Inhibition of FASN and ERα signalling during hyperglycaemia-induced matrix-specific EMT promotes breast cancer cell invasion via a caveolin-1-dependent mechanism.

Since disturbed metabolic conditions such as obesity and diabetes can be critical determinants of breast cancer progression and therapeutic failure, we aimed to determine the mechanism responsible for their pro-oncogenic effects. Using non-invasive, epithelial-like ERα-positive MCF-7 and T47D human breast cancer cells we found that hyperglycaemia induced epithelial to mesenchymal transition (EMT), a key programme responsible for the development of metastatic disease. This was demonstrated by loss of the epithelial marker E-cadherin together with increases in mesenchymal markers such as vimentin, fibronectin and the transcription factor SLUG, together with an enhancement of cell growth and invasion. These phenotypic changes were only observed with cells grown on fibronectin and not with those plated on collagen. Analyzing metabolic parameters, we found that hyperglycaemia-induced, matrix-specific EMT promoted the Warburg effect by upregulating glucose uptake, lactate release and specific glycolytic enzymes and transporters. We showed that silencing of fatty acid synthase (FASN) and the downstream ERα, which we showed previously to mediate hyperglycaemia-induced chemoresistance in these cells, resulted in suppression of cell growth: however, this also resulted in a dramatic enhancement of cell invasion and SLUG mRNA levels via a novel caveolin-1-dependent mechanism.

Hyperglycaemia-induced resistance to Docetaxel is negated by metformin: a role for IGFBP-2.

The incidence of many common cancers varies between different populations and appears to be affected by a Western lifestyle. Highly proliferative malignant cells require sufficient levels of nutrients for their anabolic activity. Therefore, targeting genes and pathways involved in metabolic pathways could yield future therapeutics. A common pathway implicated in energetic and nutritional requirements of a cell is the LKB1/AMPK pathway. Metformin is a widely studied anti-diabetic drug, which improves glycaemia in patients with type 2 diabetes by targeting this pathway. We investigated the effect of metformin on prostate cancer cell lines and evaluated its mechanism of action using DU145, LNCaP, PC3 and VCaP prostate cancer cell lines. Trypan blue dye-exclusion assay was used to assess levels of cell death. Western immunoblotting was used to determine the abundance of proteins. Insulin-like growth factor-binding protein-2 (IGFBP-2) and AMPK genes were silenced using siRNA. Effects on cell morphology were visualised using microscopy. IGFBP-2 gene expression was assessed using real-time RT-PCR. With DU145 and LNCaP cells metformin alone induced cell death, but this was reduced in hyperglycaemic conditions. Hyperglycaemia also reduced the sensitivity to Docetaxel, but this was countered by co-treatment with metformin. LKB1 was required for the activation of AMPK but was not essential to mediate the induction of cell death. An alternative pathway by which metformin exerted its action was through downregulation of IGFBP-2 in DU145 and LNCaP cells, independently of AMPK. This finding could have important implications in relation to therapeutic strategies in prostate cancer patients presenting with diabetes.

Hyperglycaemia-induced chemoresistance in breast cancer cells: role of the estrogen receptor.

Breast cancer patients with diabetes respond less well to chemotherapy; in keeping with this we determined previously that hyperglycaemia-induced chemoresistance in estrogen receptor (ERα) positive breast cancer cells and showed that this was mediated by fatty acid synthase (FASN). More recent evidence suggests that the effect of metabolic syndrome and diabetes is not the same for all subtypes of breast cancer with inferior disease-free survival and worse overall survival only found in women with ERα positive breast cancer and not for other subtypes. Here we examined the impact of hyperglycaemia on ERα negative breast cancer cells and further investigated the mechanism underlying chemoresistance in ERα with a view to identifying strategies to alleviate hyperglycaemia-induced chemoresistance. We found that hyperglycaemia-induced chemoresistance was only observed in ERα breast cancer cells and was dependent upon the expression of ERα as chemoresistance was negated when the ERα was silenced. Hyperglycaemia-induced an increase in activation and nuclear localisation of the ERα that was downstream of FASN and dependent on the activation of MAPK. We found that fulvestrant successfully negated the hyperglycaemia-induced chemoresistance, whereas tamoxifen had no effect. In summary our data suggests that the ERα may be a predictive marker of poor response to chemotherapy in breast cancer patients with diabetes. It further indicates that anti-estrogens could be an effective adjuvant to chemotherapy in such patients and indicates the importance for the personalised management of breast cancer patients with diabetes highlighting the need for clinical trials of tailored chemotherapy for diabetic patients diagnosed with ERα positive breast cancers.

