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. 2016 Mar 8;151(2):376–387. doi: 10.1093/toxsci/kfw044

Cancer Stem-Like Cells Accumulated in Nickel-Induced Malignant Transformation

Lei Wang *,†, Jia Fan †, John Andrew Hitron †, Young-Ok Son *,†, James TF Wise ‡, Ram Vinod Roy *,†, Donghern Kim †, Jin Dai †, Poyil Pratheeshkumar *,†, Zhuo Zhang †, Xianglin Shi *,†,1
PMCID: PMC4880134  PMID: 26962057

Abstract

Nickel compounds are known as human carcinogens. Chronic environmental exposure to nickel is a worldwide health concern. Although the mechanisms of nickel-induced carcinogenesis are not well understood, recent studies suggest that stem cells/cancer stem cells are likely important targets. This study examines the role of cancer stem cells in nickel-induced cell transformation. The nontransformed human bronchial epithelial cell line (Beas-2B) was chronically exposed to nickel chloride for 12 months to induce cell transformation. Nickel induced Beas-2B cell transformation, and cancer stem-like cells were enriched in nickel-transformed cell (BNiT) population. The BNiT cancer stem-like cells demonstrated enhanced self-renewal and distinctive differentiation properties. In vivo tumorigenesis studies show that BNiT cancer stem-like cells possess a high tumor-initiating capability. It was also demonstrated that superoxide dismutase 1 was involved in the accumulation of cancer stem-like cells; the regulation of superoxide dismutase 1 expression was different in transformed stem-like cells and nontransformed. Overall, the accumulation of stem-like cells and their enhanced stemness functions contribute to nickel-induced tumorigenesis. Our study provides additional insight into the mechanisms by which metals or other chemicals can induce carcinogenesis.

Keywords: nickel, carcinogenesis, cancer stem cells, superoxide dismutase (SOD), reactive oxygen species (ROS)

Chronic environmental exposure to carcinogenic metal compounds, such as nickel, is a worldwide health concern (Doll et al., 1970; Yang, 2011). Nickel-induced carcinogenesis has been well documented in cell culture and animal models (Costa, 1989). Epidemiological studies show a close relationship between nickel exposure and an increased risk of lung and nasal cancers (Leonard et al., 2004). Nickel compounds have already been classified by International Agency for Research on Cancer as class I human carcinogens (Salnikow and Zhitkovich, 2008).

Currently, the mechanism by which nickel induces human carcinogenesis is not well understood. Accumulating evidence shows that cancer stem cells (CSCs) may be related to nickel-induced carcinogenesis (Wang et al., 2012). While CSCs are generally considered to be a minor tumor subpopulation, they are responsible for tumor initiation, progression, and metastasis (Rosen and Jordan, 2009). CSCs can direct tumor invasion, metastasis, heterogeneity, and therapeutic resistance, all of which contribute to cancer progression (Gupta et al., 2009). The properties possessed by CSCs suggest that this cell population is likely involved in metal-induced carcinogenesis.

In fact, increasing evidence shows that metal-induced malignantly transformed cells demonstrate stem cell characteristics (Benbrahim-Tallaa et al., 2009; Sun et al., 2012; Tokar et al., 2010), suggesting that some association exists between metal carcinogenesis and CSCs. For example, it has been shown that chronic exposure to inorganic arsenic can induce malignant transformation (Chang et al., 2010). This transformation was associated with the acquisition of stemness properties (Chang et al., 2014). Furthermore, during the process of arsenic-induced malignant transformation, the population of CSCs was enriched. In human and rodent skin, exposure to arsenic disrupts the dynamics of stem cells in vitro and in vivo, resulting in an abundance of stem cells/CSCs (Patterson and Rice, 2007; Waalkes et al., 2008). In a similar vein, studies have shown that CSCs accumulate during cadmium-induced transformation (Benbrahim-Tallaa et al., 2009; Qu et al., 2012). Despite lack of direct evidence that nickel-induced CSCs, indirect evidence exists to suggest that CSCs are present and involved in nickel-induced malignancies (Wang et al., 2012).

Reactive oxygen species (ROS) is believed to be an important component in metal-induced carcinogenesis (Lu et al., 2005; Pan et al., 2010) and are potential mediators of metal-induced CSC formation. After exposure to carcinogenic metals, higher levels of ROS were generated and cell transformation was promoted. Once transformation was complete, ROS levels were markedly decreased (Chang et al., 2010; Zhang et al., 2015), with concentrations remaining consistently lower than those found in their parental cells. It would appear, then, that lower ROS levels or higher anti-oxidant enzyme activation act to maintain malignant phenotypes. However, there is still little information on anti-oxidant enzyme function in CSCs.

In this study, we investigated the accumulation of cancer stem-like cells after nickel-induced cell transformation and examined possible mechanisms that underlie this phenomenon.

MATERIALS AND METHODS

Materials

Nickel chloride was purchased from Sigma-Aldrich (St. Louis, MO). GAPDH, superoxide dismutase 1 (SOD1), SOD2, and β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). ALDH1A1 antibody was purchased from EMD Millipore (Billerica, MA). All other antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA). The SOD1 inhibitor LCS-1 was purchased from EMD Millipore. Matrigel was purchased from BD Biosciences (Billerica, MA).

