Changes of Coregulators, MAP Kinase Activity and p27/kip1 with Estrogen or Antiestrogen Treatment in Breast Cancer Cell Line / 한국유방암학회지

PURPOSE: Estrogen, various polypeptide hormones and growth factors are associated with the development and progression of breast cancer. Coregulatory proteins are also associated with estrogen receptor (ER) transcriptional activity and tamoxifen resistance. Therefore, it is necessary to investigate the change of coregulator mRNAs and various cell proliferation proteins and cell cycle-related proteins after treatment with estrogen or antiestrogen. METHODS: MCF-7 cells were maintained in dextran-coated charcoal stripped 10% Dulbecco's Modified Eagle Medium (DMEM). To measure the change of the coactivators' (src-1, P/CAF, CBP, AIB1) mRNAs and corepressors' (SMRT, N-coR) mRNAs, multiple PCR was carried out using specific primers. In addition, intracellular proteins related to cell proliferation and cell cycle regulation were measured by performing Western blotting after treatment with estrogen or tamoxifen. The change of mitogen activated protein kinases was also measured by performing Western after tamoxifen treatment for 4 weeks. RESULTS: Coactivator mRNAs expression rapidly decreased in 15 min after estrogen treatment but this recovered to the initial level in 3 hr. The pattern was similar for the case of tamoxifen treatment. Corepressor mRNAs expression rapidly decreased in 15 min after estrogen treatment and it remained at a lower level until 24 hr after estrogen treatment. With tamoxifen treatment, the initial response was similar to the cases of estrogen treatment, but the xpression gradually increased 3 hr after tamoxifen treatment. Treatment of estrogen induced intracellular concentrations of c-myc and Ki-67 and it increased nuclear translocation of NF-kappaB and phosphor-ERK and it decreased the intracellular cell cycle suppressor p27/kip1. Tamoxifen treatment increased nuclear p27/kip1 but it decreased c-myc, NF-kappaB and phosphor-ERK. Long-term (4 weeks) treatment of tamoxifen was associated with decrease of activated ERK and p38 but there was no change in phospho-Akt level. CONCLUSION: Estrogen induced cell proliferation and the survival pathway-related factors, but it decreased the cell cycle suppressor p27/kip1. Long-term treatment with antiestrogen tamoxifen might decrease the MAPK activities in ERalpha-expressing tumor cells.

The effect of growth factor receptor HER2 on the estrogen receptor transcriptional activities and its implications / 한국유방암학회지

PURPOSE: Until recently, breast cancer carcinogenesis has not been fully understood, but the roles of estrogen receptors(ERs) and growth factor receptors(like HER2) were known to be important. Growth factors have been shown to synergize in the E2 signaling pathway, although the actual molecular mechanism remains largely unknown. To investigate the effect of HER2 overexpression on the ERE(estrogen responsive element)-mediated transcriptional activity of the ERs, this study was designed. METHODS: NIH3T3 cells, T6-17 cells (NIH3T3 cells with stably transfected with HER2), and MCF-7 cells were maintained in dextran-coated charcoal stripped 10% Dulbecco's Modified Eagle Medium (DMEM). Transient transfection of constructs (pcDNA3-ER alpha, pcDNA3-ER beta, pERE-luc, pAP-1-luciferase, and pcDNA-HER2) into each cells was performed using the Lipofectamine PLUS(TM) system. Reporter gene assays using ERE-luciferase or AP-1-luciferase were used to measure the ER transcriptional activities after treatment with estradiol (E2) and tamoxifen. RESULTS: Reporter gene assay using ERE-luciferase in both ER alpha and ER beta, showed much less responsiveness to estrogen in HER2 overexpressing T6-17 cells than in NIH3T3 cells, but there was no remarkable difference after treatment with tamoxifen. The AP-1-mediated transcriptional activity was increased in ER beta after tamoxifen treatment, but it disappeared in HER2-expressing T6-17 cells. The responsiveness to estrogen in HER2-transfected MCF-7 cells was also slightly less than in the control MCF-7 cells, and the ERE-mediated transcriptional activity of estrogen in MCF-7 cells was decreased, in a dose-dependent manner, after HER2 transfection. CONCLUSION: Coexpression of HER2 and ER seems to make cells less responsive to estrogen stimulation, and decrease the ERE-mediated transcriptional activity in both ER alpha and ERbeta. These results suggest that the expression of HER2 reduces the estrogen dependency in cell growth and eventually induces estrogen independent-growth.

