The Milk Protein Alpha-Casein Suppresses Triple Negative Breast Cancer Stem Cell Activity Via STAT and HIF-1alpha Signalling Pathways in Breast Cancer Cells and Fibroblasts
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.
KeywordsBreast cancer stem cells Cancer activated fibroblasts HIF-1alpha. STAT
Triple negative breast cancer
Cancer associated fibroblast
Breast cancer stem cell
Recurrence free survival
Breast cancer is the most common malignancy, and the biggest cancer-related killer of women worldwide . Risk factors such as early menarche, late menopause, and late first pregnancy (after 30) are all associated with an increase in breast cancer risk [2, 3, 4]. Inversely, early first pregnancy, multiple births and extended periods of lactation confer a reduced risk of developing breast cancer . Notably, the protective effects of lactation are most profound in the aggressive triple negative subgroup [2, 3, 6], but the physiological reasons for this phenomenon remain unclear.
Triple negative breast cancer (TNBC), as defined by the lack of estrogen (ER), progesterone receptors (PR) and low expression of HER2 receptor  is the most lethal breast cancer, and there are currently no convincing targeted therapies  By definition, TNBC is not treatable by common targeted anti-cancer drugs, as they lack the receptors capable of responding to them. A signature of this subgroup is an enrichment for CD24+/ CD44+ and ALDH1 [9, 10], which confer a breast cancer stem cell phenotype . Breast cancer stem cells (BCSC) are a small population of cells that reside within the tumour mass that are capable of self-renewal, but also to repopulate all the cell types within the heterogeneous tumour mass [12, 13]. BCSC possess increased tumour initiating properties and metastatic potential . 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.
Another factor important in tumour progression is the tumour microenvironment (TME). One of the most abundant cell types in the TME are fibroblasts, which become ‘activated’ by surrounding cancer cells to acquire a ‘cancer associated fibroblast’ (CAF) phenotype [15, 16]. CAFs are phenotypically different to their normal fibroblast counterparts, and are defined by expression of alpha-smooth muscle actin (alpha-SMA) and fibroblast activation protein-alpha (FAP-alpha) [17, 18]. CAFs promote cancer progression by providing secretory factors that promote migration and invasion [19, 20], and to promote the BCSC phenotype .
Extended periods of lactation are protective against TNBC. The milk protein alpha-casein was shown to reduce tumour burden and experimental lung metastasis when over expressed in mouse Met-1 tumour cells (22). In the present study, we assessed the effects of alpha-casein on BCSC activity in vitro and found alpha-casein to significantly reduce BCSC in the triple negative MDA-MB-231 cell line. Mechanistically, we showed this activity to be mediated by HIF-1alpha, a hypoxia inducible transcription factor closely associated with the induction and maintenance of a BCSC phenotype [23, 24, 25]. We further investigated the effects of alpha-casein on the TME and show alpha-casein to also regulate HIF-1alpha signalling in CAFs. We identified STAT3 and STAT1 to be crucial transcription factors in regulating HIF-1alpha in breast cancer stem cells and CAFs respectively, and used inhibitors to demonstrate their roles in HIF-1alpha regulation and CSC activity.
Cell Lines and Culture
The human MDA-MB-231 mammary tumour cells were obtained from ATCC (ATCC® HTB-26™). The human hTERT immortalised human fibroblasts (BJ1) were obtained from CLONTECH Laboratories (CCD00309196). Cell lines were passaged for <25 passages and regularly checked for mycoplasma. Cells were cultured in complete DMEM (Sigma-Aldrich) supplemented with 10% heat inactivated foetal bovine serum, 2 mM GlutaMAX (L-alanyl-L-glutamine), 100 U/ml Penicillin and 100 μg/ml Streptomycin and incubated at 37 °C with 5% CO2. All incubations performed at 37 °C with 5% CO2.