Cell lines and cell culture

The human bronchial epithelial cell line Beas-2B was obtained from the ATCC (Rockville, MD). Nickel-induced transformed Beas-2B cells were developed by chronic exposure to 100-µM nickel chloride for 12 months according the former report (Pan et al., 2011). Cell transformation was characterized functionally by cell invasion, colony formation, and tumorigenesis assays. Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with penicillin (100 IU/ml), streptomycin (100 μg/ml), and 10% fetal bovine serum (FBS) and incubated at 37 °C with 5% carbon dioxide (CO2). Beas-2B cells were transfected with plv-AcGFP-SOD1 vector (Addgene, Cambridge, MA) using lipofectamine 2000 (Invitrigon, Grand Island, NY) as recommended by the manufacturer.

Gelatin zymography assay

The activity of MMP-9 in the medium was measured by gelatin zymography protease assay as previously described (Wang et al., 2010). Briefly, 5 × 105 cells were seeded into a 6-well cell culture plate and maintained to 80% confluence. Fresh serum-free medium was added to each well, followed by 24 h incubation. Equivalent volumes of cultured media were mixed with sample buffer without boiling or reduction and then subjected to 0.1% gelatin-8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. After electrophoresis, gels were washed with 2.5% Triton X-100 and incubated in zymogram developing buffer (50 mM Tris-HCl [pH 8.0], 5 mM CaCl2, and 0.2 mol/l NaCl) at 37 °C for 12 h. Gels were stained with coomassie brilliant blue R-250 and the bands were quantified by image-pro plus 6.0 software. Three independent experiments were performed.

Invasion assay

Invasion assays were carried out using Boyden chambers consisting of 24-well cell culture inserts (8 μM pore size) (BD Biosciences). Briefly, the surface of the top chamber was coated with 100 μl Matrigel (100 μg/ml). Then the upper chambers were seeded with 1 × 104 cells/well in 100 μl serum-free DMEM and DMEM with 10% FBS was added to the bottom chambers. After 24 h, cells on the top surface of the filter were scraped using a cotton swab, while cells on the bottom surface (invasive cells) were fixed with cold 4% paraformaldehyde and stained with crystal violet. Invasive cells were imaged using an inverted microscope and quantified by manual counting. Three independent experiments were performed.

Colony formation assay

Colony formation assay were performed to test long term cell growth capability. Briefly, cells were seeded in triplicate (1000 cells per well of 6-well cell culture plate) and incubated overnight at 37 °C for cell attachment and maintained with regular medium replacements. After 1 week, cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min and stained with crystal violet (0.5% crystal violet, 1% paraformaldehyde, and 20% methanol in PBS) for 30 min. Colonies on each plate were identified and counted under microscope. Three independent experiments were performed.

Free-floating sphere formation assay

The self-renewal capability of cells was determined by an in vitro tumor sphere formation assay. Briefly, 5000 cells were seeded into ultralow-attachment 6-well plates (Corning life sciences, Tewksbury, MA) in serum-free DMEM/F12 (Invitrogen, Grand Island, NY) medium containing 0.4% BSA, 20 ng/ml epidermal growth factor (EGF, Invitrogen), and 20 ng/ml basic fibroblast growth factor (Invitrogen). Fresh medium was added every 3 days. For secondary sphere formation analysis, primary spheres were dissociated into single cells using Accutase (Life Technologies, Grand Island, NY) and then seeded as primary sphere formation. Tumor spheres were counted and photographed under microscope. Three independent experiments were performed.

Sphere differentiation assay

The differentiation capability of stem-like cells was determined by an in vitro sphere differentiation assay. Briefly, spheres were seeded onto or into Matrigel in 24-well plates, then 250 µl medium with 10% serum was added. The medium was carefully changed every 3 days. Cells were imaged using inverted microscope at days 1, 2, 7 and 14. Three independent experiments were performed.

Western blot analysis

Whole-cell extracts were prepared by adding radioimmunoprecipitation assay buffer (RIPA buffer) (Cell Signaling Technology) containing protease inhibitor cocktail. Protein concentrations were determined by using coomassie (Bradford) protein assay reagent (Thermo, Rockford, IL). Proteins were subjected to SDS-PAGE electrophoresis and transferred to nitrocellulose membranes. The membranes were then probed with primary antibodies as indicated, followed by incubation with horseradish peroxidase conjugated secondary antibodies (Pierce, Rockford, IL). Proteins of interest were visualized using a Chemiluminescent Detection Kit (Pierce). The blots were exposed to Hyperfilm (Amersham Pharmacia Biotech, Piscataway, NJ). The bands were quantified by image-pro plus 6.0 software with normalized with loading control. Three independent experiments were performed.

Immunofluorescence microscopy

Spheres were transferred to chamber slides incubated for 2 h to allow attachment to the slides. The chamber slides were then washed with PBS and the spheres were fixed in 4% paraformaldehyde for 10 min. A 1% glycine/0.5 Triton X-100 solution was used to permeabilize cells for 15 min. Samples were blocked with 5% bovine serum albumin for 1 h and incubated with primary antibody overnight at 4 °C. The cells were then washed with PBST (PBS containing 0.1% Tween-20) followed by incubation with secondary antibody for 45 min. Following incubation, cells were washed twice with PBST, then once with PBS. The slides were mounted with Vectashield mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) (Vector Labs, Burlingame, CA). Three independent experiments were performed.

