ERBB2 Promoter Research Articles

Elevated expression of wildtype RhoC promotes ErbB2- and Pik3ca-induced mammary tumor formation

Copy number gains in genes coding for Rho activating exchange factors as well as losses affecting genes coding for RhoGAP proteins are common in breast cancer (BC), suggesting that elevated Rho signaling may play an important role. Extra copies and overexpression of RHOC also occur, although a role for RhoC overexpression in driving tumor formation has not been assessed in vivo. To this end, we report on the development of a Rosa26 (R26)-targeted Cre-conditional RhoC overexpression mouse (R26RhoC). This mouse was crossed to two models for ERBB2/NEU+ breast cancer: one based on expression of an oncogenic ErbB2/Neu cDNA downstream of the endogenous ErbB2 promoter (FloxNeoNeuNT), the other, a metastatic model that is based on high-level expression from MMTV regulatory elements (NIC). RhoC overexpression dramatically enhanced mammary tumor formation in FloxNeoNeuNT mice but showed a more subtle effect in the NIC line, which forms multiple mammary tumors after a very short latency. RhoC overexpression also enhanced mammary tumor formation in an activated Pik3ca model for breast cancer (Pik3caH1047R). The transforming effect of RhoC was associated with epithelial/mesenchymal transition (EMT) in ErbB2/NeuNT and Pik3caH1047R systems. Thus, our study reveals the importance of elevated wildtype Rho protein expression as a driver of breast tumor formation and highlights the significance of Copy Number Abberations that affect Rho signalling.

ERBB2 promoter demethylation and immune cell infiltration promote a poor prognosis for cancer patients.

BackgroundReceptor tyrosine-protein kinase erbB-2 (ERBB2) expression is a critical factor for the prognosis of various cancers. ERBB2 enrichment indicates a poor prognosis in some cancer types but could be a favorable prognostic factor in others.MethodsWe analyzed DNA methylation, mRNA, protein, immune cell infiltration, and related signaling pathways using TIMER2.0, GEPIA2, STRING, and UALCAN portal datasets in tumor tissues of diverse cancer types and their matched normal tissues.ResultsERBB2 promoter demethylation increases transcript protein amplification and promotes a poor prognosis for cancer patients. ERBB2 gain-of-function procures immune cell infiltration for tumor growth and drives away T regulatory cells, which suppress or downregulate induction and proliferation of effector T cells. The downstream signaling pathways, such as tumor proliferation, ECM-related genes, and degradation of ECM, are involved in ERBB2 gene demethylation and immune activation in cancer progression.ConclusionERBB2 gene demethylation leads to a poor prognosis in cancer patients, which is strongly influenced by the composition and abundance of tumor immune cell infiltration. ERBB2 demethylation could be used in clinical practice to identify immune profiles and direct therapeutic strategies.

FOXP3 Is an X-Linked Breast Cancer Suppressor Gene and an Important Repressor of the HER-2/ErbB2 Oncogene

(Cell 129, 1275–1286; June 29, 2007) We, the editors of Cell, were contacted by the authors of this article regarding a duplication in Figure 7 that was not identified in the previous 2008 correction. Dr. Yang Liu, the corresponding author, has determined that the error was introduced during revision of the manuscript. In preparing the final figure, three images from the experiment showing that Foxp3 expression inhibits the growth of drug-resistant clones in breast cancer cell lines shown in Figure 7A—SKBR3 cells transfected with Otc cDNA, SKBR3 cells with vector, and MCF-7 cells with Foxp3—were mistakenly inserted into Figure 7D showing that ectopic expression of ErbB2 abrogated Foxp3-mediated repression. These images were incorrectly presented as TSA cells transfected with ErbB2 + pEF1 vector, ErbB2 + Foxp3, and pcDNA6 vector + Foxp3, respectively. The authors no longer have access to the original data, and consequently, a correction is not possible. Given the age of the paper and that the duplication does not compromise the conclusions of the paper, based on the information available to us at this time, we believe that no further action is warranted. FOXP3 Is an X-Linked Breast Cancer Suppressor Gene and an Important Repressor of the HER-2/ErbB2 OncogeneZuo et al.CellJune 29, 2007In BriefThe X-linked Foxp3 is a member of the forkhead/winged helix transcription factor family. Germline mutations cause lethal autoimmune diseases in males. Serendipitously, we observed that female mice heterozygous for the “scurfin” mutation of the Foxp3 gene (Foxp3sf/+) developed cancer at a high rate. The majority of the cancers were mammary carcinomas in which the wild-type Foxp3 allele was inactivated and HER-2/ErbB2 was overexpressed. Foxp3 bound and repressed the HER-2/ErbB2 promoter. Deletion, functionally significant somatic mutations, and downregulation of the FOXP3 gene were commonly found in human breast cancer samples and correlated significantly with HER-2/ErbB2 overexpression, regardless of the status of HER-2 amplification. Full-Text PDF Open Archive

