Carolyn M. Slater

Carcinogenicity of estrogens in human breast epithelial cells.

Epidemiological and clinical evidences indicate that breast cancer risk is associated with prolonged ovarian function that results in elevated circulating levels of steroid hormones. Principal among these is estrogen, which is associated with two important risk factors, early onset of menarche and late menopause. However, up to now there is no direct experimental evidence that estrogens are responsible of the initiation of human breast cancer. We postulate that if estrogens are causative agents of this disease, they should elicit in human breast epithelial cells (HBEC) genomic alterations similar to those exhibited by human breast cancers, such as DNA amplification and loss of genetic material representing tumor suppressor genes. These effects could result from binding of the hormone to its nuclear receptors (ER) or from its metabolic activation to reactive metabolites. This hypothesis was tested by treating with the natural estrogen 17β‐estradiol (E2) and the synthetic steroid diethylstilbestrol (DES) MCF‐10F cells, a HBEC line that is negative for ER. Cells treated with the chemical carcinogen benzo (a) pyrene (BP) served as a positive control of cell transformation. BP‐, E2‐, and DES‐treated MCF‐10F cells showed increases in survival efficiency and colony efficiency in agar methocel, and loss of ductulogenic capacity in collagen gel. The largest colonies were formed by BP‐treated cells, becoming progressively smaller in DES‐ and E2‐treated cells. The loss of ductulogenic capacity was maximal in BP‐, and less prominent in E2‐ and DES‐treated cells. Genomic analysis revealed that E2‐ and DES‐treated cells exhibited loss of heterozygosity in chromosomes 3 and 11, at 3p21, 3p21–21.2, 3p21.1–14.2, and 3p14.2–14.1, and at 11q23.3 and 11q23.1–25 regions, respectively. It is noteworthy that these loci are also affected in breast lesions, such as ductal hyperplasia, carcinoma in situ, and invasive carcinoma. Our data are the first ones to demonstrate that estrogens induce in HBEC phenotypic changes indicative of cell transformation and that those changes are associated with significant genomic alterations that might unravel new pathways in the initiation of breast cancer.

Altered Gene Expression in Morphologically Normal Epithelial Cells from Heterozygous Carriers of BRCA1 or BRCA2 Mutations

We hypothesized that cells bearing a single inherited “hit” in a tumor suppressor gene express an altered mRNA repertoire that may identify targets for measures that could delay or even prevent progression to carcinoma. We report here on the transcriptomes of primary breast and ovarian epithelial cells cultured from BRCA1 and BRCA2 mutation carriers and controls. Our comparison analyses identified multiple changes in gene expression, in both tissues for both mutations, which were validated independently by real-time reverse transcription-PCR analysis. Several of the differentially expressed genes had been previously proposed as cancer markers, including mammaglobin in breast cancer and serum amyloid in ovarian cancer. These findings show that heterozygosity for a mutant tumor suppressor gene can alter the expression profiles of phenotypically normal epithelial cells in a gene-specific manner; these detectable effects of “one hit” represent early molecular changes in tumorigenesis that may serve as novel biomarkers of cancer risk and as targets for chemoprevention. Cancer Prev Res; 3(1); 48–61

Loss of GATA4 and GATA6 expression specifies ovarian cancer histological subtypes and precedes neoplastic transformation of ovarian surface epithelia.

Background The family of zinc finger-containing GATA transcription factors plays critical roles in cell lineage specification during early embryonic development and organ formation. GATA4 and GATA6 were found to be frequently lost in ovarian cancer, and the loss is proposed to account for dedifferentiation of the cancer cells. Methodology/Principal Findings We further investigated the expression of GATA4 and GATA6 in ovarian surface epithelial lesions and histological subtypes of ovarian carcinomas by immunostaining. GATA4 and GATA6 were found to be absent in high percentages (80 to 90%) of serous, clear cell, and endometrioid ovarian cancer examined. In contrast, both were found positive in 11 out of 12 cases of mucinous carcinomas, suggesting the expression of the GATA factors can distinguish mucinous cancer from other histological subtypes. GATA4 was frequently lost in preneoplastic lesions such as morphologically normal inclusion cysts and epithelial hyperplasia adjacent to malignant cells. The loss of GATA6 correlates closely with neoplastic morphological transformation of ovarian surface epithelia. In culture, GATA4 expression was progressively reduced upon passaging primary ovarian surface epithelial cells, which correlated with changes in histone modification of the GATA4 locus. A reduced GATA6 gene dosage as in GATA6 (+/−) mice led to an increased pre-neoplastic changes and inclusion cysts in the ovaries, suggesting the loss of GATA6 contributes to ovarian cancer development. Conclusions/Significance This study suggests that the expression status of GATA4 and GATA6 may dictate distinct pathologic pathways leading to serous or mucinous ovarian carcinomas. The readily loss of GATA4 expression through changes in chromatin conformation suggests a potential non-phenotypic initiating event, leading to subsequent loss of GATA6, morphological transformation, and ultimate tumorigenesis.

