Cyclin-dependent kinase inhibitors and basement membrane interact to regulate breast epithelial cell differentiation and acinar morphogenesis.

OBJECTIVE: The cyclin-dependent kinase inhibitors (CDKIs), p21(CIP1) and p27(KIP1) regulate growth and differentiation in diverse tissue types. We aimed to determine whether p21(CIP1) or p27(KIP1) could induce a terminally differentiated phenotype in breast cells, and to examine if CDKI expression is regulated by basement membrane interactions. MATERIALS AND METHODS: Effects of increased CDKI expression on the phenotype of MCF-10A breast epithelial cells were examined by retroviral transduction of p21(CIP1) or p27(KIP1) cDNA. RESULTS: Overexpression of p21(CIP1) or p27(KIP1) reduced MCF-10A growth rates in monolayer cultures, altered cellular morphology and stimulated accumulation of neutral lipid droplets, suggesting partial lactational differentiation. However, markers of luminal differentiation (oestrogen and progesterone receptors, alpha-lactalbumin, beta-casein and adipophilin) were absent when examined by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Cell-basement membrane contacts are known to be essential for full mammary epithelial cell differentiation and therefore parental MCF-10A cells were cultured on a basement membrane preparation (Matrigel) in which they form acini. Immunocytochemistry showed that Ki67, the cell proliferation marker, was initially expressed at high levels and as growth decreased p27(KIP1) expression steadily increased. Surprisingly, p21(CIP1) was highest at the early stages of acinus growth and was detected in proliferating cells, as demonstrated by colocalization in dual Ki67/p21(CIP1) immunofluorescence. Overexpression of p21(CIP1) or p27(KIP1) impaired formation of acini, whereas their knockdown, using siRNA, increased acinus formation. CONCLUSION: We conclude that both p21(CIP1) and p27(KIP1) induce partial secretory differentiation of mammary cells in monolayer, but during acinus morphogenesis in 3D culture they have a highly regulated temporal expression pattern.

TPD52 and NFKB1 gene expression levels correlate with G2 chromosomal radiosensitivity in lymphocytes of women with and at risk of hereditary breast cancer.

PURPOSE: To evaluate a transcriptomic approach to identify healthy women at increased risk of breast cancer due to G2-radiosensitivity and look at transcripts that are differentially expressed between individuals. MATERIALS AND METHODS: We perform the first study to assess the association of G2 radiosensitivity with basal gene expression in cultured T-lymphocytes from 11 women with breast cancer and 12 healthy female relatives using Affymetrix GeneChips. RESULTS: Transcripts associated with radiosensitivity and breast cancer risk were predominantly involved in innate immunity and inflammation, such as interleukins and chemokines. Genes differentially expressed in radiosensitive individuals were more similarly expressed in close family members than in un-related individuals, suggesting heritability of the trait. The expression of tumour protein D52 (TPD52), a gene implicated in cell proliferation, apoptosis, and vesicle trafficking was the most strongly correlated with G2 score while nuclear factor (kappa)-B (NFKB1) was highly inversely correlated with G2 score. NFKB1 is known to be activated by irradiation and its inhibition has been previously shown to increase radiosensitivity. CONCLUSIONS: Gene expression analysis of lymphocytes may provide a quantitative measure of radiation response potential and is a promising marker of breast cancer susceptibility.

Effects of oestrogen on gene expression in epithelium and stroma of normal human breast tissue.

