Ariana Bruzzone

α2-Adrenoceptor action on cell proliferation and mammary tumour growth in mice

Breast cancer, the most common cancer in women in most countries, is a highly stressful disease. Catecholamines released during stress bind to adrenoceptors and we have recently described α2‐adrenoceptors in human breast cell lines, linked to enhanced cell proliferation. The purpose was to assess the in vivo effects of compounds acting on α2‐adrenoceptors in a reliable model of breast cancer.

Involvement of α2- and β2-adrenoceptors on breast cancer cell proliferation and tumour growth regulation.

BACKGROUND AND PURPOSE β‐Adrenoceptors are expressed in human and experimental animal breast cancer cells. However, the effect of the agonists and antagonists reported on cell proliferation and tumour growth was paradoxical, precluding their utilization as possible adjuvant therapy, mainly in the cases of refractory tumours.

Adrenoceptors: Non Conventional Target for Breast Cancer?

Epinephrine and Norepinephrine, typically released during stress bind to nine different adrenoceptors (AR) which classically control the cardiovascular and respiratory systems. New targets were described for the many agonists and antagonists developed for these AR, as the central nervous system. During the last three decades, AR expression and action on the mammary gland/breast were extensively investigated. In the cow mammary gland, good milkability was associated with low density of beta(2)-AR and high density of alpha(2)-AR. In the rat normal mammary gland, beta-AR are expressed in the epithelial cells, alveoli, ducts, and adipocytes showing an exquisite regulation by steroid hormones and prolactin. In rat dimethylbenz(a)anthracene (DMBA) tumors, a close correlation was observed between tumor growth and beta-AR concentration. beta(2)-AR were described in numerous human cell lines and breast tumors. The action of beta-adrenergic compounds on cell proliferation is contradictory. While some authors found that beta-agonists significantly inhibit cancer cell proliferation and tumor growth in mice, others described a significant reduction in DNA synthesis by beta-blockers. Also, positive effects of beta-AR on human carcinoma cell migration have been described. alpha(2)-AR are expressed in human breast cancer and non-cancer cell lines, their stimulation being associated with increased cell proliferation. In vivo clonidine increased tumor growth and alpha (2)-adrenergic antagonists completely reversed this effect. When administered alone, rauwolscine inhibited tumor growth behaving as an inverse agonist. Therefore, the numerous adrenergic beta- and alpha-AR agonists or antagonists could prove to be unexpected therapeutic options for mammary gland/ breast and mainly breast cancer.

α2-Adrenoceptors Enhance Cell Proliferation and Mammary Tumor Growth Acting Through both the Stroma and the Tumor Cells

We have previously described enhanced human breast cancer cell proliferation and mouse mammary tumor growth induced by α(2)-adrenoceptor (α(2)-AR) expression in epithelial cells. The aim of the present work was to assess if stromal fibroblasts can contribute to this effect. α(2)-AR expression was assessed by immunocytochemistry and immunohistochemistry, cell proliferation by [(3)H]-Thymidine incorporation and tumor growth by measuring with caliper. All tested mouse and human fibroblasts expressed at least two α(2)-AR subtypes and α(2)-adrenergic agonists enhanced fibroblast proliferation. In vivo, the α(2)-adrenergic agonist clonidine significantly enhanced tumor growth. The α(2)-adrenergic antagonist rauwolscine reversed this effect, but when administered alone, significantly inhibited tumor growth. Clonidine significantly stimulated cell proliferation in the epithelial-enriched fraction, the cancer associated fibroblast-enriched fraction and the co-culture of both fractions in primary cultures from both tumors (IBH-4 and IBH-6). Rauwolscine reversed clonidine stimulation in every fraction. However, when incubated alone, the inhibitory effect was observed in fractions from IBH-4 tumors but not from IBH-6 tumors. These experiments show that fibroblasts from tumor stroma are also influenced by α(2)-adrenergic compounds through the α(2)-ARs expressed in these cells. Moreover, the α(2)-adrenergic antagonist rauwolscine could eventually block in both epithelial and stromal cells, the mitogenic effect of catecholamines released during stress, providing a potential additional treatment for breast cancer patients. Chemists synthesizing adrenergic compounds should consider their action in breast cancer patients.

