Spots, blots, peaks and chips: proteomic approaches in autoimmune diseases.

During the past five years, investigations employing a variety of proteomic technologies have yielded a wealth of information on a number of autoimmune disorders. Animal models of autoimmune disease have been examined and have provided clues that can be useful in elucidating molecular pathways and mechanisms that play a role in autoimmune disorders. Human sera and body fluids have been analyzed and have resulted in the identification of autoantibodies that can be used as diagnostic markers in specific autoimmune diseases, and proteomic fingerprints of tissues and body fluids have resulted in the identification of individual proteins or patterns of protein expression that are deregulated in autoimmune diseases. The information provided by these proteomic studies are of diagnostic and therapeutic potential. This review provides an overview of the approaches used in the proteomic analyses of autoimmune disease.

Altered expression of estrogen receptor coregulators during human breast tumorigenesis.

The hypothesis that altered expression of specific coactivators/repressors of the estrogen receptor occurs during human breast tumorigenesis in vivo is examined in this study. Using in situ hybridization and reverse transcription-PCR assays, the expression of two coactivators (SRA and AIB1) and one repressor (REA) of the estrogen receptor was compared between matched breast tumors and adjacent normal human breast tissue. The levels of SRA and AIB1 mRNA were increased in tumors compared with normal tissues (n = 19; Wilcoxon matched pairs test; P < 0.01). In contrast, the expression of REA mRNA was not different between tumors and normal tissues (n = 19; Wilcoxon; P = 0.110). The ratios of AIB1:REA and SRA:REA were higher (Wilcoxon; P < 0.05) in tumors compared with normal tissues. Furthermore, SRA:AIB1 was higher (Wilcoxon; P = 0.0058) in tumors compared with normal tissues. Although our study is small, these data are consistent with the above hypothesis and suggest that such alterations may have a role in the altered estrogen action occurring during breast tumorigenesis.

Functional characteristics of a novel murine estrogen receptor-beta isoform, estrogen receptor-beta 2.

We have isolated a highly expressed splice variant mRNA of murine estrogen receptor-beta (ERbeta), mERbeta2, containing an in-frame 54 nucleotide insertion between exons 5 and 6 of wild-type mERbeta1. The predicted ERbeta2 protein contains 18 amino acids inserted in the ligand binding domain of mERbeta1. Recombinant protein generated by in vitro transcription/translation showed that mERbeta2 had markedly reduced ligand binding (K(D)=17.7+/-4.7 nM, mean+/-s.e.m., n=3) compared with mERbeta1-bound (3)H-estradiol (K(D)=0.56+/- 0.19 nM, mean+/-s.e.m., n=3). Both receptors bound similarly to palindromic estrogen responsive elements (EREs) in vitro and in vivo, and similarly bent DNA. Transcriptional activity was assessed using transient transfection analysis into a homologous murine cell line, NIH 3T3 cells. mERbeta1 transactivated ERE-tk-CAT reporter genes similarly to mERalpha, whereas mERbeta2 had little activity except at high ligand concentrations. However, under conditions in which mERbeta2 is unlikely to be ligand saturated, co-transfected mERbeta2 inhibited activity of mERalpha and possibly mERbeta1 on ERE-tk-CAT genes. Using a 'novel raloxifene responsive' gene reporter system (TGF-beta3-CAT), we found the ability of estradiol and LY117018 to activate both mERalpha and mERbeta1 on this promoter was identical, and mERbeta2 activity in the presence of either estradiol or LY117018 was only slightly less than that observed with either mERbeta1 or mERalpha. Both mERbeta1 and mERbeta2 when liganded with LY117018 inhibited transcription at a classical ERE-regulated promoter under these transfection conditions, which was in marked contrast to their stimulatory effect at the transforming growth factor-beta3 promoter. These data suggest that responsiveness of gene expression to a relatively highly expressed variant murine ERbeta isoform, mERbeta2, is both ligand and promoter specific. Determination of the relative level of expression of mERbeta1 mRNA and mERbeta2 mRNA in mouse tissues indicated predominance of mERbeta2 mRNA in some but not all tissues. These data suggest that the mERbeta2 may have some tissue-specific and promoter-specific modulatory effects.

Lumican and decorin are differentially expressed in human breast carcinoma.

