ER-negative Breast Carcinoma Research Articles

Novel Stromal Biomarkers in Human Breast Cancer Tissues Provide Evidence for the More Malignant Phenotype of Estrogen Receptor‐Negative Tumors

Research efforts were focused on genetic alterations in epithelial cancer cells. Epithelial-stromal interactions play a crucial role in cancer initiation, progression, invasion, angiogenesis, and metastasis; however, the active role of stroma in human breast tumorigenesis in relation to estrogen receptor (ER) status of epithelial cells has not been explored. Using proteomics and biochemical approaches, we identified two stromal proteins in ER-positive and ER-negative human breast cancer tissues that may affect malignant transformation in breast cancer. Two putative biomarkers, T-cell receptor alpha (TCR-α) and zinc finger and BRCA1-interacting protein with a KRAB domain (ZBRK1), were detected in leukocytes of ER-positive and endothelial cells of ER-negative tissues, respectively. Our data suggest an immunosuppressive role of leukocytes in invasive breast tumors, propose a multifunctional nature of ZBRK1 in estrogen receptor regulation and angiogenesis, and demonstrate the aggressiveness of ER-negative human breast carcinomas. This research project may identify new stromal drug targets for the treatment of breast cancer patients.

Genomic differences between estrogen receptor (ER)‐positive and ER‐negative human breast carcinoma identified by single nucleotide polymorphism array comparative genome hybridization analysis

Estrogen receptor (ER) remains one of the most important biomarkers for breast cancer subtyping and prognosis, and comparative genome hybridization has greatly contributed to the understanding of global genetic imbalance. The authors used single-nucleotide polymorphism (SNP) arrays to compare overall copy number aberrations (CNAs) as well as loss of heterozygosity (LOH) of the entire human genome in ER-positive and ER-negative breast carcinomas. DNA was extracted from frozen tumor sections of 21 breast carcinoma specimens and analyzed with a proprietary 50K XbaI SNP array. Copy number and LOH probability values were derived for each sample. Data were analyzed using bioinformatics and computational software, and permutation tests were used to estimate the significance of these values. There was a global increase in CNAs and LOH in ER-negative relative to ER-positive cancers. Gain of the long arm of chromosome 1 (1q) and 8q were the most obvious changes common in both subtypes: An increase in the chromosome 1 short arm (1p)/1q ratio was observed in ER-negative samples, and an increased 16p/16q ratio was observed in ER-positive samples. Significant CNAs (adjusted P<.05) in ER-negative relative to ER-positive tumors included 5q deletion, loss of 15q, and gain of 2p and 21q. Copy-neutral LOH (cnLOH) common to both ER-positive and ER-negative samples included 9p21, the p16 tumor suppressor locus, and 4q13, the RCHY1 (ring finger and CHY zinc finger domain-containing 1) oncogene locus. Of particular interest was an enrichment of 17q LOH among the ER-negative tumors, potentially suggesting breast cancer 1 gene (BRCA1) mutations. SNP array detected both genetic imbalances and cnLOH and was capable of discriminating ER-negative breast cancer from ER-positive breast cancer.

Abstract 1717: Combinations of retinoids and lapatinib in the treatment of Her2/Neu-positive breast carcinomas with co-amplification of the ERBB2 and RARA genes

