Rocío Soldati

The MPA mouse breast cancer model: evidence for a role of progesterone receptors in breast cancer

More than 60% of all breast neoplasias are ductal carcinomas expressing estrogen (ER) and progesterone receptors (PR). By contrast, most of the spontaneous, chemically or mouse mammary tumor virus induced tumors, as well as tumors arising in genetically modified mice do not express hormone receptors. We developed a model of breast cancer in which the administration of medroxyprogesterone acetate to BALB/c female mice induces mammary ductal carcinomas with a mean latency of 52 weeks and an incidence of about 80%. These tumors are hormone-dependent (HD), metastatic, express both ER and PR, and are maintained by syngeneic transplants. The model has been further refined to include mammary carcinomas that evolve through different stages of hormone dependence, as well as several hormone-responsive cell lines. In this review, we describe the main features of this tumor model, highlighting the role of PR as a trigger of key signaling pathways mediating tumor growth. In addition, we discuss the relevance of this model in comparison with other presently used breast cancer models pointing out its advantages and limitations and how, this model may be suitable to unravel key questions in breast cancer.

Estrogen Receptor Alpha Mediates Progestin-Induced Mammary Tumor Growth by Interacting with Progesterone Receptors at the Cyclin D1/MYC Promoters

Synthetic progesterone used in contraception drugs (progestins) can promote breast cancer growth, but the mechanisms involved are unknown. Moreover, it remains unclear whether cytoplasmic interactions between the progesterone receptor (PR) and estrogen receptor alpha (ERα) are required for PR activation. In this study, we used a murine progestin-dependent tumor to investigate the role of ERα in progestin-induced tumor cell proliferation. We found that treatment with the progestin medroxyprogesterone acetate (MPA) induced the expression and activation of ERα, as well as rapid nuclear colocalization of activated ERα with PR. Treatment with the pure antiestrogen fulvestrant to block ERα disrupted the interaction of ERα and PR in vitro and induced the regression of MPA-dependent tumor growth in vivo. ERα blockade also prevented an MPA-induced increase in CYCLIN D1 (CCND1) and MYC expression. Chromatin immunoprecipitation studies showed that MPA triggered binding of ERα and PR to the CCND1 and MYC promoters. Interestingly, blockade or RNAi-mediated silencing of ERα inhibited ERα, but not PR binding to both regulatory sequences, indicating that an interaction between ERα and PR at these sites is necessary for MPA-induced gene expression and cell proliferation. We confirmed that nuclear colocalization of both receptors also occurred in human breast cancer samples. Together, our findings argued that ERα-PR association on target gene promoters is essential for progestin-induced cell proliferation.

Antisense oligonucleotides targeting the progesterone receptor inhibit hormone-independent breast cancer growth in mice

IntroductionPrevious data from our laboratory suggested that progesterone receptors (PRs) are involved in progestin-independent growth of mammary carcinomas. To investigate this possibility further, we studied the effects of PR antisense oligodeoxynucleotides (asPR) on in vivo tumor growth.MethodBALB/c mice with subcutaneous 25 mm2 mammary carcinomas expressing estrogen receptor-α and PR were either injected intraperitoneally with 1 mg asPR every 24 or 12 hours for 5–10 days, or subcutaneously with RU 486 (6.5 mg/kg body weight) every 24 hours. Control mice received vehicle or scPR.ResultsSignificant inhibition of tumor growth as well as a significant decrease in bromodeoxyuridine uptake was observed in asPR-treated mice, which correlated with histological signs of regression and increased apoptosis. Mice treated with RU 486 experienced almost complete tumor regression. No differences were detected between vehicle-treated and scPR-treated mice. Anti-progestin-treated and asPR-treated mice were in a continuous estrous/meta-estrous state. Decreased phosphorylated extracellular signal-regulated kinase (ERK)1 and ERK2 levels and estrogen receptor-α expression were observed as late events in RU 486-treated and asPR-treated mice with regressing tumors.ConclusionWe demonstrate, for the first time, inhibition of tumor growth in vivo using asPR. Our results provide further evidence for a critical and hierarchical role of the PR pathway in mammary carcinomas.

Novel human breast cancer cell lines IBH‐4, IBH‐6, and IBH‐7 growing in nude mice

Breast cancer is the most frequent cancer in women. However, in vivo hormone receptor positive and metastatic models are scarce. The aim of the present manuscript was to assess if the novel steroid receptor positive human cell lines IBH‐4, IBH‐6, and IBH‐7 developed in our laboratory from primary infiltrant ductal carcinomas are good models to study in vivo human breast cancer. Cell lines or tumors were inoculated to nude mice in the presence or absence of hormone supplementation. Growth was analyzed by ANOVA followed by Tukey–Kramers test. Steroid hormone expression was assessed by immunohistochemistry and Western blotting. The histology of the tumors was analyzed. IBH‐4 and IBH‐6 cells were inoculated to nude mice and 100% of the injected mice developed tumors in the presence or absence of hormone treatment, although tamoxifen inhibited growth. IBH‐4 and IBH‐6 cell lines in vivo gave rise to poorly differentiated carcinomas with areas of solid growth and sarcomatoid areas showing no morphological signs of epithelial differentiation. Distinct features of malignancy were observed. IBH‐7 tumors in animals receiving estradiol were semi‐differentiated adenocarcinomas. IBH‐7 cells grew only in the presence of estradiol, but even with hormone addition, the tumor take was 20%. These tumors metastasized to the uterus and lung and vascular tumor emboli were evident. IBH‐7 tumors were invasive and able to break through the peritoneum. As a conclusion, IBH‐4 and IBH‐6 are good models for studying tumor progression, whereas IBH‐7 is a good model for tumor take, being metastatic and strictly estrogen‐dependent. J. Cell. Physiol. 219: 477–484, 2009.

Isolation of a stromal cell line from an early passage of a mouse mammary tumor line: a model for stromal parenchymal interactions.

We have developed a murine mammary tumor cell line, MC4‐L4, and after 15 passages, a spindle‐shaped population became evident. The cuboidal cells, MC4‐L4E, cloned by limit dilution, proved to be epithelial tumor cells. When inoculated in syngeneic mice, they gave rise to invasive metastatic carcinomas expressing estrogen and progesterone receptors. These tumors regressed after anti‐progestin treatment and stopped growing after 17‐β‐estradiol administration. In vitro, they were insensitive to medroxyprogesterone acetate (MPA), 17‐β‐estradiol, and EGF and were inhibited by TGFβ1. They expressed mutated p53 and estrogen receptors α; progesterone receptors were undetectable. Cells were polyploid and shared the same four common marker chromosomes present in the parental tumor in addition to an exclusive marker. Spindle‐shaped cells, MC4‐L4F, were selected by differential attachment and detachment and proved to be non‐epithelial non‐tumorigenic cells. They were cytokeratin negative, showed mesenchymal features by electron microscopy, differentiated to adipocytes when treated with an adipogenic cocktail, were stimulated by TGFβ1 and EGF, showed a wild‐type p53, and did not exhibit the marker chromosomes of the parental tumor. Although they expressed estrogen receptors α, they were insensitive to 17‐β‐estradiol in proliferation assays. Co‐cultures of both cell types had a synergic effect on progesterone receptors expression and on cell proliferation, being the epithelial cells, the most responsive ones, and 17‐β‐estradiol increased cell proliferation only in co‐cultures. Cytogenetic studies and data on p53 mutations rule out the possibility of an epithelial mesenchymal transition. Their unique characteristics make them an excellent model to be used in studies of epithelial–stromal interactions in the context of hormone responsiveness in hormone related tumors.