The basics of epithelial-mesenchymal transition (original) (raw)
Excessive epithelial cell proliferation and angiogenesis are hallmarks of the initiation and early growth of primary epithelial cancers (65). The subsequent acquisition of invasiveness, initially manifest by invasion through the basement membrane, is thought to herald the onset of the last stages of the multi-step process that leads eventually to metastatic dissemination, with life-threatening consequences. The genetic controls and biochemical mechanisms underlying the acquisition of the invasive phenotype and the subsequent systemic spread of the cancer cell have been areas of intensive research. In many of these studies, activation of an EMT program has been proposed as the critical mechanism for the acquisition of malignant phenotypes by epithelial cancer cells (66).
Many mouse studies and cell culture experiments have demonstrated that carcinoma cells can acquire a mesenchymal phenotype and express mesenchymal markers such as α-SMA, FSP1, vimentin, and desmin (67). These cells typically are seen at the invasive front of primary tumors and are considered to be the cells that eventually enter into subsequent steps of the invasion-metastasis cascade, i.e., intravasation, transport through the circulation, extravasation, formation of micrometastases, and ultimately colonization (the growth of small colonies into macroscopic metastases) (66, 68, 69).
An apparent paradox comes from the observation that the EMT-derived migratory cancer cells typically establish secondary colonies at distant sites that resemble, at the histopathological level, the primary tumor from which they arose; accordingly, they no longer exhibit the mesenchymal phenotypes ascribed to metastasizing carcinoma cells. Reconciling this behavior with the proposed role of EMT as a facilitator of metastatic dissemination requires the additional notion that metastasizing cancer cells must shed their mesenchymal phenotype via a MET during the course of secondary tumor formation (70). The tendency of disseminated cancer cells to undergo MET likely reflects the local microenvironments that they encounter after extravasation into the parenchyma of a distant organ, quite possibly the absence of the heterotypic signals they experienced in the primary tumor that were responsible for inducing the EMT in the first place (66, 71, 72). These considerations indicate that induction of an EMT is likely to be a centrally important mechanism for the progression of carcinomas to a metastatic stage and implicates MET during the subsequent colonization process (Figure 5). However, many steps of this mechanistic model still require direct experimental validation. Moreover, it remains unclear at present whether these phenomena and molecular mechanisms relate to and explain the metastatic dissemination of non-epithelial cancer cells.
Contribution of EMT to cancer progression. Progression from normal epithelium to invasive carcinoma goes through several stages. The invasive carcinoma stage involves epithelial cells losing their polarity and detaching from the basement membrane. The composition of the basement membrane also changes, altering cell-ECM interactions and signaling networks. The next step involves EMT and an angiogenic switch, facilitating the malignant phase of tumor growth. Progression from this stage to metastatic cancer also involves EMTs, enabling cancer cells to enter the circulation and exit the blood stream at a remote site, where they may form micro- and macro-metastases, which may involve METs and thus a reversion to an epithelial phenotype.
The full spectrum of signaling agents that contribute to EMTs of carcinoma cells remains unclear. One suggestion is that the genetic and epigenetic alterations undergone by cancer cells during the course of primary tumor formation render them especially responsive to EMT-inducing heterotypic signals originating in the tumor-associated stroma. Oncogenes induce senescence, and recent studies suggest that cancer cell EMTs may also play a role in preventing senescence induced by oncogenes, thereby facilitating subsequent aggressive dissemination (73–75). In the case of many carcinomas, EMT-inducing signals emanating from the tumor-associated stroma, notably HGF, EGF, PDGF, and TGF-β, appear to be responsible for the induction or functional activation in cancer cells of a series of EMT-inducing transcription factors, notably Snail, Slug, zinc finger E-box binding homeobox 1 (ZEB1), Twist, Goosecoid, and FOXC2 (66, 71, 76–79). Once expressed and activated, each of these transcription factors can act pleiotropically to choreograph the complex EMT program, more often than not with the help of other members of this cohort of transcription factors. The actual implementation by these cells of their EMT program depends on a series of intracellular signaling networks involving, among other signal-transducing proteins, ERK, MAPK, PI3K, Akt, Smads, RhoB, β-catenin, lymphoid enhancer binding factor (LEF), Ras, and c-Fos as well as cell surface proteins such as β4 integrins, α5β1 integrin, and αVβ6 integrin (80). Activation of EMT programs is also facilitated by the disruption of cell-cell adherens junctions and the cell-ECM adhesions mediated by integrins (67, 75, 81–86).
TGF-β is an important suppressor of epithelial cell proliferation and thus primary tumorigenesis. However, it is now clear that in certain contexts it can also serve as a positive regulator of tumor progression and metastasis (87–89). Thus, in vitro studies have demonstrated that TGF-β can induce an EMT in certain types of cancer cells (90). Two possible signaling pathways have been identified as mediators of TGF-β–induced EMT. The first of these involves Smad proteins, which mediate TGF-β action to induce EMTs via the ALK-5 receptor (51, 91–95). Smad-mediated signaling induced by TGF-β facilitates motility. Inhibitory Smads modulate differential effects of relevant transcription factors and cytoplasmic kinases and induce the autocrine production of TGF-β, which can further reinforce and amplify the EMT program (91, 92, 96, 97). Signaling pathways that mediate the action of β-catenin and LEF also cooperate with Smads (61, 98) in inducing an EMT (61, 99, 100). In this regard, the involvement of LEF and β-catenin in PDGF-induced EMT was recently described (98). These studies collectively demonstrate that the TGF-β/Smad/LEF/PDGF axis is an important inducer of an EMT phenotype in cancer.
