Hormonal stimulation of lactose synthetase in mammary carcinoma.

A transplantable rat mammary carcinoma (R3230AC) synthesizes significant quantities of the mammary gland enzyme lactose synthetase in the immature virgin female rat. In this hormonal environment, mammary glands do not synthesize the enzyme. Prolactin further stimulates the enzyme activity in the tumors to levels found only in mammary glands of rats in late pregnancy or during lactation.

Human breast cancer: androgen action mediated by estrogen receptor.

Growth of the human breast cancer cell line MCF-7 is enhanced by androgens, but only at pharmacological concentrations. Although physiological concentrations of androgens translocate the androgen receptor into the nucleus, no mitogenic effects are observed. By contrast, pharmacological androgens translocate not only the androgen receptor but also the estrogen receptor, and at these high doses significantly increase both DNA and estrogen-dependent protein synthesis. We therefore propose that androgens stimulate MCF-7 cell growth not through the androgen receptor but rather through the estrogen receptor.

Predicting response to endocrine therapy in human breast cancer: a hypothesis.

We hypothesize that the presence of progesterone receptors in human breast tumors may be a sensitive marker for predicting response to endocrine therapy. Progesterone receptors were found in 56 percent of tumors with estrogen receptors, but were absent in tumors without estrogen receptors. Preliminary clinical correlations show that only those breast tumors with progesterone receptors regressed after endocrine therapy.

Prolactin receptors in estrogen receptor-deficient mammary carcinoma.

We demonstrate for the first time the presence of specific high-affinity receptors for prolactin in rat mammayy carcinoma. There appear to be no significant differences between normal rat mammary tissue and the transplantable, estrogen receptor-deficient R3230AC tumor with regard to the number of binding sites, the affinity of the receptor for prolactin, or the specificity of binding.

Mammary carcinoma: a specific biochemical defect in autonomous tumors.

Rat mammary carcinoma (R3230AC) which does not regress after ovariectomy has a markedly reduced amount of cytoplasmic estradiol binding protein. Cytoplasm from the tumor fails to interact with estradiol sufficiently to permit estradiol binding to tumor chromatin. This defect can be corrected in vitro by substituting cytoplasm, containing the binding protein, from rat uterus, thus permitting estradiol binding to tumor chromatin. The results indicate that the hormonal autonomy of this carcinoma is due to a lack of estradiol binding protein and not to the inability of estradiol to interact with the cell genome.

Human breast cancer: biologically active estrogen receptor in the absence of estrogen?

The human breast cancer cell line MCF-7 does not require estrogen for growth, but paradoxically its growth is inhibited by antiestrogens. Our results show that, unlike normal target cells, MCF-7 cells carry most of their estrogen receptors in their nuclei even when these receptors are not charged with estrogens. The receptors for androgen and for progesterone, on the other hand, are localized in the cytoplasm as usual. Therefore, it is possible that the growth of these abnormal cells is stimulated by estrogen receptor in spite of the absence of the hormone and that the binding of antiestrogen molecules antagonize this stimulation.

Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.

The HER-2/neu oncogene is a member of the erbB-like oncogene family, and is related to, but distinct from, the epidermal growth factor receptor. This gene has been shown to be amplified in human breast cancer cell lines. In the current study, alterations of the gene in 189 primary human breast cancers were investigated. HER-2/neu was found to be amplified from 2- to greater than 20-fold in 30% of the tumors. Correlation of gene amplification with several disease parameters was evaluated. Amplification of the HER-2/neu gene was a significant predictor of both overall survival and time to relapse in patients with breast cancer. It retained its significance even when adjustments were made for other known prognostic factors. Moreover, HER-2/neu amplification had greater prognostic value than most currently used prognostic factors, including hormonal-receptor status, in lymph node-positive disease. These data indicate that this gene may play a role in the biologic behavior and/or pathogenesis of human breast cancer.

Estrogen receptors in human breast cancer.

