Marginal activity of progesterone receptor B (PR-B) in dogs but high incidence of mammary cancer

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Highlights

  • The canine progesterone (P4) receptor B (PR-B) has marginal transactivation potential.

  • Replacement with the human B-upstream segment (BUS) restores activity.

  • Unliganded PR-B or P4 do not change expression profiles.

  • The few genes influenced by cPR-B present some residual activity.

  • Still dogs have the highest incidence of P4-dependent mammary carcinoma.

Abstract

Progesterone plays an important role in the normal development and carcinogenesis of the mammary gland. In vitro studies have shown that the canine progesterone receptor B (cPR-B), which is essential for mammary development in the mouse, does not transactivate reporter constructs containing progesterone response elements. Therefore, the question was raised whether the cPR-B was completely devoid of transactivation potential of endogenous progesterone regulated genes.

Canine mammary cell lines expressing doxycycline-inducible cPR-B, human PR-B or a chimera in which the canine B-upstream segment (BUS) was replaced by a human BUS were treated for 24 h with doxycycline, progesterone or a combination of the two. The expression profiling was subsequently performed using a dog-specific microarray and miRNA primers.

Incubation of stably transfected cell lines with doxycycline or progesterone alone, did not change expression of any endogenous gene. Expression of activated human PR-B or the chimera of human BUS with the canine PR resulted in differential expression of >500 genes whereas the activated cPR-B regulated only a subset of 40 genes and to a limited extent. The relevance of the marginal transactivation potential or the consequence of a lack of cPR-B function for the carcinogenesis of mammary gland tumors is discussed.

Introduction

Breast cancer is the most prevalent malignant disease in women [1]. About two/third of human breast cancers are steroid hormone receptor (ER/PR) positive and treated with combinations of selective estrogen receptor modulators (SERMs), GnRH agonists and/or aromatase inhibitors [2], [3], [4], alone or in combination with third-generation cytotoxic or biological therapies [5], [6]. A significant part of women treated by adjuvant hormonal therapies become ultimately therapy resistant [7], [8] showing a relapse and development of distant metastasis.

From studies early this century on hormone-replacement therapies (HRT) it is concluded that the synthetic progestin medroxyprogesterone acetate (MPA), rather than estrogens given as conjugated equine estrogens, are the major cause of enhanced breast cancer incidence in women on HRT [9]. Since then a numerous studies reported on the effects of various synthetic progestins and the result of cessation of HRT on breast cancer incidence. Ten years after the first report by Chlebowski et al., it was stated that progesterone signaling in breast cancer was neglected for too long and is now coming into the limelight [10].

Many studies on the effect of synthetic progestins on mammary gland development and oncogenesis come from rodent experiments. The only species, however, that has comparable or even higher incidences of mammary carcinomas compared to women are female dogs [11]. Early spaying dramatically reduces the mammary cancer incidence in dogs confirming the role of sex steroids in the development of the disease [11], [12]. Canine mammary tumors are characterized by a high homology with human breast cancer with respect to tumor-related signaling pathways and they are often, at least initially, hormone dependent [13]. The majority of malignant mammary carcinomas in the dog are of epithelial origin, but also concurrent tumors of non-epithelial origin may be present and tumors are more heterogeneous as shown by histopathology [14]. This may reflect a higher variation of differentiation of cancer stem cells.

Treatment of beagle bitches with MPA resulted in mammary hyperplasia and neoplasia [15]. A concomitant rise in plasma growth hormone (GH) concentrations suggested that elevated exposure to pituitary GH was the cause of the mammary tumors in the dog. In 1995, however, we demonstrated that MPA induced local mammary expression of GH [16], [17], [18].

Progesterone plays an important role in ductal side-branching and subsequent lactogenic differentiation of the mammary gland. For its effects progesterone uses progesterone receptors (PR) which are present in two forms, a N-terminally truncated form called PR-A and a longer PR-B [19]. Studies using knock out (KO) mice have shown that, at least in mice, PR-B is essential for mammary development. The PRBKO mouse does not develop fully differentiated mammary glands whereas the PRAKO does [20], [21]. These data were the impetus for us to study the PR isoforms in the dog. We found that the dog, as well as the other members of the canidae family (wolf, red fox and raccoon dog), have mutations and insertions in the B-upstream segment (BUS) of PR-B resulting in low to absent transactivation of genes containing progesterone response elements (PRE) in their promoter region. The major mutations causing this are related to the two LxxLL motifs and a tryptophan residue that play a role in the binding of additional co-activators to the PRE complex in the promoter regions of progesterone-regulated genes [22], [23]. Despite the loss of this important signal transduction path dogs still normally develop mammary tissue and, moreover, have a high incidence of mammary neoplasia.

To further study the genes regulated by the canine PR-B we generated a canine mammary cell lines that expresses the cPR-B, human PR-B (hPR-B) or the chimera of canine PR-A and human BUS region (hBUS), under a doxycycline-sensitive promoter [22]. Here we report on the comparison of the expression profile of genes regulated by the progesterone-activated cPR-B, hPR-B and hBUS.

Section snippets

PR-B expressing cell lines and culture

Canine mammary cell line CNMm was kindly provided by Prof. N. Sasaki [24]. This cell line is derived from a metastatic regional lymph node of a canine mammary carcinoma characterized by clinical stage II and a tubular morphology. The cell line does not express progesterone receptors as shown by western blot analysis and the absence of activation of an MMTV-luciferase construct by progesterone (data not shown).

This cell line was stably transfected with a tetracycline repressor before

Results

From the cultured cells high quality RNA was isolated with RNA integrity numbers of the samples that varied from 9.2 to 10.0 (mean ± SD; 9.8 ± 0.3), indicating that these samples were excellent for gene expression analysis.

A heatmap of the expression profiles (Fig. 1) clearly showed excellent dye swabs and a close clustering of the differently expressed genes after incubation of the hPR-Band the hBUS cells with the combination of doxycycline and progesterone. The cells expressing the canine PR-B

Discussion

In a previous study we reported on the fact that the canine PR-B showed no transactivation of a PRE2-luciferase reporter construct, specific for PR-B, whereas a low transactivation potential was found on a MMTV-luciferase construct that reflects PR activation more in general [23]. In this paper we extended this study to the expression profiling of endogenous genes in a canine mammary cell lines expressing doxycycline-inducible hPR-B, cPR-B or hBUS.

In comparison to non-treated controls the

Funding

This work was supported by the Mozaiek Grant 017.004.081 (to A.G.) from the Dutch Society for Scientific Research (NWO) and by a travel grant to The Netherlands (to F.A.V.) from CAPES/Mec – Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil. The authors have no further disclosures.

Acknowledgements

We thank Marian J.A. Groot Koerkamp from the microarray facility, department of Physiological Chemistry, University Medical Centre, Utrecht for her contribution in the hybridization of the microarrays.

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