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A tEMTing target? Clinical and experimental evidence for epithelial-mesenchymal transition in the progression of cutaneous squamous cell carcinoma (a scoping systematic review)

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Cutaneous squamous cell carcinoma (cSCC) is a disease with globally rising incidence and poor prognosis for patients with advanced or metastatic disease. Epithelial-mesenchymal transition (EMT) is a driver of metastasis in many carcinomas, and cSCC is no exception. We aimed to provide a systematic overview of the clinical and experimental evidence for EMT in cSCC, with critical appraisal of type and quality of the methodology used. We then used this information as rationale for potential drug targets against advanced and metastatic cSCC. All primary literature encompassing clinical and cell-based or xenograft experimental studies reporting on the role of EMT markers or related signalling pathways in the progression of cSCC were considered. A screen of 3443 search results yielded 86 eligible studies comprising 44 experimental studies, 22 clinical studies, and 20 studies integrating both. From the clinical studies a timeline illustrating the alteration of EMT markers and related signalling was evident based on clinical progression of the disease. The experimental studies reveal connections of EMT with a multitude of factors such as genetic disorders, cancer-associated fibroblasts, and matrix remodelling via matrix metalloproteinases and urokinase plasminogen activator. Additionally, EMT was found to be closely tied to environmental factors as well as to stemness in cSCC via NFκB and β-catenin. We conclude that the canonical EGFR, canonical TGF-βR, PI3K/AKT and NFκB signalling are the four signalling pillars that induce EMT in cSCC and could be valuable therapeutic targets. Despite the complexity, EMT markers and pathways are desirable biomarkers and drug targets for the treatment of advanced or metastatic cSCC. Graphical Abstract
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Discover Oncology
Review
A tEMTing target? Clinical andexperimental evidence
forepithelial‑mesenchymal transition intheprogression ofcutaneous
squamous cell carcinoma (a scoping systematic review)
BenjaminGenenger1,2· JayR.Perry1,2· BruceAshford2,3· MarieRanson1,2
Received: 28 April 2022 / Accepted: 27 May 2022
© The Author(s) 2022 OPEN
Abstract
Cutaneous squamous cell carcinoma (cSCC) is a disease with globally rising incidence and poor prognosis for patients
with advanced or metastatic disease. Epithelial-mesenchymal transition (EMT) is a driver of metastasis in many carcino-
mas, and cSCC is no exception. We aimed to provide a systematic overview of the clinical and experimental evidence
for EMT in cSCC, with critical appraisal of type and quality of the methodology used. We then used this information as
rationale for potential drug targets against advanced and metastatic cSCC. All primary literature encompassing clinical
and cell-based or xenograft experimental studies reporting on the role of EMT markers or related signalling pathways
in the progression of cSCC were considered. A screen of 3443 search results yielded 86 eligible studies comprising 44
experimental studies, 22 clinical studies, and 20 studies integrating both. From the clinical studies a timeline illustrat-
ing the alteration of EMT markers and related signalling was evident based on clinical progression of the disease. The
experimental studies reveal connections of EMT with a multitude of factors such as genetic disorders, cancer-associated
broblasts, and matrix remodelling via matrix metalloproteinases and urokinase plasminogen activator. Additionally,
EMT was found to be closely tied to environmental factors as well as to stemness in cSCC via NFκB and β-catenin. We
conclude that the canonical EGFR, canonical TGF-βR, PI3K/AKT and NFκB signalling are the four signalling pillars that
induce EMT in cSCC and could be valuable therapeutic targets. Despite the complexity, EMT markers and pathways are
desirable biomarkers and drug targets for the treatment of advanced or metastatic cSCC.
Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s12672- 022-
00510-4.
* Benjamin Genenger, bg038@uowmail.edu.au; * Marie Ranson, mranson@uow.edu.au | 1School ofChemistry andMolecular
Bioscience, University ofWollongong, Wollongong, NSW, Australia. 2Illawarra Health andMedical Research Institute, Wollongong, NSW,
Australia. 3School ofMedicine, University ofWollongong, Wollongong, NSW, Australia.
