Expression of Long-chain Fatty Acyl-CoA Synthetase 4 in Breast and Prostate Cancers Is Associated with Sex Steroid Hormone Receptor Negativity

Previous studies have shown that key enzymes involved in lipid metabolic pathways are differentially expressed in normal compared with tumor tissues. However, the precise role played by dysregulated expression of lipid metabolic enzymes and altered lipid homeostasis in carcinogenesis remains to be established. Fatty acid synthase is overexpressed in a variety of cancers, including breast and prostate. The purpose of the present study was to examine the expression patterns of additional lipid metabolic enzymes in human breast and prostate cancers. This was accomplished by analysis of published expression databases, with confirmation by immunoblot assays. Our results indicate that the fatty acid-activating enzyme, long-chain fatty acyl-CoA synthetase 4 (ACSL4), is differentially expressed in human breast cancer as a function of estrogen receptor alpha (ER) status. In 10 separate studies, ACSL4 messenger RNA (mRNA) was overexpressed in ER-negative breast tumors. Of 50 breast cancer cell lines examined, 17 (89%) of 19 ER-positive lines were negative for ACSL4 mRNA expression and 20 (65%) of 31 ER-negative lines expressed ACSL4 mRNA. The inverse relationship between ER expression and ACSL4 expression was also observed for androgen receptor status in both breast and prostate cancers. Furthermore, loss of steroid hormone sensitivity, such as that observed in Raf1-transfected MCF-7 cells and LNCaP-AI cells, was associated with induction of ACSL4 expression. Ablation of ACSL4 expression inMDA-MB-231 breast cancer cells had no effect on cell proliferation; however, sensitivity to the cytotoxic effects of triacsin C was increased three-fold in the cells lacking ACSL4.     

Distinct genes related to drug response identified in ER positive and ER negative breast cancer cell lines

Breast cancer patients have different responses to chemotherapeutic treatments. Genes associated with drug response can provide insight to understand the mechanisms of drug resistance, identify promising therapeutic opportunities, and facilitate personalized treatment. Estrogen receptor (ER) positive and ER negative breast cancer have distinct clinical behavior and molecular properties. However, to date, few studies have rigorously assessed drug response genes in them. In this study, our goal was to systematically identify genes associated with multidrug response in ER positive and ER negative breast cancer cell lines. We tested 27 human breast cell lines for response to seven chemotherapeutic agents (cyclophosphamide, docetaxel, doxorubicin, epirubicin, fluorouracil, gemcitabine, and paclitaxel). We integrated publicly available gene expression profiles of these cell lines with their in vitro drug response patterns, then applied meta-analysis to identify genes related to multidrug response in ER positive and ER negative cells separately. One hundred eighty-eight genes were identified as related to multidrug response in ER positive and 32 genes in ER negative breast cell lines. Of these, only three genes (DBI, TOP2A, and PMVK) were common to both cell types. TOP2A was positively associated with drug response, and DBI was negatively associated with drug response. Interestingly, PMVK was positively associated with drug response in ER positive cells and negatively in ER negative cells. Functional analysis showed that while cell cycle affects drug response in both ER positive and negative cells, most biological processes that are involved in drug response are distinct. A number of signaling pathways that are uniquely enriched in ER positive cells have complex cross talk with ER signaling, while in ER negative cells, enriched pathways are related to metabolic functions. Taken together, our analysis indicates that distinct mechanisms are involved in multidrug response in ER positive and ER negative breast cells.     

Minireview: Inflammation: an instigator of more aggressive estrogen receptor (ER) positive breast cancers

Approximately 75% of breast tumors express the estrogen receptor (ER), and women with these tumors will receive endocrine therapy. Unfortunately, up to 50% of these patients will fail ER-targeted therapies due to either de novo or acquired resistance. ER-positive tumors can be classified based on gene expression profiles into Luminal A- and Luminal B-intrinsic subtypes, with distinctly different responses to endocrine therapy and overall patient outcome. However, the underlying biology causing this tumor heterogeneity has yet to become clear. This review will explore the role of inflammation as a risk factor in breast cancer as well as a player in the development of more aggressive, therapy-resistant ER-positive breast cancers. First, breast cancer risk factors, such as obesity and mammary gland involution after pregnancy, which can foster an inflammatory microenvironment within the breast, will be described. Second, inflammatory components of the tumor microenvironment, including tumor-associated macrophages and proinflammatory cytokines, which can act on nearby breast cancer cells and modulate tumor phenotype, will be explored. Finally, activation of the nuclear factor κB (NF-κB) pathway and its cross talk with ER in the regulation of key genes in the promotion of more aggressive breast cancers will be reviewed. From these multiple lines of evidence, we propose that inflammation may promote more aggressive ER-positive tumors and that combination therapy targeting both inflammation and estrogen production or actions could benefit a significant portion of women whose ER-positive breast tumors fail to respond to endocrine therapy.     