Anti-HER2/neu Peptide Producing Vector System for Biologic Therapy - Is It Possible to Mass-produce the Peptide?

A humanized monoclonal antibody against HER2 has been using in a clinical setting and has been shown to possess therapeutic properties. A mimetic peptide against HER2 was also reported to bind to the HER2 receptor with some therapeutic potential. Based on a previous report and the sequence of Herceptin, we designed oligonucleotides of anti-HER2 mimetic peptides, named V2 and V3 peptides, in order to develop a peptide- producing vector system for biologic therapy against HER2- overexpressing cancers. We also adopted the sequence of a previously reported mimetic peptide, V1 (Park BW et al. Nat. Biotechnol, 2000, 18: 194-198), as a reference peptide. We examined the effects of the V2 and V3 peptides against the HER2-overexpressing cell lines, SK-BR-3 and T6-17. Transient transfection of the construct expressing V1 and V2 inhibited cell proliferation in HER2-overexpressing cell lines by 20 - 30%, but had no effect on the HER2-negative NIH3T3 cells. The proliferation inhibition shown by V2 was slightly better than that shown by V1. Recombinant peptides V2 and V3 were produced on a large scale in an E. coli system, and the V2 peptide showed anti-HER2-specific tumor cell proliferation inhibition of 10% to 30%. Current results suggest that anti-HER2 mimetic peptides, overexpressed by a constitutive promoter or produced in an E. coli system, could specifically inhibit the proliferation of HER2-expressing cancer cells. Further efforts to augment the biologic specificity and efficacy and to develop new technologies for the purification of the peptide from the E coli system are needed.

Expression of Estrogen Receptor-beta mRNA in Various Mammary Tissues / 한국유방암학회지

PURPOSE: Estrogen signal transduction plays very important roles in both normal mammary development and neoplastic progression. Since the discovery of estrogen receptor-beta (ER-beta) there have been many controversial reports on the role of ER-beta in breast carcinogenesis and progression, and prognostic implications. ER-beta mRNA levels were investigated in various mammary tissues in order to verify the role of ER-beta expression in breast carcinogenesis. METHODS: Using messenger RNA (mRNA) in situ hybridization, we examined ER-beta expression in 60 paired normal and cancer tissues, 11 paired normal and benign breast tumor tissues, and 10 metastatic lymph nodes. We determined the intensity and extent (proportion of cells with positive hybridization) of the mRNA hybridization signals and gave scores 0 to 3; no hybridization (0), minimal (1), moderate (2), and strong (3) by the hybridization intensity and no hybridization (0), hybridization in less than 10% of cells (1), 10~50% (2), and more than 50% of cells (3) by the proportion of positively hybridized cells. Chi-square test, independent t-test or one-way ANOVA test was used for the statistical analysis and differences were considered to be significant with a p-value of less than 0.05. RESULTS: There was no statistically difference in ER-beta expression between normal and benign mammary tissues. ERbeta expression was significantly decreased in breast cancer and metastatic lymph node tissues compared with normal mammary and benign breast tumor tissues (P<0.01). The intensity and extent of ER beta expression were also significantly lower in breast cancer and metastatic lymph node tissues than in the normal mammary and benign breast tumor tissues (P<0.01). In cases of positive hybridization, the sum of scores of intensity and area were also significantly higher in normal and fibroadenoma tissues than in cancer or metastatic lymph nodes (P<0.01). CONCLUSION: ER beta transcription decreases in the process of breast cancer development, which suggests a protective role of ER beta in breast carcinogenesis.

