Leptin : linking obesity to breast cancer progression

Abstract

Epidemiological studies have shown obesity to be linked with high incidence of cancer, high tumour grades, more metastasis, drug resistance, recurrence and hence poorer outcomes in breast cancer patients. In women, obesity is linked to 20% of all cancer deaths. The molecular mechanisms are largely unknown but the reasons for the increased risk of invasive/metastatic disease with obesity may include elevated levels of hormones like leptin, Insulin-like growth factors and estrogens. Leptin is a pleiotropic hormone primarily synthesized by adipose tissues and circulating levels are higher in obese people (200ng/ml) than non-obese people (10ng/ml-30ng/ml). Leptin receptor was found to be expressed in MCF10A, MCF10AT1, MCF7 and MDA-MB-231 breast epithelial and cancer cells and it was highly expressed in more invasive breast cancer cells (28-fold higher mRNA expression in MDA-MB-231 than MCF7, P<0.01), thus leptin was chosen for further investigation. To assess the effect of leptin on metastatic capacity of breast cancer cells in vitro, MCF7 cells were treated for 14 days with leptin. Compared to untreated MCF7 cells, treatment with 200ng/ml of leptin increased breast cancer cell motility (scratch assay, 1.59-fold, P<0.001) and invasiveness (1.66-fold, P<0.001). Leptin treatment induced epithelial to mesenchymal transition (EMT), decreasing CDH1 mRNA (5.5-fold, P<0.01) and increasing SNAI2 mRNA (4.4-fold, P<0.001) compared to the untreated cells. Leptin also induced aldehyde dehydrogenase activity by 5.84-fold (Aldefluor, P<0.01) and increased colony forming capacity (mammosphere assay, 1.53-fold, P<0.01) indicating an increased cancer stem cell (CSC) like phenotype. To assess the effect of leptin treatment on metastatic capacity of breast cancer cells in vivo, the tail vein metastasis model in Rag1 knockout mice was used. Mice injected with leptin-treated MCF7 cells had significantly higher lung weight compared to mice injected with untreated MCF7 cells (1.19-fold, P<0.01) however, there were no visible metastases in lungs in either group. On evaluation with Q-PCR, human DNA was detected in the lungs from both groups of mice and the relative amount of human DNA was higher (1.33-fold) in the leptin-treated MCF7 group but this was not statistically significant. The mechanism of leptin signalling was investigated. Leptin (200ng/ml) treatment increased transforming growth factor beta-1 expression in MCF7 cells (5.4-fold, P<0.01) and co-treatment with leptin and neutralizing antibody against transforming growth factor beta-1 abrogated the effects of leptin treatment on cell migration, EMT and CSC, indicating that the induction of these characteristics was mediated via transforming growth factor beta-1. All these effects of elevated leptin treatment were also observed in MCF10AT1 cells suggesting leptin action to be independent of estrogen receptor status. These studies indicate the potential significance of elevated leptin in breast cancer progression. Further investigations are required to determine the significance of the contribution of leptin in vivo and whether targeting elevated leptin levels has any potential as a therapeutic or preventive strategy for breast cancer.