Luminal Phenotype Research Articles

Notch and Jak/STAT signaling pathways in breast carcinoma of luminal and basal/triple-negative immunophenotypes.

618 Background: Notch and Jak/STAT pathways have a role in normal cell growth and development, but activation has been reported in several neoplasms. A relationship with positive hormonal receptor (HR) breast carcinomas (BC) has been suggested. We studied the Notch and Jak/STAT pathways in a series of BC of luminal and basal/triple negative (TN) immunophenotypes to determine their involvement. Methods: 170 invasive BC of luminal A (ER and PR ≥ 50% positive nuclei; and p53 < 20% or Ki67 < 20%), luminal B (ER and/or PR <50%, and p53 ≥ 20% or Ki67 ≥ 20%), and basal/TN (ER/PR/HER2-negative ± CK5/6 ± EGFR) immunophenotypes were analyzed. Total RNA was isolated from 2-3 paraffin-embedded cylinders from preselected tumor areas, and retrotranscripted to cDNA. RT-PCR was performed by Syber Green to study the expression of Notch1, 2, 4; Jagged1 (Notch pathway), and STAT3 (Jak/STAT pathway). The primers were tested in triplicate; RT minus controls and two commercial positive controls (total human and BC RNA) were included. The quantification was normalized to β-actin gene (Spearman rank correlation coefficient: r = 0.82; p < 0.05). Relative mRNA expression levels were carried out using the ΔΔCT method. A pool of normal breast tissues was used as calibrator for luminal BC, (similar gene expression levels were found in the adjacent tissue to all luminal BC). Each basal/TN BC was correlated with their corresponding normal breast tissue. The results were analyzed statistically by Chi-square and Fisher's exact test. Results: Similar overexpression in all analyzed genes was found in both luminal subtypes. In contrast, in the basal/TN group Notch1 and Jagged1 were down-regulated (Notch1 p < 0.000, OR: 2.849 -1.492-5.439-; Jagged1 p = 0.024, OR: 1.583 -0.964- 2.600-); and no differences were found for Notch4 and Notch2. Regarding STAT3, overexpression was detected in both phenotypes. Conclusions: The Notch pathway is activated in BC of luminal phenotype but not in the basal/TN group, in which down-regulation predominates, supporting its role in HR-positive BC. Nevertheless, up-regulation of the Jak/STAT pathway is present in all analyzed subtypes. Supported by grant FIS 03/1411and FCVI- HGUA/2006. No significant financial relationships to disclose.

EGFR and HER-2/neu expression in invasive apocrine carcinoma of the breast

This study was undertaken to investigate epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2)/neu expression in a cohort of apocrine carcinomas of the breast with emphasis on the classification of the breast tumors with apocrine morphology. In total, 55 breast carcinomas morphologically diagnosed as apocrine were evaluated for the steroid receptor expression profile characteristic of normal apocrine epithelium (androgen receptor positive/estrogen receptor (ER) negative/progesterone receptor (PR) negative), and for the expression of EGFR and Her-2/neu proteins, and the copy number ratios of the genes EGFR/CEP7 and HER-2/CEP17. On the basis of the results of steroid receptors expression, 38 (69%) cases were classified as pure apocrine carcinoma (androgen receptor positive/ER negative/PR negative), whereas 17 (31%) were re-classified as apocrine-like carcinomas because they did not have the characteristic steroid receptor expression profile. Her-2/neu overexpression was observed in 54% of the cases (57% pure apocrine carcinomas vs 47% apocrine-like carcinomas). HER-2/neu gene amplification was demonstrated in 52% of all cases (54% pure apocrine carcinomas vs 46% apocrine-like carcinomas). EGFR protein (scores 1 to 3+) was detected in 62% of all cases and was expressed in a higher proportion of pure apocrine carcinomas than in the apocrine-like carcinomas group (76 vs 29%, P=0.006). In the pure apocrine carcinoma group, Her-2/neu and EGFR protein expression were inversely correlated (P=0.006, r=−0.499). EGFR gene amplification was observed in two pure apocrine carcinomas and one apocrine-like carcinoma. Polysomy 7 was commonly present in pure apocrine carcinomas (61 vs 27% of apocrine-like carcinomas; P=0.083) and showed a weak positive correlation with EGFR protein expression (P=0.025, r=0.326). Our study showed that apocrine breast carcinomas are molecularly diverse group of carcinomas. Strictly defined pure apocrine carcinomas are either HER-2-overexpressing breast carcinomas or triple-negative breast carcinomas, whereas apocrine-like carcinomas predominantly belong to the luminal phenotype. Pure apocrine carcinomas show consistent overexpression of either EGFR or HER-2/neu, which could have significant therapeutic implications.