IGF2BP2 promotes the progression of colorectal cancer through a YAP-dependent mechanism.

To explore the effect of insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2) on colorectal cancer (CRC) by recognizing the m6A modification of YAP mRNA thus activating ErbB2 expression. High expressions of IGF2BP2, YAP, and ErbB2 promoted the proliferation, migration and invasion of CRC cells and reduced their apoptosis. IGF2BP2 recognized the m6A on YAP mRNA and promoted the translation of mRNA. YAP regulated ErbB2 expression by promoting TEAD4 enrichment in ErbB2 promoter region. Therefore, IGF2BP2 promoted the expression of ErbB2 to enhance the proliferation, invasion and migration of CRC cells, to repress cell apoptosis, and to promote solid tumor formation in nude mice. IGF2BP2 activates the expression of ErbB2 by recognizing the m6A of YAP, thus affecting the cell cycle of CRC, inhibiting cell apoptosis, and promoting proliferation.

YAP transcriptionally regulates ErbB2 to promote liver cell proliferation

The Hippo signaling pathway is implicated in regulation of liver size and dysregulation of this pathway contributes to tumorigenesis. The transcriptional targets and downstream pathways of the Hippo pathway effector YAP that contribute to liver growth have yet to be well-characterized. We examined the liver transcriptome in response to YAP overexpression and identify the ErbB signaling pathway as a mediator of cell growth downstream of YAP. ErbB2 is transcriptionally regulated by YAP in both the mouse liver and in HepG2 human hepatoma cells. Knockdown of YAP or pharmacological inhibition with verteporfin reduced ERBB2 levels in HepG2 cells. Analysis of ChIP-seq data revealed enrichment of the transcription factor TEAD4 at the ERBB2 promoter. Using luciferase reporter and chromatin immunoprecipitation assays, we show that YAP and TEAD4 directly bind to and activate a regulatory element in the ErbB2 promoter in both the mouse liver and HepG2 cells. Functionally, knockdown of YAP reduced EGF-induced ERBB2-mediated HepG2 cell proliferation and PI3K/AKT activation. Our findings highlight a mechanism by which YAP exerts its effects on liver cell proliferation through the ErbB signaling pathway by directly regulating the transcription of ErbB2.

A genome-inspired, reverse selection approach to aptamer discovery

Limitations of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and related methods that depend upon combinatorial oligonucleotide libraries have hindered progress in this area. Our laboratory has introduced a new approach to aptamer discovery that uses oligonucleotides with sequences drawn from the human genome to capture proteins from biological samples. Specifically, we have focused on capture of proteins in nuclear extracts from human cell lines using G-quadruplex (G4) forming genomic sequences. Previous studies identified capture of several proteins both in vitro and in live cells by the Pu28-mer sequence from the ERBB2 promoter region. Here we provide a more comprehensive study of protein capture from BT474 and MCF7 human breast cancer cells using G4-forming sequences from the CMYC, RB, VEGF and ERBB2 human oncogene promoter regions. Mass spectrometric analysis and Western blot analysis of protein capture at oligonucleotide-modified surfaces revealed capture of nucleolin by all three of the oligonucleotides in BT474 and MCF7 cells, and also of ribosomal protein L19 (RPL19) in BT474 cells. Chromatin immunoprecipitation (ChIP) analysis confirmed the interaction of nucleolin with all three promoter sequences in MCF7 cells and with RB in BT474 cells. ChIP also revealed interactions of RPL19 with CMYC in BT474 cells and of both RPL19 and ribosomal protein L14 (RPL14) with ERBB2 in BT474 cells. These results offer the basis for development of new aptamers based on the G4 sequences from the CMYC, RB, VEGF, and ERBB2 promoters toward proteins including nucleolin, RPL19 and RPL14. These interactions also may have biological and therapeutic significance.

