Tumor cell heterogeneity has been a known fact for a long time, but evidence increasingly suggests that heterogeneity of tumors may be associated with a subpopulation of tumor-initiating cells, also called cancer stem cells (CSCs), as a subpopulation driving tumorigenesis and cancer progression [1
]. These cells represent only a small proportion of tumor mass, but seem to have the capability of dissemination and may, for still unknown reasons, reactivate from the quiescent state and cause recurrence of the disease [2
]. The fate of CSC seems to be highly dependent on their niche and state, either activating or quiescent, which may be determined by their microenvironment. This concept of the tumor being dependent on its microenvironment has been postulated early by Stephen Paget [4
], and by clinical trials demonstrating that therapeutic interventions with bisphosphonates positively impact the clinical outcome of breast cancer patients, and confirmed the importance of these interactions [5
]. Today, a wide array of evidence suggests that the network of interactions between the tumor, the microenvironment with the stroma, the extracellular matrix and the inflammatory cells bidirectionally modulate their tumorigenicity [7
]. Despite recent advances, interactions between CSC and the microenvironment are difficult to study due to a lack of optimal methods for the isolation of CSC and efficient functional assays, as well as due to a variety of proteins, enzymes, and growth/inhibition factors forming the extracellular matrix (ECM) of the tumor and the CSC niche. In vitro sphere formation assays have been shown to be suitable surrogate models to study CSC biology [9
Numerous factors govern cell growth to generate CSC, and epithelial to mesenchymal transition (EMT) is the process that strongly supports and/or generates the CSC phenotype [11
]. EMT is a process normally occurring in embryological development, but if awakened latter in the adult organism, it becomes pathological and generates mesenchymal cells with the ability to migrate [11
]. This process is reversible, but in the means of cancer, it is highly undesirable, and EMT and back to mesenchymal to epithelial transition (MET) is the process that causes metastasis [12
]. EMT is accompanied by changes in many signaling pathways, which result in differential expression of EMT transcription factors such as snail family transcriptional repressor 1 (SNAIL), snail family transcriptional repressor 2 (SLUG), twist family bHLH transcription factor 1 (TWIST1) [13
], but also Nanog homeobox (NANOG), POU class 5 homeobox 1 (OCT4), and SRY-box transcription factor 2 (SOX2) [14
]. Studies suggest that these transcription factors, especially TWIST1, can translocate to the nucleus upon increased stiffness of ECM, represented by collagen I [15
], which indicates that ECM has a role in this process.
Oxidative stress, a state of increased reactive oxygen species (ROS) production, affects all cell systems. It also represents an important factor contributing to the modulation of tumor cell and microenvironment reactions to chemotherapeutics. Increased ROS may lead to numerous consequences, such as genetic instability, one of the major characteristics of cancer, and the modification of lipids by peroxidation [16
]. Lipid peroxidation (LPO) with its end-products—reactive aldehydes—have been increasingly recognized as a biomarker of different diseases, particularly cancer, where mitochondrial HNE plays an important role [17
]. In addition, these reactive aldehydes, especially 4-hydroxy-2-nonenal (HNE), are involved in different signaling pathways influencing the cells’ fate (e.g., differentiation, proliferation, or apoptosis) [18
]. One of the signaling pathways affected by HNE is NRF2/KEAP1(Nuclear factor erythroid 2-related factor 2/Kelch-like ECH-associated protein 1) [20
]. NRF2 is an antioxidant transcription factor that is bound to KEAP1 in its inactive state. HNE binds to KEAP1 cysteines and thereby releases its inhibition of NRF2. The release of NRF2 causes its translocation to the nucleus and activation of antioxidant genes’ transcription and consequently enabling cells to survive oxidative challenge [20
The present study aimed to elucidate if oxidative stress and HNE-modified collagen I, as a representative protein of ECM, in combination with HNE-induced chronic stress influence BCSC. The changes in the frequency of BCSC, antioxidative defense system, and transcriptional and protein expression of EMT markers were evaluated. These changes indicated that different surface modifications and chronic stress may bidirectionally modulate BCSC, supporting either differentiation or stress adaptation.
