Cecilia J. Proietti

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

ABSTRACT 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 progestin-dependent 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 HRGs 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 HRGs 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.

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.

Inhibition of in vivo breast cancer growth by antisense oligodeoxynucleotides to type I insulin-like growth factor receptor mRNA involves inactivation of ErbBs, PI-3K/Akt and p42/p44 MAPK signaling pathways but not modulation of progesterone receptor activity.

The present study addresses the effect of targeting type I insulin-like growth factor receptor (IGF-IR) with antisense strategies in in vivo growth of breast cancer cells. Our research was carried out on C4HD tumors from an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in Balb/c mice. We employed two different experimental strategies. With the first one we demonstrated that direct intratumor injection of phosphorothioate antisense oligodeoxynucleotides (AS[S]ODNs) to IGF-IR mRNA resulted in a significant inhibition of C4HD tumor growth. In the second experimental strategy, we assessed the effect of intravenous (i.v.) injection of AS [S]ODN on C4HD tumor growth. This systemic treatment also resulted in significant reduction in tumor growth. The antitumor effect of IGF-IR AS[S]ODNs in both experimental protocols was due to a specific antisense mechanism, since growth inhibition was dose-dependent and no abrogation of tumor proliferation was observed in mice treated with phosphorothioate sense ODNs (S[S]ODNs). In addition, IGF-IR expression was inhibited in tumors from mice receiving AS[S]ODNs, as compared to tumors from control groups. We then investigated signal transduction pathways modulated in vivo by AS[S]ODNs treatment. Tumors from AS[S]ODN-treated mice of both intratumoral and intravenous protocols showed a significant decrease in the degree of insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation. Activation of two of the main IGF-IR signaling pathways, phosphatidylinositol 3-kinase (PI-3K)/Akt and p42/p44 mitogen-activated protein kinases (MAPK) was abolished in tumors growing in AS[S]ODN-treated animals. Moreover, ErbB-2 tyrosine phosphorylation was blocked by in vivo administration of AS[S]ODNs. On the other hand, we found no regulation of either progesterone receptor expression or activity by in vivo AS[S]ODNs administration. Our results for the first time demonstrated that breast cancer growth can be inhibited by direct in vivo administration of IGF-IR AS[S]ODNs.

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

ABSTRACT 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, ligand-independent PR function as a signaling molecule. Stat3 activation was an absolute requirement in HRG-induced 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.

Progestin-induced caveolin-1 expression mediates breast cancer cell proliferation.

Progestin regulation of gene expression was assessed in the progestin-dependent murine tumor line C4HD which requires MPA, a synthetic progestin, for in vivo growth and expresses high levels of progesterone receptor (PR). By using suppressive subtractive hybridization, caveolin-1 was identified as a gene whose expression was increased with in vivo MPA treatment. By Northern and Western blot analysis, we further confirmed that caveolin-1 mRNA and protein expression increased in MPA-treated tumors as compared with untreated tumors. When primary cultures of C4HD cells were treated in vitro with MPA, caveolin-1 levels also increased, effect that was abolished by pre-treatment with progestin antagonist RU486. In addition, MPA promoted strong caveolin-1 promoter transcriptional activation both in mouse and human breast cancer cells. We also showed that MPA regulation of caveolin-1 expression involved in activation of two signaling pathways: MAPK and PI-3K. Short-term MPA treatment of C4HD cells led to tyrosine phosphorylation of caveolin-1 protein, where Src was the kinase involved. Additionally, we showed that MPA-induced association of caveolin-1 and PR, which was detected by coimmunoprecipitation and by confocal microscopy. Finally, we proved that MPA-induced proliferation of C4HD cells was inhibited by suppression of caveolin-1 expression with antisense oligodeoxynucleotides to caveolin-1 mRNA. Furthermore, we observed that inhibition of caveolin-1 expression abrogated PR capacity to induced luciferase activity from a progesterone response element-driven reporter plasmid. Comprehensively, our results demonstrated for the first time that caveolin-1 expression is upregulated by progestin in breast cancer. We also demonstrated that caveolin-1 is a downstream effector of MPA that is partially responsible for the stimulation of growth of breast cancer cells.

