3.1 Altered junctional components

There are four common junction types that comprise the epithelial connection, namely adherent junctions, tight junctions, desmosomes, and gap junctions. One of the most known adherent junctions is the transmembrane protein E-cadherin (epithelial cadherin, encoded by CDH1 gene), whose cytoplasmic region binds with β-catenin and p120 catenin, thereby being associated with cytoskeleton. The extracellular fragment of E-cadherin provides intercellular adhesion between opposing epithelial cells in a calcium-dependent manner.¹²⁵ In normal condition, E-cadherin is one of the most important epithelial marks. Given the fact that most tumors originate from epithelial tissues, deregulation of E-cadherin has been regarded as a hallmark of tumorigenesis. Indeed, in the context of neoplasm, E-cadherin has long been proved as a tumor-suppressor gene inhibiting cancer initiation and progression. Loss of E-cadherin, often caused by epigenetic silencing or genetic mutations, is a common event in a wide range of tumors, including breast cancer, gastric cancer, colorectal cancer, liver cancer, lung cancer, and so on.^{30, 126-129} Interestingly, E-cadherin has been also shown considerable expression level in tumor metastases.¹³⁰ Moreover, a recent study indicated that E-cadherin is required for tumor metastasis by buffering oxidative stress in breast cancer.¹¹⁸ One possible explanation is that E-cadherin has to be reexpressed in MET process, which facilitates the distal colonization in the late stage of metastasis. Other adherent junctions, such as N-cadherin (neuronal cadherin, CDH2), P-cadherin (placental cadherin, CDH3), R-cadherin (retinal cadherin, CDH4), and M-cadherin (muscle cadherin, CDH15), share similar structure but their functions are distinct.¹³¹ N-cadherin is a mesenchymal mark promoting tumor metastasis, which is opposite to E-cadherin.¹³² R-cadherin is an epithelial mark resembling E-cadherin, loss of which has been shown to facilitate EMT and tumor progression.¹³³ Interestingly, the role of P-cadherin can be either tumor promoting or tumor suppressive, depending on particular context.¹³⁴ For instance, the high expression of P-cadherin was correlated with the progression of lung cancer and ovarian cancer,^{135, 136} whereas other reports showed that P-cadherin preserves epithelial barrier to inhibit the metastasis of melanoma.^{137, 138} These dual functions of P-cadherin in regulating cancer metastasis might be attributed to the distinct characteristics of different cancer types. To be specific, in response to gonadotropin-releasing hormone (GnRH), P-cadherin induces the activation of IGF-1R in a ligand-independent manner, which phosphorylates p120 catenin, thereby promoting metastasis in ovarian cancer.¹³⁶ However, other tumor types such as melanoma might not be relevant to GnRH, IGF-1R, or p120 catenin. In other words, GnRH, IGF-1R, or p120 catenin might be with very low basal level or loss of function in other cancers, leading to the disruption of this pro-metastatic signaling, therefore P-cadherin failed to activate metastasis in such cancers. In this context, P-cadherin may serve as intercellular glue to prevent metastasis. Certainly, this postulation needs to be validated by substantial evidence.

Apart from adherent junctions, the epithelial integrity is also maintained by tight junctions, which consist of claudins, zonula occludens, and others. These tight junction proteins contain several family members. For instance, 27 members are characterized in claudin family, whereas only three members are found in zonula occluden family.¹³⁹ Similar to adherent junctions, tight junctions also play crucial roles, either positive or negative, in the EMT phenotype and cancer progression. Claudin-1 was shown to promote EMT and invasion of colorectal cancer through upregulating ZEB-1, while inhibiting tumor metastasis in gastric cancer via mediating the tumor-suppressive function of RUNX3.^{140, 141} Besides, it has been reported that claudin-2 promotes tumor metastasis in breast, lung, and colorectal cancer, but is negatively associated with the high-grade pancreatic cancer.^142-146 Therefore, the specific roles of claudin proteins in EMT status and tumor progression are largely context dependent. Another tight junction proteins, Zona occludens (ZO), are also key components of epithelial tissue. There are three members found in ZO family, namely ZO-1, ZO-2, and ZO-3. Among them, ZO-1 is the most investigated one in the neoplastic context. For example, loss of ZO-1 was reported to promote metastasis of breast cancer, colorectal cancer, liver cancer, pancreatic cancer, and so on.^147-150 These observations indicate that ZO-1 mainly serves as a tumor suppressor, in contrast to the diverse functions of abovementioned claudin proteins. However, a recent study provided exceptional evidence describing that ZO-1 can activate Rac-1-mediated cytoskeletal organization, thereby promoting metastasis in colorectal cancer.¹⁵¹ More intriguingly, different tight junction proteins can be associated with each other, thereby forming complex junction architecture. For instance, the cytoplasmic region of claudins is able to bind with the PDZ domain of zonula occludens.¹⁵² Though physical association is observed, the functional link between claudins and ZO proteins remains elusive.

Desmosomes represent another form of cell junctions, which consist of desmosomal cadherins (including desmogleins and desmocollins), armadillo proteins (including plakoglobins and plakophilins), and desmoplakin. These proteins form complex structure anchoring the intermediate filaments to the plasma membrane between neighboring cells, thus maintaining epithelial integrity.¹⁵³ As tumor suppressors, downregulation of desmosomal components plays vital roles in EMT program and consequent cancer progression.¹⁵⁴ For example, impaired desmosomes were shown to promote EMT and consequent tumor progression in invasive breast cancer.¹⁵⁵ Besides, loss of desmosomes was observed in invasive pancreatic neuroendocrine tumors (PNET), and genetic deletion of desmoplakin enhanced tumor metastasis in the PNET mouse model.¹⁵⁶ Interestingly, the proper functions of desmosomes require particular posttranslational modifications on several components. Palmitoylation of plakophilin was shown to play critical roles in the assembly of desmosomes, and dephosphorylation of plakophilin-1 is able to promote epidermal carcinogenesis.^{157, 158} In fact, the tumor-suppressive roles of desmosomes were also reported in several other tumor types, including liver cancer, breast cancer, and lung cancer.^{159, 160} Even so, the oncogenic roles of desmosomal components have been also reported. For instance, desmoglein-3 is overexpressed in human head and neck cancer and associated with advanced tumor stage, and inhibition of desmoglein-3 is sufficient to mitigate tumor progression both in vitro and in vivo.¹⁶¹ In addition to desmoglein-3, the tumor-promoting functions of several other desmosomal components were recently summarized elsewhere.¹⁶² This evidence suggest that desmosomes might not be simply regarded as tumor suppressive molecules, but rather multifunctional structure during tumor development.