Dosage-dependent regulation of cell proliferation and adhesion through dual β2-adrenergic receptor/cAMP signals

The role of β‐adrenergic receptors (β‐ARs) remains controversial in normal and tumor breast. Herein we explore the cAMP signaling involved in β ‐AR‐dependent control of proliferation and adhesion of nontumor human breast cell line MCF‐10A. Low concentrations of a β‐agonist, isoproterenol (ISO), promote cell adhesion (87.5% cells remaining adherent to the plastic dishes following specific detachment vs. 35.0% in control, P<0.001), while increasing concentrations further engages an additional 36% inhibition of Erk1/2 phosphorylation (p‐Erk1/2)‐dependent cell proliferation (P<0.01). Isoproterenol dose response on cell adhesion was fitted to a 2‐site curve (EC50(1): 16.5±11.5 fM, EC50(2): 4.08 ±3.09 nM), while ISO significantly inhibited p‐Erk1/2 according to a 1‐site model (EC50: 0.25 ±0.13 nM). Using β‐AR‐selective agonist/antagonists and cAMP analogs/inhibitors, we identified a dosage‐dependent signaling in which low ISO concentrations target a β2‐AR population localized in raft microdomains and stimulate a Gs/cAMP/Epac/ adhesion‐signaling module, while higher concentrations engage a concomitant activation of another β2‐AR population outside rafts and inhibit the proliferation by a Gs/cAMP/PKA‐dependent signaling module. Our data provide a new molecular basis for the dose‐dependent switch of β‐AR signaling. This study also sheds light on a new cAMP pathway core mechanism with a single receptor triggering dual cAMP signaling controlled by PKA or Epac but with different cellular outputs.—Bruzzone, A., Saulière, A., Finana, F., Sénard, J.‐M., Lüthy, I., Galés, C. Dosage‐dependent regulation of cell proliferation and adhesion through dual β2‐adrenergic receptor/cAMP signals. FASEB J. 28, 1342–1354 (2014). www.fasebj.org

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.

The β 2-Adrenergic Agonist Salbutamol Inhibits Migration, Invasion and Metastasis of the Human Breast Cancer MDA-MB- 231 Cell Line

BACKGROUND Breast cancer is the most diagnosed and the major cause of cancer death in women worldwide. Metastasis is the main cause of these deaths. The metastatic cascade involves multiple steps and it has been described that adrenergic receptors can modulate this process at multiple levels. However, β -adrenergic action in breast cancer is controversial. We have previously shown that β-adrenergic agonists inhibit cell proliferation and tumor growth of numerous breast cancer models. OBJECTIVE The purpose of the present investigation was to evaluate adrenergic effect in parameters related to tumor progression (migration, invasion and metastases) in two human breast cancer cell lines. METHODS Migration was assessed in IBH-6 and MDA-MB-231 cells by time-lapse videomicroscopy and modified Boyden chambers. Invasion was evaluated by Transwells coated with Matrigel and expression of pro-metastatic genes was determined by RT-qPCR. Experimental metastases studies were performed by injection of the cells in the tail vein of NSG immuno-deficient mice. RESULTS In both cell lines, salbutamol (β2-agonist) and propranolol (β-blocker) significantly diminished cell migration while epinephrine exerted opposite effects. Moreover, salbutamol inhibited invasion of both breast cancer cell lines and enhanced adhesion to extracellular matrix. Salbutamol treatment was also able to decrease the expression of pro-metastatic genes in MDA-MB-231 cells. Finally, this compound decreased the number and size of MDA-MB-231 lung experimental metastases in NSG immuno- deficient mice. No effect on the establishment of IBH-6 metastases was observed. CONCLUSION Our results suggest that salbutamol could be an effective adjuvant drug for the treatment of metastatic breast cancer.

Abstract B34: A novel role for β-adrenergic receptor in mammary branching morphogenesis and its implication in breast cancer