Hyperglycaemia-induced chemoresistance of prostate cancer cells due to IGFBP2.

Clinically relevant prostate cancer (PCa) is more frequent in Westernised societies and increasingly men have co-morbidities associated with a Western lifestyle, primarily diabetes, characterised by hyperinsulinaemia and hyperglycaemia. IGFs and their binding proteins (IGFBPs) are important mediators of the effects of nutrition on growth and play a key role in the development of PCa. We used DU145, PC3 and LNCaP PCa cell lines to examine how hyperglycaemia altered their response to docetaxel. Trypan Blue dye-exclusion assay was used to determine the percentage of cell death. Protein abundance was determined using western immunoblotting. Levels of IGFBP2 were measured using an ELISA. IGFBP2 gene silencing was achieved using siRNA technology. DNA methylation was assessed using combined bisulphide restriction analysis. Acetylation status of histones H3 and H4 associated with IGFBP2 gene was assessed using chromatin immunoprecipitation assay. Hyperglycaemia reduced docetaxel-induced apoptosis by 40% for DU145 cells and by 88% for LNCaP cells. This reduced cell death was mediated by a glucose-induced up-regulation of IGFBP2, as silencing IGFBP2 negated the survival effect of high glucose. Glucose increased IGFBP2 via increasing the acetylation of histones associated with the IGFBP2 gene promoter. This finding could have important implications in relation to therapeutic strategies as epigenetic modulation could be reversible.

Insulin-like growth factor-II is produced by, signals to and is an important survival factor for the mature podocyte in man and mouse.

Podocytes are crucial for preventing the passage of albumin into the urine and, when lost, are associated with the development of albuminuria, renal failure and cardiovascular disease. Podocytes have limited capacity to regenerate, therefore pro-survival mechanisms are critically important. Insulin-like growth factor-II (IGF-II) is a potent survival and growth factor; however, its major function is thought to be in prenatal development, when circulating levels are high. IGF-II has only previously been reported to continue to be expressed in discrete regions of the brain into adulthood in rodents, with systemic levels being undetectable. Using conditionally immortalized human and ex vivo adult mouse cells of the glomerulus, we demonstrated the podocyte to be the major glomerular source and target of IGF-II; it signals to this cell via the IGF-I receptor via the PI3 kinase and MAPK pathways. Functionally, a reduction in IGF signalling causes podocyte cell death in vitro and glomerular disease in vivo in an aged IGF-II transgenic mouse that produces approximately 60% of IGF-II due to a lack of the P2 promoter of this gene. Collectively, this work reveals the fundamental importance of IGF-II in the mature podocyte for glomerular health across mammalian species.

Role of the IGF axis in prostate cancer.

