the WISP-2 gene after transfection with ER-alpha, providing further evidences that WISP-2 expression is mediated through ER-alpha. Overexpression of WISP-2 mRNA by estrogen may be accomplished by both transcriptional activation and stabilization. MCF-7 cells exposed to progesterone had a rapid but transient increase in WISP-2 expression, and PR antagonist RU38486 blocked this mRNA induction. In combination with estradiol, progesterone acted as an antagonist inhibiting the expression of WISP-2 mRNA. Moreover, disruption of WISP-2 signaling in MCF-7 cells by use of antisense oligomers caused a significant reduction in tumor cell proliferation. The results are consistent with the conclusion that WISP-2 expression is a requirement for breast tumor cells proliferation.

The expression and distribution of neuropilin-1 (NRP-1) was examined in the samples of normal human breast tissues and in non-neoplastic and neoplastic areas of breast tissue removed for carcinoma using RT-PCR as well as conventional and tissue microarrays immunohistochemical analyses. The NRP-1 mRNA expression was significantly higher in neoplastic tissues as compared to normal breast samples. Immunohistochemically, the myoepithelial cells of the mammary ducts and lobules display positive reactions for NRP-1, whereas the inner ductal and lobular epithelial cell layers failed to react. The myoepithelial cells of ducts and lobules in both neoplastic and non-neoplastic tissue specimens displayed a stronger positive reaction for NRP-1 than those in the normal breast. A positive reaction for NRP-1, but with a gradual reduction in intensity, was observed in the myoepithelial cells of ducts with atypical epithelial hyperplasia and ductal carcinoma in situ (DCIS). The reaction was undetected or minimally detected in the areas of invasive carcinoma. NRP-1 positive immunolabeling was also localized in the vascular smooth muscle cells and in some endothelial cells of the blood vessels in normal, non-neoplastic and neoplastic breast tissue samples. In areas of breast carcinoma, NRP-1 immunolabeling was more prominent in both vascular smooth muscle cells and in some endothelial cells than in similar cells in normal breast. The specificity of the newly developed antibody for NRP-1 was confirmed by in situ hybridization with DIG-labeled PCR generated probe. These results suggest that NRP-1 may be a multiple function protein in human breast and may be involved in the induction of local invasiveness of neoplasia and angiogenesis and have direct relevance to the progression of breast cancer.

Epidermal growth factor (EGF) is a mitogen for estrogen receptor (ER) -positive breast tumor cells, and it has been proven that EGF occasionally mimicked estrogen action and cross-talks with ER-A to exert its activity. Therefore, the present study was undertaken to explore whether EGF is able to modulate the expression of Wnt-1-induced signaling protein-2/connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed 5 (WISP-2/CCN5), an estrogenresponsive gene, in normal and transformed cell lines of the human breast and, if so, whether this induction is critical for EGF mitogenesis and what downstream signaling pathways are associated with this event. Here, we show that EGF-induced WISP-2 expression in ER-and EGF receptor -positive noninvasive MCF-7 breast tumor cells was dose and time dependent and that expression was modulated at transcription level. A synergism was seen in combination with estrogen. Moreover, small interfering RNA -mediated inhibition of WISP-2/CCN5 activity in MCF-7 cells resulted in abrogation of proliferation by EGF. The multiple molecular cross-talks, including the interactions between phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase signaling pathways and two diverse receptors (i.e., ER-A and EGFR), were essential in the event of EGF-induced WISP-2/CCN5 up-regulation in MCF-7 cells. Moreover, EGF action on WISP-2/CCN5 is restricted to ER-and EGFR-positive noninvasive breast tumor cells, and this

