Abstract

Triple negative breast cancer (TNBC) is the most lethal breast cancer subtype. Extended periods of lactation protect against breast cancer development, but the mechanisms underlying this protection are unknown. We examined the effects of the milk protein alpha-casein over expression in the triple negative MDA-MB-231 breast cancer cell line. The effects of recombinant alpha-casein added exogenously to MDA-MB-231 breast cancer cells, and immortalised human fibroblasts were also investigated. We used transcriptional reporters to understand the signalling pathways downstream of alpha-casein in breast cancer cells and these fibroblasts that were activated by breast cancer cells. To extend our findings to the clinical setting, we analysed public gene expression datasets to further understand the relevance of these signalling pathways in triple negative breast cancer cells and patient samples. Finally, we used small molecular inhibitors to target relevant pathways and highlight these as potential candidates for the treatment of TN breast cancer. High levels of alpha-casein gene expression were predictive of good prognosis across 263 TNBC patient tumour samples. Alpha-casein over expression or exogenous addition reduces cancer stem cell (CSC) activity. HIF-1alpha was identified to be a key downstream target of alpha-casein, in both breast cancer cells and activated fibroblasts, and STAT transcription factors to be upstream of HIF-1alpha. Interestingly, HIF-1alpha is regulated by STAT3 in breast cancer cells, but STAT1 is the regulator of HIF-1alpha in activated fibroblasts. In analysis of 573 TNBC patient samples, alpha-casein expression, inversely correlated to HIF-1alpha, STAT3 and STAT1. STAT1 and STAT3 inhibitors target HIF-1alpha signalling in activated fibroblasts and MDA-MB-231 breast cancer cells respectively, and also abrogate CSC activities. Our findings provide an explanation for the protective effects of lactation in TNBC. Clinical data correlates high alpha-casein expression with increased recurrence-free survival in TNBC patients. Mechanistically, alpha-casein reduces breast cancer stem cell activity in vitro, and STAT3 and STAT1 were identified as regulators of pro-tumorigenic HIF-1alpha signalling in breast cancer cells and fibroblasts respectively.

Highlights

  • Breast cancer is the most common malignancy, and the biggest cancer-related killer of women worldwide [1]

  • Breast cancer stem cells (BCSC) are proposed to be responsible for the chemotherapy resistance and lethal metastasis associated with Triple negative breast cancer (TNBC), and the targeting of such cells is a promising strategy in its treatment

  • This has previously reported to be a marker of breast cancer stem cells, and predicts for poor clinical outcome [28]. alpha-casein overexpression reduces the population of ALDH positive cells by nearly half (Fig. 1f)

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Summary

Introduction

Breast cancer is the most common malignancy, and the biggest cancer-related killer of women worldwide [1]. Risk factors such as early menarche, late menopause, and late first pregnancy (after 30) are all associated with an increase in. Early first pregnancy, multiple births and extended periods of lactation confer a reduced risk of developing breast cancer [5]. A signature of this subgroup is an enrichment for CD24+/ CD44+ and ALDH1 [9, 10], which confer a breast cancer stem cell phenotype [11]. BCSC are proposed to be responsible for the chemotherapy resistance and lethal metastasis associated with TNBC, and the targeting of such cells is a promising strategy in its treatment

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