Oestrogen (E) is essential for normal and cancer development in the breast, while anti-oestrogens have been shown to reduce the risk of the disease. However, little is known about the effect of E on gene expression in the normal human breast, particularly when the epithelium and stroma are intact. Previous expression profiles of the response to E have been performed on tumour cell lines, in the absence of stroma. We investigated gene expression in normal human breast tissue transplanted into 9-10-week-old female athymic nude (Balb/c nu/nu) mice. After 2 weeks, when epithelial proliferation is minimal, one-third of the mice were treated with 17beta-oestradiol (E2) to give human luteal-phase levels in the mouse, which we have previously shown to induce maximal epithelial cell proliferation. RNA was isolated from treated and untreated mice, labelled and hybridized to Affymetrix HG-U133A (human) GeneChips. Gene expression levels were generated using BioConductor implementations of the RMA and MAS5 algorithms. E2 treatment was found to represent the largest source of variation in gene expression and cross-species hybridization of mouse RNA from xenograft samples was demonstrated to be negligible. Known E2-responsive genes (such as TFF1 and AREG), and genes thought to be involved in breast cancer metastasis (including mammoglobin, KRT19 and AGR2), were upregulated in response to E treatment. Genes known to be co-expressed with E receptor alpha in breast cancer cell lines and tumours were both upregulated (XBP-1 and GREB1) and downregulated (RARRES1 and GATA3). In addition, genes that are normally expressed in the myoepithelium and extracellular matrix that maintain the tissue microenvironment were also differentially expressed. This suggests that the response to oestrogen in normal breast is highly dependent upon epithelial-stromal/myoepithelial interactions which maintain the tissue microenvironment during epithelial cell proliferation.

Dissociation between steroid receptor expression and cell proliferation in the human breast.

We have shown previously that estradiol stimulates cell proliferation and progesterone receptor (PgR) synthesis in luminal epithelial cells of the normal human breast. Approximately 10-15% of luminal epithelial cells within the normal breast express immunodetectable estrogen receptor (ER), but little is known about their distribution within lobules and their organization in relation to the smaller population of proliferating cells. Using normal human breast tissue, we show that ER-positive cells are distributed evenly throughout the mammary epithelium. Using double antibody immunofluorescence, we show that 96% of steroid receptor-positive cells synthesize both ER and PgR (n = 25). Double labeling with antibodies to either ER or PgR coupled with either [3H]thymidine histoautoradiography or with antibodies to the Ki67 proliferation antigen indicates that dividing cells are separate from those expressing the receptors (although they are often in close proximity). However, in contrast to the normal human breast, two-thirds of ER-positive human mammary tumors examined (n = 19) have a high proportion of dividing cells that are ER positive. These data are consistent with the hypothesis that cells in normal human breast epithelium are hierarchical in organization and support a model in which proliferation of ER-negative cells is controlled by paracrine factors released from ER-positive cells under the influence of estradiol. This organization may be disrupted in some tumors.

Experimental designs for estimating the parameters of the Michaelis-Menten equation from progress curves of enzyme-catalyzed reactions.

When the progress curve for an enzyme catalysed reaction follows the integrated Michaelis-Menten equation, the maximum velocity and Michaelis constant can be determined from a single such curve. In this paper, an experimental design for collecting the data is proposed which is close to optimum in the sense that it produces the smallest standard error in the estimate of the Michaelis constant. This design involves choosing an initial substrate concentration which is approx. two or three times the value of the Michaelis constant.

Isolation and characterization of human mammary stem cells.

Since stem cells are present throughout the lifetime of an organism, it is thought that they may accumulate mutations, eventually leading to cancer. In the breast, tumours are predominantly oestrogen and progesterone receptor-positive (ERalpha/PR+). We therefore studied the biology of ERalpha/PR-positive cells and their relationship to stem cells in normal human mammary epithelium. We demonstrated that ERalpha/PR-positive cells co-express the putative stem cell markers p21(CIP1/WAF1), cytokeratin (CK) 19 and Musashi-1 when examined using dual label immunofluorescence on tissue sections. Next, we isolated a Hoechst dye-effluxing 'side population' (SP) from the epithelium using flow cytometry and demonstrated them to be undifferentiated cells by lack of expression of myoepithelial and luminal cell-specific antigens such as CALLA and MUC1. Epithelial SP cells were shown to be enriched for the putative stem cell markers p21(CIP1/WAF1), Musashi-1 and ERalpha/PR-positive cells. Lastly, SP cells, compared to non-SP, were highly enriched for the capacity to produce colonies containing multiple lineages in 3D basement membrane (Matrigel) culture. We conclude that breast stem cells include two populations: a primitive ERalpha/PR-negative stem cell necessary for development and a shorter term ERalpha/PR-positive stem cell necessary for adult tissue homeostasis during menstrual cycling. We speculate these two basic stem cell types may therefore be the cells of origin for ERalpha-positive and -negative breast tumours.