Breast Cancer Resistance to Antiestrogens Is Enhanced by Increased ER Degradation and ERBB2 Expression.

Endocrine therapies effectively improve the outcomes of patients with estrogen receptor (ER)-positive breast cancer. However, the emergence of drug-resistant tumors creates a core clinical challenge. In breast cancer cells rendered resistant to the antiestrogen fulvestrant, we defined causative mechanistic roles for the transcription factor YBX1 and the levels of ER and the ERBB2 receptor. Enforced expression of YBX1 in parental cells conferred resistance against tamoxifen and fulvestrant in vitro and in vivo Furthermore, YBX1 overexpression was associated with decreased and increased levels of ER and ERBB2 expression, respectively. In antiestrogen-resistant cells, increased YBX1 phosphorylation was associated with a 4-fold higher degradation rate of ER. Notably, YBX1 bound the ER, leading to its accelerated proteasomal degradation, and induced the transcriptional activation of ERBB2. In parallel fashion, tamoxifen treatment also augmented YBX1 binding to the ERBB2 promoter to induce increased ERBB2 expression. Together, these findings define a mechanism of drug resistance through which YBX1 contributes to antiestrogen bypass in breast cancer cells. Cancer Res; 77(2); 545-56. ©2016 AACR.

Abstract 2265: Stat3 and ErbB-2 interaction in breast cancer metastasis

Abstract Metastasis is a complex multistep process, responsible for as much as 90% of cancer-related deaths, yet obtaining successful treatment for these patients remains an elusive challenge. It has long been recognized that the tyrosine kinase receptor ErbB-2 and the signal transducer and activator of transcription 3 (Stat3), two major players in the breast cancer (BC) scenario, are involved in BC metastatic dissemination through a mechanism where Stat3 acts as a downstream effector of ErbB-2 action. In addition, we and others have also disclosed the role of nuclear ErbB-2 (NErbB-2) in BC, whose presence we identified as a poor prognostic factor in MErbB-2-positive tumors. On the other hand, microRNAs are short non-coding endogenous RNAs with regulatory functions. In particular, high levels of microRNA-21 (miR-21), a well-known oncomiR, have been reported to actively promote invasion and metastasis in BC cell lines and tissues. Here, we describe a novel hierarchical interaction between Stat3, ErbB-2 and miR-21, underlying the metastatic phenotype of ErbB-2-positive BC. We disclosed that Stat3 acts as an upstream regulator of ErbB-2 expression and function. In a panel of cell lines corresponding to different BC subtypes we found that Stat3 induced ErbB-2 expression at the transcriptional level through its recruitment to response elements (called GAS) at the ErbB-2 promoter. Furthermore, we demonstrated that Stat3 co-opted NErbB-2 function, recruiting it as a coactivator, to assemble a transcriptional complex at the GAS sites of the miR-21 promoter, leading to miR-21 up-regulation. We showed that the increase in miR-21 levels resulted in the downregulation of the metastasis suppressor protein PDCD4, a well known miR-21 target. In order to assess the physiological relevance of our molecular findings, we developed an in vivo model of ErbB-2-overexpressing metastatic BC, in which Stat3 activation was inhibited by transfection with a Stat3 dominant negative variant (Stat3Y705F) or its expression was silenced by siRNAs. We demonstrated through reconstitution assays that ErbB-2 and miR-21 were necessary downstream mediators of Stat3-induced metastases development. Furthermore, we explored the clinical significance of our findings in a cohort of ErbB-2-positive primary invasive BC patients and demonstrated that Stat3 and ErbB-2 nuclear co-expression was associated with low PDCD4 expression levels, and that this correlated with the presence of nodal metastases. Our present results in experimental models and in the clinic shed light on the molecular mechanisms underlying BC metastasis and highlight targeting either Stat3 or NErbB-2 as novel therapeutic strategies for ErbB-2-positive BC patients. #: 15-A-2849-AACR Citation Format: Leandro Venturutti, Lucía V. Romero, Alejandro J. Urtreger, María F. Chervo, María F. Mercogliano, Rosalía I. Cordo Russo, Matías G. Pereyra, Gloria Inurrigarro, María C. Díaz Flaqué, Victoria Sunblad, Juan C. Roa, Pablo Guzmán, Elisa Bal de Kier-Joffe, Eduardo H. Charreau, Roxana Schillaci, Patricia V. Elizalde. Stat3 and ErbB-2 interaction in breast cancer metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2265. doi:10.1158/1538-7445.AM2015-2265

Stat3 regulates ErbB-2 expression and co-opts ErbB-2 nuclear function to induce miR-21 expression, PDCD4 downregulation and breast cancer metastasis.