Specific quantitative techniques have been used to measure the cytoplasmic estradiol-binding protein (EBP) in human mammary carcinoma tissue specimens. Sucrose gradient centrifugation reveals EBP to sediment at 8S and 4S. Variable quantities of non-specific estradiol binding occurs in the 4S region of the sucrose gradient necessitating controls to insure specificity of the estradiol protein interaction. Using dextran-coated charcoal to separate bound from free estradiol Scatchard analysis finds the dissociation constant of the estradiol EBP interaction to be approximately 2.6x10(-10) M, indicative of the very high affinity of the ligand for the EBP. Quantitation of EBP sites in 64 primary and metastatic human breast tumors demonstrates a continuous spectrum of values from 0 to 612 fmol per mg of cytoplasmic protein. Specific 8S binding in the sucrose gradient centrifugation was not detected in specimens containing less than 9.0 fmol EBP per mg cytoplasmic protein. Since data from animal breast tumors and preliminary evidence from human breast tumors indicates an excellent correlation between the presence of abundant tumor EBP and endocrine-induced breast cancer regressions, precise quantitation of EBP in all human primary tumors may prove to be an excellent prognosticator of endocrine therapy in metastatic breast cancer.

Studies on the mechanism of action of progesterone in regulation of the synthesis of specific protein.

To study the process of hormone action, we have developed an in vitro system utilizing minced oviduct from estrogen-treated chicks incubated in tissue culture medium. Progesterone added to the medium induced synthesis of a specific protein, avidin, that continued for up to 96 hr. During this period there was no increase in total oviduct protein, ovalbumin, or lysozyme, which suggests the specificity of the progesterone effect. The induction process was dependent on new protein synthesis, since cycloheximide inhibited the induction completely. Actinomycin D in doses that prevented nuclear RNA synthesis, but not general protein synthesis, inhibited avidin production 70-90%. Avidin synthesis was not affected by 5-fluorouracil. The rate of DNA synthesis examined by thymidine-(3)H pulse labeling was not stimulated during avidin induction. Hydroxyurea (an inhibitor of DNA synthesis) and colchicine (a mitotic inhibitor) did not prevent induction. Studies utilizing uridine-(3)H pulses showed an effect on rapdly labeled nuclear RNA coincident with induction. Nuclear RNA polymerase activity increased before avidin induction. Since avidin was the only new protein synthesized in response to progesterone, the early stimulation of nuclear RNA synthesis and RNA polymerase activity would suggest a mechanism of action for this steroid at the transcription level of protein synthesis.

Autoradiographic localization of prolactin receptors in 7,12-dimethylbnez[a]anthracene-induced rat mammary carcinoma.

Prolactin receptors were localized by autoradiography in 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors from rats by incubation of tumor slices with [125I]ovine prolactin. Specific prolactin binding was confined to tumor cells, and nonspecific binding was present in alveolar spaces and connective tissue. In some tumors, all cells contained receptors; in others, up to one-half the cells remained unlabeled. These results suggested that variation in receptor content in DMBA-induced mammary tumors may be caused by two distinct populations of cells--one which contains receptors and another which possesses very few receptor sites or none at all.

PIP: In this investigation, prolactin receptor sites were localized by autoradiography in 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors from rats. Tumors were removed when they reached the size of 2 X 4 cm and sliced to a thickness of .5 mm. Sections cut from the slices were incubated for 3 hours in medium 199 (1% bovine serum albumin, 5 mM CaC12, 5mM HEPES buffer) at pH 7.4 and 400,000 counts/minute of iodine-125-ovine prolactin in the presence or absence of 1 mcg of unlabeled prolactin. After further preparation and storage for 4 months in a light-tight box slides were developed, fixed and stained and then photographed. All tumors were labeled by iodine-125-ovine prolactin. Prolactin receptors were associated with only half the total cells. Specific prolactin binding was confined to tumor cells and nonspecific binding was present in alveolar spaces and connective tissue. In some tumors prolactin recepotrs were found in all tummor cells, whereas in other tumors half of the cells did not contain any receptors, or very few. Reproductions of autoradiographs illustrate findings. Results suggest that variations in receptor content of the tumors may be caused by 2 distinct populations of cells. 1 type contains many receptors and the other very fewreceptor sites, or none at all.

Sensitive detection of estrogen receptor RNA by polymerase chain reaction assay.

We developed a simplified polymerase chain reaction (PCR) technique sensitive enough to detect estrogen receptor (ER) mRNA in breast tumor specimens from 1 microgram of total RNA. We simultaneously amplified a control gene such as beta-actin as a baseline to semiquantitate ER expression. In a preliminary test of this method on a small series of breast tumors, ER message was found as expected in a number of tumors found to be ER positive by ligand binding assay, but also ER negative in one tumor assayed. The ER in this last tumor contained a single base change in the hormone binding region, compared with the MCF-7 breast tumor cell line ER. Therefore, this PCR technique may be useful in the detection and cloning of rare ER transcripts from breast tumor biopsy specimens.

