Enhanced sensitivity to rapamycin following long-term oestrogen deprivation in MCF-7, T-47-D and ZR-75-1 human breast cancer cells

Human breast cancer cells (MCF-7, T-47-D and ZR-75-1) can adapt to circumvent any reduced growth rate during long-term oestrogen deprivation, and this provides three model systems to investigate mechanisms of endocrine resistance in breast cancer. In this paper we report consistent differences in the effects of three growth inhibitors following long-term oestrogen deprivation in all three cell models. Long-term oestrogen deprivation of MCF-7, T-47-D and ZR-75-1 cells resulted in reduced growth inhibition by PD98059 (2–10 µg/ml), implying a loss of dependence on mitogen-activated protein kinase pathways for growth. The growth inhibitor LY294002 (2–10 µM) inhibited growth of both oestrogen-maintained and oestrogen-deprived cells with similar dose–responses, implying continued similar dependence on phosphoinositide 3-kinase (PI3K) pathways with no alteration after adaptation to oestrogen independent growth. However, by contrast, long-term oestrogen deprivation resulted in an increased sensitivity to growth inhibition by rapamycin, which was not reduced by readdition of oestradiol. The enhanced inhibition of long-term oestrogen-deprived MCF-7-ED, T-47-D-ED and ZR-75-1-ED cell growth by combining rapamycin with LY294002 at concentrations where each alone had little effect, offers preclinical support to the development of therapeutic combinations of rapamycin analogues with other PI3K inhibitors in endocrine-resistant breast cancer.

Fatty acid compositions of inositol and choline phospholipids of breast tumours and normal breast tissue

The fatty acid compositions of the -choline and -inositol phospholipids of breast tumours of women undergoing surgery for treatment of breast disease (malignant n = 12; benign n = 10) and normal breast tissue of women undergoing breast reduction surgery (n = 6) were determined. The fatty acid compositions of erythrocyte phospholipids were also determined in the same subjects and in an additional number of normal healthy volunteers (n = 16). Levels of oleic acid were lower in both phospholipid fractions of erythrocytes of women with breast disease and in the phosphatidylcholine fraction of breast tumours compared with normal breast tissue. Significantly higher levels of linoleic acid were found in erythrocytes of tumour-bearing subjects and a similar trend was evident in the phosphatidylcholine fraction of tumour compared with normal breast tissues. Conversely, lower levels of two of the products of linoleic acid chain elongation and desaturation, dihomogamma-linolenic and arachidonic acids, were found in the erythrocyte phospholipids of tumour-bearing subjects and in the choline phospholipids of breast tumour tissues. These data suggest that in women with breast disease, there may be inhibition of 6-desaturase, and enhanced activity of 9-desaturase, enzymes which play an important role in determining membrane phospholipid fatty acid composition. This pattern of altered fatty acid composition characteristic of erythrocyte phospholipids of tumour-bearing subjects and phosphatidylcholine of breast tumour tissue was less evident in the case of the breast tumour phosphatidylinositol in which differences other than those described were seen.

Exposure to environmental oestrogenic chemicals and breast cancer.

The human population is now exposed on a daily basis to a multitude of environmental pollutant chemicals that would not have been present a century ago, and many of these chemicals have been detected in the human breast. The fatty nature of human breast tissue makes it a particular target for lipophilic as well as hydrophilic pollutant chemicals, which may enter the human body through oral, respiratory, or dermal routes. These chemicals possess a range of endocrine-disrupting properties and genotoxic activity, but from a breast cancer perspective the greatest concern has centered around their ability to mimic or interfere with the action of estrogen. The breast is an endocrine target organ and exposure to estrogen is a known risk factor for breast cancer.