Estrogen receptor beta in breast cancer—Diagnostic and therapeutic implications

More than 10 years have passed since the discovery of the second estrogen receptor, estrogen receptor β (ERβ). It is now evident that ERα is not the only ER in breast cancer cells; in fact, ERβ is expressed in the majority of breast cancers although at lower levels than in the normal breast. In addition, ERβ is expressed in breast cancer infiltrating lymphocytes, fibroblasts and endothelial cells, all known to influence tumor growth. By overexpressing or knocking-out ERβ in breast cancer cell lines, several researchers have investigated its function with respect to proliferation and tumor growth. It appears that ERβ is anti-proliferative, in many ways antagonising the function of ERα. Furthermore, phytoestrogens have a binding-preference for ERβ and several epidemiological studies indicate a breast cancer preventing effect of this class of compounds. Tamoxifen is one of the standard, adjuvant treatments for ERα positive breast cancer, classically thought to mediate its effect through ERα. However, in several recent studies, ERβ has been described as a potential marker for tamoxifen response. In summary, experimental, epidemiological as well as diagnostic studies point towards ERβ as an important factor in breast cancer, opening up the possibility for novel ERβ-selective therapies in the treatment of breast cancer.     

Interpreting expression profiles of cancers by genome-wide survey of breadth of expression in normal tissues

A critical and difficult part of studying cancer with DNA microarrays is data interpretation. Besides the need for data analysis algorithms, integration of additional information about genes might be useful. We performed genome-wide expression profiling of 36 types of normal human tissues and identified 2503 tissue-specific genes. We then systematically studied the expression of these genes in cancers by reanalyzing a large collection of published DNA microarray datasets. We observed that the expression level of liver-specific genes in hepatocellular carcinoma (HCC) correlates with the clinically defined degree of tumor differentiation. Through unsupervised clustering of tissue-specific genes differentially expressed in tumors, we extracted expression patterns that are characteristic of individual cell types, uncovering differences in cell lineage among tumor subtypes. We were able to detect the expression signature of hepatocytes in HCC, neuron cells in medulloblastoma, glia cells in glioma, basal and luminal epithelial cells in breast tumors, and various cell types in lung cancer samples. We also demonstrated that tissue-specific expression signatures are useful in locating the origin of metastatic tumors. Our study shows that integration of each gene's breadth of expression (BOE) in normal tissues is important for biological interpretation of the expression profiles of cancers in terms of tumor differentiation, cell lineage, and metastasis.     

DNA methylation in breast cancers: Differences based on estrogen receptor status and recurrence

DNA methylation plays a role in the etiology of primary breast cancers. We analyzed paired primary and second breast tumors to elucidate the role of methylation in recurrence. Methylation profiles from paired primary and second breast tumors of 23 women were assessed using the HumanMethylation450 BeadChip. Twelve women had estrogen receptor positive (ERpos) primary and second tumors, five had estrogen receptor negative (ERneg) primary and second tumors, and six had an ERpos primary tumor but an ERneg second tumor. Stratifying tumors by occurrence revealed that the greater methylation previously associated with ERpos tumors, is more pronounced in primary tumors than in second tumors. Further, ERneg second tumors are more methylated than ERpos second tumors among women who had ERpos primary tumors. Pathway analyses using gene lists generated from comparisons of methylation in ERpos primary tumors from the paired sets with ERpos tumors from six women without recurrences, identified differences between groups based on the ER status of the second tumor. Hypermethylated genes of significantly enriched pathways were differentially associated with survival. DNA methylation profiles of ERpos primary breast tumors support the development and use of tumor methylation profiles for stratifying women with breast cancer both for prognosis and therapy.     