Breast cancer prognosis in a mixed Caucasian-Hispanic population.

Prognostic factors were compared between 1,249 Caucasian and 360 Hispanic women with breast cancer. A significantly greater proportion of Hispanic women were less than 50 years of age at diagnosis compared to Caucasian women (P less than .0001). Significantly more Hispanic women presented with tumors larger than 3 cm in diameter and with positive axillary lymph nodes than did Caucasian women (P = .004 and P = .0001, respectively). Significantly more Hispanic women were estrogen receptor (ER) negative (P = .005). However, when examined by age groups, the relationships between ethnicity and extent of disease and ER status were observed only among women over 50 years of age. Multivariate analyses demonstrated that there was no difference in survival between Caucasian and Hispanic women once adjustments were made for the number of positive lymph nodes and ER status. Although complete data were not available, it appeared that the incidence of breast cancer is lower in this population of Hispanic women than in Caucasian women.

Biological and clinical implications of heat shock protein 27,000 (Hsp27): a review.

Heat shock and other environmental and pathophysiologic stresses stimulate synthesis of heat shock proteins (Hsps). These proteins enable the cell to survive and recover from stressful conditions by as yet uncompletely understood mechanisms. Hsp27 is an important small Hsp (molecular weight, 27,000) found in human cells--both cancer cells and normal cells. This protein, besides its putative role in thermotolerance, is of special clinical interest because of recent data suggesting it may also play a role in drug resistance. In adults, Hsp27 is found particularly in several cell types such as breast, uterus, cervix, placenta, skin, and platelets. Although low-molecular-weight (small) Hsps have been found to be involved in embryogenesis of Xenopus and Drosophila, they have not been detected in human fetal organs. Regulation of expression of the Hsp gene (also known as HSPB1) has been considered a paradigm of gene regulation and is actively being studied in both prokaryotes and eukaryotes. In prokaryotes, the major Hsp genes are transcriptionally regulated by positively and negatively acting transcription factors. In eukaryotes, the genes encoding Hsps contain a regulatory DNA motif (inverted repeats of the pentameric sequence nGAAn) known as the heat shock element. Hsp27 may function as a molecular chaperone and in signal transduction pathways of different cell regulators, and Hsp27 and other Hsps may be active in development of resistance to stressful conditions and agents including cytotoxic drugs. Study findings indicate that some but not all estrogen-positive breast cancers express Hsp27, and overexpression of Hsp27 has been associated with both good and poor prognosis. In endometrial carcinomas, the presence of Hsp27 is correlated with the degree of tumor differentiation as well as with the presence of estrogen and progesterone receptors. Studies suggest, however, that detection of Hsp27 should not be considered to be a method for identifying hormone-responsive tumors or detecting estrogen receptors. Hsp27 seems to be a biochemical marker of estrogenic endometrial response. In patients with cervical cancer, Hsp27 is predominantly expressed in well-differentiated and moderately differentiated squamous cell carcinomas. In addition, expression of Hsp27 seems to be a negative prognostic factor for gastric cancer. Different isoforms of Hsp27 have been found in lymphoid tissue of patients with acute lymphoblastic leukemia, and the protein has also been associated with viral infections. These aspects are summarized and discussed in the present review.

Regulation of insulin-like growth factor-binding protein (IGFBP) expression by breast cancer cells: use of IGFBP-1 as an inhibitor of insulin-like growth factor action.

BACKGROUND: The insulin-like growth factors (IGFs) play an important role in normal growth and development. Evidence suggests they may also regulate the growth of several cancer cell types. This regulation is mediated by interactions between the receptors and ligands. There is now ample evidence to suggest that these interactions are also influenced by extracellular IGF-binding proteins (IGFBPs). Six different IGFBPs have been cloned. Some species may act to inhibit the mitogenic effects of the IGFs. Since breast cancer cells are responsive to the IGFs, it is possible that regulated expression of the IGFBPs affects tumor growth. Furthermore, inhibitory binding proteins could be used as neutralizers of IGF action. PURPOSE: We conducted this study to fully characterize the expression and hormonal regulation of IGF-binding protein expression in human MCF-7 breast cancer cells and to test the ability of purified IGFBP-1 to inhibit IGF-I action. METHODS: We used ribonuclease protection assays and Western ligand blotting to examine IGFBP expression in MCF-7 cells. The effect of IGF-I, IGFBP-1, and 17 beta-estradiol on serum-free cell growth was also studied. RESULTS: MCF-7 cells expressed IGFBP-2, IGFBP-4, and IGFBP-5 RNA and protein. These cells are dependent on estrogen for growth. In short-term culture, IGF-I can substitute for estrogen. Concomitant addition of IGF-I and estrogen enhanced stimulation above the level achieved by either factor alone. Estrogen also increased IGFBP production, making it unlikely that the IGFBPs induced by estrogen in MCF-7 cells could function as major inhibitors of IGF action. In contrast, exogenous addition of IGFBP-1 could block IGF-I-induced mitogenesis; this effect was reversible by excess IGF-I. CONCLUSIONS: The studies suggest that cancer cell growth may be regulated by endogenous IGFBP expression. Furthermore, the exogenous addition of the IGFBP-1 blocked IGF-I action and potentially could be used as a pharmacologic inhibitor of IGF action.