The major issue currently being faced in the management of prostate cancer is the inability to distinguish between indolent prostate tumors that will not present clinically from more aggressive and metastatic prostate cancers that will impact on men's lives. Only a small proportion of prostate cancers can be accounted for by unmistakable hereditary cancer syndromes and the predominant contribution to the progression of most sporadic cancers is thought to be environmental, with nutrition having the greatest influence. Population studies have clearly implicated metabolic factors as contributors to disease progression and poor response to therapy. It is well established that the IGF system is key in regulating growth and metabolism and mediates the effects of nutrition on these processes. It consists of two ligands (IGF-I and IGF-II), two receptors [type 1 IGF-IR and IGF-II/mannose 6-phosphate receptor], and six high affinity IGF-binding proteins (IGFBP-1 to -6). This review provides evidence from in vitro, in vivo, clinical and epidemiology studies that indicates an important role for the IGF axis in the development of prostate cancer and the likely role that it plays in mediating the effects of nutrition on disease progression. We suggest that the IGF axis is central to understanding how lifestyle impacts on prostate cancer and we highlight this by describing numerous strategies being developed to target this axis.

The association of childhood height, leg length and other measures of skeletal growth with adult cardiovascular disease: the Boyd-Orr cohort.

BACKGROUND: Taller adults have a reduced risk of cardiovascular disease, and there is some evidence that pre-adolescent exposures, indexed by leg length, underlie this association. Associations with other aspects of skeletal size in childhood have not previously been investigated. METHODS: We have examined associations of cardiovascular mortality and morbidity with childhood height, shoulder breadth, leg, trunk and foot length using a cohort of children whose families participated in a 1937-9 survey of diet and health followed up for 59 years. RESULTS: Altogether 2642 traced participants had at least one anthropometric measurement; a subsample (n=1043), completed the Rose angina questionnaire and provided information about doctor-diagnosed ischaemic heart disease (IHD) in 1997-8. Childhood stature was weakly inversely associated with cardiovascular mortality, and leg length was the component with the strongest associations. There was evidence from secondary analyses that childhood anthropometric measurements were inversely related to early (age <65 years) rather than late cardiovascular mortality. Childhood stature was inversely associated with self-reported IHD and associations with leg length were strongest. Associations were somewhat attenuated in models including terms for having been breastfed and socioeconomic position. CONCLUSION: Pre-adult exposures are more strongly associated with cardiovascular morbidity than mortality, and they affect premature cardiovascular mortality more than later mortality.

Insulin-like growth factor-binding protein-2 promotes prostate cancer cell growth via IGF-dependent or -independent mechanisms and reduces the efficacy of docetaxel.

BACKGROUND: The development of androgen independence, chemo-, and radioresistance are critical markers of prostate cancer progression and the predominant reasons for its high mortality. Understanding the resistance to therapy could aid the development of more effective treatments. AIM: The aim of this study is to investigate the effects of insulin-like growth factor-binding protein-2 (IGFBP-2) on prostate cancer cell proliferation and its effects on the response to docetaxel. METHODS: DU145 and PC3 cells were treated with IGFBP-2, insulin-like growth factor I (IGF-I) alone or in combination with blockade of the IGF-I receptor or integrin receptors. Cells were also treated with IGFBP-2 short interfering ribonucleic acid with or without a PTEN (phosphatase and tensin homologue deleted on chromosome 10) inhibitor or docetaxel. Tritiated thymidine incorporation was used to measure cell proliferation and Trypan blue cell counting for cell death. Levels of IGFBP-2 mRNA were measured using RT-PCR. Abundance and phosphorylation of proteins were assessed using western immunoblotting. RESULTS: The IGFBP-2 promoted cell growth in both cell lines but with PC3 cells this was in an IGF-dependent manner, whereas with DU145 cells the effect was independent of IGF receptor activation. This IGF-independent effect of IGFBP-2 was mediated by interaction with ß-1-containing integrins and a consequent increase in PTEN phosphorylation. We also determined that silencing IGFBP-2 in both cell lines increased the sensitivity of the cells to docetaxel. CONCLUSION: The IGFBP-2 has a key role in the growth of prostate cancer cells, and silencing IGFBP-2 expression reduced the resistance of these cells to docetaxel. Targeting IGFBP-2 may increase the efficacy of docetaxel.

Hyperglycaemia confers resistance to chemotherapy on breast cancer cells: the role of fatty acid synthase.

