Molecular aspects of insulin resistance, cell signaling pathways and breast cancer in relation to obesity.

A growing number of clinical studies validate a relation of insulin resistance and breast cancer in obese patients. We hypothesised that high plasma insulin levels cause aberrant insulin signalling in breast epithelial cells which may be responsible for an increase in cell proliferation, indicative of potential carcinogenesis and increased cancer progression. It was of particular interest to determine any differences of high insulin concentrations in activating the phosphoinositide-3 kinase (PI-3 kinase) pathway or the mitogen-activated protein kinase (MAP kinase) pathway, the latter being linked to increased cell proliferation. We used two cell line models to investigate the carcinogenic (MCF-10A, immortalised breast epithelial cells) and cancer progression (MDA-MB-231, ER-negative breast cancer cells) potential of insulin. Insulin treatment (100 nM, 24 h) increased cell proliferation in MCF-10A cells, but had no cell proliferative effect on MDA-MB-231 cells. Additionally expression of PCNA as marker of proliferation was tested. The use of PI-3 kinase and MAP kinase specific inhibitors (Wortmannin and PD98059, respectively) demonstrated both pathways being responsible for the observed increase in cell proliferation (MCF-10A). Simultaneous treatment with both inhibitors eliminated insulin induced cell proliferation entirely. Phosphorylation of ERK1/2 was examined as specific activity measurement of MAP kinase pathway. Insulin induced higher phosphorylation levels in MCF-10A cells than in MDA-MB-231. These preliminary results suggest that insulin may initiate carcinogenesis of breast epithelial cells by increasing cell proliferation rather than increasing cancer progression of existing tumours. These effects may be mediated by insulin activating both the PI-3 kinase and the MAP kinase signalling pathways

Insulin-induced gene expression changes in breast cancer cells and normal breast epithelial cells.

Obesity increases breast cancer incidence rates in postmenopausal women. Chronic high levels of insulin, present in the majority of obese and insulin resistant patients, may provide the growth promoting stimulus to explain this connection. In this work, the cancer progression and cancer initiating properties of high insulin levels were examined in breast cancer cells (MDA-MB-231) and breast epithelial cells (MCF-10a), respectively. High insulin levels (100 nM) induced differential changes in cell proliferation in the two cell lines used. Human Cancer PathwayFinder DNA Microarrays (SABiosciences) were used to examine gene expression changes after insulin treatment. High insulin levels increased expression of genes involved in cell cycle control (e.g. cyclin D1) and DNA damage repair (e.g. ATM) in MDA-MB 231 cells and in MCF-10a cells (e.g. cyclin E1, CDC25a). Expression of genes responsible for mediating apoptosis and cell senescence (e.g. APAF, BAD, bcl-X) was decreased after insulin treatment in MDA-MB 231 cells but the expression of the same group of genes did not change in MCF-10a cells. High insulin levels increased expression of genes encoding for signal transduction molecules (e.g. AKT1) and transcription factors (e.g. FOS, JUN, MYC), and of genes responsible for invasion and metastasis (e.g. MMP2) in MCF-10a cells whereas gene expression of the same groups of genes did not change or was decreased in MDA-MB 231 cells. These results suggest a role for insulin resistance in breast cancer initiation and progression, aggravating the potential of breast cancer to evade apoptosis, to metastasise and may promote carcinogenesis of healthy epithelial cells

Molecular aspects linking insulin resistance to breast cancer by activation of cell signalling pathways.

Recent findings suggest a connection between obesity and breast cancer. Obesity is linked with higher incidences of insulin resistance as part of the metabolic syndrome, resulting in chronically elevated insulin plasma levels. We examined the effect of high insulin concentrations (100 nM) on estrogen-receptor (ER) negative breast cancer cells (MDA-MB-231) and normal breast epithelial cells (MCF-10a). Treatment with high insulin concentrations increased insulin receptor (IR) phosphorylation significantly in both cell lines. Phosphorylation of protein kinase B (Akt), representative of PI3-kinase cell signalling pathway activation was increased by 101% (p=0.0112) in MDA-MB-231 cells and by 81% (p=0.0031) in MCF-10a cells after 10 min insulin treatment. Phosphorylation of extracellular regulated kinase 1/2 (ERK1/2), representative of MAP-kinase cell signalling pathway activation did not change in both cell lines after 10 min of insulin treatment. Cell proliferation did not change in MDA-MB-231 cells and increased by 75% (p=0.0067) in MCF-10a cells after 24 h insulin treatment. Cell proliferation was decreased in MDA-MB-231 cells by 15% (p=0.0083) after 1 h treatment with PD98059, a MAP-kinase inhibitor. In MCF-10a cells cell proliferation was decreased by 51% (

Role of carbohydrate response element-binding protein (ChREBP) in generating an aerobic metabolic phenotype and in breast cancer progression

Background: The lipogenic transcription factor carbohydrate response element-binding protein (ChREBP) may play a key role in malignant progression of breast cancer by allowing metabolic adaptations to take place in response to changes in oxygenation. Methods: Immunohistochemical analysis of ChREBP was carried out in human breast tumour tissue microarrays representative of malignant progression from normal breast through to metastatic cancer. The ChREBP protein and mRNA expressions were then analysed in a series of breast cancers for correlative analysis with common and breast-specific hypoxia signatures, and survival. Results: In invasive ductal carcinoma, ChREBP correlated significantly with mean ‘downregulated’ hypoxia scores (r=0.3, P<0.015, n=67) and in two distinct breast progression arrays, ChREBP protein also increased with malignant progression (P<0.001). However, bioinformatic analysis of a large data set (2136 cases) revealed an apparent reversal in the relationship between ChREBP mRNA level and clinical outcome – not only being significantly correlated with increased survival (log rank P<0.001), but also downregulated in malignant tissue compared with adjacent normal tissue. Conclusion: The ChREBP expression may be reflective of an aerobic metabolic phenotype that may conflict with hypoxia-induced signalling but provide a mechanism for growth at the oxygenated edge of the tumours