Anchorage-independent growth assay

Soft agar colony formation assays were performed as described previously (Chang et al., 2010). Briefly, 3 ml of 0.5% agar in DMEM supplemented with 10% FBS was spread into each well of a 6-well culture plate. A suspension (1 ml) containing 5000 cells was mixed with 2 ml of 0.5% agar-DMEM and layered on the top of the agar layer. The plates were incubated at 37°C in 5% CO2 for 2 months, and colonies larger than 20 µm were counted microscopically. Three independent experiments were performed.

Intracellular ROS levels

Accumulation of ROS in cells was measured using H2DCFDA (Invitrogen) and dihydroethidium (DHE, Invitrogen). Adherent cells and sphere-derived cells were prepared as single cell suspensions and then seeded into 96-well cell culture plates. Cells were incubated with either H2DCFDA or DHE for 30 min. The fluorescence in cells was measured using a fluorescence microplate reader (Bio-Rad, Hercules, CA).

H2O2 released from cells.

H2O2 released from cells was measured using Amplex Red Hydrogen Peroxide/Peroxidase assay kit (Invitrogen). Adherent cells and sphere-derived cells were prepared as single cell suspensions and then added into 96-well plates. Cells were then incubated with reaction mixture for 6 h. The absorbance (560 nm) was measured using a fluorescence microplate reader (Bio-Rad, Hercules, CA).

RNA isolation and real-time polymerase chain reaction

Total RNA was extracted from cells using Trizol (Invitrogen) according the manufacturer's protocol. From each sample, 1 μg of RNA was used to generate complementary DNA using the M-MLV reverse transcriptase kit (promega, Madison, WI) according to manufacturer's protocol. The real-time quantification of RNA targets was performed using a StepOnePlus real-time polymerase chain reaction (PCR) system (Applied Biosystems, Carlsbad, CA) with a SYBR green PCR master mix (Life Technologies). The primers used were 5′-CTG AAG GCC TGC ATG GAT TC-3' (forward), 5′-CCA AGT CTC CAA CAT GCC TCT C-3' (reverse) for SOD1, and 5′-TCA CCC ACA CTG TGC CCA TCT ACG A-3' (forward), 5′-CAG CGG AAC CGC TCA TTG CCA ATG G-3' (reverse) for β-actin. The resulting data were analyzed using the software provided by the manufacturer.

Immunoprecipitation

For immunoprecipitations, cells were lysed in RIPA buffer. Lysates were incubated with protein G agarose (Life Technologies) for 2 h for preclearing prior to incubation with the indicated primary antibodies for 12 h at 4°C. Immunocomplexes were collected, washed 4 times in TBST buffer (0.1% Tween-20 in Tris-Buffered Saline [TBS]), and resolved by SDS-PAGE followed by western blot analysis.

In vivo tumorigenesis and tissue immunohistochemistry

Four-week-old male athymic nude mice (Nu/Nu mice) weighing ∼20 g were purchased from Jackson Laboratories (Bar Harbor, ME) and randomized into 2 treatment groups with 8 mice in each group. Beas-2B cells (5 × 104 cells/100 μl in medium plus 50% Matrigel) were subcutaneously injected into the left flanks of nude mice and cells from Beas-2B spheres were injected into right flanks of the same group of mice. Correspondingly, BNiT cells or cells from BNiT spheres (5 × 104 cells/100 μl in medium plus 50% Matrigel) were injected into left or right flanks of another group of mice (1 out of 8 mice in this group was unfortunately lost during the treatment). Tumors were allowed to develop and grow for 4 months. Mouse body weight and tumor mass were recorded. Tumor volume was calculated using the formula volume = (length × width2) × 0.5.

For the evaluation of tumorigenicity of stem-like cells, limited diluted (1 × 104, 1 × 103, and 1 × 102) adherent cells, or sphere cells were injected, respectively, into the flank of 4-week-old male athymic nude mice. The mice were euthanized after 2 months. Mouse body weight and tumor mass were recorded. All animals were handled according to the Institutional Animal Care and Use, University of Kentucky.

Immunohistochemical analysis

Immunohistochemical staining for stemness markers CD44, ALDH1A1, and Notch1 were carried out. Slides were prepared by imaging facility/histology core of the University of Kentucky. For immunohistochemical staining, VECTASTAIN ABC Kit (Vector Labs) was used according the manufacturer's protocol. Staining results were imaged under microscope.

Statistical analysis

Each experiment included a minimum of 3 replicates for each treatment and were repeated. The data were presented as mean ± SD, and statistical comparisons among groups were performed using 1-way ANOVA followed by a Dunnett test. P value ≤.05 was considered statistically significant. Comparisons between 2 groups were performed using Student's t-test. P value ≤ .05 was considered statistically significant. Fisher exact test was used to compare tumor incidence (Jin et al., 2014).

