The PKCθ pathway participates in the aberrant accumulation of Fra-1 protein in invasive ER-negative breast cancer cells.

Fra-1 is aberrantly expressed in a large number of cancer cells and tissues, and emerging evidence suggests an important role for this Fos family protein in both oncogenesis and the progression or maintenance of many tumour types. Here, we show that the concentration of Fra-1 is high in invasive oestrogen receptor (ER)-negative (ER-) breast cancer cell lines, regardless of their Ras pathway status. All of the ER- cells express high levels of activated PKCθ, and the inhibition of PKCθ activity using RNA interference or the expression of a dominant-negative mutant results in a dramatic reduction in Fra-1 abundance. Conversely, the ectopic expression of constitutively active PKCθ leads to Fra-1 phosphorylation and accumulation in poorly invasive ER+ cells. This accumulation is due to the stabilisation of the Fra-1 protein through PKCθ signalling, whereas other members of the PKC family are ineffective. Both Ste20-related proline-alanine-rich kinase (SPAK) and ERK1/2, whose activities are upregulated by PKCθ, participate in PKCθ-driven Fra-1 stabilisation. Interestingly, their relative contributions appear to be different depending on the cell line studied. ERK1/2 signalling has a major role in ER- MDA-MB-231 cells, whereas Fra-1 accumulation occurs mainly through SPAK signalling in ER- BT549 cells. Fra-1 mutational analysis shows that the phosphorylation of S265, T223 and T230 is critical for PKCθ-driven Fra-1 stabilisation. Phosphorylation of the protein was confirmed using specific antisera against Fra-1 phosphorylated on T223 or S265. In addition, Fra-1 participates in PKCθ-induced cell invasion and is necessary for PKCθ-induced cell migration. In summary, we identified PKCθ signalling as an important regulator of Fra-1 accumulation in ER- breast cancer cells. Moreover, our results suggest that PKCθ could participate in progression of some breast cancers and could be a new therapeutic target.

Anti-growth factor activities of benzothiophenes in human breast cancer cells.

We have tested the effects of two Eli-Lilly compounds, LY 117, 018 and raloxifene, on E2-regulated and IGF-I-induced proliferation or AP-1 activity in human breast cancer cells. We now demonstrate that both molecules have strong antiestrogenic and anti-growth factor inhibitory effects in MCF7 cells. They were as potent as ICI 182, 780 and more efficient than OH-Tam to prevent estradiol action whereas their inhibition on IGF-I stimulation was less than with ICI 182, 780 and equivalent to that of OH-Tam. Moreover, raloxifene was the most efficient molecule to prevent IGF-I-induced AP-1 activity, with a significant effect observed with a concentration as low as 5 x 10(-11)M in the presence of IGF-I alone. Similar dose-response curves were obtained with a combined treatment of IGF-I and E2 with a 2log shift. Their action on IGF-I-induced proliferation was completely abrogated in MCF7 transfectants in which the expression of an antiestrogen-regulated protein tyrosine phosphatase, PTPL1, was abolished by antisense RNA transfection. Accordingly, they were both able to dose-dependently regulate the expression of PTPL1 and to interfere with the PI3-K/Akt pathway by drastically decreasing Akt phosphorylation exclusively in wild-type PTPL1 expressing cells. Our data altogether demonstrate that raloxifene has a potent inhibitory effect on IGF-I action, with a drastic effect on AP-1 triggered responses as well as on Akt phosphorylation, suggesting that it might be a useful therapeutic agent in tumors in which these signalling pathways become constitutively active.

Characterization of the physical interaction between estrogen receptor alpha and JUN proteins.

