Tumor suppressor genes in breast cancer.
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Animals↗
Biomedical subjects
S A Fuqua
Publications and source records attributed to S A Fuqua.
Analysis of loss of heterozygosity in 399 premalignant breast lesions at 15 genetic loci.
BACKGROUND: Usual ductal hyperplasia (UDH), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS) are risk factors for invasive breast cancer (IBC), suggesting that these lesions may be direct precursors of IBC. To identify genetic changes that may be important in the early development of precursor lesions and their progression to malignant or invasive disease, we examined 399 putative precursors (211 UDH, 51 ADH, 81 non-comedo DCIS, and 56 comedo DCIS) for loss of heterozygosity (LOH) at 15 polymorphic genetic loci known to exhibit high rates of loss in IBC. We also assessed the sharing of LOH by putative precursors and synchronous cancers. METHODS: The polymerase chain reaction was used to analyze DNA from microdissected archival specimens. RESULTS AND CONCLUSIONS: In hyperplasias from noncancerous breasts (i.e., without DCIS and/or IBC in analyses of hyperplasias), LOH at any given locus was rare (range, 0%-15%), although 37% of UDH and 42% of ADH lesions showed loss for at least one locus, suggesting that the development of hyperplasias can involve many different tumor suppressor genes. In DCIS from noncancerous breasts (i.e., without IBC in analyses of DCIS), LOH was common, with 70% of noncomedo lesions and 79% of comedo lesions showing at least one loss. In DCIS, substantial rates of loss (up to 37%) were observed at loci on chromosomes 16q, 17p, and 17q, suggesting that inactivated tumor suppressor genes in these regions may be important in the development of noninvasive breast cancer. When DCIS lesions from cancerous and noncancerous breasts were compared, substantially more LOH was observed in the cancerous breasts at a few loci (on chromosomes 2p, 11p, and 17q), suggesting that genetic alterations in these regions may be important in the progression to invasive disease. Among specimens harvested from cancerous breasts, 37% of UDH, 45% of ADH, 77% of noncomedo DCIS, and 80% of comedo DCIS lesions shared LOH with synchronous cancers at one locus or more, supporting the idea that the putative precursors and the cancers are genetically related.
Breast Diseases↗
Induction of heat shock protein 27 by hydroxyurea and its relationship to experimental metastasis.
Treatment of tumor cells with hydroxyurea (HU) has been shown to increase the experimental metastatic potential of these cells. We have previously described the induction of stress proteins (antioxidants) by HU in B16 murine melanoma cells and their relationship to the metastatic process. We have now investigated the induction by HU of another set of stress proteins, the heat shock proteins, and their role in experimental metastasis. HU markedly increased the cellular content of heat shock protein (hsp) 27 but not of hsp 90, 72/73, or 60 as measured by immunoblotting. The induction of hsp27 protein was preceded by a specific increase in hsp27 mRNA. Furthermore, HU-treated cells were more thermotolerant. To investigate the functional role of hsp27, human hsp27 cDNA was constitutively overexpressed in B16 cells at levels seen in HU-treated cells. In separate experiments, we induced a global increase in hsps by heat shock. Neither the hsp27 transfectants nor the heat-shocked cells demonstrated an increase in their experimental metastatic capacity. We conclude that hsp27 protein is increased by HU by the specific induction of hsp27 mRNA in B16 melanoma cells but increased hsp27 protein is not responsible for the increase in experimental metastasis. Since high levels of hsp27 are associated with metastatic disease in breast and ovarian cancers, but not in our experimental system, the functional role of hsp27 in metastasis requires further study.
Animals↗
Estrogen receptor variants.