Nuclear Entry of Activated MAPK Is Restricted in Primary Ovarian and Mammary Epithelial Cells

Background The MAPK/ERK1/2 serine kinases are primary mediators of the Ras mitogenic signaling pathway. Phosphorylation by MEK activates MAPK/ERK in the cytoplasm, and phospho-ERK is thought to enter the nucleus readily to modulate transcription. Principal Findings Here, however, we observe that in primary cultures of breast and ovarian epithelial cells, phosphorylation and activation of ERK1/2 are disassociated from nuclear translocalization and transcription of downstream targets, such as c-Fos, suggesting that nuclear translocation is limited in primary cells. Accordingly, in import assays in vitro, primary cells showed a lower import activity for ERK1/2 than cancer cells, in which activated MAPK readily translocated into the nucleus and activated c-Fos expression. Primary cells express lower levels of nuclear pore complex proteins and the nuclear transport factors, importin B1 and importin 7, which may explain the limiting ERK1/2 import found in primary cells. Additionally, reduction in expression of nucleoporin 153 by siRNA targeting reduced ERK1/2 nuclear activity in cancer cells. Conclusion ERK1/2 activation is dissociated from nuclear entry, which is a rate limiting step in primary cells and in vivo, and the restriction of nuclear entry is disrupted in transformed cells by the increased expression of nuclear pores and/or nuclear transport factors.

Identifying a Highly-Aggressive DCIS Subgroup by Studying Intra-Individual DCIS Heterogeneity among Invasive Breast Cancer Patients

The heterogeneity among multiple ductal carcinoma in situ (DCIS) lesions within the same patient also diagnosed with invasive ductal carcinoma (IDC) has not been well evaluated, leaving research implications of intra-individual DCIS heterogeneity yet to be explored. In this study formalin-fixed paraffin embedded sections from 36 patients concurrently diagnosed with DCIS and IDC were evaluated by immunohistochemistry. Ten DCIS lesions from each patient were then randomly selected and scored. Our results showed that expression of PR, HER2, Ki-67, and p16 varied significantly within DCIS lesions from a single patient (P<0.05 for PR; P<1×10−8 for HER2, Ki-67 and p16). In addition, seventy-two percent of the individuals had heterogeneous expression of at least 2/6 markers. Importantly, by evaluating the expression of promising DCIS risk biomarkers (Ki-67, p53 and p16) among different DCIS subgroups classified by comparing DCIS molecular subtypes with those of adjacent normal terminal duct lobular units (TDLU) and IDC, our results suggest the existence of a highly-aggressive DCIS subgroup, which had the same molecular subtype as the adjacent IDC but not the same subtype as the adjacent normal TDLU. By using a systematic approach, our results clearly demonstrate that intra-individual heterogeneity in DCIS is very common in patients concurrently diagnosed with IDC. Our novel findings of a DCIS subpopulation with aggressive characteristics will provide a new paradigm for mechanistic studies of breast tumor progression and also have broad implications for prevention research as heterogeneous pre-invasive lesions are present in many other cancer types.

Identifying breast cancer risk loci by global differential allele-specific expression (DASE) analysis in mammary epithelial transcriptome