Previous studies have shown that lumican is expressed and increased in the stroma of breast tumours. Lumican expression has now been examined relative to other members of the small leucine-rich proteoglycan gene family in normal and neoplastic breast tissues, to begin to determine its role in breast tumour progression. Western blot study showed that lumican protein is highly abundant relative to decorin, while biglycan and fibromodulin are only detected occasionally in breast tissues (n=15 cases). Further analysis of lumican and decorin expression performed in matched normal and tumour tissues by in situ hybridization showed that both mRNAs were expressed by similar fibroblast-like cells adjacent to epithelium. However, lumican mRNA expression was significantly increased in tumours (n=34, p<0.0001), while decorin mRNA was decreased (p=0.0002) in neoplastic relative to adjacent normal stroma. This was accompanied by a significant increase in lumican protein (n=12, p=0.0122), but not decorin. Further evidence of altered lumican expression in breast cancer was manifested by discordance between lumican mRNA and protein localization in some regions of tumours but not in adjacent morphologically normal tissues. It is concluded that lumican is the most abundant of these proteoglycans in breast tumours and that lumican and decorin are inversely regulated in association with breast tumourigenesis.

Expression of a repressor of estrogen receptor activity in human breast tumors: relationship to some known prognostic markers.

The expression of a specific repressor of estrogen receptor activity (REA) was investigated by a semiquantitative reverse transcription-PCR assay in 40 human breast tumor biopsy samples with respect to steroid hormone receptor status and other known prognostic variables. The data showed that REA expression was positively correlated with estrogen receptor (ER) levels as defined by ligand-binding assays (Spearman r = 03231; P = 0.042) and that the median level of REA mRNA was significantly (Mann-Whitney two-tailed test, P = 0.0424) higher in ER+ tumors (median = 94.5; n = 30) compared with ER- tumors (median = 645; n = 10), with no significant differences (P = 0.4988) associated with progesterone receptor status alone. In addition, REA expression was inversely correlated with tumor grade (Spearman r = -0.4375; P = 0.0054). When the tumors were divided into two groups based on grade, REA expression was significantly (Mann-Whitney two-tailed test, P = 0.0024) higher in low-grade (median = 97; n = 16) compared with high-grade (median = 76; n = 23) tumors. These results provide preliminary data suggesting that the expression of REA varies among breast tumors and is correlated with known treatment response markers and inversely correlated with a marker of breast cancer progression. REA together with ER status may be an improved marker of endocrine therapy responsiveness in human breast cancer.

Altered expression of estrogen receptor-alpha variant messenger RNAs between adjacent normal breast and breast tumor tissues.

Using semiquantitative reverse transcription-polymerase chain reaction assays, we investigated the expression of variant messenger RNAs relative to wild-type estrogen receptor (ER)-alpha messenger RNA in normal breast tissues and their adjacent matched breast tumor tissues. Higher ER variant truncated after sequences encoding exon 2 of the wild-type ER-alpha (ERC-4) messenger RNA and a lower exon 3 deleted er-alpha variant (ERD3) messenger RNA relative expression in the tumor compartment were observed in the ER-positive/PR-positive and the ER-positive subsets, respectively. A significantly higher relative expression of exon 5 deleted ER-alpha variant (ERD5) messenger RNA was observed in tumor components overall. These data demonstrate that changes in the relative expression of ER-alpha variant messenger RNAs occur between adjacent normal and neoplastic breast tissues. We suggest that these changes might be involved in the mechanisms that underlie breast carcinogenesis.

The human orphan receptor PXR messenger RNA is expressed in both normal and neoplastic breast tissue.

The expression of PXR mRNA and a variant PXR mRNA, deleted in 111 nucleotides in the ligand-binding domain, was detected by reverse transcription-PCR amplification in both normal and neoplastic human breast tissues. The level of PXR mRNA did not differ between breast tumors and their adjacent matched normal breast tissues. However, the expression of PXR mRNA did vary among breast tumors. A statistically significant inverse relationship was found between the level of PXR mRNA expression and estrogen receptor (ER) status, as defined by ligand binding analysis. The level of PXR mRNA expression in ER+ tumors (median = 22.4, n = 15) was significantly lower (P = 0.04) than the level of PXR mRNA expression in ER-tumors (median = 46.7, n = 15). No relationship with progesterone receptor status was found. These data raise the possibility that PXR has a role in human breast tissues.

Expression of the steroid receptor RNA activator in human breast tumors.

The expression of the recently described steroid receptor RNA activator (SRA) was measured by semiquantitative reverse transcription-PCR within 27 independent breast tumors, spanning a wide spectrum of grade and estrogen receptor (ER) and progesterone receptor (PR) levels. Subgroup analysis showed that SRA expression was similar in ER+/PR+ (median = 65.5, n = 8) and in ER-/PR- (median = 94.6, n = 5) tumors. Interestingly, SRA expression in these two subgroups was significantly (Mann-Whitney rank-sum test, P < 0.05) lower than that observed in ER+/PR- (median = 156.4, n = 6) and ER-/PR+ (median = 144.8, n = 8) tumors. A variant form of SRA, presenting a deletion of 203 bp within the SRA core sequence, was also observed in breast tumor tissues. The relative expression of this new SRA isoform correlated with tumor grade (Spearman coefficient r = 0.53, n = 27, P = 0.004). These data suggest that changes in the expression of SRA-related molecules occur during breast tumor progression.