Activated estrogen receptor alpha (ERalpha) modulates transcription triggered by the transcription factor activator protein-1 (AP-1), which consists of Jun-Jun homodimers and Jun-Fos heterodimers. Previous studies have demonstrated that the interference occurs without binding of ERalpha to DNA but probably results from protein.protein interactions. However, involvement of a direct interaction between ERalpha and AP-1 is still debated. Using glutathione S-transferase pull-down assays, we demonstrated that ERalpha bound directly to c-Jun and JunB but not to FOS family members, in a ligand-independent manner. The interaction could occur when c-Jun was bound onto DNA, as shown in a protein-protein-DNA assay. It implicated the C-terminal part of c-Jun and amino acids 259-302 present in the ERalpha hinge domain. ERalpha but not an ERalpha mutant deleted of amino acids 250-303 (ER241G), also associated with c-Jun in intact cells, in the presence of estradiol, as shown by two-hybrid and coimmunoprecipitation assays. We also show that ERalpha, c-Jun, and the p160 coactivator GRIP1 can form a multiprotein complex in vitro and in intact cells and that the ERalpha.c-Jun interaction could be crucial for the stability of this complex. VP16-ERalpha and c-Jun, which both interact with GRIP1, had synergistic effect on GAL4-GRIP1-induced transcription in the presence of estradiol, and this synergistic effect was not observed with the ERalpha mutant VP16-ER241G or when c-Fos, which bound GRIP1 but not ERalpha, was used instead of c-Jun. Finally, ER241G was inefficient for regulation of AP-1 activity, and an ERalpha truncation mutant encompassing the hinge domain had a dominant negative effect on ERalpha action. These results altogether demonstrate that ERalpha can bind to c-Jun in vitro and in intact cells and that this interaction, by stabilizing a multiprotein complex containing p160 coactivator, is likely to be involved in estradiol regulation of AP-1 responses.

Estrogen regulated proteases and antiproteases in ovarian and breast cancer cells.

Cathepsin D (cath-D), an estrogen-regulated protease appears mostly to increase the number of tumor cells rather than their invasion or motility through the extracellular matrix. Estradiol is mitogenic but in vitro it also inhibits invasion and motility. In this review, we discuss the mechanism of this inhibition and the hormonal regulation of other proteases and protease inhibitors possibly involved in the control of tumor cell invasion by estrogens.

Analysis of the potential contribution of estrogen receptor (ER) beta in ER cytosolic assay of breast cancer.

Estrogen receptor (ER) content is the most useful parameter for predicting hormone response therapy in breast cancer. Assays available for detecting ER in breast tumor cytosol are ligand-binding assay (LBA), which detects both ERalpha and ERbeta, and the enzymatic immunoassay (EIA), in which monoclonal antibodies are directed against ERalpha. As shown in several studies, the 2 assays correlate and both are used routinely. However, some discrepancies between the 2 assays were found and explanations remain controversial. We evaluated ERalpha and ERbeta mRNA coexpression in breast tumors in order to study whether the presence of ERbeta could account for differences between LBA and EIA in the determination of ER protein level. Using HeLa cell lines transfected with either ERalpha or ERbeta, we confirmed that EIA, using H222 and D547 monoclonal antibodies, recognizes only ERalpha expression, whereas LBA detects both isoforms. In 119 breast tumor cytosols, the correlation between ER-EIA and ER-LBA was high (r = 0.72), although some discrepancies were found. When analyzing ER mRNA expression of samples with higher LBA values, no overexpression of ERbeta mRNA relatively to ERalpha mRNA were observed. There was a difference in ERbeta/ERalpha ratio between ER-negative and ER-positive samples, with a 10-fold increased median ratio in ER-negative samples (p = 0.01). We thus confirmed that the major form of ER in breast cancer is the ERalpha at both the protein and mRNA levels. Moreover, our data do not support the hypothesis that ERbeta expression could explain differences between LBA and EIA in the determination of ER protein level.

Unliganded and liganded estrogen receptors protect against cancer invasion via different mechanisms.