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Graphical Abstract
Keywords Epithelial-mesenchymal transition· Cutaneous squamous cell carcinoma· Metastasis· Targetedtherapy·
Urokinase plasminogen activator system· Systematic review· UV-induced
Abbreviations
EMT Epithelial-mesenchymal transition
TME Tumor microenvironment
UV Ultraviolet
cSCC Cutaneous squamous cell carcinoma
RDEB Recessive dystrophic epidermolysis bullosa
AK Actinic keratosis
lacSCC/mcSCC Locally advanced/metastatic cSCC
EMT-TF Epithelial-mesenchymal transition transcription factor
TEMTIA The epithelial-mesenchymal transition international association
SES Sun exposed skin
PNI Perineural invasion
TSK Tumor specic keratinocyte
sc-cSCC Spindle-cell cSCC
SCC Cutaneous Carcinosarcoma
CAF Cancer-associated broblast
MMP Matrix metalloprotease
uPA/uPAR/uPAS Urokinase plasminogen activator/uPA receptor/uPA system
ECM Extracellular matrix
CSC Cancer stem cell
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1 Introduction
1.1 Rationale andbackground
Epithelial Mesenchymal Transition (EMT) is a process involved in tissue development, fibrosis, and cancer progres-
sion [1]. Evidence for the involvement of EMT during the invasive and metastatic process has been found in multiple
carcinomas including head and neck squamous cell carcinoma (HNSCC) [2], pancreatic carcinoma [3], gastric cancer
[4], and non-small cell lung cancer [5]. EMT has implications in therapy response and resistance to conventional
chemotherapy and radiotherapy [6]. An increasing body of evidence also provides mechanistic links between EMT
and immunosuppression in the tumor microenvironment (TME) [2, 7, 8]. Consequently, EMT markers were found
to correlate with response to immunotherapy [9] and innate resistance to immunotherapy [10]. Additionally, the
relevance of EMT markers as biomarkers for metastasis and for more accurate risk stratification of primary tumors
is under investigation [1114]. This notion is further supported by the fact that cells along the metastatic cascade
such as at the invasive front [15], and circulating tumor cells present EMT markers [14].
The role of EMT in cutaneous squamous cell carcinoma (cSCC) pathogenesis is not well defined despite an increas-
ing global incidence of cSCC. Since 1990, deaths attributed to non-melanoma skin cancer (NMSC) have more than
doubled to reach 65,000 deaths world-wide in 2017 [16]. Despite constituting only 20 percent of NMSC cases, cSCC
accounts for roughly 70 percent of all NMSC-associated deaths [17]. Risk factors, progression and therapeutic inter-
ventions for cSCC have been subject to multiple reviews [1720]. Briefly, Patient-specific risk factors for cSCC include
age, sex, skin type, and immunosuppression [19, 21]. Ultra violet (UV) radiation exposure, immunosuppression and
chronic arsenite exposure are major environmental risk factors for cSCC [22, 23]. Additionally, inherited conditions
such as xeroderma pigmentosum or epidermolysis bullosa (EB), specifically Kindler syndrome and recessive dys-
trophic EB (RDEB), favour the formation of cSCC [24, 25]. However, UV-induced cSCC is the most prevalent aetiology
and is characterised by C to T (and CC > TT) transitions and a high tumor mutational burden [26].
Clinically, UV-induced precursor lesions, known as actinic keratosis (AK), progress to cSCC [17]. A model of progres-
sion via two different pathways, the classical or differentiated pathway has been pioneered by Fernandez-Figueras
etal. [27] and was adapted by other reviewers [28]. The classical pathway involves full-thickness keratinocyte atypia
(Bowen’s disease, cSCC insitu) prior to the acquisition of invasive properties (cSCC) and is in line with a more con-
servative progression model [29]. During the more aggressive differentiated pathway, atypical keratinocytes located
only in the lower epidermal layers invade the underlying stroma. Locally advanced disease and metastatic disease
are characterized by an increasing loss of differentiation and high mortality rates [18, 30]. Early-stage cSCC can be
removed surgically with a curative rate greater than 90 percent [31]. However, recurrent cSCC, locally advanced
(lacSCC), and metastatic disease (mcSCC) are often unresectable and associated with poor prognosis and significant
morbidity [3133]. Currently, there are no reliable biomarkers to predict metastasis of primary disease and the under-
lying mechanisms are understudied [23]. While immunotherapy is an emerging treatment for lacSCC and mcSCC
with significant clinical benefits [34, 35], there are currently no reliable therapeutic biomarkers predicting response
of cSCC to immunotherapy [35]. Due to its epithelial origin, the involvement of EMT in therapy resistance including
resistance to immunotherapy, invasion, and metastasis of cSCC is plausible and the subject of ongoing research.