Mitogen-activated Protein Kinases Activities and c-erbB-2 Expression in Breast Cancer Carcinogenesis and Progression

PURPOSE: Increased level mitogen-activated protein kinase (MAPK) and activation of MAPK have been reported in human breast cancers, especially in breast cancers with HER2/neu overexpression. To understand the relationship between the MAPK protein expressions and other clinico-pathological parameters, we examined the status of MAPKs in 20 breast cancers compared to those of paired normals. METHODS: A total of 20 breast cancers and paired normal breast tissues were included in this study. Tissues were obtained at the operation room and stored at -80degrees C. Tissue proteins were extracted and the concentration was determined by Bio-Rad protein assay method. Western blot analysis were performed to determine the level of MAPKs expressions using 100 ug of tissue protein in 8%, 10%, or 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). MAPK assays were carried out by a non-radioactive method developed by Cell Signaling Tech. as recommened by the manufacturer. Clinico-pathological information was provided from the Breast Cancer Registry of Department of Surgery, Yonsei University College of Medicine. RESULTS: The levels of MAPKs were higher in 95% of breast cancers compared to those of paired normals. The levels of ERK1/2 were significantly higher in cancer tissues compared to paired normals but the activated forms were not. The levels of JNK, p38, and MKP1 proteins were significantly increased in the cancer tissue compared to the paired normals. The levels of ERK1/2 and activated ERK1/2 proteins were not different between tumor stages. There were no significant differences of the levels of ERK1/2 and activated ERK1/2 proteins between HER2-negative and HER2- positive cancers. There were significantly higher levels of activated ERK1/2 proteins in ER-positive cancers than those in ER-negative cancers (P<0.05). CONCLUSION: The levels of MAPKs, but not the activated forms, seem to be increased in breast cancer tissues compared to those of paired normals. The levels of activated MAPKs seem to be associated with estrogen receptor expression in cancer tissues.

Expression of Estrogen Receptor-beta and Its Variants in Normal Mammary and Tumor Tissues / 한국유방암학회지

PURPOSE: Since the discovery of estrogen receptor-beta(ER-beta, five C-terminal variants of ER-beta were identified. We designed this study to investigate the pattern and clinical implications of ER-betaand its splicing variants expression in normal and malignant mammary tissues. METHODS: Using reverse transcription polymerase chain reaction (RT-PCR), we examined the expression levels of ER-alpha and ER-betaand its five splicing variants (beta1, beta2, beta3, beta4, beta5) in 50 paired normal and cancer tissues. We measured the densities of RT-PCR products using Tina version 2.10 (Raytest, Germany). Firstly, the incidence and intensity of ER-alpha and ER-beta and its five splicing variants were compared. Then the expression of ER-betamRNA splicing variants was also analyzed with regard to the ER-alphaprotein expression measured by immuno-histochemical staining and the menopausal status of the patients. Chi-square test and paired samples t-test were used for statistical analysis. Differences were considered to be significant with a p-value of less than 0.05. RESULTS: The expression of ER-betamRNA variants in normal breast and cancer tissues were as follows: ER-beta2 (100%/100%), ER-beta4 (76%/74%), ER-beta5 (32%/58%), and ER-beta1 (14%/16%). ER-beta3 was not detected at all. In terms of intensity, we observed a significant decrease of ER-beta2 (P<0.001) and an increase of ER-beta5 (P=0.004) in the mRNA expression levels among breast cancers compared to the corresponding normal breast tissues. Compared to the corresponding normal tissues, a significant decrease of ER-beta2 in cancer tissues was observed in patients with ER-alpha-positive (P<0.001), with age over 50 (P=0.01), and under 50 (P=0.04) as well, but not in patients with ER-alpha-negative (P=0.48). ER-beta4 also significantly decreased in patients with ER-alpha-positive (P=0.004) and with age over 50 (P=0.07). ER-beta5 showed a significant increment only in patient aged over 50 (P=0.04). CONCLUSION: ER-alpha mRNA expression significantly increases but ER-beta mRNA expression decreases in the cancer tissues compared to the corresponding normal tissues. Among ER-beta variant forms, ER-beta2 is predominant in both normal and malignant mammary tissues and ER-beta4, ER-beta5, and ER-beta1 in descending order but ER-beta3 does not express in mammary tissues. The decrease of ER-beta2 and ER-beta4 expression is prominent in cancer tissue especially in ER-alpha-positive cancers, which suggests that ER-beta2 and ER-beta4 may possess a regulatory function in mammary carcinogenesis. Further investigations to verify the roles of ER-beta variants are mandatory.