2. Materials and Methods
2.1. Cell Line and Medium
SUM159 cells (Asterand, Royston, Hertfordshire, UK), estrogen receptor, progesterone receptor, and Her2negative cell line, with the potential of generating stem-like subpopulation were cultured as mammospheres, according to previous publications [1
]. Briefly, cells were cultured in Mammary Epithelial Basal Medium (MEBM; Lonza, Basel, Switzerland) supplemented with 10 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL epidermal growth factor (EGF, both from Peprotech, Rocky Hill, Hartford County, CT, USA), 5000 U/mL heparin (Sigma Aldrich, St Louis, MO, USA) and 20 μL/mL B27 supplement (Gibco/Invitrogen, Waltham, MA, USA) at 37 °C in a 5% CO2
humidified atmosphere. Mammospheres larger than 40 μm were collected with 40 μm nylon cell strainers (Corning Incorporated-Life sciences, Durham, N.C., USA) and used for experiments.
2.2. Collagen Coating
To test cell growth characteristics on an extracellular matrix (ECM), collagen I was used as an ECM representative protein. Collagen I (Sigma Aldrich, St Louis, MO, USA) was dissolved in acetic acid (50 mM, Kemika, Zagreb, Croatia), diluted in redistilled sterile water in a final concentration of 2 mg/mL and used in the native state or modified by 1 or 10 µM HNE (Enzo Life Sciences, Lausen, Switzerland). Depending on the type of analysis, different formats of cell culture dishes were used with the same coating conditions: Native or modified collagen to its final concentration of 5 μg/cm2
. Thus, coated cell culture dishes were left to dry in a laminar flow cabinet overnight at room temperature (RT) and subsequently sterilized under UV light for 20 min. Dot-blot analysis with HNE-histidine monoclonal antibody was applied to confirm the binding of HNE to collagen I had occurred (Supplementary Figure S1
). After confirmation that HNE did bind to histidine residues of collagen, we proceeded with evaluating the influence of collagen on measured parameters. Cells were also seeded on uncoated surfaces, further referred to as polystyrene (PS).
2.3. Cell Seeding and HNE Treatment
Mammospheres were dissociated to a single cell suspension by TrypLE (Gibco/Invitrogen Paisley, UK), and 10,000 cells/100 µL were plated in pre-coated or uncoated cell culture dishes and left to adhere for 3 h. Regardless of the cell culture dish format used, the experimental stoichiometry was maintained in all analyses. The formats of the cell culture dishes were as follows: 96-well microplates (cell viability and proliferation; TPP, Techno Plastic Products AG, Trasadingen, Switzerland); 6-well microplates (qRT-PCR, Western blot; Falcon, BD Biosciences, Franklin Lakes, NJ, USA); 8-well glass chamber slide (immunocytochemical analyses of hormone receptors; Nalgen Nunc Int, Naperville, IL, USA). Cells were then treated with different concentrations of HNE once, for a single treatment, or every 2nd day for 10 days, for multiple treatments. For cell viability and cell proliferation assays, these HNE concentrations varied from physiological (1 to 10 µM) to supraphysiological and pathological (25 to 100 µM). Controls of each growth surface were cultured without HNE. Analyses were performed after 48 h for single HNE treatments and 10 days for multiple HNE treatments as described for each analysis below. After the analysis of cell proliferation and cell viability, 10 µM HNE was selected for further analyses of putative breast cancer stem cell phenotypes, EMT marker expression, and immunocytochemical analyses of hormone receptors and antioxidative defense system. Untreated cells of each coating condition served as controls. All the mentioned analyses are described in more detail below.
2.4. Cell Viability—MTT Assay
The cell viability was determined by an MTT-based assay, EZ4U, following the manufacturer’s recommendations (Biomedica, Vienna, Austria). Briefly after the treatment, 48 and 10 days after the seeding, cells were incubated with the MTT dye for an hour, and the absorbance was measured on a plate reader at 450 nm with a reference wavelength at 620 nm (Easy-Reader 400 FW; SLT Lab Instruments, GmbH, Salzburg, Austria).
