EPITHELIAL‐TO‐MESENCHYMAL   TRANSITION   AND   IMPLICATIONS   TO   TUMOR   PROGRESSION

Upon the growing evidences demonstrating the existence of mesenchymal cell types in carcinomas⁵⁷ and in CTCs population⁵⁸, the contribution of epithelial-to-mesenchymal transition (EMT) to tumor progression and metastasis has been widely discussed especially regarding intravasation and extravasation steps in metastatic cascade. Simply, EMT is a process that cells lose their junctions and polarity, reorganize cytoskeleton via change in gene and transcription factors^‡ profiles or signaling pathways⁵⁹. It has been proposed that epithelial cancer cells gain enhanced invasiveness, motility and resistance to apoptosis through EMT, disseminate and secondary carcinoma tumors are formed by a reverse process called mesenchymal-to-epithelial transition (MET)^{60,61 ,62} (Figure 4). However there is no direct evidence that EMT is required to generate metastasis⁶³.

FIGURE 4 PROPOSED MECHANISM OF METASTATIC CANCER CELL DISSEMINATION VIA EMT AND MET (IMAGE COPIED FROM REF 61)

‡ Transcription factors: proteins controlling which genes are turned on or off by binding to DNA or another protein⁸⁹.

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In fact EMT is a fundamental phenomenon in embryogenesis and organ development, which is crucial to diversify tissue and functional organism (type1 EMT). For example, formation of the three layered embryo is a result of this process in which certain epithelial cells have plasticity between epithelial and mesenchymal states via EMT and MET. Along the development, tissue-specific function is gained by epithelial cells and supporting role of tissue by mesenchymal cells after terminal differentiation of these transformed cells⁶². Besides embryogenesis, EMT is activated in wound healing and organ fibrosis (type2 EMT) by signaling received from inflammatory cells, fibroblasts and ECM. EMT is also suggested to occur during tumor growth and cancer progression at the invasive front of the tumors enabling them to move into the bloodstream and reach to distant organs (type3 EMT). Furthermore, it is clear that all the three types of EMT are distinct from each other in different physiological contexts⁶² depending on the cell type, tissue context and signaling pathways.

Nonetheless, in all tissue contexts, EMT can be characterized by some common features;

dissolution of epithelial cell-cell junctions, loss of their apical-basal polarity, attaining front-rear polarity (unequal distribution of proteins between the front-leading edge and the back of the cell), reorganization of cytoskeleton, changes of cell shape, down regulation of epithelial gene expressions, enhanced cell motility, and ability to degrade ECM in many cases⁵⁹.

Epithelial tissues are composed of cell layers where specific cell surface proteins form cell-cell junctions maintaining epithelial integrity and separated by basal lamina from the neighboring tissues. EMT process leads to delocalization or degradation of these proteins and thus loss of apical-basal polarity. Particularly E-cadherin expression, epithelial cell-to-cell adhesion molecule that forms the cell-to-cell-cell-to-cell or cell-to-cell-substrate junctions, is reduced while N-cadherin is upregulated. N-cadherin, mesenchymal neural cell-cell adhesion molecule, has weaker interactions between each other compared to E-cadherin and it gives higher affinity against mesenchymal cells, thus motility and invasion is increased⁶⁴. This protein is already demonstrated to be associated⁶⁵ with tumor invasiveness, metastatic dissemination and poor patient prognosis in several studies.

Cells undergoing EMT gain elongations, shape changes and thus directional motility by reorganizing their cytoskeleton. Cells produce extensions so called lamellipodia, filopodia and invadopodia to facilitate movements and these extensions can express agents degrading the ECM proteins and thereby enable invasion. One of the changes in cytoskeleton occurring in the composition of intermediate filaments during EMT is the repression of cytokeratin and increase of vimentin expression, which also have role in advanced motility⁶⁶. These changes have also been reported in primary breast carcinomas tissues and have been associated

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with more aggressive behaviors of cells suggesting that disseminated tumor cells have acquired mesenchymal cells features⁶⁷.

