The concept of epithelial–mesenchymal plasticity (EMP), which describes the dynamic flux within the spectrum of phenotypic states that invasive carcinoma cells may reside, is being increasingly recognised for its role in cancer progression and therapy resistance. The myriad of events that are able to induce EMP, as well as the more recently characterised control loops, results in dynamic transitions of cancerous epithelial cells to more mesenchymal-like phenotypes through an epithelial–mesenchymal transition (EMT), as well as the reverse transition from mesenchymal phenotypes to an epithelial one. The significance of EMP, in its ability to drive local invasion, generate cancer stem cells and facilitate metastasis by the dissemination of circulating tumour cells (CTCs), highlights its importance as a targetable programme to combat cancer morbidity and mortality. The focus of this review is to consolidate the existing knowledge on the strategies currently in development to combat cancer progression via inhibition of specific facets of EMP. The prevalence of relapse due to therapy resistance and metastatic propensity that EMP endows should be considered when designing therapy regimes, and such therapies should synergise with existing chemotherapeutics to benefit efficacy. To further improve upon EMP-targeted therapies, it is imperative to devise monitoring strategies to assess the impact of such treatments on EMP-related phenomenon such as CTC burden, chemosensitivity/-resistance and micrometastasis in patients.

Introduction

Epithelial–mesenchymal plasticity in cancer

Worldwide, more than 90% of cancer deaths are attributed to the invasive spread of metastasis [1,2]. The cascade of events that drive cellular invasion and cancer dissemination and lead to tumour progression and metastatic deposition has many properties in common with the biological programme of epithelial–mesenchymal plasticity (EMP) [36]. The spectrum of EMP, often separated into the forward process of epithelial–mesenchymal transition (EMT) and the reverse process, mesenchymal–epithelial transition (MET), is a well-recognised phenomenon, first described in the developmental biology of the chick primitive streak [7]. During embryonic development, established epithelial cells transiently adopt a more motile mesenchymal phenotype in order to translocate to a new tissue site, whereby the reverse transition occurs to allow epithelial function [810]. Although initially segregated into the directional processed of EMT and MET, increasingly we have seen recognition of the dynamic flux of EMP in embryonic/developmental events [11]. As well as its requirement during development for embryo implantation and early organ development, EMP is also necessary for tissue remodelling, organ fibrosis and wound healing where the breakdown of epithelial homeostasis is required to allow new tissue formation [12,13]. This evolutionarily conserved, complex biological process is also corrupted in the patho-physiological mechanisms of cancer progression, and its therapeutic manipulation will hence be the subject of this review.

The classical view of cell polarisation as either epithelial or mesenchymal by their hallmark phenotypes has perhaps delayed the concept of EMP being widely embraced within the cancer biology field as a direct contributor to metastatic progression [14]. As with any stochastic cellular process, the subtle occurrence of EMP and its rate of switching within the tumour microenvironment make it difficult to follow directly in vivo. Therefore, much of the work defining the regulation of EMP has been performed in cultured cells. It was not until more recently that in vivo lineage labelling experiments and circulating tumour cell (CTC) detection enabled a better grasp on clinically/biologically relevant EMP phenotypes and introduced the concept of hybrid ‘metastable’ EMP states that are neither fully epithelial nor fully mesenchymal [1519]. A hybrid EMP state is seen in carcinoma model systems, in which individual cells co-express a marker of both epithelial and mesenchymal lineages [20,21], and CTCs have been shown to exhibit a spectrum of EMP states ([2225]; reviewed in [26]). For this reason, we believe EMP to be a more suitable definition for purposes of therapeutic targeting, despite much of the referenced literature addressing either EMT or MET independently. Thus, the term ‘EMP’ has been chosen in this review to reflect the bidirectional axis in which cells with EMP phenotypes discussed below exist [5,20,2729].

The cellular and phenotypic hallmarks that define EMP are a tied to the dynamic alterations that occur as carcinoma cells lose their epithelial associations and acquire more mesenchymal properties, allowing them to migrate from the primary site. These cells may then exist in a hybrid state as they migrate or re-adopt epithelial traits through a reverse, MET process. Such processes are broadly marked by the regulation of EMP transcription factors (TFs; e.g. SNAI1/2, ZEB1/2 and Twist 1/2/3), which act to control expression of epithelial adhesion molecule E-cadherin and activate a host of biological programmes that lead to cell remodelling and migration. The processes by which MET is regulated are still relatively undefined as much work to date has been performed on drivers of the forward EMT process.

Indirect clinical evidence of the EMP programme in tumour progression is prevalent, and mechanisms that regulate EMP phenotypes including migration and invasion, ultimately leading to metastatic dissemination, have been widely studied [12,16,3032]. Both EMT and MET are required for invasion, dissemination, survival and colonisation at distant sites. EMP also influences resistance to anoikis, enhances survival, creates genomic instability and induces resistance to chemotherapies (reviewed in refs [33,34]).

Cancer stem cells (CSCs) have been recognised as a distinct minor population of cells that exhibit many properties different from that of the main compartment of cancer cells. While the bulk of cancer cells are aggressive and highly proliferative, CSCs are resistant to anoikis, able to self-renew and to differentiate to independently form tumours [35]. It has been proposed that the ability of EMP to drive these CSC features, including decreased proliferation and increased DNA repair capability, ultimately leads to a tumorigenic, drug-resistant cell [36]. Such resistant ‘stem-like’ cells also do not undergo apoptosis when subjected to chemotherapy, allowing them to self-renew when treatment is withdrawn, supporting the notion of CSCs being responsible for disease recurrence [37]. Cells in the tumour periphery that associate with the surrounding stroma demonstrate phenotypes consistent with CSCs, consistent with the induction of EMP programmes [3840]. Furthermore, there is evidence to support the notion that the induction of EMP invokes CSC features that lead to local invasion prior to tumour formation in models of pancreatic cancer [19]. Importantly, the prognostic extent of EMP in CSCs is relatively under-explored, although such EMP programmes have been shown to correlate with increased metastatic potential [26,41,42]. These properties of CSCs, directly attributable to EMP de-differentiation, are thought to be major drivers of cancer recurrence. While therapies act on the bulk of a tumour, the presence of CSCs that are able to survive independently, resistant treatment and initiate tumours are undeniably problematic in combating recurrence, highlighting the importance of research into the fundamental biology of EMP, which underpins the CSC phenotype.

While it is clear that EMP affects many of aspects of cancer that make it difficult to treat and may even synergise with oncogenic cellular programmes to drive cancer progression, it is important to realise the overarching distinction that EMP has from carcinogenesis in general. Cancer is a genetic disease caused by the accumulation of somatic mutations, resulting in a functional imbalance between tumour suppressive and oncogenic signals [43]. EMP is a transient, dynamic phenotypic process controlled by drivers and repressors that are biased by the particular tumour microenvironment in which they exist. Such drivers and repressors act through epigenetic modifications, transcription factor regulation, alternative splicing of mRNA, microRNA (miRNA or miR) translational control and post-translational regulation to affect signal stability [4446]. This complexity results in a continuum of targetable intermediate states that are better defined by their phenotypic properties, including invasion, survival, clonogenicity, tumourigenicity and proliferation.

Translational research aiming to delineate the EMP features common to tumour invasion, CSCs and CTCs is therefore vital to curtail tumour metastasis and benefit overall patient outcomes. An emerging challenge is also to better comprehend clinically relevant intermediate states of EMP, and how they contribute to tumour progression for more effective therapeutic interventions [47].

EMP and therapeutic monitoring

CTCs in cancer monitoring

Research into the molecular hallmarks of EMP have given rise to many traceable phenomena in carcinoma progression in vivo. The presence of CTCs in the blood is prognostic for metastatic spread of cancers, survival outcome and therapy resistance [4851]. Several meta-analyses have shown that the presence of CTCs during treatment is significantly associated with poor patient survival in colorectal cancer [52], breast cancer [53], prostate cancer [54] and other solid cancers [5557]. Assessment of CTCs in metastatic cancers before, during and after therapies has also allowed personalised treatment to be tailored to individual patients [5860]. Considerable research is being undertaken to explore the use of CTCs for longitudinal monitoring of metastatic burden and therapy responses of cancer patients. The success of CTC isolation strategies remains an issue due to the technically challenging nature of low CTC prevalence, high blood cell background and EMP issues that may dilute the epithelial differential from blood cells. CTCs are incredibly rare in most malignancies (<1 CTC per ml of blood), severely affecting the ability to isolate a pure CTC population. This, combined with issues surrounding CTC viability and the presence of dying or senescent CTCs, makes their analysis exceedingly difficult [61].

Although CTCs are hypothesised to be instrumental in cancer metastasis, recent evidence has suggested that CTCs disseminate into the bloodstream at a relatively early stage, in parallel to the primary tumour growth [62,63]. Several molecules and their signalling pathways have emerged as novel markers to trace CTC origins in various cancer types [64]. For instance, CK7 was found to be a potential marker for CTCs from lung carcinoma [65], while TTF1 is expressed in CTCs of thyroid and lung cancer patients [66]. To date, several high-resolution genomic and transcriptomic approaches have been performed to dissect and characterise these cells from various cancer types [18,6769]. While the precise mechanisms of CTC genesis remain elusive, the biology of CTCs is the subject of intense research. Moreover, use of ‘liquid biopsy’ from blood to monitor disease state and therapy response by CTC quantification and subsequent molecular characterisation is gaining popularity. Initial CTC methods relied mostly on epithelial marker expression, predominantly employing EpCAM as a CTC marker to facilitate enrichment and subsequent identification. The use of label-free isolation methods based on CTC morphology has more recently allowed a more comprehensive analysis of EMP status in CTC populations not defined by limited markers [61]. Such isolation strategies include the use of filtration and microfluidic devices to avoid biases in the immunoselective approaches against CTC that may exhibit varying marker expression.

There is considerable evidence of EMP in CTCs [27,70] and ex vivo CTC cultures [24]. Such cell populations have been shown to be heterogeneous, harbouring diverse biological and molecular characteristics surrounding EMP [71]. CTC subpopulations have thus been grouped into epithelial CTCs, mesenchymal CTCs and biophenotypic epithelial/mesenchymal CTCs [72], the latter highlighting the hybrid/metastable EMP state. CTCs have been characterised using varying epithelial and mesenchymal markers, including EpCAM, vimentin, N-cadherin, cytokeratins (CKs) and E-cadherin [23,73,74], as well as CSC markers such as CD133, CD44 and CD24 [75]. RNA in situ hybridisation strategies using probes specific for epithelial or mesenchymal markers have been devised to distinguish between the two subpopulations in CTCs, aiming to elucidate the extent of hybrid EMP states in metastatic breast cancer [18,24].

miRNA in situ hybridisation uses a locked nucleic acid-based probe combined with immune-magnetic selection based on CK expression and immunocytochemistry to detect CTCs expressing miR-21, an onco-miR marker found in CTCs with an EMP phenotype. This method is effective in distinguishing CTCs from leukocytes, since haematopoietic cells do not express miR-21 [76]. Wu et al. demonstrated the use of a cell marker enrichment technique named ‘CanPatrol’ to identify CTCs isolated using ScreenCell® filtration. CTCs were assessed using multiple EMP markers, such as VIM, TWIST, EPCAM, CK 8/18/19, as well as the leukocyte marker CD45. They observed an increased presence of CTCs and CTC microemboli with a mesenchymal phenotype in metastatic breast cancers [77]. Multimarker transcriptional assessment of early breast cancer CTCs isolated by density gradient enrichment followed by negative selection using CD45 showed more frequent expression of mesenchymal markers (VIM, SNAI1 and UPAR) in subjects with positive lymph node metastasis [78].

EMP status of CTCs is being evaluated in many clinical studies. CTCs from peripheral blood and paired tumour biopsies are being evaluated to assess changes in EMP and stemness in a phase I trial of AB-16B5, a monoclonal antibody (mAb) against EMP target clusterin (secreted CLU) in patients with advanced solid malignancies (NCT02412462). Aspirin has been found to down-regulate SNAI2, increase E-cadherin (CDH1) expression and inhibit EMP in NSCLC (non-small-cell lung cancer) [79]. Clinical trials are currently being conducted to evaluate the effect of aspirin on CTC number and subtype in metastatic breast cancer and colorectal cancer (NCT02602938). Another clinical trial measured the ratio of epithelial or mesenchymal markers in CTCs using fluorescent in situ hybridisation to predict the pathological status in prostate cancer (NCT02940977). The EMP status of CTCs and the non-invasive nature of sampling from cancer patients make them ideal for monitoring the efficacy of EMP-targeted therapies. Indeed, the trials discussed above are supportive of the clinical utility of such a strategy.

EMP-associated gene expression in tumours

Several EMP pathway members have been shown to be significant prognostic indicators of cancer progression and patient survival. In fact, EMP drivers and downstream transcription factors have been extensively studied to validate their potential as prognostic markers for clinical evaluation. Commonly evaluated markers indicative of EMP status include E-cadherin, N-cadherin and vimentin. The transition of cell junction associations mediated by E-cadherin to N-cadherin, also referred to as the E-/N-cadherin switch, describes the concomitant yet functionally opposing roles of the two cadherins. During EMP, the down-regulation of E-cadherin and up-regulation of N-cadherin trigger alterations in cell–cell detachment, migration, invasion and stemness [8082]. In a univariate survival analysis, cadherin switching significantly associated with tumour progression and decreased patient survival [83], supporting the involvement of such a switch in the metastatic process [84]. Not surprisingly, Kaplan–Meier survival analysis of high N-cadherin expression in the primary tumour showed significantly lower overall and disease-free survival compared with patients with low N-cadherin expression in colorectal cancer [85], supporting the use of N-cadherin as a prognostic indicator in colorectal cancer [86]. Furthermore, N-cadherin expression in bladder cancer was shown to be a similarly prognostic [87]. It was absent in normal urothelium, appeared in stage pT1, and increased throughout pT stage progression.