Abstract 4226: A luminal epithelial cell line isolated from PTEN−/−;P53−/− mouse protospheres forms adenocarcinoma in vivo

Abstract Pten deletion in the mouse prostate epithelium results in the expansion of a prostatic stem/progenitor cell subpopulation, which co-fractionates with basal epithelial cells. Progenitor cells in the luminal layer of Pten null prostates also have been suggested as tumor initiating cells. Here we describe the isolation of a protosphere-derived, androgen independent cell line with a luminal epithelial phenotype that displays properties of prostate progenitor cells and forms adenocarcinoma in-vivo. The cell line was generated from Rosa26-CreERPtenfl/flP53fl/fl prostate cells. Cells were plated to form protospheres, which originate from stem/progenitor cells, and the deletion of Pten and p53 was induced by tamoxifen. After spheres were serially passaged for 3 generations, a monolayer cell line capable of growth in minimal serum-free media was established. A clonal line also was generated from the parental cell line. In order to determine the differentiated cell phenotype of the parental and the clonal cell lines, cells were stained over multiple generations for markers of epithelial (CK5, CK8, CK14) and neuroendocrine (beta 3 tubulin) cell lineages. The cell line is entirely CK8 positive with a minor component of CK8/CK14 double positive cells. Cells are capable of forming protospheres both in Matrigel and in suspension, a property of stem/progenitor cells. In addition, based on lentiviral reporter assays, a minor population of cells in the parental and the clonal cell line expressed Nanog, Oct4 and Notch dependent activity. Interestingly, Nanog and Oct4 positive cells showed higher sphere forming ability as compared to negative cells. In order to investigate their tumorigenic potential, the parental and the cloned lines were injected subcutaneously into athymic nude mice. Visible tumor formation was observed after 2 weeks, and histological analysis revealed adenocarcinoma and poorly differentiated carcinoma. In addition to CK8+ cells within the tumors, there were rare CK5+ and neuroendocrine cells, suggesting plasticity in the differentiation potential. In conclusion, we have isolated a prostate luminal epithelial cell line and derivative clones with a defined background (Pten and p53 null) that have stem-like and tumor initiating properties in vitro and in vivo. Future experiments are aimed at deciphering the molecular mechanisms regulating growth and differentiation downstream of Pten and p53 loss. 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 4226.

Cytotoxic Effects Induced by Docetaxel, Gefitinib, and Cyclopamine on Side Population and Nonside Population Cell Fractions from Human Invasive Prostate Cancer Cells

The present study has been undertaken to establish the therapeutic benefit of cotargeting epidermal growth factor receptor (EGFR) and sonic hedgehog pathways by using gefitinib and cyclopamine, respectively, for improving the efficacy of the current chemotherapeutic drug docetaxel to counteract the prostate cancer progression from locally invasive to metastatic and recurrent disease stages. The data from immuofluorescence analyses revealed that EGFR/Tyr(1173)-pEGFR, sonic hedgehog ligand, smoothened coreceptor, and GLI-1 were colocalized with the CD133(+) stem cell-like marker in a small subpopulation of prostate cancer cells. These signaling molecules were also present in the bulk tumor mass of CD133(-) prostate cancer cells with a luminal phenotype detected in patient's adenocarcinoma tissues. Importantly, the results revealed that the CD133(+)/CD44(high)/AR(-/low) side population (SP) cell fraction endowed with a high self-renewal potential isolated from tumorigenic and invasive WPE1-NB26 cells by the Hoechst dye technique was insensitive to the current chemotherapeutic drug, docetaxel. In contrast, the docetaxel treatment induced significant antiproliferative and apoptotic effects on the CD133(-)/CD44(low)/AR(+) non-SP cell fraction isolated from the WPE1-NB26 cell line. Of therapeutic interest, the results have also indicated that combined docetaxel, gefitinib, and cyclopamine induced greater antiproliferative and apoptotic effects on SP and non-SP cell fractions isolated from WPE1-NB26 cells than individual drugs or two-drug combinations. Altogether, these observations suggest that EGFR and sonic hedgehog cascades may represent the potential therapeutic targets of great clinical interest to eradicate the total prostate cancer cell mass and improve the current docetaxel-based therapies against locally advanced and invasive prostate cancers, and thereby prevent metastases and disease relapse.