RESULTS

Cells chronically exposed to nickel exhibit malignant characteristics

Pronounced invasiveness, fast growth, and the ability to form anchorage-independent colonies are among the characteristics of malignant cells. The increased invasiveness is due, in part, to the expression of matrix metalloproteinases (MMPs), which degrade extracellular matrix proteins and enable cancer cell invasion and distant metastasis (Hanahan and Weinberg, 2011). Beas-2B cells were chronically exposed to 100 µM nickel chloride for a period of 12 months. Cells chronically exposed to nickel exhibited robust increase in the activity of secreted MMP-9 (Figure 1A). Compared with passage matched Beas-2B cells, the invasive capability of cells chronically exposed to nickel was much higher (Figure 1B).

FIG. 1.

FIG. 1.

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Cells chronically exposed to nickel exhibit malignant characteristics. Cells chronically exposed to nickel were generated by exposure to 100 µM nickel chloride for 12 months. (A) Image (top) and graphic representation (bottom) of zymography demonstrating activity of MMP-9 secreted by nontransformed cells (Beas-2B) and cells with chronic nickel exposure (BNiT). Bars, SD (*P < .05 compared to Beas-2B cells). (B) Photomicrograph and graphic representation of cell invasion by Beas-2B and BNiT cells. Bars, SD (*P < .05 compared to Beas-2B cells) (×200 magnification). (C) Colony formation of Beas-2B and BNiT cells. Bars, SD (*P < .05 compared to Beas-2B cells). (D) Athymic nude mice were inoculated with Beas-2B or BNiT (5 × 104 cells per mouse, subcutaneously, 8 mice per group) for 4 months. Photomicrographs of the xenografts are shown in the upper panel and xenograft weight with tumor incidence is shown on lower panel. Bars, SD.

Colony formation is another important characteristic of malignant cells (Hanahan and Weinberg, 2011). Cells chronically exposed to nickel generated more of colonies than those without nickel exposure (Figure 1C). In addition, the in vivo tumorigenesis assay confirmed that cells chronically exposed to nickel gain the properties of malignant transformation and are able to initiate tumorigenesis (Figure 1D). Taken together these results suggest that cells chronically exposed to nickel acquire cancer cell properties. Thus, these exposed cells have been termed Beas-2B nickel-transformed cells (BNiT).

Nickel-transformed cells acquire cancer stemness properties

A common characteristic of CSCs in culture is the formation of floating “spheres” of viable cells, with stem cells being enriched. This floating sphere structure has been utilized for the identification and isolation of CSCs (Khan et al., 2015). To evaluate the presence of stem cells/CSCs in Beas-2B and BNiT cultures the floating sphere formation assay was performed. BNiT cells had an increase in both the number and size of free-floating primary spheres relative to Beas-2B cells. The secondary spheres, obtained by harvest and re-plating the initial spheres, are also increased in both number and size for BNiT cells compare to Beas-2B secondary spheres. Additionally, there was a decrease in the number and size of Beas-2B secondary spheres as compared with its primary spheres, whereas the number of BNiT secondary spheres was slightly higher than for BNiT primary spheres (Figure 2A). The increased sphere number, sphere size, and primary/secondary sphere ratio in BNiT cells indicate that BNiT cancer stem-like cells obtain enhanced self-renewal capacity.

FIG. 2.

FIG. 2.

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Cancer stem-like properties in nickel-transformed cells. (A) Photomicrograph and graphic representation of free-floating sphere number and diameter formed by Beas-2B cells and BNiT cells, respectively (bar = 50 μm). Primary spheres were recovered, dissociated into single cells, and then used for secondary sphere assays. Statistical analysis of sphere formation was shown as sphere number and sphere diameter. Bars, SD (*P < .05 compared to Beas-2B primary spheres). (B) Photomicrographs demonstrating differentiation of Beas-2B and BNiT spheres on Matrigel and in Matrigel are displayed (bar = 20 μm).

Differentiation is another major property of stem cells/CSCs (Nguyen et al., 2012). To investigate differentiation capability, both Beas-2B spheres and BNiT spheres were seeded in Matrigel and incubated with complete medium. Morphologic changes were monitored microscopically at days 0, 2, 7, and 14. In Beas-2B spheres, epithelial-like cells were found extending from the sphere at day 2 and had grown to near-confluence by day 14. In BNiT spheres, no epithelial-like cells were found. Instead, the cells in the BNiT spheres maintained a rounded appearance until day 14 although some of the spheres stopped growing (Figure 2B). When spheres were instead seeded over a Matrigel layer, the differentiation pattern changed. For Beas-2B spheres, cells kept their rounded shape until day 14 and grew slightly larger. In BNiT spheres, tube-like structures extended from the spheres at day 7 and formed a perfect network structure by day 14 (Figure 2B). The distinctly different morphologies between Beas-2B and BNiT spheres suggest that after chronic nickel exposure, the differentiation potential of these cells had changed. Based on the characteristics of stemness, cells in BNiT spheres were identified as cancer stem-like cells and that this change can be attributed to the chronic nickel exposure.