1.2 Objectives
We aimed to provide a systematic review of the nature, extent, and quality of the evidence, from both experimen-
tal (in vitro and xenograft models) and clinical studies examining EMT in cSCC progression. This included a critical
appraisal of the cell line-based models, markers and assays used to study EMT in cSCC based on the EMT International
Association (TEMTIA) guidelines for experimental studies on EMT [36]. Their statement provides definitions to unify
terminology as well as guidelines for cell line-based studies to adequately support claims of observed EMT. Research-
ers are urged to underpin findings of EMT in a combinatorial approach involving both EMT markers (e.g. E-cadherin,
cytokeratins, integrins, vimentin), as well as changes in cellular properties (e.g. loss of cell–cell interactions, increased
motility, decreased adhesion). The statement further highlights the complex non-linear nature of the process and
delineates EMT from linked but distinct processes such as differentiation, stemness, survival, and metabolism. The
statement concludes with the importance of EMT heterogeneity in tumor progression and the metastatic cascade
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and outlines the implications as a therapeutically targetable process. For clinical studies, all studies investigating at
least one epithelial and mesenchymal marker or underlying signalling molecules with experimental validation were
considered. In clinical research, the expression of EMT markers is sufficient as a proof for EMT due to the inherent link
with the invasive biology of cancer. Based on our synthesis of information describing the basic signalling pathways
driving EMT in cSCC progression and past efforts of modulating EMT in cSCC therapeutically, this review also evalu-
ated the potential for targeting EMT as a valid therapeutic strategy for lacSCC and mcSCC.
2 Methods
This review was planned and conducted in accordance with the Preferred Reporting Items for Systematic Reviews and
Meta-analysis extension for Scoping Reviews (PRISMA-ScR) [37]. A complete checklist is attached as Supplementary File
1. This study has not been registered in databases for the registration of systematic literature reviews. However, to avoid
duplicates, Open Science Framework, PROSPERO, and Cochrane databases were checked for similar protocols and stud-
ies prior to commencement and submission of the work.
All primary literature on studying the role of EMT in the progression of cSCC were considered regardless of aetiology.
English and peer-reviewed studies published before 26/10/2021 were included if they provided clinical data or experi-
mental data generated using human-derived models such as cell lines or xenograft models. Murine models were excluded
as they represent either a dierent disease aetiology (i.e. chemically induced models), dierent species of origin, and an
extensive body of literature beyond the scope of this review.
The search was conducted using PubMed and Medline (EBSCOhost) as subject specic databases as well as Scopus
and Web of Science as general databases. Epithelial-Mesenchymal Transition, Cell plasticity, and Skin neoplasms were
identied as relevant Medical Subject Headings (MeSH). The search string for each database can be accessed in Sup-
plementary File 2. The search results were exported and uploaded to the platform Rayyan.ai [38] where the duplicates
were removed and the record screening was conducted. To ensure maximum coverage, the bibliography of relevant
reviews were scanned for eligible studies.
For clinical investigations, the stage during the clinical progression, the EMT markers investigated, and observed
tendencies were extracted and presented as Fig.2. For studies using cell lines, information about the model used, loca-
tion/aetiology of the respective model, entity investigated, as well as interventions or alterations made were identied.
Additionally, the EMT markers, EMT-associated properties, and EMT-transcription factors (EMT-TFs) involved were charted.
The extracted methodology was assessed for compliance with the guidelines on EMT research proposed by TEMTIA (Sup-
plementary table2) [36]. A study was rated compliant to the TEMTIA guidelines if it incorporates at least one epithelial
marker (e.g. E-cadherin) and one mesenchymal marker (e.g. Vimentin, Fibronectin), as well as investigation of at least
one EMT-related property (e.g. migration, invasion). The integration of EMT-TFs was optional. Limitations of this study
include incomplete or insucient indexing of manuscripts that may have prevented the identication of all possible
eligible studies. Additionally, this study is subject to publication bias as drug candidates eliciting the opposite eect or
no eect are unlikely to be published [39].
3 Results
3.1 Included studies
The screening of 3443 search results yielded 86 eligible studies (Fig.1). Those excluded covered either a dierent disease
(melanoma, BCC, cervical SCC, HNSCC), were secondary literature (review articles, book chapters), were unrelated to EMT,
and/or used inducible murine models (DMBA, TPA, UV ). Eligible studies encompassed 44 cell line-based (experimental)
studies, 22 clinical studies, and 20 studies integrating both cell line work and clinical investigation (Supplementary
Table1 and 2).
