Enhancer of the rudimentary gene homologue (ERH) expression pattern in sporadic human breast cancer and normal breast tissue
© Zafrakas et al; licensee BioMed Central Ltd. 2008
Received: 22 January 2008
Accepted: 23 May 2008
Published: 23 May 2008
The human gene ERH (Enhancer of the Rudimentary gene Homologue) has previously been identified by in silico analysis of four million ESTs as a gene differentially expressed in breast cancer. The biological function of ERH protein has not been fully elucidated, however functions in cell cycle progression, pyrimidine metabolism a possible interaction with p21(Cip1/Waf1) via the Ciz1 zinc finger protein have been suggested. The aim of the present study was a systematic characterization of ERH expression in human breast cancer in order to evaluate possible clinical applications of this molecule.
The expression pattern of ERH was analyzed using multiple tissue northern blots (MTN) on a panel of 16 normal human tissues and two sets of malignant/normal breast and ovarian tissue samples. ERH expression was further analyzed in breast cancer and normal breast tissues and in tumorigenic as well as non-tumorigenic breast cancer cell lines, using quantitative RT-PCR and non-radioisotopic in situ hybridization (ISH).
Among normal human tissues, ERH expression was most abundant in testis, heart, ovary, prostate, and liver. In the two MTN sets of malignant/normal breast and ovarian tissue,ERH was clearly more abundantly expressed in all tumours than in normal tissue samples. Quantitative RT-PCR analyses showed that ERH expression was significantly more abundant in tumorigenic than in non-tumorigenic breast cancer cell lines (4.5-fold; p = 0.05, two-tailed Mann-Whitney U-test); the same trend was noted in a set of 25 primary invasive breast cancers and 16 normal breast tissue samples (2.5-fold; p = 0.1). These findings were further confirmed by non-radioisotopic ISH in human breast cancer and normal breast tissue.
ERH expression is clearly up-regulated in malignant as compared with benign breast cells both in primary human breast cancer and in cell models of breast cancer. Since similar results were obtained for ovarian cancer, ERH overexpression may be implicated in the initiation and/or progression of certain human malignancies. Further studies on large breast cancer tissue cohorts should determine whether ERH could function as a prognostic factor or even a drug target in the treatment of human breast cancer.
The human ERH (Enhancer of the Rudimentary gene Homologue) gene encodes a protein highly conserved among eukaryotes. It has been identified after comparison of human ESTs with known genes in public databases, as a gene highly homologous to the enhancer of the rudimentary gene (DROER) in Drosophila melanogaster . The human ERH consists of 797 nucleotides, including an open reading frame of 312 nucleotides, encoding a protein of 104 amino acids. ERH has been mapped to the chromosomal band 7q34 by fluorescence in situ hybridization, and its expression was originally found in all normal human tissues examined . Details on the purification and crystallization of the human ERH protein have been reported recently .
Intriguingly, the enhancer of the rudimentary gene – named ERH in humans, er(h) in all non-human species, DROER in Drosophila and XERH in Xenopus – is highly conserved among vertebrates, invertebrates, and plants with various orthologs identified, while there are no homologous sequences known within the same species [3–5]. The human and mouse coding regions are 93% identical, and the amino acid sequence of their proteins are completely identical to each other, as well as to that of the frog (Xenopus laevis) [3, 4]. Furthermore, the human ERH protein has a 79.8% identity in amino acid sequence to that of D. melanogaster . Similarly impressive is the conservation of hydrophobic amino acids: Of the 27 positions occupied by hydrophobic amino acids in DROER, 25 (93%) are conserved in the mosquito and human, 23 (85%) in the nematode (C. elegans), and 20 (74%) in the Arapidopsis thaliana protein .