Altered Expression of Neurotensin Receptors Is Associated with the Differentiation State of Prostate Cancer

In prostate cancer, traditional treatments such as androgen response manipulation often provide only temporary resolution of disease, with emergence of a more aggressive, androgen-independent tumor following initial therapy. To treat recurrent disease, cell surface proteins that are specifically overexpressed on malignant cells may be useful for generating targeted therapeutics. Recent evidence suggests that neurotensin receptors (NTR) are recruited in advanced prostate cancer as an alternative growth pathway in the absence of androgens. In this study, we assessed the potential use of these receptors as targets by analyzing NTR expression patterns in human prostate cell lines and primary prostate tumor cell cultures derived from patient samples. In primary tumor cell cultures, NTR1 was upregulated in cells with a basal phenotype (cytokeratin 1/5/10/14+), whereas NTR2 and NTR3 were upregulated in cells with luminal phenotype (cytokeratin 18+). Similar patterns of NTR expression occurred in benign prostate tissue sections, implicating differentiation state as a basis for the differences observed in tumor cell lines. Our findings support the use of NTRs as tools for therapeutic targeting in prostate cancers composed of both poorly differentiated and/or well-differentiated cells.

Our Inner Mouse: Genomic Insights from Mouse Mammary Cancer Models To Guide Pre-clinical Testing

Abstract Accelerating the pre-clinical testing of novel therapies or combination therapies is an important goal of translational research. This is particularly true for identifying potentially new therapeutic approaches for triple-negative (TN) breast cancer (lacking expression of ER, PR and her2) for which no specifically targeted therapies currently exist. This sub-type of breast cancer, which generally falls into a basal category, has a poorer prognosis than breast cancers expressing ER/PR with a luminal phenotype. Most basal TN breast tumors have lost normal function of the tumor suppressor genes p53 and Rb. Using genetically-engineered mouse tumor models where p53 and Rb have been functionally inactivated through over-expression of SV40 T/t-antigens, we have identified a core gene expression (Tag) signature of about 150 genes involved in cell proliferation, cell replication, DNA repair, and apoptosis that is very relevant to human epithelial cancer. This signature, which is highly represented in the basal TN, C3(1)/SV40 T/t-antigen (Tag) mouse model of breast cancer, segregates human breast cancers with the basal TN phenotype and poor prognosis from luminal type tumors with a better prognosis. Based upon this cross-species analysis, we are determining whether genes within the Tag signature are potential therapeutic targets for basal TN breast cancer using human basal breast cancer cell lines and cell lines derived from the C3(1)/Tag tumors. This is initially being assessed using siRNA, drugs that inhibit specific targets within the signature, drugs in clinical use and combinations of these to find potentially novel therapeutic approaches for basal TN tumors. Additionally, in vivo studies are being performed using both human xenograft and Tag tumor transplant models for preclinical testing of selected therapies. Unlike studies using human xenografts in immunocompromised mice, experiments using Tag tumor transplants can be performed in mice with an intact immune system, adding an important dimension to these pre-clinical studies. Our approach demonstrates the values of cross-species genomic analyses to identify an appropriate model of basal TN breast cancer for pre-clinical testing, potential therapeutic targets, and an accelerated means of performing pre-clinical studies. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr BS1-1.

The molecular underpinning of lobular histological growth pattern: a genome‐wide transcriptomic analysis of invasive lobular carcinomas and grade‐ and molecular subtype‐matched invasive ductal carcinomas of no special type

Invasive lobular carcinoma (ILC) is the most frequent special type of breast cancer. The majority of these tumours are of low histological grade, express hormone receptors, and lack HER2 expression. The pleomorphic variant of ILCs (PLCs) is characterized by atypical cells with pleomorphic nuclei and is reported to have an aggressive clinical behaviour. Expression profiling studies have demonstrated that classic ILCs preferentially display a luminal phenotype, whereas PLCs may be of luminal, HER2 or molecular apocrine subtypes. The aims of this study were two-fold: to determine the transcriptomic characteristics of lobular carcinomas and to define the genome-wide transcriptomic differences between classic ILCs and PLCs. To define the transcriptomic characteristics of ILCs, minimizing the impact of histological grade and molecular subtype on the analysis, we subjected a series of grade- and molecular subtype-matched ILCs and invasive ductal carcinomas (IDCs) to genome-wide gene expression profiling using oligonucleotide microarrays. Hierarchical clustering analysis demonstrated that ILCs formed a separate cluster and a supervised analysis revealed that 5.8% of the transcriptionally regulated genes were significantly differentially expressed in ILCs compared to grade- and molecular subtype-matched IDCs. ILCs displayed down-regulation of E-cadherin and of genes related to actin cytoskeleton remodelling, protein ubiquitin, DNA repair, cell adhesion, TGF-beta signalling; and up-regulation of transcription factors/immediate early genes, lipid/prostaglandin biosynthesis genes, and cell migration-associated genes. Supervised analysis of classic ILCs and PLCs demonstrated that less than 0.1% of genes were significantly differentially expressed between these tumour subtypes. Our results demonstrate that ILCs differ from grade- and molecular subtype-matched IDCs in the expression of genes related to cell adhesion, cell-to-cell signalling, and actin cytoskeleton signalling. However, classic ILCs and PLCs are remarkably similar at the molecular level and should be considered as part of a spectrum of lesions.