3.2 Characteristics ofstudies
Of the 64 studies encompassing cell-line models identied in the search (Supplementary Table2), 79% were compli-
ant with the guidelines suggested by TEMTIA and 13% provided insucient evidence for their claims of EMT [33]. The
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remaining 8% were marked as not applicable due to study scope and design (e.g. dierential gene expression analy-
sis). However, investigation of spatial and/or transcriptional information beyond the pure protein levels of markers as
obtained by Western Blotting was rare. In addition, integration of EMT-TFs and linking of specic transcription factors to
their inuences on EMT markers and properties in keratinocytes is infrequent.
While many of the experimental studies met TEMTIA guidelines, the cell line models used to investigate EMT in cSCC
(Table1) misrepresent the patient population and aetiology. The most frequently used model (40%) is the vulva-derived
A431 cell line. The location of the primary tumor makes UV involvement (the most frequent cause of cSCC) in the aetiology
unlikely, as has been noted by others [40]. Male sex is a risk factor for developing cSCC accounting for a 1.5- to twofold
increase in incidence and increased mortality rates [41]. The two most frequently used models, A431 and SCC-13, are
both derived from female donors and account for 57% of studies reversing the clinically observed male to female ratio.
This may be of concern due to hormonal dierences between the sexes and the potential role of hormonal regulation
of EMT in cSCC [4245].
Fig. 1 Selection of the sources
of evidence
Table 1 Cell line models used
to study EMT in cSCC (used
in > 5% of studies)
M: male; F: female
Model Frequency of studies (%) Location of tissue of origin/Patient characteristics
A431 40 Vulva (F, 85)
SCC-13 17 Face (F, 56)
HaCaT 15 Upper back/Transformed (As/ Ras) (M, 62)
MET1/2/4 14 hand/ hand/ left axillary lymph node (M, 45)
SCL1 9 Face, Caucasian (F, 74)
SCC12 9 Face (M, 60)
HSC-5 6 Location unknown/Japanese (M, 75)
NHEK/ PHK 6 Normal adult skin/Variable Location(HPV/
ectopic protein expression)
SCC-IC1 5 Right temple (M, 75)
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There were no uniform set of markers used to assess EMT. A listing for each study specifying marker and methods
is available in Supplementary Table1 and 2. The most frequently employed epithelial and mesenchymal markers were
E-cadherin, Cytokeratins, and EpCAM as well as Vimentin, N-Cadherin, Fibronectin and α-SMA actin, respectively. Some-
times other non-canonical EMT markers were employed to assess other closely linked properties or processes such as
disassembly of cellular junctions (Girdin, α-/βcatenin, ZO1, Desmoglein 3), dierentiation and stemness (KLF4, involucrin,
CD133, CD44), and ECM remodelling (MMPs, uPAR, PAI-1). EMT marker analysis was primarily performed via Western
blotting (84%), immunouorescence (39%), and RT-qPCR (22%). Morphological changes towards a more spindle like
phenotype are were used as starting point to prompt further investigation. Migration and invasion were frequently
assessed using transwell migration assays, scratch wound assays, transwell migration assays into Matrigel coating, or
organotypic assays. Cell adhesion was rarely evaluated but when done, used either a trypsinization assay or bead-based
cell traction force assays. EMT property related assays were often paired with assays assessing stemness (colony/spheroid/
tumor-forming assays) or proliferation (MTS/ MTT, CCK8, counting).
Of the many reports screened that included clinical samples, only 22 purely clinical investigational studies and 20
integrating both clinical investigation and cell line investigated EMT or related signalling in cSCC. A listing of each of
these eligible studies specifying marker, methods and tissue comparisons is available in Supplementary 1. Again, as for
experimental studies, the markers used to assess EMT were varied and included mRNA, proteins and miRNA/lncRNA
expression analyses by comparison most often to normal or matched skin. Synthesis of this data in a timeline of cSCC
progression is summarized in Fig.2 and discussed below.
