María C. Díaz Flaqué

Progesterone Receptor Induces ErbB-2 Nuclear Translocation To Promote Breast Cancer Growth via a Novel Transcriptional Effect: ErbB-2 Function as a Coactivator of Stat3

ABSTRACT Progesterone receptor (PR) and ErbB-2 bidirectional cross talk participates in breast cancer development. Here, we identified a new mechanism of the PR and ErbB-2 interaction involving the PR induction of ErbB-2 nuclear translocation and the assembly of a transcriptional complex in which ErbB-2 acts as a coactivator of Stat3. We also highlighted that the function of ErbB-2 as a Stat3 coactivator drives progestin-induced cyclin D1 promoter activation. Notably, PR is also recruited together with Stat3 and ErbB-2 to the cyclin D1 promoter, unraveling a new and unexpected nonclassical PR genomic mechanism. The assembly of the nuclear Stat3/ErbB-2 transcriptional complex plays a key role in the proliferation of breast tumors with functional PR and ErbB-2. Our findings reveal a novel therapeutic intervention for PR- and ErbB-2-positive breast tumors via the specific blockage of ErbB-2 nuclear translocation.

Integrin αvβ3 acting as membrane receptor for thyroid hormones mediates angiogenesis in malignant T cells

The interaction of lymphoid tumor cells with components of the extracellular matrix via integrin αvβ3 allows tumor survival and growth. This integrin was demonstrated to be the membrane receptor for thyroid hormones (THs) in several tissues. We found that THs, acting as soluble integrin αvβ3 ligands, activated growth-related signaling pathways in T-cell lymphomas (TCLs). Specifically, TH-activated αvβ3 integrin signaling promoted TCL proliferation and angiogenesis, in part, via the upregulation of vascular endothelial growth factor (VEGF). Consequently, genetic or pharmacologic inhibition of integrin αvβ3 decreased VEGF production and induced TCL cell death in vitro and in human xenograft models. In sum, we show that integrin αvβ3 transduces prosurvival signals into TCL nuclei, suggesting a novel mechanism for the endocrine modulation of TCL pathophysiology. Targeting this mechanism could constitute an effective and potentially low-toxicity chemotherapy-free treatment of TCL patients.

Abstract 1549: Targeting Stat3 induces senescence in breast cancer cells and elicits an immune response inhibiting tumor growth and metastasis

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Having in mind that Stat3 inhibition in tumor cells induces the expression of chemokines and pro-inflammatory cytokines, we proposed the use of Stat3-inhibited breast cancer cells as a source of immunogens to induce an anti-tumor immune response. We have demonstrated that the administration of irradiated breast cancer cells that express a dominant negative (DN) form of Stat3 (Stat3Y705F-breast cancer cells) provides protection against the murine progestin-dependent C4HD tumor, through the activation of CD4+ T cells and cytotoxic natural killer (NK) cells. To extend our results to different breast cancer models, we worked with the hormone independent 4T1 mammary carcinoma cell line that displays constitutive activation of Stat3. Immunization with irradiated Stat3Y705F-4T1 cells prevented wild-type 4T1 tumor development in 50% of the challenged mice (P<0.001), and the tumor-bearing mice displayed tumors of smaller size (81% decrease in tumor volume, P<0.001) when compared to mice injected with pcDNA3.1-4T1 cells. Moreover, the number of metastasis per lung decreased by 90% in Stat3Y705F-4T1-immunized animals (P<0.05). When we analyzed the tumor milieu composition by flow cytometry, we observed that Stat3Y705F-4T1 immunized animals displayed an increase in the percentage of tumor infiltrating NK cells (CD3-DX5+) and a decrease in tumor infiltrating T regulatory lymphocytes (CD4+CD25+FoxP3+), compared to pcDNA3.1-4T1-immunized mice. In order to evaluate if this vaccination may be effective in a therapeutic setting, we immunized mice with irradiated Stat3Y705F-4T1 or pcDNA3.1-4T1 cells, 4, 11 and 18 days after challenging with 4T1 cells. On day 35, we observed a significant decrease on tumor volume and growth rate in Stat3Y705F-4T1 cell-immunized animals, when compared to mice immunized with pcDNA3.1-4T1 cells (37.5%, P<0.05) and a decrease in the number of metastasis per lung (P<0.05). On the other hand, cellular senescence is an important mechanism of tumor regression upon oncogene inactivation that leads to the secretion of pro-inflammatory cytokines that resemble the ones we found after blocking Stat3. Therefore, we wondered whether Stat3 inhibition could drive a senescence program. Inhibition of Stat3 in murine C4HD and 4T1 cells by transfection with Stat3Y705F, or Stat3 silencing by siRNA, resulted in increased senescence-associated-β-galactosidase (SA-α-gal) accumulation and increased expression of the senescence-associated markers p15INK4b and p16INK4a. As cellular senescence is associated to chromatin changes, we studied heterochromatin formation and observed an increase of trimethyl-K4 histone H3 upon Stat3 inhibition. As a whole, our findings indicate that Stat3 inhibition in breast cancer cells induce an increase in immunogenicity capable of eliciting an anti tumor immune response, presumably through the activation of a senescence program. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1549. doi:1538-7445.AM2012-1549