Estrogen receptor (ER)3 gene expression in breast epithelium is an intricately regulated event. The human ER gene is transcribed from at least three different promoters which are expressed in a cell- and tissue-specific manner, and result in mRNA isoforms with unique 5'-untranslated exons. The ER is overexpressed in about two thirds of breast tumors, and even in early premalignant breast lesions compared with adjacent normal breast epithelium. Furthermore, normal breast epithelium as well as breast cancer tissue contains alternatively spliced ER mRNA variants where single or multiple exons are skipped. It is still unclear if any or all of the ER mRNA splicing variants are translated in vivo, and if a change in the balance of ER variants could effect tumor development and progression to hormone-independent growth. Although infrequent in primary breast cancer, single amino acid changes within the ER in metastatic disease which might influence cell proliferation may also contribute to neoplastic progression of the mammary epithelium.
Breast Neoplasms↗
Identification of genes expressed in the rat prostate that are modulated differently by castration and Finasteride treatment.
In mammals, testosterone and 5alpha-dihydrotestosterone (DHT) are the principal male hormones (androgens). Testosterone is the most abundant circulating androgen, and is converted in specific tissues to DHT by the 5alpha-reductase enzymes. Although each of these androgens binds to the same receptor protein (androgen receptor, AR), each exerts biologically distinct effects. Theories to explain the specific effects of testosterone and DHT have centered on kinetic differences of binding of androgens to the receptor or differences in the metabolic fates of the two hormones. In the current experiments, differential display PCR (ddPCR) was used to identify genes regulated differently by testosterone and DHT. Adult male rats were treated as follows: castrated, treated with Finasteride (an inhibitor of 5alpha-reductase) or left intact for ten days. RNA was prepared from the dissected prostates of these animals and used for ddPCR. Genes exhibiting four distinct patterns of regulation were observed among the mRNAs. Class 1 genes showed equivalent expression in intact and Finasteride-treated animals, but were absent in castrated animals (mRNAs D1, D2, D6, D10). Class 2 genes showed higher expression in intact animals, intermediate levels following Finasteride treatment, but were absent in castrated animals (mRNA D8). Two classes of gene were particularly intriguing: class 3 showed gene expression only in the intact animal (mRNA D7, D9) and class 4 showed increased gene expression following Finasteride treatment (mRNA D3). While the patterns observed for some of these genes (e.g. D8) suggest that the different biological effects of testosterone and DHT may be due to the lower affinity of the AR for testosterone and limiting tissue concentrations of androgen, our results also suggest that some genes expressed in the rat prostate may be regulated in fundamentally different ways in response to testosterone and DHT.
5-alpha Reductase Inhibitors↗
Novel nuclear matrix protein HET binds to and influences activity of the HSP27 promoter in human breast cancer cells.
Since the small heat shock protein hsp27 enhances both growth and drug resistance in breast cancer cells, and is a bad prognostic factor in certain subsets of breast cancer patients, we have characterized the transcriptional regulation of hsp27, with the long-term goal of targeting its expression clinically. The majority of the promoter activity resides in the most proximal 200 bp. This region contains an imperfect estrogen response element (ERE) that is separated by a 13-bp spacer that contains a TATA box. Gel-shift analysis revealed the binding of a protein (termed HET for Hsp27-ERE-TATA-binding protein) to this region that was neither the estrogen receptor nor TATA-binding protein. We cloned a complete cDNA (2.9 kb) for HET from an MCF-7 cDNA library. To confirm the identity of the HET clone, we expressed a partial HET clone as a glutathione S-transferase fusion protein, and showed binding to the hsp27 promoter fragment in gel-retardation assays. The HET clone is almost identical to a recently published scaffold attachment factor (SAF-B) cloned from a HeLa cell cDNA library. Scaffold attachment factors are a subset of nuclear matrix proteins (NMP) that interact with matrix attachment regions. Analyzing how HET could act as a regulator of hsp27 transcription and as a SAF/NMP, we studied its subnuclear localization and its effect on hsp27 transcription in human breast cancer cells. We were able to show that HET is localized in the nuclear matrix in various breast cancer cell lines. Furthermore, in transient transfection assays using hsp27 promoter-luciferase reporter constructs, HET overexpression resulted in a dose-dependent decrease of hsp27 promoter activity in several cell lines.