Analysis of aluminium content and iron homeostasis in nipple aspirate fluids from healthy women and breast cancer-affected patients

Aluminium is not a physiological component of the breast but has been measured recently in human breast tissues and breast cyst fluids at levels above those found in blood serum or milk. Since the presence of aluminium can lead to iron dyshomeostasis, levels of aluminium and iron-binding proteins (ferritin, transferrin) were measured in nipple aspirate fluid (NAF), a fluid present in the breast duct tree and mirroring the breast microenvironment. NAFs were collected noninvasively from healthy women (NoCancer; n = 16) and breast cancer-affected women (Cancer; n = 19), and compared with levels in serum (n = 15) and milk (n = 45) from healthy subjects. The mean level of aluminium, measured by ICP-mass spectrometry, was significantly higher in Cancer NAF (268.4 ± 28.1 μg l−1; n = 19) than in NoCancer NAF (131.3 ± 9.6 μg l−1; n = 16; P < 0.0001). The mean level of ferritin, measured through immunoassay, was also found to be higher in Cancer NAF (280.0 ± 32.3 μg l−1) than in NoCancer NAF (55.5 ± 7.2 μg l−1), and furthermore, a positive correlation was found between levels of aluminium and ferritin in the Cancer NAF (correlation coefficient R = 0.94, P < 0.001). These results may suggest a role for raised levels of aluminium and modulation of proteins that regulate iron homeostasis as biomarkers for identification of women at higher risk of developing breast cancer. The reasons for the high levels of aluminium in NAF remain unknown but possibilities include either exposure to aluminium-based antiperspirant salts in the adjacent underarm area and/or preferential accumulation of aluminium by breast tissues.

Plasma lipids and prolactin in patients with breast cancer

In a comparative study of pre- and postmenopausal women with benign and malignant breast disease, a number of differences were observed in circulating plasma prolactin and lipid concentrations. Plasma lipids, phospholipids, triglycerides, cholesterol and free fatty acids were all higher in blood obtained from breast cancer patients prior to surgery. HDL-Cholesterol levels were significantly lower in these patients. These differences remained when the patient groups were sub-divided according to menopausal status. Plasma prolactin concentrations were also found to be higher in cancer compared with non-cancer patients, this effect being more marked in premenopausal than in postmenopausal patients. Premenopausal patients with invasive or poorly differentiated disease had significantly higher prolactin levels than those with non-invasive disease. No correlations were found between plasma prolactin and any of the lipid fractions.

Crosstalk with insulin and dependence on PI3K/Akt/mTOR rather than MAPK pathways in upregulation of basal growth following long-term oestrogen deprivation in three human breast cancer cell lines

Background: MCF-7, T-47-D, ZR-75-1 human breast cancer cell lines are dependent on oestrogen for growth but can adapt to grow during long-term oestrogen deprivation. This serves as a model for identification of therapeutic targets in endocrine-resistant breast cancer. Methods: An overlooked complication of this model is that it involves more than non-addition of oestrogen, and inadequate attention has been given to separating molecular events associated with each of the culture manipulations. Results: Insulin and oestradiol were shown to protect MCF-7 cells against upregulation of basal growth, demonstrating a crosstalk in the growth adaptation process. Increased phosphorylation of p44/42MAPK and c-Raf reflected removal of insulin from the medium and proliferation of all three cell lines was inhibited to a lesser extent by PD98059 and U0126 following long-term oestrogen/insulin withdrawal, demonstrating a reduced dependence on the MAPK pathway. By contrast, long-term oestrogen/insulin deprivation did not alter levels of phosphorylated Akt and did not alter the dose-response of growth inhibition with LY294002 in any of the three cell lines. The IGF1R inhibitor picropodophyllin inhibited growth of all MCF-7 cells but only in the long-term oestrogen/insulin-deprived cells was this paralleled by reduction in phosphorylated p70S6K, a downstream target of mTOR. Long-term oestrogen/insulin-deprived MCF-7 cells had higher levels of phosphorylated p70S6K and developed increased sensitivity to growth inhibition by rapamycin. Conclusions: The greater sensitivity to growth inhibition by rapamycin in all three cell lines following long-term oestrogen/insulin deprivation suggests rapamycin-based therapies might be more effective in breast cancers with acquired oestrogen resistance. Keywords Akt, breast cancer cells, endocrine resistance, insulin, MAPK, MCF-7 cells, mTOR, oestrogen, oestrogen-deprived, PI3K, picropodophyllin, rapamycin, T-47-D cells, ZR-75-1 cells

