Association of Over-Expressed Estrogen Receptor Alpha with Development of Tamoxifen Resistant Hyperplasia and Adenocarcinomas in Genetically Engineered Mice

Abstract

Background: Estrogen receptor alpha (ERα) and cyclin D1 are frequently co-expressed in human breast cancer. Some, but not all, studies link tamoxifen resistance to co-expression of cyclin D1 and ERα. In mice over-expression of either cyclin D1 or ERα in mammary epithelial cells is sufficient to induce mammary hyperplasia. Cyclin D1 over-expression in mice leads to mammary adenocarcinoma associated with activated estrogen signaling pathways. ERα over-expression in mice leads to mammary hyperplasia and cancer. Significantly, disease development in these mice is abrogated by loss of cyclin D1.

Methods: Genetically engineered mouse models were used to determine whether or not ERα over-expression demonstrated cooperativity with cyclin D1 over-expression in cancer development, reaction to the chemical carcinogen DMBA, or tamoxifen response.

Results: Adding ERα over-expression to cyclin D1 over-expression increased the prevalence of hyperplasia but not cancer. Single dose DMBA exposure did not increase cancer prevalence in any of the genotypes although cyclin D1 over-expressing mice demonstrated a significant increase in hyperplasia. Tamoxifen treatment was initiated at both young and older ages to test for genotype-specific differences in response. Although normal ductal structures regressed in all genotypes at both younger and older ages, tamoxifen did not significantly reduce the prevalence of either hyperplasia or cancer in any of the genotypes. All of the cancers that developed were hormone receptor positive, including those that developed on tamoxifen, and all showed expression of nuclear-localized cyclin D1. In summary, development of tamoxifen resistant hyperplasia and cancer was associated with expression of ERα and cyclin D1.

Conclusion: These preclinical models will be useful to test strategies for overcoming tamoxifen resistance, perhaps by simultaneously targeting cell cycle regulatory pathways associated with cyclin D1.

Keywords: Breast cancer; Cyclin D1; ERα; Hyperplasia, Tamoxifen; Mouse models.

Figure 1. Prevalence of hyperplastic alveolar nodules (HANs) and invasive adenocarcinomas in one-year-old mice over-expressing ERα (CERM), cyclin D1 (D1), both ERα and D1 (CERM/D1) mice in no-treatment and DMBA and tamoxifen intervention groups

(A) Bar graphs comparing the prevalence of at least one HAN, multiple HANs, and invasive cancers in one-year-old CERM, CERM/D1, D1 and WT mice that received no treatment intervention. (B) Bar graphs comparing the prevalence of at least one HAN, multiple HANs, and invasive cancers in one-year-old CERM, CERM/D1, and D1 mice that were exposed to a single-dose of DMBA intervention at four months of age. (C) Bar graphs comparing the prevalence of at least one HAN, multiple HANs, and invasive cancers in one-year-old CERM, CERM/D1, D1 and WT mice that received tamoxifen treatment intervention at ten months of age. * indicates statistically significant differences (Fisher’s exact test, one tailed, p≤0.05).

Figure 2. Representative mammary gland whole mounts without and with tamoxifen treatment in six-month and one-year old CERM, CERM/D1, D1 and WT mice

Representative whole mount images of six-month and one-year old CERM, CERM/D1, D1 and WT mice treated with tamoxifen for two months demonstrating uniform ductal regression in all genotypes when treated at four months of age and presence of multiple HANs in CERM and CERM/D1 mice when treated at ten months of age (arrows and insets).

Figure 3. Invasive adenocarcinomas demonstrated expression of hormone receptors and cyclin D1

Representative panels illustrating H&E staining and immunohistochemistry for ERα, PgR, cyclin D1, Ki67 and ErbB2 in mammary adenocarcinomas from CERM, CERM/D1 and D1 mice. Arrows indicate representative stained cells with either nuclear (ERα, PgR, cyclin D1, Ki67) or higher intensity 2+ membrane (ErbB2) staining. Size markers are indicated on all panels.