Epithelial to mesenchymal transition (EMT) is a paradigm of cell plasticity, thought to be crucial for embryogenesis, injury repair, organ fibrosis, and malignant progression 6, 7
. EMT is characterized by reversible loss of epithelial characteristics coupled with gain of mesenchymal properties, including transition from epitheliod to stellate morphology, loss of cell polarity, enhanced motility, and synthesis of extracellular matrix remodeling proteins. EMT is thought to be reversible under most circumstances, a process called mesenchymal to epithelial transition (MET).
Although it has been appreciated for decades that EMT is crucial for proper development of various embryonic tissues, over the past few years numerous publications have also highlighted the importance of EMT in cancer, and have suggested that EMT may be a driving force behind invasion, metastasis, and chemoresistance in many carcinomas including pancreatic ductal adenocarcinoma (PDAC) 8, 9
Much of what we know regarding the signaling pathways and molecules involved in regulating EMT are derived from in vitro
cell culture studies, due to the ease of studying pure populations of epithelial cells undergoing synchronous EMT. In the decades since the first in vitro
description of EMT by Elizabeth Hay, 10
much of our current understanding of EMT has been obtained by treatment of cultured epithelial cells with various extracellular signaling molecules that trigger EMT, such as transforming growth factor-beta (TGF-β).. These signaling molecules activate pathways that up-regulate expression of certain “master regulatory” transcription factors, including Snail, Slug, Zeb1, Twist, and SIP1, that converge upon the promoter of E-cadherin (and likely other epithelial-specific genes) to repress transcription 6, 7
. E-cadherin is a vital component of epithelial desmosomes, and loss of E-cadherin triggers the disassembly of adherens junctions. This results in loss of cell contacts that normally maintain epithelial differentiation and inhibit induction of EMT. Indeed, overexpression of master regulatory factors or knockdown of E-cadherin expression by RNAi is sufficient to induce EMT in the absence of exogenous signaling molecules 11
. In addition to master regulatory factors, several recent studies in cell culture have also implicated other molecular processes that regulate EMT, including chromatin remodeling and microRNAs, many of which also regulate expression of E-cadherin via interplay with the master transcription factors.
The in vivo
study of EMT in mice is hampered by the lack of known biological situations where homogenous populations of cells undergo synchronous EMT. Many studies to date have focused on whether or not the EMT process and the factors that control it are active in human cancers transplanted into mouse models. Several lines of evidence from these models support a role for EMT in malignant progression. First, single cells that break apart from the bulk of well-differentiated tumors at their invasive fronts undergo EMT in models of breast cancer12
. Second, many human carcinomas (including PDAC) with poorly differentiated and/or spindled morphology often display features of EMT and are usually (although not always) more aggressive than their well-differentiated counterparts, and this behavior is often associated with loss of E-cadherin and changes in EMT master regulatory transcription factors 13–15
. Third, there is accumulating evidence that cells undergoing EMT may constitute the fraction of tumor initiating cells (TICs) in some carcinomas. Isolation of cells with properties ascribed to TICs from bulk tumors has shown that these cells display features of EMT 16
, and differentiated cells induced to undergo EMT acquire TIC properties in culture and in mice 17, 18
. Indeed, compounds that eradicate cells undergoing EMT also eradicate TIC subpopulations 18
. Finally, PDAC cells with properties of EMT are highly chemoresistant (e.g. to gemcitabine) when compared to differentiated cells19
. Chemoresistance is a property often ascribed to TICs, and chemoresistant TICs are thought to be present in human PDAC20, 21
. Indeed, treatment of PDAC cells with gemcitabine seems to greatly enrich the fraction of TIC-like cells, presumably via selecting for EMT20, 22–24
Thus, experimental studies suggest that EMT or some EMT-like process does play a crucial role in propagation, malignant progression, and chemoresistance of many cancers including PDAC, at least in cell culture and in mouse models. Does the same hold true for PDAC in human patients, where tumor evolves within its native human host rather than an immunodeficient murine host environment?
A broad range of degrees of differentiation can be observed for human invasive PDAC in histologic sections. Poorly and undifferentiated carcinomas, as the names suggest, feebly recapitulate the glandular differentiation of non-neoplastic ductal cells25
. The neoplastic cells, instead, are mononuclear or spindle shaped with aggressive clinical behavior. These mononuclear and spindle-shaped cells often display immunolabeling patterns consistent with EMT, including loss of E-cadherin 14
. For these types of poorly differentiated tumors, it has been hypothesized that these neoplastic cells are genetically driven into an irreversible EMT state while retaining the ability to proliferate26
, via bypassing EMT-driven growth arrest.