The prognosis for women with breast cancer is adversely affected by the comorbidities of obesity and diabetes mellitus (DM), which are conditions associated with elevated levels of circulating fatty acids, hyperglycaemia and hyperinsulinaemia. We investigated the effects of exposure of non-malignant and malignant human breast epithelial cells to elevated levels of fatty acids and glucose on their growth, survival and response to chemotherapeutic agents. We found that palmitate induced cell death in the non-malignant cells but not in the malignant cells, which was abrogated through the inhibition of ceramide production and by oleate but not by IGF1. Fatty acid synthase (FAS) is responsible for the de novo synthesis of fatty acids from sugars, and is over-expressed in many epithelial cancers. Abundance of FAS was higher in malignant cells than in non-malignant cells, and was up-regulated by IGF1 in both cell types. IGF-induced growth of non-malignant cells was unaffected by suppression of FAS expression, whereas that of malignant cells was blocked as was their resistance to palmitate-induced cell death. Palmitate did not affect cell proliferation, whereas oleate promoted the growth of non-malignant cells but had the opposite effect, that is, inhibition of IGF1-induced growth of malignant cells. However, when the phosphatidylinositol 3-kinase pathway was inhibited, oleate enhanced IGF1-induced growth in both cell types. Hyperglycaemia conferred resistance on malignant cells, but not on non-malignant cells, to chemotherapy-induced cell death. This resistance was overcome by inhibiting FAS or ceramide production. Understanding the mechanisms involved in the associations between obesity, DM and breast cancer may lead to more effective treatment regimens and new therapeutic targets.

Childhood dairy and calcium intake and cardiovascular mortality in adulthood: 65-year follow-up of the Boyd Orr cohort.

BACKGROUND: Dairy consumption in childhood may have long-term effects on cardiovascular mortality through influencing the development of risk factors or programming effects. OBJECTIVE: To investigate whether dairy and calcium consumption in childhood is associated with adult mortality due to coronary heart disease (CHD), stroke and all causes. METHODS: In 1937-9, 4999 children in England and Scotland participated in a study of family food consumption, assessed from 7-day household food inventories. Cause of death was ascertained between 1948 and 2005 in 4374 traced cohort members with complete data. Per capita household intake estimates for dairy products and calcium were used as proxies for individual intake. RESULTS: No strong evidence that a family diet in childhood high in dairy products was associated with CHD or stroke mortality was found. However, childhood calcium intake was inversely associated with stroke mortality (multivariable adjusted hazard ratio (HR) for highest versus lowest calcium group: 0.41; 95% confidence interval (CI) 0.16 to 1.05; p for trend = 0.04), but not CHD mortality. All-cause mortality was lowest in those with the highest family dairy (HR = 0.77; 95% CI 0.61 to 0.98; p for trend = 0.04) and calcium intake (HR = 0.77, 95% CI 0.60 to 0.98; p for trend = 0.05). CONCLUSIONS: Children whose family diet in the 1930s was high in calcium were at reduced risk of death from stroke. Furthermore, childhood diets rich in dairy or calcium were associated with lower all-cause mortality in adulthood. Replication in other study populations is needed to determine whether residual confounding explains part of these findings.

IGF-II and IGFBP-2 differentially regulate PTEN in human breast cancer cells.

The dual-function phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is the second most frequently mutated gene in human cancers. PTEN counteracts the functions of many growth factors, the most prevalent of which is insulin-like growth factor II (IGF-II). PTEN expression is stimulated by IGF-II forming a feedback loop. Investigating IGF-binding protein (IGFBP) modulation of IGF-II actions on MCF-7 breast cancer cells, we found that IGFBP-2 also regulates PTEN. The MCF-7 cells were not responsive to high doses of IGF-II due to induction of PTEN, which was not observed with an IGF-II-analog that does not bind to IGFBPs or in the presence of an inhibitor that prevents IGFs associating with IGFBPs. These cells predominantly produce IGFBP-2: blocking IGFBP-2 with a specific antibody, or preventing IGFBP-2 binding to integrins, restored the induction of PTEN and the cells were non-responsive to high doses of the IGF-II-analog. Our findings indicate that breast cancer cells do not respond to high doses of IGF-II due to induction of PTEN, but IGFBP-2, when free from IGF-II can suppress PTEN. Levels of IGFBP-2 are elevated frequently in human tumors: its ability to regulate PTEN could have important implications in relation to therapeutic strategies targeting growth factor pathways.

