The estrogen receptor alpha (ERa) plays a critical role in the pathogenesis and clinical behavior of breast cancer. To obtain further insights into the molecular basis of estrogen-dependent forms of this malignancy, we used real-time quantitative reverse transcription (RT)-PCR to compare the mRNA expression of 560 selected genes in ERa-positive and ERa-negative breast tumors. Fifty-one (9.1%) of the 560 genes were significantly upregulated in ERa-positive breast tumors compared with ERa-negative breast tumors. In addition to well-known ERa-induced genes (PGR, TFF1/PS2, BCL2, ERBB4, CCND1, etc.) and genes recently identified by cDNA microarray-based approaches (GATA3, TFF3, MYB, STC2, HPN/HEPSIN, FOXA1, XBP1, SLC39A6/LIV-1, etc.), an appreciable number of novel genes were identified, many of, which were weakly expressed. This validates the use of large-scale real-time RT-PCR as a method complementary to cDNA microarrays for molecular tumor profiling. Most of the new genes identified here encoded secreted proteins (SEMA3B and CLU), growth factors (BDNF, FGF2 and EGF), growth factor receptors (IL6ST, PTPRT, RET, VEGFR1 and FGFR2) or metabolic enzymes (CYP2B6, CA12, ACADSB, NAT1, LRBA, SLC7A2 and SULT2B1). Importantly, we also identified a large number of genes encoding proteins with either pro-apoptotic (PUMA, NOXA and TATP73) or anti-apoptotic properties (BCL2, DNTP73 and TRAILR3). Surprisingly, only a small proportion of the 51 genes identified in breast tumor biopsy specimens were confirmed to be ERa-regulated and/or E2-regulated in vitro (cultured cell lines). Therefore, this study identified a limited number of genes and signaling pathways, which better delineate the role of ERa in breast cancer. Some of the genes identified here could be useful for diagnosis or for predicting endocrine responsiveness, and could form the basis for novel therapeutic strategies.

The recent cloning of a second estrogen receptor (ER), designated ERb, has prompted a reevaluation of the role of ERs in breast cancer. We have developed and validated a real-time RT ± PCR assay to quantify ERa and ERb gene expression at the mRNA level in a series of 131 patients with unilateral invasive primary breast cancer. Although ERb expression showed wide variations in tumor tissues, its range (nearly three orders of magnitude) was smaller than that of ERa (nearly four orders of magnitude), suggesting that ERb is more tightly controlled than ERa. We observed a negative correlation between ERa and ERb expression.`ERa-negative' tumors (containing very low ERa mRNA levels) were associated with SBR histopathological grade III, RB1 underexpression and ERBB2 overexpression, con®rming that ERa negativity delineates poorly di erentiated tumors. The amount of ERa mRNA (but not that of ERb mRNA) increased with age and was consequently higher in postmenopausal patients' tumors. Expression of ERa (but not that of ERb) also correlated strongly with progesterone receptor (PR) and PS2 expression, suggesting that ERa has stronger transcriptional activity than ERb towards genes containing an ERE (estrogen response element) in their promoters. Interestingly, we found a negative correlation between the expression of ERb (but not ERa) and CCND1, which contains an AP1 element but not an ERE in its promoter. Taken together, these data con®rm that ERa and ERb play di erent roles in breast cancer, partly by mediating the transcription of various genes via di erent types of DNA enhancer. PR and PS2 seem to be mainly ERaresponsive genes, whereas CCND1 may be mainly ERbresponsive. Our ®ndings also underline the need for a reliable method, providing full range of quantitative values, to determine ERa and ERb status in the clinical setting. Oncogene (2001) 20, 8109 ± 8115.

IntroductionBreast cancer growth is regulated by estrogen, which acts by binding to its estrogen receptor alpha (ERα). The presence of ERα in breast tumors is used as a biological marker to identify patients who may respond to endocrine agents such as tamoxifen. However, one-half of the patients with ERα-positive tumors fail to respond favorably to antiestrogen treatment [1,2]. ABCC1 = ATP binding cassette C1 isoform; AUC = area under the curve; CYP2A6 = cytochrome P450 2A6 isoform; CYP2B6 = cytochrome P450 2B6 isoform; ERα = estrogen receptor alpha; ESR1/ERα = estrogen receptor alpha; FMO5 = flavin-containing monooxygenase 5 isoform; GSTM3 = glutathione S-transferase M3 isoform; MKI67 = proliferation-related Ki-67 antigen; NAT1 = N-acetyltransferase 1 isoform; PCR = polymerase chain reaction; ROC = receiver-operating characteristic; RT = reverse transcriptase; SULT2B1 = sulfotransferase 2B1 isoform; TBP = TATA box-binding protein. AbstractIntroduction: Little is known of the function and clinical significance of intratumoral dysregulation of xenobioticmetabolizing enzyme expression in breast cancer. One molecular mechanism proposed to explain tamoxifen resistance is altered tamoxifen metabolism and bioavailability.

The PEA3/E1AF/ETV4 gene encodes an Ets-related transcription factor that is expressed in the epithelial cells of the mammary gland. Previous reports have shown that PEA3 can up-regulate promoter activities of many genes associated with tumorigenesis. A significant fraction of those encode matrix metalloproteinases (MMP genes) required for degradation of the extracellular matrix. To better obtain a molecular characterization of PEA3 expression in sporadic breast cancer, we quantified PEA3 mRNA by means of real-time reverse transcriptase-polymerase chain reaction assay in a large series of human primary breast tumors. PEA3 expression showed wide variations in tumor tissues, being under-expressed in 30 of 130 (23.1%) and over-expressed in 18 of 130 (13.8%) compared with normal breast tissues. High PEA3 mRNA levels correlated significantly with Scarff-Bloom-Richardson histopathological grade III (P = 0.018) but not with poor prognosis, suggesting that PEA3 is a marker of tumor aggressiveness rather than a prognostic factor in human breast cancer. We also observed positive links between the expression of PEA3 and those of MKI67 and ERBB2 (P = 0.034 and P = 0.045, respectively) and an inverse relationship with ERalpha (P = 0.0016). Our results do not support recent findings suggesting that PEA3 could be a tumor-suppressor gene that can act therapeutically in ERBB2 over-expressed tumors. Our results also suggest major roles of the MMP2, NRG1 and CGB genes (which encode type I gelatinase, heregulin and human chorionic gonadotropin beta subunit, respectively) in the PEA3 pathway dysregulation observed in breast cancer. Taken together, the data confirm the role of the PEA3 gene in breast tumorigenesis, and suggest the existence of numerous other still unknown genes transactivated by the PEA3 transcription factor.