Proteomic characterization of Her2/neu-overexpressing breast cancer cells

The receptor tyrosine kinase HER2 is an oncogene amplified in invasive breast cancer and its overexpression in mammary epithelial cell lines is a strong determinant of a tumorigenic phenotype. Accordingly, HER2-overexpressing mammary tumors are commonly indicative of a poor prognosis in patients. Several quantitative proteomic studies have employed two-dimensional gel electrophoresis in combination with MS/MS, which provides only limited information about the molecular mechanisms underlying HER2/neu signaling. In the present study, we used a SILAC-based approach to compare the proteomic profile of normal breast epithelial cells with that of Her2/neu-overexpressing mammary epithelial cells, isolated from primary mammary tumors arising in mouse mammary tumor virus-Her2/neu transgenic mice. We identified 23 proteins with relevant annotated functions in breast cancer, showing a substantial differential expression. This included overexpression of creatine kinase, retinol-binding protein 1, thymosin 4 and tumor protein D52, which correlated with the tumorigenic phenotype of Her2-overexpressing cells. The differential expression pattern of two genes, gelsolin and retinol binding protein 1, was further validated in normal and tumor tissues. Finally, an in silico analysis of published cancer microarray data sets revealed a 23-gene signature, which can be used to predict the probability of metastasis-free survival in breast cancer patients.     

Estrogen receptor mediated inhibition of cancer cell invasion and motility: An overview

In this overview of results from our laboratory, we address the question of the role of estrogens during early steps of metastasis, involving cell invasion through the basement membrane and cell motility. The motility of several estrogen receptor (ER) positive breast (MCF7, T47D) and ovarian (BG-1, SKOV3, PEO4) cancer cell lines was studied using a modified Boyden chamber assay. We observed, in all cases, estradiol induced inhibition of cancer cell invasion and motility. A similar inhibitory effect of estradiol was found when the wild-type ER was stably transfected in the ER-negative MDA-MB231 cells and 3Y1-Ad12 cancer cells. The mechanism of this inhibitory effect is unknown. In ovarian cancer, however, it may involve intermediary proteins such as fibulin-1, an extracellular matrix protein that strongly interacts with fibronectin and which is induced by estrogen and secreted by ovarian cancer cells. We conclude that estrogens in ER-positive breast and ovarian cancers have a dual effect, since they stimulate tumor growth but inhibit invasion and motility. This may be consistent with the good initial prognostic value of ER-positive breast cancers compared to ER negative breast cancers noted in several clinical studies.     

Expression of insulin-like growth factor binding proteins in human breast cancer correlates with estrogen receptor status

The insulin-like growth factors (IGFs) have been implicated in the growth regulation of human breast cancer. Since the IGFs are associated with specific binding proteins (IGFBPs) which may modulate receptor/ligand interactions, production of IGFBPs by breast cancer cells could alter their IGF-dependent growth. This study examined the expression of IGFBPs 4, 5, and 6 in eight breast cancer cell lines (BCCLs) using ribonuclease (RNase) protection assays. IGFBP-4 mRNA was detected in all BCCLs studied. IGFBP-5 expression was higher in estrogen receptor (ER) positive cells, while IGFBP-6 mRNA was detected in only two ER negative BCCLs. We also found that E₂ treatment enhanced the expression of IGFBPs 2, 4, and 5 in T47-D cells. We next studied IGFBP mRNA expression in 40 primary breast tumors. All tumors expressed mRNA for IGFBPs 2–6 but none expressed IGFBP-1 message. IGFBP-3 expression was higher in ER negative tumors, while that of IGFBP-4 and -5 was higher in ER positive specimens. These differences were statistically significant (P < .05). Ligand blot analysis of tumor extracts confirmed the presence of IGFBPs in breast cancer tissues. Thus, differential IGFBP expression in ER positive and negative tumors suggests an important role for this protein in breast cancer biology.     