While estrogens are mitogenic in breast cancer cells, the presence of estrogen receptor a (ERalpha) clinically indicates a favorable prognosis in breast carcinoma. To improve our understanding of ERalpha action in breast cancer, we used an original in vitro method, which combines transient transfection and Matrigel invasion assays to examine its effects on cell invasiveness. ERalpha expression in MDA-MB-231 breast cancer cells reduced their invasiveness by 3-fold in the absence of hormone and by 7-fold in its presence. Integrity of hormone and DNA-binding domains and activating function 2 were required for estradiol-induced inhibition, suggesting that transcriptional activation of estrogen target genes was involved. In contrast, these domains were dispensable for hormone-independent inhibition. Analysis of deletion mutants of ERalpha indicated that amino acids 179-215, containing the N-terminal zinc finger of the DNA-binding domain, were required for ligand-independent receptor action. Among different members of the nuclear receptor family, only unliganded ERalpha and ERbeta reduced invasion. Calreticulin, a Ca2+-binding protein that could interact with amino acids 206-211 of ERalpha, reversed hormone-independent ERalpha inhibition of invasion. However, since calreticulin alone also inhibited invasion, we propose that this protein probably prevents ERalpha interaction with another unidentified invasion-regulating factor. The inhibitor role of the unliganded ER was also suggested in three ERalpha-positive cell lines, where ERalpha content was inversely correlated with cell migration. We conclude that ERalpha protects against cancer invasion in its unliganded form, probably by protein-protein interactions with the N-terminal zinc finger region, and after hormone binding by activation of specific gene transcription.

FRA-1 expression level modulates regulation of activator protein-1 activity by estradiol in breast cancer cells.

We compared the effect of estradiol on activator protein-1 (AP-1) activity in estrogen receptor positive (ER alpha+) and estrogen receptor negative (ER alpha-) human breast cancer cell lines transiently transfected with the AP-1-responsive reporter plasmid AP-1-TK-CAT and an ER alpha expression vector. While estradiol increased AP-1 activity in the ER alpha+ cell lines MCF7, ZR75.1, and T47D, it decreased (MDA-MB231 and BT20 cells) or had no significant effect (MDA-MB435 cells) on AP-1-mediated transcription in ER alpha- cells. Estradiol also inhibited AP-1 activity in ER alpha-MDA-MB231 cells stably transfected with ER alpha and in which ER alpha levels are close to those found in MCF7. Use of ER alpha mutant expression vectors demonstrated that the DNA-binding domain of ER alpha was needed for stimulation or inhibition of AP-1 activity by estradiol but suggested that ER alpha binding to estrogen-responsive elements was not required for these effects. Changes in regulation paralleled quantitative and qualitative changes in protein binding to AP-1 sites, as demonstrated by gel shift assay: protein binding was greater and DNA/protein complexes migrated faster for ER alpha--than for ER alpha+ cells. In fact, by Northern blot, a high level of Fra-1 mRNA was found in BT20 and MDA-MB231 cells as compared with ER alpha+ cells, and MDA-MB435 cells showed an intermediary level of expression. The differential expression of Fra-1 in MCF7 and MDA-MB231 cells was confirmed at the protein level by supershift experiments. In addition, overexpression of Fra-1 in MCF7 cells decreased the positive effect of estradiol while inhibition of Fra-1 expression in MDA-MB231 cells, by transient transfection of the Fra-1 antisense expression vector, abolished the negative effect of the hormone. In conclusion, we demonstrated that ER alpha- breast cancer cell lines differ from ER+ cells by a high level of AP-1 DNA-binding activity due, at least in part, to high Fra-1 constitutive expression. High Fra-1 concentration is crucial for the negative regulation of AP-1 activity by estradiol and thus may take part in estradiol-induced inhibition of cell proliferation in ER alpha- breast cancer cells transfected with ER alpha expression construct.

Retinoid regulation of human cathepsin D gene expression.