Cancer stem-like cells have increased stemness marker expression

Stemness markers are important features in the study of CSCs, as they useful in CSC identification and isolation. To identify stemness markers in BNiT cells, western blot analysis was performed. Compared with Beas-2B, BNiT cells expressed higher levels of the stemness markers ALDH1A1, CD44, and Notch1. Additionally, both Beas-2B spheres and BNiT sphere showed higher expression of all 3 stemness markers when compared to their respective nonsphered cell lines. As expected, cells from BNiT spheres expressed these markers at higher levels than cells from Beas-2B spheres (Figure 3A). Immunofluorescence analysis of Beas-2B spheres and BNiT spheres revealed high and widespread expression of stemness markers in the BNiT spheres (Figure 3B). These results demonstrate that stemness markers, including ALDH1A1, CD44, and Notch1, are highly expressed in nickel-transformed cells, and especially in BNiT cancer stem-like cells. To further investigate the stemness characteristics of BNiT spheres, several self-renewal factors were investigated. As shown in Figure 3C, in BNiT spheres, the expressions of Sox-2, Klf4, and Oct4 were higher than those obtained from Beas-2B spheres. The immunofluorescence staining of spheres confirmed these results (Figure 3D). Thus, these results substantiate the presence of cancer stem-like cells in BNiT cells. These results also demonstrate that the cells in the BNiT spheres possess more properties associated with stemness than the cells in the Beas-2B spheres.

FIG. 3.

FIG. 3.

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Stemness markers are identified in nickel-transformed cells and stem-like cells. (A) Image of western blot analysis for adherent and sphere cell lysates using antibodies against stemness marker including ALDH1A1, CD44, Notch1, and loading control GAPDH. (B) Photomicrographs of immunofluorescence staining for stemness markers in Beas-2B and BNiT spheres (bar = 50 μm). (C) Images of western blot analysis using antibodies against the self-renewal proteins Sox2, Klf4, Oct4, and loading control GAPDH. (D) Photomicrographs demonstrating immunofluorescence staining for self-renewal proteins in Beas-2B and BNiT spheres (bar = 50 μm).

Cancer stem-like cells obtain an aggressive phenotype

CSCs are known to contribute to the malignant behavior of cancers (Tang et al., 2007). To identify the characteristics of BNiT cancer stem-like cells, western blot analysis and zymography, invasion, and soft agar colony formation assays were performed. Cells from BNiT spheres showed an increased MMP-9 activity when compared with cells from Beas-2B spheres (Figure 4A). Western blot analysis confirmed increased MMP-9 protein levels in cells from BNiT spheres (Figure 4B). Not surprisingly, the invasive capability of cells from BNiT spheres was also higher than that in cells from Beas-2B spheres (Figure 4C). Soft agar colony formation assays were used to measure the capacity for anchorage-independent growth, a characteristic of malignant cells. The soft agar colony formation capability of cells from BNiT spheres was approximately 4-fold higher than that observed with cells from Beas-2B spheres (Figure 4D). Taken together, these results indicate that BNiT cancer stem-like cells have an aggressive malignant phenotype.

FIG. 4.

FIG. 4.

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Aggressive phenotypes of cancer stem-like cells. Cells from Beas-2B and BNiT spheres were analyzed for malignant characteristics. (A) Image (top) and graphic representation (bottom) of zymography demonstrating MMP-9 activity secreted by Beas-2B spheres and BNiT spheres. Bars, SD (*P < .05 compared to Beas-2B sphere). (B) Image of western blot analysis using antibodies against MMP-1, MMP-2, MMP-9, and loading control GAPDH. (C) Photomicrograph and graphic representation of invasion by cells from Beas-2B and BNiT spheres. Bars, SD (*P < .05 compared to Beas-2B sphere) (× 200 magnification). (D) Photomicrograph and graphic representation of anchorage-independent growth by cells from Beas-2B and BNiT spheres by the formation of large colonies in soft agar. Bars, SD (*P < .05 compared to Beas-2B sphere) (bar = 20 μm).

Cancer stem-like cells have an elevated tumor-initiating capability

The ability to initiate tumor formation and growth is the gold standard of CSCs (Visvader and Lindeman, 2008) . To determine the tumorigenic capability of cells in the Beas-2B and BNiT spheres, in vivo tumorigenesis assays were performed. Athymic nude mice were inoculated with cells from either Beas-2B sphere or BNiT spheres (5 × 104 cells per mouse, right side, subcutaneous, n  =  8 per group) and maintained for 4 months. As shown in Figure 5A, cells from BNiT spheres have a higher capability to form tumors with a tumor incidence of 7 out of 7, while cells from Beas-2B spheres had a tumor incidence of 2 out of 8. However, the “xenografts” generated from Beas-2B spheres were too small to be measured in size and weight. To further determine the tumor-initiating capability of cancer stem-like cells, athymic nude mice were inoculated with limiting dilutions of either BNiT or BNiT sphere cells (1 × 104, 1 × 103, or 1 × 102 cells per mouse, per side, subcutaneous) and observed for 2 months. As shown in Figures 5B and C, when the inoculated cell number decreased, tumor incidence and tumor size decreased for both cell lines although the tumorigenic capability of cells from BNiT spheres was higher than that of BNiT cells. Tumor tissue sections subjected to staining revealed that the stemness markers CD44, ALDH1A1, and Notch1 were highly expressed in BNiT sphere-derived xenografts compare to those derived from BNiT (Figure 5D). These results suggest that cells from BNiT spheres acquire a higher tumor initiation capability in vivo than the cells obtained from an initial nickel exposure.