Childhood diet and insulin-like growth factors in adulthood: 65-year follow-up of the Boyd Orr Cohort.

OBJECTIVE: High levels of insulin-like growth factor-I (IGF-I) are associated with an increased cancer risk and reduce risk of diabetes and coronary heart disease. We investigated associations of diet in childhood, in particular energy intake, with the IGF system in adulthood to determine if IGF-I - disease associations could be linked to early nutrition. DESIGN: Retrospective cohort study. SETTING: Sixteen survey centres in England and Scotland that originally participated in the Carnegie (Boyd Orr) Survey of Diet and Health in Pre-War Britain, 1937-1939. SUBJECTS: Seven hundred and twenty-eight participants (679 with complete data) in the Boyd Orr cohort. METHODS: Participants were originally surveyed between 1937 and 1939 (at median age 5.8 years; inter-quartile range: 2.9-9.6) and were followed up for 65 years. Dietary exposure in childhood was assessed from 7-day household food inventories. Outcomes are expressed as regression coefficients for the change in IGF per standard deviation increased childhood nutrient or food intake, as derived from levels of household consumption. RESULTS: In fully-adjusted models, energy-rich family diets in childhood were not associated with IGF-I (regression coefficient: 0.9 ng/ml; 95% confidence interval (CI): -1.8, 3.7), IGF-II, IGF binding proteins (IGFBP)-2 or IGFBP-3 in adulthood. IGF-I was associated inversely with childhood family-diets high in milk (-2.5 ng/ml; -5.1, 0.1; P=0.05) and positively with vegetable-rich diets (3.5 ng/ml; 0.9, 6.1; P=0.009). IGF-I was not associated with family diets rich in protein, carbohydrates, fats, calcium, meat or fruit. IGF-II, IGFBP-2 and IGFBP-3 were not related to childhood family diet. CONCLUSIONS: This study suggests that energy-rich family diets in childhood do not program the IGF system in adulthood. As childhood diet was based on household consumption, however, measurement error may obscure individual-level diet-IGF associations. The associations of milk- and vegetable-rich family diets in childhood with IGF-I could be chance findings, but nevertheless are consistent with recent publications and warrant further investigation.

IGF1, growth pathway polymorphisms and schizophrenia: a pooling study.

It has been hypothesized that insulin-like growth factors (IGFs) and components of the growth-hormone (GH)-IGF axis may underlie reported associations of poor fetal and childhood growth with schizophrenia. We have investigated the association of schizophrenia with 16 SNPs spanning the IGF1 gene with an inter-marker distance of approximately 2-3 kb. We also examined associations with four common functional polymorphisms of genes involved in aspects of the GH-IGF system--the IGF1 receptor (IGF1R), insulin receptor substrate (IRS1), growth hormone (GH1), and IGF binding protein-3 (IGFBP3). The study was based on an analysis of pooled DNA samples from 648 UK and Irish cases of schizophrenia and 712 blood donor controls and of 297 Bulgarian parent offspring trios. In replicated pool analyses, none of the 16 SNPs in IGF1 nor the 4 key SNPs in the other growth pathway genes were associated with schizophrenia. SNP coverage of IGF1 was extensive, so our findings do not support a major role for IGF-I in the aetiology of schizophrenia.

Insulin-like growth factor binding protein 3 has opposing actions on malignant and nonmalignant breast epithelial cells that are each reversible and dependent upon cholesterol-stabilized integrin receptor complexes.