Abstract The current study represents proof of principle for the use of all-trans retinoic acid (ATRA) alone or in combination with the tyrosine kinase inhibitor, lapatinib, in the management of a subset of Her2/Neu-positive breast carcinomas. First-line treatment of Her2/Neu-positive breast carcinoma is based on the monoclonal antibody, trastuzumab, while specific tyrosine kinase inhibitors, such as lapatinib, are used as second-line agents. Novel and more effective therapeutic strategies are called for this type of cancer. ATRA and derivatives (retinoids) have been proposed in the chemoprevention and treatment of breast carcinoma. Retinoids activate specific nuclear receptors (RARs and RXRs), which mediate the therapeutic effects of these agents. The beneficial activity of retinoids is limited to ER-positive breast cancer cells, possibly as a consequence of RARα induction by estrogens. A significant fraction of Her2/Neu-positive breast carcinomas are ER-negative, hence this type of cancer is predicted to be refractory to retinoids. Her2/Neu-positivity is very often the result of an amplification of the corresponding gene (ERBB2), which maps to chromosome 17, approximately 650 kilobases upstream of the locus encoding RARα (RARA). Given this background, we evaluated whether a proportion of Her2/Neu-positive and ER-negative breast carcinomas are characterized by co-amplification of ERBB2 and RARA, using real-time PCR on genomic DNA extracted from paraffin sections from 60 patients. We observed that 28% of the cases showed co-amplification of ERBB2 and RARA, while the amplicon did not contain the latter in the remaining fraction of patients. RARA amplification was associated with increased levels of the corresponding RARα protein, as determined by immuno-histochemistry. To evaluate whether RARA co-amplification and consequent RARα over-expression was associated with retinoid sensitivity, we turned to a panel of representative breast carcinoma cell lines. We observed that the growth of two cell lines, SKBR3 and UACC812, characterized by ERBB2 and RARA co-amplification, was inhibited by ATRA. Growth inhibition was accompanied by differentiating and pro-senescence responses to the retinoid. All these phenomena were mediated by RARα, as demonstrated by the use of selective retinoid agonists and by silencing of the receptor by siRNAs. Similar effects were not observed in MDA-MB-453 and HCC1569 cells, which carry a simple ERBB2 amplicon and are totally refractory to the various effects of ATRA. In SKBR3 and UACC812, inhibition of Her2/Neu tyrosine kinase activity by lapatinib, led to a dramatic enhancement of all the retinoid responses, demonstrating strong synergism between the two compounds. Gene expression profiling and mechanistic studies in SKBR3 and UACC812 indicated a two-way cross-talk between Her2/Neu and RARα. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 1717.

Abstract 4878: Bioactive dietary supplements reactivate ER expression in ER-negative breast cancer cells by epigenetic mechanisms involving active chromatin modification

Abstract Breast cancer is the second leading cause of cancer-related death among women in the United States. One of the important classifications of breast tumors is based on the presence or absence of the estrogen receptor (ER). While the majority of breast cancers are ER-positive, approximately 30% are ER-negative. Patients with ER-positive breast cancer receive hormonal therapy using either tamoxifen or aromatase inhibitors, and have a better prognosis. However, treatment of patients with ER-negative tumor is challenging due to the poor response to hormonal therapies in the absence ER expression. Many studies have demonstrated the chemopreventive properties of green tea polyphenols (GTPs) and sulforaphane (SFN) through inhibition of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activity, respectively. In the present study, we have observed that GTPs and SFN dose-dependently inhibit the cellular proliferation in both ER-positive and ER-negative human breast carcinoma MCF-7 and MDA-MB-231 cells, respectively. Treatment of ER-negative breast cancer cells with GTPs and SFN alone or in combination leads to the reactivation of ER mRNA expression. The combination of 20 µg/mL GTPs and 5 µM SFN was found to be the optimal dose of ER reactivation at 3 days in MDA-MB-231 cells. The reactivation of ER expression was consistently correlated with ER promoter hypomethylation and hyperacetylation. Chromatin immunoprecipitation (ChIP) analysis of the ER promoter revealed that GTPs and SFN decreased the level of dimethyl H3K9, but increased the acetylation by increasing the level of acetyl H3, acetyl H4 and acetyl H3K9. In addition, a combination of GTPs and SFN significantly inhibited DNMT and HDAC activity, which might have an important role in the reactivation of ER expression in MDA-MB-231 cells. The combination of GTPs- and SFN-mediated inhibition of DNMT activity and increase of acetylation underscores the importance of DNMTs and HDACs in maintaining the repressive environment at target genes such as ER. In addition, treatment with tamoxifen in combination with GTPs and SFN significantly increased both cell death and inhibition of cellular proliferation in MDA-MB-231 cells in comparison to treatment with tamoxifen alone. Collectively, our findings suggest that HDAC inhibitors combined with demethylating agents are a promising strategy for the epigenetic reactivation of ER expression for the effective treatment of ER-negative breast cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 4878.

Abstract 3291: Upregulation of Insulin Receptor Substrate-2 (IRS-2) expression by hypoxia