3.3 Synthesis ofresults
3.3.1 Clinical progression ofcSCC isparalleled byincreased acquisition ofamesenchymal phenotype andEMT markers
Human epidermal keratinocytes display alterations of surface markers and transcription factors consistent with an EMT-
phenotype early during the clinical progression. Bakshi etal. [46] reported reduced E-cadherin levels paralleled with
increased Snail, Twist and Slug expression in sun-exposedskin (SES) vs non-sun-exposed skin (NSES). Others have found
that miR-497 levels are signicantly reduced in SES vs NSES. miR-497 is a negative modulator of the EMT markers Slug,
N-cadherin and Vimentin, a direct repressor of SERPINE1 and inhibitor of migratory properties in cSCC cell lines [47]. In
cSCC, Snail and Slug levels are negatively correlated with E-cadherin levels [4850].
While, an increasing body of evidence suggests that EMT is the determining factor for the progression of AK and
Bowen’s disease to cSCC, dierences between the classical and dierentiated pathway have been reported. Saenz-Sardà
etal. [51] found signicantly lower expression of EMT markers Vimentin, E-cadherin, and membranous β-catenin in cSCC
arising through the dierentiated pathway when compared to the classical pathway. Additionally, the proliferation marker
Ki67 was signicantly increased in cSCC arising through the classical pathway [51]. Additional dierences between the
pathways include increased miR-31 and MMP levels in the dierentiated pathway [52]. Nevertheless, the expression of
markers at the invasive front of cSCC and Bowen’s disease acquiring de novo invasive ability suggest a pivotal role of the
EMT process in facilitating invasion in cSCC [51, 53].
The importance of EMT in cSCC is further supported by increased expression of EMT-TFs and mesenchymal markers
including Snail, Slug, ZEB1, Twist, Podoplanin, and Vimentin [46, 48, 49, 5459] with increasing progression of the disease
and increasing loss of dierentiation. The gain of mesenchymal markers and properties is complemented by loss of epi-
thelial markers (Involucrin, E-cadherin, KLF4, Cytokeratin, Claudin1) and apparent disassembly of cellular junctions [46, 53,
5557, 6068]. For example, Girdin, an adherence junction protein closely linked to E-Cadherin, has been associated with
collective migration. Furthermore, Girdin expression is correlated to well-dierentiated cSCC but is lost in poorly dieren-
tiated cSCC [69]. Toll etal. [58] linked cSCC expressing Vimentin, Twist, ZEB1, nuclear βcatenin and podoplanin to lymph
node metastasis in a study cohort comprising tumors from 146 patients. Additionally, Vimentin levels correlated with
recurrence, disease specic death, tumor stage, perineural invasion (PNI), desmoplasia, and dierentiation. Podoplanin,
despite not being a classical EMT-marker, was also identied by another independent study as predictor of regression-
free survival [54]. Perineural invasion (PNI) is an established predictor of recurrence, metastasis, and poor prognosis in
cSCC [70]. Brugière etal. [67] mapped cells with EMT-features to the site of PNI and increased neurotrophin signalling.
This evidence links EMT to both lympho-vascular and PNI, two hallmarks of advanced disease and poor patient outcome.
The OVOL transcription factors 1 and 2 antagonize EMT-TFs and hence are important negative regulators of EMT
[71]. OVOL1 and 2 protein levels are upregulated in benign precursor lesions of cSCC, such as AKs and Bowen’s disease
(cSCC insitu) compared to cSCC [49, 50]. Compared to AKs, OVOL1/2 expression is lost (or reduced) in cSCCs and this is
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associated with increased expression of EMT markers, Vimentin and Zeb1 [49]. Additionally, Murata [49] found a signicant
inverse association between OVOL2 and Zeb1 levels using 30 AK and 30 cSCC samples and increased ZEB1 expression
upon OVOL1 and OVOL2 knockdown in A431 cells. Furthermore, Ito etal. [50] report signicantly reduced c-Myc levels
and invasiveness in A431 cell upon OVOL1 knockdown. Interestingly. Ito etal. [50] could not conrm the loss OVOL2
with progression from Bowens disease to cSCC. Rather, they report OVOL2 translocation to the cytoplasm in cSCC, which
hinders OVOL2 to act as a transcription factor. Altogether, these studies suggest a role of OVOL transcription factors as
the last guardian against malignancy in pre-neoplastic lesions.