IGF-binding protein (IGFBP)-3 is generally considered to have actions that counterbalance those of IGFs and is therefore being developed as a cancer treatment. In breast tumors, however, high levels are associated with aggressive tumors and poor prognosis. Consistent with this we have demonstrated that although IGFBP-3 and a non-IGF-binding fragment (serine phosphorylation domain peptide) reduced attachment and enhanced apoptosis of Hs578T breast cancer cells cultured on collagen or laminin, it promoted their attachment and survival on fibronectin, which is abundant in the matrix of aggressive tumors. We have now examined the factors that determine whether IGFBP-3 has positive or negative actions on breast epithelial cells. IGFBP-3 also promoted survival of Hs578T cells in the presence of an antibody to the beta1-integrin subunit or when cholesterol-stabilized complexes were disrupted. These actions were blocked by IGF-I or a MAPK inhibitor. Serine phosphorylation domain peptide had similar actions on MCF-7 cells that were again reversed on fibronectin or with disruption of cholesterol-stabilized complexes and blocked by the beta1-integrin antibody. In contrast, IGFBP-3 promoted growth and survival for nonmalignant MCF-10A cells, but these effects were again reversed on fibronectin and blocked by the beta1 antibody or a MAPK inhibitor or by disruption of cholesterol-stabilized complexes. On Hs578T cells, IGFBP-3 bound to caveolin-1 and beta1-integrins, enhancing their aggregation, the recruitment of focal adhesion kinase, and the activation of MAPK. In summary, with three breast epithelial cell lines, IGFBP-3 had positive or negative effects on growth and survival dependent upon the status of cholesterol-stabilized integrin receptor complexes.

Insulin-like growth factors (IGF) I and II utilize different calcium signaling pathways in a primary human parathyroid cell culture model.

BACKGROUND: In most cell types, influx of calcium (Ca2+) induces a growth or secretory response. The opposite occurs in parathyroid (PTH), cells where there is an inverse relationship between intracellular Ca2+ concentration and PTH secretion. We have examined the effects of calcium channel and metabolism modulators on insulin-like growth factors (IGFs) in a parathyroid cell culture model. METHODS: Cell cultures were prepared from 9 patients undergoing operation for hyperparathyroidism. Following adhesion, the cells were transferred to serum-free medium and dosed with IGF I, II +/- ethyleneglycol-bis(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), nifedipine, nickel, 2-aminoethoxy-diphenylborate (2-APB), or dantrolene. Proliferation (96 hours) was assessed by measuring tritiated thymidine incorporation and PTH release (1 and 3 hours) assayed by IRMA. RESULTS: Both IGF I and II increased DNA synthesis to 162.8% +/- 10.6% (SEM) and 131.1% +/- 7.7%, respectively (P < 0.05). EGTA at 0.2 mmol (ionized Ca2+ 0.2 mmol) did not affect the response to both IGFs. EGTA at 2 mmol (ionized Ca2+ 0 mmol) reduced the DNA synthesis of IGF I and II to 29% and 26%, respectively (P < 0.05). Nifedipine and nickel (nonspecific Ca2+ channel blocker) were equally potent in negating the mitogenic effects of both IGFs. 2-APB (IP3R blocker) reduced the basal DNA synthesis to 51.3% +/- 8.4% but had no effect on either IGF. Dantrolene (ryanodine receptor blocker) negated IGF II induced mitogenisis (74.2% +/- 6.7%) and partially inhibited IGF I mitogenesis (123% +/- 6%) (P < 0.05). The rate of PTH secretion was greater after IGF II stimulation than after IGF I stimulation. CONCLUSIONS: IGFs I and II induce mitogenesis by different calcium signaling pathways. These data suggest that parathyroid cells may utilize different calcium signaling pathways to distinguish growth factors and serum calcium changes.

Characterisation of morbidity in a UK, hospital based, obesity clinic.