Breast Neoplasms↗
Quercetin inhibits heat shock protein induction but not heat shock factor DNA-binding in human breast carcinoma cells.
The flavonoid quercetin inhibits the heat-induced synthesis of heat shock proteins (hsps) in a variety of cell lines. To determine whether quercetin could inhibit hsp expression in breast cancer cells, we used the human breast cancer cell line, MDA-MB-231. Treatment of these cells with quercetin decreased the heat-induced synthesis of hsp27 and hsp70. However, inhibition of hsp expression did not correspond with the reduced ability of heat shock transcription factors (HSFs) to bind DNA. Furthermore, while quercetin treatment inhibited HSF2 expression, it only slightly affected HSF1 expression in breast cancer cells. In contrast, quercetin inhibited both HSF DNA-binding activity and HSF expression in HeLa cells. Our studies suggest that quercetin's action is cell-type specific, and in breast cancer cells may involve regulation of HSF transcriptional activity, rather than regulation of its DNA-binding activity.
Binding Sites↗
MCF7/LCC9: an antiestrogen-resistant MCF-7 variant in which acquired resistance to the steroidal antiestrogen ICI 182,780 confers an early cross-resistance to the nonsteroidal antiestrogen tamoxifen.
Acquired resistance to antiestrogens is a major problem in the clinical management of initially endocrine responsive metastatic breast cancer. We have shown previously that estrogen-independent and -responsive MCF7/LCC1 human breast cancer cells selected for resistance to the triphenylethylene tamoxifen produce a variant (MCF7/LCC2) that retains sensitivity to the steroidal antiestrogen ICI 182,780 (N. Brunner et al., Cancer Res., 53: 3229-3232, 1993). We have now applied stepwise selections in vitro from 10 pM to 1 microM ICI 182,780 against MCF7/LCC1 and obtained a stable ICI 182,780-resistant variant designated MCF7/LCC9. In contrast to 4-hydroxytamoxifen-selected MCF7/LCC2 cells, MCF7/LCC9 cells exhibit full cross-resistance to tamoxifen, despite never having been exposed to this drug. Significantly, tamoxifen cross-resistance arose early in the selection, appearing following selection against only 0.1 nM ICI 182,780. Although limited resistance to ICI 182,780 also was observed, full ICI 182,780 resistance was not detected until the selective pressure increased to 100 nM ICI 182,780. Cross-resistance to tamoxifen persisted throughout these additional selections. Despite their antiestrogen cross-resistance, MCF7/LCC9 cells retain a level of estrogen receptor expression comparable to that of their parental MCF7/LCC1 cells. Whereas MCF7/LCC1 cells retain an estrogen-inducible expression of progesterone receptors, MCF7/LCC9 cells exhibit an up-regulated expression of both progesterone receptor mRNA and protein that is no longer estrogen responsive. Estrogen-independent and -responsive components of the MCF7/LCC9 phenotype are apparent in vivo. These cells form slowly growing tumors in ovariectomized athymic nude mice but respond mitogenically upon estrogenic supplementation. The in vivo growth of MCF7/LCC9 tumors is not affected by treatment with ICI 182,780. Although there is some evidence of tamoxifen stimulation of tumor growth, this did not reach statistical significance. If this pattern of cross-resistance occurs in some breast cancer patients, administering triphenylethylene antiestrogens as a first-line therapy with a cross-over to steroidal compounds upon recurrence may be advantageous.
Animals↗
An estrogen receptor mutant with strong hormone-independent activity from a metastatic breast cancer.