Stable Cell Line Generation
Transcriptional response elements used in luciferase reporter constructs
Cignal lenti ISRE reporter (luc) CLS-008 L
Type I IFN
Cignal lenti GAS reporter (luc) CLS-009 L
Type II IFN
Cignal lenti STAT3 (luc) CLS-6028 L
Cignal lenti NF-κB (luc) CLS-013 L
Cigna lenti HIF-1α (luc) CLS-007 L
Production of Conditioned Media
2 × 104 cells / cm3 were cultured in 7.5 ml/ cm3 of low serum (Nu-serum) media for 48 H. media was collected and centrifuged to remove debris and supernatant collected.
Human alpha-casein antibody F20.14 (Abcam ab47972). STAT1 #9172, Phospho-STAT1 (Tyr 701) #9167, Phospho-STAT3 (Tyr 705) #9131 purchased from Cell Signalling (1:1000). β-actin loading control (Sigma-Aldrich A2228) (1:10000).
Cell lysates were collected using RIPA lysis buffer (Sigma-Aldrich) with cOmplete ULTRA protease inhibitor (Roche) and PhosSTOP (Roche) phosphatase inhibitor. 20 μg protein sample was loaded onto 12% polyacrylamide gel and transferred to nitrocellulose membranes. Membrane was blocked in 5% bovine serum albumin for 1 h at room temperature and then probed with primary antibody, diluted 1:1000 in blocking solution, and incubated at 4 °C overnight. Anti-mouse or anti-rabbit secondary antibodies (1:500) were added and incubated at room temperature for 1 h. Protein was detected using SuperSignal™ West Pico or SuperSignal™ West Dura chemiluminescent Substrate (ThermoFisher) and developed on Kodak film.
Non-adherent mammosphere culture plates were made by covering clear 6 well plates with 2 ml poly-HEMA and incubated at 60 °C for 3 days. Cells were syringed through a 25 gauge needle to provide cells in single cell suspension. A 1:1 volume of Trypan blue was used to count viable cells, and 5 × 103 single cells added to each well in DMEM/F-12 (Sigma-Aldrich) with B27 supplement, 20 ng/ml EGF and 100 U/ml Penicillin and 100 μg/ml Streptomycin. Plates were then incubated for 5 days, and spheres >50 μM counted at 10 x magnification. The mammosphere formation efficiency (MFE) was calculated by dividing the number of spheres >50 μM after 5 days by the number of cells seeded, and is expressed as a percentage.
2 × 105 cells were incubated with or without drug treatment for 48 h. 2 × 105 were re-suspended in 1 ml Dublecco’s PBS. Dry Aldefluor reagent was reconstituted as per manufacturers’ instructions. 5 μl Aldefluor reagent was added and 500 μl sample removed and added to a fresh eppendorf tube containing 10 μl DEAB to stop the reaction. This is the control sample. Cells were then incubated at 37 °C, 5% CO2 for 40 min, and centrifuged at 4 °C. Cell pellets were then re-suspended in 500 μl Aldefluor assay buffer and analysed using the BD Laser Fortessa cell analyser (Biosciences).
Luciferase Reporter Assays
5 × 103 reporter cells were seeded in triplicate in 96 well black plates and incubated overnight. Culture medium was removed and cells washed with PBS, and 100 μl control media, conditioned media or drug treatment was then added. Plates were then incubated for 24, 48 or 72 h as per experimental design. Reporter cells were fluorescently labelled to normalise luciferase activity. Following fluorescence reading, cells were lysed and luciferase activity measure using the Promega luciferase system and read on a IVIS® luminometer (PerkinElmer).
1 × 104 cells per well were seeded in triplicate in 96 well black plates, and incubated for 8 h for cells to adhere. 20 μl PrestoBlue® reagent was added and incubated for 1 h and fluorescence (excitation 544 nm, emission 621–10 nm) measured using a FLUOstart Omega microplate reader (BMG Labtech) for T = 0 timepoint. Further plates are incubated for 24, 48 and 72 h, and fluorescence measured as before. Fluorescence readings were normalised by T = 0 h.