Mammaglobin, a potential marker of breast cancer nodal metastasis.

The Mammaglobin gene, a breast-specific member of the uteroglobin gene family, has been previously identified as being overexpressed in some breast tumours, but the cellular origin and relationship to tumour progression are unknown. Using a subtractive hybridization approach, mammaglobin mRNA has also been found to be overexpressed in the in situ compared to the invasive element within an individual breast tumour. Further study by in situ hybridization performed in 13 breast tumours, selected to include normal, in situ, and invasive primary tumour elements, and in most cases axillary lymph node metastases, revealed that mammaglobin expression occurs in all elements, is restricted to epithelial cells, and is significantly increased in tumour cells compared with normal cells ( p< 0.04). Analysis of mammaglobin expression within 20 independent primary breast tumours and their corresponding axillary lymph nodes revealed that all 13 lymph nodes positive and none of the seven nodes negative for metastatic breast carcinoma by histology were mammaglobin-positive by reverse transcription-polymerase chain reaction (RT-PCR) ( p=0.0001). These results suggest that mammaglobin could be a marker of axillary lymph node breast metastases.

Altered expression of exon 6 deleted progesterone receptor variant mRNA between normal human breast and breast tumour tissues.

The progesterone receptor (PR) is an important prognostic marker in breast cancer as well as a marker of responsiveness to endocrine therapies. The presence of several exon-deleted PR variant mRNAs in both normal and neoplastic breast samples has recently been reported. Amongst them, a variant mRNA deleted in exon 6 (D6-PR mRNA) that if translated would encode a truncated PR-like protein missing the hormone binding domain and one of the transactivating domains of the wild-type PR protein. In order to determine whether changes in D6-PR variant expression could occur during tumorigenesis, its expression was investigated by reverse transcription and polymerase chain reaction in ten normal reduction mammoplasty samples, nine breast tumours with high PR levels (> 100 fmol mg(-1) protein) and eight breast tumours with low PR levels (< 15 fmol mg(-1) protein), as determined by ligand binding assay. The relative expression of D6-PR to wild-type PR mRNA was lower (P< 0.01 ) in normal than in all tumour breast samples. Moreover, a trend to lower (P < 0.1) relative D6-PR expression was observed in high PR tumours, compared to low PR tumours. These data suggest that increased expression of D6-PR occurs during tumorigenesis.

Oestrogen receptor-alpha variant mRNA expression in primary human breast tumours and matched lymph node metastases.

We have shown previously that the relative expression of a truncated oestrogen receptor-alpha variant mRNA (ER clone 4) is significantly increased in axillary node-positive primary breast tumours compared with node-negative tumours. In this study, we have examined the relative expression of clone 4-truncated, exon 5-deleted and exon 7-deleted oestrogen receptor-alpha variant mRNAs in 15 primary breast tumour samples and in synchronous axillary lymph node metastases. Overall, there were no significant differences between the primary tumours and the matched metastases in the relative expression of these three specific variant mRNAs. Furthermore, the pattern of all deleted oestrogen receptor-alpha variant mRNAs appeared conserved between any primary and its matched secondary tumour.

Expression of estrogen receptor beta1, beta2, and beta5 messenger RNAs in human breast tissue.

A triple-primer PCR assay was developed, based on the coamplification of estrogen receptor (ER)-beta1, -beta2, and -beta5 cDNAs, to investigate the relative expressions of the corresponding mRNAs in breast cancer lines and in 53 independent breast tumors. The expression of ER-beta2 and ER-beta5 mRNAs was higher than that of ER-beta1 mRNA in both cancer cell lines and breast tumors. In breast tumors, increases in the ER-beta2:ER-beta1 and ER-beta5:ER-beta1 mRNA expression ratios were observed, which positively correlated with the level of tumor inflammation and tumor grade, respectively. A trend toward an increase of these ratios was also found in tumors, as compared to the normal adjacent breast tissue available for 13 cases. Our data suggest that changes in the relative expression of ER-beta1, -beta2, and -beta5 mRNAs occur during breast tumorigenesis and tumor progression.

Estrogen receptor-beta messenger RNA expression in human breast tumor biopsies: relationship to steroid receptor status and regulation by progestins.

