Tamoxifen resistance and Her2/neu expression in an aged, irradiated rat breast carcinoma model

Abstract

Clear links have been established between occupational or therapeutic radiation exposure and breast cancer. Tamoxifen chemoprevention following radiation exposure may be able to reduce the risk of developing breast cancer later in life. In order to model carcinogenesis in this setting, an in vivo model of tamoxifen chemoprevention and tamoxifen failure in a radiation-induced rat mammary carcinoma model was characterized. Two hundred and twenty-seven 60-day-old female rats received whole body or sham exposure to ionizing radiation. Thirty days later long-term, continuous, tamoxifen chemoprevention was initiated in half the population and all animals were monitored over three and a half years for the development of mammary tumors. Mammary tumors were surgically removed and carcinomas were histologically identified and characterized. Results showed that tamoxifen chemoprevention decreased the incidence and prolonged the latency of radiation-induced mammary carcinomas. However, many individuals receiving tamoxifen chemoprevention developed their first carcinoma very late in life. These carcinomas shared morphological features distinct from the majority of carcinomas that developed in the absence of tamoxifen chemoprevention. Analyses of cell lines established from these carcinomas and immunohistochemistry of tumor sections revealed that the highest levels of Her2/neu expression were associated with in vivo tamoxifen exposure. Treatment of rat mammary carcinoma cells with an anti-rat Her2/neu monoclonal antibody (MAb 7.16.4) inhibited cell growth and this effect was more pronounced in the presence of tamoxifen. These studies suggest that carcinoma growth driven by the Her2/neu pathway may be associated with tamoxifen chemoprevention failure in the rat mammary carcinoma model. Additionally, strategies combining targeted Her2/neu antibodies, vaccines or drugs with estrogen pathway modification may be more effective in reducing breast cancer chemoprevention failures.

Fig. 1:. Time to First Carcinoma and Cumulative Incidence

Fig. 2:. Mean Mammary Carcinoma Latency

Fig. 3:. Example - Immunohistochemical Analysis of Her-2/neu Expression in a Rat Mammary Tumor

Paraffin embedded mammary tumor tissue taken from rat #538 was analyzed as described in Table 2 legend and the Methodology Section and presented here as an example. Brownish pigment is indicative of the presence of erbB-2/neu expression. A: IgG2a control antibody, B: Anti-rat Her-2/neu monoclonal antibody, 7.16.4.

Fig. 4:. Relative Her-2/neu Cell-Surface Expression

Flow cytometric analysis was performed on RMT cell lines reacted with MAb 7.16.4 to determine their relative levels of erbB-2/neu expression. The mean peak fluorescence of erbB-2 transfected NIH 3T3 cells was 162 +/−33 (not shown). Three groups from each cell line were independently assayed and the mean of the peak fluorescence mean is graphed with error bars showing the standard deviation. * mean peak fluorescence was significantly greater than all the other cell lines presented ** mean peak fluorescence was significantly less than RMT 18, 50, and 56. ANOVA (p<0.05), Tukey 95% Simultaneous Confidence Interval.

Fig. 5:. Focus Formation Assay of Rat Mammary Tumor Cell Lines

Cells were seeded at a density of 150 cells/plate in 10mm cell culture dishes and grown for 9 days in 10% Fetal Bovine Serum/DMEM. At the end of this growth period the media was removed from the cells and they were stained with 1mg/ml p-Iodonitrotetrazolium violet overnight. Numbers correspond to rat mammary tumor (RMT) cell line

Fig. 6:. In Vitro RMT Cell Growth Response to Tamoxifen

RMTs 50, 56, and 91 were exposed to the indicated concentrations of tamoxifen for 5 days. The MTT assay was performed to determine relative numbers of viable cells in each group. Replicates of 5 were analyzed for each treatment group and the means +/− standard deviations are represented. The assay was repeated and similar results were obtained (data not shown). * mean absorbance (540nm) of 5 uM tamoxifen treated cells was significantly less than untreated cells. ANOVA (p<0.05), Fisher 95% Independent Confidence Interval.

Fig. 7:. Estrogen Receptor Protein Expression

Western Blot analysis was performed on cell lysates as described in the Materials & Methods section of the text. The expression of the estrogen receptor forms was standardized to β-actin expression by dividing the Scion digitized band intensities of each ER form by their respective re-blotted β- actin intensities (Ratio 76 and 70 Kd). Arrows depict 76 and 70Kd ER forms.

Fig. 8. In Vitro RMT Cell Growth Response to Anti-Her-2/neu MAb 7.16.4 Treatment

RMTs 50, 56, and 91 were seeded in 4 replicate plates and incubated with the indicated concentrations of MAb 7.16.4. The MTT assay was performed on days 4, 5, 6 and 7 to determine relative numbers of viable cells in each group. Replicates of 5 were analyzed for each treatment group on each day and the means +/− standard deviations are represented. * mean absorbance (540nm) was significantly less than other treatment groups. ANOVA (p<0.05), Tukey 95% Simultaneous Confidence Interval.

Fig. 9:. In Vitro RMT Cell Growth Response to Combined Tamoxifen and Anti-Her-2/neu MAb 7.16.4 Treatment

RMT 50 (A) and RMT 56 (B & C) were seeded in 5 replicate plates and incubated with 0.25 uM tamoxifen (T), 50 ug/ml MAb 7.16.4 (7), 50 ug/ml IgG2a, without antibody (Ab-), or any combination of these as indicated. C) 0.5 uM tamoxifen, 200 ug/ml MAb 7.16.4, and 200 ug/ml IgG2a were used. The MTT assay was performed on one of the plates at each of the specified days to determine the relative numbers of viable cells in each group. Replicates of 5 were analyzed for each treatment group on each day and the means are represented. * mean absorbance (540nm) was significantly less than other treatment groups. ANOVA (p<0.05), Tukey 95% Simultaneous Confidence Interval.