Genomic profiling of breast tumours in relation to BRCAabnormalities and phenotypes

IntroductionGermline mutations in the BRCA1 and BRCA2 genes account for a considerable fraction of familial predisposition to breast cancer. Somatic mutations in BRCA1 and BRCA2 have not been found and the involvement of these genes in sporadic tumour development therefore remains unclear.MethodsThe study group consisted of 67 primary breast tumours with and without BRCA1 or BRCA2 abnormalities. Genomic alterations were profiled by high-resolution (~7 kbp) comparative genome hybridisation (CGH) microarrays. Tumour phenotypes were analysed by immunohistochemistry on tissue microarrays using selected biomarkers (ER, PR, HER-2, EGFR, CK5/6, CK8, CK18).ResultsClassification of genomic profiles through cluster analysis revealed four subgroups, three of which displayed high genomic instability indices (GII). Two of these GII-high subgroups were enriched with either BRCA1- or BRCA2-related tumours whereas the third was not BRCA-related. The BRCA1-related subgroup mostly displayed non-luminal phenotypes, of which basal-like were most prominent, whereas the other two genomic instability subgroups BRCA2- and GII-high-III (non-BRCA), were almost entirely of luminal phenotype. Analysis of genome architecture patterns revealed similarities between the BRCA1- and BRCA2 subgroups, with long deletions being prominent. This contrasts with the third instability subgroup, not BRCA-related, where small gains were more prominent.ConclusionsThe results suggest that BRCA1- and BRCA2-related tumours develop largely through distinct genetic pathways in terms of the regions altered while also displaying distinct phenotypes. Importantly, we show that the development of a subset of sporadic tumours is similar to that of either familial BRCA1- or BRCA2 tumours. Despite their differences, we observed clear similarities between the BRCA1- and BRCA2-related subgroups reflected in the type of genomic alterations acquired with deletions of long DNA segments being prominent. This suggests similarities in the mechanisms promoting genomic instability for BRCA1- and BRCA2-associated tumours, possibly relating to deficiency in DNA repair through homologous recombination. Indeed, this feature characterized both familial and sporadic tumours displaying BRCA1- or BRCA2-like spectrums of genomic alterations. The importance of these findings lies in the potential benefit from targeted therapy, through the use of agents leading to DNA double-strand breaks such as PARP inhibitors (olaparib) and cisplatin, for a much larger group of patients than the few BRCA1 and BRCA2 germline mutation carriers.

Role of FOXC1 in regulation of basal-like/triple-negative breast cancer

11016 Background: Class identification studies have proposed 3 prognostically relevant molecular subtypes of breast cancer: luminal, HER2 and basal-like. The latter is associated with poor prognosis but its molecular basis is not clear. We hypothesized a direct correlation between FOXC1 expression and basal-like breast cancer. Methods: Expression of FOXC1, CK5, CK14, EGFR, c-Kit, αB-crystallin, ITGB4 and FOXC2 in basal-like breast cancer was examined using publicly available microarray datasets. A molecular signature of 40 genes sharing co-ordinate up or down regulation with FOXC1 was identified on one microarray (49 patients) and validated on 5 other microarrays (1,232 patients). The clinical significance of FOXC1 gene expression and the FOXC1 gene signature was evaluated using censored survival data. FOXC1 protein expression was assessed by immunohistochemistry (IHC) of a 96-sample breast cancer tissue microarray. Normal breast epithelial, luminal and basal breast cancer cells transfected with FOXC1 vectors were evaluated for cell proliferation, migration and invasion. Results: FOXC1 was found to be consistently and exclusively upregulated in basal-like triple negative breast cancer and was associated with poor overall survival (p&lt;0.0001). The FOXC1 gene signature accurately predicted the basal-like phenotype. IHC analysis of FOXC1 protein expression in human breast cancers confirmed its potential to be used as a clinical biomarker of basal-like breast cancer. Normal breast epithelial cells and luminal breast cancer cells with low or no FOXC1 expression underwent epithelial-to-mesenchymal transition and displayed increased cellular proliferation, migration, invasion, and expression of basal cell markers when FOXC1 was overexpressed. In contrast, knockdown of FOXC1 by shRNA in basal-like breast cancer cells conferred luminal phenotype. Breast cancer progression-linked signaling pathways like NF-κB and p38MAPK were significantly stimulated in basal-like breast cancer as well as by in vitro FOXC1 overexpression. Conclusions: FOXC1 is a dominant determinant of the basal-like phenotype of breast cancer. We propose FOXC1 to be the single best molecular marker of and a potential therapeutic target for basal-like / triple negative breast cancer. No significant financial relationships to disclose.

