Fibroblast growth factor signalling in mouse mammary gland development.

Fibroblast growth factors (Fgfs) and their receptors are important intercellular signalling molecules involved in many aspects of animal development. The aberrant expression of the Fgfs or the inappropriate activation of their cell surface receptors have been implicated in tumorigenesis. Here, we describe the evidence that as well as playing a critical role in the formation of the mammary primordia during embryogenesis, signalling by Fgfs is necessary for optimal lobuloalveolar development of the mouse mammary gland during pregnancy.

Fibroblast growth factor signalling and cyclin D1 function are necessary for normal mammary gland development during pregnancy. A transgenic mouse approach.

A number of growth factors, growth factor receptors and cell cycle regulatory proteins have been implicated in the genesis of mammary carcinomas both in animal models as well as in human breast tumour samples. Studies on the development of the mammary gland has revealed that several of the proto-oncogenes, or their closely related gene-family members, have a function in the normal growth and differentiation of the gland. In this review the role of fibroblast growth factor signalling and the critical requirement for the cell cycle regulator, cyclin D1 is discussed with respect to their normal function in mammary gland development and abnormal role in mammary carcinogenesis.

Mammary gland oncogenes as indicators of pathways important in mammary gland development.

The identification of dominant acting proto-oncogenes in mammary tumors from mice and humans has highlighted a number of signal transduction pathways that have subsequently been shown to have a role in normal mammary growth and differentiation. Here we describe the use of two different transgenic mouse strategies to investigate the function of two of these signalling pathways in the normal growth and differentiation of the mouse mammary gland during pregnancy.

Tyrosine kinase signalling in breast cancer: fibroblast growth factors and their receptors.

The fibroblast growth factors [Fgfs (murine), FGFs (human)] constitute a large family of ligands that signal through a class of cell-surface tyrosine kinase receptors. Fgf signalling has been associated in vitro with cellular differentiation as well as mitogenic and motogenic responses. In vivo, Fgfs are critical for animal development, and some have potent angiogenic properties. Several Fgfs have been identified as oncogenes in murine mammary cancer, where their deregulation is associated with proviral insertions of the mouse mammary tumour virus (MMTV). Thus, in some mammary tumours of MMTV-infected mouse strains, integration of viral genomic DNA into the somatic DNA of mammary epithelial cells was found to have caused the inappropriate expression of members of this family of growth factors. Although examination of human breast cancers has shown an altered expression of FGFs or of their receptors in some tumours, their role in the causation of breast disease is unclear and remains controversial.

Impaired mammary gland development in Cyl-1(-/-) mice during pregnancy and lactation is epithelial cell autonomous.

A specific defect of mice lacking cyclin D1 (Cyl-1(-/-)) is impaired development of the mammary gland during pregnancy. Here we show that when tissue from Cyl-1(-/-) mammary gland was transplanted into empty mammary fat pad of wild-type mice, the abnormal phenotype was maintained, indicating that it is epithelial cell autonomous. Nevertheless, in pregnancy the early proliferative response, which is characterized by extensive side branching, still occurs in the absence of cyclin D1. However, the response is atypical due to a marked reduction in the formation of accompanying alveoli. This reduction and delay in alveolar development persists throughout pregnancy. Moreover, although prolactin synthesis and release appear to be normal, lactogenesis is severely compromised. Consistent with the appearance of numerous side branches, progesterone receptor expression was readily detected in the mammary tissue of pregnant Cyl-1(-/-) mice, although there was a significant change in the ratio of the two (A and B) receptor isoforms. In Cyl-1(-/-) mammary glands during late pregnancy there was a decrease in the abundance of total and phosphorylated Stat5a, as well as delayed onset and substantial diminution of milk protein expression. The biochemical analysis suggests that there is a cumulative delay in growth and differentiation of the mammary gland during pregnancy that results in a severely compromised gland when, at parturition, further development is curtailed by the abrupt change in hormonal milieu.

Amplification, expression, and steroid regulation of the preprogalanin gene in human breast cancer.