AIM: To identify clinical features which predict those most at risk of co-morbidities within an obesity clinic. METHODS: Children attending an obesity clinic had fasting glucose, insulin, and lipids measured prior to a standard oral glucose tolerance test (OGTT). History and examination established birth weight, family history of type 2 diabetes/obesity, pubertal status, and presence of acanthosis nigricans. Central and total fat mass was estimated by bio-impedance. RESULTS: Of the 126 children evaluated, 10.3% (n = 13) had impaired glucose tolerance (IGT); the majority (n = 11) of these would not have been identified on fasting glucose alone. Those with IGT were more likely to have a parental history of type 2 diabetes (relative risk 3.5). IGT was not associated with acanthosis nigricans. Twenty five per cent (n = 19) of those evaluated (n = 75) had evidence of the "metabolic syndrome" (MS). HDL cholesterol and triglyceride levels were related to insulin sensitivity (HOMA-R); HDL cholesterol was also related to birth weight SDS. We observed a trend for those with MS to have a lower birth weight SDS. The severity of obesity did not influence the likelihood of IGT or MS. CONCLUSIONS: Significant numbers of obese children have associated co-morbidities. Analysis of fasting blood glucose samples alone is not satisfactory to adequately evaluate glucose homoeostasis. The overall level of obesity does not predict co-morbidities. Special attention should be given to those with parental diabetes and a history of low birth weight who are more likely to have IGT and abnormal lipid profiles respectively.

A prospective study of serum insulin-like growth factor-I (IGF-I), IGF-II, IGF-binding protein-3 and breast cancer risk.

The associations between serum concentrations of insulin-like growth factor-I (IGF-I), IGF-II and IGF-binding proteins (IGFBP)-3 and risk of breast cancer were investigated in a nested case-control study involving 117 cases (70 premenopausal and 47 postmenopausal at blood collection) and 350 matched controls within a cohort of women from the island of Guernsey, UK. Women using exogenous hormones at the time of blood collection were excluded. Premenopausal women in the top vs bottom third of serum IGF-I concentration had a nonsignificantly increased risk for breast cancer after adjustment for IGFBP-3 (odds ratio (OR) 1.71; 95% confidence interval (CI): 0.74-3.95; test for linear trend, P=0.21). Serum IGFBP-3 was associated with a reduction in risk in premenopausal women after adjustment for IGF-I (top third vs the bottom third: OR 0.49; 95% CI: 0.21-1.12, P for trend=0.07). Neither IGF-I nor IGFBP-3 was associated with risk in postmenopausal women and serum IGF-II concentration was not associated with risk in pre- or postmenopausal women. These data are compatible with the hypothesis that premenopausal women with a relatively high circulating concentration of IGF-I and low IGFBP-3 are at an increased risk of developing breast cancer.

Prolactin acts as a potent survival factor for human breast cancer cell lines.

Human breast cancer is the leading cause of cancer death in women from Western societies, and a large study of the epidemiology demonstrated strong associations between human prolactin and risk of breast cancer. Using established models of apoptosis of human breast cancer cell lines, we assessed the role of prolactin in breast cancer cell growth and survival. We showed that prolactin had no effect on the metabolic activity or total cell number of any cell lines. We confirmed endogenous prolactin production by these cells and that the levels varied. In the presence of a prolactin-neutralising antibody, each of the cell lines responded with the induction of apoptosis as opposed to growth inhibition. The sensitivity of the cell lines to the physiological inducer of apoptosis, C2-ceramide, appeared relative to the levels of endogenous prolactin that they contained. We then showed that exogenously added prolactin acted as a potent survival factor against apoptosis in all the cell lines examined. In addition, we demonstrated that a prolactin-neutralising antibody in combination with C2-ceramide caused an anticipated, additive increase in cell death. This study demonstrated that prolactin protects human breast cancer cell lines against apoptosis and this may have important implications for cancer treatment.