The proliferation of normal human breast tissue implanted into athymic nude mice is stimulated by estrogen but not progesterone.

In order to resolve the question of which ovarian steroid stimulates normal human mammary epithelial cell proliferation, we have implanted pieces of normal human breast tissue subcutaneously into athymic nude mice. These mice were then treated with slow-release pellets containing estradiol (E2) or progesterone (P) such that serum levels of E2 and P were increased to those seen in normal women. The proliferative activity of the tissue implants was assessed by uptake of tritiated thymidine and steroid receptor expression was measured immunocytochemically. Insertion of a 2 mg E2 pellet 14 days after tissue implantation increased the thymidine labeling index (TLI) from a median of 0.4% (n = 34) to a median of 2.1% after 7 days (n = 43; P < 0.001 by Mann Whitney U test). In contrast, treatment with a P pellet (4 mg) had no effect upon the TLI whereas P (4 mg) in combination with E2 (2 mg) had no effect over and above that of E2 alone. There was a significant correlation between the increase in TLI and either the E2 content of the pellets (P < 0.001 by linear regression) or the serum E2 levels achieved (P < 0.001). Expression of the P receptor was increased 15- to 20-fold by E2 treatment. We conclude that E2 is sufficient to stimulate human breast epithelial cell proliferation at physiologically relevant concentrations and that P does not affect proliferation either alone or after E2 priming.

P27(KIP1) expression indicates that steroid receptor-positive cells are a non-proliferating, differentiated subpopulation of the normal human breast epithelium.

To test the hypothesis that steroid receptor-expressing cells are derived from the proliferative population, we examined expression of the p27(KIP1) inhibitor of cyclin-dependent kinase activity (a differentiation marker) while tracking the fate of proliferating cells in normal human breast tissue implanted into athymic nude mice using tritiated thymidine [3H]-dT. We identified a small number of cells that appeared to have divided just once before switching on p27(KIP1) expression. p27(KIP1)+ve cells also expressed steroid receptors, but not the Ki67 proliferation-associated antigen. These data support the hypothesis that steroid receptor-expressing cells are a differentiated population within the normal human breast epithelium.

Abnormal regulation of the oestrogen receptor in benign breast lesions.