2.5. Cell Proliferation—3H-thymidine Incorporation Assay (3HT)
The assay was based on the incorporation of radioactively labeled thymidine to the replicating DNA. The assay was performed as described previously [3
]. Briefly, cells were treated as described in the previous chapter. 3
H-thymidine (1 µCi/well) was added to each well 24 h or 9 days after HNE treatment(s) and left for 24 h to allow thymidine incorporation into the DNA. Cells were then harvested, and the rate of 3
H-thymidine incorporation was measured on a Wallac 1904 DSA liquid scintillation counter (Perkin Elmer, Waltham, MA, USA).
2.6. Flow Cytometry Analyses of Putative Breast Cancer Stem Cell Phenotypes
For analyses of putative breast cancer stem cell markers, cells were treated as described above. After 10 days, cells were collected from culture dishes with accutase (PAA Laboratories GmbH, Pasching, Austria). Cells were then incubated for 5 min at 37 °C and rinsed twice with phosphate-buffered saline (PBS). Cells forming mammospheres during the experiments were singularized with TrypLE and finally resuspended in MEBM with supplementation for further analyses.
For the Aldefluor assay, cells were washed, counted, and finally resuspended in Aldefluor buffer [21
]. To measure aldehyde dehydrogenase (ALDH) activity, the Aldefluor assay (STEMCELL Technologies, Grenoble, France) was performed according to the manufacturer’s instructions and as previously published [21
]. Briefly, 2 sets of samples with the Aldefluor substrate BODIPY-aminoacetaldehyde (BAAA) were prepared: (a) control: With diethylaminobenzaldehyde (DEAB, the specific inhibitor of ALDH) and (b) sample: Without DEAB. Controls were used for establishing the background fluorescence of these cells and defining the ALDH-positive region on the Fluorescence Channel 1 (FL1*) vs. the SSC dot plot. The absence of DEAB in the sample group converted BAAA to its fluorescent product, BODIPY-aminoacetate (BAA), defining the ALDH-positive population.
For analyses of CD44 and CD24 expression, cells were incubated with horse serum dilute 1:20 in 6% bovine serum albumin (BSA)/PBS for 30 min. Aliquots of antibodies anti-CD44 Allophycocyanin and anti-CD24 Fluorescein isothiocyanate (BD Bioscience, Schwechat, Austria) at a dilution of 1:40 in a final concentration of 0.08 µg/mL and 5 µg/mL, respectively, were then added and the samples were incubated at 4 °C for 30 min. The cells were washed and stored at 4 °C in the dark until the acquisition on the flow cytometer was performed. The protocol was performed as previously published [2
]. Cells without staining and isotype controls, all from BD Bioscience, were integrated as controls in all experiments.
All samples were assayed on an LSRII flow cytometer (BD Bioscience), and the data were analyzed with the DIVA software version 8.0.1 (BD Bioscience Concorde Business Park 1/E/1/7, Schwechat, Austria).
2.7. Immunocytochemical Analyses of Hormone Receptors
For immunocytochemical analyses, cells were treated as described above. After 10 days, cells were fixed in ice-cold methanol for 20 min, dried, and stored until the staining. Cells were subjected to the antigen retrieval using Tris-EDTA solution, pH 9.0, by heating at 85 °C for 10 min to enable correct epitope folding. The monoclonal mouse anti-human estrogen receptor α (M7047, clone 1D5, DAKO, Glostrup, Denmark) and monoclonal mouse anti-human progesterone receptor (M3569, clone PgR636, DAKO, Glostrup, Denmark), both diluted to 1:50 in 1% BSA/PBS, were used. The secondary antibody EnVision (DAKO, Glostrup, Denmark), was used as recommended by the manufacturer. Finally, the reaction was visualized by DAB (3,3-diaminobenzidine tetrahydrochloride in organic solvent). Nuclei were counterstained by hematoxylin. The positive reaction was evaluated and scored by a trained pathologist (S.Š.) in a blinded manner.