The GALN gene encodes the preprogalanin protein that is cleaved to liberate the galanin peptide, a neuropeptide and tumor cell mitogen, and the galanin message-associated peptide, which is of unknown function. GALN is located at chromosome 11q13, a frequently amplified locus in diverse tumor types including breast cancer. To determine whether GALN may contribute to the tumor phenotype resulting from 11q13 amplification, we examined GALN amplification and preprogalanin mRNA levels in breast tumors and cell lines. GALN was amplified in a subset of breast tumors and cell lines that carried 11q13 amplifications. Preprogalanin mRNA was expressed in the majority of breast cancer cell lines, but Northern analysis failed to demonstrate a relationship between GALN amplification and preprogalanin mRNA levels. Eight of eight estrogen receptor-positive cell lines expressed detectable preprogalanin mRNA, and further investigation showed that preprogalanin mRNA was increased by treatment with estradiol and progestin and decreased by the removal of serum or treatment with antiestrogens. Thus, GALN amplification is unlikely to contribute to the phenotype conferred by 11q13 amplification in breast cancer, but preprogalanin mRNA is expressed by breast cancer cells and is under steroid hormone control in estrogen receptor-positive cells, opening the wider question of the role of this steroid-regulated neuropeptide in the normal and cancerous breast.

Mice lacking cyclin D1 are small and show defects in eye and mammary gland development.

Using homologous recombination, mice lacking cyclin D1 were generated by replacing most of the first exon of the Cyl-1 gene with sequences encoding neomycin resistance. Cyl-1(-1-) mice were viable and fertile but consistently smaller than their heterozygous or wild-type littermates. The nullizygous animals also showed two distinctive abnormalities: a severe retinopathy caused by impaired development of all layers of the retina and, in the mammary gland during pregnancy, a marked reduction in acinar development accompanied by a failure to lactate. Approximately 50% of animals also had a malformation of the jaw that manifested itself as a misalignment of the incisor teeth. Mouse embryo fibroblasts isolated from 14 day nullizygous, heterozygous, or wild-type embryos and grown under standard conditions showed similar cell-cycle and growth characteristics. Thus although cyclin D1 kinase activity may facilitate G1 progression, it is not essential for the development of most tissues and organs, and only a few specialized cell lineages are demonstrably sensitive to its absence.

Amplification of chromosome band 11q13 and a role for cyclin D1 in human breast cancer.

In this paper we describe how research on the mouse mammary tumor virus model of breast cancer resulted in the identification of an amplified region of DNA on human chromosome 11 band q13. This amplification occurs in approximately 15% of primary breast cancers. Several candidate oncogenes map within the amplicon but by analysing expression of these genes a strong case can be made for a role for cyclin D1 in tumorigenesis. Immunohistochemical staining indicates that cyclin D1 is expressed at elevated levels in around 40% of breast cancers, including those with the 11q13 amplification. The potential function of cyclin D1 as a regulator of early cell division cycle events would be consistent with a role in neoplasia.

Chromosome 11q13 markers and D-type cyclins in breast cancer.

One in six primary human breast cancers has DNA amplification centered on the cyclin D1 gene (CCND1) on chromosome 11q13. This genetic abnormality is preferentially associated with estrogen-receptor positive tumors and may define a sub-class of patients with an adverse prognosis. Although CCND1 has the credentials of a cellular oncogene, being a target for chromosomal translocation and retroviral integration, the 11q13 amplicon encompasses several other markers and CCND1 is not the only candidate for the key gene on the amplified DNA. To assess their relative importance, we have constructed a physical map of the amplified DNA and compared the extent and frequency of amplification across the region. Since it is likely that the gene providing the selective force for amplification will be expressed at elevated levels, we have also examined expression of both RNA and protein. By these criteria, cyclin D1 remains the strongest candidate for the key oncogene on the amplicon and we are currently investigating the functional consequences of its over-expression.

Amplification and overexpression of cyclin D1 in breast cancer detected by immunohistochemical staining.

Immunohistochemical staining with a monoclonal antibody against human cyclin D1 can be used to identify breast cancers that have an amplification of the q13 region of chromosome 11. In general, the intensity of staining is directly proportional to the degree of DNA amplification. In two unusual tumors, in which the CCND1 locus is highly amplified but staining is relatively weak, it appears that the DNA has undergone rearrangement and that the amplified/rearranged CCND1 allele may have reduced transcriptional activity. More significantly, the immunohistochemical technique identifies additional tumors in which the cyclin D1 gene is overexpressed with only marginal or undetectable increases in copy number, implying that other mechanisms can lead to deregulated expression. These results suggest that the frequency of overexpression is much higher than previously concluded from DNA-based analyses and that more than one-third of human breast cancers may contain excessive levels of cyclin D1. The technique we describe should facilitate the detection of this abnormality in a clinical setting and clarify its prognostic significance.

Chromosome 11q13 abnormalities in human breast cancer.