Loss of E-cadherin expression is indicative of invasive lobular carcinoma (ILC) [88]. The American Cancer Society has endorsed the use of E-cadherin to distinguish invasive ductal carcinoma from ILC. Germline E-cadherin mutations have also been reported to drive progression of hereditary diffuse gastric cancer [89] and breast cancer [90]. Single-nucleotide polymorphism profiling has also indicated that certain E-cadherin genetic variants are predictive of patient survival in breast cancer [91]. There is still, however, lack of a clear correlation between E-cadherin expression and other prognostic parameters in other breast cancers, such as tumour size, tumour grade, ER, PR and HER-2 expression status [92]. Meta-analysis of E-cadherin expression across other cancers correlated with certain pathological features in a prognostic capacity, revealing that down-regulation of E-cadherin in colorectal cancer significantly predicted poor clinical outcome in Asian, but not in European patients. [93].

The intermediate filament protein vimentin is generally expressed in mesenchymal cells, or mesenchymal-shifted cells, which have enhanced invasive potential, and overexpression of vimentin is significantly correlated with tumour growth, invasion and poor prognosis in several carcinomas [94]. While research is supportive of the role that vimentin overexpression has in predicting recurrence, distant metastasis and reduced survival in gastric cancer [95], methylation-mediated suppression of vimentin in also detected in colorectal cancer cells [96]. In a study to identify markers of metastasis, vimentin was significantly up-regulated in melanoma, with an exceptionally high up-regulation in haematogenous metastasis, suggesting its value as a metastatic predictor [97]. Additional meta-analysis in NSCLC has also strongly implicated vimentin overexpression in poor prognosis [98]. Vimentin, therefore, is largely prognostic in numerous cancer types and may serve as a marker of invasive potential or disease aggressiveness.

The importance of therapeutic targeting of EMP

As well as the prognostic power of the EMP phenomenon mentioned above, direct functional evidence of the EMP programme in tumour progression is also prevalent, albeit not universally. The invasive front of human tumours is typically characterised by the presence of fibroblast-like cells expressing mesenchymal markers such as vimentin, fibronectin and N-cadherin, with decreased expression of junction adhesion molecule E-cadherin and increased nuclear β-catenin [99103]. In vivo lineage labelling and intravital microscopy studies are also supportive of the contribution of EMP to metastatic spread [19,104]. While these models are strongly indicative of its role in driving cancer invasion, the direct contribution of EMP to metastasis in vivo has recently been challenged [105,106]. Genetic ablation of EMP transcription factors, Snai1 or Twist, in a mouse model of pancreatic ductal adenocarcinoma (PDAC) showed that metastatic dissemination was unaffected, which the authors use as evidence against the requirement for EMP in tumour progression [106]. Such evidence has since been called into question, as it was not shown that complete suppression of the EMP programme was produced by Snai1 or Twist deletion [107]. Indeed, it has been shown that EMP transcription factor, Zeb1, is responsible for driving EMP in this model of PDAC development [108]. Zeb1-depleted tumours were better differentiated, indicating less local invasion, and showed significantly reduced metastasis when compared with control PDAC mice [108]. This is in direct contrast with depletion of Twist1 or Snai1 not affecting metastasis in the same model, highlighting the importance of recognising the context- and tissue-specific drivers of EMP. In a MMTV-PyMT murine model of breast cancer, Fsp-1 reporters used to mark epithelial cells that entered into an EMP programme did not provide strong evidence for the requirement of EMP in metastatic colonisation [105]. Again, the use of single-gene reporters not representative of the EMP spectrum may be a limitation in this approach, leaving the debate open for further clarification. These studies belie a wealth of data supporting a functional role of either EMT or MET or both in cancer progression (reviewed in refs [16,31,109111]).

It is, nonetheless, widely recognised that the mesenchymal and stem-like properties associated with EMP are implicated in chemoresistance in vitro and in vivo [105,106,112116]. The contribution of EMP towards resistance of apoptosis, anoikis, increased DNA repair and up-regulation of drug efflux transporters likely underpins its role in chemoresistance [17,117,118]. This has spurred much interest in addressing EMP with adjuvant therapies to increase efficacy of existing treatment regimes. Treating cancers with a single chemotherapeutic approach often results in alternative pathways being activated to give rise to multidrug-resistant cancer cell populations [119]. Hence, combining EMP-directed treatments to synergise with current chemotherapeutics and induce chemosensitivity is a logical step. It is worth noting that due to significant redundancy in the complexity and heterogeneity of EMP triggers, it is likely that multiple pathways would need to be inhibited to gain control over cellular EMP phenotypes to address carcinoma progression. Such therapies would aim to target specific attributes or pathways of EMP and are consolidated in this review as different strategies: (i) inhibitors of EMP signalling pathway members, monoclonal antibodies targeting EMP regulators; (ii) transcriptional control of EMP drivers by epigenetic modifiers; (iii) translational control by miRNA; (iv) post-translational control of EMP pathway members; (v) repurposing drugs to target EMP; (vi) EMP-targeting vaccines and (vii) nutraceuticals (Figure 1).

Major strategies for targeting EMP.

Figure 1.
Major strategies for targeting EMP.

Three major strategies for inhibiting cancer progression and recurrence in relevance to EMP are (1) agents that can target the EMP spectrum where cells are in multiple transition states; (2) agents that can selectively kill mesenchymal phenotype and (3) agents/compounds that can lead to MET. CTC, circulating tumour cells; ECM, extracellular cell matrix; EMP, epithelial–mesenchymal plasticity; EGF, epidermal growth factor; N-cad, N-cadherin; TGF, transforming growth factor; Vim, vimentin.

Figure 1.
Major strategies for targeting EMP.

Three major strategies for inhibiting cancer progression and recurrence in relevance to EMP are (1) agents that can target the EMP spectrum where cells are in multiple transition states; (2) agents that can selectively kill mesenchymal phenotype and (3) agents/compounds that can lead to MET. CTC, circulating tumour cells; ECM, extracellular cell matrix; EMP, epithelial–mesenchymal plasticity; EGF, epidermal growth factor; N-cad, N-cadherin; TGF, transforming growth factor; Vim, vimentin.

Inhibitors of EMP signalling pathway members

Well-known exogenous stimuli trigger paracrine and autocrine signalling to induce carcinoma cells to undergo a context-dependent shift away from an epithelial phenotype, including members of TGF-β [120], EGF [121], FGF [122], PDGF [123], HGF [124], IGF [125], interleukin-6 (IL-6) [126] and BMP. Cross-talk in downstream signalling is extensive, resulting in involvement of WNT, NOTCH, HIPPO, Janus-activated kinase (JAK)-STAT, AP-1, NF-κB and PI3K/AKT mechanisms that act to dysregulate the epithelial function and promote migration and chemoresistance [8,127,128]. Moreover, these pathways have been implicated in many of the hallmarks of cancer, such that significant research and effort has gone into defining these pathways and identifying agents that may serve as functional inhibitors [129]. Indeed, such pathways lend themselves to very specific inhibitors which, interfere with one or more steps in the cascade. Many molecules targeted at different steps in these pathways have been identified and validated in preclinical settings (Table 1). Many of these factors and pathways are involved in pathological processes in addition to EMP, making it difficult to isolate their EMP-specific impact. Even so, targeting these pathways may synergise to inhibit multiple pathways that are corrupted in cancer, thereby enhancing the response to conventional cytotoxic treatments.