Thirty tumors from metastatic breast cancer patients were screened for mutations in the estrogen receptor (ER) gene using single-strand conformation polymorphism and sequence analysis. Three missense mutations, Ser47Thr, Lys531Glu, and Tyr537Asn, were identified in these lesions. To investigate these mutated ERs or altered transcriptional activation function, expression vectors containing wild-type (wt) and mutant ERs were constructed and cotransfected with different estrogen response element reporter gene constructs into HeLa cells and MDA-MB-231 human breast cancer cells. The first two ER mutants were similar to wt ER. However, the Tyr537Asn ER mutant possessed a potent, estradiol-independent transcriptional activity, as compared to wt ER. Moreover, the constitutive activity of the Tyr537Asn ER mutant was virtually unaffected by estradiol, tamoxifen, or the pure antiestrogen ICI 164,384. Tyr537 is located at the beginning of exon 8 in the COOH-terminal portion of the hormone-binding domain of the ER, to which dimerization and transcription activation functions have also been ascribed. It has been identified as a phosphorylation site implicated in hormone binding, dimerization, and hormone-dependent transcriptional activity. Our results suggest that the Tyr537Asn substitution induces conformational changes in the ER that might mimic hormone binding, not affecting the ability of the receptor to dimerize, but conferring a constitutive transactivation function to the receptor. If present in other metastatic breast tumors, this naturally occurring ER mutant may contribute to breast cancer progression and/or hormone resistance.
Breast Neoplasms↗
Auto-regulation of the estrogen receptor promoter.
The presence or absence of estrogen receptor (ER) plays a key role in the diagnosis and treatment of breast tumors. It is known that patients with breast tumors classified as ER-positive have a better prognosis. Observations such as this have led us to explore the question of what makes some breast tumors overexpress ER whereas others express either very low levels or none at all. To begin a study of ER regulation, we first chose to examine a 200 bp region of the ER promoter located immediately upstream from the transcribed sequence of the human ER gene. We found that this region of the ER promoter contained basal activity when transiently transfected into ER-negative HeLa cells. ER promoter activity was further increased by co-transfection of a wild-type ER expression vector, and this increased activity was hormone-dependent. Several ER deletion mutant constructs were also able to increase the activity of the ER promoter fragment, but none could support equivalent activity as was seen with the full-length ER. Therefore, we conclude that the ER can contribute to its own expression, and we hypothesize that this auto-regulation may contribute to its overexpression in some breast tumors.
Autocrine Communication↗
A point mutation in the human estrogen receptor gene is associated with the expression of an abnormal estrogen receptor mRNA containing a 69 novel nucleotide insertion.
A novel ER-like mRNA containing a 69 nucleotide insertion precisely between exon 5 and 6 sequences was previously identified in human breast cancer biopsy samples. Data are presented which suggest that the 69 nucleotide sequence is normally present in intron 5 of the human estrogen receptor gene. The region corresponding to and surrounding this 69 nucleotide sequence was cloned and the nucleotide sequence determined. Cloning and sequencing of the corresponding region in genomic DNA isolated from a breast tumor expressing the 69 nucleotide inserted ER mRNA, revealed an A-->G point mutation immediately 3' to the 69 nucleotide sequence. This point mutation resulted in the generation of a consensus splice donor site. A consensus splice acceptor site sequence is normally present immediately 5' to the 69 nucleotide sequence. These data are consistent with the 69 nucleotide sequence being recognized as an exon by the splicing machinery, and resulting in processing of a mature ER mRNA containing the 69 nucleotide insert.
Base Sequence↗
The small heat shock protein hsp27 increases invasiveness but decreases motility of breast cancer cells.