Gap junctions are ion channels formed by connexin, pannexin, and innexin proteins, which provide both adherence and direct intercellular communication between neighboring cells.¹⁶³ Among them, connexins are the most investigated channels. Similar to other junction proteins, connexins play dual roles in tumorigenesis. It has been reported that connexins are overexpressed in tumors, such as connexin-26 in pancreatic cancer and colorectal cancer.^{164, 165} However, it has been also shown that connexins can serve as tumor suppressors, including connexin-43 and connexin-45 in colorectal cancer.^{166, 167} Moreover, high expression of connexins can predict either better or poor prognosis in cancer patients. For instance, overexpression of connexin-43 prolongs the survival of patients with prostate cancer, breast cancer, and colorectal cancer,^{166, 168, 169} whereas accelerating death of patients with bladder cancer, esophageal squamous cell carcinoma, and oral squamous cell carcinoma.^170-172 Not surprisingly, gap junction-regulated EMT and tumor progression are largely dependent on their ion channel function, which provide communication between nearby cells, in addition to their adherent effect. It has been reported that gap junctions among U2OS cells failed to inhibit EMT, but gap junctions between U2OS cells and osteoblasts did, suggesting that gap junctions inhibit EMT through U2OS-osteoblast communication rather than merely intercellular glue.¹⁷³ Moreover, gap junction was shown to amplify potassium currents, thereby establishing electrochemical communication between neuron and glioma. This effect significantly promotes glioma progression, which can be abrogated by gap junction antagonists.¹⁷⁴ Thus, targeting gap junctions can be a potential therapeutic strategy for cancer treatment.

3.2 Cytoskeleton reorganization

As mentioned above, cell–cell junctions are linked with actin cytoskeleton to form epithelial architecture, suggesting the vital roles of cytoskeleton organization during EMT program. Indeed, junction proteins are associated with Rho GTPases, which are dominantly responsible for the organization of actin cytoskeleton.^{10, 175} The activity of Rho GTPases is finely tuned by three classes of regulators, namely guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs).¹⁷⁶ In response to adherent or growth factor signals, Rho GTPases are activated by the exchange of GDP with GTP, and this process is catalyzed by GEFs. In contrast, Rho GTPases can be inactivated by GAP-mediated hydrolyzation of GTP into GDP. GDIs controls the subcellular localization of Rho GTPases by forming protein complex. To date, 20 Rho GTPase family members are identified, among which RhoA, Rac1, and Cdc42 are the most documented, especially in neoplastic context.^{177, 178} For example, RhoA has been found to be overexpressed in colorectal cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, and so on.^179-181 The upregulation of Rac1 can be observed in prostate cancer, gastric cancer, breast cancer and leukemia.^{179, 181-183} Cdc42 was reported to be overexpressed in colorectal cancer, breast cancer, lung cancer, and melanoma.^{179, 184-186} Interestingly, RhoA gene was shown to be rarely amplified but frequently deleted in a wide range of tumors according to The Cancer Genome Atlas dataset, suggesting its tumor suppressive role which is somewhat contradictory to most literatures.¹⁷⁷ Rho GTPases regulate actin polymerization through complex mechanisms, thereby organizing cytoskeleton. In this process, globular actin (G-actin) is polymerized to form filamentous actin (F-actin), whereas Arp2/3 complex is one of the key molecular machines enabling this.¹⁸⁷ For example, Rac1 can bind with the nucleation promoting factor WAVE, which activates Arp2/3 to generate branched actin networks.^{187, 188} Similarly, Cdc42 interacts with N-WASP, leading to the activation of Arp2/3 thus playing critical role in actin polymerization.^{189, 190} Aberrant formation of F-actin in cancer cells is closely correlated with EMT and metastasis of a variety of tumors, including hepatocellular carcinoma, glioblastoma, pancreatic cancer, bladder cancer, breast cancer, and so on.^{30, 191-197} Besides, the antagonists for actin polymerization, latrunculin A/B, have been shown anticancer effects through disrupting the formation of F-actin.^{30, 198-200} This evidence indicates that the organization of actin cytoskeleton network profoundly affects EMT and tumor progression.

Cytoskeleton reorganization frequently leads to the formation of membrane protrusions, namely lamellipodia, filopodia, invadopodias, and podosomes, thus contributing to the migration and invasion of tumor cells. Lamellipodia and filopodia are defined as sheet-like and spike-like extensions, respectively. Both protrusions are present on the leading edge of migrating cells, which determine the movement direction of cells.²⁰¹ Invadopodia appears on the ventral surface of membrane, and often involved in the degradation of ECM via MMPs.²⁰² Podosomes are similar to, but less effective in ECM degradation than invadopodia.^{203, 204} These membrane protrusions can be visualized from microscopy, thus being useful marks for EMT. Compelling evidence suggests the crucial roles of membrane protrusions in EMT and tumor progression. For instance, formation of filopodia encouraged by EMT program facilitates both initiation and metastatic colonization in breast cancer.²⁰⁵ Besides, invadopodia is correlated with the EMT program and consequent metastasis in a variety of tumors, including hepatocellular carcinoma, breast cancer, bladder cancer, and so on, suggesting invadopodia as a potential prognostic marker for tumor metastasis.^206-210 Given their critical roles in tumor metastasis, inhibitors targeting these protrusions can be of therapeutic value. For example, lidocaine has been shown to reduce metastatic dissemination of breast cancer by inhibiting the formation of invadopodia.²¹¹ However, the formation and turnover of these membrane protrusions are highly dynamic and the duration ranges from minutes to hours, targeting these structures might be with off-target effects thus waiting for further investigation.

4.1 Snail

Snail proteins include Snail1 (Snail) and Snail2 (Slug), which promote EMT and metastasis in a variety of cancer through the epigenetic regulation. Briefly, Snail harbors four zinc-finger motifs in its carboxy-terminal region, which bind with the E-box DNA sequence of target genes.²⁰ This event facilitates the recruitment of the polycomb repressive complex 2 (PRC2), which contains the methyltransferase enhancer of zeste homolog 2 (EZH2). The recruitment of PRC2 leads to DNA methylation as well as repressive histone modifications such as H3K9me2, H3K9me3, and H3K27me3 on the promoter of target genes, resulting in epigenetic silencing.²²² A variety of junction proteins are repressed through this mechanism, including E-cadherin, occludin, ZO-1, claudin-3, claudin-5, claudin-7, and so on.^{83, 223-226} Interestingly, PRC2 also induces active marks, such as H3K4me3.²²⁷ This mark might be associated with the MET program, or the increase of mesenchymal proteins including N-cadherin and MMP-9.^{228, 229} In addition to PRC2, Snail also recruits other epigenetic modifiers such as histone deacetylases (HDACs) and lysine-specific histone demethylase 1 (LSD1), thereby regulating EMT program.^230-232 For example, Snail has been found to form protein complex with HDAC1 and HDAC2 at the promoter of E-cadherin, leading to the deacetylation and consequent repression of E-cadherin, which results in the EMT and migration of breast cancer, pancreatic cancer, and nasopharyngeal cancer.^233-236 To date, the mechanisms underlying the preference of Snail to different epigenetic modifiers remain unclear.