Table 1
Agents that target stimuli and signalling pathways associated with EMT
Target class Functional class Name Cancer type Clinical status In vivo or In vitro effects observed 
Inhibitors of extracellular mediators and their corresponding receptors 
TGF-β–TGF-β receptor inhibitors Monoclonal antibody GC-1008 (Fresolimumab) Glioma, metastatic melanoma, RCC, MBC NCT01472731; NCT00356460; NCT01401062 
 Antisense oligonucleotide AP-12009 (Trabedersen) Pancreatic neoplasms, melanoma and CRC NCT00844064, NCT00761280, NCT00431561 Blocks proliferation and migration of pancreatic cancer cells 
 Small-molecule inhibitor LY-573636 (tasisulam sodium) Unresectable/metastatic NSCLC, unresectable/metastatic soft tissue sarcoma, unresectable/metastatic melanoma NCT00363766; NCT00383292; NCT00490451 Inhibits tumour invasion 
 Tyrosine kinase inhibitor LY-2152799 (Galunisertib) Breast, hepatocellular carcinoma, metastatic pancreatic cancer, prostrate cancer, malignant glioma NCT02538471; NCT01682187; NCT02452008; NCT02734160; NCT02423343 Anti-CSC activity 
 TGF-β receptor inhibitor TEW-7197 Solid cancers NCT02160106, NCT02178358 Inhibits EMP in TGF-β-treated breast cancer cells and 4T1 orthotopic-grafted mice 
 TGF-β1 receptor kinase inhibitor SD-208, SD-093 MBC Preclinical Inhibits growth, invasiveness and enhances immunogenicity of murine and human glioma cells in vitro and in vivo 
 TGF-β1 and 2 receptor kinase inhibitor EW-7203, EW-7197, IN-1130 Breast, lung metastases Preclinical Blocks TGF-β1-mediated EMP in mammary epithelial cells 
 Inhibition of ATP binding to ALK5 kinase SB-431542 Glioma Preclinical Attenuates the tumour-promoting effects of TGF-β, including TGF-β-induced EMP, cell motility, migration, invasion and VEGF secretion in human cancer cell lines 
IL-6/IL-6R inhibitors Monoclonal antibody Siltuximab (CNTO-328, Tocilizumab) Metastatic prostate cancer, metastatic RCC, refractory multiple myeloma NCT00433446; NCT00265135; NCT00401843 Reduces the survival and/or self-renewal of head and neck cancer stem-like cells in vivo 
HGF/MET inhibitors Monoclonal antibody Rilotumumab Gastric cancer NCT02137343 (recently terminated) 
  SAIT301 Solid tumours NCT02296879 Reduces the invasion and migration of nasopharyngeal cancer cells 
PDGF/PDGFR inhibitors Tyrosine kinase inhibitor  Axitinib Prostate cancer, metastatic RCC, salivary gland cancers, advanced solid tumours NCT01409200; NCT02579811; NCT02857712; NCT01999972 Promotes MET in cervical cancer cells 
  Nilotinibx Thyroid cancer, relapsed solid tumour, CML NCT01788982; NCT02379416; NCT02973711 
 Antisense oligonucleotide ISIS 5132 (CGP 69846A) MBC NCT00003236 
EGF/EGFR inhibitors Tyrosine kinase inhibitor Icotinib Nasopharyngeal carcinoma NCT01534585 Reduces EMP in squamous cell carcinomas 
  Lapatinib Head and neck cancer NCT01711658 Induces MET in squamous cell carcinomas 
  Afatinib (BIBW2992) Head and neck cancer, brain cancer, metastatic chordoma, NSCLC NCT01427478; NCT02423525; NCT03083678; NCT01953913 Induces MET in HNSCC 
  Dacomitinib (PF00299804) NSCLC, advanced KRAS mutant malignancies, oesophagus carcinoma; EGFRmut solid malignancies NCT01918761; NCT02039336; NCT02353936; NCT02108964 Attenuates migration and invasion, and reduces EMP expression markers in epithelial ovarian cancer 
  Poziotinib (HM781-36B) NSCLC, MBC NCT03066206; NCT02659514; NCT02544997; NCT02979821 
  AP26113 NSCLC NCT02737501; NCT02706626 
 Monoclonal antibody Panitumumab (ABX-EGF mAb) Metastatic CRC, advanced solid tumours NCT02613221; NCT02301962; NCT01061788 
  Necitumumab (IMC-IIF8) NSCLC NCT02496663; NCT02392507 
  Nimotuzumab (h-R3) Brainstem tumour, oesophageal squamous cell carcinoma NCT02672241; NCT02272699 EGF-stimulated EMP was suppressed in adenoid cystic carcinoma 
  HLX07 Advanced solid cancers NCT02648490 
  Matuzumab (EMD72000) Recurrent ovarian cancer, oesophago-gastric cancer NCT00073541; NCT00215644 
IGF/IGFR inhibitors Monoclonal antibody IGF-1R:MK-0646 (Dalotuzumab) Advanced pancreatic cancer NCT00769483 
FGF/FGFR inhibitors Tyrosine kinase inhibitor Lenvatinib Advanced solid tumours, salivary gland carcinoma, anaplastic thyroid cancer NCT02640508; NCT02860936; NCT02657369 
  Nintedanib (BIBF1120) Advanced solid cancer, pancreatic cancer, thyroid cancer, breast cancer NCT02835833; NCT02902484; NCT01788982; NCT02389764 
  Pazopanib Metastatic RCC, recurrent ovarian cancer, advanced soft tissue sarcoma NCT01521715; NCT01600573; NCT01975519 Blocks EMP progression 
  Ponatinib Acute myelogenous leukemia, glioblastoma, NSCLC NCT02428543; NCT02478164; NCT01813734 Reduction in EMP, suppressing TGF-β1/Smad3 pathway 
TNFα inhbitors Monoclonal antibody Infliximab RCC, melanoma, lung cancer NCT02763761 Inhibits EMP in obliterative bronchiolitis 
 Monoclonal antibody Etanercept (Enbrel) MBC, recurrent ovarian cancer Preclinical 
Hedgehog/Smoothened inhibitors Smoothened antagonists (small-molecule inhibitor) BMS-833923 Solid tumours, metastatic cancer NCT01413906; NCT00670189 
  Vismodegib Basal cell carcinoma, metastatic pancreas, metastatic CRC NCT01201915; NCT01088815; NCT00636610 Targets CSC pathways in gastric cancer, tumour growth, cell motility and invasiveness 
  Sonidegib (LDE225) Advanced gastroesophageal adenocarcinoma, multiple myeloma NCT02138929; NCT02086552 Inhibits EMP in animal models of prostate cancer 
  Taladegib (LY2940680) Metastatic solid tumours, localised adenocarcinoma of the oesophagus NCT02530437; NCT02784795 
Notch/Notch ligand (Delta-like and Jagged) inhibitors Monoclonal antibody Notch2/Notch3: OMP-59R5 (Tarextumab) SCLC, pancreatic cancer, solid tumours NCT01859741; NCT01647828; NCT01277146 Delayed tumour recurrence, decrease CSC frequency and modulated function of tumour vasculature 
  DLL4: OMP-21M18 (Demcizumab) Pancreatic cancer, ovarian cancer, NSCLC, CRC, solid tumours NCT01189968; NCT01952249; NCT02259582; NCT01189942; NCT00744562 Anti-CSC compound 
  DLL4: MEDI0639 Advanced solid tumours NCT01577745 Modulates endothelial cell function and angiogenesis in vivo 
 Small-molecule inhibitor γ-secretase inhibitor: MK0752 Advanced breast cancer, solid tumour NCT00106145; NCT0645333 
  γ-secretase inhibitor: LY3039478 Advanced solid tumour NCT02784795 
  γ-secretase inhibitor DAPT Tamoxifen-resistant breast cancer, gastric cancer Preclinical Reduced micrometastatic tumour burden in mice 
  γ-secretase inhibitor: PF-03084014 Advanced solid tumour malignancies NCT02955446 Reverses the EMP phenotype and diminish the TICs in breast cancer models 
  PAN-Notch inhibitor BMS-906024 Metastatic solid tumours NCT01653470 
WNT/Frizzled inhibitors Monoclonal antibody OMP-18R5 (Vantictumab) NSCLC, pancreatic cancer, breast cancer, solid tumours NCT01957007; NCT02005315; NCT01973309; NCT01345201 Reduces tumour-initiating cell frequency 
 Wnt5a mimetic Foxy-5 Metastatic breast, colon and prostrate NCT02655952 Impair migration of epithelial cancer cells 
 Peptidomimetics CWP232291 Acute myeloid leukemia NCT01398462 Targets β-catenin for degradation and inhibits the expression of cell cycle and anti-apoptotic genes such as cyclin D1 and survivin 
 Inhibition of post-translational palmitoylation of Wnt ligands ETC-1922159 Solid cancers NCT02521844 Loss of cell cycle, stem cell and proliferation genes, and an increase in differentiation markers (differentiation therapy) 
 Decoy receptor for Wnt OMP-54F28 Solid cancers NCT01608867 Impedes the growth of numerous solid tumour types and selectively reduced CSCs 
 Porcupine inhibitor LGK974 Solid malignancies NCT01351103 Prolongs metastasis-free survival in a mouse model of Ewing sarcoma and inhibits migration of ES cell lines through the suppression of expression of multiple transcriptional regulators of EMP 
 NTR1 antagonist SR48692 HCC Preclinical Inhibits metastasis in mice 
 Inhibits the recruiting of β-catenin with its co-activator CBP PRI-724 Metastatic pancreatic, advanced myeloid malignancies, metastatic CRC NCT01764477; NCT01606579; NCT02413853 CSC-targeting drugs 
Inhibitors of intracellular signalling pathways 
SRC inhibitors Tyrosine Kinase inhibitor Ponatinib (AP23464) CML NCT02467270 
  Dasatinib (BMS-354825) Haematologic malignancies, advanced solid tumours NCT01643603; NCT01609816; NCT02389309 Blocks TGF-β1-induced EMP in PDAC cell lines 
  Bosutinib (SKI-606) Malignant solid tumours NCT00195260 Reduces tumour growth, invasion, EMP and distant metastasis in a mouse model of thyroid cancer 
FAK inhibitors Tyrosine Kinase inhibitor Defactinib (VS-6063) NSCLC, solid tumours, ovarian cancer, relapsed malignant mesothelioma NCT01951690; NCT01943292; NCT01778803; NCT02372227 Targets mesothelioma CSCs, reversal of drug resistance 
 Small-molecule inhibitor PF-00562271 Advanced non-haematological malignancies NCT00666926 (discontinue) 
PI3K/AKT/mTOR inhibitors PI3K inhibitor Idelalisib CLL NCT02662296 
 AKT inhibitor AZD5363 MBC, malignant female reproductive system neoplasm NCT02423603; NCT02208375 Tumour growth inhibition 
  Temsirolimus Advanced cancer NCT01529593; NCT02389309 Addition with the autophagy inhibitor (chloroquine) to EMP-induced cells decreases their viability 
 Tyrosine kinase inhibitor CX-4945 Cholangiocarcinoma NCT02128282 Blocks TGF-β1-induced EMP in A549 human lung adenocarcinoma cells 
 Tyrosine kinase inhibitor VS-5584 Relapsed malignant mesothelioma, lymphoma, advanced non-haematological malignancies NCT02372227; NCT01991938 Targets CSC in breast cancer models 
 AURKA/SYK Tyrosine kinase inhibitor Midostaurin Leukaemia, CRC NCT01282502 Post-EMP breast cancer cell-specific drug 
AXL inhibitors Tyrosine Kinase inhibitor BGB324 AML, NSCLC NCT02488408; NCT02424617 Blocks EMP 
  Glesatinib (MGCD265) NSCLC NCT02544633 
RAS/RAF/MAPK inhibitors BRAF inhibitors Vemurafenib and dabrafenib Metastatic melanoma NCT02052193 
 A RAF inhibitor Sorafenib RCC and HCC NCT01444807; NCT02733809 EMP suppression in HCC 
 MEK inhibitor Trametinib Melanoma NCT02858921 EMP modulator in ACHN cells 
Inhibitors of transcription factors that indirectly induce EMP 
JAK and STAT3 inhibitors Small-molecule inhibitor STAT3 inhibitor: OPB-31121 Solid tumours NCT00955812 
  STAT3: BB1608 (Napabucasin) Advanced CRC, refractory haematological malignancies, mesothelioma, advanced glioblastoma NCT01830621; NCT02352558; NCT02347917; NCT02315534 Blocks spherogenesis of CSCs, kills CSCs and down-regulates CSC-associated genes 
 JAK2 and STAT3 inhibitor WP1066 Malignant glioma and brain metastasis from melanoma NCT01904123 Reverses EMP in HNSCC 
Compounds acting on epigenetic modulators 
Histone deacetylase inhibitor  Vorinostat Advanced breast cancer, AML NCT01118975; NCT00368875; NCT02412475 Exerts EMP reversal effects 
  Romidepsin Relapsed lymphoid malignancies, T-cell lymphoma NCT01998035; NCT01638533; NCT01738594 
  Mocetinostat Advanced solid tumors and NSCLC NCT02805660; NCT02954991 Supresses EMP and induces chemosensitivity in breast and pancreatic cell lines 
  Panobinostat Unresectable III/IV melanoma, glioma NCT02032810; NCT02717455 Modulates EMP markers in HCC 
Histone demethylase inhibitor LSD1 inhibitor Tranylcypromine AML NCT02717884 Supresses EMP and invasion 
  GSK2879552 SCLC, AML NCT02034123; NCT02177812 Supresses EMP and invasion 
Histone methyl transferases inhibitor EZH2 inhibitor GSK503 Melanoma Preclinical 
  JQEZ5 Lung adenocarcinomas Preclinical 
  E7438 (Tazemetostat, EPZ-6438) Haematological and solid tumours, sarcoma, melanoma, mesothelioma NCT01897571; NCT02601950; NCT02860286 
 EZH1/2 inhibitor DS-3201b Non-Hodgkin's lymphoma NCT02732275 
Inhibitors of stimuli from the tumour microenvironment 
HIF-1α inhibitors Antisense oligonucleotide EZN-2698 Advanced solid tumours, metastatic RCC Preclinical Tumour cell growth inhibition, down-regulation of HIF-1α target genes and impaired ability of HUVEC cells to form tubes in vitro 
 Small-molecule inhibitor PX-478 Advanced metastatic cancer NCT00522652 In vitro and in vivo inhibition of EMP in ESCC 
  PT2385 RCC NCT02293980 Reduces tumour growth and tumour vascular area in VHL−/− clear cell renal cell carcinoma (ccRCC) patient derived xenografts 
  Digoxin MBC, head and neck cancer, prostate cancer NCT01887288; NCT02906800; NCT01162135 Inhibits LOX and blocks metastatic niche formation in the lungs of MDA-MB — 231 tumour-bearing mice 
 Clusterins Humanised monoclonal antibody against secreted clusterin (sCLU) AB-16B5 Advanced solid tumours NCT02412462 Inhibits TGF-β-induced EMP in several HCC tumour cell lines 
Target class Functional class Name Cancer type Clinical status In vivo or In vitro effects observed 
Inhibitors of extracellular mediators and their corresponding receptors 
TGF-β–TGF-β receptor inhibitors Monoclonal antibody GC-1008 (Fresolimumab) Glioma, metastatic melanoma, RCC, MBC NCT01472731; NCT00356460; NCT01401062 
 Antisense oligonucleotide AP-12009 (Trabedersen) Pancreatic neoplasms, melanoma and CRC NCT00844064, NCT00761280, NCT00431561 Blocks proliferation and migration of pancreatic cancer cells 
 Small-molecule inhibitor LY-573636 (tasisulam sodium) Unresectable/metastatic NSCLC, unresectable/metastatic soft tissue sarcoma, unresectable/metastatic melanoma NCT00363766; NCT00383292; NCT00490451 Inhibits tumour invasion 
 Tyrosine kinase inhibitor LY-2152799 (Galunisertib) Breast, hepatocellular carcinoma, metastatic pancreatic cancer, prostrate cancer, malignant glioma NCT02538471; NCT01682187; NCT02452008; NCT02734160; NCT02423343 Anti-CSC activity 
 TGF-β receptor inhibitor TEW-7197 Solid cancers NCT02160106, NCT02178358 Inhibits EMP in TGF-β-treated breast cancer cells and 4T1 orthotopic-grafted mice 
 TGF-β1 receptor kinase inhibitor SD-208, SD-093 MBC Preclinical Inhibits growth, invasiveness and enhances immunogenicity of murine and human glioma cells in vitro and in vivo 
 TGF-β1 and 2 receptor kinase inhibitor EW-7203, EW-7197, IN-1130 Breast, lung metastases Preclinical Blocks TGF-β1-mediated EMP in mammary epithelial cells 
 Inhibition of ATP binding to ALK5 kinase SB-431542 Glioma Preclinical Attenuates the tumour-promoting effects of TGF-β, including TGF-β-induced EMP, cell motility, migration, invasion and VEGF secretion in human cancer cell lines 
IL-6/IL-6R inhibitors Monoclonal antibody Siltuximab (CNTO-328, Tocilizumab) Metastatic prostate cancer, metastatic RCC, refractory multiple myeloma NCT00433446; NCT00265135; NCT00401843 Reduces the survival and/or self-renewal of head and neck cancer stem-like cells in vivo 
HGF/MET inhibitors Monoclonal antibody Rilotumumab Gastric cancer NCT02137343 (recently terminated) 
  SAIT301 Solid tumours NCT02296879 Reduces the invasion and migration of nasopharyngeal cancer cells 
PDGF/PDGFR inhibitors Tyrosine kinase inhibitor  Axitinib Prostate cancer, metastatic RCC, salivary gland cancers, advanced solid tumours NCT01409200; NCT02579811; NCT02857712; NCT01999972 Promotes MET in cervical cancer cells 
  Nilotinibx Thyroid cancer, relapsed solid tumour, CML NCT01788982; NCT02379416; NCT02973711 
 Antisense oligonucleotide ISIS 5132 (CGP 69846A) MBC NCT00003236 
EGF/EGFR inhibitors Tyrosine kinase inhibitor Icotinib Nasopharyngeal carcinoma NCT01534585 Reduces EMP in squamous cell carcinomas 
  Lapatinib Head and neck cancer NCT01711658 Induces MET in squamous cell carcinomas 
  Afatinib (BIBW2992) Head and neck cancer, brain cancer, metastatic chordoma, NSCLC NCT01427478; NCT02423525; NCT03083678; NCT01953913 Induces MET in HNSCC 
  Dacomitinib (PF00299804) NSCLC, advanced KRAS mutant malignancies, oesophagus carcinoma; EGFRmut solid malignancies NCT01918761; NCT02039336; NCT02353936; NCT02108964 Attenuates migration and invasion, and reduces EMP expression markers in epithelial ovarian cancer 
  Poziotinib (HM781-36B) NSCLC, MBC NCT03066206; NCT02659514; NCT02544997; NCT02979821 
  AP26113 NSCLC NCT02737501; NCT02706626 
 Monoclonal antibody Panitumumab (ABX-EGF mAb) Metastatic CRC, advanced solid tumours NCT02613221; NCT02301962; NCT01061788 
  Necitumumab (IMC-IIF8) NSCLC NCT02496663; NCT02392507 
  Nimotuzumab (h-R3) Brainstem tumour, oesophageal squamous cell carcinoma NCT02672241; NCT02272699 EGF-stimulated EMP was suppressed in adenoid cystic carcinoma 
  HLX07 Advanced solid cancers NCT02648490 
  Matuzumab (EMD72000) Recurrent ovarian cancer, oesophago-gastric cancer NCT00073541; NCT00215644 
IGF/IGFR inhibitors Monoclonal antibody IGF-1R:MK-0646 (Dalotuzumab) Advanced pancreatic cancer NCT00769483 
FGF/FGFR inhibitors Tyrosine kinase inhibitor Lenvatinib Advanced solid tumours, salivary gland carcinoma, anaplastic thyroid cancer NCT02640508; NCT02860936; NCT02657369 
  Nintedanib (BIBF1120) Advanced solid cancer, pancreatic cancer, thyroid cancer, breast cancer NCT02835833; NCT02902484; NCT01788982; NCT02389764 
  Pazopanib Metastatic RCC, recurrent ovarian cancer, advanced soft tissue sarcoma NCT01521715; NCT01600573; NCT01975519 Blocks EMP progression 
  Ponatinib Acute myelogenous leukemia, glioblastoma, NSCLC NCT02428543; NCT02478164; NCT01813734 Reduction in EMP, suppressing TGF-β1/Smad3 pathway 
TNFα inhbitors Monoclonal antibody Infliximab RCC, melanoma, lung cancer NCT02763761 Inhibits EMP in obliterative bronchiolitis 
 Monoclonal antibody Etanercept (Enbrel) MBC, recurrent ovarian cancer Preclinical 
Hedgehog/Smoothened inhibitors Smoothened antagonists (small-molecule inhibitor) BMS-833923 Solid tumours, metastatic cancer NCT01413906; NCT00670189 
  Vismodegib Basal cell carcinoma, metastatic pancreas, metastatic CRC NCT01201915; NCT01088815; NCT00636610 Targets CSC pathways in gastric cancer, tumour growth, cell motility and invasiveness 
  Sonidegib (LDE225) Advanced gastroesophageal adenocarcinoma, multiple myeloma NCT02138929; NCT02086552 Inhibits EMP in animal models of prostate cancer 
  Taladegib (LY2940680) Metastatic solid tumours, localised adenocarcinoma of the oesophagus NCT02530437; NCT02784795 
Notch/Notch ligand (Delta-like and Jagged) inhibitors Monoclonal antibody Notch2/Notch3: OMP-59R5 (Tarextumab) SCLC, pancreatic cancer, solid tumours NCT01859741; NCT01647828; NCT01277146 Delayed tumour recurrence, decrease CSC frequency and modulated function of tumour vasculature 
  DLL4: OMP-21M18 (Demcizumab) Pancreatic cancer, ovarian cancer, NSCLC, CRC, solid tumours NCT01189968; NCT01952249; NCT02259582; NCT01189942; NCT00744562 Anti-CSC compound 
  DLL4: MEDI0639 Advanced solid tumours NCT01577745 Modulates endothelial cell function and angiogenesis in vivo 
 Small-molecule inhibitor γ-secretase inhibitor: MK0752 Advanced breast cancer, solid tumour NCT00106145; NCT0645333 
  γ-secretase inhibitor: LY3039478 Advanced solid tumour NCT02784795 
  γ-secretase inhibitor DAPT Tamoxifen-resistant breast cancer, gastric cancer Preclinical Reduced micrometastatic tumour burden in mice 
  γ-secretase inhibitor: PF-03084014 Advanced solid tumour malignancies NCT02955446 Reverses the EMP phenotype and diminish the TICs in breast cancer models 
  PAN-Notch inhibitor BMS-906024 Metastatic solid tumours NCT01653470 
WNT/Frizzled inhibitors Monoclonal antibody OMP-18R5 (Vantictumab) NSCLC, pancreatic cancer, breast cancer, solid tumours NCT01957007; NCT02005315; NCT01973309; NCT01345201 Reduces tumour-initiating cell frequency 
 Wnt5a mimetic Foxy-5 Metastatic breast, colon and prostrate NCT02655952 Impair migration of epithelial cancer cells 
 Peptidomimetics CWP232291 Acute myeloid leukemia NCT01398462 Targets β-catenin for degradation and inhibits the expression of cell cycle and anti-apoptotic genes such as cyclin D1 and survivin 
 Inhibition of post-translational palmitoylation of Wnt ligands ETC-1922159 Solid cancers NCT02521844 Loss of cell cycle, stem cell and proliferation genes, and an increase in differentiation markers (differentiation therapy) 
 Decoy receptor for Wnt OMP-54F28 Solid cancers NCT01608867 Impedes the growth of numerous solid tumour types and selectively reduced CSCs 
 Porcupine inhibitor LGK974 Solid malignancies NCT01351103 Prolongs metastasis-free survival in a mouse model of Ewing sarcoma and inhibits migration of ES cell lines through the suppression of expression of multiple transcriptional regulators of EMP 
 NTR1 antagonist SR48692 HCC Preclinical Inhibits metastasis in mice 
 Inhibits the recruiting of β-catenin with its co-activator CBP PRI-724 Metastatic pancreatic, advanced myeloid malignancies, metastatic CRC NCT01764477; NCT01606579; NCT02413853 CSC-targeting drugs 
Inhibitors of intracellular signalling pathways 
SRC inhibitors Tyrosine Kinase inhibitor Ponatinib (AP23464) CML NCT02467270 
  Dasatinib (BMS-354825) Haematologic malignancies, advanced solid tumours NCT01643603; NCT01609816; NCT02389309 Blocks TGF-β1-induced EMP in PDAC cell lines 
  Bosutinib (SKI-606) Malignant solid tumours NCT00195260 Reduces tumour growth, invasion, EMP and distant metastasis in a mouse model of thyroid cancer 
FAK inhibitors Tyrosine Kinase inhibitor Defactinib (VS-6063) NSCLC, solid tumours, ovarian cancer, relapsed malignant mesothelioma NCT01951690; NCT01943292; NCT01778803; NCT02372227 Targets mesothelioma CSCs, reversal of drug resistance 
 Small-molecule inhibitor PF-00562271 Advanced non-haematological malignancies NCT00666926 (discontinue) 
PI3K/AKT/mTOR inhibitors PI3K inhibitor Idelalisib CLL NCT02662296 
 AKT inhibitor AZD5363 MBC, malignant female reproductive system neoplasm NCT02423603; NCT02208375 Tumour growth inhibition 
  Temsirolimus Advanced cancer NCT01529593; NCT02389309 Addition with the autophagy inhibitor (chloroquine) to EMP-induced cells decreases their viability 
 Tyrosine kinase inhibitor CX-4945 Cholangiocarcinoma NCT02128282 Blocks TGF-β1-induced EMP in A549 human lung adenocarcinoma cells 
 Tyrosine kinase inhibitor VS-5584 Relapsed malignant mesothelioma, lymphoma, advanced non-haematological malignancies NCT02372227; NCT01991938 Targets CSC in breast cancer models 
 AURKA/SYK Tyrosine kinase inhibitor Midostaurin Leukaemia, CRC NCT01282502 Post-EMP breast cancer cell-specific drug 
AXL inhibitors Tyrosine Kinase inhibitor BGB324 AML, NSCLC NCT02488408; NCT02424617 Blocks EMP 
  Glesatinib (MGCD265) NSCLC NCT02544633 
RAS/RAF/MAPK inhibitors BRAF inhibitors Vemurafenib and dabrafenib Metastatic melanoma NCT02052193 
 A RAF inhibitor Sorafenib RCC and HCC NCT01444807; NCT02733809 EMP suppression in HCC 
 MEK inhibitor Trametinib Melanoma NCT02858921 EMP modulator in ACHN cells 
Inhibitors of transcription factors that indirectly induce EMP 
JAK and STAT3 inhibitors Small-molecule inhibitor STAT3 inhibitor: OPB-31121 Solid tumours NCT00955812 
  STAT3: BB1608 (Napabucasin) Advanced CRC, refractory haematological malignancies, mesothelioma, advanced glioblastoma NCT01830621; NCT02352558; NCT02347917; NCT02315534 Blocks spherogenesis of CSCs, kills CSCs and down-regulates CSC-associated genes 
 JAK2 and STAT3 inhibitor WP1066 Malignant glioma and brain metastasis from melanoma NCT01904123 Reverses EMP in HNSCC 
Compounds acting on epigenetic modulators 
Histone deacetylase inhibitor  Vorinostat Advanced breast cancer, AML NCT01118975; NCT00368875; NCT02412475 Exerts EMP reversal effects 
  Romidepsin Relapsed lymphoid malignancies, T-cell lymphoma NCT01998035; NCT01638533; NCT01738594 
  Mocetinostat Advanced solid tumors and NSCLC NCT02805660; NCT02954991 Supresses EMP and induces chemosensitivity in breast and pancreatic cell lines 
  Panobinostat Unresectable III/IV melanoma, glioma NCT02032810; NCT02717455 Modulates EMP markers in HCC 
Histone demethylase inhibitor LSD1 inhibitor Tranylcypromine AML NCT02717884 Supresses EMP and invasion 
  GSK2879552 SCLC, AML NCT02034123; NCT02177812 Supresses EMP and invasion 
Histone methyl transferases inhibitor EZH2 inhibitor GSK503 Melanoma Preclinical 
  JQEZ5 Lung adenocarcinomas Preclinical 
  E7438 (Tazemetostat, EPZ-6438) Haematological and solid tumours, sarcoma, melanoma, mesothelioma NCT01897571; NCT02601950; NCT02860286 
 EZH1/2 inhibitor DS-3201b Non-Hodgkin's lymphoma NCT02732275 
Inhibitors of stimuli from the tumour microenvironment 
HIF-1α inhibitors Antisense oligonucleotide EZN-2698 Advanced solid tumours, metastatic RCC Preclinical Tumour cell growth inhibition, down-regulation of HIF-1α target genes and impaired ability of HUVEC cells to form tubes in vitro 
 Small-molecule inhibitor PX-478 Advanced metastatic cancer NCT00522652 In vitro and in vivo inhibition of EMP in ESCC 
  PT2385 RCC NCT02293980 Reduces tumour growth and tumour vascular area in VHL−/− clear cell renal cell carcinoma (ccRCC) patient derived xenografts 
  Digoxin MBC, head and neck cancer, prostate cancer NCT01887288; NCT02906800; NCT01162135 Inhibits LOX and blocks metastatic niche formation in the lungs of MDA-MB — 231 tumour-bearing mice 
 Clusterins Humanised monoclonal antibody against secreted clusterin (sCLU) AB-16B5 Advanced solid tumours NCT02412462 Inhibits TGF-β-induced EMP in several HCC tumour cell lines 