Transactivation of ErbB-2 induced by tumor necrosis factor α promotes NF-κB activation and breast cancer cell proliferation

Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine which, acting locally, induces tumor growth. Accumulating evidence, including our findings, showed that TNFα is mitogenic in breast cancer cells in vitro and in vivo. In the present study, we explored TNFα involvement on highly aggressive ErbB-2-overexpressing breast cancer cells. We found that TNFα induces ErbB-2 phosphorylation in mouse breast cancer C4HD cells and in the human breast cancer cell lines SK-BR-3 and BT-474. ErbB-2 phosphorylation at Tyr877 residue was mediated by TNFα-induced c-Src activation. Moreover, TNFα promoted ErbB-2/ErbB-3 heterocomplex formation, Akt activation and NF-κB transcriptional activation. Inhibition of ErbB-2 by addition of AG825, an epidermal growth factor receptor/ErbB-2-tyrosine kinase inhibitor, or knockdown of ErbB-2 by RNA interference strategy, blocked TNFα-induced NF-κB activation and proliferation. However, the humanized monoclonal antibody anti-ErbB-2 Herceptin could not inhibit TNFα ability to promote breast cancer growth. Interestingly, our work disclosed that TNFα is able to transactivate ErbB-2 and use it as an obligatory downstream signaling molecule in the generation of mitogenic signals. As TNFα has been shown to be present in the tumor microenvironment of a significant proportion of human infiltrating breast cancers, our findings would have clinical implication in ErbB-2-positive breast cancer treatment.

Targeting Stat3 Induces Senescence in Tumor Cells and Elicits Prophylactic and Therapeutic Immune Responses against Breast Cancer Growth Mediated by NK Cells and CD4+ T Cells

Aberrant Stat3 activation and signaling contribute to malignant transformation by promoting cell cycle progression, inhibiting apoptosis, and mediating tumor immune evasion. Stat3 inhibition in tumor cells induces the expression of chemokines and proinflammatory cytokines, so we proposed to apply Stat3-inhibited breast cancer cells as a source of immunogens to induce an antitumor immune response. Studies were performed in two murine breast cancer models in which Stat3 is activated: progestin-dependent C4HD cells and 4T1 cells. We immunized BALB/c mice with irradiated cancer cells previously transfected with a dominant-negative Stat3 vector (Stat3Y705F) in either a prophylactic or a therapeutic manner. Prophylactic administration of breast cancer cells transfected with Stat3Y705F (Stat3Y705F-breast cancer cells) inhibited primary tumor growth compared with administration of empty vector-transfected cells in both models. In the 4T1 model, 50% of the challenged mice were tumor free, and the incidence of metastasis decreased by 90%. In vivo assays of C4HD tumors showed that the antitumor immune response involves the participation of CD4+ T cells and cytotoxic NK cells. Therapeutic immunization with Stat3Y705F-breast cancer cells inhibited tumor growth, promoted tumor cell differentiation, and decreased metastasis. Furthermore, inhibition of Stat3 activation in breast cancer cells induced cellular senescence, contributing to their immunogenic phenotype. In this work, we provide preclinical proof of concept that ablating Stat3 signaling in breast cancer cells results in an effective immunotherapy against breast cancer growth and metastasis. Moreover, our findings showing that Stat3 inactivation results in induction of a cellular senescence program disclose a potential mechanism for immunotherapy research.

Immunization with Murine Breast Cancer Cells Treated with Antisense Oligodeoxynucleotides to Type I Insulin-Like Growth Factor Receptor Induced an Antitumoral Effect Mediated by a CD8+ Response Involving Fas/Fas Ligand Cytotoxic Pathway

We have demonstrated that in vivo administration of phosphorothioate antisense oligodeoxynucleotides (AS[S]ODNs) to type I insulin-like growth factor receptor (IGF-IR) mRNA resulted in inhibition of C4HD breast cancer growth in BALB/c mice. The present study focused on whether in vivo administration of C4HD tumor cells pretreated with IGF-IR AS[S]ODN and irradiated could provide protection against C4HD wild-type tumor challenge and also on elucidating the mechanism mediating this effect. Our results showed that mice immunized with IGF-IR AS[S]ODN-treated C4HD cells experienced a growth inhibition of 53.4%, 61.6%, and 60.2% when compared with PBS-treated mice, wild-type C4HD cell-injected mice, or phosphorothioate sense oligodeoxynucleotide-treated C4HD cell-injected mice, respectively. The protective effect was C4HD-specific, because no cross-protection was observed against other syngeneic mammary tumor lines. The lack of protection against tumor formation in nude mice indicated that T cells were involved in the antitumoral response. Furthermore, cytotoxicity and splenocyte proliferation assays demonstrated that a cellular CD8+-dependent immune response, acting through the Fas/Fas ligand death pathway, could be mediating the antitumor effect induced by immunization with AS[S]ODN-treated cells. Immunization also induced splenocytes to produce Ag-dependent IFN-γ, indicating the presence of a type 1 response. We demonstrated for the first time that IGF-IR AS[S]ODN treatment of breast cancer cells induced expression of CD86 and heat shock protein 70 molecules, both involved in the induction of the immunogenic phenotype. Immunization with these tumor immunogens imparted protection against parental tumor growth through activation of a specific immune response.

