Effect of medroxyprogesterone acetate (MPA) and serum factors on cell proliferation in primary cultures of an MPA-induced mammary adenocarcinoma

Rosemblit

Béguelin

et al. 2009

Self Cite

Cross talk between the steroid hormone receptors for estrogen and progesterone (PR) and the ErbB family of receptor tyrosine kinases appears to be a hallmark of breast cancer growth, but its underlying mechanism remains poorly explored. Here we have highlighted signal transducer and activator of transcription 3 (Stat3) as a key protein activated by heregulin (HRG), a ligand of the ErbB receptors, through co-opted, ligandindependent PR function as a signaling molecule. Stat3 activation was an absolute requirement in HRGinduced mammary tumor growth, and targeting Stat3 effectively inhibited growth of breast cancer cells with activated HRG/ErbB-2 and PR. Our findings unravel a novel potential therapeutic intervention in PR-and ErbB-2-positive breast tumors, involving the specific blockage of PR signaling activity.Accumulated evidence indicates that the ErbB family (epidermal growth factor receptor [EGFR]/ErbB-1, ErbB-2, ErbB-3, and ErbB-4) of receptor tyrosine kinases is involved in breast cancer growth. Particularly, ErbB-2 overexpression in breast tumor samples is associated with increased metastatic potential and a poor prognosis (34). A large number of ligands for ErbBs have been described, including all isoforms of heregulins (HRG), which bind to ErbB-3 and ErbB-4 and recognize EGFR and ErbB-2 as coreceptors (35). Although the molecular mechanisms through which ErbBs control breast cancer growth have yet to be elucidated fully, the activation of mitogenic intracellular signaling, such as the p42/p44 mitogenactivated protein kinase (MAPK) pathway, seems to be causally involved in ErbB-induced breast cancer proliferation (27). Notably, several research works, including our own with progesterone receptor (PR) and HRG (20), have demonstrated that unliganded steroid hormone receptors (SHR) could indeed be transcriptionally activated through cross talk with ErbB signaling pathways (22, 39), indicating that another likely mechanism through which ErbBs induce breast cancer proliferation is by ligand-independent activation of SHR. On the other hand, several reports as well as our own findings with HRG/ErbBs and PR have shown that steroid hormones are able to activate ErbBs in breast cancer (2, 9, 21). Bidirectional cross talk between steroid hormones and ErbB signaling pathways therefore seems to be a hallmark of mammary cancer growth.Over the last few years, a unique family of proteins, the signal transducers and activators of transcription (Stats), were found to be involved in cross talk with both steroid hormones and ErbB receptors (30,38). Particularly, accumulating evidence has revealed that one of the members of the Stat family, Stat3, plays a key role in breast tumorigenesis (38).We already demonstrated that HRG, acting via ErbB-2, induced growth in C4HD cells from an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female BALB/c mice (2). Notably, we found that HRG proliferative effects in both C4HD cells (2) and ...

Section: Resultsmentioning

confidence: 60%

Activation of Stat3 by Heregulin/ErbB-2 through the Co-Option of Progesterone Receptor Signaling Drives Breast Cancer Growth

Rosemblit

Béguelin

et al. 2009

Self Cite

“…Primary cultures of the C4HD murine mammary tumor line (3,4,25,44) were used. As we have already described in detail (19), C4HD epithelial cells derive from a ductal, progestin-dependent mammary tumor line. C4HD cells require MPA administration to proliferate and express high levels of PR (19).…”

Section: Resultsmentioning

confidence: 99%

“…As we have already described in detail (19), C4HD epithelial cells derive from a ductal, progestin-dependent mammary tumor line. C4HD cells require MPA administration to proliferate and express high levels of PR (19). We first investigated Stat3 expression and tyrosine phosphorylation in C4HD cells.…”

Section: Resultsmentioning

confidence: 99%

Progestins Induce Transcriptional Activation of Signal Transducer and Activator of Transcription 3 (Stat3) via a Jak- and Src-Dependent Mechanism in Breast Cancer Cells