Retinoic acid (RA) regulation of human cathepsin D (cath D) gene expression was investigated in this study. RA enhanced cath D mRNA levels in a concentration-dependent manner in MCF-7 human breast carcinoma cells. RA regulation of cath D mRNA levels was predominantly transcriptional because RA also increased cath D gene core promoter activity. Upon further characterization of the core promoter we localized RA responsive region to proximal 112-bp. The proximal 112-bp region of cath D gene promoter harbours several retinoid response element (RARE)-like sequences. In gel shift experiments the sequence between -100 and -74 nucleotides in the CD112 region carrying imperfect direct repeat and a palindrome competed with RARE for binding to RAR/RXRs. These sequences, however, exhibited binding to protein complexes which could not be competed with unlabeled RARE or up-shifted with RAR/RXR-specific antibodies. We conclude that RA predominantly regulates cath D gene expression from the proximal 112-bp of the promoter region, but this regulation appears indirect.

Genomic cross-talk between the estrogen receptor and growth factor regulatory pathways in estrogen target tissues.

Cell proliferation and differentiation are modulated by both estrogens and growth factors. Even though these two kinds of effectors interact with receptors localized in different compartments of the cell, their nuclear end points are often on the same genes. We review here the first evidence for transcriptional interference between these two regulatory pathways which might be essential in understanding the control of cell proliferation and invasion in human estrogen-responsive cancers.

Differential effect of forms A and B of human progesterone receptor on estradiol-dependent transcription.

In addition to stimulation of the target gene fatty-acid synthetase, the synthetic progestin R5020 strongly inhibited estradiol-induced pS2 and cathepsin D mRNA levels in MCF7 human breast cancer cells as shown by Northern blot analysis. Inhibition was half-maximal with 30 pM R5020, and the antiprogestin RU486 had only a weak effect. Two human progesterone receptor isoforms have been described; isoform A is a truncated form of isoform B and lacks the 164 N-terminal amino acids. We hypothesized that the two isoforms could have a differential capacity to transrepress estrogen-induced responses. Therefore, in MDA-MB231 cells containing no progesterone and estrogen receptors, we transiently transfected progesterone receptor expression vectors coding for form B (hPR1 or hPR0) or form A (hPR2) along with the estrogen receptor expression vector HEO. We show that R5020 inhibited estradiol-induced transcription of the pS2-CAT reporter plasmid only in cells selectively expressing isoform B. The same results were obtained when progesterone receptor isoforms were overexpressed in MCF7, Ishikawa, HeLa, or NIH-3T3 cells. Transrepression was dependent on the promoter context since the extent of inhibition by isoform B was higher when evaluated with pS2 or cathepsin D nonpalindromic estrogen-responsive element-mediated transcription than with the perfect palindromic form of the vitellogenin gene. Isoform A was inefficient regardless of the reporter construct used. Inhibition varied with the isoform ratio, and isoform B had a dominant effect, with > 70% inhibition measured in cells transfected with the same amount of both progesterone receptor isoforms. Progestin repressed only one of the two transcription activation functions of the estrogen receptor, AF-2, which corresponds to the hormone-binding domain. We conclude that differential expression of progesterone receptor isoforms could be responsible for a tissue-specific inhibition of estrogen target genes by progestins.

Synthetic antiestrogens modulate induction of pS2 and cathepsin-D messenger ribonucleic acid by growth factors and adenosine 3',5'-monophosphate in MCF7 cells.

In MCF7 human breast cancer cells, the antiestrogens 4-hydroxy-tamoxifen and ICI 164,384 inhibit the mitogenic activity of epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I). These growth factors also stimulate the expression of cathepsin-D and pS2 genes. Therefore, we studied the effects of antiestrogens on growth factor induction of pS2 and cathepsin-D mRNA. The two antiestrogens strongly inhibited the transcriptional induction of pS2 by growth factors. On the contrary, estradiol and IGF-I or EGF had an additive effect on pS2 mRNA accumulation. Growth factor induction of cathepsin-D was also inhibited by ICI 164,384. By contrast, 4-hydroxytamoxifen had an agonist effect on cathepsin-D and an additive effect on IGF-I-induced mRNA. When 12-O-tetradecanoylphorbol-13-acetate or 8-bromo-cAMP (8-Br-cAMP) was used instead of growth factors, similar effects of 4-hydroxytamoxifen and ICI 164,384 were obtained on pS2 (12-O-tetradecanoylphorbol-13-acetate and 8-Br-cAMP) and cathepsin-D (8-Br-cAMP) induction. A mechanism based on the classical competitive inhibition by antiestrogens of estrogen binding and action on the estrogen receptor was very unlikely, as 1) no antigrowth factor activity was obtained with R5020, which was a potent inhibitor of estrogen induction of pS2 and cathepsin-D mRNA; 2) in the Ishikawa endometrial cancer cell line, the cathepsin-D gene is unresponsive to estrogen, but was inhibited by antiestrogen after its induction by EGF or 8-Br-cAMP; and 3) the residual estrogen concentration in cells was too low to induce the expression of estrogen-specific genes. However, antiestrogens did not inhibit the expression of all genes induced by growth factors, as they were without effect on IGF-I induction of glyceraldehyde-3-phosphate dehydrogenase mRNA. These results demonstrate that antiestrogens can modulate the transcription of some growth factor-induced genes and strongly suggest that this effect is not due to interference with residual estrogens.