EMT is also dependent on the remodeling of ECM and cell interactions between ECM.

Epithelial cells communicate with the basement membrane whereas mesenchymal cells with different ECM. For example, increased expression of integrin proteins receiving signals from ECM related to mesenchymal phenotype can further interact with collagen and contribute to the loss of E-cadherin⁶⁸. Similarly increased proteases enzymes such as the matrix metalloproteinases MMP2 and MMP9 have been correlated with integrin changes leading to degradation of ECM proteins.

These hallmarks of EMT initiation could be activated in several ways whether genetically or epigenetically. There are several transcription factors having a significant role in EMT such as SNAIL, SLUG, TWIST and zinc-finger E-box binding (ZEB)⁵⁹ regulating the repression of epithelial genes and activating the mesenchymal genes in a cooperative way. Their involvement in EMT process depends on the tissue type and their role in signaling pathway. Non-coding miRNAs have been showed to activate EMT by selectively binding mRNA and thereby inhibiting or promoting the degradation of proteins that regulate the process. It could also be triggered by a variety of other mechanisms such as hypoxia, tumor-ECM interactions and growth factor signaling⁶⁹.

Despite the fact that EMT has an obvious role in acquisition of high level of malignancy, the importance of EMT in human tumors is a subject of debate given the lack of evidences due to experimental challenges^{63, 70,71}. The main concerns about this issue could be summarized as following. Many studies demonstrating the EMT are performed in transgenic mouse model that do not necessarily represent the reality of human tumors^,71,72. Besides, there are already evidences showing that phenotypic shift of cells from mesenchymal to epithelial state (MET) occurs supporting the EMT role in metastasis⁷³. However, there is a need for more clear evidence demonstrating how one tumor cell has abandoned one state and committed to the other at lineage level characterized by both epithelial and mesenchymal lineage markers, not only showing the expression of related EMT proteins in epithelial cancer cells. Similarly, the evidences showing EMT related expression of transcription factors located at the invasive front of tumor⁶¹ and EMT markers observed in CTC are favoring the EMT-induced dissemination, but this again does not indicate that cells has comprised the whole EMT steps. The lack of evidence whether the cells have gone EMT, completed the whole metastatic process and switch their phenotype by MET and colonize was mainly due to lack of robust markers differentiating mesenchymal tumor cells from stromal cells and as well as difficulty to track and image single cells in noninvasive ways⁷⁴. A very recent study, on the contrary to EMT/MET hypothesis, was

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able to show that EMT is not required for lung metastasis in breast cancer models by using cell linage tracing approach. Nonetheless, contribution of EMT for chemoresistance was demonstrated clearly by observation of recurrent EMT-derived metastasis after therapy⁶³. Yet such kind of studies should be applied to other kind of cancers with different metastatic sites to see how globally this pattern is observed and still investigating and blocking EMT in tumor progression hold great importance in terms of therapy efficacy.

Another intriguing concept is the acquisition of stem cell properties when the cells undergo EMT. There is now increasing number of evidence that upon EMT induction, cells gain stem cells traits both in normal and cancer cells, and vice versa, stem cell populations exhibit a variety of EMT markers^33,42. Several different EMT inducers have been shown to support this concept in cooperation alone or mutually with another^{42, 75, 76}. For example non-tumorigenic subset of CD44^low/CD24⁺ cell could give rise to tumorigenic population of CD44⁺/CD24^-/low cell by EMT induction through Ras/MAPK signaling pathway and treatment with TGFβ accelerating the process⁷⁵. Also stem cell concept is further supported by the formation of mammospheres only with CD44⁺/CD24^-/low population. However, the link between CSC and EMT is not clear whether EMT is necessary for generating stem cells or CSC benefit EMT for differentiation and advance in tumor progression⁷⁷.

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