Abstract Insulin Receptor Substrate-2 (IRS-2) belongs to the IRS family of cytoplasmic adaptor proteins that mediate signaling from cell surface receptors, including growth factor, cytokine and integrin receptors. Many of these receptors have been implicated in cancer. Although the IRS proteins are highly homologous in structure and have some complementary functions, growing evidence supports that the IRS proteins have unique roles in cancer. IRS-1 has been shown to promote tumor cell proliferation, while IRS-2 has been positively associated with tumor cell motility and invasion. Previously, our lab demonstrated that Polyoma Middle T-derived Irs-2−/− mouse mammary tumors are significantly less metastatic, and Irs-1−/− tumors significantly more metastatic, than their wild-type counterparts. In recent studies, we observed that IRS-2 expression increases in response to hypoxia in both metastatic mouse mammary tumor cells and poorly differentiated ER-negative metastatic human breast carcinoma cells. In contrast, IRS-1 expression does not increase in response to hypoxia, supporting the notion of their non-overlapping functions. Hypoxia promotes the adaptation and resistance of cancer cells to chemo- and radiation therapy, and also promotes tumor cell survival, invasion and metastasis by selecting for aggressive tumor cells that can survive under stressful low oxygen conditions. We have also shown that IRS-2 upregulation in response to hypoxia promotes tumor cell viability and invasion, selecting for tumor cells with increased metastatic potential. To understand how IRS-2 is regulated by hypoxia, we identified a minimal 200 bp region of the IRS-2 promoter located 700 bp upstream from the translation start site that is hypoxia-responsive. Mutation of an E-box binding site within this region that has been previously implicated in regulating IRS-2 in response to hormone stimulation inhibits hypoxic upregulation of IRS-2. We are currently investigating the factors that bind to this E-box to regulate IRS-2 expression. In addition, we have established that HIF is required for IRS-2 upregulation in hypoxic environments, and we are investigating if HIF acts in a direct or indirect manner to regulate IRS-2 expression. Identifying the mechanism by which IRS-2 expression is regulated by hypoxia is essential for developing novel methods to inhibit IRS-2 expression to suppress metastasis. 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 3291.

Abstract B13: TIA1+ and FOXP3+ tumor infiltrating lymphocytes are positive prognostic factors in estrogen receptor negative breast cancer

Abstract Background: Mounting evidence indicates that tumor-specific host immunity is associated with favorable prognosis in a variety of cancer settings. However, the role of immunity in breast cancer remains controversial. The purpose of this study was to determine the significance of tumor infiltrating leukocytes (TIL) with respect to pathological features and patient survival in estrogen receptor alpha (ER) negative breast cancer. Patients and Methods: Tissue microarrays and immunohistochemistry were used to assess and categorize TIL in a cohort of 255 ER-negative invasive ductal breast carcinomas. Cases were registered by the Manitoba Breast Tumor Bank during the years 1988 to 2000 and had a minimum follow-up of 36 months. Stromal and intraepithelial TIL expressing the markers CD3, CD8, CD4, TIA1, CD25, FOXP3, CD20, CD68, and myeloperoxidase (MPO) were quantified by an experienced pathologist (PHW) and evaluated for associations with pathological parameters and survival. The median value for each marker was used as a cut-point to define high versus low cases. Results: TIL expressing CD4, FOXP3, TIA1, CD20, or MPO were significantly associated with improved overall and disease-free survival (DFS). A combination of high TIA1+ and FOXP3+ TIL, termed IR (immune response)-high, was superior to any single TIL marker and was significantly associated with DFS in multivariate analysis involving the following covariates: nodal status, patient age, tumor size, tumor grade, Her2/PR expression, and adjuvant treatment (HR = 0.478, 95% CI 0.246–0.932; P = 0.030). IR associated with DFS principally in patients with axillary lymph node metastasis (P = 0.0019 versus P = 0.2659 for node-negative patients) and in those who did not receive adjuvant radiation or chemotherapy (P &amp;lt; 0.0001 versus P = 0.0489 for adjuvant-treated patients). Similarly, nodal status and adjuvant therapy had prognostic value only for patients with low IR. Indeed, the outcomes of IR-high cases with or without lymph node metastasis were nearly identical (10 year DFS rates of 65% and 69%, respectively; P = 0.6503), as were those of IR-high patients who did or did not receive adjuvant therapy (10 year DFS rates of 68% and 67%, respectively; P = 0.7997). Unlike standard prognostic features such as nodal status (P = 0.7941), IR correlated with DFS at times greater than five years after diagnosis (P = 0.0084). Conclusions: High levels of TIL are indicative of favorable prognosis in ER-negative breast cancer. The prognostic effects of TIL are particularly relevant for node-positive patients and those not treated with adjuvant radiation or chemotherapy. These findings suggest that in ER-negative disease, the survival benefits of adjuvant therapy may be restricted to patients with poor tumor-specific immune responses. Assessment of TIL may improve overall prognostic prediction for ER-negative breast cancer and may be useful in identifying patients who are likely to respond to adjuvant regimens. Furthermore, our data suggest that ER-negative breast cancer may be a strong candidate for the design and deployment of novel immunotherapies. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B13

Distinctive Expression of Cancer/Testis-X Antigens in a Subset of ER Negative Breast Carcinomas.