The role of miR-206 in the epidermal growth factor (EGF) induced repression of estrogen receptor-alpha (ERalpha) signaling and a luminal phenotype in MCF-7 breast cancer cells.

Epidermal growth factor (EGF) receptor (EGFR)/MAPK signaling can induce a switch in MCF-7 breast cancer cells, from an estrogen receptor (ER)alpha-positive, Luminal-A phenotype, to an ERalpha-negative, Basal-like phenotype. Although mechanisms for this switch remain obscure, Basal-like cancers are typically high grade and confer a poorer clinical prognosis. We previously reported that miR-206 and ERalpha repress each other's expression in MCF-7 cells in a double-negative feedback loop. We show herein that miR-206 coordinately targets mRNAs encoding the coactivator proteins steroid receptor coactivator (SRC)-1 and SRC-3, and the transcription factor GATA-3, all of which contribute to estrogenic signaling and a Luminal-A phenotype. Overexpression of miR-206 repressed estrogen-mediated responses in MCF-7 cells, even in the presence of ERalpha encoded by an mRNA lacking a 3'-untranslated region, suggesting miR-206 affects estrogen signaling by targeting mRNAs encoding ERalpha-associated coregulatory proteins. Furthermore, EGF treatments enhanced miR-206 levels in MCF-7 cells and ERalpha-negative, EGFR-positive MDA-MB-231 cells, whereas EGFR small interfering RNA, or PD153035, an EGFR inhibitor, or U0126, a MAPK kinase inhibitor, significantly reduced miR-206 levels in MDA-MB-231 cells. Blocking EGF-induced enhancement of miR-206 with antagomiR-206 abrogated the EGF-inhibitory effect on ERalpha, SRC-1, and SRC-3 levels, and on estrogen response element-luciferase activity, indicating that EGFR signaling represses estrogenic responses in MCF-7 cells by enhancing miR-206 activity. Elevated miR-206 levels in MCF-7 cells ultimately resulted in reduced cell proliferation, enhanced apoptosis, and reduced expression of multiple estrogen-responsive genes. In conclusion, miR-206 contributes to EGFR-mediated abrogation of estrogenic responses in MCF-7 cells, contributes to a Luminal-A- to Basal-like phenotypic switch, and may be a measure of EGFR response within Basal-like breast tumors.

Tiling Path Genomic Profiling of Grade 3 Invasive Ductal Breast Cancers

To characterize the molecular genetic profiles of grade 3 invasive ductal carcinomas of no special type using high-resolution microarray-based comparative genomic hybridization (aCGH) and to identify recurrent amplicons harboring putative therapeutic targets associated with luminal, HER-2, and basal-like tumor phenotypes. Ninety-five grade 3 invasive ductal carcinomas of no special type were classified into luminal, HER-2, and basal-like subgroups using a previously validated immunohistochemical panel. Tumor samples were microdissected and subjected to aCGH using a tiling path 32K BAC array platform. Selected regions of recurrent amplification were validated by means of in situ hybridization. Expression of genes pertaining to selected amplicons was investigated using quantitative real-time PCR and gene silencing was done using previously validated short hairpin RNA constructs. We show that basal-like and HER-2 tumors are characterized by "sawtooth" and "firestorm" genetic patterns, respectively, whereas luminal cancers were more heterogeneous. Apart from confirming known amplifications associated with basal-like (1q21, 10p, and 12p), luminal (8p12, 11q13, and 11q14), and HER-2 (17q12) cancers, we identified previously unreported recurrent amplifications associated with each molecular subgroup: 19q12 in basal-like, 1q32.1 in luminal, and 14q12 in HER-2 cancers. PPM1D gene amplification (17q23.2) was found in 20% and 8% of HER-2 and luminal cancers, respectively. Silencing of PPM1D by short hairpin RNA resulted in selective loss of viability in tumor cell lines harboring the 17q23.2 amplification. Our results show the power of aCGH analysis in unraveling the genetic profiles of specific subgroups of cancer and for the identification of novel therapeutic targets.

Loss of 16q in high grade breast cancer is associated with estrogen receptor status: Evidence for progression in tumors with a luminal phenotype?