Transwell Migration Assay
5 × 104 cells were added to the top of 8 μM pore cell culture inserts (Transwells) (Falcon) placed in 24-well plates in serum-free media. Cells were allowed to migrate through the membrane for 6 h at 37 °C, towards 10% foetal bovine serum placed in the bottom wells to serve as chemoattractant. Non-invasive cells were removed from the top of the wells using cotton swabs, and migrated cells stained with crystal violet solution. After 20 min, wells were washed in H20 and membranes left to dry overnight. The number of migrated cells was calculated using a bright-field microscope, where the number of cells in six fields of view per membrane (20x objective) were counted. An average number was calculated over 3 independent experiments, in duplicate, carried out on independent days.
All graphs were produced in Graphpad Prism 7, and this software was used for statistical analysis. Data are shown using the standard error of the mean (±SEM), taken over 3 biological replicates with 2 or 3 technical replicates. Statistical significance was calculated using parametric testing, assuming equal variance, with un-paired Student’s t test used to confirm statistical significance. Statistical significance was governed as a p value < = 0.05. Gene expression levels of alpha-casein signalling genes were retrieved from 17 Affymetrix datasets integrated to remove batch effects as previously described . Similarly, Affymetrix gene expression data from three breast cancer cell line panel studies were integrated as described previously . Cox proportional hazards survival analysis was performed for all possible points-of-separation (low–high cut-off points) for each gene (Pearce DA, Nirmal AJ, Freeman TC, Sims AH. Continuous biomarker assessment by exhaustive survival analysis. bioRxiv 2017. doi: https://doi.org/10.1101/208660). Gene expression levels of alpha-casein between normal and invasive ductal carcinoma were explored with dataset GSE10780 .
Alpha-Casein Expression Correlates with Improved Clinical Outcome in Triple Negative Breast Cancer, and Reduces Breast Cancer Stem Cell Activity In Vitro
Next, we tested the effect of alpha-casein overexpression on ALDH enzyme activity using the Aldefluor assay. This has previously reported to be a marker of breast cancer stem cells, and predicts for poor clinical outcome . alpha-casein overexpression reduces the population of ALDH positive cells by nearly half (Fig. 1f). These results suggest the anti-tumourigenic effects of alpha-casein in vivo  may partly be explained by the ability of alpha-casein to inhibit cancer stem cell activity.
Alpha-Casein Functions Mechanistically to Target pro-Tumorigenic HIF-1alpha Signalling in Breast Cancer Cells and Fibroblasts
We next wanted to investigate the cellular signalling mechanisms responsible for the inhibitory effects of alpha-casein on BCSC activity. HIF-1alpha stabilisation and upregulation promotes and maintains the BCSC phenotype via multiple mechanisms (Mathieu et al, 2011; Schwab et al, 2012; Zhang et al, 2016). NF-kB is a second well -established signalling pathway that is upregulated in breast cancer, and known to regulate SC activity (Iliopoulos et al, 2009; Iliopoulos et al, 2011; Rinkenbaugh and Baldwin, 2016; Shostak and Chariot, 2011). Thus, we sought to investigate alpha-casein effects on these key CSC signalling pathways in breast cancer.
Alpha-Casein Inhibits STAT3 and STAT1 in Breast Cancer Cells and Fibroblasts Respectively
STAT3 is a strong candidate for HIF-1alpha regulation in breast cancer cells. Previous studies in breast cancer cells showed that STAT3 can regulate transcription of HIF-1alpha target genes directly , but that STAT3 and HIF-1alpha also work cooperatively to regulate HIF-1alpha target genes via the formation of an ‘enhansome complex’ with CREB binding protein and p300 .
Alpha-Casein Expression Inversely Correlates with STAT1, STAT3 and HIF-1alpha in Basal Breast Cancer
STAT3 and STAT1 Function Upstream of HIF-1alpha in Breast Cancer Cells and Fibroblasts Respectively
Targeting STAT3 Reduces BCSC Activity
TNBC is the most aggressive breast cancer subtype, and is associated with poor clinical outcome. A major factor governing this lethality is the propensity of TNBC to recur following chemotherapy [33, 34], metastasise [35, 36], and the lack of targeted therapy. One of the major factors responsible for this chemotherapy resistance, and thus high recurrence rates in TNBC is the enrichment of BCSC, particularly in this subtype [9, 10]. Furthermore, chemotherapy, which is the current mainstay treatment for TNBC, has been shown to further enrich for cells with a BCSC phenotype [37, 38], with the activation of the hypoxia inducible factor HIF-1alpha being key in this response .