BACKGROUND: In normal breast tissue the oestrogen receptor (ER) and the proliferation associated antigen Ki67 are negatively associated, indicating that ER+ cells are non-dividing, or that the receptor is downregulated as cells enter cycle. This relation is completely or partially lost in many ER+ breast cancers and in in situ proliferations associated with an increased cancer risk, where coexpression of the two markers is often found. AIMS: To determine whether similar changes can be identified in other risk associated breast lesions. PATIENTS/METHODS: Paraffin wax blocks from 12 cases of lactational change, 21 apocrine metaplasias, 22 duct ectasias, 20 sclerosing adenosis, 20 fibroadenomas, 19 phyllodes tumours, 20 radial scars, 21 papillomas (15 solitary and six multiple), 15 gynaecomastias, and nine postmortem male breast tissues were retrieved. Immunohistochemistry was used to determine the expression of ER and dual labelling immunofluorescence was used to detect cells expressing both ER and Ki67. RESULTS: Increased numbers of ER+ cells were seen in sclerosing adenosis, radial scars, papillomas, fibroadenomas, and phyllodes tumours but not in apocrine cysts (where no ER+ cells were detected) or duct ectasia (where normal numbers were found). As in the normal breast, the proportion of ER+ cells increased with age in all lesions with the exception of fibroadenomas. Coexpression of ER and Ki67 was found in an increased proportion of cells of all risk associated lesions studied. ER+ cells were less likely to be dividing than ER- cells in all cases, although this was significant only for sclerosing adenosis. The data on sclerosing adenosis, radial scars, papillomas, and fibroadenomas are comparable with those reported previously in hyperplasia of usual type, whereas those in duct ectasia are similar to those of the normal breast. The findings in all lesions, however, differed from those in ductal carcinoma in situ, where proportions of ER+ and ER+/Ki67+ cells are higher and the relation between ER+ cell numbers and age is lost. Thus, the nature and degree of dysregulation of ER in benign breast lesions is broadly in accordance with the degree of risk of developing breast cancer with which they are associated. In gynaecomastia, the proportions of ER+ and ER+/Ki67+ cells were comparable with those seen in benign female breast lesions, but changes with age were not observed. However, the changes in gynaecomastia were similar to those seen in normal male breast. CONCLUSION: These findings are in keeping with the contention that the dissociation of ER and Ki67 expression is a very early change in the pathway to many breast cancers. However, this change might only have preneoplastic importance in the hormonal milieu of the female breast.

The primary use of endocrine therapies.

Primary endocrine therapy is potentially superior to primary chemotherapy in patients with ER-positive tumors. The ability to give endocrine therapy perioperatively may be a better test than chemotherapy of the hypothesis that the events accompanying surgery affect prognosis. However, a major problem in all studies of primary endocrine therapy is that there has been no clear experimental test of preoperative versus postoperative therapy. This is because the major thrust of treatment has been in the elderly with the purpose of determining whether surgery can be avoided altogether. The fact that in ER-positive tumors primary endocrine therapy is associated with similar response rates to chemotherapy make it an attractive therapy for older women. This is the group where adjuvant chemotherapy has not been adequately tested (> or = 70 years of age). In contradistinction, adjuvant endocrine therapy shows marked survival benefits in patients with ER-positive tumors in these age groups (Table 1). It appears likely that primary endocrine therapy will allow breast conservation and prognostic information as is seen with chemotherapy. A major question which requires answering is whether primary endocrine therapy will improve survival more than adjuvant therapy alone. Although ER status is a good marker of responsiveness, its specificity, in particular, is not optimal. The ability to assess the dynamic effects of primary endocrine therapy by sequential biopsy and measurement of biological responses to oestrogen deprivation may allow us to predict precisely the patients likely to benefit from treatment. This clinical scenario allows us to use other potentially useful assessments such as the non-invasive estimation of angiogenesis using quantitative imaging techniques of blood flow. The newer anti-estrogens and aromatase inhibitors appear ideally suited to primary therapy since they have rapid and profound inhibitory activities, few or no agonist effects, and low side effect profiles. A preoperative trial of Faslodex is planned by the EORTC and another with Arimidex is under consideration by the ATAC (Arimidex, tamoxifen and combined) Trialist Group (Fig. 7). The precise design of these studies will require considerable thought.

Tissue-engineered rotator cuff tendon using porcine small intestine submucosa. Histologic and mechanical evaluation in dogs.

