Patricia V. Elizalde

Activation of ErbB-2 via a hierarchical interaction between ErbB-2 and type I insulin-like growth factor receptor in mammary tumor cells.

The present study focused on interactions between signaling pathways activated by progestins and by type I and II receptor tyrosine kinases (RTKs) in mammary tumors. An experimental model in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in Balb/c mice was used. MPA-stimulated proliferation, both in vivo and in vitro, of progestin-dependent tumors induced up-regulation of ErbB-2 protein levels and tyrosine phosphorylation of this receptor. Combinations of antisense oligodeoxynucleotides (ASODNs) directed to ErbB-2 mRNA with ASODNs directed to the insulin-like growth factor-I receptor (IGF-IR) were used to study the effect of the simultaneous block of these receptors on the MPA-induced proliferation of epithelial cells from the progestin-dependent C4HD line. Neither synergistic nor additive effects on the inhibition of MPA-induced proliferation of C4HD cells were observed as a result of the combination of these ASODNs. Suppression of IGF-IR expression by ASODNs resulted in complete abrogation of MPA-induced phosphorylation of ErbB-2 in C4HD cells, whereas blockage of ErbB-2 did not affect IGF-IR phosphorylation. These results show the existence of a hierarchical interaction between IGF-IR and ErbB-2, by means of which IGF-IR directs ErbB-2 phosphorylation. We demonstrated, for the first time, that this hierarchical interaction involves physical association of both receptors, resulting in the formation of a heteromeric complex. Furthermore, confocal laser microscopy experiments demonstrated that MPA was able to induce co-localization of ErbB-2 and IGF-IR. This hetero-oligomer was also found in MCF-7 human breast cancer cells in which association of IGF-IR and ErbB-2 was induced by heregulin and IGF-I.

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.

Varying patterns of expression of insulin-like growth factors I and II and their receptors in murine mammary adenocarcinomas of different metastasizing ability.

We studied the expression of insulin‐like growth factors I (IGF‐I) and II (IGF‐II) and their receptors (IGF‐R) in 2 related murine mammary adenocarcinoma in vivo lines, M3 and MM3, with different metastasizing ability. We further investigated the effects of IGFs on the secretion of a key enzyme in the metastatic cascade, the urokinase‐type plasminogen activator (uPA) in M3 and MM3 cells. M3 is a spontaneous mammary tumor originated in BALB/c mice, with a 40% incidence of lung metastases. MM3 variant, obtained by successive s.c. implants of M3 lung metastases into syngeneic mice, shows a 95% incidence of lung metastases. Similar levels of expression of IGF‐I protein were found in M3 and MM3 tumors, whereas IGF‐II expression was 4‐fold higher in MM3. RNAse protection assays showed similar levels of IGF‐I mRNA in M3 and MM3 tumors and revealed a 4‐fold increase in IGF‐II transcripts in MM3 tumors compared with M3. Authentic IGF‐I and II messages were also found in primary cultures of M3 and MM3 cells. IGF‐I mRNA levels were similar in both cultures and, as described for solid tumors a 5‐fold increase in IGF‐II message was detected in MM3 cells. The presence of type I and mannose‐6‐phosphate (Man‐6P)/type II IGF‐R was demonstrated in both M3 and MM3 tumors. A 2‐fold increase of type I IGF‐R was detected in MM3 tumors compared with M3. Man‐6P/type II IGF‐R levels were 2‐fold lower in MM3 tumors than in M3. As observed in tumor membranes, type I IGF‐R concentrations were higher and Man‐6P/type II IGF‐R lower in cultures of MM3 epithelial cells compared with MM3 cells. In addition, we found that IGF‐I enhanced secreted uPA activity in both M3 and MM3 cells while IGF‐II only stimulated uPA secretion in MM3 cells.

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.

Heregulin inhibits proliferation via ERKs and phosphatidyl-inositol 3-kinase activation but regulates urokinase plasminogen activator independently of these pathways in metastatic mammary tumor cells.