Estradiol increases and anti-estrogens antagonize the growth factor-induced activator protein-1 activity in MCF7 breast cancer cells without affecting c-fos and c-jun synthesis.

In estrogen receptor positive human breast cancer cells, anti-estrogens inhibit the mitogenic effect of growth factors in the absence of estrogens. As activator protein-1 (AP-1) activity is one of the first nuclear events following growth factor receptor activation, we studied the effects of estrogens and anti-estrogens on growth factor-induced AP-1 activity using transient transfection of the AP-1-responsive gene (AP-1)4-TK-CAT into MCF7 cells. The growth factor-induced AP-1 response was increased by estradiol and inhibited by anti-estrogens in conditions where growth factor-induced c-fos and c-jun mRNA levels were unchanged by hormone and anti-hormone treatments. The same regulations were obtained when the AP-1 response was directly induced by co-transfection of c-fos and c-jun expression vectors. Co-transfection of the wild-type estrogen receptor HEGO amplified both effects. Inhibition of AP-1 activity by anti-estrogens was unlikely to be explained by the presence of residual estrogens in MCF7 cells. (i) anti-estrogens inhibited AP-1 activity in conditions where they had no effect on basal ERE-mediated activity levels, whereas estradiol was as efficient in stimulating both activities. (ii) The relative efficacy of the two anti-estrogens, OH-tamoxifen and ICI 164,384 in inhibiting these two activities was different; OH-tamoxifen was more efficient in inhibiting ERE-mediated activity, whereas ICI 164,384 was more efficient in trans-repressing AP-1-mediated activity. We conclude that in conditions where c-fos and c-jun syntheses were not affected, the estrogen receptor cooperated with growth factors to stimulate the AP-1 response when activated by estrogens but inhibited AP-1-mediated transcription when occupied by anti-estrogens.

Progestin-induced fatty acid synthetase in human mammary tumors: from molecular to clinical studies.

Fatty acid synthetase (FAS) is one of the first well-characterized progestin-induced proteins with available antibodies and cDNA. This paper reviews basic studies on FAS regulation in human breast cancer cell lines and recent data on the possible clinical significance of this new marker of hormone responsiveness in mammary cancer and benign breast diseases.

Computer-aided quantification of RNA levels detected by in situ hybridization of tissue sections.

A semi-automatic program, designed for non-computer scientists, was developed for quantification of RNA levels detected by in situ hybridization in heterogeneous tissues. A video camera was used to acquire microscopic images of autoradiographed tissue sections which are then digitized on a video monitor for semi-automated quantification of silver grains. We describe a data entry and analysis procedure for systematic quantification of RNA levels in which about 300 cells per tissue sample can be analysed within 10 min. When compared with visual counting, computer-aided quantification was found to be more objective and reliable, with the highest variation coefficient between individuals being 7.5% using computer-aided quantification, compared to 24% with manual counting of the same section areas. A comparative study of c-myc oncogene expression in 11 mammary adenocarcinomas from 3 independent experiments showed the good reproducibility of results using the computer-aided method, with an 18% maximum variation between experiments. The program, with its simple user-interface, reliability and rapidity, is convenient for measuring specific genetic expression levels in clinical studies requiring large numbers of specimens.