The expression and function of Snail can be regulated at transcriptional level, posttranscriptional level, and posttranslational level. The transcription of Snail involves other transcription factors, such as NF-κB, YY1, and even Snail itself.^237-239 Particularly, several transcription factors capable of regulating Snail are the components of embryogenesis-related pathways, including YAP (component of Hippo pathway), Gli1 (Hedgehog), Smad (TGFβ), and many others.^240-242 This connection between EMT and embryonic development signaling will be discussed in the following section. The regulation of Snail at the posttranscriptional level is evidenced by N6-Methyladenosine (m6A) modification on its mRNA.²⁴³ Briefly, the methyltransferase-like 3 (METTL3) induces m6A modification in Snail CDS, which can be recognized by YTH domain-containing family protein 1 (YTHDF1).²⁴³ This event facilitates polysome-mediated translation of Snail, leading to EMT and metastasis of tumor cells.²⁴³ Besides, it has been reported that UDP-glucose enhances the stability of Snail mRNA, resulting in the overexpression of Snail and the metastasis of lung cancer.²⁴⁴ In terms of its posttranslational regulation, the most known mechanisms are phosphorylation and ubiquitination. For instance, GSK-3β-mediated phosphorylation of Snail at its first motif promotes its ubiquitination and subsequent degradation, whereas phosphorylation at its second motif leads to its cytoplasmic retention.²⁴⁵ In contrast, phosphorylation of Snail at Ser249 by the PAR-atypical protein kinase C (aPKC) inhibits the ubiquitination of Snail, thus promoting tumor metastasis.²⁴⁶ Besides, the ubiquitin-specific protease 3 (USP3) has been shown to stabilize Snail via deubiquitination.²⁴⁷ Interestingly, aforementioned transcription factor, NF-κB, also regulates the ubiquitination of Snail.²⁴⁸ Another member of Snail protein family, Slug, is also regulated by ubiquitination. It has been shown that several deubiquitinases, including USP5, USP10, USP20, counteract the ubiquitination of Slug therefore enhancing its protein stability.^249-251 Moreover, recent study showed that Slug can be SUMOylated through the interaction with Ubc9 and SUMO-1, which enhances the transcriptional repression activity of Slug and promotes the progression of lung cancer.²⁵²

4.2 Twist

Twist protein family consists of two members Twist1 and Twist2, which belong to the basic helix–loop–helix (bHLH) transcription factors.²⁵³ Based on their structural similarities and functional redundancy, we mainly discuss Twist1 here although the differences between them have been reviewed elsewhere.²⁵⁴ As one of important EMT-TFs, Twist1 has long been associated with cancer progression. For instance, knockout of Twist1 was shown to abrogate tumor metastasis in breast cancer.²⁵⁵ Similar to Snail, Twist1 represses the epithelial marks such as E-cadherin, α-catenin, γ-catenin and upregulates mesenchymal marks including vimentin, fibronectin, N-cadherin, leading to EMT and cancer progression.^{9, 256} Aforementioned epigenetic repressing complex, PRC2, can be also recruited by Twist1 to induce the H3K27me3 modification at Ink4A/Arf locus, thus preventing the senescence of mesenchymal stem cells.²⁵⁷ Besides, Twist1-mediated EMT program involves other epigenetic modifiers, such as the methyltransferase SET8, the PRC1 component BMI1, and the NuRD transcriptional repressive complex. For example, during breast cancer metastasis, Twist1 interacts with SET8 thus inducing a H4K20 monomethylation mark at the promoter of E-cadherin and N-cadherin, which downregulates and upregulates the transcription, respectively.²⁵⁸ Besides, Twist recruits BMI1 to repress the transcription of E-cadherin, and this effect is correlated with the poor prognosis of head and neck cancers.²⁵⁹ Furthermore, it has been reported that Twist1 can recruit NuRD complex, which contains HDAC1/2, to decrease the acetylation of H3K9 at Foxa1 promoter, thereby repressing the expression of Foxa1.²⁶⁰ This event contributes to the Twist1-induced metastasis of breast cancer, but less responsible for Twist1-induced EMT phenotype of breast cancer cells, which is interesting.²⁶⁰

Compelling evidence has suggested that Twist1 can be regulated at transcriptional level and posttranslational level. In neuroblastoma, both N-Myc and c-Myc proteins physically associate with the promoter of Twist1, thus activating its transcription.²⁶¹ Other transcription factors, such as Sox12 and Sox13, can also transcribe Twist1 thus leading to EMT and metastasis of HCC cells.^{262, 263} In contrast, the bHLH transcription factors BHLHE40 and BHLHE41 inhibit the transcription of Twist1.²⁶⁴ The posttranslational modifications on Twist1 include phosphorylation and ubiquitination. For example, PTEN with K27-linked polyubiquitination (PTEN^K27-polyUB) can dephosphorylate Twist1 at Ser123, resulting in the nuclear translocation of Twist1 and subsequent EMT phenotype.²⁶⁵ In terms of the ubiquitination, the E3 ligase Pirh2 is able to prime Twist1 for degradation.²⁶⁶ Interestingly, the RING-finger E3 ligase RNF8 promotes the K63-linked ubiquitination of Twist1 at K38, which enhances, but not decreases, the protein stability of Twist1, leading to its nuclear localization thereby playing critical roles in cancer drug resistance.²⁶⁷ It is worth noting that there is causal relationship between the phosphorylation and ubiquitination of Twist1, as evidenced by the facts that protein kinase Cα (PKCα)-mediated phosphorylation of Twist1 at Ser144 diminishes the ubiquitination of Twist1, whereas AKT1-mediated phosphorylation of Twist1 at Ser42, Tyr121, and Ser123 enhances its ubiquitination.^{268, 269} Sometimes, these posttranslational modifications regulate Twist1 activity through affecting protein–protein interaction. For instance, LYN-mediated phosphorylation of Twist1 leads to the dissociation between Twist1 and its cytoplasmic anchor G3BP2, resulting in the nuclear translocation of Twist1.^{270, 271} This event underlies the nature of Twsit1 as a mechanomediator in response to mechanical cues such as matrix stiffness.^{270, 271} Besides, the association between Twist1 and another binding partner TGIF1 is able to inhibit Twist1, whereas this inhibitory effect can be abolished by the phosphorylation of TGIF1 in pancreatic ductal adenocarcinoma.²⁷² Interestingly, Twist1 can bind with itself to form a homodimer, regulating fibroblast activation, cell migration, and embryonic development.^{273, 274}