FIG. 5.

FIG. 5.

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Tumorigenic capability of cancer stem-like cells. (A) Athymic nude mice were inoculated with Beas-2B spheres and BNiT sphere cells (5 × 104 cells per mouse, subcutaneously, 8 mice per group) for 4 months. The graphic representations are of the xenograft weight and volume with tumor incidence indicated in the upper panel (NA, not applicable). Bars, SD. Tumor incidence was analyzed by Fisher exact test. (B) Athymic nude mice were inoculated with BNiT and BNiT sphere cells (1 × 104, 1 × 103, or 1 × 102 cells per mouse, subcutaneously, 8 mice per group) for 2 months. Images are of the xenografts with the tumor incidence indicated. Tumor incidence was analyzed by Fisher exact test. (C) The graphic representations are of xenograft weight and volume from (B). Bars, SD (*P < .05 compared to BNiT cells). (D) Photomicrographs demonstrate immunohistochemical staining in xenograft tissue sections from BNiT and BNiT sphere cells. Results revealed that stemness markers CD44, ALDH1A1, and Notch1 are highly expressed in BNiT sphere-derived xenograft (bar = 20 μm).

SOD1 expression increases cancer stem-like cell phenotype

ROS play an important role in nickel-mediated carcinogenesis (Lee et al., 2012). It is well documented that ROS levels are suppressed in CSCs (Diehn et al., 2009). To determine the ROS level in adherent cells and sphere-derived cells, cells were incubated with the fluorescent dyes 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and DHE to measure hydrogen peroxide (H2O2) and superoxide anion radical (O2•-), respectively. The results demonstrate that accumulated ROS levels in transformed cells were lower than those of nontransformed cells and that ROS levels in sphere-derived cells were lower than those of adherent cells, with ROS levels in BNiT sphere-derived cells were the lowest of all 4 cell groups (Figure 6A). H2O2 released from cells as measured by Amplex Red Hydrogen Peroxide/Peroxidase assay kit is shown in Figure 6B. To understand the underlying cause for the decreased ROS levels in cancer stem-like cells, expression levels of several important anti-oxidant enzymes were investigated by western blot. Catalase, SOD1, and superoxide dismutase 2 (SOD2) all exhibited higher expression in cells from BNiT spheres (Figure 6C), with SOD1 level being dramatically higher. These results indicate that SOD1 may play an important role in keeping ROS levels low in CSCs. When Beas-2B and BNiT cells were treated with the SOD1 specific inhibitor (LCS-1), the number and size of floating free spheres generated by both cell lines were decreased (Figs. 6D and E). When SOD1 was overexpressed in Beas-2B cells, both sphere number and size increased (Figs. 6G and H). These results indicate that O2•− works to decrease cancer stemness properties in nickel-transformed cells.

FIG. 6.

FIG. 6.

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Superoxide dismutase 1 (SOD1) contributes to cancer stem-like cells phenotype. (A) Graphic representation of adherent cells and sphere-derived cells were incubated with H2DCFDA or DHE. The fluorescence in cells was measured with a fluorescence microplate reader. Bars, SD (*P < .05 compared to Beas-2B cells). (B) Graphic representation of H2O2 released from cells measured by Amplex Red Hydrogen Peroxide/Peroxidase assay kit. Bars, SD (*P < .05 compared to Beas-2B cells). (C) Image of western blot analysis of lysates from adherent and sphere cells using antibodies against the anti-oxidant enzymes including Catalase, SOD1, and SOD2. (D) Photomicrographs of free-floating spheres formed by Beas-2B and BNiT cells that were treated with/without SOD1 inhibitor LCS-1 (bar = 50 μm). (E) Graphic representation of sphere number and diameter from (D) (NA,  not applicable). Bars, SD (*P < .05 compared to nontreatment cells). (F) Image of western blot demonstrating the presence of eGFP-SOD1 protein after transfection into Beas-2B cells. Western blot analysis was carried out using SOD1, GFP, and β-actin antibodies. (G) Photomicrograph of free-floating spheres formed by Beas-2B and SOD1 over-expressed cells (bar = 50 μm). (H) Graphic representation of sphere number and diameter from (G). Bars, SD (*P < .05 compared to nonSOD1 transfected cells).