Abstract Background: Cancer/testis-X antigens are a multigene family that are predominantly expressed in human germ line cells, with little or no expression in somatic adult tissues, but become aberrantly activated in various malignancies. Some such CT-X antigens represent ideal targets for cancer immunotherapy and have already been used in clinical testing. In contrast to melanomas, bladder, lung, ovarian and hepatocellular carcinomas which show higher levels of CT-X antigen expression, the reports in breast cancers have been inconclusive to date and a comprehensive gene expression and clinicopathological analysis has yet to be performed.Material and Methods: Using sequencing data as well as nine publicly available gene expression data sets, we analyzed the expression of Cancer/testis-X antigens in more than 1900 primary breast cancers. Complementary analysis was performed on three tissue microarrays comprising a total of 201 primary breast carcinomas and 53 brain metastases. Clinical information on the ER-, PR-, HER2, Ki67, p53, EGFR and basal markers was available for statistical analysis.Results: A significantly higher expression of Cancer/testis-X antigens was found in ER negative breast carcinomas over different data sets with a concordant gene expression pattern of several Cancer/testis-X antigens. Members of the MAGEA family and NY-ESO-1/CTAG1B were consistently the most prevalent. Immunohistochemical analyses confirmed a significant correlation of MAGEA family and NY-ESO-1/CTAG1B with ER negative (pValue &amp;lt; 0.0001), PR negative (pValue &amp;lt; 0.01) and Ki67 staining (pValue &amp;lt; 0.0001). Many of these tumors were also positive for basal markers.Discussion: Previous studies of Cancer/testis-X antigens in breast have focused on ER positive cancers, in smaller subsets and provided inconclusive results. Using comprehensive gene expression data sets and tissue microarrays, we have demonstrated a significant association of MAGEA family and NY-ESO-1/CTAG1B with ER/PR negative breast cancer. Since these cancers represent a subgroup for which less therapeutic modalities are available, we propose the use of MAGEA and NY-ESO-1/CTAG1B cancer vaccines in the adjuvant setting as an approach to restricting tumor growth and metastases. Clinical trails using MAGEA and NY-ESO-1/CTAG1B are warranted. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3139.

EGFR over-expression and activation in high HER2, ER negative breast cancer cell line induces trastuzumab resistance

HER2 is gene amplified or over-expressed in 20-25% of breast cancers resulting in elevated HER2 activation. Trastuzumab (Herceptin), a humanized monoclonal antibody, targets activated HER2 and is clinically effective in HER2-over-expressing breast cancers. However, despite prolonged survival, treated breast cancer patients develop resistance. Resistance to trastuzumab occurs upon inactivation of HER2 regulatory proteins or upon up-regulation of alternative receptors. In particular, elevated levels of EGFR, present in estrogen receptor (ER) positive, trastuzumab-resistant BT-474 xenografts caused, a trastuzumab-resistant phenotype (Ritter et al. Clin Cancer Res 13:4909-4919, 2007). However, the role of EGFR in acquired trastuzumab resistance in ER negative cell models is not well defined. In this study, SKBR3 cell line clones expressing EGFR were generated to examine the role of EGFR over-expression on trastuzumab sensitivity in an, ER-negative breast carcinoma cell line. A stable clone, SKBR3/EGFR (clone 4) expressing moderate levels of EGFR remained sensitive to trastuzumab, whereas a stable clone, SKBR3/EGFR (clone 5) expressing high levels of EGFR, became resistant to trastuzumab. Depletion of EGFR by EGFR small-interfering RNAs in the SKBR3/EGFR (clone 5) reversed trastuzumab resistance. However, the SKBR3/EGFR (clone 5) cell line remained sensitive to lapatinib, an EGFR/HER2 inhibitor. Biochemical analysis using co-immunoprecipitation and proximity-based quantitative VeraTag assays demonstrated that high levels of EGFR phosphorylation, EGFR/EGFR homo-dimerization, and EGFR/HER2 hetero-dimerization were present in the trastuzumab-resistant cells. We conclude that EGFR over-expression can mediate trastuzumab resistance in both ER positive and ER negative cells and hypothesize that a threshold level of EGFR, in the absence of autocrine ligand production, is required to induce the resistant phenotype.

