M. De Bortoli

Tamoxifen up-regulates c-erbB-2 expression in oestrogen-responsive breast cancer cells in vitro

Expression of the c-erbB-2 proto-oncogene is inhibited by oestrogens in oestrogen-responsive human breast cancer cells, at both mRNA and protein level. Here we report that, where the regulation of c-erbB-2 is concerned, tamoxifen displays a full anti-oestrogenic activity, enhancing the expression of c-erbB-2 in oestrogen receptor-positive cells cultured with untreated fetal calf serum or reversing the inhibitory effect of added oestrogens. Meanwhile, tamoxifen strongly inhibited cell growth. Tamoxifen was inactive on both c-erbB-2 expression and growth of oestrogen receptor-negative cells. These results may have important implications to explain occasional failure of tamoxifen therapy in oestrogen receptor-positive breast cancers.

Hormonal regulation of c-erbB-2 oncogene expression in breast cancer cells

Expression of the c-erbB-2 (neu, HER-2) oncogene is found to be subjected to hormonal and developmental regulation in normal as well as neoplastic mammary cells. We have previously reported that estrogens inhibit c-erbB-2 expression at both the mRNA and protein level in estrogen receptor (ER)-positive, but not in ER-negative, breast cancer cell lines. Reversion of c-erbB-2 inhibition is seen with tamoxifen. The effect on c-erbB-2 expression of several other hormones and factors, which influence mammary cell growth and differentiation, has been studied. Our observations indicate that, in normal and neoplastic mammary cells, c-erbB-2 expression is inversely related to cell proliferation. While estrogens, anti-estrogens and cAMP clearly regulate c-erbB-2 mRNA levels, epidermal growth factor dramatically decreases the c-erbB-2 protein without affecting the level of c-erbB-2 mRNA. Therefore, different signals converging in terms of cell proliferation regulate c-erbB-2 expression by different molecular mechanisms.

DNA chips: the future of biomarkers

DNA chips are small, solid supports such as microscope slides onto which thousands of cDNAs or oligonucleotides are arrayed, representing known genes or simply EST clones, or covering the entire sequence of a gene with all its possible mutations. Fluorescently labeled DNA or RNA extracted from tissues is hybridized to the array. Laser scanning of the chip permits quantitative evaluation of each individual complementary sequence present in the sample. DNA chip technology is currently being proposed for qualitative and quantitative applications, firstly for the detection of point mutations, small deletions and insertions in genes involved in human diseases or affected during cancer progression; secondly, to determine on a genome-wide basis the pattern of gene expression in tumors, as well as in a number of experimental situations. The extraordinary power of DNA chips will have a strong impact on medicine in the near future, both in the molecular characterization of tumors and genetic diseases and in drug discovery and evaluation. Quantitative applications will soon spread through all fields of biology.

Quantitative real-time RT-PCR analysis of eight novel estrogen-regulated genes in breast cancer

BACKGROUND Biological markers capable of predicting the risk of recurrence and the response to treatment in breast cancer are eagerly awaited. Estrogen and progesterone receptors (ER, PgR) in tumor cells mark cancers that are more likely to respond to endocrine treatment, but up to 40% of such patients do not respond. Here, the expression of a group of estrogen-regulated genes, previously identified by microarray analysis of in vitro models, was measured in breast tumors and possible associations with other clinicopathological variables were investigated. METHODS The expression of CD24, CD44, HAT-1, BAK-1, G1P3, TIEG, NRP-1 and RXRalpha was measured by quantitative real-time RT-PCR on RNA from eighteen primary breast tumors. Statistical analyses were used to identify correlations among the eight genes and the available clinicopathological data. RESULTS Variable expression levels of all the genes were observed in all the samples examined. Significant associations of CD24 with tumor size, CD44 with lymph node invasion, and HAT-1 and BAK-1 with ER positivity were found. The possible combinatorial value of these genes was assessed. Unsupervised hierarchical clustering analysis demonstrated that the expression profile of these genes was able to predict ER status with an acceptable approximation. CONCLUSIONS Eight novel potential markers for breast cancer have been preliminarily characterized. As expected from in vitro data, their expression is able to discriminate ER- versus ER+ tumors.

Role of coactivators and corepressors in steroid and nuclear receptor signaling: potential markers of tumor growth and drug sensitivity.

Nuclear receptors regulate target gene expression in response to steroid and thyroid hormones, retinoids, vitamin D and other ligands. These ligand-dependent transcription factors function by contacting various nuclear cooperating proteins, called coactivators and corepressors, which mediate local chromatin remodeling as well as communication with the basal transcriptional apparatus. Nuclear receptors and their coregulatory proteins play a role in cancer and other diseases, one leading example being the estrogen receptor pathway in breast cancer. Coregulators are often present in limiting amounts in cell nuclei and modifications of their level of expression and/or structure lead to alterations in nuclear receptor functioning, which may be as pronounced as a complete inversion of signaling, i.e. from stimulating to repressing certain genes in response to an identical stimulus. In addition, hemizygous knock-out of certain coactivator genes has been demonstrated to produce cancer-prone phenotypes in mice. Thus, assessment of coactivator and corepressor expression and structure in tumors may turn out to be essential to determine the role of nuclear receptors in cancer and to predict prognosis and response to therapy.