Abbreviations: CRC, colorectal cancer; CLL, chronic lymphocytic leukaemia; CML, chronic myeloid leukaemia; CSC, cancer stem cells; EMP, epithelial–mesenchymal transition; ESCC, oesophageal squamous cell cancer; FAK, focal adhesion kinase; FGF, fibroblast growth factor; FGFR, FGF receptor; HCC, hepatocellular carcinoma; HGF, hepatocyte growth factor; HIF-1α, hypoxia-inducible factor 1α; HNSCC, head and neck squamous cell cancer; IL-6, interleukin-6; IL-6R, IL-6 receptor; JAK, Janus kinase; LOX, lysyl oxidase; LOXL2, LOX-like protein 2; mAb, monoclonal antibody; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; MBC, metastatic breast cancer; miR, microRNA; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor κB; NSCLC, non-small-cell lung cancer; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PI3K, phosphoinositide 3-kinase; RCC, renal cell carcinoma; STAT3, signal transducer and activator of transcription 3; TGF-β, transforming growth factor-β.

*The clinical trial information was obtained from the ClinicalTrials.gov database. Wherever, for the different search terms, the recovery resulted in a lot of studies, only the studies in the current open stage of recruitment and for advanced cancers were selected. X reflects that no direct correlation of drug molecule with respect to EMP and CSC inhibition is proven yet.

TGF-β inhibitors are some of the most extensively trialled agents, as TGF-β signalling is heavily implicated in tumour progression, EMP induction and an ability to augment adaptive immune responses against tumours [130,131]. TGF-β ligands, their receptors and subsequent signal transduction mechanisms have been targeted using strategies involving small-molecule inhibitors, antisense oligonucleotides, blocking monoclonal antibodies and receptor tyrosine kinase inhibitors (RTKIs) to block TGF-β-associated signalling. Efficacy of the TGF-β RTKI LY2157299 was also validated in glioblastoma patients with no reported cardiovascular toxicity [132], which has historically delayed the clinical development of TGF-β antagonists [133]. LY2157299 is being evaluated in many ongoing trials in metastatic pancreatic cancer, recurrent glioblastoma, hepatocellular carcinoma, androgen receptor castration-resistant prostate cancer and triple-negative breast cancer, reflecting the promise that this antagonist holds for aiding treatment of solid malignancies. TEW 7197 (NCT02160106) is a small-molecule inhibitor that blocks TGF-β R1 from phosphorylating its intracellular substrates and is being trialled in combination with LY2157299 in HCC (hepatocellular carcinoma) [134] (NCT02178358).

Safety, efficacy and tolerability of AP12009 (Trabedersen), an 18-oligomer antisense phosphorothioate oligodeoxynucleotide that blocks the production of TGF-β2, was confirmed in advanced pancreatic, melanoma and colorectal cancers overproducing TGF-β2 (NCT00761280). Additional trials using AP12009 have also been undertaken to treat solid tumours and showed small increases in overall patient survival (NCT00844064) [135,136]. Phase IIb clinical trials (NCT00431561) have also been completed in refractory glioma patients, validating its ability to improve the 2- to 3-year survival rate [137]. Many other pathways involved in EMP signalling have been extensively characterised in the preclinical setting, including PI3K–AKT–mTOR (mammalian targeted by rapamycin) [138], RAF–RAS–MAPK [139], Wnt–β-catenin [140], NOTCH [141], Hedgehog [142] and Axl–Gas6 (growth arrest-specific 6) pathways [143,144]. Inhibition of these pathways has been reported to prevent and reverse EMP in some models, and thus may serve as targets for the development of specific inhibitors [145148].

Tamoxifen-resistant MCF-7 breast cancer cells have showed constitutive STAT3 phosphorylation, and NOTCH inhibition with the inhibitor DAPT showed decreased STAT3, as well as a reduction of micrometastatic burden in mice [149]. Inhibition of FGFR signalling in chemoresistant breast cancer cells sensitised them to lapatinib, suggesting that FGFR signalling has a role in maintaining a resistant mesenchymal phenotype in breast cancer cells [150]. NTS/NTR1 was shown to activate Wnt/β-catenin signalling in HCC cells, leading to EMP and invasion in vitro. NTR1 antagonist SR48692 blocked such signalling and also inhibited metastasis in mice [151].

Tyrosine kinase inhibitors affecting PI3K–mTOR, FAK (focal adhesion kinase)–SRC and AXL are also being investigated as therapies in preclinical and clinical studies for their potential to affect control upon EMP programmes [152155]. Concerns surrounding the clinical use of TKIs involve the undesired effects they may have on apoptotic pathways [156], up-regulation of ATP-binding cassette (ABC) drug efflux transporters, as well as the possibility that point mutations within kinases that may render them resistant to such treatment.