Rhomboid domain containing 2 ( RHBDD2): A novel cancer-related gene over-expressed in breast cancer

In the course of breast cancer global gene expression studies, we identified an uncharacterized gene known as RHBDD2 ( Rhomboid domain containing 2) to be markedly over-expressed in primary tumors from patients with recurrent disease. In this study, we identified RHBDD2 mRNA and protein expression significantly elevated in breast carcinomas compared with normal breast samples as analyzed by SAGE ( n = 46) and immunohistochemistry ( n = 213). Interestingly, specimens displaying RHBDD2 over-expression were predominantly advanced stage III breast carcinomas ( p = 0.001). Western-blot, RT-PCR and cDNA sequencing analyses allowed us to identify two RHBDD2 alternatively spliced mRNA isoforms expressed in breast cancer cell lines. We further investigated the occurrence and frequency of gene amplification and over-expression affecting RHBDD2 in 131 breast samples. RHBDD2 gene amplification was detected in 21% of 98 invasive breast carcinomas analyzed. However, no RHBDD2 amplification was detected in normal breast tissues ( n = 17) or breast benign lesions ( n = 16) ( p = 0.014). Interestingly, siRNA-mediated silencing of RHBDD2 expression results in a decrease of MCF7 breast cancer cells proliferation compared with the corresponding controls ( p = 0.001). In addition, analysis of publicly available gene expression data showed a strong association between high RHBDD2 expression and decreased overall survival ( p = 0.0023), relapse-free survival ( p = 0.0013), and metastasis-free interval ( p = 0.006) in patients with primary ER-negative breast carcinomas. In conclusion, our findings suggest that RHBDD2 over-expression behaves as an indicator of poor prognosis and may play a role facilitating breast cancer progression.

Nek2 as a novel molecular target for the treatment of breast carcinoma

We investigated the role of Nek2, a member of the serine-threonine kinase family, in the tumorigenic growth of breast carcinoma. Increased expression of Nek2 was observed in all breast carcinoma cell lines examined (BT20, BT474, Hs578T, MCF7, MDA-MB-231, T47D, and ZR-75-1) by immunoblotting. By treatment with Nek2 short interfering RNA (siRNA), expression of Nek2 was clearly decreased in both estrogen receptor (ER)-positive (MCF7) and ER-negative (MDA-MB-231) breast carcinoma cell lines. Cell growth, colony formation in soft agar, and in vitro invasiveness of these cell lines were substantially suppressed by Nek2 siRNA treatment. In a xenograft nude mouse model with subcutaneous implantation of MCF7 or MDA-MB-231, subcutaneous injection of Nek2 siRNA around the tumor nodules resulted in a reduction of tumor size compared with those of control siRNA injection. Taken together, Nek2 appears to play a pivotal role in tumorigenic growth of breast carcinoma cells, and could be a useful therapeutic molecular target for the treatment of breast carcinoma both in ER-positive and ER-negative cases.

Downregulation of EZH2 decreases growth of estrogen receptor-negative invasive breast carcinoma and requires BRCA1

Increased levels of EZH2, a critical regulator of cellular memory, are associated with negative estrogen receptor (ER) expression and disease progression in breast cancer. High levels of EZH2 signal the presence of metastasis and poor outcome in breast cancer patients. To test the hypothesis that deregulation of EZH2 contributes to ER negative breast cancer progression, EZH2 expression was inhibited in ER negative breast cancer cells MDA-MB-231 and CAL51 using a lentivirus system. EZH2 knockdown decreased proliferation and delayed the G2/M cell cycle transition, while not affecting apoptosis. In vivo, EZH2 down-regulation significantly decreased breast xenograft growth and improved survival. EZH2 knockdown up regulated BRCA1 protein. Of note, BRCA1 knockdown was sufficient to rescue the effects of EZH2 down-regulation in proliferation, G2/M arrest, and on the levels of hyperphosphorlated mitotic Cdc25C and Cyclin B1 proteins, crucial for entry into mitosis. Invasive ER negative breast carcinomas show significant overexpression of EZH2 and down-regulation of BRCA1 proteins. Taken together, we show that EZH2 plays a role in ER negative breast cancer progression in vivo and in vitro, and that BRCA1 is required for the proliferative effects of EZH2. Blockade of EZH2 may provide a prime target to prevent and/or halt ER negative breast cancer progression.

