A fixed partial epithelial‐mesenchymal transition (EMT)... : Hepatology (original) (raw)

Potential conflict of interest: Nothing to report.

See Article on page 934

Supported by the Russian Science Foundation (grant no.: 14‐15‐00318).

The research article by Matak et al.1 provides a good pretext for discussion of two hypotheses that concerns the carcinogenesis associated with the pathology of epithelial‐mesenchymal transition (EMT) and the “stochastic phenotype switching” phenomenon described by Matak et al.1

Carcinogenesis is classically considered as a process of the origin of the neoplastic clone through the appearance of mutations, escape from apoptosis and immune control, and acquisition of the immortalization and unlimited proliferation. The malignancy of the neoplastic clone is a result of the occurrence of new mutations that endow cells with the ability to invade, intravasate, survival in the circulation, interact with cells and molecules of the premetastatic niche, percept proliferation stimuli, form stroma in the metastatic niche, and develop a clinically detectable macrometastasis. The carcinogenesis according to this scenario is a long and slow process and discussed in the gradual and punctuated models of cancer origin and evolution.2

The hypothesis proposed here postulates that regardless of factors inducing EMT (e.g., transforming growth factor beta) in the stem (or nonstem) cell, genetic or epigenetic changes occurring because of intrinsic or external stimuli may establish a partial EMT and make the hybrid (epithelial‐mesenchymal; EM) phenotype stabile (Fig. 1A). It results in the appearance of the neoplastic clone with a couple of features attributed to mesenchymal cells: the invasive phenotype, avoiding apoptosis, and resistance to hypoxia and toxic effects, including chemotherapeutic agents.3 The maintenance of stemness or acquisition of stem‐like traits in the process of EMT makes the neoplastic clone capable to respond to proliferation stimuli. Carcinogenesis through the irreversible EMT probably does not require driver mutations.4 In contrast to the “classical” model of carcinogenesis, cancer origin through the irreversible EMT represents a rapid process because it leads simultaneously both to the appearance of the neoplastic clone and acquisition of the ability to invade and to metastasis. We believe that carcinogenesis associated with the irreversible EMT underlies cases when carcinomas are clinically manifested by not the primary tumors, but by lymph node and distant metastases (metastases with unknown primary tumors).

Figure 1:

Hypothetical involvement of a partial EMT in cancer. (A) The hypothesis of carcinogenesis associated with the irreversible EMT: Tumors or metastases from unknown primary tumors can originate from stem cells with the fixed hybrid EM phenotype, which migrated in the favorable sites—pretumor or premetastatic niches. The irreversibility of the hybrid EM phenotype is established by any genetic or epigenetic alterations (e.g., mutations/hypermethylation in the CDH1 gene). Most likely, nonstem cells undergoing a partial EMT can also produce tumors and metastases. (B) The hypothesis of “stochastic phenotype switching”: Tumor cells with the hybrid EM phenotype are able to self‐renewal and to produce epithelial or mesenchymal cells through quick reprogramming phenotype during asymmetric division.

Which mechanisms could be involved in the irreversibility of a hybrid EM phenotype of normal stem or nonstem cells and are there examples of this phenomenon? The recent studies based on the mathematical approaches predicted the role of OVOL transcription factors and the inhibitor of Notch intercellular signaling—Numb in the induction and maintenance of a partial EMT.5 In addition, mutations in the cadherin 1 (CDH1) gene and the absence of E‐cadherin or production of its altered variant may provoke a cascade of molecular events that lead to permanent activation of EMT transcription factors and maintenance of the mesenchymal phenotype.6 The similar effect can be observed in case of irreversible epigenetic silencing of the CDH1 gene.7 In fact, these mechanisms can be also implicated in carcinogenesis associated with the irreversible EMT.

The results by Matak et al.1 can be also regarded as an illustration of how phenotypes of three types of subclones obtained from sarcomatoid cholangiocarcinoma are established and maintained. The first subclone was keratin 7 positive (K7+) and always produced K7+ cells as well as K7 heterogeneous subclone (K7het). The second subclone was K7− and yielded only K7− cells. And the third K7het subclone comprised simultaneously K7+ and K7− cells. Seventy percent of the K7het subclone was represented by the mixture of cells with stable K7+ and K7− phenotypes that were actually the same as two aforementioned subclones. Only 30% of the K7het population consisted of cells that were able to switch their phenotypes stochastically and produce all K7 types of cells. In general, K7het subclone was predominantly represented by K7− cells, whereas K7+ cells were single.1