Some studies have successfully made the leap to clinical translation. Chemical library screening of MCF10A cells overexpressing TWIST revealed the potential of kinase inhibitor midostaurin in blocking EMP. It was shown to block Aurora A (AURKA) and spleen tyrosine kinase (SYK) activity, in turn abrogating STAT3 signalling associated with EMP phenotypes and causing selective apoptosis of these cells [157]. It is currently in clinical trial to treat leukaemia and advanced colorectal cancer (NCT01282502). Kinase inhibitor VS-5584 blocks PI3K isoforms from complexing with mTOR1/2 and significantly reduces xenograft CSC production, as well as synergising with cisplatin to slow tumour growth and delay metastasis in vivo. It is also currently recruiting in phase I trial for the treatment of lymphomas (NCT01991938).

CLU is transcriptionally activated by TWIST1 and has been shown to potentiate TGF-β-induced EMP in vitro and in vivo [158160]. The subsequent development of a humanised monoclonal antibody (AB-16B5) to inhibit the activity of secreted CLU has led to its trial in treating advanced solid malignancies (NCT02412462). This could potentially be used in conjunction with current frontline treatments, such as the epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erolitinib, to which patients are known to develop drug resistance [161], and notably, EMP was associated with erlotinib resistance in NSCLC [162,163], highlighting the potential benefit of using these CLU-targeted therapies in combination with conventional chemotherapies prior to emergence of resistance. Several interventional studies are in early recruitment stages aiming to target therapies based on personalised patient genomics and high-throughput drug sensitivity assays to target advanced refractory solid tumours, lymphomas, leukaemias and multiple myelomas (NCT02465060, NCT02788201 and NCT02551718).

Given the broad range of cellular contexts and signalling pathways that are known to regulate EMP, many of which are considered important to many cancer attributes and thus already being considered for therapeutic development, this is a major strategy for EMP inhibition. EMP inhibition by targeting such general cancer regulators must be considered in the broader context, and in most cases, we do not know the degree to which EMP contributes to the respective oncobiology, and thus the degree to which EMP inhibition participates in the overall efficacy affecting tumour growth, metastatic progression and therapy resistance. Through iterative assessment of EMP in the trials mentioned above, we will gain a better understanding of these key issues.

Monoclonal antibodies targeting EMP regulators

Additional avenues to regulate EMP may also include the use of mAbs to target carcinoma progression through activation of immune responses to antibody-dependent cellular cytotoxicity. A phase II trial of a human anti-TGF-β antibody (fresolimumab) in combination with radiotherapy is being conducted in 24 metastatic breast cancer patients (NCT01401062). Etanercept (Enbrel), a recombinant humanised mAb, and Infliximab, a chimeric human–mouse mAb devised against TNF-α, have shown promising results in disease stabilisation in various advanced metastatic cancers [164,165]. Siltuximab, an anti-IL-6 chimeric mAb, has also shown encouraging results for the treatment of metastatic renal cell carcinoma in phase I/II clinical trials (NCT 00265135) [166].

AXL RTK is overexpressed during EMP induction and has been implicated in drug resistance, invasion and proliferation of many cancers. Its inhibition has been shown to sensitise mesenchymal cells to anti-mitotic drugs [144,146]. Preclinical validation of an antibody targeting the AXL receptor in pancreatic cancer has been performed [167]. Therapeutic mAbs against RTK ROR1 have also been designed, despite its restricted cell surface expression in paediatric B-lineage acute lymphoblastic leukaemia [168]. ROR1 considers a CSC marker and its expression is associated with EMP phenotypes. Treatment using humanised mAb against ROR1 inhibited ovarian cancer cell engraftment in mice and also reduced the number of metastatic lesions in mouse models of breast adenocarcinomas [169,170]. mAbs designed against several CSC-specific biomolecules, such as Delta-like protein 4 (DLL4), Notch 2, Notch 3 and Frizzled receptors, have shown promising results in preclinical studies [171173] and are being further investigated in clinical trials in combination therapies (Table 1).

Transcriptional control of EMP drivers by epigenetic modifiers

Targeting of epigenetic modification represents an emerging therapeutic window to reversibly control gene expression. Histone-modifying enzymes which act to relax or compress chromatin structure can either activate or repress gene transcription, respectively, by modifying residues in the histone complex. Core histones H3 and H4 are most commonly targeted for regulation by acetylation or methylation. Histone acetyltransferases (HATs) acetylate lysine residues K9 or K14 on histone H3, generally activating transcription, while histone deacetylases (HDACs) catalyse their removal and act to repress transcription. Conversely, histone methyltransferases (HMTs) generally supress transcription and histone demethylases (KDMs) allow transcription to proceed. H3K9, H3K27 and H4K20 methylation are associated with transcription repression, whereas methylation of H3K4 and H3K36, as well as more general acetylation of H3 and H4, are generally indicative of gene activation. Owing to their pivotal roles in numerous disease states, major programmes are underway to discover and develop inhibitors of epigenetic regulators.

Targeting histone demethylases

LSD1 (KDM1A) was the first discovered and is the best characterised histone demethylase, and is required for SNAI1-mediated repression of epithelial genes during EMP. It acts by removing methylation of H3K4m2 marks following SNAI1 transcription factor binding, thus affecting the suppression of gene transcription [174]. Contradictory studies showing that the LSD1 complex acts to supress EMP and invasion [175] highlight the difficulty in attributing direct phenotypes to epigenetic regulation due to the multiple histone substrates on which these enzymes act. Although not specifically targeting its role in SNAI1-induced EMP, LSD1 inhibitors are in clinical trial for other anti-tumour properties and may provide insights into the EMP-related activities upon review and parallel analysis. The LSD1 inhibitor Tranylcypromine is currently in phase I clinical trial for acute myeloid leukaemia (AML) in combination therapy with all-trans retinoic acid (NCT02717884). GSK2879552, another selective LSD1 inhibitor, is in clinical trial in patients with relapsed or refractory small-cell lung cancer (SCLC) (NCT02034123), as well as AML (NCT02177812). Other solid tumours and advanced malignancies are being tested with the LSD1 inhibitor INCB059872 (NCT02712905). Thus, multiple LSD1/KDM1A inhibitor trials, in which EMP could be playing an integral role, are ongoing; however, the contribution of EMP modulation remains to be identified.

Histone methyltransferases

EZH2 is a catalytic component of the Polycomb repressive complex 2 and has been shown to be highly expressed in several aggressive cancer types [176], where its expression inversely correlates with patient survival [177]. EZH2 is responsible for trimethylation of lysine 27 on histone H3 (H3K27me3), an epigenomic transcriptional suppression mark that has been shown to contribute to oncogenesis and metastasis. Most notably, it acts to repress epithelial genes, including E-cadherin, and induce EMP upon induction by TGF-β through transcription factor SOX4, in vitro and in vivo [178,179]. This SOX4/EZH2 expression axis has been observed in pancreatic and breast cancer patient tissues [180,181].

EZH2 is silenced by miR-101, and high mir-101 levels in endometrial cancer cells inversely correlated with EZH2 expression, enforcing an epithelial phenotype and inhibiting migration and invasion of endometrial cancer cells in vitro [182]. Similarly, EZH2-mediated gene suppression promoted EMP and cisplatin-resistance in epithelial ovarian cancer [183]. Genetic ablation of EZH2 or inhibition by GSK503 also abolished metastasis in mouse models of cutaneous melanoma [184]. EZH2 has been shown to associate with the 3′-end of the lncRNA MALAT1, an association that was required for E-cadherin repression and oxaliplatin-resistance in CRC (colorectal cancer) cells, and was also shown to negatively regulate CLDN14, which is required for tight junction complexes in HCC cells [185]. Inhibition of EZH2 in multiple myeloma cells promoted expression of tumour suppressive genes in vitro and in vivo [186], and the novel inhibitor JQEZ5 was shown to promote regression of lung adenocarcinomas in vivo. Taken together, these data highlight the validity of approaches to inhibit EZH2 activity to repress EMP and the importance of developing specific inhibitors for such EMP processes.

Other members of the HMT family are also under investigation for their roles in metastasis and are known to affect EMP. SET8 monomethylates H4K20me1 in prostate cancer cells and was shown to associate with ZEB1 to repress E-cadherin and activate vimentin expression [187]. G9a, another HMT, has been implicated in chemoresistance [188,189], and its association with patient outcome is being verified in patient cancers (NCT01271764), although its involvement in EMP is not yet known. Thus, while EMP phenotypes are not primary endpoints of current clinical trials involving HMT inhibitors, these trials may yield subtle insights into epigenetic regulation of EMP. The EZH2 inhibitor E7438 (Tazemetostat, EPZ-6438) is currently in trial against both haematological and solid tumours (NCT01897571). Tazemetostat is under trial to treat sarcoma, melanoma (NCT02601950) and malignant mesothelioma (NCT02860286), all of which are mesenchymal, so it will be interesting to see whether any epithelial change is seen in the treated cancer cells. DS-3201b, a dual EZH1/2 inhibitor, is being evaluated in non-Hodgkin's lymphoma (NCT02732275).

HDAC inhibitors

Not surprisingly, given the EMP implications of histone modifications mentioned above, Class I HDACs are also heavily implicated in regulating EMP. A recent screen of HDAC inhibitors that up-regulated E-cadherin expression identified several active compounds, all of which had selective activity for class I HDACs [190]. A fold increase in E-cadherin transcription far surpassed the fold reduction in EMP TFs, suggesting that class I HDAC inhibitory compounds exhibited direct epigenetic regulation of E-cadherin. Treatment of mesenchymal-like cells using class I HDAC inhibitors such as vorinostat, mocetinostat and entinostat showed reduced anchorage-independent growth and spheroid formation, both of which have been associated with EMP. Of note in the present study, epithelial cells showed some up-regulation of EMP TFs upon class I HDAC inhibitor treatment, despite small increases in E-cadherin, whereas mesenchymal-like cells showed strong re-epithelialisation, suggesting context-specific effects of the HDAC inhibitor compounds in opposite directions. This again supports the notion that heterogeneity of histone complexes with genomic loci in the cell underpins such context-dependent effects, and gives credence to the universal appeal of targeting the plasticisers in combatting EMP. Many HDACs inhibitors are being widely tested as anti-cancer therapies [191], and so their adoption to validate in vitro effects on EMP phenotypes is a logical step.

Several specific EMP modulations have already been reported with various HDAC inhibitors. Mocetinostat interfered with ZEB1 expression, restoring miR-203 expression, suppressing EMP and inducing chemosensitivity in breast and pancreatic cell lines [192]. Pan HDAC class 1/II inhibitor vorinostat inhibited EMP and reduced chemoresistance in biliary tract cancer, inhibiting SMAD4 translocation to the nucleus [193]. Valproic acid supressed EMP in TE9 oesophageal cancer cells [194]; however, trichostatin A and valproic acid induced EMP in colon carcinoma cells [195], with induction of SNAI1 [196], again highlighting context-depended mechanisms of the action of such compounds.

Trials combining paclitaxel and bevacizumab with vorinostat in breast cancer are being investigated for synergistic effects in combating metastatic deposits (NCT00368875). Vorinostat in combination with bevacizumab, targeting hypoxia-inducible factor 1α/VEGF signalling, has been tested in clear cell renal carcinoma and showed limited toxicity but required further investigation to show efficacy compared with bevacizumab alone (NCT00324870) [197]. Vorinostat in combination with carboplatin/paclitaxel was shown to enhance efficacy against NCSLC [198], and this association is being investigated in several clinical trials (NCT01281176, NCT00616967 and NCT00481078). Neoadjuvant trials combining vorinostat with gemcitabine combination therapies are currently underway in patients with pancreatic cancer (NCT02349867) [199]. Combination treatments to inhibit both DNA methyltransferases and histone deacetylases, by decitabine plus vorinostat, respectively, are also in trial to overcome drug resistance and relapse in AML (NCT02412475). The chemopreventative effects of valproic acid are also being assessed in head and neck squamous cell cancer (NCT02608736).

Novel approaches to epigenomic regulation

Emerging technologies revolving around the use of CRISPR/Cas9 systems to affect gene control are an exciting prospect to provide targeted therapies. Once the groundwork to properly characterise the epigenetic regulation of candidate molecules is complete, specific regions can be targeted for activation or repression. DCas9 DNA methyltransferase fusion proteins have been developed to allow targeted imprinting of genomic loci [200]. Similarly, guided approaches to control histone modifications by Cas9–HAT [201] or Cas9–histone methylases [202] have also been developed and provide an excellent opportunity to explore the translational uses of such technologies.

Of particular relevance to the present study, regulation of EMP is mediated and/or reinforced by histone modification and DNA methylation [15,203,204], making this an important approach, especially given the opposing roles played by different EMP states. It is possible that blocking the invasive stem-like and chemoresistant mesenchymal state could, in certain circumstances, drive the colonising potential associated with the epithelial state. Reciprocally, targeting the epithelial colonisation state may drive early invasive events and therapy resistance. Targeting the epigenetic ‘plasticisers’ may offer advantages that circumvent this problem that may be seen if either of the end-states are targeted.

Targeting translational control by microRNAs

Genomic and functional studies have increasingly highlighted the importance of miRs in cancer [205]. Their frequent dysregulation is responsible for a myriad of deleterious downstream phenotypes due to their ability to regulate several target transcripts, thereby affecting multiple signalling networks simultaneously. Disruptions in the expression of the enzymes responsible for miR biogenesis, DROSHA, DICER and AGO2, and the selective deregulation of miR expression is observed across diverse cancer types [206211]. The tumour suppressive and oncogenic roles of miRs and their dysregulation in cancer have been reviewed extensively elsewhere [212221].