SOD1 is up-regulated by both transcriptional and posttranslational pathways in BNiT spheres

Real-time PCR was performed to measure the mRNA level of SOD1 in both adherent and sphere cells. The results show that SOD1 mRNA levels in BNiT, Beas-2B sphere, and BNiT sphere cells were all higher than that observed in Beas-2B cells, which was consistent with the western blot results (Figs. 6B and 7A). In addition, several potential transcriptional factors which may regulate SOD1 were investigated, including Nrf2, C/EBP, IκB, and TCF1. The results show that Nrf2 and C/EBP were accumulated in BNiT and BNiT spheres (Figure 7B). IκB, which inhibits NF-κB, was highly expressed in Beas-2B cells, and its expression was much lower in cells from BNiT spheres. These results suggest that the high SOD1 mRNA levels in cells from BNiT spheres may be coregulated by multiple pathways. The level of transcriptional factor TCF1 was higher in Beas-2B spheres than in BNiT spheres, suggesting that transcriptional regulation in nontransformed spheres is different from that observed in transformed spheres (Figure 7B). Beyond transcriptional regulation, SOD1 protein expression can also be regulated by posttranslational regulation (Milani et al., 2011). SOD1 posttranslational regulation was measured by a protein degradation assay. The eGFP-SOD1-fused protein vectors transfected Beas-2B and BNiT spheres were treated with MG132 and chloroquine to inhibit proteasome and lysosome activity, respectively. The eGFP-SOD1 fusion protein was precipitated with GFP antibody followed by western blot analysis using antibody raised against ubiquitin. Results show that chloroquine-treated BNiT sphere cells exhibited a similar ubiquitination protein level as untreated BNiT sphere cells, suggesting that SOD1 protein lysosomal degradation was blocked in BNiT spheres (Figure 7C). Endogenous SOD1 ubiquitination assay showed the similar results (Figure 7D). Taken together, these data suggest that the high SOD1 expression in BNiT spheres is regulated by both transcriptional and posttranslational pathways.

FIG. 7.

FIG. 7.

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Superoxide dismutase 1 (SOD1) is regulated by both transcriptional and posttranslational pathways. (A) Graphic representation of SOD1 mRNA measured by real-time polymerase chain reaction (PCR). Data were normalized to the GAPDH mRNA level. Bars, SD (*P < .05 compared to Beas-2B cells). (B) Image of western blot analysis for the signaling molecules Nrf2, IkB, C/EBP, and TCF1 from lysates of parental and sphere cells. (C) eGFP-SOD1 overexpressing Beas-2B and BNiT spheres were treated with MG132 and chloroquine, respectively. The eGFP-SOD1 was precipitated with GFP antibody followed with western blot analysis using antibody against ubiquitin. (D) The endogenous SOD1 was precipitated with SOD1 antibody followed with western blot analysis using antibody against ubiquitin.

DISCUSSION

Nickel compounds are classified as a toxin and human carcinogen. Various mechanisms of nickel-induced carcinogenesis have been proposed, including elevated oxidative stress (Lu et al., 2005), increased induction of DNA damage (Costa, 1991), differential modification of epigenetic factors (Salnikow and Zhitkovich, 2008), and activation of oncogenic pathways. Despite intensive investigation, the role of CSC in nickel-induced carcinogenesis is still unclear. Investigation of CSCs is an intensely studied topic in carcinogenesis. The CSC hypothesis is an attractive model to explain tumor initiation, growth, progression, and metastasis. CSCs potentially play a crucial role in metal-induced carcinogenesis (Wang et al., 2012). During carcinogenesis, a small subset of pluripotent/stem-like cells are exposed to carcinogens, these cells then potentially develop into CSCs, leading to carcinogenic progression (Pardal et al., 2003; Sullivan et al., 2010; Wicha et al., 2006).

Previous studies have demonstrated that metal-induced malignantly transformed cells obtain stem cell-like properties (Benbrahim-Tallaa et al., 2009). Although there are few reports on the relationship between CSCs and chronic nickel exposure, evidence from studies using other carcinogenic metals such as arsenic and cadmium suggest that CSCs could be a mechanism for nickel-induced carcinogenesis. In this study, chronic treatment of Beas-2B cells with nickel chloride noticeably increased sphere formation. These spheres exhibited multiple distinct characteristics of CSCs, including increased anchorage-independent colony formation, disordered differentiation, enhanced invasiveness, and higher tumorigenicity. These results demonstrate that chronic nickel exposure induces the accumulation of cancer stem-like properties. Our study also shows that the self-renewal capability in BNiT cells was higher than that observed in Beas-2B cells. The increased sphere-formation rate observed in BNiT cells suggests that these transformed cells have an increased population of cancer stem-like cells. Since sphere size is positively correlated with stem cell self-renewal (Bisson and Prowse, 2009), the increased sphere size formed by BNiT cells indicates that the transformed cells have an enhanced self-renewal capability. This enhanced self-renewal capability promotes more secondary spheres for BNiT than those formed by Beas-2B. Additionally, the size and number of secondary BNiT spheres are only slightly increased when compared with BNiT primary spheres, since the self-renewal processes are already in place, and the BNiT cells have enhanced self-renewal ability when compared to Beas-2B cells.

Cell differentiation is another important characteristic of stem cells/CSCs. Our results show that BNiT spheres were able to differentiate into tube-like structures, which did not occur with Beas-2B spheres. Tube-like structures always appear in vascular endothelial cells when cultured on Matrigel. These results suggest that BNiT stem-like cells may obtain some vascular endothelial-like differentiation potential. These vascular endothelial-like differentiation features may contribute to tumor angiogenesis, metastasis, and progression. Aberrant self-renewal and differentiation are involved in tumor initiation, maintenance, and progression. The enhanced self-renewal and distinctive differentiation of BNiT spheres suggest that cancer stem-like cells in these spheres are highly tumorigenic, a hypothesis which was verified by tumorigenesis studies using the xenograft model.