Membrane overexpression of the receptor tyrosine kinase ErbB-2 (MErbB-2) accounts for a clinically aggressive breast cancer (BC) subtype (ErbB-2-positive) with increased incidence of metastases. We and others demonstrated that nuclear ErbB-2 (NErbB-2) also plays a key role in BC and is a poor prognostic factor in ErbB-2-positive tumors. The signal transducer and activator of transcription 3 (Stat3), another player in BC, has been recognized as a downstream mediator of MErbB-2 action in BC metastasis. Here, we revealed an unanticipated novel direction of the ErbB-2 and Stat3 interaction underlying BC metastasis. We found that Stat3 binds to its response elements (GAS) at the ErbB-2 promoter to upregulate ErbB-2 transcription in metastatic, ErbB-2-positive BC. We validated these results in several BC subtypes displaying metastatic and non-metastatic ability, highlighting Stat3 general role as upstream regulator of ErbB-2 expression in BC. Moreover, we showed that Stat3 co-opts NErbB-2 function by recruiting ErbB-2 as its coactivator at the GAS sites in the promoter of microRNA-21 (miR-21), a metastasis-promoting microRNA (miRNA). Using an ErbB-2 nuclear localization domain mutant and a constitutively activated ErbB-2 variant, we found that NErbB-2 role as a Stat3 coactivator and also its direct role as transcription factor upregulate miR-21 in BC. This reveals a novel function of NErbB-2 as a regulator of miRNAs expression. Increased levels of miR-21, in turn, downregulate the expression of the metastasis-suppressor protein programmed cell death 4 (PDCD4), a validated miR-21 target. Using an in vivo model of metastatic ErbB-2-postive BC, in which we silenced Stat3 and reconstituted ErbB-2 or miR-21 expression, we showed that both are downstream mediators of Stat3-driven metastasis. Supporting the clinical relevance of our results, we found an inverse correlation between ErbB-2/Stat3 nuclear co-expression and PDCD4 expression in ErbB-2-positive primary invasive BCs. Our findings identify Stat3 and NErbB-2 as novel therapeutic targets to inhibit ErbB-2-positive BC metastasis.

Chromatin Modifications Sequentially Enhance ErbB2 Expression in ErbB2-Positive Breast Cancers

ErbB2 gene amplification occurs in 20%-25% of breast cancers, and its therapeutic targeting has markedly improved survival of patients with breast cancer in the adjuvant setting. However, resistance to these therapies can develop. Because epigenetic mechanisms can importantly influence oncogene expression and be druggable as well, we investigated histone modifications that influence ErbB2 overexpression, independent of gene amplification. We demonstrate here that ErbB2-overexpressing breast carcinomas acquire the H3K4me3 mark on the erbB2 promoter and that receptor-amplified tumors further acquire the H3K9ac mark, which is dependent on H3K4me3 mark acquisition. Targeting WD repeat domain 5 (Wdr5), which is absolutely required for H3K4me3 enrichment, decreased ErbB2 overexpression, associated with a decrease in the H3K4me3 mark on the erbB2 promoter. Of note, Wdr5 silencing cooperated with trastuzumab or chemotherapy in specifically inhibiting the growth of ErbB2-positive breast tumor cells. Thus, our studies illuminate epigenetic steps in the selection for ErbB2 activation.

Abstract 3643: Fetal exposure to diethylstilbestrol and DNA methylation in adult women.