Both the aberrant down-regulation of tumour suppressive miRs and overexpression of oncogenic miRs in cancer progression lend themselves to the notion of miR-based therapies to restore cellular homeostasis. Their ability to regulate multiple mRNA targets, and hence pathways, makes them promising candidates with which to modulate complex cellular phenotypes in vivo. Hence, the introduction of synthetic miRs (miR mimics) to restore miR activity and antisense miRs (anti-miRs) to suppress miR availability are feasible methods to manipulate miRs in vivo. Such approaches also represent an opportunity to redirect a target cell's phenotype by manipulating its own endogenous processes and may produce favourable outcomes when compared with current cytotoxic chemotherapies in terms of side effects. miRs may also offer a prognostic and/or predictive value in guiding disease status and treatment.

Although miRNAs have been shown to regulate many phenotypes relevant to carcinogenesis [222], several have a specific and robust implication in EMP, and as such metastasis and chemoresistance in vivo [218,223]. The miRs discussed below and summarised in Table 2 highlight the diversity of pathways upon which they act to regulate EMP.

Table 2
Consolidated information of miRNAs targeting EMP factors
miRNA Cancer cell type Target EMP regulation References 
10b Breast, bladder, ovarian, osteosarcoma, endometrial, colorectal, nasopharyngeal, NSCLC, pancreatic HOXD1, KLF4, E-Cad 3′-UTR Positive [333336
15b Oral SCC TRIM14 Negative [337
20 CRC SMAD4 Positive [338
21 Ovarian, prostate, gastric, CCA, ccRCC, NSCLC, breast PDCD4, SPRY2, SMAD7, PIK3R1 Positive [339345
23b HCC PYK2 Negative [346
26 Doctaxel-resistant NSCLC, HCC EZH2, SMAD1 Negative [347,348
27a Oral SCC, gastric YAP1, APC Negative [349,350
30a NSCLC, CRC SNAI1, ITGB3 Negative [351,352
32 NSCLC TWIST1 Negative [353
34 Breast, CRC, prostate, NSCLC SNAI1, BCL-2, SIRT1 Positive [237
92 Bladder GSK3 Positive [354
93 Breast, lung, endometrial WNK1, NEDD4L Positive [355,356
101 Endometrial, oral SCC, pancreatic, NSCLC, ovarian EZH2, MCL-1, FOS Negative [182,357,358
106 CRC PRRX1 Negative [359
124 NSCLC, endometrial, cervical, CRC, HCC, breast, prostate IQGAP1, AEG-1, Slug, Zeb1 Negative [360366
132/212 Prostate SOX4 Negative [367
135 NSCLC KLF8 Negative [368
138 NSCLC, SCC ZEB2, VIM, EZH2 Negative [369,370
144 HCC, breast SMAD4, ZEB1/2 Negative [371,372
145 Cervical, pancreatic, osteosarcoma, CRC, breast, renal SIP1, ANG2, ERG, LASP1, MRP1, ANGPT2, NEDD9 Negative [373375,377,378
150 NSCLC FOX04 Positive [379
151-3 Breast TWIST1 Negative [380
154 NSCLC, prostate ZEB2, HMGA2 Negative [381,382
182 Breast, CRC SMAD7, FoxF2, β-TrCP2, TRIM8 Positive [376,383385
187-3p HCC S100A4 Negative [386
195 Prostate FGF2 Negative [387
200 Breast, HCC, CRC, pancreatic ZEB1 Positive [45,246,247
203, 211 Cervical, ovarian, HNSCC, NSCLC, breast MUC4, NUAK1, SMAD3, ZEB2 Negative [388392
204 Oesophageal, breast, osteosarcoma, gastric, NSCLC FOXM1, Six1, Sirt1 Negative [393398
206 NSCLC MET Negative [399
214 Cholangiocarcinoma, cervical Twist, ARL2 Negative [400,401
218 Gastric, renal WASF3, CAV2 Negative [402,403
296 CRC, ovarian S100A4 Negative [404,405
300 CRC, HNSCC P53, TWIST1 Positive/Negative [406,407
367 PDAC SMAD7 Positive [408
375 CRC, prostate SP1, YAP1 Negative [28,409
410 Breast SNAI1 Negative [410
424-5p Oesophageal SCC SMAD7 Positive [411
449 HCC SOX4 Negative [412
484 Cervical ZEB1, SMAD2 Negative [413
486-5p Prostate SNAI1 Negative [414
497 CRC FRA-1 Negative [415
543 Prostate RKIP Positive [416
564 Breast AKT2, GNA12, GYS1, SRF Negative [417
580 Breast TWIST1 Negative [418
598 CRC JAG1 Negative [419
616 HCC PTEN Positive [420
655 Breast PRRX1 Negative [421
671 Breast FOXM1 Negative [422
1271 Gastric, PDAC FOXQ1, ZEB1, TWIST1 Negative [423,424
4775 CRC SMAD7 Positive [425
miRNA Cancer cell type Target EMP regulation References 
10b Breast, bladder, ovarian, osteosarcoma, endometrial, colorectal, nasopharyngeal, NSCLC, pancreatic HOXD1, KLF4, E-Cad 3′-UTR Positive [333336
15b Oral SCC TRIM14 Negative [337
20 CRC SMAD4 Positive [338
21 Ovarian, prostate, gastric, CCA, ccRCC, NSCLC, breast PDCD4, SPRY2, SMAD7, PIK3R1 Positive [339345
23b HCC PYK2 Negative [346
26 Doctaxel-resistant NSCLC, HCC EZH2, SMAD1 Negative [347,348
27a Oral SCC, gastric YAP1, APC Negative [349,350
30a NSCLC, CRC SNAI1, ITGB3 Negative [351,352
32 NSCLC TWIST1 Negative [353
34 Breast, CRC, prostate, NSCLC SNAI1, BCL-2, SIRT1 Positive [237
92 Bladder GSK3 Positive [354
93 Breast, lung, endometrial WNK1, NEDD4L Positive [355,356
101 Endometrial, oral SCC, pancreatic, NSCLC, ovarian EZH2, MCL-1, FOS Negative [182,357,358
106 CRC PRRX1 Negative [359
124 NSCLC, endometrial, cervical, CRC, HCC, breast, prostate IQGAP1, AEG-1, Slug, Zeb1 Negative [360366
132/212 Prostate SOX4 Negative [367
135 NSCLC KLF8 Negative [368
138 NSCLC, SCC ZEB2, VIM, EZH2 Negative [369,370
144 HCC, breast SMAD4, ZEB1/2 Negative [371,372
145 Cervical, pancreatic, osteosarcoma, CRC, breast, renal SIP1, ANG2, ERG, LASP1, MRP1, ANGPT2, NEDD9 Negative [373375,377,378
150 NSCLC FOX04 Positive [379
151-3 Breast TWIST1 Negative [380
154 NSCLC, prostate ZEB2, HMGA2 Negative [381,382
182 Breast, CRC SMAD7, FoxF2, β-TrCP2, TRIM8 Positive [376,383385
187-3p HCC S100A4 Negative [386
195 Prostate FGF2 Negative [387
200 Breast, HCC, CRC, pancreatic ZEB1 Positive [45,246,247
203, 211 Cervical, ovarian, HNSCC, NSCLC, breast MUC4, NUAK1, SMAD3, ZEB2 Negative [388392
204 Oesophageal, breast, osteosarcoma, gastric, NSCLC FOXM1, Six1, Sirt1 Negative [393398
206 NSCLC MET Negative [399
214 Cholangiocarcinoma, cervical Twist, ARL2 Negative [400,401
218 Gastric, renal WASF3, CAV2 Negative [402,403
296 CRC, ovarian S100A4 Negative [404,405
300 CRC, HNSCC P53, TWIST1 Positive/Negative [406,407
367 PDAC SMAD7 Positive [408
375 CRC, prostate SP1, YAP1 Negative [28,409
410 Breast SNAI1 Negative [410
424-5p Oesophageal SCC SMAD7 Positive [411
449 HCC SOX4 Negative [412
484 Cervical ZEB1, SMAD2 Negative [413
486-5p Prostate SNAI1 Negative [414
497 CRC FRA-1 Negative [415
543 Prostate RKIP Positive [416
564 Breast AKT2, GNA12, GYS1, SRF Negative [417
580 Breast TWIST1 Negative [418
598 CRC JAG1 Negative [419
616 HCC PTEN Positive [420
655 Breast PRRX1 Negative [421
671 Breast FOXM1 Negative [422
1271 Gastric, PDAC FOXQ1, ZEB1, TWIST1 Negative [423,424
4775 CRC SMAD7 Positive [425

Antisense miRs or miR mimics are synthetically synthesised double- or single-stranded oligos that are chemically modified in locked nucleic or peptide nucleic acid formats that result in more stable interaction with their mRNA targets. miR sponges are an additional anti-miR format, which possess multiple complimentary sites to the targeted mRNA, resulting in greater efficacy to down-regulate miR activity [224]. Owing to the nature of RNA stability, the use of miRs as treatments requires that they be chemically stabilised and encapsulated to protect them from degradation. Several strategies exist that aim to protect the miRNAs and target them to cancer cells, while minimising toxicity or adverse immune responses to the delivery vehicle.

Polymers, which electrostatically coat the nucleic acid, improve stability in circulation and allow accumulation at the target site. Such vehicles commonly used for delivery in vivo include PEI and PEG derivatives. These delivery techniques are reviewed elsewhere [225]; however, notable vehicles aside from that used in the MRX34 formulation have been developed for gene therapy approaches. Modified PEI–PEG conjugates featuring MMP2 cleavable peptide sequences have been developed to allow specific release of their miR cargo at tumour sites high in MMP2 activity in vivo [226]. Bacterially derived, EDV nanocell, an EGFR antibody-targeted vehicle, has also been recently developed to deliver nucleotide-based therapies [227]. Trials using such a vehicle to target mesothelioma and NSCLC and deliver mir-16 mimics are recruiting (NCT02369198) and offer a novel approach for gene therapy delivery.

Mir-34 and -200 families

MiR-34 family miRNAs are well studied and are the most advanced in terms of clinical application. MiR-34 family members are transcriptionally activated by tumour suppressor P53, which plays a central role in the DNA damage response, and expression of both these molecules is frequently lost in cancer. The miR-34 a/b/c family regulates translation of cell cycle genes CDK4, CDK6, anti-apoptosis proteins BCL-2 and SIRT1, immune evasion proteins PD1/PDL1 as well as metastasis-associated genes MET, BMI1, CD44, CD133, OLFM4, WNT 1/3, MEK1, HDAC1, Nanog, AXL, NOTCH and MYC [228,229]. Importantly, mir-34 has been shown to regulate SNAI1, thereby directly controlling the loss of the epithelial phenotype [230]. MiR-34 has been shown to affect these processes in models of breast cancer, colon cancer [231], prostate cancer [232], chondrosarcoma, NSCLC [233], B-cell lymphoma [234] and pancreatic cancer [235]. Given the extent of support for miR-34's role in cancer, it is therefore not surprising that mimics have been developed to restore its tumour suppressive function. Mimics that restore endogenous miR-34 activity have been tested in vitro [232] and in preclinical models [236], spurring the development of a liposomal miR-34 formulation, MRX34. This mimic has been tested in phase 1 clinical trials (NCT01829971), with further pharmacokinetic studies in melanoma patients proposed (NCT02862145). Although the breadth of miR-34's targets has been the likely driver of miR-34 mimic development, it has been identified in a double feedback repressive loop with SNAI1 [237], such that parallel assessment of clinical trial for EMP is warranted.

MRX34 is a double-stranded miR-34a mimic, encapsulated in an amphoteric lipid formulation, NOV40, which is positively charged at low pH, allowing electrostatic adherence and release within the tumour microenvironment. Anionic properties at neutral pH found in the bloodstream minimise toxicity and adherence to endothelial lining of the vasculature. Such formulations possess a long half-life in blood, with good delivery to liver, bone marrow, spleen and lung when injected i.v. [238]. Results released by the trial group up until September 2016 show reduction in miR-34a targets FOXP1 and BCL2 in peripheral blood; however, the trial was halted due to severe adverse immune reactions [239]. While further investigation is required to delineate the cause of such adverse reactions, the liposomal carrier has been used in a separate anti BCL-2 trial with minimal adverse effects [240], pointing to perhaps the use of double-stranded DNA triggering innate immune and inflammatory responses [241,242]. Other immune-related adverse events could be due to action of miR-34a suppressing PD-1/PDL1 and affecting immune checkpoint blockade [243].

MiR-200 family expression has also been heavily implicated for its role in maintaining an epithelial phenotype. Consisting of five members spread over two genomic clusters, miR-200a/b/429 and miR-200c/141, they have been shown to negatively regulate ZEB1 and suppress EMP phenotypes [45,244,245]. TGF-β signalling silences miR-200 expression [246], and this silencing induces EMP through the loss of ZEB1 repression [244,247]. Ectopic expression of miR-200 drives MET through reversal of the same mechanism [244,245,247]. Once induced, ZEB1 binds the miR-200 promoter, repressing its expression and furthering EMP [248]. MiR-192 has been shown to co-operate in a similar mechanism, acting to repress ZEB2. Somewhat surprisingly, despite much in vitro and preclinical interest in miR-200, stable mimics have not yet been developed.

Given the selective loss of miRs in cancers, and the essential regulatory functions they play for immediate control of RNA processing, miRs represent a very attractive strategy for selective cancer therapy. The implication of miR-34 in EMP regulation offers an opportunity to assess the extent to which this sensitive metric is affected by miR-34-targetted approaches. The EMP specificity of the miR-200 family in particular, although not developed therapeutically at this stage, may shed more light on the specific role that EMP targeting may play in isolation of other major cancer activities as those underpinning the original development of miR-34-targeted approaches.

Post-translational control of EMP pathway members

Post-translational modifications (PTMs) of proteins within the cell are responsible for a suite of directive signalling actions, including guiding subcellular localisation, activity, binding associations and degradation. Phosphorylation can trigger proteins to be recruited for nuclear import to affect gene transcription or conversely be exported from the nucleus to repress its effect on gene transcription. Similarly, phosphorylation can act to protect from, or enable, ubiquitin-mediated degradation. Such functional effects of PTMs are important in an EMP signalling context, as they can guide gene transcription and signalling stability of key regulators. Most research, to date, has focussed on the PTMs of EMP TFs that regulate their nuclear localisation and transcriptional activity, and selective targeting of the kinases involved may yield therapeutic benefits.