2-Methoxyestradiol (2-ME(2)), a promising anticancer drug, induces growth arrest and apoptosis in various androgen-dependent (LNCaP) and -independent (DU145 and PC-3) prostate cancer cell lines. Moreover, flow cytometric analysis indicated a novel dual impact of 2-ME(2) on the cell division cycle of prostate cancer cells. Chronic exposure of high doses of 2-ME(2) enhance the accumulation of cells in S and G2/M phases, while cell numbers in the G1 phase were reduced significantly by this treatment. Because cyclin B1 overexpression, induction of cdc2 phosphorylation, and its regulatory proteins wee1 and phospho-cdc25C (interphase and mitotic forms) by 2-ME(2) treatment correlated with the induction of apoptosis, growth arrest at the G2/M phase, and accumulation of the S phase, we reasoned that cyclin B1 and cdc2 phosphorylation and its upstream regulatory molecular networks may be associated with the ultimate impacts of 2-ME(2). Because phosphorylation of cdc2 and upregulation of wee1 by 2-ME(2) can be abolished by both extracellular receptor kinase (ERK) inhibitor (U0126) and c-Jun N-terminal kinase (JNK) inhibitor (SP600125), our studies indicate that the 2-ME(2)-induced upregulation of wee1 and subsequent cdc2 phosphorylation are mediated through mitogen-activated protein kinase (MAPK)-ERK-JNK signaling pathways.

2-Methoxyestradiol (2-ME2) was reported to elicit both stimulation and inhibition of tumor angiogenesis and growth depending on the dosage used. However, the mechanism(s) of the biphasic action of 2-ME2 has been elusive. Here we describe a regulatory role of vascular endothelial growth factor-A (VEGF-A) in the biphasic effects on estrogen receptor (ER)+ GH3 rat pituitary tumor cells and MCF-7 human breast tumor cells depending on the dosage of 2-ME2 used. We observed that acute exposure to 2-ME2, irrespective of dosage, did not alter cellular proliferation, but enhanced the VEGF-A mRNA level. As the treatment duration increased, biphasic effect was elicited. A concentration of 1 microM 2-ME2 increased both cell proliferation and VEGF-A levels in these cells, whereas higher doses exhibited reversed impact. A low dose of 2-ME2 also increased the VEGF-A mRNA expression in ER-alpha-transfected human mammary epithelial cells (HMECs). The effect was reversed in ER- cells. The enhanced expression of VEGF-A mRNA could be blocked by the pure estrogen antagonist, ICI 182,780, and reveal that the upregulation of VEGF-A expression by 2-ME2 is mediated through ER-alpha. Furthermore, the biphasic effect of 2-ME2 on cell proliferation can be modulated by administrating VEGF-A antibodies or VEGF-A proteins. Studies also demonstrate that the VEGF-A protein, induced by 2-ME2, is functionally active and upregulates the proliferation of adjacent endothelial cells.

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PMA and active phorbol esters stimulate the proliferation of various tumor cells, including ER-positive human breast tumor cell lines. However, the specific signaling pathways involved in the PMA-induced mitogenic effect on breast tumor cells have not been fully elucidated. In the present study, we explored the mechanisms associated with the mitogenic influence of PMA on breast tumor cells. Following an acute exposure (i.e., within 2 to 6 h) to PMA (50 nM), a mitogenic effect was observed on WISP-2/CCN5-positive breast tumor cell lines, including MCF-7, ZR-75-1 and SKBR-3 cells, and induction of WISP-2/CCN5 mRNA expression paralleled the observed mitogenic proliferation. This effect was undetected in WISP-2/CCN5 negative MDA-MB-231 breast tumor cells or human mammary epithelial cells with or without ER-alpha transfection. The mitogenic effect of PMA was perturbed by short hairpin RNA (shRNA)-mediated inhibition of WISP-2/CCN5 signaling in MCF-7 cells. Moreover, the upregulation of WISP-2/CCN5 by PMA is not ER dependent but is instead mediated through a complex PKCalpha-MAPK/ERK and SAPK/JNK signaling pathway, which leads to growth stimulation of MCF-7 breast tumor cells. These series of experiments provide the first evidence that WISP-2/CCN5 is a novel signaling molecule that critically participates in the mitogenic action of PMA on noninvasive, WISP-2/CCN5-positive breast tumor cells through PKCalpha-dependent, multiple molecular signal transduction pathways.