Breast cancer presents as either estrogen receptor A (ERA) positive or negative, with ERA+ tumors responding to antiestrogen therapy and having a better prognosis. By themselves, mRNA expression signatures of estrogen regulation in ERA+ breast cancer cells do not account for the vast molecular differences observed between ERA+ and ERAÀ cancers. In ERAÀ tumors, overexpression of epidermal growth factor receptor (EGFR) or c-erbB-2, leading to increased growth factor signaling, is observed such that mitogen-activated protein (MAP) kinase (MAPK) is significantly hyperactivated compared with ERA+ breast cancer. In ERA+/progesterone receptor-positive, estrogen-dependent MCF-7 breast cancer cells, we stably overexpressed EGFR or constitutively active erbB-2, Raf, or MAP/extracellular signalregulated kinase kinase, resulting in cell lines exhibiting hyperactivation of MAPK, estrogen-independent growth, and the reversible down-regulation of ERA expression. By global mRNA profiling, we found a ''MAPK signature'' of f400 genes consistently up-regulated or down-regulated in each of the MAPK+ cell lines. In several independent profile data sets of human breast tumors, the in vitro MAPK signature was able to accurately distinguish ER+ from ERÀ tumors. In addition, our in vitro mRNA profile data revealed distinct mRNA signatures specific to either erbB-2 or EGFR activation. A subset of breast tumor profiles was found to share extensive similarities with either the erbB-2-specific or the EGFRspecific signatures. Our results confirm that increased MAPK activation causes loss of ERA expression and suggest that hyperactivation of MAPK plays a role in the generation of the ERAÀ phenotype in breast cancer. These MAPK+ cell lines are excellent models for investigating the underlying mechanisms behind the ERAÀ phenotype. (Cancer Res 2006; 66(7): 3903-11)

Purpose: In breast cancer, the presence of estrogen receptor a (ER) denotes a better prognosis and response to antiestrogen therapy. Lack of ERa correlates with overexpression of epidermal growth factor receptor or c-erbB-2. We have shown that hyperactivation of mitogen-activated protein kinase (MAPK)

Objective. To use noninvasive magnetic resonance imaging (MRI), biochemical analyses, and mechanical testing of engineered neocartilage grown in a hollowfiber bioreactor (HFBR) to establish tissue properties, and to test the hypothesis that MRI can be used to monitor biochemical and biomechanical properties of neocartilage.Methods. Chondrocytes from day 16 embryonic chick sterna were inoculated into an HFBR and maintained for up to 4 weeks with and without exposure to chondroitinase ABC. The fixed-charge density (FCD) of the cartilage was determined using the MRI gadolinium exclusion method. The sulfated glycosaminoglycan (S-GAG), hydroxyproline, and DNA contents were determined using biochemical procedures, while dynamic and equilibrium moduli were determined from mechanical indentation tests.Results. S-GAG content, tissue cross-sectional area, and equilibrium modulus of the neocartilage increased with development time. There was a gradient of S-GAG content across the length of control neocartilage at the 4-week time point, with higher values being found toward the inflow region. Exposure to chondroitinase ABC resulted in a decrease in tissue area, negative FCD, proteoglycan content, and equilibrium and dynamic moduli. The treated bioreactors displayed a lengthwise variation in S-GAG content, with higher values toward the outflow end. Linear correlations were established among FCD, proteoglycan content, and biomechanical properties.Conclusion. HFBR-derived neocartilage showed regional variation in S-GAG content under control conditions, and in the decrease of S-GAG in response to enzyme treatment. In addition, the results support the hypothesis that tissue parameters derived from MRI can be used to noninvasively monitor focal neocartilage formation and biochemical and biomechanical properties.