To determine its efficacy in stimulating the regeneration of a rotator cuff tendon, an implant of 10-ply porcine small intestinal submucosa was used to replace a completely resected infraspinatus tendon in 21 adult mongrel dogs. The contralateral infraspinatus tendon was elevated and then reattached to the greater tubercle with sutures to mimic conventional repair (sham operation). Mechanical evaluations were performed at 0, 3, and 6 months (five specimens at each time period). Histologic comparisons were made at 3 and 6 months (three specimens). At both times, the gross appearance, histologic continuity, and failure mode of the constructs mimicked those of sham-operated and native infraspinatus tendons, thus suggesting host tissue ingrowth and implant remodeling with solid integration of the regenerated tissue to muscular and bony interfaces. Tissue ingrowth occurred without histologic evidence of foreign body or immune-mediated reactions or adhesions to peripheral tissues. Sham operations simulated tendon mobilization and reimplantation procedures routinely performed to treat chronic rotator cuff tendon injuries. Although the ultimate strength of small intestinal submucosa-regenerated tendons was significantly less than that of native infraspinatus tendons (P < 0.001), it was similar to that of reimplanted tendons at 3 (P > 0.05) and 6 months (P > 0.05).

Tumour-promoting activity of altered WWP1 expression in breast cancer and its utility as a prognostic indicator.

WWP1 is a ubiquitin ligase, associated with the post-translational regulation of several tumour-promoting and tumour suppressor proteins. Here we show that WWP1 expression is up-regulated in a subset of breast tumour cell lines and primary breast tumours. We overexpressed WWP1 in MCF10A breast epithelial cells and demonstrated increased cell growth and anchorage-independent colony formation. RNAi knockdown of WWP1 expression in T47D and MCF7 breast tumour cell lines reduced anchorage-independent colony formation. We used WWP1 protein expression levels, in combination with its sub-cellular localization, to classify breast tumours into four categories. Surprisingly, a category with low/absent WWP1 expression displayed a consistently worse prognosis compared with WWP1-expressing tumours. Importantly, the association with disease-free survival was independent of the status of other commonly used prognostic indicators. Thus, WWP1 is a prognostic marker and may be a potential therapeutic target for a subset of breast tumours.

Mammary development, carcinomas and progesterone: role of Wnt signalling.

The mammary gland begins development during embryogenesis but after exposure to hormonal changes during puberty and pregnancy undergoes extensive further development. Hormonal changes are key regulators in the cycles of proliferation, differentiation, apoptosis and remodelling associated with pregnancy, lactation and involution following weaning. These developmental processes within the breast epithelium can be explained by the presence of a long-lived population of tissue-specific stem cells. The longevity of these stem cells makes them susceptible to accumulating genetic change and consequent transformation. The ovarian steroid progesterone, acting via the secreted factor Wnt4, is known to be essential for side branching of the mammary gland. One function of Wnt proteins is self-renewal of adult tissue stem cells, suggesting that progesterone may exert its effects within the breast, at least partly, by regulating the mammary stem cell population.

Breast stem cells and cancer.

Recent results have increased our understanding of normal stem cells and the signalling pathways which regulate them during the development of the mammary gland. Tumours in many tissues are now thought to develop from dysregulated stem cells and depend on activated stem cell self-renewal pathways such as Notch for their tumourigenic capacity. These cancer stem cells are recognised by specific cell surface proteins that they express and their capacity to grow tumours in vivo or spheres in vitro. We have described human breast DCIS mammospheres grown from cancer stem cells and demonstrated their dependence on the EGF and Notch receptor pathways. Stem cell self-renewal pathways such as these may represent novel therapeutic targets to prevent recurrence of pre-invasive and invasive breast cancer.

Effects of oestrogens and anti-oestrogens on normal breast tissue from women bearing BRCA1 and BRCA2 mutations.

