We found innate resistance and hyper-adaptability to arsenite toxicity in WPE-stem cells compared with their mature, heterogeneous parental cell line, RWPE-1. This resistance and hyper-adaptability was associated with higher mRNA and protein expression of several antiapoptotic, stress-related, and arsenic adaptation factors and lower expression of proapoptotic factors. We also found that by the point of arsenite-induced malignant transformation (4
), a stem cell survival selection advantage has occurred in the RWPE-1 cells that manifested itself as an arsenite-specific overabundance of CSC-like cells. These data support the hypothesis that, as part of its carcinogenic mechanism, arsenic targets stem cells for transformation, which subsequently produces cancers enriched in CSC-like cells.
For environmental chemical carcinogenesis to occur, tumor-forming cells must survive the primary exposure and any subsequent chronic exposure and retain the ability to propagate. WPE-stem cells were innately much more resistant than their mature parental RWPE-1 cells to arsenite-induced apoptosis, which is similar to the generalized apoptotic resistance of many cancer cells (13
) and likely provides a survival advantage during initial chemical exposure. Furthermore, in general, the progressive acquisition of apoptotic resistance contributes to tumor development and progression (11
). In this study, both the parental and stem cell lines showed acquired resistance (adapted) to arsenite-induced cytotoxicity following 6 weeks of exposure to an environmentally relevant level of arsenite [5 μM (32
)] compared with nonadapted cells. However, the stem cells adapted to a greater extent than the parental cells, presumably because they selectively expressed many factors that contribute to apoptotic resistance, which indicates arsenite hyper-adaptability of WPE-stem cells.
The factors that contribute to apoptotic resistance are well documented (11
). Caspases are proapoptotic molecules that are critical to apoptotic commitment and execution. Reduced caspase activity can perturb or even prevent apoptosis and is common in apoptotic-resistant cancers (11
). The Bcl2 family of proteins is considered a main intracellular regulator of the apoptotic process; some Bcl2 family members inhibit apoptosis (ie, Bcl2, Bcl2l1), whereas others (eg, Bax) promote apoptosis. Higher ratios of Bcl2 or Bcl2l1 to Bax are associated with resistance to apoptosis (37
), and Bcl-2 overexpression has been shown to protect stem cells against apoptosis, increase their frequency, and confer them with a competitive repopulation advantage (48
). Furthermore, MT can protect cells that are exposed to metals, including arsenic, and perturb metal-induced apoptosis (16
). Poor MT expression is associated with increased sensitivity to arsenic (39
). ABC transporter proteins (ie, ABCC1, ABCC2), GSTP1, and GSH are associated with arsenic tolerance and generalized inhibition of apoptosis (16
). In addition, ABCC1, ABCC2, GSTP1, and GSH work together to efflux arsenic out of cells (40
), a key element in arsenic adaptation (16
). HIF1A, HMOX1, SOD1, and NFE2L2 play important roles in innate resistance and/or adaptation to arsenic by preventing arsenic-induced oxidative damage and apoptosis (46
). Increased expression of PRODH, a stress-induced enzyme that metabolizes proline and generates ATP, can maintain cellular energy levels and help cancer cells adapt to nutrient and energy limitations (42
). Expression of all of these factors enhanced the intrinsic resistance of WPE-stem cells to arsenite compared with mature parental RWPE-1 cells and, taken together, would likely increase the survival selection advantage of WPE-stem cells during malignant transformation. Most cells, including RWPE-1, adapt to arsenite during periods of low-level chronic exposure (18
). However, in this study, the stem cells displayed greater adaptation to arsenite than the mature parental cells.
Our data clearly show that WPE-stem cells possess an inherent capacity to survive arsenite exposure and to continue with self-renewal in the face of continuous arsenite exposure, making them ideal candidates to acquire the lesions necessary for malignant transformation, thereby becoming cells that drive tumorigenesis in arsenite-transformed RWPE-1 cells (4
). Tumor-derived cancer cell lines can form free-floating spheres that contain chemoresistant and malignant CSC-like cells (30
), and many cancer cell lines that are cultured for a long time form CSC-containing holoclones (45
). The arsenite-induced malignant transformant, CAsE-PE, was the only isogenic RWPE-1 transformant that formed free-floating spheres at a higher rate than control cells and produced floating cells that formed holoclones that could be repeatedly subcloned and propagated for multiple passages, indicating that these cells possess a greater self-renewal capacity consistent with a stem cell or CSC nature (8
). Moreover, we showed that CAsE-PE holoclones overexpress multiple common stem cell and CSC markers, including BMI1
, and PROM1
. Clonogenic assays such as colony formation in soft agar have long been used to enrich for tumor-initiating cells, and the cells that form colonies in such assays are generally considered to be CSCs (29
). Only floating cells from CAsE-PE cells formed colonies in soft agar, whereas those from RWPE-1 cells transformed by inorganic cadmium and the direct-acting organic carcinogen MNU did not. Taken together, these data demonstrate that the selection of stem cells and the ability to greatly increase their numbers during malignant transformation is a potentially unique mechanistic feature of arsenic.
Some potential limitations of this study include the use of molecular markers to identify CSCs. Although biomarkers are widely used to identify CSCs in tumors, this practice is still somewhat controversial (59
). Analysis of additional prostate CSC biomarkers (eg, CD117, Sca1) in the isogenic RWPE-1 transformants could further support the role of these cells in prostate carcinogenesis. Although in this and other (9
) studies, arsenic appears to target a stem cell population during carcinogenesis, similar in vitro or in vivo studies are needed to determine if this phenomenon is generalizable to all targets of arsenic carcinogenesis. Finally, all analyses in this study were done in cultured cell lines. Similar studies using arsenic-induced carcinogenesis in whole animals and humans are needed to determine if these results translate to in vivo conditions.
In summary, the prostate stem cell line, WPE-stem, showed inherent resistance to apoptosis and hyper-adaptability to arsenite that was likely because of its enhanced protective molecular responses compared with the mature cell line from which it was derived. The consequence of this increased resistance to apoptosis and arsenite hyper-adaptability when arsenite transformed the heterogeneous mature RWPE-1 line was an increase in the number of CSC-like cells, suggesting that a selection for stem cells had occurred. The similarities between stem cells and cancer cells, coupled with the accumulating evidence of the key role of CSCs in carcinogenesis (8
), indicate that the apoptotic resistance that characterizes most tumor cells is also an intrinsic characteristic of WPE-stem cells in response to arsenic. The innate resistance and hyper-adaptability in prostate stem cells observed in this study indicate that these cells, compared with their mature counterparts, could more readily survive arsenic exposure yet retain the critical quality of self-renewal essential to tumor formation. Emerging data indicate that normal stem cells are highly protected from the effects of toxicants and that CSCs represent a similarly small, yet highly chemoresistant, population (20
). The characteristics of the WPE-stem cells that provide for arsenite resistance essentially mirror those in cells malignantly transformed with arsenic (4
). These observations fortify the contention that arsenic likely targets cells that have a stem or progenitor phenotype during malignant transformation.