Salatino

Rosemblit

et al. 2005

Self Cite

117

137

Progesterone regulates diverse biological effects in a broad range of tissues, mostly by interaction with the classical progesterone receptor (PR), a member of the nuclear receptor superfamily of ligand-dependent transcription factors. Particularly in the mammary gland, progesterone plays a key role in the control of cell proliferation and differentiation (31 and references within). Accumulated evidence also indicates that progestins are involved in controlling mammary gland tumorigenesis, both in women and in animal models (6,8,15,21,24,26,31,32,48). Although the mechanisms by which progestins stimulate growth of breast cancer cells have not been completely deciphered, several lines of evidence (6, 26, 33), including our own work (3,4,25,44), have shown that convergence between progestins and growth factor (GF) signaling pathways mediates proliferative effects of progestins in mammary tumor cells.In addition to their direct transcriptional effects, rapid or nongenomic biological effects of progestins have been described in several species, including fish, amphibian, and mammalian (27). Whether the recently cloned membrane PR from humans and other vertebrates (59) is involved in mediating nongenomic progestin effects in mammalians remains to be elucidated. Nongenomic effects of progestins in breast cancer cells have been unraveled by startling reports from Auricchio and coworkers (5,13,33), who demonstrated that progestin treatment of human breast cancer T47D cells activates the signal-transducing c-Src/p21 ras /MAPK pathway, which results in cell proliferation (5,13,33). Progestin activation of the c-Src/p21 ras /MAPK pathway has also been described by Edwards and coworkers (6), who further explored mechanisms involved in progestin modulation of c-Src activity.Over the last years, a unique family of proteins, the signal transducers and activators of transcription (Stats), was found to be involved in cross talks with both steroid hormones and

“…Primary cultures of epithelial cells from C4HD tumors, growing in MPA-treated mice, were performed as previously described (21). In brief, tumors were aseptically removed, minced, and washed with DMEM-F12 (Dulbecco modified Eagle medium-Ham F12 [1:1], without phenol red, with 100 U of penicillin/ml, and with 100 g of streptomycin/ml).…”

Section: Animals and Tumorsmentioning

confidence: 99%

“…Enzyme action was stopped by adding DMEM-F12 plus 5% heat-inactivated fetal calf serum (FCS; Gen S.A., Buenos Aires, Argentina). Epithelial and fibroblastic cells were separated as previously described (21). Briefly, the cell suspension was resuspended in 15 ml of DMEM-F12 plus 10% FCS and allowed to sediment for 20 min.…”

Section: Animals and Tumorsmentioning

confidence: 99%

Heregulin Induces Transcriptional Activation of the Progesterone Receptor by a Mechanism That Requires Functional ErbB-2 and Mitogen-Activated Protein Kinase Activation in Breast Cancer Cells

Labriola

Salatino

et al. 2003

Self Cite

The present study addresses the capacity of heregulin (HRG), a ligand of type I receptor tyrosine kinases, to transactivate the progesterone receptor (PR). For this purpose, we studied, on the one hand, an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female BALB/c mice and, on the other hand, the human breast cancer cell line T47D. HRG was able to exquisitely regulate biochemical attributes of PR in a way that mimicked PR activation by progestins. Thus, HRG treatment of primary cultures of epithelial cells of the progestindependent C4HD murine mammary tumor line and of T47D cells induced a decrease of protein levels of PRA and -B isoforms and the downregulation of progesterone-binding sites. HRG also promoted a significant increase in the percentage of PR localized in the nucleus in both cell types. DNA mobility shift assay revealed that HRG was able to induce PR binding to a progesterone response element (PRE) in C4HD and T47D cells. Transient transfections of C4HD and T47D cells with a plasmid containing a PRE upstream of a chloramphenicol acetyltransferase (CAT) gene demonstrated that HRG promoted a significant increase in CAT activity. In order to assess the molecular mechanisms underlying PR transactivation by HRG, we blocked ErbB-2 expression in C4HD and T47D cells by using antisense oligodeoxynucleotides to ErbB-2 mRNA, which resulted in the abolishment of HRG's capacity to induce PR binding to a PRE, as well as CAT activity in the transient-transfection assays. Although the inhibition of HRG binding to ErbB-3 by an anti-ErbB-3 monoclonal antibody suppressed HRG-induced PR activation, the abolishment of HRG binding to ErbB-4 had no effect on HRG activation of PR. To investigate the role of mitogen-activated protein kinases (MAPKs), we used the selective MEK1/MAPK inhibitor PD98059. Blockage of MAPK activation resulted in complete abrogation of HRG's capacity to induce PR binding to a PRE, as well as CAT activity. Finally, we demonstrate here for the first time that HRG-activated MAPK can phosphorylate both human and mouse PR in vitro.Recent evidence has clearly shown the presence of cross talks between steroid hormone and growth factor (GF) signaling pathways, which were previously thought to be distinctly separate processes. Members of the steroid receptor superfamily are heavily phosphorylated proteins that, upon ligand binding, act as nuclear transcription factors (for a review, see reference 72). Particularly, progesterone receptor (PR), the focus of the present work, is phosphorylated in the absence of hormone and undergoes an increase in phosphorylation upon hormonal stimulation (19,20,67). Although the functional role of PR phosphorylation remains elusive, increased evidence indicates that it plays a role in the regulation of PR transcriptional activity (3,7,14,22,40,66). On the other hand, most GFs bind to transmembrane receptors that carry an intrinsic activity of tyrosine kinase. Tyrosine resid...