The genomic profile of HER2‐amplified breast cancers: the influence of ER status

Expression profiling studies have suggested that HER2-amplified breast cancers constitute a heterogeneous group that may be subdivided according to their ER status: HER2-amplified ER-positive breast carcinomas that fall into the luminal B cluster; and HER2-amplified ER-negative cancers which form a distinct molecular subgroup, known as the erbB2 or HER2 subgroup. ER-negative breast cancer differs significantly from ER-positive disease in the pattern, type, and complexity of genetic aberrations. Here we have compared the genomic profiles of ER-positive and ER-negative HER2-amplified cancers using tiling path microarray-based comparative genomic hybridization (aCGH). Validation of the differentially amplified regions was performed in an independent series of 70 HER2-amplified breast cancers. Although HER2-amplified cancers had remarkably complex patterns of molecular genetic aberrations, ER-positive and ER-negative HER2-amplified breast carcinomas shared most molecular genetic features as defined by aCGH. Genome-wide Fisher's exact test analysis revealed that less than 1.5% of the genome was significantly differentially gained or lost in ER-positive versus ER-negative HER2-amplified cancers. However, two regions of amplification were significantly associated with ER-positive carcinomas, one of which mapped to 17q21.2 and encompassed GJC1, IGFBP4, TNS4, and TOP2A. Chromogenic in situ hybridization analysis of an independent validation series confirmed the association between ER status and TOP2A amplification. In conclusion, although hormone receptor status does not determine the overall genetic profile of HER2-amplified breast cancers, specific genetic aberrations may be characteristic of subgroups of HER2 breast cancers.

HMGB1 expression in hormonally independent breast carcinoma

14655 Background: Estrogen and estrogen receptors (ER) play key roles in normal breast development and breast cancer progression; this was observed more than 100 years ago. Hormonal receptor expression correlates with cellular turnover rates, nuclear grade, degree of histological differentiation, and disease-free intervals. The function of ER is modulated by binding of ER to estrogen response elements and mediated by additional cellular proteins. High mobility group box protein1( HMGB1) is a nuclear protein that has been implicated in modulating ER gene interactions, particularly DNA bending. Here we explore HMGB1 expression in breast cancer cell lines. Methods: ER- negative breast cancer cell lines MCF7/adr, MDA-MB-231, BT-20, and ER-positive breast cancer cell lines MCF7/cst, MDA-MB-13, and BT-474 were obtained from the American Type Culture Collection (Rockville, USA). HMGB1 levels were evaluated by quantitative immunoblotting technique. The detection limit of HMGB1 was achieved at concentrations above 1 ng/ml. Each experiment was repeated at least five times to ensure reproducibility. Results: ER-negative breast cancer cell lines expressed HMGB1 levels 3–4 times higher than ER-positive breast cancer cell lines, as shown in Table 1. Conclusion: To our knowledge this is the first report to show elevated expression of HMGB1 expression in ER-negative breast carcinoma. These variations may contribute to different the biological characteristics and therapeutic responses among breast tumors. Table 1: HMGB1 expression is elevated in ER-negative breast cancer ER− ER+ 450 100 325 200 430 110 Mean 401.67 136.67 Standard Deviation 54.82 44.97 Standard Error of the Mean 31.65 44.97 Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Critical Therapeutics Inc. (CRTX)

C-Myc overexpression and endocrine resistance in breast cancer

The oncoprotein c-Myc is frequently overexpressed in breast cancer and ectopic expression in breast cancer cell lines attenuates responses to antiestrogen treatment. Here, we review preliminary data aimed at further elucidating a potential role for c-Myc in clinical endocrine resistance in breast cancer. Immunohistochemical and semi-quantitative PCR revealed that c-Myc protein and c- myc mRNA were frequently overexpressed in both ER-positive and ER-negative breast carcinoma. Furthermore, both constitutive and inducible c-Myc overexpression in MCF-7 breast cancer cell lines markedly reduced their sensitivity to the growth inhibitory effects of the pure antiestrogen ICI 182,780. In order to identify potential downstream targets of c-Myc that mediate this effect, Affymetrix microarrays were employed to examine the patterns of gene expression shared by MCF-7 cells stimulated by estrogen, or by induction of c-Myc. Approximately 50% of estrogen target genes identified 6 h after treatment were also regulated by c-Myc. One novel target, EMU4, was transcriptionally regulated by c-Myc. In addition, there was a strong correlation between c- myc and EMU4 mRNA expression in a battery of breast cancer cell lines. These data confirm that c-Myc overexpression is a common event in breast cancer, and that this is associated with resistance to antiestrogens in vitro. Furthermore, the development of an experimental paradigm for the discovery of c-Myc and estrogen target genes associated with endocrine resistance provides a framework for the discovery and validation of genes involved in estrogen signalling, and c-Myc-mediated-antiestrogen resistance.

