The notion that miRNAs may play a role in tumors was first supported by studies showing that certain miRNAs are critically involved in the regulation of cellular differentiation, proliferation, and apoptosis (Miska, 2005
). Although substantial evidence exists in support of a tumor suppressive effect for miRNAs, this concept is established largely based on the findings from studies using tumor cells and tissues. Our results reveal a causal role for loss of miR-200b expression in arsenic-caused malignant transformation of immortalized HBECs and subsequent tumor formation. These findings provide additional novel evidence indicating that individual miRNAs can function as tumor suppressors by suppressing carcinogen-caused human cell malignant transformation and tumor formation.
EMT refers to a program during normal embryonic development featuring a loss of epithelial properties, such as cell adhesion and expression of the epithelial marker E-cadherin and an acquisition of mesenchymal properties such as increased cell motility and expression of the mesenchymal marker vimentin (Lee et al., 2006
). EMT is now widely viewed as an important step in tumor invasion and metastasis (Thiery et al., 2009
). However, EMT has not been regarded to be involved in normal cell malignant transformation—the initiation of tumorigenesis. It was recently reported that loss of expression of miR-200 family members in tumor cells is associated with a mesenchymal morphology, and reexpressing miR-200s in tumor cells causes mesenchymal to epithelial transition (Gregory et al., 2008
; Korpal et al., 2008
). We found that chronic arsenite exposure can trigger the expression of the EMT-inducing transcription factors ZEB1 and ZEB2 and cause depletion of miR-200s, resulting in EMT and malignant transformation. Forcing expression of ZEB1 alone in nontransformed cells is sufficient to phenocopy the effect of 16 weeks of arsenite exposure, depleting miR-200s and inducing EMT and malignant transformation. Furthermore, stably expressing miR-200b is capable of abolishing ZEB1 expression, completely reversing and preventing EMT and cell malignant transformation resulting from arsenic exposure. Together, these results suggest that ZEB1 is not only the major factor that causes depletion of miR-200 but also the important miR-200b target that mediates EMT and normal cell malignant transformation resulting from arsenic exposure. It was recently reported that EMT generates cells with properties of stem cells (Mani et al., 2008
) and ZEB1, which induces EMT, also promotes tumorigenicity of human pancreatic cancer cells (Wellner et al., 2009
). It is thus highly likely that in addition to promoting tumor invasion and metastasis, ZEB1-mediated EMT may also be critically involved in the initiation of tumorigenesis by promoting cell malignant transformation.
It is interesting to note that arsenite exposure induces ZEB1 and ZEB2, depletes miR-200s, and causes EMT and cell malignant transformation only in p53low
HBECs but not in p53-intact HBECs. A recent study showed that arsenic acts specifically on p53-compromised human lung cancer cells and immortalized lung epithelial cells to induce centrosomal abnormalities and soft agar colony formation (Liao et al., 2007
). Another recent study reported that expression of a mutant p53 was required to expand an EGF receptor-overexpressing esophageal cellular subpopulation capable of undergoing ZEB-mediated EMT (Ohashi et al., 2010
). The findings from this and other recent studies suggest that loss of p53 function is essential for the expansion of epithelial cells that obtain mesenchymal characteristics through induction of ZEB expression. Perhaps induction of ZEB expression in epithelial cells with normal levels of p53 causes its activation, which in turn results in cell cycle arrest and/or apoptosis, thus limiting EMT and protecting against ZEB-induced transformation. Further studies are warranted to determine how p53 regulates ZEB-mediated EMT.
How does arsenic exposure cause depletion of miR-200? Although abnormal miRNA expression is common in human tumors, the detailed underlying mechanisms of miRNA deregulation are still largely unknown. It has been recently reported that increased DNA methylation plays important roles in silencing the expression of the miR-200 family and miR-205 (Vrba et al., 2010
; Wiklund et al., 2011
), and decreased DNA methylation is associated with overexpression of miR-200a and miR-200b (Li et al., 2010
). We found that arsenic exposure depletes the miR-200s through induction of ZEB1 and ZEB2 and DNA methylation. Although the mechanism of ZEB1 and ZEB2 induction by arsenic exposure remains to be determined, our finding has important implications in metal carcinogenesis. It is well accepted that arsenic is not a typical carcinogen and probably exerts its carcinogenic effect mainly through altering cell signaling and gene expression (Hernandez et al., 2009
; Ren et al., 2011
). It is thus likely that a similar mechanism of depleting the expression of miR-200s may also be exerted by other nontypical metal carcinogens such as nickel and cadmium. Further studies to determine the mechanism by which arsenite exposure induces the expression of ZEB1 and ZEB2 are warranted.
These findings potentially have important clinical implications. Our results and other recent findings (Ceppi et al., 2010
; Gibbons et al., 2009
) reveal that loss of miR-200b and 200c expression is common in human lung cancer cells. Because miR-200b and miR-200c are highly expressed in normal HBECs and overexpressing miR-200b showed no toxic effect on normal HBEC viability and proliferation, our finding that reexpression of miR-200b completely reverses arsenite-induced cell transformation, and tumor formation provides a good rationale to explore the therapeutic effect of miR-200b for lung tumors caused by arsenic exposure and probably by other exposures. Indeed, it was recently reported that forcing expression of the miR-200b cluster in lung tumor cells derived from mice that develop metastatic lung adenocarcinoma owing to expression of mutant K-ras and p53 abrogated the capacity of these tumor cells to undergo EMT, invade, and metastasize (Gibbons et al., 2009
). Our findings establish a causal role for loss of miR-200b expression in arsenic-caused human lung cell malignant transformation and tumor formation, suggesting that miR-200b may be a useful therapeutic target for lung cancer resulting from arsenic exposure.