4.3 ZEB

ZEB is potent EMT inducer, which represses epithelial marks including E-cadherin, ZO-1, claudin-1, desmoplakin, and is implicated in various type of cancer such as gastric cancer, leukemia, squamous cell carcinoma, liver cancer, colorectal cancer, and so on.^{82, 140, 150, 159, 275-278} There are two members in vertebrate ZEB protein family, namely ZEB1 and ZEB2, which share structural and functional similarities but are also distinguished by considerable differences. For example, a switch from ZEB2 to ZEB1 promotes the progression of melanoma, suggesting these two ZEB proteins function in an opposite manner under this condition.⁴ These diverse effects of ZEB1 and ZEB2 can be explained by different epigenetic modifiers recruited by them. ZEB1 binds p300 to activate transcription, whereas ZEB2 interacts with C-terminal-binding protein (CTBP) to silence the target genes.²⁷⁹ In addition, ZEB1 has been shown to recruit the SWI/SNF chromatin-remodeling protein BRG1, thereby repressing E-cadherin independently of CTBP.²⁸⁰ Moreover, ZEB1 can also recruit NuRD complex, which contains HDAC1/2, to promote the EMT and tumor progression in pancreatic cancer and lung cancer.^{281, 282} Similarly, the NuRD complex can also recruited by ZEB2, thus regulating the metastasis of breast cancer and the differentiation of neural cells.^{283, 284}

A variety of transcription factors have been found to activate ZEB1, such as Snail1, Twist, ETS1, and myocyte enhancer factor 2A (MEF2A).^{285, 286} In addition to this transcriptional regulation, ZEB1 is also regulated at the protein level, such as phosphorylation. For instance, ERK phosphorylates ZEB1 at Thr867, which inhibits its nuclear translocation, DNA binding, and function of transcriptional repression.²⁸⁷ This ERK–ZEB1 axis is involved in the progression of various tumors, including lung cancer, breast cancer, liver cancer, prostate cancer, ovarian cancer, and glioblastoma.^288-293 Interestingly, ERK also activates ZEB1 through upregulating aforementioned transcription factor ETS1, suggesting that ERK can regulate ZEB1 in either direct or indirect manner.²⁹⁴ Moreover, ZEB1 has been also reported to be phosphorylated by ATM at S585, which enhances its protein stability in breast cancer cells.²⁹⁵ Similar to other EMT-TFs, ZEB1 can also be regulated by ubiquitination. For example, the E3 ligase tripartite motif-containing 26 (TRIM26) downregulates ZEB1 through ubiquitination-mediated protein degradation, whereas USP39 acts as a deubiquitinase to stabilize ZEB1.²⁹⁶ Thus, TRIM26 and USP39 function in an antagonistic manner to coordinate the fate of liver cancer.²⁹⁶ Besides, the ubiquitination of ZEB1 involves additional E3 ubiquitin ligases such as checkpoint with Forkhead and ring finger domains (CHFR), F-box only protein 45 (FBXO45), and deubiquitinases including USP51 and USP43.^297-300 As to ZEB2, the regulation of protein stability is associated with E3 ubiquitin ligases FBXW7 and TRIM14.^{301, 302} These observations indicate that the regulation of ZEB proteins is rather complex in tumor progression.

4.4 Novel EMT-regulating transcription factors

As a rapidly evolving field, more transcription factors have been identified as novel regulators of EMT, such as PRRX1 and Sox. PRRX1 cooperates with Twist1 to induce EMT during embryogenesis and tumor invasion, but overexpression of PRRX1 is associated with a favorable prognosis in breast cancer patients.⁹⁹ Mechanistically, loss of PRRX1 induces MET, which is required for metastatic colonization of breast cancer cells.⁹⁹ Moreover, PRRX1 loss-mediated MET also confers cancer cells with stemness, leading to drug resistance thus further explaining why PRRX1 overexpression predicts good prognosis.^{99, 303} However, opposite finding showed that upregulated PRRX1 induces EMT, cancer stemness, metastasis, and poor prognosis in colorectal cancer, suggesting the role of PRRX1 is highly context-dependent in different tumor types.¹⁰⁰ This contradiction might be attributed to the diverse functions of PRRX1 isoforms, namely PRRX1b that activates EMT, whereas PRRX1a that activates MET, and this isoform switching underlies tumor invasion and metastatic colonization in pancreatic cancer.³⁰⁴ Sox protein family consists of over 20 members in vertebrates, many of which are involved in tumor initiation and progression.³⁰⁵ Numerous studies indicated that Sox proteins regulate EMT via classical EMT-TFs. For instance, Sox13 transcriptionally activates Twist, thereby promoting EMT in liver cancer.²⁶³ Interestingly, several Sox proteins have been shown to modulate EMT through either direct or indirect way. Sox4 binds with the promoter of Slug to activate its transcription, leading to EMT in uterine carcinosarcoma.³⁰⁶ Alternatively, Sox4 can also directly transcribe N-cadherin, thus inducing EMT independent of classical EMT-TFs.¹⁰¹ Another Sox protein, Sox9, can directly bind with the promoters of claudin-1 and ZEB1 to modulate their transcription, suggesting both direct and indirect regulation of EMT by Sox9.¹⁰²

5.1 TGFβ

TGFβ signaling is activated by the interaction between TGFβ ligands and receptors (type I and type II) on the cell membrane. Next, type I receptor is phosphorylated by type II receptor, which then phosphorylates Smad proteins, the key transcription factors mediating the biological consequence of this signaling. There are eight members in Smad protein family with different roles. Briefly, Smad1/2/3/4/5/8 positively regulate TGFβ signaling through the formation of activated Smad complexes, which translocate into nucleus to initiate gene transcription. In contrast, Smad6/7 negatively regulate TGFβ signaling via preventing the formation of activated Smad complexes and facilitating the degradation of TGFβ receptors.³¹⁵ In the context of neoplasm, although TGFβ signaling can be both oncogenic and tumor suppressive, a variety of tumors benefit from activated TGFβ signaling, and targeting TGFβ signaling can be a promising therapeutic strategy for cancer treatment.^{316, 317} One of the mechanisms is that TGFβ signaling regulates EMT program of tumor cells, thus promoting cancer progression (Figure 4). For example, TGFβ–Smad signaling has been shown to activate the expression of Snail1, leading to EMT and proliferation of lung cancer cells.²⁴ Not surprisingly, Smad proteins play central roles in TGFβ-mediated EMT program. Indeed, Smads physically associate with EMT-TFs such as Snail, Twist, ZEB to form an EMT-promoting Smad complex (EPSC; e.g., Snail1–Smad3/4 complex), which represses the transcription of epithelial marks while increasing the expression of mesenchymal marks.^{224, 318} Besides, TGFβ induces long noncoding RNA (lncRNA)-ATB, which acts as a competing endogenous RNA (ceRNA) to competitively bind with miR-200s. This effect increases the expression of ZEB1/2, leading to EMT and metastasis of liver cancer.³¹⁹ In contrast, several tumor suppressors exhibit their antimetastatic function through inhibiting TGFβ signaling, including circular RNA circPTK2 and lncRNA SMASR.^{320, 321} Interestingly, TGFβ signaling has been also demonstrated tumor-suppressive via a lethal EMT.³²² This observation is in line with the context-dependent roles of TGFβ signaling.