Heat shock protein hsp70 in patients with axillary lymph node-negative breast cancer: prognostic implications.

BACKGROUND: Cell synthesis of heat shock (stress-response) proteins is increased by a variety of environmental and pathophysiological stressful conditions. The 70-kd heat shock protein (hsp70) is thought to be involved in protein-protein interactions including those of the protein products of the human c-myc oncogene and the p53 (also known as TP53) tumor suppressor gene. PURPOSE: The purpose of this study was to investigate whether elevated hsp70 expression may be an indicator of biological stress experienced by a breast cancer and may, therefore, predict disease outcome. METHODS: Levels of hsp70 were determined by Western blot analysis in primary breast tumors from patients with negative axillary lymph nodes. We performed exploratory data analyses on a set of 162 primary breast cancers and constructed prognostic indexes of hsp70 expression levels. The optimal cutpoint for hsp70 expression was considered to be the value yielding the greatest separation for disease-free survival for the resulting two groups of patients. That cutpoint was then validated in a set of 345 tumors by univariate and multivariate analyses. Data were analyzed for overall survival, disease-free survival, tumor size, and patient age, as well as estrogen receptor and progesterone receptor status, ploidy (DNA content), and percentage of cells in S phase as determined by flow cytometry. RESULTS: Expression of hsp70 emerged as a useful prognostic factor, both in univariate and in multivariate analyses. Patients whose tumors had high expression of hsp70 had significantly shorter disease-free survival (P = .006). The other statistically significant factors were S-phase fraction (P = .008) and tumor size (P = .01). For patients who received adjuvant therapy, hsp70 was the only independent predictor of disease recurrence (P = .05). For those with tumors 1-3 cm in diameter, hsp70 (P = .008) and S-phase fraction (P = .02) were statistically significant predictors of recurrence. CONCLUSIONS: Measurement of hsp70 expression in primary tumors from patients with node-negative breast cancer may be useful in identifying patients at high risk for disease recurrence and thus may affect decisions regarding treatment after surgery. IMPLICATIONS: Future studies should be performed to determine if detection of hsp70 by immunohistochemistry can be used to predict clinical outcome and to better understand the relationships between hsp70 and the effects of various treatment modalities.

Association of p53 protein expression with tumor cell proliferation rate and clinical outcome in node-negative breast cancer.