The anti-progestin RU486 stabilizes the progestin-induced fatty acid synthetase mRNA but does not stimulate its transcription.

Progestins induce fatty acid synthetase (FAS) in breast cancer cell lines, both increasing its gene transcription and mRNA stabilization (Joyeux, C., Rochefort, H., and Chalbos, D. (1989) Mol. Endocrinol. 4, 681-686). In vitro run-on transcription assays show that RU486, in contrast to progestin, inhibits FAS transcription by 40-50%. Moreover and surprisingly, anti-progestin RU486 also stabilizes FAS mRNA 3- to 4-fold in MCF7 cells as measured by chase experiments in the presence of actinomycin D or cordycepin or after short cell labeling with [3H]uridine. Dexamethasone is inefficient in increasing the half-life of FAS mRNA in MCF7 cells. RU486 had no effect on MDA-MB 231 cells which contain glucocorticoid but no progesterone receptors, indicating that the progesterone receptor is implicated in this regulation. RU486-induced mRNA stabilization allows delayed accumulation of FAS mRNA. These results indicate that the progesterone receptor can be activated separately to stimulate gene transcription or stabilize mRNA.

PIP: The mechanism of action of RU-486 on the progesterone receptor was examined in MCF7 breast cancer cells in vitro, using messenger RNA (mRNA) for the enzyme fatty acid synthetase (FAS) as an indicator. Transcription and half-life of FAS mRNA were assayed by Northern Blot hybridization, with the progestin R5020 as a reference, and the C3 and actin clones as negative controls. Incubating MCF7 breast cancer cells for 5 or 48 hours with RU-486 decreased FAS gene transcription, and inhibited synthesis of its mRNA 50%. Paradoxically, RU-486 lengthened the half-life of FAS mRNA from 6 to 24 hours, as demonstrated by short-term cell labeling with tritiated uridine in the presence of DNA synthesis inhibitors actinomycin D or cordycepin. This treatment did not stabilize mRNA for C3 or actin. The half-size of FAS mRNA was increased from 6 to 26 hours as measured by a pulse-chase experiment. The steady state level of FAS mRNA doubled in a 2-day incubation. The fact that the glucocorticoid dexamethasone did not stabilize FAS mRNA in MDA-MB23 breast cancer cells suggested that the observed stabilization of mRNA in the RU-486 experiments is mediated by the progesterone receptor. This is the 1st demonstration that a steroid hormone antagonist prevents transcription of a regulated gene, but stabilizes its mRNA, suggesting 2 different regulatory steps.

Regulation of fatty acid synthetase ribonucleic acid in the human endometrium during the menstrual cycle.

Fatty acid synthetase (FAS) is induced by progesterone in MCF7 and T47D breast cancer cell lines. We studied a possible in vivo regulation of expression of this gene by looking for FAS RNA in human endometrial biopsies at various periods of the menstrual cycle, using a cloned cDNA FAS probe. By Northern blot analysis, we detected the 8-kilobase FAS RNA throughout the cycle in 7 uterine samples. RNA in situ hybridization analysis of frozen sections from 22 endometrial biopsies showed that FAS RNA was present during follicular and luteal phases of the menstrual cycle in stromal and epithelial cells. RNA levels were quantified by counting autoradiographic silver grains using a computer-aided image analyzer. FAS RNA levels were significantly higher in epithelial cells than in fibroblasts (P less than 2 x 10(-5]. Furthermore, in both cell types, mean FAS RNA concentrations were higher in biopsies removed during the luteal phase than the follicular phase of the menstrual cycle (P = 2 x 10(-3) and 9 x 10(-5), respectively). A 2- to 3-fold increase in FAS RNA levels between days 8-14 and days 22-24 was detected in 2 normal patients who had previously undergone 2 successive biopsies. This increase was not observed in 2 patients with low plasma estradiol and progesterone concentrations, indicating a probable dysovulation. We conclude that FAS normally increases in both stromal and epithelial endometrial cells during the luteal phase. This increase is probably due to progesterone, which implies that FAS is induced in normal endometrium, as demonstrated in breast cancer.