5.2 Wnt

Wnt signaling exert its biological effects mainly through the control of transcription cofactor β-catenin. Briefly, in the quiescent state, β-catenin is ubiquitinated and inhibited by GSK-3β complex. When activated, Wnt proteins bind with the Frizzled receptor, leading to the recruitment of coreceptor LRP5/6. This event leads to the activation of Dishevelled (Dvl), which inhibits GSK-3β thus protecting β-catenin from degradation. Then, stabilized β-catenin translocates into nucleus, where it interacts with transcription factor LEF/TCF to activate transcription.^323-325 In terms of oncology, Wnt signaling is overall oncogenic, which allows malignant proliferation and metastasis of cancer cells, and this process involves the induction of EMT program³²⁶ (Figure 5). For instance, Her2 positive early disseminated cancer cells can enter into a partial EMT state via activation of Wnt signaling, thereby initiating metastasis in breast cancer.³²⁷ Mechanistically, the EMT-promoting function of Wnt pathway is largely attributed to β-catenin/LEF/TCF-mediated transcription control. This is supported by the fact that β-catenin/TCF4 can bind to the promoter of ZEB1 and increase its transcription, leading to the EMT and metastasis of colorectal cancer.³²⁸ Similarly, β-catenin/TCF3 and β-catenin/LEF1 interact with and activate the promoter of Snail and Twist, respectively.^329-331 As a consequent, Snail can physically associated with β-catenin to form a transcription complex, which activates Wnt target genes independent of TCFs.³³² In contrast, autophagic degradation of β-catenin has been shown to inhibit EMT and abrogate the metastasis of colorectal cancer.³³³ Interestingly, Wnt signaling can also promote EMT through a noncanonical way independent of β-catenin. For example, the Wnt receptor Frizzled2 drives EMT and tumor metastasis in liver cancer via Fyn and Stat3, and this process is not affected by pharmacological inhibition of β-catenin.²⁷ In addition, EMT-TFs can in turn activate Wnt signaling, forming a positive feedback loop to proceed EMT program. It has been shown that ZEB1 can repress the expression of miR200A, leading to the reactivation of β-catenin.^{334, 335} Besides, Twist binds with the promoter of Wnt5a to increase its transcription, which activates Wnt signaling thus promoting breast cancer metastasis.³³⁶

5.3 Hedgehog

Hedgehog signaling is activated by the binding of hedgehog ligands with the receptor Patched (PTCH1). This binding leads to the activation of membrane GPCR-like protein Smoothened (SMO). In the absence of activated SMO, the transcription factor complex Gli1/2/3 is processed by proteasome, during which the active Gli1/2 is degraded, whereas the repressive Gli3 is preserved, resulting in transcriptional repression. When SMO is activated in response to Hedgehog signals, Gli1/2/3 is differentially processed to yield an active Gli1/2, which translocates into nucleus to initiate transcription.^{337, 338} Similar to other developmental pathways, Hedgehog signaling is tightly correlated with EMT program (Figure 6). For instance, aberrant activation of Hedgehog signaling promotes EMT of immature ductular cells, the accumulation of which results in fibrosis, cirrhosis and other liver diseases.^{29, 339, 340} In the field of oncology, Hedgehog signaling has long been implicated in cancer progression, at least partially due to its critical role in the regulation of EMT program of cancer cells.^341-343 In human cholangiocarcinoma tissues, Hedgehog ligand is highly expressed to repress E-cadherin, leading to a EMT phenotype and elevated viability of tumor cells.³⁴⁴ Besides, Hedgehog signaling-mediated EMT, characterized by the overexpression of vimentin, Snail, N-cadherin, and repression of E-cadherin, ZO-1, has been also reported in the metastasis of bladder cancer and breast cancer.^{345, 346} Inhibition of Hedgehog signaling with the administration of Vismodegib, the antagonist for PTCH1, is able to suppress EMT and cell proliferation of castration-resistant prostate cancer.³⁴⁷ Compelling evidence suggest that Hedgehog signaling promotes EMT via the key transcription factor Gli. Indeed, the EMT-TF Snail is a transcriptional target of Gli1.²⁴¹ Stabilization of Gli1, which is mediated by the deubiquitinase USP37, has been shown to activate EMT and increase invasiveness in breast cancer.³⁴⁸ Interestingly, USP37 also catalyzes the deubiquitination of Snail.³⁴⁹ Moreover, both Gli and Snail can be ubiquitinated by E3 ligase β-TrCP.^{245, 350} Therefore, EMT program and Hedgehog signaling are connected via sharing protein turnover machinery. In turn, loss of E-cadherin or activation of EMT-TFs can activate Gli, thus probably forming a positive signaling circuit to sustain EMT phenotype.^{351, 352} Briefly, EMT-TFs activate Six1, which stimulate Hedgehog signaling in neighboring tumor cells, but how E-cadherin deficiency activates Gli remains elusive.^{351, 352} Although most literatures indicate that Hedgehog signaling positively regulates the EMT program and tumor progression, the contrary findings were also reported that describe that Gli1 binds with the promoter of E-cadherin to activate its transcription, thereby inhibiting EMT.³⁵³ Besides, downregulation of Gli1 results in the disassembly of adherens junctions, leading to EMT and cell migration in pancreatic ductal adenocarcinoma.³⁵³ This observation suggests a context-dependent role of Hedgehog signaling in EMT and cancer progression.

5.4 Hippo

The core of Hippo signaling is a kinase cascade that negatively regulates the activity of transcription cofactors YAP/TAZ. Given the oncogenic nature of YAP/TAZ, the Hippo signaling is generally regarded as a tumor-suppressive pathway. Briefly, active Hippo signaling is characterized by phosphorylation of MST1/2–SAV1 complex, which phosphorylates LATS1/2–MOB1 complex. Then, phosphorylated LATS1/2 induce the phosphorylation of YAP/TAZ, leading to their cytoplasmic retention or ubiquitin-mediated degradation. When Hippo signaling is inactivated, dephosphorylated YAP/TAZ can translocate into nucleus, where they interact with transcription factors TEAD1/2/3/4 to initiate transcription.^{354, 355} YAP/TAZ have been demonstrated potent EMT inducers promoting the progression of a variety of cancer (Figure 7). For instance, YAP is able to promote EMT and tumor metastasis by downregulating E-cadherin and remodeling cytoskeleton in renal cancer and nasopharyngeal carcinoma.^{356, 357} Mechanistically, YAP interacts with several EMT-TFs, including ZEB1, Snail, and Slug to form a transcriptional complex. This event results in a functional switch of EMT-TFs from transcriptional repressors to transcriptional activators, which activates tumor-promoting genes involved in EMT, tissue regeneration, and cancer metastasis.^{216, 358-361} During EMT, the activation of YAP can be attributed to multiple mechanisms. For example, the catalytic subunit of protein phosphatase 2A (PP2Ac)-mediated dephosphorylation of YAP facilitates YAP nuclear translocation, leading to EMT and metastasis of HCC.³⁶² Besides, excessive formation of filamentous actin (F-actin) leads to the dephosphorylation of LATS1, resulting in YAP stabilization and consequent liver cancer metastasis.³⁰ Interestingly, YAP can either positively or negatively regulate the formation of F-actin, suggesting this process is highly context-dependent.^{363, 364} Besides, the activity of YAP can also be regulated at the RNA level. N6-methyladenosine (m6A) modification on the YAP mRNA can be recognized by YTHDF1 and YTHDF2, which facilitates the translation and decay of YAP mRNA, respectively.³⁶⁵ Moreover, Hippo signaling can be cross-linked with other developmental pathways, such as TGFβ and Wnt, to coordinate EMT program. It has been shown that YAP can prevent GSK3β-mediated Smad3 degradation, thereby promoting Smad3-induced EMT.³⁶⁶ Furthermore, YAP is physically associated with β-catenin to form a transcriptional complex with TEAD4 in nucleus, leading to the overexpression of EMT-TFs including Slug and Twist in breast cancer.³⁶⁷ It is worth noting that TEAD4 can regulate EMT and promote metastasis by directly transcribing vimentin without the binding with YAP in colorectal cancer, which is interesting.³⁶⁸