Aluminium and human breast diseases

The human breast is exposed to aluminium from many sources including diet and personal care products, but dermal application of aluminium-based antiperspirant salts provides a local long-term source of exposure. Recent measurements have shown that aluminium is present in both tissue and fat of the human breast but at levels which vary both between breasts and between tissue samples from the same breast. We have recently found increased levels of aluminium in noninvasively collected nipple aspirate fluids taken from breast cancer patients (mean 268±28 g/l) compared with control healthy subjects (mean 131±10 g/l) providing evidence of raised aluminium levels in the breast microenvironment when cancer is present. The measurement of higher levels of aluminium in type I human breast cyst fluids (median 150g/l) compared with human serum (median 6g/l) or human milk (median 25g/l) warrants further investigation into any possible role of aluminium in development of this benign breast disease. Emerging evidence for aluminium in several breast structures now requires biomarkers of aluminium action in order to ascertain whether the presence of aluminium has any biological impact. To this end, we report raised levels of proteins that modulate iron homeostasis (ferritin, transferrin) in parallel with raised aluminium in nipple aspirate fluids in vivo, and we report overexpression of mRNA for several S100 calcium binding proteins following long-term exposure of MCF-7 human breast cancer cells in vitro to aluminium chlorhydrate.

Measurement of paraben concentrations in human breast tissue at serial locations across the breast from axilla to sternum

The concentrations of five esters of p-hydroxybenzoic acid (parabens) were measured using HPLC-MS/MS at four serial locations across the human breast from axilla to sternum using human breast tissue collected from 40 mastectomies for primary breast cancer in England between 2005 and 2008. One or more paraben esters were quantifiable in 158/160 (99%) of the tissue samples and in 96/160 (60%) all five esters were measured. Variation was notable with respect to individual paraben esters, location within one breast and similar locations in different breasts. Overall median values in nanograms per gram tissue for the 160 tissue samples were highest for n-propylparaben [16.8 (range 0–2052.7)] and methylparaben [16.6 (range 0–5102.9)]; levels were lower for n-butylparaben [5.8 (range 0–95.4)], ethylparaben [3.4 (range 0–499.7)] and isobutylparaben 2.1 (range 0–802.9). The overall median value for total paraben was 85.5 ng g−1 tissue (range 0–5134.5). The source of the paraben cannot be identified, but paraben was measured in the 7/40 patients who reported never having used underarm cosmetics in their lifetime. No correlations were found between paraben concentrations and age of patient (37–91 years), length of breast feeding (0–23 months), tumour location or tumour oestrogen receptor content. In view of the disproportionate incidence of breast cancer in the upper outer quadrant, paraben concentrations were compared across the four regions of the breast: n-propylparaben was found at significantly higher levels in the axilla than mid (P = 0.004 Wilcoxon matched pairs) or medial (P = 0.021 Wilcoxon matched pairs) regions (P = 0.010 Friedman ANOVA).

Effect of patient age on management decisions in breast cancer: consensus from a national consultation

This qualitative study investigated the attitudes, perceptions, and practices of breast cancer specialists with reference to the effect of patient age on management decisions in breast cancer, and attempted to identify national consensus on this issue. One hundred thirty-three relevant specialists, including 75 surgeons and 43 oncologists, participated in a virtual consultation using e-mailed questionnaires and open-ended discussion documents, culminating in the development of proposed consensus statements sent to participants for validation. A strong consensus was seen in favor of incorporating minimum standards of diagnostic services, treatment, and care for older patients with breast cancer into relevant national guidance, endorsed by professional bodies. Similarly, an overwhelming majority of participants agreed that simple, evidence-based protocols or guidelines on standardizing assessment of biological and chronological age should be produced by the National Institute for Health and Clinical Excellence and the Scottish Medicines Consortium, developed in collaboration with specialist oncogeriatricians, and endorsed by professional bodies. A further recommendation that all breast cancer patient treatment and diagnostic procedures be undertaken in light of up-to-date, relevant scientific data met with majority support. This study was successful in gauging national specialist opinion regarding the effect of patient age on management decisions in breast cancer in the U.K.