Abstract In utero diethylstilbestrol (DES) exposure has long term consequences including increased risk of vaginal and breast cancer. Epigenetic factors are suspected to contribute to some of these increased risks and animal studies show DES-mediated effects on DNA methylation in both uterine tissue and blood. However, there are currently no studies examining the effect of fetal DES exposure on DNA methylation in humans. Therefore, we investigated differences in DNA methylation between women exposed to DES in utero compared to unexposed women. All our study subjects were participants in the Sister Study -a prospective cohort focused on environmental and familial risk factors for breast cancer and other diseases. Within this study 1,156 women (2.3%) said that they were definitely or probably exposed to DES in utero. In a special sub study (including both women who reported positive exposure and women who reported no exposure), we confirmed exposure history with the woman's mother and from those with matching mother daughter reports we randomly selected 100 exposed and 100 unexposed women. We analyzed DNA methylation patterns in blood using the Illumina 450k array to assess methylation at 473,844 CpG sites. Primary analysis did not reveal any differentially methylated CpGs (dmCpGs) below the false discovery rate (FDR, q<0.05), nonetheless there were about 8000 dmCpGs with p-values <0.05. Stratification of women into subgroups -based on menopausal status, fertility related diagnoses or hormone intake- did not alter the results. Previous work using mouse models shows gene specific changes in gene expression and DNA methylation following perinatal exposure to DES. When we compared these expression changes in ten genes to changes in promoter methylation in our data we found an inverse correlation between gene expression in mouse and the direction of methylation in our samples. This was especially evident in the ERBB2 promoter region. We also examined the subset of genes known to directly interact with DES: in four of the seven direct DES targets (AR, TTR, AFP, and GLI3) we found a majority of the 5’ CpGs had lower methylation levels in exposed women, although these differences were not statistically significant. This is the first study investigating genome wide methylation changes in women subjected to an unusual and very potent prenatal exposure that is known to have severe consequences into adulthood. Study weaknesses include lack of information regarding dose and exact timing of exposure as well as use of whole blood instead of uterine target tissue, but we still observe changes at p<0.05. If DNA methylation changes are detectable in blood decades after exposure this strengthens the idea that DES exposure leads to epigenetic changes and should be possible to verify even further using samples from uterine or vaginal tissue. Citation Format: S Sophia Harlid, Zongli Xu, Vijayalakshmi Panduri, Aimee D'Aloisio, Lisa DeRoo, Dale Sandler, Jack A. Taylor. Fetal exposure to diethylstilbestrol and DNA methylation in adult women. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3643. doi:10.1158/1538-7445.AM2013-3643

Loss of lkb1 Expression Reduces the Latency of ErbB2-Mediated Mammary Gland Tumorigenesis, Promoting Changes in Metabolic Pathways

The tumor suppressor kinase LKB1 is mutated in a broad range of cancers however, the role of LKB1 mammary gland tumorigenesis is not fully understood. Evaluation of human breast cancer tissue microarrays, indicate that 31% of HER2 positive samples lacked LKB1 expression. To expand on these observations, we crossed STK11fl/fl mice with mice genetically engineered to express activated Neu/HER2-MMTV-Cre (NIC) under the endogenous Erbb2 promoter, to generate STK11−/−/NIC mice. In these mice, the loss of lkb1 expression reduced the latency of ErbB2-mediated tumorigenesis compared to the latency of tumorigenesis in NIC mice alone. Analysis of STK11−/−/NIC mammary tumors revealed hyperactivation of mammalian target of rapamycin (mTOR) through both mTORC1 and mTORC2 pathways as determined by the phosphorylation status of ribosomal protein S6 and AKT. Furthermore, STK11−/−/NIC mammary tumors had elevated ATP levels along with changes in metabolic enzymes and metabolites. The treatment of primary mammary tumor cells with specific mTOR inhibitors AZD8055 and Torin1, that target both mTOR complexes, attenuated mTOR activity and decreased expression of glycolytic enzymes. Our findings underscore the existence of a molecular interplay between LKB1-AMPK-mTORC1 and ErbB2-AKT-mTORC2 pathways with mTOR at its epicenter, suggestive that loss of LKB1 expression may serve as a marker for hyperactivated mTOR in HER2 positive breast cancer and warranting further investigation into therapeutics that target LKB1-AMPK-mTOR and glycolytic pathways.