6.1 Redox regulation of cell junctions and cytoskeleton

ROS increase endothelial permeability through disrupting the integrity of cellular junctions between endothelial cells. For instance, ROS induced by the metabolic intermediate, 4-hydroxy-2-nonenal, has been shown to modulate adherens junctions, tight junctions and integrins, leading to dysfunction of endothelial barrier.⁴⁰⁴ In line with it, another study showed that protein tyrosine phosphatase SHP2 is a critical factor preserving the integrity of endothelial barrier.⁴⁰⁵ In response to lipopolysaccharide (LPS)-induced oxidative stress, SHP2 is oxidized at its Cys459, leading to its inactivation.⁴⁰⁵ This event activates Fyn-related kinase, which phosphorylates several junction proteins, resulting in the disruption of endothelial adherens junction.⁴⁰⁵ Besides, ROS-mediated endothelial permeability also involves the S-glutathionylation of Rac1, a small Rho GTPase that regulates cytoskeleton.⁴⁰⁶ Metabolic stress-induced S-glutathionylation of Rac1 at Cys81 and Cys157 can inactivate Rac1, leading to the reorganization of cytoskeleton network and consequent vascular permeability in the aorta.⁴⁰⁶ Interestingly, β-actin can be oxidized at Cys374, suggesting a direct regulation of cytoskeleton by redox signaling.⁴⁰⁷ In intestinal epithelial cells, the protein level of E-cadherin can be decreased in response to TNF-α-induced oxidative stress, while its mRNA level is not changed.⁴⁰⁸ This observation suggests that ROS-mediated loss of E-cadherin might be at least partially independent of conventional epigenetic mechanisms by EMT-TFs.⁴⁰⁸ In addition to E-cadherin, many other EMT marks have been shown to be regulated by oxidative stress, including claudins, occludin, zonula occludens, α-SMA, and vimentin.^{409, 410}

6.2 Regulation of EMT-TFs by redox sensors

As mentioned above, EMT-TFs play crucial roles in EMT program. Though most EMT-TFs do not seem to undergo direct redox modification, they are actually regulated by other redox sensors. The transcription factor, activator protein-1 (AP-1), is a typical redox sensor involved in EMT program. In response to oxidative stress, cysteines between its subunits are oxidized to form intermolecular disulfide bond, which decreases its DNA binding affinity.^411-413 AP-1 has been shown to directly bind with the promoter of Snail and ZEB2, thus activating their transcription, leading to EMT and metastasis of skin cancer, cervical cancer, and breast cancer.^414-417 Besides, AP-1 can also physically interact with Snail and Twist, forming a transcriptional complex to induce EMT.^{418, 419} Another redox sensor, prolyl hydroxylase (PHD), can be inactivated by the oxidation of cysteines in its catalytic domain.⁴²⁰ This event leads to the activation of HIF-1α, which transcribes ZEB1 and Twist to induce EMT and promote tumor metastasis.^421-423 HIF-1α can also improve the protein stability of Snail, thereby promoting cancer metastasis.⁴²⁴ In addition, the redox sensor IKKγ (also known as NEMO) can be activated via ROS-induced disulfide bond formation between Cys54 and Cys347.⁴²⁵ Activated IKKγ phosphorylates IκB, leading to the dissociation between IκB and NF-κB. This effect results in the activation of NF-κB, which promotes the transcription of several EMT-TFs.⁴²⁶ Moreover, FOX, class O (FOXO) proteins are a group of redox sensitive transcription factors, which play vital roles in cellular antioxidant defense.^{378, 427} In response to ROS, FOXO4 and transportin-1 can form protein complex through intermolecular disulfide bond, promoting the nuclear translocation of FOXO4, which is required for the transcription of SOD2 and subsequent ROS elimination.⁴²⁸ Simultaneously, FOXO proteins are largely involved in cancer progression, notably the EMT program.⁴²⁹ For example, hypoxia-induced oxidative stress promotes the phosphorylation and activation of FOXO1, which directly binds with the promoter of Twist to increase its transcription, leading to EMT and metastasis of prostate cancer.⁴³⁰ Besides, FOXO1 can also upregulate ZEB1 during EMT process.⁴³¹ Another FOXO family member, FOXO3a, has been shown to inhibit β-catenin via upregulating miR-34 or directly protein binding.⁴³² This effect abolishes the β-catenin-mediated transcription of ZEB1, thus repressing EMT in prostate cancer.⁴³² Interestingly, FOXO proteins might regulate EMT independent of classical EMT-TFs. For instance, FOXO1 mRNA serves as a competitive endogenous RNA (ceRNA), whose 3′UTR region can be targeted by miR-9.⁴³³ This event protects E-cadherin mRNA from miR-9-mediated degradation, resulting in maintenance of E-cadherin expression thus inhibiting breast cancer metastasis.⁴³³

7.1 Metabolic reprogramming

Cancer cells undergo metabolic reprogramming to meet the energy and nutrient demand, which enables the sustaining proliferation in adverse condition. One of the well-known metabolic reprograming is the “Warburg effect” describing that tumor cells utilize glycolysis rather than oxidative phosphorylation even in a normoxia condition, termed as “aerobic glycolysis.”⁴³⁵ During EMT process, tumor cells reprogram their glucose metabolism and lipid metabolism to maintain aggressive behaviors. Specifically, the key EMT-TF Snail can regulate the transcription of enzymes in glucose metabolism. For instance, Snail represses the expression of phosphofructokinase PFKP, thus switching the glucose flux toward pentose phosphate pathway.⁴³⁶ Besides, Snail-mediated silencing of fructose-1,6-biphosphatase induces glycolysis, which maintains stemness and invasiveness of basal-like breast cancer cells.⁴³⁷ However, it has been also reported that metastatic cancer cells prefer oxidative phosphorylation to glycolysis, which might be due to the decease of glucose uptake and consequent energy crisis in CTCs.⁴³⁸ Therefore, tumor cells probably tend to utilize oxidative phosphorylation to achieve more efficient ATP production rather than glycolysis during EMT.⁴³⁹ This concept is supported by the finding that nuclear phosphoglycerate kinase 1 drives EMT and metastasis by oxidative phosphorylation in Smad4-deficient pancreatic ductal adenocarcinoma cells.⁴⁴⁰ Notably, aberrant glucose metabolism in turn affects EMT through regulating EMT-TFs, such as Snail. It has been shown that hyperglycemic condition induces an O-GlcNAc modification at Ser112 on Snail1, thereby enhancing its protein stability to facilitate EMT program in breast cancer cells.⁴⁴¹