Amplification of markers centred on band q13 of human chromosome 11 is a consistent feature in a subset of oestrogen receptor positive breast cancers. Although the amplification was initially scored via FGF3/INT2, which has strong credentials as a mammary oncogene, current data suggest that some other gene on 11q13 provides the driving force for amplification. Here we have reviewed our understanding of the amplified DNA, the genes it encompasses and the evidence in favour of two candidate oncogenes, CCND1 and EMS1. As well as being among the most frequently amplified markers in the region, these genes are expressed at elevated levels as a consequence of amplification, and their predicted functions would be consistent with a role in tumorigenesis. Irrespective of the final conclusions regarding their biological relevance, the overexpression of CCND1 or EMS1 should provide a more amenable assay for the amplification and help to clarify its clinical significance.

Nonuniform expression of a mouse mammary tumor virus-driven int-2/Fgf-3 transgene in pregnancy-responsive breast tumors.

We have developed transgenic mice in which expression of the mouse int-2/Fgf-3 gene is regulated by a single long terminal repeat from mouse mammary tumor virus. Such mice contain and transmit a replica of the activated int-2/Fgf-3 allele present in a spontaneous mammary tumor from a BR6 mouse. Although free of infectious mouse mammary tumor virus and with a different genetic background, the transgenic mice develop pregnancy-responsive mammary epithelial proliferations that are similar to the early stages of tumorigenesis in the BR6 strain. Histological examination revealed that most of these tumors showed pronounced tubular and acinar structures, features usually associated with morphological differentiation. In some cases, the tumors were locally invasive, causing disruption of the dermis which manifested itself as local hair loss. In situ hybridization showed that patterns of transgene expression in the abnormal glands were markedly nonuniform. In contrast, mouse mammary tumor virus-induced neoplasms showed more uniform expression of int-2/Fgf-3, as did the urogenital epithelial proliferations that occur among males of this transgenic line. These data suggest that mammary tumors in virally infected animals may depend primarily on autocrine stimulation by the int-2/Fgf-3 gene product, whereas both autocrine and paracrine mechanisms may contribute to tumors and hyperplasias found in transgenic animals.

D11S287, a putative oncogene on chromosome 11q13, is amplified and expressed in squamous cell and mammary carcinomas and linked to BCL-1.

Approximately 15 to 20% of primary breast cancers and an even higher proportion of squamous cell carcinomas of the head and neck show amplification of DNA markers on band q13 of human chromosome 11. However, known genes within the amplified region, such as the FGF-related oncogenes INT-2 and HST-1, are very rarely expressed in these tumors. Here we show that another candidate oncogene, designated D11S287, implicated in the pathogenesis of parathyroid adenomas, is also amplified in breast cancers. Significantly, it is consistently coamplified with INT-2 and HST-1 in 36 out of 202 primary tumors, including one case in which the amplified unit did not encompass the translocation breakpoint marker BCL-1. This implies that D11S287 is on the same side of the breakpoint as INT-2, and pulsed-field gel electrophoresis indicates that D11S287 is less than 250 kb from the BCL-1 marker. Since D11S287 RNA was present at elevated levels in a group of tumors and cell lines in which the 11q13 region is amplified, it may be the key oncogene on this amplified unit, and could also be activated by BCL-1 translocations.

Gene amplification on chromosome band 11q13 and oestrogen receptor status in breast cancer.

We have analysed DNA from 183 primary breast cancers for amplification or rearrangement of a number of cellular proto-oncogenes, focusing primarily on a cluster of markers on the long arm of chromosome 11. Two of these oncogenes, INT2 and HST1, both of which encode members of the fibroblast growth factor family, are implicated in the generation of virally induced mammary tumours in mice. Here we confirm earlier reports that the q13 region of chromosome 11, in which these genes are tandemly linked, is modestly amplified in approximately 15% of primary human breast cancers. This amplification is confined, with one exception, to cases in which the oestrogen receptor (ER) levels are in excess of 20 fmol/mg protein (P = 0.001). However, DNA amplification does not usually result in detectable expression of either the INT2 or HST1 gene. The data imply that some other gene in the vicinity must contribute to the development of a subset of ER-positive tumours and that assessing the amplification of this region of DNA may be of value in defining a separate category of ER-positive tumour.

Effects of a monoclonal antibody against progesterone, on embryo transport, development and implantation in laboratory mice.