Negative transcriptional control of ERBB2 gene by MBP-1 and HDAC1: diagnostic implications in breast cancer

BackgroundThe human ERBB2 gene is frequently amplified in breast tumors, and its high expression is associated with poor prognosis. We previously reported a significant inverse correlation between Myc promoter-binding protein-1 (MBP-1) and ERBB2 expression in primary breast invasive ductal carcinoma (IDC). MBP-1 is a transcriptional repressor of the c-MYC gene that acts by binding to the P2 promoter; only one other direct target of MBP-1, the COX2 gene, has been identified so far.MethodsTo gain new insights into the functional relationship linking MBP-1 and ERBB2 in breast cancer, we have investigated the effects of MBP-1 expression on endogenous ERBB2 transcript and protein levels, as well as on transcription promoter activity, by transient-transfection of SKBr3 cells. Reporter gene and chromatin immunoprecipitation assays were used to dissect the ERBB2 promoter and identify functional MBP-1 target sequences. We also investigated the relative expression of MBP-1 and HDAC1 in IDC and normal breast tissues by immunoblot analysis and immunohistochemistry.ResultsTransfection experiments and chromatin immunoprecipitation assays in SKBr3 cells indicated that MBP-1 negatively regulates the ERBB2 gene by binding to a genomic region between nucleotide −514 and −262 of the proximal promoter; consistent with this, a concomitant recruitment of HDAC1 and loss of acetylated histone H4 was observed. In addition, we found high expression of MBP-1 and HDAC1 in normal tissues and a statistically significant inverse correlation with ErbB2 expression in the paired tumor samples.ConclusionsAltogether, our in vitro and in vivo data indicate that the ERBB2 gene is a novel MBP-1 target, and immunohistochemistry analysis of primary tumors suggests that the concomitant high expression of MBP-1 and HDAC1 may be considered a diagnostic marker of cancer progression for breast IDC.

ErbB3-binding protein EBP1 decreases ErbB2 levels via a transcriptional mechanism

Ectopic expression of EBP1, an ErbB3-interacting protein, reduces the expression of the ErbB2 protein and mRNA. However, the mechanism of EBP1-induced decrease in ErbB2 mRNA levels has not yet been determined. Since EBP1 affects both transcriptional and post-transcriptional processes, we evaluated the ability of EBP1 to regulate ErbB2 transcription and RNA stability. We discovered that while wild-type EBP1 decreased the activity of a proximal ErbB2 promoter, EBP1 mutants unable to interact with the Sin3A transcriptional repressor inhibited activity to a lesser extent. EBP1 also decreased the activity of distal ErbB2 promoters. Chromatin immunoprecipitation analysis indicated that EBP1 bound both distal and proximal endogenous ErbB2 promoters in serum-starved conditions. The ErbB3 ligand heregulin (HRG) at growth-promoting concentrations reduced EBP1 binding to the ErbB2 promoter. Although endogenous EBP1 bound ErbB2 mRNA, EBP1 overexpression or ablation of EBP1 protein by shRNA failed to alter ErbB2 mRNA stability. These results suggest that the major effect of EBP1 on ErbB2 mRNA levels is at the transcriptional level.

Capture and identification of proteins that bind to a GGA-rich sequence from the ERBB2 gene promoter region

The ERBB2 gene (HER2/neu) is overexpressed in many human breast cancers. It is an important therapeutic target and its product protein is a key biomarker for breast cancer. A 28-bp GGA repeat sequence (Pu28-mer) in the nuclease hypersensitive site of the ERBB2 promoter region may play an important role in the regulation of ERBB2 transcription, possibly involving the formation of a G-quadruplex. In order to investigate this possibility, an affinity MALDI-MS approach was used for in vitro protein capture from nuclear extracts from cultured MCF-7 and BT-474 cancer cells at Pu28-mer and control oligonucleotide-modified surfaces. Captured proteins from MCF-7 cells were analyzed by LC-MS/MS. Based on these results, Western blot was then used to interrogate captured proteins from both MCF-7 and the Her-2/neu-positive BT-474 cells. Results support the formation of a G-quadruplex by Pu28-mer, indicated by circular dichroism spectroscopy, that selectively captures transcription factors including Ku70, Ku80, PURA, nucleolin, and hnRNP K. Chromatin immunoprecipitation confirmed binding of Ku70, Ku80, PURA, and nucleolin to ERBB2 promoter in the live BT-474 cells. These findings may lead to a better understanding of the role of non-duplex DNA structures in gene regulation and provide a more complete picture of the regulation of ErbB2 expression in breast cancer. The results also provide a blueprint for development of "genome-inspired" aptamers based on the Pu28-mer sequence for in vitro and in vivo detection of proteins related to regulation of ERBB2 gene expression and breast cancer.