The mammary gland develops from embryogenesis to infancy-puberty and adulthood, by the input of circulating hormones. At birth the gland is composed of a rudimentary ductal system that grows allometrically until puberty (4-weeks in mice). Later on, estrogens, growth hormone and insulin like growth factor induce expansive proliferation. Normal breast has three types of lobules, type 1, 2 and 3. Full term pregnancy and lactation results in the development of lobule type 3 into type 4, preparing the gland for lactation. This transformation is known to inhibit carcinogenic initiation through the induction of differentiation. β2-adrenergic receptors (β2-AR) have been well characterized in several human breast cell lines (normal and tumor) and in tissue samples. Recently, we have demonstrated that β2-AR expression and stimulation are associated with a benign breast tumor cell phenotype, reducing proliferation and cell migration, and increasing adhesion, suggesting that this receptor might be an important factor during tumor progression. In order to understand the implications of β2-AR on breast development and cancer, we evaluated β-activation in experimental models of normal and tumor breast, using cell lines, cultures 3D and in vivo approaches. Non tumor breast cell line MCF-10A cultured during 15 days on matrigel, formed gland-like organoids. Tubular structures mimicking mammary ducts were recognized, with type 1 and 2 lobules. No sign of lumen was observed. When treated with the β-agonist Isoproterenol (1 μM ISO) MCF-10A cells showed an increase in type 2 and 3 lobules (p Later, Balb/c female virgin mice, weaning and 2 months old were treated daily with ISO 1 mg/Kg during 15 days. Control groups received saline solution. Mice were sacrificed and the 4th mammary glands were removed for Whole Mount and hematoxylin and eosin staining. Interestingly, ISO induced a statistically significant difference in mammary gland branching in weaning (150±32 vs 57±21 branches/field in controls, p Finally, we tested ISO in a mouse model of mammary ductal carcinoma. Surprisingly, tumors exhibited newly rough lumens and changes in cell polarity towards them, reinforcing the differentiating role of ISO also in a pathological context. In conclusion, β-AR stimulation seems to be involved in normal mammary development, leading to a terminal stage phenotype. In agreement with this, the analysis of a publicly available MicroArray dataset (GSE8191) showed an increase in β-AR expression levels during lactation. This study highlights a possible physiological role of β-AR in the development of mammary gland. Understanding the function of β-AR in normal development is crucial to elucidate its role in breast cancer and its possible use as a therapeutic target. Citation Format: Lucia Gargiulo, Maria May, Ezequiel Mariano Rivero, Lydon John, Caroline Lamb, Claudia Lanari, Isabel Alicia Luthy, Ariana Bruzzone. A novel role for β-adrenergic receptor in mammary branching morphogenesis and its implication in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B34.

Abstract 1047: Beta adrenergic action on human breast cancer cell lines

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Breast cancer, the most frequent cancer among women in the majority of countries, is also a stressful disease. The principal effectors of the stress system include the epinephrine (EPI) and norepinephrine which bind to α1-, α2- and β-adrenoceptors (AR). We have described α2-AR in human tumor and non-tumor breast cell lines, associated with increased cell proliferation and tumor growth. We have also studied the effect of β-AR agonists and antagonists in different breast cancer experimental models. When animals were treated with the agonist isoproterenol (ISO) and/or salbutamol (SALB), tumor growth was significantly reduced correlating with Erk 1/2 phosphorylation status in whole protein tumor samples by Western blotting. The aims of the present work were: to assess the expression of β2-AR in human breast cancer cell lines (IBH-4, IBH-6, MDA-MB-231, MCF-7, among others) by RT-PCR and immunofluorescence (IF) techniques; to study the biological effect of β-AR agonists by means of proliferation assays and compare these results with the effect of the natural agonist (EPI); to find out the signaling pathways involved in reduced Erk phosphorylation induced by β-AR stimulation. β2-AR expression was detected by RT-PCR, using GAPDH as a control. A positive staining was seen in all cells by IF. After the incubation of the cells with increasing doses (from 10−13M to 10−6M) of the β-AR agonist (ISO or SALB), a significant reduction in [3H]-thymidine incorporation to the cells nuclei was observed as compared to control values (expressed as percentage of control incorporation). IBH-4: ISO 0.1μM, 75.87±8.49% vs control 100±7.18% p<0.05; SALB 0.1nM, 63.86±6.42% vs 100±7.79% p<0.05. IBH-6: ISO 0.1μM, 67.02±6.11% vs control 100±6.64% p<0.05; SALB 0.1nM, 59.88±5.62% vs control 100±7.34% p<0.05. A similar inhibition was detected in MDA-MB-231, MCF-7 and HS-578T cell lines. In all the cell lines studied, the adrenergic agonist EPI exerted a stimulating cell proliferation effect. Erk1/2 phosphorylation was significantly inhibited by SALB or ISO studied by Western blotting. Erk1/2 phosphorylation decreased when the cells were treated with 8-Br-cAMP, or the PKA specific analog (6-Bnz) and this effect was mimic with forskolin. The effect on Erk1/2 phosphorylation was reversed by H-89 (PKA inhibitor) in the presence and absence of β-agonists. We conclude that both ISO and SALB, acting through β-AR, inhibit cell proliferation. On the other hand, EPI increases cell proliferation acting through α2-AR. We propose that in the cell lines studied, the adrenergic compounds act via classic Gs pathways, augmenting cAMP and activating PKA and not EPAC. The results are promising and in agreement with the in vivo experiments presented before. These compounds, devoid of serious side-effects, could eventually be employed to inhibit tumor growth. In the future, it might be useful to analyse the metastatic potential of the cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1047. doi:10.1158/1538-7445.AM2011-1047