A monoclonal antibody against progesterone (11P27) given on Day 2 of pregnancy interrupted pregnancy in BALB/c mice but not in BCF1 mice. The reason for this strain difference remains unclear, although it may involve discrimination by the recipient's immune system. The effects in BALB/c mice were reversed by progestin treatment. A dose of 5 nmol, which completely blocked implantation, had no significant effects on embryo transport and development on Day 4. A dose of 10 nmol did not increase the proportion of abnormal embryos, even though it accelerated tubal transport and increased embryo loss. No tubal retention was evident; most remaining embryos reached the uterus at the normal time. The transformation of morulae to blastocysts was only slightly delayed in 11P27-treated mice, and transfer experiments showed no decrease in embryo viability. The antibody appeared to act by blocking actions of endogenous progesterone on the uterus: uteri of 11P27-treated mice failed to develop a decidual cell reaction to intrauterine oil, and embryos from untreated donors failed to implant in 11P27-treated recipients. Antagonism of progesterone by antibody treatment prevented implantation in BALB/c mice apparently by actions on the uterus rather than on the embryo.

Measurement of human corticosteroid binding globulin by enzyme-linked immunoassay.

Thirteen monoclonal antibodies have been raised against corticosteroid binding globulin (CBG). From four of those with highest affinity for the antigen, two were selected for development of a sandwich enzyme-linked immunoassay (ELISA). The sensitivity of the assay was such that 0.7 fmol CBG could be detected. Levels of the binding protein in men (740 +/- 67 nmol/l) and women (690 +/- 103 nmol/l) were not significantly different, while those found during the third trimester of pregnancy (1500 +/- 423 nmol/l) were approximately twice these levels. CBG denatured by heating to 60 degrees C could not be detected by the ELISA.

Urinary epidermal growth factor excretion and breast cancer risk.

The amount of urinary epidermal growth factor (EGF) excreted was determined in 350 normal women of whom 37 subsequently developed breast cancer. These were a group of women selected on a case-control basis from 5000 volunteers who had participated in a prospective epidemiological study. Urinary EGF excretion was not correlated with known risk factors such as age at menarche or menopause, age at first or last full-term child or parity. Neither was it associated with day or length of menstrual cycle, breast mammographic parenchymal pattern or the blood concentration of prolactin, dehydroepiandrosterone or its sulphate ester. Univariate analysis indicated that the amount of urinary EGF was significantly correlated with urinary creatinine (P less than 0.001), age (P less than 0.001), urinary androsterone (P less than 0.02) or aetiocholanolone (P less than 0.02), height (P less than 0.05) and weight (P less than 0.05). However, multivariate analysis showed that the amount of urinary EGF was correlated only with creatinine excretion (P less than 0.001) and age (P less than 0.001) and that the significance of the other correlations were probably due to the confounding influence of creatinine.

Salivary oestradiol and progesterone levels in premenopausal women with breast cancer.

The concentrations of oestradiol and progesterone have been measured in salivary specimens collected daily over a complete menstrual cycle in 12 patients with operable breast cancer and 12 normal control volunteers. There was no significant difference (P greater than 0.05) for either hormone between these two groups. Both showed a mid-cycle rise in oestradiol levels followed by a smaller but sustained increase during the luteal phase. The progesterone concentration increased markedly during the luteal phase of the cycle. Total or non-protein bound oestradiol levels measured in blood samples from 19 normal women were both linearly correlated (P less than 0.001) with the concentration of oestradiol in matched saliva samples. The amount of free oestradiol in blood was about twice that found in saliva.

A solid-phase radioimmunoassay of serum dehydroepiandrosterone sulphate using a monoclonal antibody.

A monoclonal antibody directed against dehydroepiandrosterone, but with high affinity for dehydroepiandrosterone sulphate (DHA-S), has been used to develop a solid phase radioimmunoassay for measuring serum DHA-S. The antibody was covalently linked to polyacrylamide microbeads with no change in binding characteristics. The procedure requires only the chromatography of serum on anion-exchange cellulose before assaying the equivalent of 0.25 microliter serum. The method is precise, accurate and specific and can detect 19.5 pg of DHA-S. Serum DHA-S levels measured by this method were in good agreement with those found in a validated radioimmunoassay method involving hydrolysis. The method is quick and one operator could assay 50 blood specimens per day. DHA-S levels in serum from 50 men and 86 women were in agreement with those in the literature. With the availability of theoretically limitless quantities of consistently high quality monoclonal antibodies the advantages of developing solid phase radioimmunoassays for steroids is discussed.