Establishment of two basal-like breast cancer cell lines with extremely low tumorigenicity from Taiwanese premenopausal women

The research of carcinogenetic mechanisms of breast cancer in different ethnic backgrounds is an interesting field, as clinical features of breast cancers vary among races. High premenopausal incidence is distinctive in East-Asian breast cancer. However, human cell lines derived from Asian primary breast tumor are rare. To provide alternative cell line models with a relevant genetic background, we aimed to establish breast cancer cell lines from Taiwanese patients of Han-Chinese ethnicity. Fresh tissue from mammary tumors were digested into organoids, plated and grown in basal serum-free medium of human mammary epithelial cells (HuMEC) with supplements. Cells were further enriched by positive selection with CD326 (epithelial cell adhesion molecule; EpCAM)-coated micro-magnetic beads. Two breast cancer cell lines derived from premenopausal women were successfully established by this method, and named Chang-Gung Breast Cancer 01 (CGBC 01) and 02 (CGBC 02). These two cell lines had a similar phenotype with weak expression of estrogen receptor (ER), progesterone receptor (PR), and without amplification of receptor tyrosine protein kinase erbB-2 (HER2/neu). Genome-wide Single Nucleotide Polymorphism (SNP) array showed multiple copy number alterations in both cell lines. Based on gene expression profiles, CGBC 01 and 02 were clustered into basal-like subtype with reference to the breast cancer cell line gene expression database. The tumorigenicity of both cell lines was extremely low in both anchorage-independence assay and transplantation into the mammary fat pads of nude mice. CGBC 01 and CGBC 02 are low tumorigenic breast cancer cell lines, established from Han-Chinese premenopausal breast cancer patients, which serve as in vitro models in studying the biological features of Asian breast cancer.     

Folate Receptor-α (FOLR1) Expression and Function in Triple Negative Tumors

Folate receptor alpha (FOLR1) has been identified as a potential prognostic and therapeutic target in a number of cancers. A correlation has been shown between intense overexpression of FOLR1 in breast tumors and poor prognosis, yet there is limited examination of the distribution of FOLR1 across clinically relevant breast cancer subtypes. To explore this further, we used RNA-seq data from multiple patient cohorts to analyze the distribution of FOLR1 mRNA across breast cancer subtypes comprised of estrogen receptor positive (ER+), human epidermal growth factor receptor positive (HER2+), and triple negative (TNBC) tumors. FOLR1 expression varied within breast tumor subtypes; triple negative/basal tumors were significantly associated with increased expression of FOLR1 mRNA, compared to ER+ and HER2+ tumors. However, subsets of high level FOLR1 expressing tumors were observed in all clinical subtypes. These observations were supported by immunohistochemical analysis of tissue microarrays, with the largest number of 3+ positive tumors and highest H-scores of any subtype represented by triple negatives, and lowest by ER+ tumors. FOLR1 expression did not correlate to common clinicopathological parameters such as tumor stage and nodal status. To delineate the importance of FOLR1 overexpression in triple negative cancers, RNA-interference was used to deplete FOLR1 in overexpressing triple negative cell breast lines. Loss of FOLR1 resulted in growth inhibition, whereas FOLR1 overexpression promoted folate uptake and growth advantage in low folate conditions. Taken together, our data suggests patients with triple negative cancers expressing high FOLR1 expression represent an important population of patients that may benefit from targeted anti-FOLR1 therapy. This may prove particularly helpful for a large number of patients who would typically be classified as triple negative and who to this point have been left without any targeted treatment options.     

Heterogeneity of ERα and ErbB2 Status in Cell Lines and Circulating Tumor Cells of Metastatic Breast Cancer Patients

Hormone therapy and anti-ErbB2 therapies are prescribed according to the hormone receptor [estrogen receptor α (ERα)/progesterone receptor] and ErbB2 status of the initial tumor, but it appears that circulating tumor cells (CTCs) and, consequently, the metastatic cells may have a different receptor status. As an attempt to meet the crucial need for identification of the subpopulation of patients that will benefit from more individualized therapies, rapidly evolving therapies should allow a profiling of the tumors and/or of the CTCs. We established a triple fluorescence staining using eight cell lines to visualize the CTCs (cytokeratin detection) and then to define their individual ERα and ErbB2 status. Afterward, we used this method for blood samples from 26 metastatic breast cancer patients. We identified major differences of ERα levels between the cell lines and even within one cell line. For the metastatic patients, we detected and characterized CTCs in 38.5% of the patients with a total of 92 CTCs. We could demonstrate that at least 69.6% of the CTCs exhibit an ERα and/or ErbB2 status different from the status of the primary tumor and that the CTCs from only 30% of the patients had no change of receptor status. Strikingly, heterogeneities of the status, aggregation, and size clearly appear within the CTCs. The data we generated outline the importance of a profiling not only of tumors but also of CTCs to establish individualized treatments. CTCs may then appear as new prognosis and treatment marker for both metastatic and adjuvant breast cancers.