Hormonal Regulation of Type I Receptor Tyrosine Kinase Expression in the Mammary Gland

Hormones guide mammary gland development and differentiation by regulating the expression of local growth factors and their receptors at the cell surface. In line with this principle the expression of the epidermal growth factor receptor (EGFR)3 and ErbB2 receptors varies in the mammary gland during pregnancy, following the changing hormonal profile. In breast cancer, expression of EGFR and ErbB2 is clearly related to the absence of estrogen and progesterone receptors. In breast cancer cells in vitro, the expression of these receptors is modulated by hormones and other growth-modulatory reagents. Moreover, transcriptional regulation of both EGFR and ERBB2 by estrogens has been demonstrated. The action of hormones may therefore result in the differential availability of individual ErbB family members at the cell surface, in this way determining the specific response of the cell to EGF-like factors and heregulins.

Targeting of the adaptor protein Tab2 as a novel approach to revert tamoxifen resistance in breast cancer cells

Pharmacological resistance is a serious threat to the clinical success of hormone therapy for breast cancer. The antiproliferative response to antagonistic drugs such as tamoxifen (Tam) critically depends on the recruitment of NCoR/SMRT corepressors to estrogen receptor alpha (ERα) bound to estrogen target genes. Under certain circumstances, as demonstrated in the case of interleukin-1β (IL-1β) treatment, the protein Tab2 interacts with ERα/NCoR and causes dismissal of NCoR from these genes, leading to loss of the antiproliferative response. In Tam-resistant (TamR) ER-positive breast cancer cells, we observed that Tab2 presents a shift in mobility on sodium dodecyl sulfate--PAGE (SDS-PAGE) similar to that seen in MCF7 wt upon stimulation with IL-1β, suggesting constitutive activation. Accordingly, TamR treatment with Tab2-specific short interfering RNA, restored the antiproliferative response to Tam in these cells. As Tab2 is known to directly interact with the N-terminal domain of ERα, we synthesized a peptide composed of a 14-aa motif of this domain, which effectively competes with ERα/Tab2 interaction in pull-down and co-immunoprecipitation experiments, fused to the carrier TAT peptide to allow internalization. Treatment of TamR cells with this peptide resulted in partial recovery of the antiproliferative response to Tam, suggesting a strategy to revert pharmacological resistance in breast cancer. Silencing of Tab2 in TamR cells by siRNA caused modulation of a gene set related to the control of cell cycle and extensively connected to BRCA1 in a functional network. These genes were able to discern two groups of patients, from a published data set of Tam-treated breast cancer profiles, with significantly different disease-free survival. Altogether, our data implicate Tab2 as a mediator of resistance to endocrine therapy and as a potential new target to reverse pharmacological resistance and potentiate antiestrogen action.

Na+/K+-ATPase from Xenopus laevis (Daudin) kidney and epidermis: high sensitivity towards regulatory compounds.

Na+/K+-ATPase was prepared from Xenopus laevis epidermis. Purification was obtained by ultracentrifugation on sucrose discontinuous gradient. The maximum of enzyme-containing membranes was concentrated in the denser sucrose layer, exhibiting a good and long-lasting activity (specific activity about 55 mumoles of ATP hydrolyzed/mg of protein/hour). The Kd for the ouabain of kidney and epidermis enzymes were very low (in purified preparations respectively 16 nM for kidney enzyme and 4 nM for skin enzyme), indicating a very high sensitivity toward the cardioglycoside. The dose-response graphs of kidney and skin Na+/K+-ATPase vs ouabain concentrations show that at ouabain concentrations ranging from 1 nM and 1 pM the inhibition elicited by the cardioglycoside disappears and is replaced by an activatory effect. At cardioglycoside concentrations higher than 1 nM, the graphs show the typical inhibition curve.

Activatory effect of two cardioglycosides on Cavia cobaya kidney Na+/K+-ATPase activity

Ouabain and K-strophanthoside promote an enhancement of Na+/K+-ATPase activity in a range of cardioglycoside concentrations from 100 nM to 100 pM, with a maximum (+30%) between 10 and 4 nM. Binding experiments with [3H]ouabain show upward-curved Scatchard plots and evidence two intrinsic affinity constants for the ligand: (a) High-affinity constant: 350 nM (microsomes) and 15 nM (purified enzyme). (b) Low-affinity constant: 2100 nM (microsomes) and 890 nM (purified enzyme). The reaction velocity trend indicates that at ouabain concentrations higher than 20 nM but lower than the minimal inhibiting level, the enhanced reaction velocity is tending towards the control values.

Na+-K+-ATPase from Xenopus laevis (Daudin) kidney—purification and characterization

1. 1. Na+-K+-ATPase was prepared from Xenopus laevis kidney. Purification was obtained by ultracentrifugation on sucrose discontinuous gradient. The maximum of enzyme containing membranes was concentrated in the denser sucrose layer. 2. 2. The Km for ATP was lower, and the Ki for ouabain was very much lower compared with the enzyme from Bufo bufo kidney (0.24 mM and 0.025 μM respectively). 3. 3. The optimum pH was 7.2 and the optimum temperature indicated two peaks at 37°C and 45°C. 4. 4. The K12 for Na+ and for K+ were respectively 20.6 and 1.6 mM.