Studies of gene regulated by estrogen in breast cancer 1 (GREB1) have focused on mRNA levels with limited evidence about GREB1 protein expression in normal and breast cancer cells. A monoclonal antibody that recognizes GREB1 protein in breast tissues could be applied to correlate protein expression with established mRNA expression data. A hybridoma expressing a murine monoclonal antibody targeting a 119 amino acid peptide specific to human GREB1 was generated. The novel monoclonal GREB1 antibody (GREB1ab) was validated for use in Western blotting as well as immunohistochemical (IHC) applications. GREB1ab detects a 216 kDa protein corresponding to GREB1 in estrogen receptor alpha (ERalpha+) breast cancer cells as well as ERalpha- breast cancer cells transduced with a GREB1 expression vector. GREB1ab specificity was verified using an ERalpha antagonist to prevent GREB1 induction as well as a silencing siRNA targeting GREB1 mRNA. GREB1ab was further validated for detection of GREB1 by IHC in breast cancer cell lines and breast tissue microarrays (TMA). ERalpha+ cell lines were observed to express GREB1 while ERalpha- cell lines did not express detectable levels of the protein. Using breast cancer tissue whole sections, IHC with the GREB1ab identified protein expression in ERalpha+ breast cancer tissue as well as normal breast tissue, with little GREB1 expression in ERalpha- breast cancer tissue. Furthermore, these data indicate that GREB1 mRNA expression correlates well with protein expression. The novel monoclonal GREB1ab is specific for GREB1 protein. This antibody will serve as a tool for investigations focused on the expression, distribution, and function of GREB1 in normal breast and breast cancer tissues.

It has been well established that GREB1 mRNA expression is induced by estrogen in breast cancers and that GREB1 is critically involved in the estrogen-induced growth of breast cancer. To date, all studies of GREB1 have focused on mRNA levels using PCR and array technologies with very little known about GREB1 protein expression in normal and breast cancer cells. The lack of a specific antibody to GREB1 has inhibited protein-based investigations in target cells. Our collaborative research group has generated a novel monoclonal GREB1 antibody (GREB1ab) for research use in Western blotting as well as immunohistochemical (IHC) applications.Our team created a hybridoma expressing a murine monoclonal antibody against a 119 amino acid peptide specific to human GREB1. By Western Blot Analysis, GREB1ab detects a 216 kD protein corresponding to GREB1a in ER+ breast cancer cells expressing GREB1 as well as cells transfected with a GREB1a expression plasmid. GREB1ab specificity was verified using ICI 182,780, an estrogen receptor antagonist to prevent GREB1 induction, as well as silencing siRNA targeting GREB1 mRNA expression. GREB1 protein expression was reduced in MCF-7 cells treated with estrogen plus ICI 182,780 compared with that of estrogen treatment. There was no detectable GREB1 protein expression when GREB1 mRNA is silenced by siRNA at 48 hours. In a time course study, inhibition of GREB1 protein occurred as early as 24 hours and lasted up to 72 hours. Thus, by Western Blot Analysis, the monoclonal GREB1ab is specific for GREB1 and can be employed to detect changes in GREB1 expression in breast cancer cell lines under various experimental conditions.GREB1ab was validated for detection of GREB1 by IHC in breast cancer cell lines and breast tissue microarrays (TMA). IHC staining with GREB1ab revealed GREB1 was strongly expressed in the ERα-positive breast cancer cell line, MCF-7, with weak GREB1 expression in ERα-negative MDA-231 cells. A panel of breast cancer cell lines was screened for endogenous expression of GREB1 protein in a TMA format using IHC. As expected, ER-positive cell lines (n=5) were observed to express GREB1 while ER-negative cell lines (n=11) did not express detectable levels. Using breast cancer tissue whole sections, IHC with the GREB1ab indicated protein expression in ERα positive breast cancer tissue as well as normal breast tissue, with little GREB1 expression in ERα negative breast cancer tissue.The monoclonal GREB1ab is specific for GREB1 protein. This antibody will serve as a tool for investigations focused on the expression, distribution and function of GREB1 in normal breast and breast cancer tissues. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2126.