Evidence for the involvement of a second TGF-β–induced pathway in EMT is also compelling. More specifically, some data indicate that p38 MAPK and RhoA mediate an autocrine TGF-β–induced EMT in NMuMG mouse mammary epithelial cells (96, 101). This process also requires integrin β1–mediated signaling and the activation of latent TGF-β by αVβ6 integrin (96, 101). Fibulin-5, an ECM molecule, augments TGF-β–induced EMT in a MAPK-dependent mechanism (102). TGF-β can induce an EMT in Ras-transformed hepatocytes, mammary epithelial cells (via MAPK), and MDCK cells; at the same time, Ras-activated PI3K inhibits TGF-β–induced apoptosis to facilitate this transition (103–106). Evidence for these connections comes from observations that ERK/MAPK and PI3K/Akt pathways mediate Ras mutant–induced EMT, and that such an EMT is reversed by either wild-type Ras or MAPK kinase 1 (MEK1) inhibitors (106). In this regard, Raf also mediates TGF-β–induced EMT and promotes invasiveness of cancer cells. In mouse models of skin carcinoma and human colon cancer, the absence of TGF-β receptor expression actually confers better prognosis (107, 108). The connection between inflammation and EMT was demonstrated when COX-2 was shown to inactivate Smad signaling and enhance EMT stimulated by TGF-β through a PGE2-dependent mechanism (109). Changes in the expression of certain cell polarity proteins may also play an important role in TGF-β–induced EMT, since evidence of a role for partitioning-defective protein 6 (Par6) in this process is emerging (66, 110).
The connection between loss of E-cadherin expression by cancer cells and passage through an EMT has been established by many studies (111, 112). For example, induction of the c-Fos oncogene in normal mouse mammary epithelial cell lines induces an EMT and is associated with a decrease in E-cadherin expression (99). Moreover, epithelial cell adhesion complexes reorganize and cell proliferation is suppressed when the full-length or the cytoplasmic portion of E-cadherin (containing the β-catenin binding site) is ectopically expressed in cells that have passed through an EMT, causing such cells to lose their mesenchymal phenotype (99, 113). Sequestration of β-catenin in the cytoplasm is important for the preservation of epithelial features of cancer cells, and acquisition of the mesenchymal phenotype correlates with the movement of β-catenin to the nucleus, where it becomes part of Tcf/LEF complexes (100, 114). Such β-catenin accumulation in the nucleus, which is often associated with loss of E-cadherin expression, correlates with susceptibility to enter into an EMT and acquisition of an invasive phenotype (61, 66). Thus, cells that lose cell surface E-cadherin become more responsive to induction of an EMT by various growth factors (61).
Some studies have demonstrated that the epigenetic control of E-cadherin and β-catenin/LEF activity is important in establishing the metastatic potential of cancer cells (115–118). Cell lines that lack E-cadherin show increased tumorigenicity and metastasis when transferred into immunodeficient mice (118). E-cadherin expression levels vary dramatically in different human tumors, and an inverse relationship between levels of E-cadherin and patient survival has been documented (117). In this regard, mutations in the E-cadherin gene have been identified in cancer cells, making them more susceptible to EMT and metastasis (115, 116). A more thorough analysis of such mutations and their correlation to metastatic progression is still required.
The central role played by E-cadherin loss in the EMT program is further illustrated by the actions of several EMT-inducing transcription factors that facilitate acquisition of a mesenchymal phenotype, such as Snail and Slug, as well as those encoding two key zinc finger–containing basic helix-loop-helix transcription factors, survival of motor neuron protein interacting protein 1 (SIP1) and E12 (also known as E47-E2A). These transcription factors are induced by TGF-β exposure and, once expressed, repress E-cadherin expression (78). Snail also facilitates an invasive phenotype in mice (31). Loss of E-cadherin promotes Wnt signaling and is associated with high levels of Snail in the nucleus (119). SIP1 represses E-cadherin expression and binds, along with Snail, to the E-cadherin promoter in an overlapping fashion (120, 121). The expression of Snail and E-cadherin correlates inversely with the prognosis of patients suffering from breast cancer or oral squamous cell carcinoma (119, 122). The use of gene expression analyses to compare expression of genes in metastatic and non-metastatic mouse breast cancer cell lines has led to the identification of Twist and Goosecoid as important genes that facilitate EMT and induce metastasis (85, 123). Some have reported that matrix-degrading enzymes such as MMP-3 facilitate EMT by inducing genomic instability via Rac1b and ROS (124).
Noncoding microRNAs are also components of the cellular signaling circuitry that regulates the EMT program. For example, microRNA 200 (miR200) and miR205 inhibit the repressors of E-cadherin expression, ZEB1 and ZEB2, and thereby help in maintaining the epithelial cell phenotype (125–129). In breast carcinoma, a loss of miR200 correlates with increased expression of vimentin and a decrease in the levels of E-cadherin in cancer cells (125–127, 129). Acting in the opposite direction, miR21 is upregulated in many cancers and facilitates TGF-β–induced EMT (130). Interestingly, CD44hiCD20lo cells purified from normal and malignant breast cancer tissue exhibit features of an EMT, and human cancer cells induced to undergo EMT exhibit stem cell–like properties and increased metastatic potential (84). Therefore, EMT may play a role in the generation of high-grade invasive cells with stem cell–like features, and the latter phenotype, which includes self-renewal potential, may facilitate the formation of secondary tumors by disseminating cancer cells, a notion that still requires direct demonstration.