Effects of progestins and menstrual cycle on fatty acid synthetase and progesterone receptor in human mammary glands.

Fatty acid synthetase (FAS) is induced by progestins in human breast cancer cell lines. To study its regulation in normal mammary glands, the FAS level was estimated by immunohistochemistry, using the biotin-streptavidin method, in ducts and lobules of normal tissues adjacent to nonproliferative benign breast lesions collected by biopsy. Rabbit polyclonal antibodies to human FAS specifically recognized the 250-kDa FAS from MCF7 cells, as shown by Western immunoblotting. An excess of purified FAS totally switched off FAS immunostaining of R5020-treated MCF7 cells, demonstrating the validity of FAS immunocytochemical detection. FAS labeling was quantified using a computer-aided image analyzer (SAMBA 2005) in 18 patients receiving progestin therapy from the 15th to the 25th day of the menstrual cycle and 26 untreated patients. In the 2 groups, FAS staining, absent of fibroblasts, was observed in the cytoplasm of epithelial cells. It was higher in lobules than in ducts and increased significantly from the follicular to the luteal phase in both structures. Progestin treatment increased FAS expression in both structures. Using monoclonal antibodies, progesterone receptor expression was measured in frozen serial sections. In patients receiving progestin treatment, the progesterone receptor level increased from the beginning of the cycle to day 14 and then decreased during the second part of the menstrual cycle, probably down-regulated by progestin, indicating a regulation similar to that in the endometrium. We conclude that FAS is induced by progestins in the ducts and lobules of human normal mammary glands as it is in human breast cancer cells. FAS may, therefore, be useful for studying the effect of progesterone in normal human mammary glands.

Expression of the progestin-induced fatty acid synthetase in benign mastopathies and breast cancer as measured by RNA in situ hybridization.

We have shown previously that fatty acid synthetase (FAS) is specifically induced by progestins in human breast cancer cell lines. To test the potential value of FAS as a clinical marker in breast diseases, we measured FAS expression in frozen sections of 22 benign and 27 malignant mammary tumors using in situ hybridization with the [35S]UTP alpha S-labeled FAS anti-sense mRNA. The hybridized RNA was quantified with an IMSTAR computerized image analyzer. We found FAS RNA in epithelial cells, but no labeling was detected in the connective tissue. In breast cancer, we found no correlation between FAS expression and estrogen receptor and progesterone receptor concentrations or status. However, the level of FAS was significantly (P less than .02) higher in premenopausal than in post-menopausal patients and increased with the grade of tumor differentiation (P less than .005 between the poorly and well-differentiated tumors). In benign mastopathies, high levels of FAS RNA were found in some cysts (mostly with apocrine metaplasia). In lobules, the FAS RNA level increased proportionally to the degree of proliferation determined by histological examination (P less than .015) and correlated with the H4 histone level measured in an adjacent section using in situ hybridization (r = 0.85, P less than .001). In ductal structures, a lower correlation (r = 0.64, P less than .01) was found between FAS and H4 RNA levels. We conclude that FAS RNA is overexpressed in some mammary tumors and may be useful in predicting high-risk mastopathies and less aggressive breast cancers.

Regulation of fatty acid synthetase by progesterone in normal and tumoral human mammary glands.

Progesterone and estrogens play an important role in the control of growth, differentiation and function of mammary epithelial cells. Their mechanism of action can be studied in human metastatic breast cancer cell lines (MCF7, T47D, ZR75-1...) that contain progesterone and estrogen receptors. We used this system to try to define progestin-regulated human genes which would permit to study progestin-regulation of gene expression in cell culture and to develop clinical markers of progestin-responsiveness. This paper summarizes our investigation of the progestin-regulated 250K protein, recently identified as human fatty acid synthetase (FAS).