BackgroundThe significant mortality associated with breast cancer (BCa) suggests a need to improve current research strategies to identify new genes that predispose women to breast cancer. Differential allele-specific expression (DASE) has been shown to contribute to phenotypic variables in humans and recently to the pathogenesis of cancer. We previously reported that nonsense-mediated mRNA decay (NMD) could lead to DASE of BRCA1/2, which is associated with elevated susceptibility to breast cancer. In addition to truncation mutations, multiple genetic and epigenetic factors can contribute to DASE, and we propose that DASE is a functional index for cis-acting regulatory variants and pathogenic mutations, and that global analysis of DASE in breast cancer precursor tissues can be used to identify novel causative alleles for breast cancer susceptibility.ResultsTo test our hypothesis, we employed the Illumina® Omni1-Quad BeadChip in paired genomic DNA (gDNA) and double-stranded cDNA (ds-cDNA) samples prepared from eight BCa patient-derived normal mammary epithelial lines (HMEC). We filtered original array data according to heterozygous genotype calls and calculated DASE values using the Log ratio of cDNA allele intensity, which was normalized to the corresponding gDNA. We developed two statistical methods, SNP- and gene-based approaches, which allowed us to identify a list of 60 candidate DASE loci (DASE ≥ 2.00, P ≤ 0.01, FDR ≤ 0.05) by both methods. Ingenuity Pathway Analysis of DASE loci revealed one major breast cancer-relevant interaction network, which includes two known cancer causative genes, ZNF331 (DASE = 2.31, P = 0.0018, FDR = 0.040) and USP6 (DASE = 4.80, P = 0.0013, FDR = 0.013), and a breast cancer causative gene, DMBT1 (DASE=2.03, P = 0.0017, FDR = 0.014). Sequence analysis of a 5′ RACE product of DMBT1 demonstrated that rs2981745, a putative breast cancer risk locus, appears to be one of the causal variants leading to DASE in DMBT1.ConclusionsOur study demonstrated for the first time that global DASE analysis is a powerful new approach to identify breast cancer risk allele(s).

Identifying putative breast cancer-associated long intergenic non-coding RNA loci by high density SNP array analysis.

Recent high-throughput transcript discoveries have yielded a growing recognition of long intergenic non-coding RNAs (lincRNAs), a class of arbitrarily defined transcripts (>200 nt) that are primarily produced from the intergenic space. lincRNAs have been increasingly acknowledged for their expressional dynamics and likely functional associations with cancers. However, differential gene dosage of lincRNA genes between cancer genomes is less studied. By using the high-density Human Omni5-Quad BeadChips (Illumina), we investigated genomic copy number aberrations in a set of seven tumor-normal paired primary human mammary epithelial cells (HMECs) established from patients with invasive ductal carcinoma. This Beadchip platform includes a total of 2,435,915 SNP loci dispersed at an average interval of ~700 nt throughout the intergenic region of the human genome. We mapped annotated or putative lincRNA genes to a subset of 332,539 SNP loci, which were included in our analysis for lincRNA-associated copy number variations (CNV). We have identified 122 lincRNAs, which were affected by somatic CNV with overlapped aberrations ranging from 0.14% to 100% in length. lincRNA-associated aberrations were detected predominantly with copy number losses and preferential clustering to the ends of chromosomes. Interestingly, lincRNA genes appear to be less susceptible to CNV in comparison to both protein-coding and intergenic regions (CNV affected segments in percentage: 1.8%, 37.5%, and 60.6%, respectively). In summary, our study established a novel approach utilizing high-resolution SNP array to identify lincRNA candidates, which could functionally link to tumorigenesis, and provide new strategies for the diagnosis and treatment of breast cancer.

The Protein Encoded by the CCDC170 Breast Cancer Gene Functions to Organize the Golgi-Microtubule Network

Genome-Wide Association Studies (GWAS) and subsequent fine-mapping studies (> 50) have implicated single nucleotide polymorphisms (SNPs) located at the CCDC170/C6ORF97-ESR1 locus (6q25.1) as being associated with the risk of breast cancer. Surprisingly, our analysis using genome-wide differential allele-specific expression (DASE), an indicator for breast cancer susceptibility, suggested that the genetic alterations of CCDC170, but not ESR1, account for GWAS-associated breast cancer risk at this locus. Breast cancer-associated CCDC170 nonsense mutations and rearrangements have also been detected, with the latter being specifically implicated in driving breast cancer. Here we report that the wild type CCDC170 protein localizes to the region of the Golgi apparatus and binds Golgi-associated microtubules (MTs), and that breast cancer-linked truncations of CCDC170 result in loss of Golgi localization. Overexpression of wild type CCDC170 triggers Golgi reorganization, and enhances Golgi-associated MT stabilization and acetyltransferase ATAT1-dependent α-tubulin acetylation. Golgi-derived MTs regulate cellular polarity and motility, and we provide evidence that dysregulation of CCDC170 affects polarized cell migration. Taken together, our findings demonstrate that CCDC170 plays an essential role in Golgi-associated MT organization and stabilization, and implicate a mechanism for how perturbations in the CCDC170 gene may contribute to the hallmark changes in cell polarity and motility seen in breast cancer.