Early growth response gene 1 (EGR1) is deleted in estrogen receptor‐negative human breast carcinoma

Patients with estrogen receptor (ER)-positive and ER-negative breast carcinomas differ in terms of disease progression, treatment regimens, and prognosis. The mechanism underlying the biologic differences of the two groups is understood poorly. Array comparative genome hybridization (CGH) on breast carcinoma subtypes demonstrated a consistent association between loss in regions of chromosome 5q and ER-negative tumors. It was shown previously that early growth response gene 1 (EGR1) on 5q acts like a tumor-suppressor gene, with its expression repressed in breast carcinomas. To test the hypothesis that EGR1 is deleted differentially in ER-negative versus ER-positive breast carcinomas, fluorescence in situ hybridization was employed in this study to determine the EGR1 deletion status in 50 breast carcinoma specimens. Deletion status was measured by the signal ratio of EGR1/chromosome 5. Linear regression was used to assess the results. The mean EGR1/chromosome 5 ratio for the ER-negative group was significantly lower compared with the same ratio for the ER-positive group (P < 0.001). Although grade alone was not significant for predicting the ratio, the interaction between ER status and grade was significant (P < 0.01): For ER-negative specimens, the higher the grade, the lower the EGR1/chromosome 5 ratio. The EGR1 gene appeared to be deleted in ER-negative human breast carcinomas. Egr-1 may contribute to the pathogenesis of ER-negative breast carcinomas versus ER-positive breast carcinomas.

Myoepithelial markers are expressed in at least 29% of oestrogen receptor negative invasive breast carcinoma

Around 20% of invasive breast carcinoma are oestrogen receptor alpha (ER) negative. Theoretically, this negativity could be either due to the result of downregulation of ER expression in the tumour cells, or the result of the tumour being derived from or differentiating towards cells which normally lack that expression. Normal basal, including myoepithelial, cells of the breast are ERnegative. CD10, smooth muscle actin and S100 are markers of these basal cells that can be used for their demonstration in routinely processed sections. This study was aimed at comparing the incidence of positivity for three myoepithelial markers in ER-negative and ER-positive invasive breast carcinoma. We have examined sections of 117 cases of breast carcinoma, including 77 ER-negative and 40 ER-positive cases, for the expression of CD10, smooth muscle actin and S100, using the avidin–biotin complex immunoperoxidase technique. A tumour was considered positive if more than 10% of the tumour cells were positively stained. In all, 36 (47%) ER-negative tumours were positive for one or more of these myoepithelial markers. The percentage of positively stained tumour cells varied between 30 and 100%. Of the 40 ER-positive tumours, only three (8%) were positive; two for S100 and one for actin, with none being positive for CD10. If cases stained only with S100 are excluded, as some of these may represent luminal differentiation, definite myoepithelial differentiation seems to be present in 29% (22/77) of ER-negative tumours as compared with 2.5% (1/40) of ER-positive tumours; a difference which is highly significant (P<0.001). It is suggested that at least 29% of ER-negative invasive breast carcinomas may be derived from or differentiating along the direction of basal nonconventional luminal epithelial breast cells that normally lack the expression of ER but totally or partially express various myoepithelial markers. Such tumours might need a different therapeutic approach.

Oncogenic role of the ubiquitin ligase subunit Skp2 in human breast cancer

Estrogen receptor (ER) expression and Her-2 amplification define specific subsets of breast tumors for which specific therapies exist. The S-phase kinase-associated protein Skp2 is required for the ubiquitin-mediated degradation of the cdk-inhibitor p27 and is a bona fide proto-oncoprotein. Using microarray analysis and immunohistochemistry, we determined that higher levels of Skp2 are present more frequently in ER-negative tumors than in ER-positive cases. Interestingly, the subset of ER-negative breast carcinomas overexpressing Skp2 are also characterized by high tumor grade, negativity for Her-2, basal-like phenotype, high expression of certain cell cycle regulatory genes, and low levels of p27 protein. We also found that Skp2 expression is cell adhesion-dependent in normal human mammary epithelial cells but not in breast cancer cells and that an inhibition of Skp2 induces a decrease of adhesion-independent growth in both ER-positive and ER-negative cancer cells. Finally, forced expression of Skp2 abolished effects of antiestrogens, suggesting that deregulated Skp2 expression might play a role in the development of resistance to antiestrogens. We conclude that Skp2 has oncogenic potential in breast epithelial cells and is overexpressed in a subset of breast carcinomas (ER- and Her-2 negative) for which Skp2 inhibitors may represent a valid therapeutic option.