Heregulin (HRG) and type I receptor tyrosine kinase (RTK) expression was investigated in the highly invasive and metastatic LM3 cell line, our previously described model of metastasis for mammary cancer (Bal de Kier Joffe et al. [1986] Invasion Metastasis 6:302–12; Urtreger et al. [1997] Int J Oncol 11:489–96). Although LM3 cells do not express HRG, they exhibit high levels of ErbB‐2 and ErbB‐3 as well as moderate expression of ErbB‐4. Addition of exogenous HRGβ1 resulted in inhibition of both proliferation and migration of LM3 cells. HRGβ1 was also able to decrease the activity of urokinase‐type plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP‐9), 2 key enzymes in the invasion and metastatic cascade. HRGβ1 treatment of LM3 cells induced tyrosine phosphorylation of ErbB‐2, ErbB‐3 and ErbB‐4 as well as the formation of ErbB‐2/ErbB‐3 and ErbB‐2/ErbB‐4 heterodimers. Assessment of the signaling pathways involved in HRGβ1 action indicated that the addition of HRGβ1 to LM3 cells resulted in activation of phosphatidylinositol 3‐ kinase (PI‐3K) and in strong induction of the association of the p85 subunit of PI‐3K with ErbB‐3. HRGβ1 also caused the rapid activation of ERK1/ERK2 and Stat3 and Stat5 (signal transducers and activators of transcription [STAT]). This is the first demonstration of the ability of HRGβ1 to activate STATs in mammary tumor cells. Blockage of PI‐3K activity with its chemical inhibitor wortmannin, or of MEK1/ERKs activity with PD98059, resulted in suppression of the ability of HRGβ1 to inhibit LM3 cell growth. Notwithstanding the suppression of these 2 signaling pathways, HRGβ1 still proved capable of inhibiting uPA activity. Therefore, our results provide evidence that signaling pathways involved in HRGβ1‐induced proliferation appear to be distinct from those involved in HRGβ1 regulation of uPA, a protease that plays a pivotal role in invasion and metastasis.

Small Interfering RNA Targeted to IGF-IR Delays Tumor Growth and Induces Proinflammatory Cytokines in a Mouse Breast Cancer Model

Insulin-like growth factor I (IGF-I) and its type I receptor (IGF-IR) play significant roles in tumorigenesis and in immune response. Here, we wanted to know whether an RNA interference approach targeted to IGF-IR could be used for specific antitumor immunostimulation in a breast cancer model. For that, we evaluated short interfering RNA (siRNAs) for inhibition of in vivo tumor growth and immunological stimulation in immunocompetent mice. We designed 2′-O-methyl-modified siRNAs to inhibit expression of IGF-IR in two murine breast cancer cell lines (EMT6, C4HD). Cell transfection of IGF-IR siRNAs decreased proliferation, diminished phosphorylation of downstream signaling pathway proteins, AKT and ERK, and caused a G0/G1 cell cycle block. The IGF-IR silencing also induced secretion of two proinflammatory cytokines, TNF- α and IFN-γ. When we transfected C4HD cells with siRNAs targeting IGF-IR, mammary tumor growth was strongly delayed in syngenic mice. Histology of developing tumors in mice grafted with IGF-IR siRNA treated C4HD cells revealed a low mitotic index, and infiltration of lymphocytes and polymorphonuclear neutrophils, suggesting activation of an antitumor immune response. When we used C4HD cells treated with siRNA as an immunogen, we observed an increase in delayed-type hypersensitivity and the presence of cytotoxic splenocytes against wild-type C4HD cells, indicative of evolving immune response. Our findings show that silencing IGF-IR using synthetic siRNA bearing 2′-O-methyl nucleotides may offer a new clinical approach for treatment of mammary tumors expressing IGF-IR. Interestingly, our work also suggests that crosstalk between IGF-I axis and antitumor immune response can mobilize proinflammatory cytokines.

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.