Transcription factors SNAI1/2, TWIST1/2 and ZEB1/2 directly regulate EMP in cancer cells. They have been well characterised and are extensively reviewed elsewhere [6,249256]. While there is evidence for redundancy of these core transcription factors in inducing EMP across cancer types, in vitro and preclinical models support their inhibition as valid clinical approaches to modulate metastasis and chemoresistance [257].

Of note, SNAI1/2 and TWIST1 are highly unstable proteins, tightly controlled by ubiquitation processes. SNAI1 phosphorylation by PAK1 facilitates its entry into the nucleus, inducing suppression of E-cadherin [258]. LATS2 phosphorylates SNAI1, stabilising and retaining it within the nucleus [259]; however, PKD1 phosphorylates SNAI1 at S-11, resulting in nuclear export and binding to 14-3-3σ, thereby blocking EMP [260]. SNAI1 phosphorylation at S-97 and S-101 by GSK3β induces nuclear export, where it is targeted by β-TrCP for proteasomal degradation [261263]. SNAI2 lacks a GSK3β-binding motif, and it has been shown to be targeted by MDM2 e3 ligase for regulation [264]. Both TWIST and SNAI1 have GSK3β motifs and so are regulated by β-TrCP E3 ligases for degradation. FBXL14 ligase regulates EMP transcription factors SNAI1, SNAI2 and TWIST1 [265,266]. Development of inhibitors for these kinase interactions with their substrates, while cumbersome, may allow tight control of these EMP processes.

MAPK, AKT, GSK3β and IKKβ phosphorylate TWIST1, recruiting FBXL14 or β-TrCP E3 ligases for its ubiquitination and subsequent degradation [265268], and the AKT kinase inhibitor Mk-2206 was shown to stabilise TWIST1, enhancing EMP in breast cancer cells. This could be reversed by resveratrol, inducing TWIST1 to be targeted by β-TrCP for ubiquitination and degradation, in turn reversing EMP [269].

Bromodomain and extraterminal (BET) family proteins bind acetylated lysines on histones and activate gene transcription, and this mechanism is often corrupted to drive oncogenes in cancer. Despite seemly opposing effects, inhibitors of BET and HDAC have been shown to synergise to kill Myc-induced mouse lymphoma [270] and mouse models of PDAC [271].

Acetylation of TWIST at K73/76 was shown to recruit the BRD4/P-TEFβ/RNA-Pol II transcription complex, driving WNT5a expression and WNT5a-associated EMP processes. The small-molecule inhibitor of BRD4, JQ1, which targets the bromodomain responsible for binding WNT5a promoter, suppresses EMP and CSC phenotypes [272]. JQ1 also suppressed tumour growth in xenograft models of PDAC [273] as well as blocked acinar to ductal metaplasia and PanIN formation [271].

Overall, the breadth of catalysis required to regulate EMP lends itself to very specific regulation. The development of specific inhibitors to permit such regulation, however, requires a very fine resolution understanding of the biochemical interactions at play and thus require much further investigation before a therapy may be reached.

Repurposing drugs to target EMP

Current medications already approved to treat human disease are attractive prospects to be repurposed as anti-cancer treatments since their safety and toxicity profile is already established. Several examples affecting EMP are discussed below.

Zidovudine, an anti-viral drug, was demonstrated to inhibit EMP in gemcitabine-resistant pancreatic cancer cells by inhibiting the AKT/GSK3/SNAI1 pathway, significantly reducing tumour formation in a mouse xenograft model [274]. The antimalarial drug, chloroquine, was also demonstrated to play a role in the elimination of CSC traits in pre-malignant lesions [275]. It was shown to act in triple-negative breast cancer through deregulation of JAK2 and DNA methyltransferase 1, as well as through inhibition of CXCR4 and Hedgehog signalling in pancreatic CSCs [276,277].

Metformin, an anti-diabetic compound, was shown to attenuate EMP and induce chemosensitivity by down-regulating key EMP TFs, ZEB1, TWIST1 and SNAI2 [278281]. It was shown to inhibit TGF-β signalling, activating the AMP-activated protein kinase (AMPK) pathway and attenuating ERK signalling, resulting in reduced HDAC activity, subsequent hypomethylation of the E-cadherin promoter and an enforced epithelial phenotype [282]. Metformin also inhibited EMP by blocking IL-6/STAT3 signalling in lung adenocarcinoma [283] and reduced tumour growth in a mouse xenograft model when co-administered with doxorubicin [279]. It was shown to activate AMPK and subsequently inhibit mTOR activity, which reversed the mesenchymal phenotype of cancer cells [281,284,285]. Studies are underway to investigate the efficacy of metformin combined with gefitinib to treat NSCLC (NCT01864681). Metformin is currently a favourable choice in impeding tumour progression [275] as more than 200 human clinical trials have attested to its tolerability.

High-throughput screens to identify ‘repurposable’ small molecules against EMP-induced chemoresistance have also been performed. Salinomycin, a potassium ionophore antibiotic, displayed more than a 100-fold efficacy when compared with conventional paclitaxel treatment against populations of breast cancer stem-like cells, also inhibiting tumour formation in mice [286]. Salinomycin was further shown to overcome doxorubicin resistance in HCC cells and breast cancer cells; however, its mechanism of action remains to be elucidated [287,288]. Several other high-throughput screens for selective activity against EMP-associated stemness have also been undertaken, yielding several active compounds. Examples of these include Withaferin A [289], the antimalarial Artesunate [290], HDAC inhibitor 1-benzylsulfonyl indoline and an acyl hydrazone scaffold [291]. Such advanced screening platforms allow rapid identification of compounds for novel uses, benefiting preclinical investigation by providing a robust phenotype prior to further investigation.

Agents described in this strategy have been identified for their effects on the EMP phenotype; however, they undoubtedly affect a host of cancer hallmarks which may or may not be EMP-associated. Only time and further studies will tell the respective role of EMP regulation in any anti-cancer efficacy that has been or may be seen; however, their ability to perturb EMP supports their potential therapeutic efficacy.

EMP-targeting vaccines

Novel vaccines are becoming an increasingly sought after approach to curtail complex diseases. The principal action of vaccines are to tailor immune cell responses to desired target antigens specific for an undesired cellular process, killing the host cell. A remarkable antitumor effect on lung metastasis was demonstrated in mice vaccinated with recombinant TWIST1 from heat-inactivated yeast [292]. After vaccination, they confirmed that both CD4+ and CD8+ T-cell responses specific for TWIST1 were induced. To extend this approach, non-canonical EMP-TF Brachyury, which is overexpressed in advanced gastrointestinal, bladder, kidney, ovary, uterus and testicular carcinomas, has been successfully targeted in initial clinical trials in patients with advanced tumours (NCT01519817). Subsequent phase II trials in combination with radiotherapy are underway in chordoma patients (NCT02383498) [293296].

Other EMP-targeted vaccines, many as combinatorial therapies, are also in preclinical research and may have translational impacts. These include vaccines designed against EMP drivers Muc1, PINCH-1 and Gastrin-releasing peptide [297299]. Combination immunotherapies proposed by Jiang et al. [300] include the use of curcumin with a fibroblast activation protein α vaccine. This vaccine aims to target EMP-inducing fibroblasts that secrete TNF-α and was shown to boost immune response to melanoma cells implanted in mice. Advantages of such vaccine approaches are that they may provide more tolerability in patients treated with combinatorial chemotherapies.

In light of the current explosive interest and success with immunotherapies, EMP-targeting vaccines, such as Brachyury as described above and other that have likely an impact on EMP, hold great hope and promise.

Nutraceuticals

Nutraceuticals are pharmaceutical grade, natural products and phytochemicals that exhibit pharmacological properties. Several of these compounds have been investigated for their use as potential anti-cancer agents and have hence been shown to affect cellular signalling pathways that affect EMP. Studies aiming to assess these nutraceuticals for their functional roles are ongoing, and a better understanding of their metabolic stability, bio-availability and accumulation at tumour sites is needed. Approaches are being pursued to improve their pharmacodynamics through packaging with various polymeric materials [301]. Those implicated in modulating EMP are discussed below and in Table 3.

Table 3
List of nutraceuticals targeting inducers and associated signalling pathways regulated in EMP
Natural product Derived from Targets Functional endpoint Cancer References 
β-Elemene Herbal medicine Curcuma wenyujin Down-regulates N-cad, CD133, β-catenin and up-regulates E-cad Regulates stemness and reversal of EMT Glioblastoma cancer [426
  TGF-β1/Smad3 pathway Inhibits EMT Breast cancer [427
Curcumin Turmeric BMI1, SUZ12 and EZH2 miRNA induces EMT suppression and chemosensitization to 5-fluorouracil Colorectal cancer [428
EGCG Green tea PI3K/Akt, ERK, HGF and IGF-1 Inhibits cancer cell proliferation Breast cancer, prostate cancer [429
  TNF-α Attenuates pain and inflammation Bone cancer [430
Fucoidan Brown seaweed polysaccharide TGFR/Smad/Snail, Slug, Twist and EMT axes Reduces tumour cell proliferation and metastasis Breast cancer [431
Genistein Soybeans TH1 and MDSC-associated cytokines Immunomodulation via suppressing cytokines Prostate cancer [432
  Akt, GSK-3 Inhibits cancer cell proliferation Prostate cancer [433
  Osteopontin Inhibits metastasis Prostate cancer [434
Lycopene Tomato Akt/mTOR and IGF-1 attenuates carcinogenesis Liver cancer, castration-resistant prostate cancer [435,436
  Met, β-Catenin, mTOR, miR-199a/b, miR 214 Inhibits carcinogen-initiated inflammation Liver cancer [437
Moscatilin Component of orchid Dendrobrium loddigessi Vimentin, Slug and Snail/Akt-Twist-dependent pathway Inhibits EMT and sensitizes anoikis Breast cancer [438
Punicalagin Pomegranate IGF-I/Akt/mTOR Inhibits cancer cell proliferation Prostate cancer [439
Quercetin Flavonoid in many fruits, vegetables and grain Twist, N-cad and Vimentin Inhibits EMT, self-renewal capacity and invasion Head and neck cancer [440
Resveratrol Polyphenol from grapes Metastasis-associated protein 1, TGF-β1/Smads, PTEN/Akt pathway Suppresses EMT Colorectal cancer, prostate cancer [320,441
Silibinin Milk thistle extract IGFβ-3 and IGF Inhibits advanced carcinoma growth Prostate cancer [442
  Snail, ZEB and N-cadherin Suppresses EMT-driven Erolitinib resistance Non-small-cell lung cancer [303
Sulforaphane Broccoli sprout Wnt/β-catenin pathway, ALDH-positive cells Inhibits CSC differentiation Breast cancer [315
  NF-κB and TGF-β Regulates apoptosis, metastasis and angiogenesis Prostate cancer [313
  Twist and Vimentin Inhibits self-renewal capacity of CSCs Pancreatic cancer [314
  TGF-β1, EGF and IGF pathway Perturbs oncogenic signalling pathway Prostate cancer [311
Thymoquinone Plant ingredient Nigella sativa Twist1 Inhibits cancer cell growth, migration and invasion Breast cancer [443
Natural product Derived from Targets Functional endpoint Cancer References 
β-Elemene Herbal medicine Curcuma wenyujin Down-regulates N-cad, CD133, β-catenin and up-regulates E-cad Regulates stemness and reversal of EMT Glioblastoma cancer [426
  TGF-β1/Smad3 pathway Inhibits EMT Breast cancer [427
Curcumin Turmeric BMI1, SUZ12 and EZH2 miRNA induces EMT suppression and chemosensitization to 5-fluorouracil Colorectal cancer [428
EGCG Green tea PI3K/Akt, ERK, HGF and IGF-1 Inhibits cancer cell proliferation Breast cancer, prostate cancer [429
  TNF-α Attenuates pain and inflammation Bone cancer [430
Fucoidan Brown seaweed polysaccharide TGFR/Smad/Snail, Slug, Twist and EMT axes Reduces tumour cell proliferation and metastasis Breast cancer [431
Genistein Soybeans TH1 and MDSC-associated cytokines Immunomodulation via suppressing cytokines Prostate cancer [432
  Akt, GSK-3 Inhibits cancer cell proliferation Prostate cancer [433
  Osteopontin Inhibits metastasis Prostate cancer [434
Lycopene Tomato Akt/mTOR and IGF-1 attenuates carcinogenesis Liver cancer, castration-resistant prostate cancer [435,436
  Met, β-Catenin, mTOR, miR-199a/b, miR 214 Inhibits carcinogen-initiated inflammation Liver cancer [437
Moscatilin Component of orchid Dendrobrium loddigessi Vimentin, Slug and Snail/Akt-Twist-dependent pathway Inhibits EMT and sensitizes anoikis Breast cancer [438
Punicalagin Pomegranate IGF-I/Akt/mTOR Inhibits cancer cell proliferation Prostate cancer [439
Quercetin Flavonoid in many fruits, vegetables and grain Twist, N-cad and Vimentin Inhibits EMT, self-renewal capacity and invasion Head and neck cancer [440
Resveratrol Polyphenol from grapes Metastasis-associated protein 1, TGF-β1/Smads, PTEN/Akt pathway Suppresses EMT Colorectal cancer, prostate cancer [320,441
Silibinin Milk thistle extract IGFβ-3 and IGF Inhibits advanced carcinoma growth Prostate cancer [442
  Snail, ZEB and N-cadherin Suppresses EMT-driven Erolitinib resistance Non-small-cell lung cancer [303
Sulforaphane Broccoli sprout Wnt/β-catenin pathway, ALDH-positive cells Inhibits CSC differentiation Breast cancer [315
  NF-κB and TGF-β Regulates apoptosis, metastasis and angiogenesis Prostate cancer [313
  Twist and Vimentin Inhibits self-renewal capacity of CSCs Pancreatic cancer [314
  TGF-β1, EGF and IGF pathway Perturbs oncogenic signalling pathway Prostate cancer [311
Thymoquinone Plant ingredient Nigella sativa Twist1 Inhibits cancer cell growth, migration and invasion Breast cancer [443

Silibinin

A milk thistle extract silymarin and its constituent silibinin have been shown to exert significant anti-neoplastic effects in different models of skin, breast, lung, colon, bladder, prostate and kidney carcinomas [302]. Silibinin reversed the high miR-21/low miR-200c miRNA signature associated with EMP and repressed mesenchymal markers SNAI1, ZEB1 and N-cadherin, effectively causing MET [303]. Silibinin also acts as an antagonist of STAT3, a major driver of EMP in model systems [304]. A water-soluble, silibinin-rich, milk thistle extract has been observed to reduce tumour volumes by 50% in erlotinib-refractory NSCLC xenografts [304]. Furthermore, its potential clinical utility is being evaluated as Siliphos® in a Phase II clinical trial as adjunct therapy with erlotinib in EGFR mutant NSCLC (NCT02146118). It is also in Phase I trials with oral green tea extract (NCT01239095) in colorectal cancer patients.