Abstract 4991: Identifying a highly-aggressive DCIS subgroup by studying intra-individual DCIS heterogeneity among invasive breast cancer patients

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Detection rates for ductal carcinoma in situ (DCIS) have dramatically increased because of the widespread use of mammographic screening. The heterogeneity among multiple DCIS lesions within the same patient, also diagnosed with invasive ductal carcinoma (IDC), has not been well evaluated, leaving clinical and research implications of this intra-individual heterogeneity of DCIS yet to be explored. In the study presented here, we tested the hypothesis that classification of heterogeneous DCIS based on adjacent IDC and normal terminal duct lobular units (TDLU) in patients concurrently diagnosed with IDC and DCIS helps to identify DCIS subgroups which have different degrees of “aggressiveness”. Experimental Designs and Results: Ten DCIS lesions from each of 36 patients concurrently diagnosed with DCIS and IDC were randomly selected and scored with IHC biomarkers. Our results showed that expression of PR, HER2, Ki-67 and p16 varied significantly in DCIS lesions from a single patient (P 2.0, P<0.05]. A majority of genes were upregulated in type IIb DCIS and more than 50% of genes are cancer-relevant (P < 10-8∼-2). Importantly, a group of differentially expressed genes in subgroup IIb including CD44, IFIT1, STAT1, MLH1, PARP9 and BRIP1 were confirmed by qPCR validation and likely play important roles in DCIS progression based on previous findings, which further support that Type IIb DCIS may represent a highly aggressive subgroup. Conclusions: By using a systematic approach, our results clearly demonstrate that intra-individual heterogeneity in DCIS is very common in patients concurrently diagnosed with IDC. Our novel findings of a DCIS subpopulation with high-aggressiveness will provide a new paradigm for mechanistic studies of breast tumor progression and also have broad implications for prevention research as heterogeneous pre-invasive lesions are present in many other cancer types. This work was kindly supported by the Susan G. Komen for the Cure (KG100274), and Eileen Stein Jacoby Fund. Citation Format: Zhengyu Jiang, Dana Pape-Zambito, Karthik Devarajan, Hong Wu, Carolyn Slater, Lauren Dolinsky, Mary Daly, Xiaowei Chen. Identifying a highly-aggressive DCIS subgroup by studying intra-individual DCIS heterogeneity among invasive breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4991. doi:10.1158/1538-7445.AM2014-4991

Chromosome 17p13.2 transfer reverts transformation phenotypes and fas-mediated apoptosis in breast epithelial cells

Transformation of the human breast epithelial cells (HBEC) MCF‐10F with the carcinogen benz(a)pyrene (BP) into BP1‐E cells resulted in the loss of the chromosome 17 p13.2 locus (D17S796 marker) and formation of colonies in agar‐methocel (colony efficiency (CE)), loss of ductulogenic capacity in collagen matrix, and resistance to anti‐Fas monoclonal antibody (Mab)‐induced apoptosis. For testing the role of that specific region of chromosome 17 in the expression of transformation phenotypes, we transferred chromosome 17 from mouse fibroblast donors to BP1‐E cells. Chromosome 11 was used as negative control. After G418 selection, nine clones each were randomly selected from BP1‐E‐11neo and BP1‐E‐17neo hybrids, respectively, and tested for the presence of the donor chromosomes by fluorescent in situ hybridization and polymerase chain reaction‐based restriction fragment length polymorphism (PCR‐RFLP) analyses. Sensitivity to Fas Mab–induced apoptosis and evaluation of transformation phenotype expression were tested in MCF‐10F, BP1‐E, and nine BP1‐E‐11neo and BP1‐E‐17neo clones each. Six BP1‐E‐17neo clones exhibited a reversion of transformation phenotypes and a dose dependent sensitivity to Fas Mab‐induced apoptosis, behaving similarly to MCF‐10F cells. All BP1‐E‐11neo, and three BP1‐E‐17neo cell clones, like BP1‐E cells, retained a high CE, loss of ductulogenic capacity, and were resistant to all Fas Mab doses tested. Genomic analysis revealed that those six BP1‐E‐17neo clones that were Fas‐sensitive and reverted their transformed phenotypes had retained the 17p13.2 (D17S796 marker) region, whereas it was absent in all resistant clones, indicating that the expression of transformation phenotypes and the sensitivity of the cells to Fas‐mediated apoptosis were under the control of genes located in this region.