Abstract 1063: The ErbB3/4 receptor ligand Heregulin negatively regulates ErbB2 and ErbB3 receptor levels

Abstract ErbB2 is overexpressed in 30% of breast cancers and its overexpression is associated with poor prognosis. In addition, the ErbB2/3 heterodimer forms the most potent mitogenic receptor in vitro and is key to the proliferation of breast cancer cells. Despite the importance of this heterodimer in breast cancer progression, the negative regulation of these ErbB tyrosine kinases is still poorly understood. We demonstrate here for the first time that the ErbB3/4 ligand Heregulin (HRG) reduces mRNA and protein levels of both ErbB2 and ErbB3 in human breast cancer cell lines. We have found that after 24h of HRG treatment, ErbB2 expression decreased at both the RNA and protein levels in ErbB2 overexpressing AU565, BT474 and LTLT-Ca breast cancer cell lines. In contrast, EGFR levels were unaffected by HRG treatment. An initial decline in steady state mRNA levels was first noted at 1 hour after HRG treatment. ErbB3 levels also decreased in LTLT-Ca cells after HRG treatment. Lapatinib, which inhibits ErbB2 and EGFR kinase activity and HRG induced-ErbB3 phosphorylation, diminished the HRG- induced decrease in ErbB2 and ErbB3 in AU565 and LTLT-Ca cells. This finding suggests that the kinase activity of EGFR/ErbB2 may be involved in the HRG-induced down-regulation. HRG did not change the activity of ErbB2 promoter reporter constructs spanning 215 kb to 6007 kb upstream of the ErbB2 transcription start site as indicated by luciferase assays. An ongoing study suggests that HRG could be involved in decreasing ErbB2 mRNA stability. Our findings suggest that resistance to Lapatinib may be due in part to the fact that inhibition of ErbB2/ EGFR kinase activity dampens the negative effect of HRG on ErbB2 expression, resulting in elevated levels of ErbB2 in breast cancer cells. 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 1063. doi:1538-7445.AM2012-1063

Loss of the 14-3-3σ Tumor Suppressor Is a Critical Event in ErbB2-Mediated Tumor Progression

14-3-3σ is a putative tumor suppressor involved in cell-cycle progression and epithelial polarity. We demonstrate that loss of one or both copies of the conditional 14-3-3σ allele results in accelerated mammary and salivary tumorigenesis in mice expressing an activated erbB2 oncogene under the endogenous erbB2 promoter. Significantly, the majority of tumors bearing a single conditional 14-3-3σ allele lose expression of the remaining 14-3-3σ allele, which is associated with epigenetic methylation of the 14-3-3σ locus. In addition to accelerated tumor onset, in a mouse mammary tumor virus-driven ErbB2 tumor model, loss of 14-3-3σ results in enhanced metastatic phenotype that is correlated with loss of cellular junctions. Taken together, these results provide compelling evidence that 14-3-3σ is a potent tumor suppressor involved in ErbB2-driven breast cancer initiation and metastasis. 14-3-3σ has been identified as a normal mammary epithelial cell marker frequently downregulated during neoplastic development. Consistent with its potential role as a tumor suppressor, we demonstrate that targeted disruption of 14-3-3σ in a number of epithelial tissues can profoundly impact both the initiation and metastatic phases of ErbB2-mediated tumor progression through modulation of a number of distinct signaling networks.

A feedback loop between BEX2 and ErbB2 mediated by c‐Jun signaling in breast cancer

BEX2 is a member of brain expressed X-linked gene family that is differentially expressed in primary breast tumors. We have previously demonstrated that BEX2 expression protects breast cancer cells against mitochondrial apoptosis and G1 cell cycle arrest. In addition, we have shown that BEX2 is a c-Jun target gene and, in turn, regulates the phosphorylation of c-Jun in breast cancer cells. In our study, we investigated BEX2 protein expression in a tissue microarray cohort of 225 breast tissue samples with known clinical, pathological and biomarker information. We observed that BEX2 protein was overexpressed in ∼50% of malignant breast tumors compared to only 7% of benign breast samples. Notably, BEX2 positive tumors identified a subset of breast cancers with the overexpression of ErbB2 and phosphorylated c-Jun proteins. Furthermore, using in vitro models, we demonstrated that the mechanism of this association is a functional feedback loop involving ErbB2, c-Jun and BEX2 in breast cancer cells. In this feedback loop, ErbB2 overexpression results in an induction of c-Jun and BEX2 expression. Importantly, ErbB2 induction of BEX2 expression was abrogated by a dominant-negative mutant of c-Jun, suggesting that this effect was mediated through the regulation of c-Jun signaling. In turn, the overexpression of BEX2 led to an increase in both c-Jun-mediated induction of ErbB2 and c-Jun binding to the ErbB2 promoter in MCF-7 cells. Our study suggests that BEX2 protein is overexpressed in approximately half of breast cancers and has a positive feedback loop with ErbB2 mediated by c-Jun signaling in breast cancer cells.