Abstract 2285: Stat3 modulates heregulin (HRG)-induced progesterone receptor (PR) transcriptional activation

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The signal transducer and activator of transcription (Stat) family of proteins was found to be involved in crosstalks with both steroid hormones and type I receptor tyrosine kinases (RTKs) signaling pathways. We have previously demonstrated that HRG, a ligand of RTKs, transactivates PR both in C4HD cells from an experimental model of hormonal carcinogenesis in which MPA induced mammary adenocarcinomas in Balb/c mice, and in the human breast cancer cell line T47D (Mol Cell Biol. 2003 Feb;23(3):1095-111). We have also shown that HRG induces Stat3 tyrosine phosphorylation and transcriptional activation by a mechanism that requires PR signaling (Mol Cell Biol. 2009 Mar;29(5):1249-65). In the present work we explored whether Stat3 acting as a coactivator, could modulate ligand-independent activation of PR by HRG. Assessment of the expression of the progestin-regulated gene bcl-X showed that HRG treatment of C4HD cells resulted in an increase in bcl-X protein levels. This effect was completely abolished when Stat3 expression was silenced using siRNAs. HRG treatment of cells transfected with a luciferase reporter plasmid under the control of the murine bcl-X promoter which contains two progesterone response elements (PREs), resulted in an increase in luciferase activity. HRG had no effect on PR transcriptional activation when cells were transfected with a mutant vector containing a deletion spanning both PREs in bcl-X promoter. We assessed the specific association of Stat3 and PR to the PRE region of bcl-X promoter in the context of living cells by performing Chromatin Immunoprecipitation (chIP) Assays. We found that HRG treatment of primary cultures of C4HD cells induced PR and Stat3 recruitment to the bcl-X promoter. HRG also induced PR and Stat3 occupancy of the stably integrated MMTV promoter in T47D-Cat0 breast cancer cells. By performing sequential chIP assays we showed that HRG induced simultaneous PR and Stat3 occupancy of the bcl-X promoter region. We explored Stat3 role in the non-classical mechanism of action of PR, where PR is recruited to p21 promoter indirectly through interaction with Sp1 transcription factor. Interestingly, when cells were treated with HRG, we detected Stat3 binding to the Sp1 binding sites of the PR-regulated p21 promoter, together with Sp1 and PR. These results provide the first evidence that Stat3 modulates ligand-independent activation of PR by HRG in breast cancer 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 2285. doi:10.1158/1538-7445.AM2011-2285