Using the in silico method electronic Northern (eNorthern) for RNA expression profiling, we have previously identified a genetic signature containing hundreds of candidate genes differentially expressed in breast and ovarian cancer . Characterization of a subset of these candidate genes, by cDNA dot blot using cancer profiling arrays, real-time RT-PCR, non radioisotopic RNA in situ hybridization (ISH) and immunohistochemistry has been reported elsewhere [7–11]. ERH was identified by this in silico approach among other genes, and this gave us the impetus to further study its expression in human breast cancer. In the present study, we present a systematic expression analysis of ERH in a panel of breast cancer cell lines and malignant and normal human breast tissue samples using Northern blot, quantitative RT-PCR and non-radioisotopic RNA ISH.
Tissue Specimens and RNA extraction
Baseline characteristics of primary breast carcinomas (n = 25)
Age at diagnosis
median 63.7 years (range 35–83 years)
= 50 years
Lymph node statusb
Estrogen receptor status
negative (IRSc 0–2)
positive (IRS 3–12)
Progesterone receptor status
negative (IRS 0–2)
positive (IRS 3–12)
Cell lines and RNA extraction
The non-tumorigenic breast cancer cell lines MCF12A and MCF10A, and five tumorigenic breast cancer cell lines (MCF7, SKBR3, T47D, ZR75-1, and BT-20) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured under recommended conditions. RNA from cell lines was extracted using the Trizol protocol (see above).
RNA expression analysis by northern blot in normal and malignant human tissues
ERH expression was analyzed by multiple tissue northern blots (MTN) in a panel of 16 normal tissues, a set of four matched breast cancer/normal breast, and a set of four matched ovarian cancer/normal ovarian tissue samples (Clontech, Heidelberg, Germany). The following normal tissues were analyzed: heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood leukocytes. The breast cancer MTN contained four pairs of invasive ductal carcinoma and matched normal breast tissue from four female patients (51, 36, 47, and 45 years old). The ovarian cancer MTN contained four pairs of malignant and normal ovarian tissue from four female patients (age 48 – serous papillary cystadenocarcinoma; age 30 – papillary cystadenocarcinoma; age 42 – granulosa-theca cell tumour; age 28 – adenocarcinoma).
Hybridization was performed using 25 ng of a gene-specific 32P-labeled DNA probe derived from a Unigene cDNA clone [GenBank accession number W33000]. This gene-specific cDNA fragment was radiolabelled using a Megaprime labelling kit (Amersham Biosciences, Braunschweig, Germany), hybridized overnight at 68°C using ExpressHyb Hybridization Solution (Clontech, Heidelberg, Germany), washed, and exposed to Kodak XAR-5 X-ray film with an intensifying screen (Eastman Kodak Co, Rochester, NY).
Primers and probes used in real-time RT-PCR
Non-radioisotopic RNA in situ hybridization
Non-radioisotopic RNA ISH was performed as previously described [7, 9]. In brief, riboprobes were obtained from plasmids containing cDNA inserts from the same clones used for array hybridization, linearized with restriction enzymes. Probes were digoxigenin-labeled using the Dig RNA labelling kit (Roche Applied Science, Mannheim, Germany). Paraffin embedded tissue specimens were deparaffinized, re-hydrated, washed two times in PBS, and processed according to the manufacturer's instructions (Roche Applied Science, Mannheim, Germany). Hybridized probes were detected using alkaline phosphatase conjugated anti-DIG antibodies and BM Purple as substrate (Roche Applied Science, Mannheim, Germany). After nuclear fast red counter staining (containing 5% aluminium sulphate; VWR International, Dublin, Ireland) sections were examined by a pathologist.
In order to compare the delta CT values of the real time RT-PCR results between specific groups the non-parametric Mann-Whitney-U-test was used.
