Arsenic exposure transforms human epithelial stem/progenitor cells into a cancer stem-like phenotype

Abstract

Background: Inorganic arsenic is a ubiquitous environmental carcinogen affecting millions of people worldwide. Evolving theory predicts that normal stem cells (NSCs) are transformed into cancer stem cells (CSCs) that then drive oncogenesis. In humans, arsenic is carcinogenic in the urogenital system (UGS), including the bladder and potentially the prostate, whereas in mice arsenic induces multi-organ UGS cancers, indicating that UGS NSCs may represent targets for carcino-genic initiation. However, proof of emergence of CSCs induced by arsenic in a stem cell population is not available.

Methods: We continuously exposed the human prostate epithelial stem/progenitor cell line WPE-stem to an environmentally relevant level of arsenic (5 microM) in vitro and determined the acquired cancer phenotype.

Results: WPE-stem cells rapidly acquired a malignant CSC-like phenotype by 18 weeks of exposure, becoming highly invasive, losing contact inhibition, and hyper-secreting matrix metalloproteinase-9. When hetero-transplanted, these cells (designated As-CSC) formed highly pleomorphic, aggressive tumors with immature epithelial- and mesenchymal-like cells, suggesting a highly pluripotent cell of origin. Consistent with tumor-derived CSCs, As-CSCs formed abundant free-floating spheres enriched in CSC-like cells, as confirmed by molecular analysis and the fact that only these floating cells formed xeno-graft tumors. An early loss of NSC self-renewal gene expression (p63, ABCG2, BMI-1, SHH, OCT-4, NOTCH-1) during arsenite exposure was sub-sequently reversed as the tumor suppressor gene PTEN was progressively suppressed and the CSC-like phenotype acquired.

Conclusions: Arsenite transforms prostate epithelial stem/progenitor cells into CSC-like cells, indicating that it can produce CSCs from a model NSC population.

Figure 1

In vitro metrics of transformation in chronic arsenite-treated (5 μM) WPE-stem cells. (A) Secreted MMP-9 activity. Inset: representative zymogram (18 weeks). (B) Invasion; the A549 human lung carcinoma line was used as a positive control. (C) CFE; average numbers of colonies were 334 (control), 374 (15 weeks), and 504 (18 weeks). (D) Ductal/glandular-like structures produced by WPE-stem cells (top) or As-CSCs (bottom). Bars = 50 μm. See “Materials and Methods” for details. Numerical data represent mean and 95% CI (n = 3). *p < 0.05 compared with control..

Figure 2

Xenograft tumors from arsenite-transformed WPE-stem cells. (A, B) Representative sections showing the highly undifferentiated, pleomorphic nature of tumors after As-CSC inoculation. Areas of red blood cells indicative of probable vasculature disruption, including potential point of access, are visible; bars = 100 μm in A and 50 μm in B. B is a representative tumor arising from As-CSC inoculation at a higher magnification than A. (C) Immunohistologic section showing K5, a prostate epithelial SC marker, in an As-CSC xenograft tumor, with strong, heterogeneous distribution in the epithelial and mesenchymal cells; bar = 25 μm. (D) Tumor incidence after inoculation under the renal capsule with control (n = 10) or arsenite-treated cells (18 weeks) (n = 17). *p < 0.05.

Figure 3

CSC-like characteristics in arsenite-treated and control cells. (A) Free-floating, viable spheres formed by arsenite-treated WPE-stem cells (top; bar = 100 μm) and sphere quantitation in control or arsenite-treated cells after malignant transformation (bottom). (B) Secreted MMP-9 activity from sphere and adherent cells from control and arsenite-treated cells after malignant transformation (bottom); the zymogram (top) shows MMP-9 activity. (C) Increased CFE in arsenite-transformed spheres after malignant transformation. Average numbers of colonies were 313 (control spheres), 637 (arsenite spheres), 326 (control adherent), and 479 (arsenite adherent). Data are percent increase compared with controls (mean and 95% CI; n = 3). (D) Tumor incidence in mice (n = 10/group) inoculated with control cells, arsenite-exposed adherent sphere cells, or arsenite-exposed sphere cells. *p < 0.05 compared with control.

Figure 4

Expression of p63, an NSC gene associated with maintenance and self-renewal, during arsenite-induced malignant transformation of WPE-stem cells. p63 transcript (A) and protein (B) levels decreased at 9 weeks but increased again to control levels by point of malignant transformation (18 weeks). Data represent mean and 95% CI (n = 3–6). (C) Immunocytochemical images for p63 consistent with Western blots show that WPE-stem cells remained a homogeneous population during transformation; bars = 50 μm. *p < 0.05 compared with time-matched controls (A) or baseline levels (B).

Figure 5

Expression of NSC genes associated with maintenance, self-renewal, and differentiation during arsenite-induced malignant transformation of WPE-stem cells. Transcript levels for BMI-1 (A), ABCG2 (B), SHH (C), OCT-4 (D), NOTCH-1 (E), and K5 (F) all show the same loss then reactivation of expression as that seen with p63, indicating that a common pattern of alterations in an NSC-like transcription program involving self-renewal occurs during arsenite-induced progression of NSCs to CSCs. Data represent mean and 95% CI (n = 3–6). *p < 0.05 compared with time-matched controls.

Figure 6

K18 expression during arsenic-induced malignant transformation of WPE-stem cells. (A) K18 transcript levels during chronic arsenic exposure show early, marked increases followed by a decrease back toward control levels by the time of malignant transformation (18 weeks; n = 6). (B) Quantitative analysis of K18 protein levels during malignant transformation (n = 3) shows that the trend is consistent with transcript levels and is opposite that seen for SC markers (Figures 4 and 5). Data represent mean and 95% CI. *p < 0.05 compared with time-matched controls (A) or baseline levels (B).

Figure 7

PTEN expression during arsenite-induced malignant transformation of WPE-stem cells. PTEN transcript levels show a progressive and marked decrease starting at 9 weeks of arsenite treatment. Data represent mean and 95% CI (n = 6). *p < 0.05 compared with time-matched controls.