It must be noted that “stochastic phenotype switching” observed in the K7het subclone is not related to the activity of cancer stem cells. This statement is supported by the lack of expression of stem cell markers both in the primary culture and the cholangiocarcinoma samples.1 In our opinion, “stochastic phenotype switching” can be explained by EMT. Albeit Matak et al. indicated the lack of the correlation between EMT markers and K7 expression in different subclones, expression of EMT genes was detected in the sarcomatoid component of cholangiocarcinoma, which was K7 negative.1 In addition, the researchers discuss that transdifferentiation of the K7het subclone cannot be associated with EMT because external triggers were absent in vitro. However, we think that this work does not consider the fact that introduction of cholangiocarcinoma cells that are used to maintain the metabolism through the histohematic barrier to culture may result in EMT induction with the purpose of the adaptation to new conditions. Another reason of a cell‐autonomous EMT process may be oncogenic activation of signaling pathways, for example, because of mutations in the TP53 and KRAS genes.8 Probably, K7+ and K7− cells comprising 30% of the K7het subclone may be in a partial EMT and show hybrid EM phenotypes. Besides other reasons mentioned below, this assumption is resulted from the observations by Matak et al. that K7− subclones display E‐cadherin down‐regulation and fibronectin up‐regulation and are residing in a more mesenchymal state.1

Hybrid EM cells are well known to show similarity with stem cells. These cells express stem cell markers and are capable of self‐renewal (asymmetric division) and to form mammospheres.3 Thus, EMT is considered as an instrument for amplifying the pool of stem cells through EMT induction both in normal cells and stem cells themselves with the following: their switching from asymmetric to symmetric division and rapid generation of daughter stem cells.3

Interestingly, K7‐distinct subclones significantly differed in the tumorigenic potential. The highest ability to generate tumors in nonobese diabetic/severe combined immunodeficiency mice was observed for K7het subclone. These results are not surprising if one thinks of a high similarity between hybrid EM and stem cells. Moreover, the previous studies also showed higher tumorigenic and metastable potential of hybrid cells.3 Of note, only K7− cells of the heterogeneous subclone displayed tumorigenicity, whereas K7+ cells had undergone necrotic changes.

It is possible that “stochastic phenotype switching” occurs in the process of asymmetric division when the way to more epithelial or mesenchymal phenotype is chosen (Fig. 1B). In fact, asymmetric division allows reprogramming cell phenotype quickly and effectively. Although the exact mechanisms involved in “stochastic phenotype switching” are unknown, it is clear that they are not related to DNA methylation. Nevertheless, the fixation of epithelial and mesenchymal phenotypes and the maintenance of their stability is associated with hypo‐ and hypermethylation, respectively, in the genome regions that are responsible for the epitheliality.1

In the tumor, asymmetric division and “stochastic phenotype switching” can be observed in hybrid EM tumor cells that may acquire a stable mesenchymal phenotype and associated invasive, apoptosis‐resistant, and other features. Such mechanism of the appearance of the sarcomatoid (mesenchymal) subclone can be universal for the so‐called spindle cell carcinomas (lung, kidney, etc.). Similarly, in EMT‐associated carcinogenesis, asymmetric division of the normal stem or nonstem cell undergoing a partial EMT may be a locus minoris for the occurrence of genetic/epigenetic changes and the irreversible fixation of a hybrid EM phenotype.

Taken together, the data presented here show that hybrid EM cells can be involved both in carcinogenesis and cancer progression. The irreversible “programming” of a hybrid EM state in the stem or nonstem cell may lead to the appearance of the neoplastic clone and the simultaneous acquisition of invasiveness and high metastatic potential (the hypothesis of EMT‐associated carcinogenesis). In cancer progression, hybrid EM tumor cells switch stochastically their phenotypes during asymmetrical division and produce cells with pronounced and stable epithelial or mesenchymal features (the hypothesis of “stochastic phenotype switching”). In fact, transdifferentiation through asymmetric division allows to reach mesenchymality or regain epithelial features quickly without EMT advancement or the involvement of mesenchymal‐epithelial transition, respectively. Overall, hybrid EM cells represent a useful model to investigate the mechanisms of cancer origin and progression and a promising target for novel prognostic and therapeutic approaches in cancer medicine.

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7. Hsiao SM, Chen MW, Chen CA, Chien MH, Hua KT, Hsiao M, et al. The H3K9 methyltransferase G9a represses E‐cadherin and is associated with myometrial invasion in endometrial cancer. Ann Surg Oncol 2015;22(Suppl 3):S1556‐S1565.

8. Larsen JE, Nathan V, Osborne JK, Farrow RK, Deb D, Sullivan JP, et al. ZEB1 drives epithelial‐to‐mesenchymal transition in lung cancer. J Clin Invest 2016;126:3219‐3235.

Author names in bold designate shared co‐first authorship.