Curcumin

The active component of turmeric, curcumin, has been reported to possess multiple activities on apoptosis, cell adhesion and invasion in vitro [305]. Curcumin treatment has been reported to induce degradation of the EMP-related cell adhesion proteins β-catenin, E-cadherin and adenomatous polyposis coli (APC) in HCT-116 cells, where this degradation could be blocked by caspase-3 inhibition [306]. Curcumin, as well as its analogue hydroxycinnamaldehyde, was also shown to inhibit EMP and prevent lung metastasis in a mouse model of breast cancer, via inactivation of SNAI1-dependent NF-κβ signalling [307,308]. Curcumin has been implicated in the inhibition of several Wnt/β-catenin pathway members responsible for down-regulation of E-cadherin [309]. Administration of the derivative difluorinated curcumin, resulted in tumour inhibition in mouse xenograft models of pancreatic cancer with reduced expression of EZH2, Notch-1, CD44, EpCAM and Nanog [310].

Sulforaphane

Sulforaphane is an isothiocyanate generated in the gut from broccoli, which, along with other cruciferous vegetables, has been shown to reduce both the incidence and progression of prostate cancer [311]. It has a multifaceted role in inhibiting carcinogenesis through its effects on the PI3K/AKT and NF-κβ pathways, and modulated EMP, angiogenesis and metastasis via induction of TNF-related apoptosis-inducing ligand R1/DR4, inhibiting the expression of β-catenin, vimentin, TWIST1 and ZEB1 and activating caspase-mediated apoptosis [312314]. Signalling events downstream from androgens, TGF-β, insulin/IGF and EGF were perturbed in biopsy samples from high-grade prostatic intraepithelial neoplasia (HGPIN) lesions in patients treated for 12 months with a broccoli-rich diet compared with a pea-rich diet (NCT00535977), potentially mediated by covalent interactions of these agonists with sulforaphane, as observed in parallel biochemical studies [311]. Sulforaphane has also been shown to inhibit breast and pancreatic CSCs [314,315].

Resveratrol

Resveratrol is a phytoalexin present in mulberries, peanuts, grapes and red wine. It has several biological activities as an antioxidant, antibacterial, anti-inflammatory and cardioprotective agent, as well as anti-cancer properties in breast, prostate, colon, thyroid, skin and pancreatic cancers [316]. This promiscuous molecule has the capability to strongly interact with ∼20 proteins involved in diverse inflammation, metabolism and cell cycle-related pathways [317]. Resveratrol has also been implicated in enforcing an epithelial phenotype in prostate cancer [318] and is reported to inhibit lung and colorectal cancer invasion and metastasis by suppressing TGF-β1/SMAD signalling [319,320]. Resveratrol was also reported to inhibit pancreatic CSC shedding in humans and in K-Ras (G12D) transgenic mice by inhibiting pluripotency-maintaining factors (Nanog, Sox-2, c-Myc and Oct-4) and the drug-resistance gene ABCG2 [321]. A major advantage of using resveratrol as an adjunct therapy is that, to date, no apparent toxicity has been reported in clinical models [321].

Much of the community looks to complementary and alternative medicines rather than manufactured agents, thus the activity nutraceutical has growing relevance in our society. In reality, many of our current pharmaceuticals had their roots in natural products, such that this also represents a very viable approach in the early genesis of newer, potentially more potent forms of such activity. As with many of the agents above, the nutraceuticals discussed here are not EMP-specific, and further work is needed to determine the precise role that EMP alteration may play in the activity seen.

Discussion (caveats and approaches)

Significant advancements have been made in EMP-centric cancer research, but there are some crucial issues that need to be addressed before specifically targeting EMP in earnest. Complexities in designing interventions targeting EMP and CSC phenotypes lie in the lack of clear understanding of the events which predispose cells to enter into such a cell state, as well as the extensive interactions between EMP and many other cancer processes. As demonstrated in this review, genetic instability, epigenetic modification, miRNA regulation and the tumour microenvironment all play integral roles in initiating and controlling the EMP programme, but have additional and often stronger effects in other cancer hallmarks. That said, their effects on EMP, in addition to or as part of their effects on other cancer hallmarks, add to the support for their therapeutic potential, and ultimately, it is the overall effect, safety and side effects that will determine their utility. Although the role of EMP in metastatic spread remains somewhat controversial, a wealth of data to support such a role far outweighs the lack of supportive evidence in two recent studies [105,106]. Moreover, there seems strong consensus that EMP contributes to therapy resistance, so between these two, there is great incentive to consider targeting EMP along the lines described above, and the major approaches have been summarised. These approaches cannot be considered in isolation of the broader outcome-orientated mission of therapeutic development, especially as EMP is implicated in many of the established cancer hallmarks.

Among the many approaches summarised above, inhibition of TGF-β and STAT3 signalling are excellent candidates to date, as they are constitutive key players of EMP induction in many carcinomas [322]. Acquired resistance through mutation or alternative pathway switching is a major hindrance in current drug treatment regimens [156,323,324]. Clinical trialling of a mAb rilotumumab to target the HGF–MET pathway in advanced gastric cancer provided an example of such acquired resistance and resulted in the Phase III trial being discontinued (NCT02137343). It is, therefore, increasingly important to design treatments targeted to pathways that are less able to be bypassed by such resistance mechanisms. Additional complexity lies in targeting growth factor ligands that stimulate EMP as they often act in a context-specific manner as both oncogenic and tumour suppressive. Such temporal variation in their roles during tumour progression is well demonstrated for TGF-β, Wnt–β-catenin and Notch pathways [325327].

The choice of EMP inhibitor requires a significant level of insights into the cross-section of tumour's characteristics, and this information may be used to target relevant EMP drivers; however, further investigation is needed to better understand such cancer- and context-dependent driver specificity. The plastic nature of EMP requires that compounds collectively target the cross-section of cellular phenotypes, including epithelial cells that are in early stages of EMP, as well as developed tumours that have already acquired mesenchymal or persistent intermediate phenotype, which may require different classes of EMP inhibitors. Remarkably, the extent of EMP can be quantified using a universal scoring system developed across different tumour types and patient samples by Thiery and co-workers [328]. The systematic classification of tumour subtypes and CTCs based on their EMP score may prove an invaluable tool in the design of personalised medicines to counter such properties.

Redundancy in the pathways targeted by EMP inhibitors may lead to resistance through switching of alternative mechanistic drivers. The design of EMP inhibitors is thus a daunting task due to the multitude of cross-talk between signalling networks that may act to propagate EMP. The combinations of EMP inhibitors that act on different EMP mechanisms are therefore central to their success as treatments. The combination of Src and FAK inhibitors, dasatinib and capecitabine, respectively, has showed significant promise in Phase I studies against advanced breast cancer, when compared with their monotherapy trials [155,329].

Enforcing an epithelial phenotype to suppress EMP carries with it the potential consequence of driving micrometastasis that may arise from the mesenchymal-like cells that have already evaded the primary tumour. Such de-differentiation therapy [110] may temporarily promote metastatic colonisation within organs not associated with the typical metastatic cascade, and such derivatives may possess unknown pathology. Moreover, such an MET could result in a metastatic growth that possesses a residual EMP memory. This may endow a more locally invasive phenotype with a predisposition to be easily stimulated towards EMP, or an inherent resistance to the chemotherapeutic challenges previously encountered. As a result, compounds that selectively neutralise mesenchymal phenotypes may be advantageous when compared with such de-differentiation approaches. Interestingly, eribulin, an agent derived from sponges which is approved for third-line therapy against metastatic breast cancer and liposarcoma, was recently shown to promote epithelial traits in EMP-affected cancer cells [330,331]. It has also been shown to enhance vascular flow in preclinical models, which together with forced epithelialisation may explain the increase efficacy of eribulin compared with other microtubule inhibitory drugs alone (taxanes, etc.). In particular, eribulin has been shown to selectively reduce new metastatic deposits rather than established metastatic deposits in clinical trials (NCT00388726) [332], consistent with its targeted effects against EMP-activated carcinoma cells.

Importantly, EMP and CSC programmes display many similarities with the EMP required for development and wound healing [147], and so drugs acting to inhibit the Notch, Wnt, Hedgehog pathway might have the capacity to induce unknown side effects. Nevertheless, the classes of EMP inhibitors discussed in the present study have great potential in leveraging multiple factors against metastatic complications, chemoresistance and the overall cancer progression, at least in part through EMP modulation.

Abbreviations

     
  • ABC

    ATP-binding cassette

  •  
  • AML

    acute myeloid leukaemia

  •  
  • AMPK

    AMP-activated protein kinase

  •  
  • anti-miRs

    antisense miRs

  •  
  • AP-1

    AP-1 transcription factor subunit

  •  
  • APC

    adenomatous polyposis coli

  •  
  • AXL

    AXL receptor tyrosine kinase

  •  
  • BET

    bromodomain and extraterminal

  •  
  • BMP

    bone morphogenetic protein

  •  
  • CK

    cytokeratin

  •  
  • CLL

    chronic lymphocytic leukaemia

  •  
  • CLU

    Clusterin

  •  
  • CML

    chronic myeloid leukaemia

  •  
  • CRC

    colorectal cancer

  •  
  • CSC

    cancer stem cells

  •  
  • CTC

    circulating tumour cell

  •  
  • EGF

    epidermal growth factor

  •  
  • EGFR

    epidermal growth factor receptor

  •  
  • EMP

    epithelial–mesenchymal transition

  •  
  • EMT

    epithelial–mesenchymal transition

  •  
  • EpCAM

    epithelial cell adhesion molecule

  •  
  • ER

    estrogen receptor

  •  
  • ESCC

    oesophageal squamous cell cancer

  •  
  • FAK

    focal adhesion kinase

  •  
  • FGF

    fibroblast growth factor

  •  
  • FGFR

    FGF receptor

  •  
  • GSK3β

    glycogen synthase kinase 3 beta

  •  
  • HATs

    histone acetyltransferases

  •  
  • HCC

    hepatocellular carcinoma

  •  
  • HDACs

    histone deacetylases

  •  
  • HER-2

    Erb-B2 receptor tyrosine kinase 2

  •  
  • HGF

    hepatocyte growth factor

  •  
  • HIF-1α

    hypoxia-inducible factor 1α

  •  
  • HIPPO

    HIPPO signal transduction pathway

  •  
  • HMTs

    histone methyltransferases

  •  
  • HNSCC

    head and neck squamous cell cancer

  •  
  • IGF

    insulin like growth factor

  •  
  • IKKβ

    inhibitor of nuclear factor κB kinase subunit beta

  •  
  • ILC

    invasive lobular carcinoma

  •  
  • IL-6

    interleukin-6

  •  
  • IL-6R

    IL-6 receptor

  •  
  • JAK

    Janus kinase

  •  
  • LOX

    lysyl oxidase

  •  
  • LOXL2

    LOX-like protein 2

  •  
  • LSD1

    lysine demethylase 1A

  •  
  • mAb

    monoclonal antibody

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MBC

    metastatic breast cancer

  •  
  • MEK

    MAPK/ERK kinase

  •  
  • MET

    mesenchymal–epithelial transition

  •  
  • miR

    microRNA

  •  
  • MMP

    matrix metalloproteinase

  •  
  • mTOR

    mammalian target of rapamycin

  •  
  • NF-κB

    nuclear factor κB

  •  
  • NOTCH

    NOTCH signal transduction pathway

  •  
  • NSCLC

    non-small-cell lung cancer

  •  
  • NTS/NTR1

    neurotensin/neurotensin receptor

  •  
  • PDAC

    pancreatic ductal adenocarcinoma

  •  
  • PDGF

    platelet-derived growth factor

  •  
  • PDGFR

    PDGF receptor

  •  
  • PEI

    polyethylenimine

  •  
  • PEG

    polyethylene glycol

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • PI3K/AKT

    PI3K/AKT signal transduction pathway

  •  
  • PR

    progesterone receptor

  •  
  • PTMs

    post-translational modifications

  •  
  • RCC

    renal cell carcinoma

  •  
  • RTKIs

    receptor tyrosine kinase inhibitors

  •  
  • SCLC

    small-cell lung cancer

  •  
  • SRC

    SRC proto-oncogene, non-receptor tyrosine kinase

  •  
  • STAT

    signal transducer and activator of transcription

  •  
  • TEFβ

    PAR BZIP transcription factor

  •  
  • TFs

    transcription factors

  •  
  • TGF-β

    transforming growth factor-β

  •  
  • TTF1

    transcription termination factor 1

  •  
  • VEGF

    vascular endothelial growth factor A

  •  
  • WNT

    Wnt family member

  •  
  • ZEB1

    zinc finger E-box binding homeobox 1

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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