BACKGROUND: The p53 (also known as TP53) tumor suppressor gene encodes for a nuclear phosphoprotein thought to regulate proliferation of normal cells. Most p53 mutations result in a nonfunctional protein that accumulates in tumor cell nuclei. These common mutations appear to be involved in the development and/or progression of several neoplastic diseases including human breast cancer. PURPOSE: Our purpose was to investigate the relationships between levels of mutant p53 protein expression, tumor cell proliferation rate, and clinical outcome in patients with node-negative breast cancer. METHODS: Expression of mutant p53 protein was evaluated by frozen-section immunohistochemistry (IHC) and light microscopy in 700 breast cancers from axillary lymph node-negative patients with long-term follow-up (median, 54 months). The immunostaining signal was expressed as the sum of scores representing the proportion and staining intensity of negative and positive tumor cell nuclei (ranges, 0 and 2-8, respectively). Statistical comparisons were made between levels of p53 protein expression and disease-free survival, overall survival, and tumor proliferation rate expressed as the percentage of cells in the S phase (%S phase) as determined by flow cytometry. RESULTS: Of the 700 tumors, 362 (52%) showed positive nuclear immunostaining (IHC score > 0). Proliferation rates were significantly higher (P = .0001) in positive tumors (median %S phase, 7.1%) than in negative tumors (4.1%). In a univariate cutpoint analysis, negative tumors (n = 388) versus low-positive tumors (IHC score = 2-6; n = 263) versus high-positive tumors (IHC score > 6; n = 99) showed progressively reduced disease-free survival (80% versus 72% versus 58% at 5 years, respectively; P < or = .05 for all pairwise comparisons). Analogous results for overall survival were 88% versus 84% versus 74%; only the result for negative versus high positive tumors was significant (P = .003). In a multivariate analysis, expression of p53 protein and high %S phase were independently associated with reduced disease-free survival (P = .008 and .01, respectively). CONCLUSIONS: Expression of mutant p53 protein was associated with high tumor proliferation rate, early disease recurrence, and early death in node-negative breast cancer. Despite the strong direct correlation between accumulation of p53 protein and tumor proliferation rate, both factors were independently associated with poor prognosis, suggesting that p53 may have other biological functions in addition to cell-cycle regulation. IMPLICATIONS: This test, when combined with other prognostic factors, may enhance our ability to identify node-negative breast cancer patients at high risk for early disease recurrence and/or death, for whom the use of adjuvant chemotherapy is unequivocally justified.

Progesterone receptor gene restriction fragment length polymorphisms in human breast tumors.

We examined the progesterone receptor (PgR) gene in tissue from both primary human breast tumors and normal placentas, detecting restriction fragment length polymorphisms (RFLPs) with the restriction endonucleases Pst I/Sst I and HindIII. There was a general agreement of the Pst I and Sst I polymorphisms in any individual tumor, suggesting that they define two alleles in the human PgR locus, one being characterized by a deletion of about 300 base pairs with respect to the other. Both primary human breast tumor specimens (n = 36) and human term placentas (n = 48) displayed similar allele frequencies and typical mendelian distribution of these Pst I/Sst I alleles. The previously reported HindIII PgR RFLP was also investigated in 132 breast tumors. The HindIII PgR gene RFLP did not display typical mendelian distribution in the breast tumors; the factors affecting the HindIII allele frequencies are presently unknown. Neither the HindIII RFLP nor the deletion defined by Pst I and Sst I correlated with PgR expression as determined by a ligand-binding assay, suggesting that neither is related to the heterogeneity of PgR expression seen in breast tumors.

Steroid receptors in human breast cancer.

The measurement of cytoplasmic estrogen receptor in tumors from patients with breast cancer is now well established. Potential uses include prognosis of early recurrence following mastectomy, stratifying patients for adjuvant therapies, and selecting or rejecting endocrine therapy in advanced breast cancer. The use of progesterone receptor measurements to improve our selection process has a good theoretical basis, and early reports now emerging indicate that the presence of both estrogen and progesterone receptor in a breast tumor predicts a high response rate to endocrine therapy. Further work in this area is required, however, since patients with estrogen receptor but not progesterone receptor still have an appreciable response rate.

New strategies for investigating antiestrogen action in breast cancer.

The major difference observed by us between estrogen and antiestrogen pathways in breast cancer cells occurs immediately after translocation. We have called the loss of nuclear receptor following estradiol administration "processing" and have hypothesized that is is a prerequisite for estrogen stimulation in breast cancer cells and that failure of processing relates to antiestrogen effects. In the very near future, monoclonal antibody technology could be used to dissect the processing of nuclear receptor and shed now light on the mechanism of antiestrogen action.

Follow-up study of HER-2/neu amplification in primary breast cancer.

Amplification of the HER-2/neu oncogene was determined in 362 tumors from patients with primary breast cancer (185 node-positive patients and 177 node-negative patients). The overall amplification rate was 33% (30% for node-negative patients; 31% for patients with 1-3 positive nodes; 40% for patients with greater than 3 positive nodes). Gene copy number was not associated with axillary lymph node status, steroid receptor status, or patient age but was weakly correlated with the size of the primary tumor. Amplification of the HER-2/neu gene did not correlate with either disease-free or overall survival in univariate or multivariate analyses. The results were unambiguously negative for patients with node-negative disease. Although the univariate results for node-positive patients were marginally significant (P = 0.07), the significance was not retained in multivariate analyses. Thus, while HER-2/neu amplification may be biologically important in primary breast cancer, it will only be of marginal utility as a prognostic factor for predicting clinical outcome.