Expression analysis using multiple tissue Northern blots
Cellular localization of ERH mRNA
Thus far, a variety of different functions have been attributed to the ERH protein, including enhancement of pyrimidine biosynthesis, a role in cell cycle regulation, cell growth, repression of transcription and interaction with p21(Cip1/Waf1) [4, 12–14]. Moreover, little is known about the role of ERH in human malignancies. In the present study, the gene expression pattern of ERH has been systematically analyzed in normal human tissues, breast cancer cell lines and a panel of malignant and normal human breast and ovarian tissue samples using three independent methods. Our analysis provides useful insights regarding the still inconsistently defined biological role of ERH. It is furthermore the first study to supply a systematic data set concerning ERH expression in a human malignancy.
The Enhancer of rudimentary gene was first discovered in Drosophila melanogaster: Mutations of the rudimentary gene (r), encoding a multifunctional protein for the first three enzymatic activities of the pyrimidine biosynthetic pathway, lead to a characteristic truncation of the wings. Mutation of another gene led to more severely truncated wings in the background of r-mutations, and thus this gene was named Enhancer of the rudimentary . Later studies provided experimental evidence suggesting that the wild-type ERH protein is a transcriptional (co-)repressor [4, 15, 16] and its activity is not restricted to the pyrimidine biosynthetic pathway. Another hypothesis was based on the observation that ERH is only weakly expressed in non-dividing cell lines of hepatocytes while it is abundantly expressed in fibroblast and hepatoma cell lines, suggesting that ERH might have a function necessary for normal cellular proliferation .
However, our findings do not support a key role of ERH in cellular proliferation, since ERH seems to be heterogeneously expressed in normal tissues expected to be transcriptionally active (more abundant expression in testis, ovary, prostate, and liver, but lower expression in placenta, kidney, pancreas, small intestine and colon – see Figure 1), as well as heterogeneously expressed in terminally differentiated tissues expected to have low transcriptional activity (very weak expression in lung, brain and peripheral blood leucocytes, but abundant in heart and skeletal muscle – see Figure 1).
Based on our finding that ERH is abundantly expressed in the majority of normal human tissues analyzed, it does not appear to be an ideal therapeutic drug target in human cancer treatment, but still such a role cannot be completely excluded. On the other hand, more abundant ERH expression in tumorigenic as compared with non-tumorigenic breast cancer cell lines (see Figure 3) and the trend of higher expression in malignant as compared with normal tissue samples (see Figure 4) suggest that ERH could be possibly used as a prognostic factor in breast cancer. Since similar results were obtained for ovarian cancer (see Figure 2), the expression pattern and the prognostic role of ERH in breast cancer and gynecologic malignancies awaits evaluation in future studies. Furthermore, this expression pattern suggests that ERH might be implicated in carcinogenesis and tumour-progression and this should be further investigated in appropriately designed functional studies.
ERH expression is clearly up-regulated in tumorigenic as compared with non-tumorigenic breast cancer cell lines (found by quantitative RT-PCR), and in malignant as compared with normal breast tissue samples (confirmed by three independent methods, i.e. MTN, quantitative RT-PCR, and non-radioisotopic ISH). These findings suggest that ERH is progressively up-regulated with tumour progression, and thus it could be used as a prognostic factor in breast cancer. A similar expression pattern was also found in ovarian cancer (by MTN), suggesting that ERH overexpression might be implicated in the initiation and/or progression of other human malignancies as well. Further studies on large breast cancer tissue cohorts are necessary in order to investigate whether ERH could function as a prognostic factor or even a drug target in the treatment of human breast cancer, while functional studies should delineate its possible role in carcinogenesis and tumour-progression.
- ERH Enhancer of the Rudimentary gene Homologue (Homo sapiens :
enhancer of the rudimentary gene homologue (species other than Homo sapiens)
Drosophila Enhancer of Rudimentary
Expressed Sequence Tag
Multiple Tissue Northern blot
Reverse Transcription – Polymerase Chain Reaction
In Situ Hybridization
- GAPDH :
- r rudimentary gene (Drosophila melanogaster :
Xenopus Enhancer of Rudimentary.
The study was supported by the German Ministry for Education and Research (BMBF grant 01KW0404 to E. Dahl) as part of the German Human Genome Project (DHGP).
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