Loss of the long arm of chromosome 16 (16q) is observed in the vast majority of low grade/grade I (GI) invasive ductal carcinomas of no special type (IDC-NSTs), whereas this event is uncommonly seen in high grade/grade III (GIII) IDC-NSTs. Together with data on the pathology and genetics of breast cancer recurrences, this has led to the proposal that GI and GIII breast cancers evolve through distinct genetic pathways and that progression from GI to GIII is an unlikely biological phenomenon. We compared the genomic profiles of GIII-IDC-NSTs with 16q whole arm loss (16qWL) according to estrogen receptor (ER) status. 16qWL was found in 36.5% of cases and was significantly associated with ER expression and luminal phenotype. ER+ GIII-IDC-NSTs with 16qWL displayed significantly higher levels of genomic instability than ER+ IDC-NSTs without 16qWL. Furthermore, ER+ and ER- IDC-NSTs stratified according to the presence of 16qWL harbored distinct patterns of genetic aberrations. Interestingly, ER+/16qWL tumors displayed genetic features usually found in tumors with homologous DNA repair defects and significantly more frequently harbored heterozygous loss of BRCA2 than the remaining ER+ cancers. Our results demonstrate that approximately one third of GIII tumors harbor 16qWL, confirming that progression from low to high grade breast cancer is not found in the majority of breast cancers. 16qWL was significantly more prevalent in ER+/luminal GIII-IDC-NSTs. Given that GI breast cancers harbor a luminal phenotype, our results suggest that if progression from GI to GIII breast cancer does happen, it may preferentially occur in breast cancers of luminal phenotype.

The chemoresistant population of luminal subtype human breast cancer cells expresses a basal phenotype.

Abstract Abstract #42 Background: We recently identified rare cells that express cytokeratin 5 (CK5+) and lack estrogen and progesterone receptors (ER– and PR–), in solid tumor xenografts derived from ER+PR+ T47D luminal A subtype human breast cancer cells (Horwitz et al, 2008 PNAS 105: 5774). These rare CK5+ cells have the capacity to re-form the majority of ER+PR+CK18+ cells in luminal tumor xenografts, and recapitulate the original tumor heterogeneity. CK5 is a marker of the poor prognosis basal-like subtype of human breast cancers. Additionally, CK5 is postulated to be a marker of stem/progenitor like cells in the human breast, and we have shown that CK5+ cells co-express the breast cancer stem cell marker CD44. We postulated that these rare basal-like CK5+ cells represent a potential chemoresistant subpopulation within luminal tumors. To test this, we used T47D and MCF7 cells, both of which have been classified by genetic profiling as luminal A (ER+PR+CK18+HER2–).&amp;#x2028; Methods: Cells were plated at subconfluent densities in serum-containing media and treated with either vehicle or cyclophosphamide (1mM), 5-fluorouracil (150μM) or docetaxel (11nM) for 72 hours; these doses were determined to yield a one to two-log kill. Residual chemoresistant cells were fixed: i) immediately following chemotherapy, or ii) after colony repopulation for 7 days. The chemoresistant subpopulation was characterized by immunocytochemistry for markers of basal/stem and luminal phenotypes, including CK5, ER, PR, CK18, and CD44.&amp;#x2028; Results: At baseline, as well as in controls, both cell lines were ER+PR+CK18+ and ER–PR–CK5+ cells were rare or undetectable. In response to treatment with each of the three drugs, the surviving population was enriched in ER–CK5+ cells. PR expression was variable (+/–) in CK5+ cells. These chemoresistant cells were able to repopulate colonies that expressed ER+PR+CK18+ and resembled the original cells by having a predominant luminal phenotype, with only rare/undetectable receptor negative, CK5+ basal-like cells.&amp;#x2028; Conclusion: We conclude that luminal subtype human breast cancer cells contain a rare basal/stem-like chemoresistant subpopulation that is capable of repopulating a luminal tumor after therapy. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 42.

The Prognostic Value of the Proliferation Marker Phosphohistone H3 (PPH3) in Luminal, Basal-Like and Triple Negative Phenotype Invasive Lymph Node-Negative Breast Cancer

Purpose: Prognostic comparison of phosphohistone-H3 (PPH3) with Cytokeratin 5/6 and/or 14 positive (= basal-CK), triple (ER, PR, HER2)-negative (= TNP) and basal-like (= TNP and basal-CK positive) phenotype in invasive breast cancers.Patients and Methods: Classical variables, PPH3, ER, PR, basal-CK and HER2 in 240 T1–2N0M0 patients under 71 years.Results: TNP and basal-like cancers had higher PPH3 expression than the other cancers (mean 48 versus 11, P &lt; 0.001). Fifteen percent of the patients in the whole group, but 32–38% of TNP and basal-like cancers recurred.With multivariate analysis, PPH3 &lt; 13 (n = 156) versus ≥ 13 (n = 84 = 35% of all cases) was the strongest and only prognosticator (10-year survival 96% and 64%, P ≤ 0.001, Hazard ratio = 9.0).Conclusion: PPH3 is the strongest prognosticators in luminal, Triple negative and basal-like T1–2N0M0 invasive breast cancers.

