The findings presented here describe an unexpected convergence of two lines of recent research; stem cells have been documented in a variety of normal tissues and, more recently, this paradigm was extended to neoplasias (Clarke et al., 2006
; Reya et al., 2001
). Independent of these discoveries, the epithelial-mesenchymal transition (EMT) has been described over the past decade as a cell-biological program that is required for the remodeling of cells and tissues during embryogenesis, during certain types of wound healing, and during the acquisition of malignant traits by carcinoma cells (Hay, 2005
; Thiery, 2003
). None of these findings hinted at a link between these two sets of phenomena.
The discovery that the EMT generates cells with many of the properties of self-renewing stem cells holds the promise of resolving a major problem in cancer biology. Many types of cancer cells leaving primary carcinomas appear to rely on the EMT program to facilitate execution of most of the steps of the invasion-metastasis cascade (Thiery, 2003
). However, the last step, termed colonization, which involves the growth of micrometastases into macroscopic metastases, has represented a conceptual dilemma: If the vast majority of cells leaving a primary tumor and disseminating to distant sites lack self-renewal capability, their ability to found macroscopic metastases is compromised from the outset because of their limited proliferative potential. This factor would dictate that successful formation of a macroscopic metastasis is an exceedingly rare event. This problem may be addressed, at least in part, by the present findings, since the EMT program that enables cancer cells to disseminate from a primary tumor also promotes their self-renewal capability. Indeed, just such a connection between metastasis and stem-cell state has previously been proposed in a speculative voice as a means of enabling the formation of macroscopic metastases (Brabletz et al., 2005
While the present work indicates that the EMT confers many of the properties of the normal and neoplastic stem cell state, it does not yet demonstrate all of them. In the case of normal mammary stem cells, the case for the EMT leading to a stem-cell state has been supported, only indirectly: the EMT leads to great increases in the number of self-renewing cells that can initiate the seeding of mammospheres and, as shown by others and ourselves, mammospheres are enriched in stem cells and can seed entire mammary epithelial trees when implanted into cleared mammary fat pads (Supplementary Table 3
) (Liao et al., 2007
; Liu et al., 2006
; Moraes et al., 2007
). A more direct demonstration of this point will depend on a transient induction of an EMT in explanted mammary epithelial cells, and the demonstration that, at various limiting dilutions, such an induction leads to substantial increases in mammary gland-repopulating cells upon implantation into cleared mammary fat-pads.
In principle, some of the presently observed behaviors of the HMLE human mammary epithelial cells might be attributable to the introduced genes that were used previously to immortalize these cells, specifically hTERT, which encodes the catalytic subunit of the human telomerase holoenzyme, as well the SV40 early region. For example, these genes might facilitate the reprogramming of the HMLE cells initiated by EMT-inducing transcription factors such as Snail or Twist. However, we observe exactly the same responses to Snail or Twist in non-immortalized primary mammary epithelial cells, which indicates that the hTERT and SV04 T antigens appear to have no effect on the induction of the EMT.
An analogous demonstration of the connection between the EMT and the generation of neoplastic stem cells is also still required. We have shown here that the induction of an EMT in transformed human mammary epithelial cells yields cells with a CD44high
antigenic phenotype; others have shown, and we have confirmed, that such cells are greatly enriched in tumor-initiating cells. (Al-Hajj et al., 2003
); Guo et al, ms. in preparation). We have also demonstrated that the number of tumor-initiating cells is increased by at least two orders of magnitude if transformed cells were forced to constitutively express either a Twist or Snail EMT-inducing transcription factor. However, in the end a more definitive test of the connections between the EMT and tumor-initiating ability will come from inducing an EMT through activation of Twist-ER or Snail-ER in transformed cells ex vivo
followed by tests of their tumorigenicity in vivo
in the absence of ongoing Twist or Snail activity.
We note that the transient induction in mammary epithelial cells of the EMT yields great increases in their ability to form mammospheres. Since this capability can be transmitted through repeated cycles of serial passage of mammosphere-forming cells from one culture to the next, it seems apparent that the transient induction of an EMT creates a heritable, self-renewing state that continues to be manifest in the distant lineal descendants of a cell that is initially forced to undergo an EMT, indeed long after the EMT-inducing stimulus has been removed.
The presently described connections appear to hold a number of implications for the biology of epithelial cells. Thus, they suggest that the stem cells of certain epithelial organs show many of the attributes of the mesenchymal cell state. Moreover, the transient induction of an EMT in large populations of cancer cells may make possible the generation of relatively unlimited numbers of cancer stem cells, whose biology may then be studied with far greater facility. Perhaps more importantly, the use of genetic or pharmacologic techniques to transiently induce an EMT in large populations of differentiated normal epithelial cells may provide a means of generating at will large numbers of tissue-reconstituting, normal epithelial stem cells.