While these observations in humans would at first appear to support the hypothesis that EMT occurs in human PDAC and other cancers, pathologic examination of those PDACs with well-differentiated morphology reveal two observations that appear to run contrary to this25
First, not all aggressive PDACs are poorly differentiated. Despite the dismal prognosis associated with pancreatic cancer, many PDACs, including those that metastasize and kill patients, are remarkably well-differentiated (). This is one of the great paradoxes of pancreatic pathology- that such an aggressive neoplasm is often so well-differentiated. Second, the most invasive components of some pancreatic cancers still form nearly perfect glands. This includes adenocarcinoma that has colonized perineural spaces (), blood vessels, and even in distant metastatic sites (e.g. “lepidic growth” in lungs). Simply put, in many cases, the overtly malignant PDAC cells in nerves, vessels and metastatic sites are not poorly differentiated or spindle-shaped mesenchymal appearing cells. They are the opposite. They are so well-differentiated that they are often even impossible to distinguish histologically from non-neoplastic glands except for their abnormal location25
. In addition, expression of E-cadherin is nearly always intact in these cells ().
Figure 1 Infiltrating ductal adenocarcinoma of the pancreas. Well-differentiated carcinoma in the muscularis propria of the duodenum (A) and around nerves (B). Note how the neoplastic cells closely recapitulate normal ducts. C) Immunolabeling of for e-cadherin (more ...)
How then can we put these two observations together? How can we reconcile the importance of EMT for malignant behavior in mouse models and poorly differentiated human PDAC, with the finding that many highly aggressive pancreatic adenocarcinomas in humans can be extremely well-differentiated? Why aren’t these cells undergoing EMT and/or poorly-differentiated or spindled upon invasion and metastasis? One explanation is that EMT is simply not required for aggressive behavior for all carcinomas in human microenvironments, well-differentiated ones in particular. In some mouse models and human cancers, carcinomas may invade collectively without infiltration of single EMT cells at the invasive tumor front27
, and retention of their epithelial cell adhesion molecules may be required for invasion and growth in vessels, nerves, and sites of distant metastasis. One could even envision a scenario that once vascular invasion is achieved, these well-differentiated cells may utilize adhesion molecules to “hitch a ride” to distant metastatic sites by attaching to blood cells or platelets. Even simpler, a metastatic process that depends on tightly adherent malignant cells circulating in the bloodstream as a neoplastic bolus may offer a much more efficient means by which carcinoma can lodge and colonize a metastatic site than a process based on EMT and single cell dissemination. Thus, at first glance it would appear that EMT does not explain malignant behavior in well-differentiated PDAC in humans.
However, in a recent elegant review, Thomas Brabletz outlined recent evidence that EMT coupled with the reverse
process MET, may play a crucial role in malignant progression of well-differentiated carcinomas26
. First, although most, if not all, neoplastic cells in well-differentiated carcinomas appear epithelial, rare individual cells that appear to be undergoing EMT can be observed in histologic sections and even isolated from these tumors (). As in mouse models, it is conceivable that these rare EMT-like cells may underlie single cell invasion into stroma, nerves, and vessels, and are also required for dissemination into the bloodstream. However, efficient colonization and growth within locally invaded tissues and sites of distant metastasis may require reversal of the process, mesenchymal to epithelial transition (MET), thereby producing well-differentiated cells that are more efficient at populating a site once EMT has allowed access. Indeed, it is well-known that cells undergoing EMT are typically growth arrested and therefore incapable dividing to form a mass, whereas fully differentiated carcinoma cells readily enter the cell cycle. Also, cell surface adhesion molecules required for epithelial attachments to each other and other cells are lost during EMT, which could also inhibit growth within stroma, nerves, and vessels, as well as disrupt attachments to other tumor cells required to produce mass lesions. Careful examination of histologic sections of locally invasive and/or metastatic well-differentiated PDACs for rare cells undergoing EMT and MET could help resolve these questions.
An understanding of the role of EMT and MET in humans has implications for therapies targeting EMT, since these treatments would only eradicate a small number of cells at the invasive front of tumors and those that are disseminating in the bloodstream. They would not treat the bulk of the invasive primary pancreatic carcinoma or well-differentiated cells in established metastasis. Obviously, if any malignant cell in a well-differentiated PDAC is capable of undergoing EMT, then combination therapy targeted toward both EMT and well-differentiated cells would be required. Clearly, EMT raises many provocative questions for pancreatic cancer pathology that will need to be addressed with studies in model systems and in man.