The prognostic value of the proliferation marker phosphohistone H3 (PPH3) in luminal, basal-like and triple negative phenotype invasive lymph node-negative breast cancer.

Purpose: Prognostic comparison of phosphohistone-H3 (PPH3) with Cytokeratin 5/6 and/or 14 positive (= basal-CK), triple (ER, PR, HER2)-negative (= TNP) and basal-like (= TNP and basal-CK positive) phenotype in invasive breast cancers. Patients and Methods: Classical variables, PPH3, ER, PR, basal-CK and HER2 in 240 T1–2N0M0 patients under 71 years. Results: TNP and basal-like cancers had higher PPH3 expression than the other cancers (mean 48 versus 11, P < 0.001). Fifteen percent of the patients in the whole group, but 32–38% of TNP and basal-like cancers recurred.With multivariate analysis, PPH3 < 13 (n = 156) versus ≥ 13 (n = 84 = 35% of all cases) was the strongest and only prognosticator (10-year survival 96% and 64%, P ≤ 0.001, Hazard ratio = 9.0). Conclusion: PPH3 is the strongest prognosticators in luminal, Triple negative and basal-like T1–2N0M0 invasive breast cancers.

Breast cancer cells expressing stem cell markers CD44+ CD24lo are eliminated by Numb-1 peptide-activated T cells

Cancer stem cells (CSC) are resistant to chemo- and radiotherapy. To eliminate cells with phenotypic markers of CSC-like we characterized: (1) expression of CD44, CD24, CD133 and MIC-A/B (NKG2 receptors) in breast (MCF7) and ovarian (SK-OV-3) cells resistant to gemcitabine (GEM), paclitaxel (PTX) and 5-fluorouracil (5-FU) and (2) their elimination by Numb- and Notch-peptide activated CTL. The number of cells in all populations with the luminal CSC phenotype [epithelial specific antigen(+) (ESA) CD44(hi) CD24(lo), CD44(hi) CD133(+), and CD133(+) CD24(lo)] increased in drug-resistant MCF7 and SK-OV-3 cells. Similarly, the number of cells with expressed MIC-A/B increased 4 times in drug-resistant tumor cells compared with drug-sensitive cells. GEM(Res) MCF7 cells had lower levels of the Notch-1-extracellular domain (NECD) and Notch trans-membrane intracellular domain (TMIC) than GEM(Sens) MCF7. The levels of Numb, and Numb-L-[P]-Ser(265) were similar in GEM(Res) and GEM(Sens) MCF7 cells. Only the levels of Numb-L (long)-Ser(295) decreased slightly. This finding suggests that Notch-1 cleavage to TMIC is inhibited in GEM(Res) MCF7 cells. PBMC activated by natural immunogenic peptides Notch-1 (2112-2120) and Numb-1 (87-95) eliminated NICD(positive), CD24(hi) CD24(lo) MCF7 cells. It is likely that the immunogenic Numb-1 peptide in MCF7 cells originated from Numb, [P]-lated by an unknown kinase, because staurosporine but not wortmannin and MAPK-inhibitors decreased peptide presentation. Numb and Notch are antagonistic proteins which degrade each other to stop and activate cell proliferation, respectively. Their peptides are presented alternatively. Targeting both antagonistic proteins should be useful to prevent metastases in patients whose tumors are resistant to conventional treatments.

Involvement of MicroRNAs in Breast Cancer

MicroRNAs regulate numerous aspects of normal and pathologic cellular processes, including cancer. Breast cancer is a heterogeneous form of cancer that is derived from mammary epithelial cells. This review discusses the involvement of microRNAs in the regulation of normal mammary epithelial stem cells, their differentiation into basal and luminal phenotypes, and their control of breast cancer stem cells, also referred to as tumor-initiating cells. In the second section, we summarize the findings of differential microRNA expression in normal versus breast tumor tissue and among the various subtypes of breast cancer (primarily luminal, basal-like, and HER2). In the third and fourth sections of the review, specific mRNA targets of microRNAs in breast cancer are discussed, including those encoding the estrogen receptor-alpha and epidermal growth factor receptor, as well as survival, tumor suppressor, and cell-cycle-related proteins. Finally, the involvement of microRNAs in the promotion and suppression of breast cancer metastasis is reviewed. The studies presented herein provide a rationale for the design of therapeutic agents that target specific microRNAs in the treatment of breast cancer. Hopefully, this review will provide an impetus for more studies on the role of microRNAs in the regulation of normal mammary gland development and function.