Clinical relevance of ErbB-2/HER2 nuclear expression in breast cancer

BackgroundThe biological relevance of nuclear ErbB-2/HER2 (NuclErbB-2) presence in breast tumors remains unexplored. In this study we assessed the clinical significance of ErbB-2 nuclear localization in primary invasive breast cancer. The reporting recommendations for tumor marker prognostic studies (REMARK) guidelines were used as reference.MethodsTissue microarrays from a cohort of 273 primary invasive breast carcinomas from women living in Chile, a Latin American country, were examined for membrane (MembErbB-2) and NuclErbB-2 expression by an immunofluorescence (IF) protocol we developed. ErbB-2 expression was also evaluated by immunohistochemistry (IHC) with a series of antibodies. Correlation between NuclErbB-2 and MembErbB-2, and between NuclErbB-2 and clinicopathological characteristics of tumors was studied. The prognostic value of NuclErbB-2 in overall survival (OS) was evaluated using Kaplan-Meier method, and Cox model was used to explore NuclErbB-2 as independent prognostic factor for OS.ResultsThe IF protocol we developed showed significantly higher sensitivity for detection of NuclErbB-2 than IHC procedures, while its specificity and sensitivity to detect MembErbB-2 were comparable to those of IHC procedures. We found 33.6% NuclErbB-2 positivity, 14.2% MembErbB-2 overexpression by IF, and 13.0% MembErbB-2 prevalence by IHC in our cohort. We identified NuclErbB-2 positivity as a significant independent predictor of worse OS in patients with MembErbB-2 overexpression. NuclErbB-2 was also a biomarker of lower OS in tumors that overexpress MembErbB-2 and lack steroid hormone receptors.ConclusionsWe revealed a novel role for NuclErbB-2 as an independent prognostic factor of poor clinical outcome in MembErbB-2-positive breast tumors. Our work indicates that patients presenting NuclErbB-2 may need new therapeutic strategies involving specific blockage of ErbB-2 nuclear migration.

Progesterone receptor assembly of a transcriptional complex along with activator protein 1, signal transducer and activator of transcription 3 and ErbB-2 governs breast cancer growth and predicts response to endocrine therapy

IntroductionThe role of the progesterone receptor (PR) in breast cancer remains a major clinical challenge. Although PR induces mammary tumor growth, its presence in breast tumors is a marker of good prognosis. We investigated coordinated PR rapid and nonclassical transcriptional effects governing breast cancer growth and endocrine therapy resistance.MethodsWe used breast cancer cell lines expressing wild-type and mutant PRs, cells sensitive and resistant to endocrine therapy, a variety of molecular and cellular biology approaches, in vitro proliferation studies and preclinical models to explore PR regulation of cyclin D1 expression, tumor growth, and response to endocrine therapy. We investigated the clinical significance of activator protein 1 (AP-1) and PR interaction in a cohort of 99 PR-positive breast tumors by an immunofluorescence protocol we developed. The prognostic value of AP-1/PR nuclear colocalization in overall survival (OS) was evaluated using Kaplan-Meier method, and Cox model was used to explore said colocalization as an independent prognostic factor for OS.ResultsWe demonstrated that at the cyclin D1 promoter and through coordinated rapid and transcriptional effects, progestin induces the assembly of a transcriptional complex among AP-1, Stat3, PR, and ErbB-2 which functions as an enhanceosome to drive breast cancer growth. Our studies in a cohort of human breast tumors identified PR and AP-1 nuclear interaction as a marker of good prognosis and better OS in patients treated with tamoxifen (Tam), an anti-estrogen receptor therapy. Rationale for this finding was provided by our demonstration that Tam inhibits rapid and genomic PR effects, rendering breast cancer cells sensitive to its antiproliferative effects.ConclusionsWe here provided novel insight into the paradox of PR action as well as new tools to identify the subgroup of ER+/PR + patients unlikely to respond to ER-targeted therapies.