2.8. Real-Time Quantitative PCR (qRT-PCR) Analyses of EMT Markers
After the cell treatment for 10 days, total RNA was extracted using a TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the recommendation provided by the manufacturer. Nanodrop was used to quantify and asses the assay for purity (ThermoScientific, Waltham, MA, USA). The reverse transcription of one microgram of total RNA was performed using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) following the instructions of the manufacturer. LightCycler 480 (Roche) was used to perform qRT-PCR. Reactions were performed in 20 μL of total volume, consisting of 1× SYBR Green I Master Mix (Roche), 20 nanograms of cDNA as well as 25 μM of each primer (final concentration). All qRT-PCR reactions were conducted in duplicate, and afterward, the values of the quantification cycle were averaged. The comparative Ct method was utilized in the calculation of gene expression. Beta-2-microglobulin (B2M) and lactate dehydrogenase A (LDHA) were used as reference genes with the following primer sequences: B2M forward 5′TGCTGTCTCCATGTTTGATGTATCT 3′, B2M reverse 5′ TCTCTGCTCCCCACCTCTAAGT 3′ (NM_004048.3), LDHA forward 5′ TGTAGCAGATTTGGCAGAGAG 3′, LDHA reverse 5′ CATCATCCTTTATTCCGTAAAGAC 3′ (NM_005566.4). Primer sequences for fibronectin (FN), vimentin (VIM), N-cadherin (N CAD), SNAIL, SLUG, and TWIST were previously published [23
2.9. ROS and Antioxidant Measurements
For ROS and antioxidant measurements, cells were treated as described above. On the 10th day of experiments, cells were incubated with 2′,7′–dichlorofluorescin diacetate (DCFDA) to allow the dye to overload the cells. Excess DCFDA was removed after 60 min when the cells were either incubated with medium alone (control) or with 10 µM HNE. ROS were measured with a Cary Eclipse Fluorescence Spectrophotometer (Varian Australia Pty Ltd., Mulgrave, Victoria, Australia) at λex 500 nm and λem 529 nm.
For antioxidant measurements, cells were detached from the surface by TrypLE, and pelleted by centrifugation at the end of the 10-day treatment. Mammospheres were pelleted and dry pellets of all the experimental groups were stored till analyses. Prior to analyses, cells were lysed by 4 freeze/thaw cycles and afterward were centrifuged to remove cellular debris. Protein levels were then determined according to Bradford [24
]. The catalase activity was measured according to the method by Goth with some modifications [25
]. The activity of catalase was expressed as units per milligram of proteins in cell lysate (U mg–1
For the total GSH content, samples were diluted to 0.03 mg/mL and assayed using a modification of the Tietze method [26
]. Concentrations of total GSH were expressed as µM of GSH per milligram of total protein (nmol mg–1
2.10. Western Blot
In order to perform Western blot analyses, cells were treated for 10 days, as described above. After 10 days, the cells were lysed in RIPA buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)) containing protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany). The protein concentration of the thus obtained supernatant was quantified according to the Bradford method [24
] by measuring absorbance at 595 nm using the microplate reader Multiskan EX (Thermo Electron Corporation, Shanghai, China) and interpolating from the standard curve. Protein samples were mixed with Laemmli buffer, boiled for 5 min at 95 °C and 40 µg of total proteins were resolved on the Tris-glycine SDS-PAGE gels (9% or 10%) and transferred to nitrocellulose membranes (Roti®
-NC, Carl Roth, Karlsruhe, Germany). Membranes were stained with Ponceau S solution (Sigma Aldrich, St. Louis, MI, USA) for evaluation of transfer efficacy and scanned. Following blocking with 5% nonfat milk (Cell Signaling Technology (CST), Danvers, MA, USA) in Tris-buffered saline (TBS; 50 mM Tris-Cl, 150 mM NaCl, pH 7.6) containing 0.1% Tween-20 for 1 h, membranes were incubated with primary antibodies overnight at +4 °C. The primary antibodies used were: Rabbit monoclonal antibodies for NRF2 (CST:#12721), SLUG (CST:#9585), SNAIL (CST:#3879), NANOG (CST:#4903), OCT4 (CST:#2840), GAPDH (CST:#5174); rabbit polyclonal antibody for TWIST (Santa Cruz Biotechnology, sc-15393); mouse monoclonal antibody for Vimentin (Dako, M0725, Glostrup, Denmark). After incubation with horseradish peroxidase-conjugated secondary species-specific antibodies, immunoreactive bands were visualized using the SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA) and Alliance 4.7 (UVITEC, Cambridge, UK). The analysis software Image Studio Lite (LI-COR, Lincoln, NE, USA) was used for quantification of levels of protein expression. Normalization was made with total proteins (Ponceau S staining) and with GAPDH as a loading control. Results are expressed as relative expression according to non-treated mammospheres (PS 0).