When the level of estrogen receptor (ER)-beta mRNA in tumors, determined by reverse transcription-PCR, was assessed according to either ER status or PR status alone, determined by ligand binding assays, the level of ER-beta mRNA was significantly lower in PR+ tumors compared with PR- tumors (P = 0.036), and no association with ER status was found. Subgroup analysis showed that ER-beta mRNA expression in ER+/PR+ breast tumors was significantly less than in ER+/PR- (P = 0.009), ER-/PR+ (P = 0.029), and ER-/PR- (P = 0.023) groups. Interestingly, the ER-beta mRNA expression was specifically decreased by progestin in T47D breast cancer cells. The data suggest the possibility that expression of ER-beta in human breast tumors is a marker of endocrine therapy responsiveness.

Estrogen receptor-alpha mRNA variants in murine and human tissues.

A side-by-side comparison of several normal mouse and human tissues was undertaken in order to determine if exon-deleted variant ER-alpha mRNAs are expressed in the mouse. The data showed that the complex pattern of ER-alpha alternative splicing that is detected in multiple human tissues was not apparent in murine tissues. Only low levels of an exon-4 deleted ER-alpha transcript were detected in murine tissues, although multiple relatively abundant exon-deleted ER-alpha transcripts were detected in human tissues. The data support a species-specific difference in the expression of ER-alpha variant mRNAs between mouse and human.

Influence of estrogen receptor variants on the determination of ER status in human breast cancer.

Determination of estrogen receptor alpha (ER) status in breast cancer is an important predictive factor for clinical response to endocrine therapy. We have recently shown that discrepancies in ER status determined by immunohistochemical assay (ER-IHA) can occur between amino-terminal (1D5) and carboxyl-terminal (AER-311) targeted ER antibodies and that those tumors which demonstrate discordance are associated with increased expression of truncated ER variant mRNAs. In this study, we have explored this observation to examine if ER variant expression can exert a direct effect on ER-IHA or whether this association is attributable to the characteristics of the antibodies. ER negative cos-1 cells were transfected with expression vectors containing wild type ER (wt-ER) and/or a frequently expressed truncated variant, ER-clone-4 variant. We found that ER-IHA performed with the same N- and C-terminal targeting ER antibodies on cos-1 cells expressing wt-ER alone demonstrated no difference in signals by western blot (P > 0.1). However, co-expression of wt-ER and the truncated ER-clone-4 variant, resulted in discordant IHA results with relatively higher ER-IHA H-scores from N-terminal antibodies (P < 0.03). Furthermore, re-examination of a subset of breast tumors previously studied by ER-IHA showed persistent concordance in 4/5 cases and persistent differences in 3/5 cases with a different pair of ER antibodies. We conclude that the presence of truncated ER variant proteins can interfere with the interpretation of ER status determined by IHA and that this may account for some of the inconsistencies between ER status and response to endocrine therapy.

The pathophysiological role of estrogen receptor variants in human breast cancer.

The accumulated evidence supports the expression of estrogen receptor variants at both the mRNA and protein levels. The relative level of expression of some estrogen receptor variant mRNAs and possibly progesterone receptor variant mRNAs is altered during breast tumorigenesis and breast cancer progression. The altered expression of estrogen receptor variants may effect estrogen signal transduction as well as the interpretation of assays where the estimation of estrogen receptor levels is used as a guide to treatment strategies and prognosis.

Altered estrogen receptor alpha and beta messenger RNA expression during human breast tumorigenesis.

Using a multiplex reverse transcription-PCR assay, we compared the relative expression of estrogen receptor (ER) alpha and ER-beta mRNA between adjacent samples of normal breast tissue and matched primary breast tumors obtained from 18 different patients. Within this cohort, 7 tumors were ER negative, and 11 tumors were ER positive, as determined by the ligand binding assay. No differences in the ratio of ER-alpha:ER-beta expression were observed in the ER-negative cohort. However, in the ER-positive cohort, a significantly (P < 0.02) higher ER-alpha:ER-beta ratio was observed in the tumor compared with that of the normal tissue component. Our data revealed that the increase in the ER-alpha:ER-beta ratio was due primarily to a significant (P < 0.05) increase in ER-alpha mRNA expression in conjunction with a lower ER-beta mRNA expression in the tumor compared with that of the normal compartment in some, but not all, ER-positive cases. These results suggest that the role of ER-alpha- and ER-beta-driven pathways and/or their interaction change during breast tumorigenesis.

Estrogen receptor-beta mRNA variants in human and murine tissues.

Estrogen receptor (ER)-beta mRNA splice variants have been identified in human breast tumors as well as normal human and mouse ovarian, uterine and mammary tissues. In both species transcripts deleted in exons 5 or 6, or 5 + 6 have been characterized by RT-PCR followed by cloning and sequencing. In mouse tissues an ER-beta transcript containing 54 nucleotides inserted in frame between exons 5 and 6 was identified. Interestingly, no equivalent of the mouse inserted transcript was detected in any of the four human tissues analyzed.