Lipid metabolism is closely related with cancer metastasis, as evidenced by the finding that the metabolic shift toward fatty acid oxidation is critical for tumor metastasis to lymph nodes.⁴⁴² Besides, the formation of lipid droplets has been found to promote metastasis of tumor cells.⁴⁴³ Given the direct connection between EMT and metastasis, it is not surprising that lipid metabolism plays vital roles during EMT program. Indeed, EMT-like morphological changes require the alterations in membrane lipid composition, which is enabled at least partially through the fatty acid translocase CD36.^{444, 445} In addition, the formation of invadopodia, a classical EMT-like morphological alteration, is induced by the reprogramming of lipid metabolism during the progression of liver cancer.⁴⁴⁶ Apart from regulation of membrane dynamics, lipid metabolism also regulates the fate of EMT cells through ferroptosis, a cell death form induced by lipid peroxidation. Cancer cells in completed mesenchymal state that lack of E-cadherin expression largely suffer from severe oxidative stress, making them highly vulnerable to ferroptosis.¹¹⁸ Nevertheless, lymph protects those metastatic cancer cells from ferroptosis by reliving oxidative stress, which is dependent on the abundant oleic acid, a kind of unsaturated fatty acid.³⁷³ This evidence suggests that metabolism with different lipid substrates can diversely regulate ferroptosis, cell survival, and metastasis during EMT. Furthermore, different lipid metabolism enzymes also regulate metastasis in contrary ways. For example, the lipid metabolism enzyme ATP-citrate lyase can promote the metastasis of colorectal cancer and liver cancer.^{447, 448} In contrast, another lipid metabolism enzyme, acyl-CoA synthetase long-chain family member 4, sensitizes cancer cells to ferroptosis by esterifying arachidonic acid and adrenic acid into phosphatidylethanolamine.⁴⁴⁹ According to the profound impacts of lipid metabolism in EMT and metastasis of cancer, targeting lipid metabolism can be a potential therapeutic strategy for cancer treatment. For instance, inhibiting lipid metabolism by simvastatin-based nanomedicine has been shown to reverse EMT and overcome cancer drug resistance.⁴⁵⁰

7.2 Cancer stem cell property

The capacity of tumor initiation is partly driven by the activation of the EMT program because active EMT-TFs confer numerous properties of stemness on those with cell plasticity. Indeed, aberrant expression of EMT-TFs, such as Twist1, SIP-1, and Snail, in cancer cells contributes to the acquisition of stem-cell features, as evidenced by the increased self-renewal ability in vitro and tumor propagation potential in vivo.^{42, 437, 451, 452} Moreover, ZEB1, as an EMT activator, has been identified to enhance tumorigenic and colonization capacity by stabilizing the stem cell marker Sox2 in a miR-200c-mediated manner in pancreatic cancer derived from a KPC model with mutant Kras and p53.¹⁷ Similar results have been demonstrated in glioblastoma, where ZEB1 contributes to tumor initiation by orchestrating a series of stemness effectors, including SOX2, OLIG2, and CD133/PROM1.⁴⁵² Additionally, the results from breast cancer indicate that the EMT regulators ZEB1/2 and Bmi1 promote stemness toward tumorigenesis and metastasis via TET-Family-dependent epigenetic modulation. Mechanistically, miR-22 induces chromatin remodeling by decreasing 5hmC levels by inhibiting the TET family, therefore leading to epigenetic silencing of miR-200, which is involved in the expression of ZEB1/2 and Bmi1.⁴⁵³ Recently, a novel signaling axis, the basic helix–loop–helix (bHLH) transcription factor E2A with Snail1, was confirmed to participate in the maintenance of breast cancer stemness, facilitating tumor initiation, metastasis, and drug resistance.⁴⁵⁴ It has also been reported that deletion of the protocadherin FAT1 induces a hybrid EMT state in skin squamous cell carcinoma and lung tumors by triggering not only CAMK2–CD44–SRC axis-mediated YAP1 activation and ZEB1 expression but also EZH2-regulated SOX2 expression, thus resulting in maximal stemness and accelerated cancer progression.¹¹⁴

In addition to the involvement of tumorigenesis, EMT-mediated stemness is also identified as a key driver of acquired drug resistance and subsequent tumor relapse after drug holidays.³⁵ Accumulated evidence reveals that cancer cells in EMT activation seem to undergo dedifferentiation similar to stem cells, showing upregulation of antiapoptotic and drug efflux proteins.^{455, 456} For instance, multidrug resistance (MDR), especially to gemcitabine, in pancreatic cancer is closely correlated with the activation of the EMT-like transcription factor ZEB1.⁴⁵⁷ In detail, solute carrier family 39 member 4, which functions to modulate the intracellular zinc concentration, induces the expression of the zinc-dependent EMT protein ZEB1 by stimulating STAT3 activation. Consequently, ZEB1-induced ITG A3/B1 triggers integrin α3β1 signaling and subsequent c-Jun-N-terminal kinase signaling, therefore inhibiting the expression of gemcitabine transporter ENT1.⁴⁵⁷ Similarly, SRF/MRTF-mediated signaling is involved in the mesenchymal transition of melanoma with RAC1P29S by upregulation of the EMT-TFs Snai2 and Jun, driving a low apoptosis rate and BRAFi resistance.⁴⁵⁸

Of note, various cancer therapies, including chemo-, radio-, and immunotherapy, can also result in an EMT-mediated stress adoption mechanism, which was recently defined as one feature of the drug-tolerant persister (DTP) state.⁴⁵⁹ These residual cells with EMT characteristics govern the nongenetic mechanisms to enter the dormant state against a wide spectrum of antiproliferation treatments.⁴⁶⁰ For example, Matthew and colleagues indicate that high-mesenchymal persister cancer cells derived from drug treatments hold more resistance to tyrosine kinase inhibitor therapy and chemotherapy.⁴⁶¹ Furthermore, the features of EMT activation, upregulation of vimentin, and downregulation of E-cadherin have also been examined in MDR persister cells, which develop DTP phenotypes after canonical MDR reversal treatment.⁴⁶² Notably, observations in patients with breast cancer or mice with lung cancer suggest a strong underlying connection among EMT, stemness, and drug resistance by analyzing the expression of EMT markers in posttherapy specimens, showing an activated EMT program and elevated chemoresistance-related proteins.^{115, 463}

7.3 Tumor-promoting inflammation

The progression of cancer is largely dependent on the interaction between tumors and tumor microenvironment, especially local inflammatory status. Inflammatory microenvironment is composed by a variety of cells, including tumor cells, stromal cells, tumor-associated macrophages (TAM), cancer-associated fibroblasts, myeloid-derived suppressive cells, T cells, monocytes, dendritic cells, adipocytes, and so on. Inflammatory response is required for immunosurveillance to kill cancer cells; however, it can also promote cancer progression. For example, macrophages can be polarized into M2 tumor-promoting ones. During cancer progression, EMT program can regulate the function of inflammatory and immune cells, whereas several inflammatory cytokines are potent inducers of EMT.⁴⁵ As such, EMT program and inflammatory response are affected by each other thus forming complex network, which leads to cancer regression or progression.