Abstract 2281: GATA3 and progestin interaction in mammary cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL GATA3 is a Master Transcription Factor crucial in mammary gland development and differentiation. Due to the role of progesterone in mammary gland development and in the etiology and progression of breast cancer, we explored the regulation of GATA3 by the synthetic progestin medroxyprogesterone acetate (MPA) in breast cancer cells. The experiments were performed in C4HD cells from an experimental model of hormonal carcinogenesis in which MPA induced mammary adenocarcinomas in Balb/c mice, and in the human breast cancer cell line T47D. Our results indicate that MPA produces GATA3 downregulation, as measured by Western Blot (WB) experiments, and this effect is abolished by the progestin antagonist RU486. In order to assess MPA regulation of GATA3 mRNA levels we performed Real Time PCR, and observed that MPA induced GATA3 transcriptional downregulation. By performing in silico analysis, we detected that GATA3 aminoacidic sequence contains a PEST sequence, recognized as a site for protein degradation. This finding lead us to explore whether MPA affects GATA3 post-translational regulation. For that purpose we treated T47D cells with cicloheximide, and a reduction of GATA3 half-life was observed with MPA treatment. To evaluate whether the B isoform of Progesterone Receptor (PR-B) was sufficient to maintain GATA3 downregulation, we used T47D – YB cell line, which expresses only PR-B. As assessed by WB analysis, PR-B proved to be sufficient for MPA-induced GATA3 downregulation. Remarkably, MPA treatment of T47D-Y- DBD cells, which express a mutant form of PR that is unable to bind to DNA or to tether with other transcription factors, was able to cause GATA3 downregulation. This result suggests that GATA3 downregulation occurs via PR citoplasmatic signaling. In order to dissect the signaling pathway involved in MPA regulation of GATA3 expression, cells were incubated with U0126 + MPA. We found that the addition of U0126 abrogated GATA3 dowregulation by MPA. This result suggests that ERK 1/2 phosphorylation is necessary to maintain this regulation. However, when Src phosphorylation was prevented, together with Src downstream target ERK 1/2 phosphorylation, GATA3 downregulation persisted. This result points to an off-target effect of U0126 inhibitor, probably preventing ERK 5 phosphorylation, as it was previously reported. Finally, we performed in silico analysis of cyclin D1 gene proximal promoter and found three potential GATA3 binding sites. To assess the effect of GATA3 on cyclin D1 expression, we overexpressed human GATA3 in T47D cells, and measured cyclin D1 levels by WB. Overexpression of GATA3 resulted in lower cyclin D1 levels. This study shows for the first time GATA3 downregulation by MPA in breast cancer cells, both at transcriptional and post-translational levels. It also sheds light over the PR mechanism involved in this process and the signaling pathway potentially implicated. Finally, we demonstrate that GATA3 overexpression affects cyclin D1 levels. 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 2281. doi:10.1158/1538-7445.AM2011-2281

Abstract 1718: ErbB-2 acts as coactivator of Stat3 in heregulin-induced breast cancer growth

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We have previously demonstrated that heregulin (HRG), a ligand for ErbB receptors, activates signal transducer and activator of transcription 3 (Stat3) in primary cultures of ErbB-2 overexpressing C4HD cells, from a murine progestin-dependent mammary tumor. ErbB-2 activity is an absolute requirement in the mechanisms of HRG stimulation of Stat3 activity. Recent findings have demonstrated ErbB-2 nuclear migration and its function as a transcription factor. In this work, we studied whether HRG induces ErbB-2 nuclear migration and its interaction with Stat3 in breast cancer cells. We demonstrated that HRG induces ErbB-2 nuclear migration and colocalization with Stat3 in C4HD and human T47D breast cancer cells. Cyclin D1 is a cancer-related gene that contains Stat3 binding sites (GAS sites) but lacks ErbB-2 response elements (HAS sites). By chromatin immunoprecipitation assays (ChIP) and sequential ChIP, we demonstrated that HRG induces in vivo binding of Stat3 and ErbB-2 to the GAS sites of the cyclin D1 promoter. This finding prompted us to evaluate the ability of HRG to regulate cyclin D1 expression. By inhibiting ErbB-2 and Stat3 activation or silencing their expression, we demonstrated that ErbB-2 and Stat3 participate in the mechanism of HRG-induced cyclin D1 protein expression. Next, we explored whether HRG induces cyclin D1 promoter directly via Stat3 binding to its response elements. C4HD and T47D cells transiently transfected with a cyclin D1 promoter luciferase construct showed an enhanced transcriptional activity with HRG treatment. Overexpression of increasing amounts of ErbB-2 wt resulted in a dose-dependent ErbB-2 capacity to enhance Stat3 transcriptional activity induced by HRG. On the other hand, transfection with increasing amounts of an ErbB-2 mutant that is defective in nuclear entry but retains its cell-surface location and functions (ErbB-2ΔNLS) resulted in abrogation of HRG-induced Stat3 activation of cyclin D1 promoter. Finally, we addressed the effect of targeting ErbB-2 in in vivo HRG-dependent growth of C4HD breast tumors. Transfection of C4HD cells with the ErbB-2ΔNLS expression vector significantly inhibited these cells’ ability to form tumors in syngeneic mice. Taken together, these results suggest a new role of ErbB-2 as a coactivator in the mechanism of HRG-induced transcriptional activation of Stat3. We also found nuclear ErbB-2 to be a requisite for HRG stimulation of in vitro and in vivo breast cancer growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1718.