2.11. Statistical Analysis
All experiments were performed in at least 2 independent experiments with technical quadruplicates. For both single and multiple HNE treatments, inhibitory concentrations of 50% (IC50) were calculated using non-linear regression curve fitting log (inhibitor) vs. response and variable slope with a least square (ordinary) fit, using GraphPadPrism 5 software (GraphPad Software, San Diego, CA, USA). Statistical analyses were performed using two-way ANOVA with Tukey’s post hoc test. Values of p < 0.05 were considered significant.
Cells can, to a certain extent, adapt to numerous stress conditions, and, therefore, the aim of this study was to evaluate whether oxidative stress caused by lipid peroxidation representative end-product HNE has the capability to cause specific molecular changes of tumor cells and impact the frequency of BCSC. Numerous factors may affect tumors, such as oxidative stress, which is a risk factor in tumor initiation and proliferation but can modify tumor microenvironment components, such as proteins and cells, which can further affect tumors. Additionally, a subpopulation of tumor cells, CSC, are increasingly recognized as the main factor of tumor growth and recurrence. Until now, these factors were studied separately. Our findings suggest that HNE modifications of collagen I, in combination with chronic exposure to HNE, may cause changes in the distribution of putative BCSC. Oxidative stress may cause either cell differentiation or, when chronic, an increase of BCSC population and up-regulation of EMT markers.
We have studied the influence of oxidative stress and lipid peroxidation on breast cancer cell line SUM159, modeling both the direct influence of HNE and combinations with modifications of collagen I. The microenvironment of each tumor is unique and the changes in this environment due to inflammation and oxidation processes are complex. Therefore, it is challenging to model these modifications. Oxidative stress is involved in mutagenesis, which is a driving force of (breast) cancer initiation and progression, especially in hereditary breast cancer, where the mere loss of BRCA1 increases ROS [29
]. Therefore, it is not surprising that oxidative stress and lipid peroxidation biomarkers are changed in breast cancer patients [30
]. HNE is also recognized as a biomarker of oxidative stress, and as such, is involved in (breast) cancer progression [31
]. In accordance with its role are concentrations found in human plasma, where concentrations ranging from 0.1 µM to 1 µM are considered physiological, while 1 µM to 10 µM are considered as ”where pathology begins” [34
]. Taken that hereditary mutations in breast cancer, as well as conventional cancer treatment strategies, such as chemo- and radiotherapy, cause increases in ROS, which can, in turn, cause lipid peroxidation and HNE formation, these oxidative processes may affect numerous signaling molecules such as HNE activation of NRF2 transcription factor. In order to study the influence of ECM, we have chosen collagen I, as it can influence some of the EMT markers [15
]. We show in our study that collagen may act as a protective agent on SUM159 cell viability, regardless of previous HNE modifications of the collagen. In acute HNE treatment, cell viability was affected at rather higher HNE concentrations (IC50
about 100 µM HNE), whereas proliferation was inhibited already at lower HNE concentrations (25 µM), thereby indicating modulation of cell growth and survival. Previously, we observed similar effects of HNE with collagen oxidized by hydroxyl radical instead of HNE [35
], indicating that oxidative modifications of collagen I are an important factor when studying cell responses to different stimuli or inhibition factors.