Mounting evidence indicate that EMT can be regulated by inflammation. For instance, pancreatitis has been found to promote EMT of premalignant pancreatic epithelial cells, leading to their dissemination to liver, and this process can be abolished by the administration of immunosuppressive agent dexamethasone.⁴⁶⁴ Generally, inflammation-induced EMT is largely attributed to proinflammatory cytokines, such as NF-κB, interleukins (ILs), interferons, chemokines, and others. The level of IL-22 is increased during pancreatitis, which promotes EMT of pancreatic ductal adenocarcinoma cells and subsequent tumor progression.⁴⁶⁵ Another IL, IL-6, facilitates the transformation of normal liver stem cells into metastatic CSCs by inducing EMT.⁴⁶⁶ Interestingly, IL-35 derived from M2 TAMs has been reported to promote MET of metastatic tumor cells, thus resulting in distant colonization.⁴⁶⁷ Similar tumor-promoting effects were also observed in other proinflammatory factors, such as M2 TAM-derived chemokine CCL-22 and natural killer cell-derived interferon IFN-γ, both of which promote the progression of liver cancer via induing EMT.^{468, 469} Mechanistically, these cytokines might regulate the expression of EMT-TFs, thus affecting EMT program. For instance, the pro-inflammatory cytokine NF-κB is able to inhibit the ubiquitination and degradation of Snail, thereby mediating inflammation-induced tumor metastasis.²⁴⁸

In contrast to the large amount of literature describing inflammation-regulated EMT, the investigation of EMT-regulated inflammation and immune response is rather limited. It has been shown that EMT can change the antigens present in tumor surface, recruit M2 TAMs instead of M1 macrophages, thus contributing to the immunosuppression in breast cancer, and this process is probably enabled by the release of cytokine granulocyte-macrophage colony-stimulating factor.^{470, 471} Besides, the EMT-TF ZEB2 can regulate cytokine signaling thus playing critical role in hematopoietic differentiation and myeloid diseases.²⁷⁶ Interestingly, tumor cells undergoing EMT have been reported to increase susceptibility to natural killer cells, which enhance immunosurveillance to inhibiting metastasis in lung cancer.⁴⁷² This observation suggests that inflammatory or immune responses are not always tumor promoting, but can provide beneficial for cancer patients in some circumstances if not being hijacked by tumor cells.

8.1 Clinical diagnosis by identifying EMT markers

Given the crucial role of the EMT program in tumorigenesis and development, the identification of EMT-involved biomarkers has accordingly emerged as a diagnostic approach, which is used to match personalized therapies or adjust the treatment intensity. Indeed, an increasing number of clinical cases have indicated that EMT multi-marker combination analysis by immunohistochemistry or immunofluorescence is a key part of tumor stratification or companion diagnostics for patients with different tumor types.^{473, 474} (Table 1)

In colorectal cancer, miR-200c, a known EMT mediator that has been investigated in preclinical models, is examined in 45 pairs of primary tumor tissues and corresponding matched liver metastases. Expectedly, altered expression of miR-200c, which has low expression in the invasive front of primary cancer tissues and high expression in liver metastasis tissues, showed a strong association with various EMT-related proteins, such as ZEB1, E-cadherin, and vimentin, suggesting a potential metastatic indicator for patients.⁴⁷⁵ In line with this, the results from a longitudinal follow-up study based on 185 stage II/III colorectal cancer patients solidly demonstrated a relationship between a high combined EMT score and poor prognosis.⁴⁷⁶ Furthermore, several EMT genes, including CDH11, MMP2, and ZEB1, have been identified with higher expression in invasive breast cancer than in pure ductal carcinoma in situ, indicating that EMT activation is related to a high risk of invasive disease in all subtypes of breast cancer, especially in ER-negative disease.⁴⁷⁷ A study investigating prostate cancer patients treated pre- and postradiotherapy revealed that downregulation of E-cadherin combined with upregulation of N-cadherin and vimentin was correlated with biochemical recurrence, providing an independent predictive factor for treatment response to radiotherapy.⁴⁷⁸ Recently, Navas and colleagues proposed a validated, high-resolution digital microscopic immunofluorescence assay for assessing the EMT phenotype of various human tumor specimens. Their study revealed that β-catenin+ cancer cells accompanied by the induction of E-cadherin and vimentin exist in various metastatic patients. Notably, the expression of stemness markers, such as CD133, CD44v6, ALDH1, and NANOG, is also examined in some cases, implying a standardized assessment for clinical monitoring of EMT-mediated therapy adaptation.⁴⁷⁴

In addition to tissue biopsy, liquid biopsies are another promising method for clinical diagnosis and can also be used to analyze EMT characteristics of tumors, mainly CTCs from the blood of cancer patients. For example, a follow-up study comprising 226 blood samples of 39 patients with metastatic breast cancer showed that the expression rates of EMT-related proteins and the stemness marker ALDH1 were 62 and 44%, respectively, in CTCs of patients with no response to therapy. In contrast, the expression rates in responders are 10 and 5%, respectively.⁴⁷⁹ Likewise, through investigating related protein expression in CTCs isolated from blood, Ning et al. demonstrated that the coexpression pattern of EMT markers (PI3Kα and/or Akt-2) and stemness markers (ALDH1) predicts significantly shortened progression-free survival and overall survival (OS) in metastatic colorectal cancer patients.⁴⁸⁰ To date, the clinical trials of CTCs with EMT features are ongoing or have already been activated for diagnosing or treating patients with breast, prostate, pancreatic, and colorectal cancers (NCT04265274, NCT04021394, NCT04323917, and NCT04323813, respectively).

8.2 Therapeutic interventions targeting the EMT program

With the increasing in-depth knowledge of detailed mechanisms modulating the EMT program, the identification of novel agents targeting this program has been an irreversible trend. Thus far, three reasonable strategies aimed at preventing the initiation of EMT activation, selectively eliminating mesenchymal-like cancer cells, and reversing the EMT process by inducing the MET program all seem to be promising for controlling EMT-mediated tumorigenesis and therapeutic tolerance. However, which approach is the best one with long-term efficacy in the clinic remains controversial. In this section, we present various EMT-targeted drugs based on the approaches mentioned above and list the related clinical trials (Table 2).