There is considerable interest in whether anti-oestrogens can be used to prevent breast cancer in women bearing mutations in the BRCA1 and BRCA2 genes. The effects of oestradiol (E2), tamoxifen (TAM) and fulvestrant (FUL) on proliferation and steroid receptor expression were assessed in normal breast epithelium taken from women at varying risks of breast cancer and implanted into athymic nude mice, which were treated with E2 in the presence and absence of TAM or FUL. Tissue samples were taken at various time points thereafter for assessment of proliferative activity and expression of oestrogen and progesterone receptors (ERalpha and PgR) by immunohistochemistry. Oestradiol increased proliferation in the breast epithelium from women carrying mutations in the BRCA1/2 genes, those otherwise at increased risk and those at population risk of breast cancer. This increase was reduced by both TAM and FUL in all risk groups. In the absence of E2, PgR expression was reduced in all risk groups but significantly more so in the BRCA-mutated groups. Subsequent E2 treatment caused a rapid, complete induction of PgR expression in the population-risk group but not in the high-risk or BRCA-mutated groups in which PgR induction was significantly delayed. These data suggest that the mechanisms by which E2 induces breast epithelial PgR expression are impaired in BRCA1/2 mutation carriers, whereas those regulating proliferation remain intact. We conclude that early anti-oestrogen treatment should prevent breast cancer in very high-risk women.

Activated c-SRC in ductal carcinoma in situ correlates with high tumour grade, high proliferation and HER2 positivity.

Overexpression and/or activity of c-Src non-receptor tyrosine kinase is associated with progression of several human epithelial cancers including breast cancer. c-Src activity in 'pure' ductal carcinoma in situ (DCIS) was measured to assess whether this predicts recurrence and/or correlates with HER2 expression and other clinical parameters. Activated c-Src levels were evaluated in DCIS biopsies from 129 women, with median follow-up at 60 months. High levels of activated c-Src correlated with HER2 positivity, high tumour grade, comedo necrosis and elevated epithelial proliferation. In univariate analysis, high activated c-Src level associated with lower recurrence-free survival at 5 years (P=0.011). Thus, high c-Src activity may identify a subset of DCIS with high risk of recurrence or progression to invasive cancer where therapeutics targeting c-Src may benefit this patient subset.

Human breast cell proliferation and its relationship to steroid receptor expression.

The steroidal regulation of proliferation and differentiation in the rodent mammary gland is well described, but how ovarian hormones regulate these processes in the human remains poorly understood. To investigate this, we developed the athymic nude mouse model in which intact normal human breast tissue is grafted subcutaneously and treated with estrogen and/or progesterone at human physiological serum levels. We demonstrated, first, that estrogen and not progesterone is the major epithelial cell mitogen in the adult non-pregnant, non-lactating breast, second, that estrogen induces progesterone receptor (PR) expression and, third, that PR expression is maximally induced at low estrogen concentrations while a higher amount of estrogen was required to induce proliferation. These data raised the question of whether one cell type possessed differential responses to high and low estrogen concentrations or whether PR expression and proliferation occurred in two cell populations. Using double-label immunofluorescence, we demonstrated that steroid receptor expression and cell proliferation (Ki67 antigen) occurred in separate cell populations in normal human breast epithelium, and that cells expressing the estrogen receptor-alpha (ERalpha) invariably contained the PR. We also found that this dissociation between steroid receptor expression and cell proliferation in normal epithelium was disrupted at an early stage in breast tumor formation. Recent findings presented herein support the proposal that some ERalpha/PR-positive epithelial cells are quiescent breast stem cells that act as 'steroid hormone sensors'. Such hormone sensor cells are likely to secrete positive or negative paracrine/juxtacrine factors dependent on the prevailing estrogen or progesterone concentration to influence the proliferative activity of adjacent ERalpha/PR-negative epithelial cells.

Epithelial stem cells in the mammary gland: casting light into dark corners.

The epithelial structures of the human breast or the mouse mammary gland are derived from a relatively small number of multipotent, tissue-specific stem cells, of which we are surprisingly ignorant. We do not know how many are required to produce a complete mammary gland, how many times they divide during the process, where they are situated in the gland, or even what they look like. We want to know the answers to these questions, not just to satisfy intellectual curiosity, but also because the answers may shed light on the evolution of breast cancer. Now, studies carried out by Kordon and Smith at the National Cancer Institute have pointed the way toward a new understanding of mammary stem cells and their progeny.