As expected, chronic HNE treatment had a higher impact on cells. Interestingly, proliferation was generally lower in tested cultures than in acute stress, with the exception of 25 µM HNE. This decrease in the proliferation rate after 10 days could be a consequence of increased cell density. In support of this conclusion is the proliferation burst with 25 µM HNE, indicating that these cells adapted and survived the treatment, and, due to the initial decrease in proliferation, now were not spatially limited to grow. Notably, 1 µM HNE, which was considered the physiological concentration, caused differentiation, observed by a decrease in BCSC markers and an increase in hormone receptors, effects that have been described for colon cancer cells and HL-60 cells as well [36
]. Interestingly, the BCSC marker that we show here, ALDH activity, is the enzyme that can detoxify HNE, particularly ALDH2, which is located in the mitochondrial matrix [17
Next, we aimed to investigate the influence of chronic stress and HNE modifications of collagen on the expression and protein levels of EMT markers as well as antioxidant parameters measured by catalase activity, GSH levels, and NRF2 antioxidant transcription factor level. Interestingly, although collagen itself, regardless of HNE pretreatment, lowered ALDH, it did not influence EMT markers in the same manner. For example, fibronectin expression was unaffected by different growth surfaces nor by HNE treatment. A similar pattern of expression and protein levels was observed for vimentin, which was increasing with HNE pretreatment concentrations. SLUG was not affected by both mRNA and protein levels. Similarly, SNAIL expression pattern changes were not followed by changes in protein level. Interestingly, expression patterns of TWIST and OCT4 were not followed by protein levels, which were higher in cells on collagen, native, or HNE-pretreated, than on PS. EMT was recognized as an important factor in cancer progression because it represented a conversion between differentiated epithelial cells into migratory mesenchymal cancer cells [38
]. The plasticity of CSC enabled them to follow transition traits between EMT and MET, thereby contributing to the metastatic potential of the primary tumor [39
]. While many studies link EMT and cancer development and malignancy [40
], the influence of oxidative stress/ROS and reactive aldehydes are simply not investigated enough [41
]. Numerous factors can stimulate these transitions, and, as shown here, one of them may be chronic oxidative stress.
It was shown previously that EMT might be abolished by the addition of antioxidant curcumin, underscoring the possible role of redox signaling in this process [42
]. Therefore, in addition to EMT markers, the levels of GSH, catalase activity, and ROS were measured after HNE treatment and the antioxidant transcription factor NRF2. Interestingly, while catalase activity was the highest in control mammospheres on PS, and decreased by growth on collagen, native or HNE pretreated, GSH levels were significantly increased by both HNE treatment and growth on collagen. It is not surprising that GSH levels were increased by HNE as this is the major scavenger of HNE, and the first step in HNE detoxification [43
], while the thioredoxin system is inhibited by HNE and does not contribute to its detoxification [44
]. Finally, and in support of GSH increase, were the levels of ROS and NRF2. In all control groups, ROS were at the same level, while the addition of HNE increased ROS, which was additionally increased by HNE pretreated collagen. Following the ROS pattern, growth on collagen increased NRF2 levels, and HNE treatments additionally increased NRF2 on native collagen and collagen pretreated with 10 µM of HNE. HNE is known to activate NRF2 by releasing it from KEAP1 inhibition, and once NRF2 is freed, it translocates to the nucleus [20
]. In the nucleus, NRF2 activates transcription of antioxidant genes, among which are glutamate-cysteine ligase, catalytic subunit, and glutamate-cysteine ligase, a modifier subunit, and an enzyme which catalyzes the first step in GSH synthesis [20
Finally, a recent study indicated that EMT is not the limiting factor for metastasis, but contributes greatly to chemoresistance [45
]. Taking all the results into account, our findings indicate that under chronic stress, EMT markers remain elevated and in combination with elevated antioxidant factors such as GSH and NRF2, which can contribute to the maintenance of the BCSC phenotype and therapy resistance.