Upholding a role for EMT in breast cancer metastasis

Abstract

The cell-biological program termed the epithelial-to-mesenchymal transition (EMT) has been invoked as a critical component of the meta static process. Contrastingly, Fischer et al.1 recently reported that in two genetically engineered mouse models of mammary tumour deve lopment, carcinoma cells could metastasize without activating EMT programs. However, as detailed below, we find their evidence that EMT programs were not expressed in these primary tumours to be insufficient. Therefore, the contribution of EMT to carcinoma meta stasis could not be ruled out in their analysis. There is a Reply to this Comment by Fischer, K. R. et al. Nature 547, http://dx.doi.org/10.1038/nature22817 (2017). It has been well-established that the EMT is not a single, stereotypical program2–4. Instead, it represents a group of related cellbiological programs, each of which confers certain mesenchymal traits on epithelial cells. Its variability stems from the facts that (1) it can be induced by expression of multiple alternative transcription factors5–9; (2) cells activating an EMT program often proceed only part-way towards a fully mesenchymal state, acquiring only a fraction of various mesenchymal markers2,10,11 and such partial and reversible activation of EMT has been shown to be critical for metastasis12–15; and (3) EMT programs may be manifested in different ways in different tissues2–4,12. These considerations help to illuminate the EMT programs analysed in the publications by Fischer et al.1, who concluded that metastasis from primary tumours occurred in the absence of EMT activation. Fischer et al.1 employed the Cre/CreER lineage-tracing method to track carcinoma cells that have undergone EMT activation. In such a genetic tracing protocol, the promoter of interest drives the expression of the Cre recombinase, which in turn inflicts a stable genetic mark on the genome of a cell and its lineal descendants. A derivation of this basic protocol involves a CreER protein, which only functions when a ligand of the oestrogen receptor (ER), in this case tamoxifen, is present. Hence, the marking of a cell depends on the concomitant presence of CreER expression and experimentally supplemented tamoxifen. In the present case, the authors used a Cre-activatable GFP transgene; accordingly, transient actions of Cre/CreER would permanently turn off an RFP marker and activate a GFP reporter. By monitoring the expression of GFP, Fischer et al.1 would therefore be able to determine whether an ancestor of these cells had expressed Cre/CreER. In order for the lineage tracing system to effectively mark carcinoma cells that have undergone EMT activation, it needs to meet, in our view, at least two criteria (Fig. 1a). First, the Cre/CreER driver needs to be expressed in most if not all of the cells that transiently undergo EMT activation. Second, once expressed, the Cre/CreER protein needs to activate the GFP reporter in most if not all of the carcinoma cells where Cre/CreER is expressed. Fischer et al.1 chose to use transgenic Cre/CreER lines driven by the promoters of two genes, Fsp1 and Vim, that are known to be expressed in certain versions of the EMT program. For most of their analyses, they used an Fsp1-cre transgene. The authors portrayed Fsp1 as a “critical gatekeeping gene” of EMT initiation, based on a publication showing that Fsp1 is required in order for renal proximal tubular epithelial cells to undergo an EMT in vitro in response to TGFβ16. If this portrayal of the gatekeeper role of Fsp1 were accurate, this would qualify its expression as a sensitive indicator of EMT activation. However, given the variable manifestations of the EMT program in various tissues2–4,14, there is no reason to believe that a marker of EMT activation in renal tubular cells will serve as a useful indicator in unrelated tissues, in this case the mammary epithelium. In fact, there is abundant evidence that Fsp1 is not an integral component of all EMT programs17, as Fsp1-knockout mice are capable of undergoing all stages of developmental EMT and are viable and fertile18. Moreover, in our own studies, while we could indeed