Because of the properties of stem cells and the limitations of technology in stem cell research, it is difficult to get direct evidence to support the hypothesis that stem cells/CSCs play a key role in nickel-induced carcinogenesis. However, our results show that cancer stem-like cells are accumulated in nickel-transformed cells. This indirect evidence suggests that stem cells/CSCs are involved in metal-induced transformation. Thus, we can conservatively draw the conclusion that accumulation of cancer stem-like cells as well as enhanced self-renewal and modified differentiation of cancer stem-like cells contribute to nickel-induced tumorigenesis.

SOD1, an antioxidant enzyme which scavenges superoxide anion radicals, is important for various cellular functions. This study shows that cancer stem-like cell accumulation was positively correlated with SOD1 expression level. We found that elevated SOD1 contributed to the formation of floating spheres. Low ROS levels in CSCs suggest that SOD1 plays an important role in maintaining cancer stemness properties. In nickel-transformed cells, ROS are at lower levels than in nontransformed cells (Zhang et al., 2015). High expression of SOD1 seems to be a driving force in maintaining lower ROS levels in cancer stem-like cells. According to previous studies, SOD1 protein level is controlled at both transcriptional and posttranslational levels. The human SOD1 promoter has been well studied and binding sites for AP1, C/EBPs, Nrf2, and NF-κB transcription factors were identified and verified by functional studies (Milani et al., 2011). In this study, we found that transcriptional factors such as Nrf2, NF-κB, and C/EBP associate more in BNiT spheres than Beas-2B spheres. These transcriptional factors would up-regulate SOD1 expression in BNiT spheres. Interestingly, transcriptional regulation of SOD1 in Beas-2B spheres is somehow different from that in BNiT spheres. The protein levels of TCF1 are higher in Beasa-2B spheres than those from BNiT sphere. The different transcriptional regulation of SOD1 between nontransformed and transformed stem-like cells suggests that chronic nickel exposure could affect the transcriptional regulation network of stem cells. Moreover, posttranslational regulation is also different between BNiT spheres and Beas-2B spheres; lysosomal degradation of SOD1 was blocked in BNiT spheres, suggesting that decreased autophagy flux may be involved in SOD1 up-regulation. Other studies report that nickel exposure stabilized Oct4, a cell self-renewal factor (Yao et al., 2014). The accumulation of SOD1 in BNiT stem-like cells is coregulated by multiple transcription factors and protein degradation. The stabilization of Oct4 and SOD1 provide indirect evidence that nickel functions on certain proteins to contribute to the maintenance of cell stemness.

Although stemness markers are widely used to identify CSCs in tumors, this practice is still somewhat controversial (Rosen and Jordan, 2009). The heterogeneity among tumors and within tumor subtypes makes it difficult to discover unique markers for different CSCs. However, some common stemness markers such as CD24, CD44, CD133, and CD166 have been identified as CSC markers from several kinds of cancers, including breast, lung, pancreas, and colorectal (Magee et al., 2012). In this study, to identify potential cancer stemness markers in BNiT cells a series of reported stemness markers were tested. Except for ALDH1A1, CD44, and Notch1, no other positive markers were identified. High expressions of ALDH1A1, CD44, and Notch1 in BNiT spheres suggest that these proteins are potential markers of nickel-induced CSCs. These stemness markers induced by chronic nickel exposure might be used for risk assessment of nickel-mediated carcinogenesis.

Oct4 is considered a pluripotent gene required for the self-renewal and maintenance of stem cells. Oct4 and other stemness factors such as Sox2 and Klf4 form a transcriptional network that controls pluripotency in stem cells (Yao et al., 2014). Takahashi et al. successfully generated induced pluripotent stem cells by introducing 4 factors: Oct3/4, Sox2, Klf4, and c-Myc. This study demonstrates that these factors are essential for the stemness of embryonic stem cells, stem cells, induced pluripotent cells, and CSCs (Takahashi et al., 2007). BNiT sphere cells exhibit high expression of Oct4, Sox2, and Klf4, which confirms that the spheres exhibit stemness characteristics beyond the enhanced self-renewal capability. The high expression of Oct4 is consistent with a recent report that nickel compounds contributed to Oct4 stabilization (Yao et al., 2014).

In conclusion, we present compelling evidence for the hypothesis that cancer stem-like cells are accumulated in malignant transformed cells induced by chronic nickel exposure. These cancer stem-like cells also acquire more aggressive malignant phenotypes and higher tumor initiating capabilities. On a cellular level, our study verifies that enhanced self-renewal and distinctive differentiation promote cancer stem-like cell accumulation. At the molecular level, our study demonstrates that SOD1 is the driving force for the accumulation of cancer stem-like cells. Our study indicates that cancer stem-like cells may be, at least partly, responsible for nickel-induced malignant transformation, and we offer a novel understanding of nickel-induced tumorigenesis.

ACKNOWLEDGMENT

We thank Hong Lin for her technical help.

FUNDING

National Institutes of Health (R01ES021771, R01ES025515, R01ES020870, and R01ES017244).

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