PubMed · 11518378
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G Tsavellas, A Huang. 2001. Get knotted!. https://pubmed.ncbi.nlm.nih.gov/11518378/
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Elevated expression of human epidermal growth factor receptor 2 (HER2) is known to alter cell signaling and behavioral responses implicated in tumor progression. However, multiple diverse mechanisms may be involved in these overall effects, including signaling by HER2 itself, modulation of signaling by epidermal growth factor receptor (EGFR), and modification of trafficking dynamics for both EGFR and HER2. Because these processes are so tightly interrelated, the net effect of HER2 overexpression is difficult to reliably attribute to any single particular mechanism. To take an important first step toward dissecting the effects of HER2 overexpression on cell responses in terms of the various specific underlying mechanisms, we have developed and validated a quantitative model of the relevant trafficking processes. We then use our model for successful prediction of EGFR and HER2 level and location changes attributable to HER2 overexpression in 184A1 human mammary epithelial cells expressing a series of HER2 levels by retroviral infection. Model predictions are based on our independent experimental measurement of key trafficking parameters for both EGFR and HER2. In terms of trafficking processes, HER2 overexpression reduces the EGFR internalization rate constant and increases the fraction of EGFR recycled. Consequently, our model successfully predicts that HER2 increases the overall level of activated EGFR by both enhancing its recycling and reducing its internalization, but it increases activated EGFR localization at the cell surface almost solely by its reduction of internalization. Furthermore, the model also successfully predicts the effects of monoclonal antibody 2C4, which interferes with HER2/EGFR heterodimerization, on EGFR and HER2 levels and compartmental locations. We anticipate that this model should ultimately be useful in parsing the relative contributions of direct effects of HER2 via signaling vis-a-vis indirect effects of HER2 via modification of EGFR signaling.
The transcription factor CCAAT/enhancer binding protein (C/EBP)beta is critical for normal growth and differentiation of the mammary gland. The intronless C/EBPbeta gene encodes a single mRNA that produces three protein isoforms, C/EBPbeta-1, -2, and -3, which share a common basic-leucine zipper domain at their C-terminus, but are distinguished at their N-termini by the in-frame methionine codon used to initiate translation. Although C/EBPbeta-1 and -2 are both transactivators, they likely perform distinct functions in mammary epithelial cells. C/EBPbeta-1 is the only isoform detected in normal human mammary tissue. In breast cancer cell lines, C/EBPbeta-1 is absent, and the C/EBPbeta-2 transactivator is expressed. Moreover, our data suggest that C/EBPbeta-2 is upregulated in human primary breast tumors. To assess C/EBPbeta-2's ability to participate in the transformation process, we generated recombinant retrovirus selectively encoding epitope-tagged C/EBPbeta-2. Strikingly, 10 days after infecting a normal human mammary epithelial cell line (MCF10A) with C/EBPbeta-2 virus, transformed subcultures were readily generated. Specifically, C/EBPbeta-2-overexpressing MCF10A cells form foci, gain anchorage independence, express markers associated with having undergone an epithelial-to-mesenchymal transition, and acquire an invasive phenotype. These studies, and our previous observations, provide supportive evidence that deregulated expression of C/EBPbeta-2 contributes to malignant conversion of the human breast.
A new and reliable HPLC method for the quantitation of daidzein, equol, and genistein in human breast tissue has been developed. The method was applied to biopsies from women undergoing breast reductions, who, prior to surgery, had ingested either a soy isoflavone preparation or a placebo tablet. The results were compared with data collected for urine and serum of the same subjects using standard methods. The limits of detection in the breast tissue homogenate were 24.7 nmol/l for daidzein, 148.0 nmol/l for equol, and 28.4 nmol/l for genistein (S/N of 3). The chromatographic limits of quantitation were 62.5 nmol/l for daidzein and genistein, and 125.0 nmol/l for equol, for which the accuracies were 86.0%, 83.6%, and 81.8%, respectively. The coefficients of variation of these measurements were all below 20% (11.1% for daidzein, 16.4% for genistein, and 13.2% for equol). The sample preparation comprised a concentration step and the absolute limits of quantitation were, therefore, 4.7 nmol/l, 18.8 nmol/l, and 0.94 nmol/l for daidzein and genistein, and 9.4 nmol/l, 37.5 nmol/l, and 1.9 nmol/l for equol in urine, serum, and breast tissue homogenate, respectively. Recoveries were between 70% (+/-5.6%) in breast tissue homogenate and 100% (+/-14.1%) in urine and serum for all three compounds. Equol (less than 1 micromol/l homogenate) was found to be the predominant phytoestrogen in breast tissue and its concentrations exceeded those in serum. The concentrations of phytoestrogens were at least 100-fold higher in urine than in serum and breast tissue.