The inhibitory effects of the milk protein alpha-casein on mouse mammary tumour burden and experimental lung metastasis in vivo have previously been described . There are a number of possible explanations why extended lactation could be protective, for example, limiting the exposure of the breast to the inflammatory environment of involution, or preventing potentially pre-cancerous cells from proliferating. If lactation is having a protective effect, it must be preventing transformation from normal to pre-cancerous cell, progression from pre-cancerous to tumour cell, or preventing growth and metastasis of the tumour. In this study, we have modelled the final lethal stage, where acquisition of a CSC phenotype promotes invasion and metastasis and provided functional explanation for these findings, where alpha-casein disrupts the CSC activity of MDA-MB-231 TNBC cells. In future studies it will therefore be important to test the effects of casein on earlier stages of progression.
A role of HIF-1alpha in the propagation and maintenance of this CSC phenotype has been reported [23, 24, 40], and this study further supports these ideas. We investigated the effects of alpha-casein on HIF-1alpha activation and signalling and found it to inhibit HIF1-alpha in TNBC cells, and that STAT3 is a regulator of HIF-1alpha signalling in this context. We used small molecule inhibitors of STAT3 to show its importance in the activation of HIF-1alpha in TNBC cells and downstream BCSC activity, where STAT3 and HIF-1alpha inhibition results in a > 70% reduction in CSC characteristics. STAT3 has previously been implicated in the promotion of CSC activity in hepatocellular carcinoma via activation of the Notch signalling pathway . Herein we have found an alternative mechanism of STAT3 mediated CSC activation in breast cancer, where STAT3 phosphorylation at Y705 and concurrent activation increases BCSC via HIF-1alpha.
Interestingly, alpha-casein also inhibits HIF-1alpha signalling in fibroblasts that have been activated by breast cancer cells, but the mechanism of regulation is via a different STAT transcription factor in these cells, STAT1. As the adoption of this CAF phenotype via HIF-1alpha serves to promote cancer progression by providing metabolites and nutrients to energy-demanding cancer cells [41, 42] and promoting a CSC phenotype [23, 24, 40], alpha-casein may also function via a feedforward inhibition of HIF-1alpha. Here, inhibition of HIF-1alpha signalling decreases the CSC activity, propagation and invasiveness of breast cancer cells, which in turn reduces CAFs in the surrounding stroma.
Thus, alpha-casein has a dual role on inhibiting breast cancer progression. We propose a mechanism (Fig. 6) whereby alpha-casein directly inhibits the CSC activity of transformed breast cancer cells via inhibition of STAT3 and HIF-1alpha. Alpha-casein also inhibits HIF-1alpha in CAFs to abrogate the pro-tumorigenic reciprocal relationship between CAFs and BCSCs and ultimately cancer progression and metastasis.
We have provided mechanistic evidence as to how extended periods of lactation can potentially protect against TNBC. In the present study, we describe a new role of the milk protein alpha-casein as a negative regulator of CSC activity and transformation to a CAF phenotype in TNBC via HIF-1alpha (Fig. 6). This work indicates that targeting STAT1 and STAT3 using small molecule drugs could be beneficial to patients in the clinic.
We thank Priti Kalra, Ernestina DeFrancesco and Gloria Bonuccelli for technical assistance.
KELG designed experiments, acquired, analysed and interpreted the data and drafted the manuscript, NJH performed some mammosphere experiments, RL designed experiments, AHS analysed gene expression data, RBC designed experiments, interpreted data and critically revised the manuscript.
We acknowledge Breast Cancer Now (Grant MAN/Q2) and the NIHR Manchester Biomedical Research Centre (IS-BRC-1215-20007) for funding this project.
Compliance with Ethical Standards
Ethics Approval and Consent to Participate
Consent for Publication
We have agreement from all authors (Kirsten Garner, Rebecca Lamb, Andy Sims and Robert Clarke) to submit this manuscript for publication.
Conflict of Interest
The authors declare that they have no competing interests.
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