The small heat shock protein hsp27 is often expressed at high levels in clinical breast tumors; however, its biological role in this disease still remains unclear. Several laboratories have recently shown that hsp27 expression is associated with aggressive tumor behavior. We hypothesized that hsp27 may influence the metastatic tumor process since this is part of tumor 'aggressiveness'. Therefore, we stably transfected breast cancer cell lines with sense (MDA-MB-231) and antisense (MDA-MB-435) hsp27 constructs, respectively, and examined various cellular aspects associated with the metastatic process. We found that hsp27-overexpressing clones lost their protrusive morphology, but exhibited higher membrane ruffling as compared to low expressing cells. hsp27 overexpression also resulted in decreased cell motility, but invasiveness, adhesion, and growth in Matrigel were all significantly increased. Conversely, antisense suppression of hsp27 expression resulted in increased cell motility, but decreased in vitro invasiveness. The direct correlation of hsp27 levels with metastasis was confirmed by an in vivo assay measuring the number of lung metastases in mice injected with hsp27-transfected cells. Thus, we conclude that hsp27 overexpression may influence the invasive and metastatic potential of human breast cancer cells.
Animals↗
Basal regulatory promoter elements of the hsp27 gene in human breast cancer cells.
The small human heat shock protein hsp27 has been shown to play important roles in diverse cellular processes such as actin polymerization, thermotolerance, growth, and chemotherapeutic drug resistance. Two breast cancer cell lines MCF-7 and MDA-MB-231 were used as a model to study the molecular mechanisms important for basal hsp27 promoter transcriptional activity. A genomic clone containing 1.1 kb of the hsp27 promoter was sequenced and the regulatory elements were characterized. The first 200 bp within this 5'-flanking region holds the majority of the transcriptional activity, according to transient transfection assays using a series of hsp27 promoter deletion fragments in luciferase reporter vectors. The basal activity of this fragment is largely confined to a G/C-rich region containing overlapping SP1 and AP2 transcription factor binding sites.
Base Sequence↗
Multiple splicing variants of the estrogen receptor are present in individual human breast tumors.
Transcript variants of the estrogen receptor (ER) were investigated in 109 primary breast tumors using reverse transcription-polymerase chain reaction (RT-PCR) and primers allowing analysis of each internal exon. A high incidence of different ER variants was observed, each individual tumor often manifesting multiple variants coexisting with corresponding wildtype (wt) ER. These variants, by sequence analysis confirmed to represent exon splicing deletions, included ER deltaE2 (found in 41% of the tumors examined), ER deltaE3 (74%), ER deltaE4 (72%), ER deltaE5 (66%), and ER deltaE7 (88%). No evidence of transcripts lacking exon 6 was found, although a ER deltaE5,7 variant manifesting simultaneous deletion of exons 5 and 7 was observed. The presence of specific ER variants was not significantly correlated to the status of ER and progesterone receptor (PgR) protein expression, as assessed by routine analysis, although a trend towards a higher incidence of ER deltaE3 and increased expression of ER deltaE7 in ER+/PgR- tumors was observed, suggesting a dominant inhibitory effect on normal ER function to be involved. Moreover, ER deltaE4 was more common in ER+ tumors, possibly due to a cytoplasmic sequestring of this variant lacking a nuclear localization sequence. The presence of ER variants was not associated to clinicopathological variables, and equally frequent in tumors from patients having recurred or remained recurrence-free during adjuvant tamoxifen therapy. In conclusion, although ER splicing variants are abundant in breast cancer, the present study provides no evidence for a direct role of these ER variants in tumor development and tamoxifen resistance. It remains possible, however, that minor cell clones within the tumor, undetected by analysis of tumor homogenates, displaying extreme difference in content of ER variants, could be selected for during therapy or metastasis.
Adult↗
RNA from air-dried frozen sections for RT-PCR and differential display.
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Adenocarcinoma↗
The small heat shock protein HSP27 is not an independent prognostic marker in axillary lymph node-negative breast cancer patients.