Oncogenic role of the ubiquitin ligase subunit Skp2 in human breast cancer

Estrogen receptor (ER) expression and Her-2 amplification define specific subsets of breast tumors for which specific therapies exist. The S-phase kinase-associated protein Skp2 is required for the ubiquitin-mediated degradation of the cdk-inhibitor p27 and is a bona fide proto-oncoprotein. Using microarray analysis and immunohistochemistry, we determined that higher levels of Skp2 are present more frequently in ER-negative tumors than in ER-positive cases. Interestingly, the subset of ER-negative breast carcinomas overexpressing Skp2 are also characterized by high tumor grade, negativity for Her-2, basal-like phenotype, high expression of certain cell cycle regulatory genes, and low levels of p27 protein. We also found that Skp2 expression is cell adhesion-dependent in normal human mammary epithelial cells but not in breast cancer cells and that an inhibition of Skp2 induces a decrease of adhesion-independent growth in both ER-positive and ER-negative cancer cells. Finally, forced expression of Skp2 abolished effects of antiestrogens, suggesting that deregulated Skp2 expression might play a role in the development of resistance to antiestrogens. We conclude that Skp2 has oncogenic potential in breast epithelial cells and is overexpressed in a subset of breast carcinomas (ER- and Her-2 negative) for which Skp2 inhibitors may represent a valid therapeutic option.

Oncogenic role of the ubiquitin ligase subunit Skp2 in human breast cancer.

Estrogen receptor (ER) expression and Her-2 amplification define specific subsets of breast tumors for which specific therapies exist. The S-phase kinase-associated protein Skp2 is required for the ubiquitin-mediated degradation of the cdk-inhibitor p27 and is a bona fide proto-oncoprotein. Using microarray analysis and immunohistochemistry, we determined that higher levels of Skp2 are present more frequently in ER-negative tumors than in ER-positive cases. Interestingly, the subset of ER-negative breast carcinomas overexpressing Skp2 are also characterized by high tumor grade, negativity for Her-2, basal-like phenotype, high expression of certain cell cycle regulatory genes, and low levels of p27 protein. We also found that Skp2 expression is cell adhesion-dependent in normal human mammary epithelial cells but not in breast cancer cells and that an inhibition of Skp2 induces a decrease of adhesion-independent growth in both ER-positive and ER-negative cancer cells. Finally, forced expression of Skp2 abolished effects of antiestrogens, suggesting that deregulated Skp2 expression might play a role in the development of resistance to antiestrogens. We conclude that Skp2 has oncogenic potential in breast epithelial cells and is overexpressed in a subset of breast carcinomas (ER- and Her-2 negative) for which Skp2 inhibitors may represent a valid therapeutic option.

Increased choline kinase activity in human breast carcinomas: clinical evidence for a potential novel antitumor strategy.

Choline kinase (ChoK) and its product, phosphocholine (PCho), have been implicated in human carcinogenesis. Inhibition of this enzyme has been shown to be an efficient antitumor strategy in vivo. The aim of this study was to assess the relationship between the regulation of ChoK and clinical features in patients with breast cancer. To that end, normal and tumoral tissues from 53 patients with breast carcinomas were analysed for ChoK activity and expression, and compared to some clinical parameters. We report a relevant increase in ChoK activity in 38.5% of the tumoral tissues analysed when compared to the normal levels in healthy tissues. Furthermore, some clinical features were found significant versus ChoK status. First, an association of choline enzymatic activity with histological tumor grade was observed (P<0.001). In addition, increased ChoK activity was also associated with ER-negative breast carcinomas (P=0.037). A significant association between ChoK overexpression and both high histologic tumor grade (P=0.017) and ER-negative tumors (P=0.003) was found. Finally, ChoK overexpression was found in 17% of the samples and all corresponded with those that display the highest increase in ChoK activity (P<0.001). Here we provide evidence that ChoK may be related to the development of human breast cancer, suggesting that this finding may constitute the basis for the development of a novel antitumoral strategy for these patients.