Alternative TFAP2A isoforms have distinct activities in breast cancer

IntroductionAP-2α is a transcription factor implicated in the regulation of differentiation and proliferation in certain tissues, including the mammary gland. In breast tumours, continued expression of AP-2α has been correlated with a better prognosis, but this is hard to reconcile with a reported role in the upregulation of the ERBB2 oncogene. The existence of TFAP2A isoforms, deriving from alternative first exons and differing in their N-terminal sequence, has been described in some mammals, but their relative abundance and activity has not been investigated in the human breast.MethodsExpression levels of four TFAP2A isoforms were assayed at the level of RNA and protein (via the generation of isoform-specific antibodies) in a panel of breast tumour cell lines and in tissue from normal breast and primary tumour samples. Expression constructs for each isoform were used in reporter assays with synthetic and natural promoters (cyclin D3 and ERBB2) to compare the activation and repression activity of the isoforms.ResultsWe demonstrate that the two isoforms AP-2α 1b and AP-2α 1c, in addition to the originally cloned, AP-2α 1a, are conserved throughout evolution in vertebrates. Moreover, we show that isoform 1c in particular is expressed at levels at least on a par with the 1a isoform in breast epithelial lines and tissues and may be more highly expressed in tamoxifen resistant tumours. The isoforms share a similar transactivation mechanism involving the recruitment of the adaptors CITED2 or 4 and the transactivators p300 or CBP. However, isoform 1b and 1c are stronger transactivators of the ERBB2 promoter than isoform 1a. In contrast, AP-2α 1a is the only isoform able to act as a repressor, an activity that requires an intact sumoylation motif present within the N-terminus of the protein, and which the other two isoforms lack.ConclusionsOur findings suggest that TFAP2A isoforms may be differentially regulated during breast tumourigenesis and this, coupled with differences in their transcriptional activity, may impact on tumour responses to tamoxifen therapy. These data also have implications for the interpretation of tumour studies that seek to correlate outcomes with TFAP2A expression level.

The ErbB3 binding protein EBP1 regulates ErbB2 protein levels and tamoxifen sensitivity in breast cancer cells

The ErbB2/3 heterodimer plays a critical role in breast cancer progression and in the development of endocrine resistance. EBP1, an ErbB3 binding protein, inhibits HRG-stimulated breast cancer growth, decreases ErbB2 protein levels and contributes to tamoxifen sensitivity. We report here that ectopic expression of EBP1 in Estrogen Receptor (ER) positive breast cancers that express ErbB2 at both high and low levels decreased ErbB2 protein levels. ErbB2 protein expression was also increased in mammary glands of Ebp1 knock out mice. To define the mechanism of ErbB2 down regulation, we examined the effects of EBP1 on ErbB2 mRNA levels, transcription of the ErbB2 gene and ErbB2 protein stability. We found that ectopic expression of EBP1 decreased steady state levels of endogenous ErbB2 mRNA in all cell lines tested. EBP1 overexpression decreased the activity of an ErbB2 promoter reporter in cells which overxpress ErbB2. However, reporter activity was unchanged or increased in cells which express low endogenous levels of ErbB2. We also found that ectopic expression of EBP1 accelerated ErbB2 protein degradation and enhanced ErbB2 ubiquitination in cells which express both low and high levels of ErbB2. Treatment with proteasome inhibitors prevented this decrease in ErbB2 protein levels. Ablation of EBP1 expression led to tamoxifen resistance that was abrogated by inhibition of ErbB2 activity. These results suggest that EBP1 inhibits expression of ErbB2 protein levels by multiple mechanisms and that EBP1's effects on tamoxifen sensitivity are mediated in part by its ability to modulate ErbB2 levels.