Heat shock protein 27 (hsp27) belongs to the family of heat shock proteins and is thought to be involved in thermotolerance, cell proliferation, drug resistance, and chaperone processes. The aim of this study was to investigate whether hsp27 levels are correlated with clinical outcome in axillary lymph node-negative breast cancer patients. We describe a Western blot study measuring hsp27 levels in 425 patients and an immunohistochemistry (IHC) study analyzing 788 patients. Results obtained by both methods were concordant. Univariate survival analysis was performed considering hsp27 either as an optimally dichotomized variable or as a continuous variable. Additional data include age at biopsy, tumor size, estrogen receptor (ER) and progesterone receptor status, tumor ploidy and percentage of cells in S phase, and adjuvant therapy. hsp27 levels correlated positively with ER status (P = 0.0001 in Western blot and IHC study), progesterone receptor status (P = 0.0001 in Western blot and IHC study), and aneuploidy (Western blot study, P = 0.0012; IHC study, P = 0.0004) but not with tumor size (Western blot study, P = 0.69; IHC, P = 0.53) or S phase (Western blot study, P = 0.19; IHC study, P = 0.38). Overall, there was no relationship between hsp27 expression and disease-free survival (Western blot study, P = 0.70/0.54; IHC, P = 0.47/0.30) or overall survival (Western blot study, P = 0.16/0.15; IHC, P = 0.46/0.78). Exploratory subset analyses defined by ER status and use of adjuvant treatment indicated that in ER+/untreated patients, high hsp27 levels correlated modestly with shorter disease-free survival (Western blot, P = 0.04/0.04; IHC, P = 0.11/0. 03). hsp27 is not a useful prognostic marker for the clinic in axillary lymph node-negative patients. However, the finding of modest prognostic value of hsp27 in the subgroup of ER+/untreated patients raises new questions about the biological function of hsp27 in breast cancer.
Adult↗
Differential expression of wild-type and variant ER mRNAs by stocks of MCF-7 breast cancer cells may account for differences in estrogen responsiveness.
The MCF-7 human breast cancer cell line is routinely used as a model system for the study of ER+, estrogen-responsive human breast cancer. Although most investigators have found these cells to be estrogen-responsive, there are reports that some stocks of MCF-7 cells may be either insensitive or may display decreased sensitivity to the mitogenic effects of estrogen. We report here that differences in estrogen responsiveness appear to be related to varying ratios of wild type to variant ER mRNAs. MCF-7 stocks which express a high ratio of wild type to variant ER transcripts are more responsive to the mitogenic effects of physiological concentrations of estradiol than stocks which express an elevated ratio of exon 5 deletion variant to wild type ER transcripts.
Breast Neoplasms↗
Molecular aspects of estrogen receptor variants in breast cancer.
Measurements of the estrogen receptor (ER) and the estrogen-induced progesterone receptor (PgR) are used by most clinicians as indicators of both overall prognosis and likelihood of response to endocrine therapy. Patients with ER+/PgR+ tumors have the highest likelihood of response; conversely, patients with ER-/PgR-tumors have the lowest likelihood of response. Unfortunately, most patients treated successfully with endocrine therapy eventually develop endocrine-resistant disease recurrence. In an effort to study potential mechanisms of endocrine resistance, we have studied discordant ER-/PgR+ tumors, in which the normally estrogen-regulated PgR gene is induced in the apparent absence of ER. Our laboratory has previously cloned, from ER-/PgR+ tumors, a variant ER mRNA precisely missing the sequence corresponding to ER exon 5, and has demonstrated that the truncated protein product translated from this variant RNA is capable of constitutively inducing the expression of an estrogen-responsive reporter gene in a yeast expression vector system (Fuqua et al. Cancer Res 51:105-109, 1991). In the present report we describe further experiments to characterize the activity and biological consequences of expression of this variant ER in human breast cancer cells. We have stably transfected MCF-7 human breast cancer cells with a mammalian expression vector for the exon 5 deletion variant ER. These transfected cells produce a truncated ER protein of the expected 40 kDa size. Cells expressing the exon 5 ER deletion variant constitutively express PgR, and manifest increased anchorage-independent colony formation in the absence of estrogen.(ABSTRACT TRUNCATED AT 250 WORDS)
Breast Neoplasms↗