Increased wound-response revealed by proteomics of estrogen receptor-negative breast cancers

Besides the mitogenic action of estrogens in breast cancer cells there is also evidence that estrogen receptors mediate protective, anti-invasive effects. The aim of this project was to identify downstream ER-dependent proteins differentially expressed in fresh-frozen ER-positive and –negative breast cancer. Tissue sections from eight ER-positive and eight ER-negative cryo-preserved breast cancer specimen were analysed by differential multiplex ProteoTope imaging of 2D-PAGE gels. This method is based on labeling proteins with chemically identical iodine radio-isotopes (I–125, I–131) and provides a direct quantitative and multi color differential proteome display at the low-attomole level. Spots were matched across gels, and their intensities were analysed relative to ER status. The statistically most significant differential protein spots were identified by mass spectrometry using picked co-migrating non-radioactive tumor proteins. In total, proteins from 325 spots were identified by MALDI-TOF PMF with MASCOT scores greater than 70. 72 spots represented 16 proteins that were identified in more than one protein spot. Proteins significantly differentially abundant across all pools at the 0.1% level were consistent with previously published literature. We observed elevated levels of keratins 19, 18 and 8 in ER-positive tumors suggesting a luminal phenotype for ER-positive tumors. Additionally, proteins associated with increased inflammation and wound response were observed in ER-negative tumors. Our data are consistent with previously published literature, suggesting an altered keratin pool associated with increased inflammation and wound response in ER-negative tumors.

A novel tissue-slice culture model for non-malignant human prostate

A novel tissue culture system was established for modeling the non-neoplastic human prostate in vitro. Precision-cut prostate slices were cultivated in culture plates with a gas-permeable base in a novel serum-free mixture. Cultivated specimens was evaluated by an immunohistochemical analysis of cytokeratins 18 and 14, androgen receptor (AR), prostate specific antigen (PSA), prostate acid phosphatase (PAP), and the endothelial cell marker von Willebrand factor. Epithelial viability in the presence and absence of dihydrotestosterone (DHT) was also assessed. Satisfactory maintenance of glandular cytoarchitecture was observed in the presence of DHT with approximately half of the glands displaying a columnar or cuboidal phenotype and an intact layer of basal cells. In the absence of DHT, the corresponding percentage was significantly lower. The occurrence of involutive changes and epithelial cell death was significantly higher in the absence of DHT. Glandular and stromal cells maintained their capacity to express AR. PSA and PAP were expressed throughout the culture period, albeit at a lower level than in uncultured tissue. The viability of endothelial cells differed markedly between individual samples. During culture, the tissue slices became covered with epithelial cells originating from glands that were cut open during tissue slicing. This cell layer consisted of a stratified basal compartment overlaid by cells with a luminal phenotype. The present culture system provides a novel in vitro setting in which to study normal human prostate biology and pathobiology and may help to obviate problems related to the use of established cancer cell lines and animal models.

Integrated Genomic and Transcriptomic Analysis of Ductal Carcinoma In situ of the Breast

To gain insight into genomic and transcriptomic subtypes of ductal carcinomas in situ of the breast (DCIS). We did a combined phenotypic and genomic analysis of a series of 57 DCIS integrated with gene expression profile analysis for 26 of the 57 cases. Thirty-two DCIS exhibited a luminal phenotype; 21 were ERBB2 positive, and 4 were ERBB2/estrogen receptor (ER) negative with 1 harboring a bona fide basal-like phenotype. Based on a CGH analysis, genomic types were identified in this series of DCIS with the 1q gain/16q loss combination observed in 3 luminal DCIS, the mixed amplifier pattern including all ERBB2, 12 luminal and 2 ERBB2(-)/ER(-) DCIS, and the complex copy number alteration profile encompassing 14 luminal and 1 ERBB2(-)/ER(-) DCIS. Eight cases (8 of 57; 14%) presented a TP53 mutation, all being amplifiers. Unsupervised analysis of gene expression profiles of 26 of the 57 DCIS showed that luminal and ERBB2-amplified, ER-negative cases clustered separately. We further investigated the effect of high and low copy number changes on gene expression. Strikingly, amplicons but also low copy number changes especially on 1q, 8q, and 16q in DCIS regulated the expression of a subset of genes in a very similar way to that recently described in invasive ductal carcinomas. These combined approaches show that the molecular heterogeneity of breast ductal carcinomas exists already in in situ lesions and further indicate that DCIS and invasive ductal carcinomas share genomic alterations with a similar effect on gene expression profile.