Ji etal. [15] resolved the spatial architecture of 10 cSCC with matched normal skin using a combination of single
cell-RNA sequencing, spatial transcriptomics, and multiplexed ion beam imaging. They identied a subpopulation of
keratinocytes exclusive to tumor tissue. These tumor-specic keratinocytes (TSKs) are located towards the invasive edge
Fig. 2 Summary of EMT markers and related signalling during the clinical progression of cSCC as determined through eligible studies
included in this review (see supplementary table1). In the progression of normal skin to cSCC the number of EMT markers steadily increases
as the regulatory barriers fall and underlying pathways become activated. In cSCC the expression of EMT markers is by no means homog-
enous. Consequently, the contextual nature of the presented evidence needs to be considered (see footnotes). scSCC and CCS are rare
extreme forms of cSCC often only reported on a case basis. Hence, + and − denote the quality of the immunohistochemical analysis for
these disease states [76]
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of the tumor and possess an EMT-like signature. TSKs express markers of EMT (Vimentin, ITGA5) despite lacking the expres-
sion of classical EMT-transcription factors (excluding SLUG). Single-cell regulatory network interference and clustering
nominated AP1 and ETS transcription factors as regulators of EMT in TSKs. Additionally, their preferential localization
at the invading edge infers invasive migratory properties when compared to their basal, dierentiating and cycling
counterparts. Furthermore, TSKs display a broad spectrum of EMT markers reecting the high complexity and plasticity
underlying the EMT continuum.
Even further along the epithelial-mesenchymal spectrum than cSCC are spindle cell cSCC (sc-cSCC), a poorly dierenti-
ated form of cSCC with a characteristic mesenchymal phenotype [72]. Nakamura etal. [62] reported a case of cSCC mim-
icking an atypical broxanthoma staining positive for Vimentin and Snail whilst staining negative for Cytokeratin. Iwata
etal. [63] reported cases of sc-cSCC with complete loss of E-Cadherin, p120-catenin, and Desmoglein-3. More recently,
Shimokawa etal. [61] showed increased nuclear staining of Snail, increased cytoplasmic Vimentin, and decreased levels
of E-cadherin paralleled by signicant reduction of COX2 levels in six sc-SCC compared to three non-sc-cSCC. Combined
with the case reports, this warrants the consideration of sc-SCC as a tumor displaying advanced features of EMT and
clinical progression of cSCC. Even more advanced, the position of cutaneous carcinosarcomas (CCS), biphasic tumors
constituting of both a mesenchymal and epidermal component, in this clinical progression remains elusive [73]. The
involvement of EMT in the formation of these tumors is up for debate [65, 74, 75]. Matching genotypes of both phases
and EpCAM positive staining of mesenchymal and epidermal components both point towards singular epidermal origin
of CCS and infer a quasi-full EMT as potential mechanism [65]. However fascinating, the rarity of sc-cSCC and CCS makes
them less relevant in the overall picture [72, 73].
3.3.2 Genetic disorders associated withincreased frequency ofaggressive cSCC facilitate EMT viaaltered cell–matrix
interactions
Epidermolysis bullosa (EB) is a group of conditions that are associated with early onset and rapid progression of cSCC
[24, 77]. Kindler syndrome is caused by mutations in the FERMT1 gene [78]. FERMT1 codes for Kindlin-1, a protein co-
localizing at focal adhesions and involved in the activation of their receptor functions. Lack of Kindlin-1 is associated
with dysregulated integrin signalling, cell adhesion and migration [79]. Immortalized patient-derived kindlin-decient
keratinocytes display reduced cell–cell adhesion, cell–matrix adhesion, and epithelial markers. Conversely, induction
of mesenchymal markers, ECM components and proteases point towards a more mesenchymal phenotype [80]. Con-
icting data by Ji etal. [15] identies FERMT1 as an essential transducer of integrin signalling in TSKs and amplication
across many cancers. Additional studies suggest a role for FERMT1 in regulating EMT across multiple cancers including
[8183]. In cSCC, evidence points towards a contextual involvement of Kindlin-1 in EMT regulation and warrants further
investigation [15, 80].
RDEB patients bear loss-of-function mutations in the COL7A1 gene and produce highly aggressive and metastatic cSCC
[77, 84]. Knockdown of ColVII in the non-RDEB cSCC cell lines, Met1 and SCC-IC1, increased migration and invasion by
enhancing EMT and preventing dierentiation [85]. In xenografts derived from ColVII knockdown SCC-IC1 cells, recombi-
nant human ColVII reduced the eects of the ColVII deciency. In addition to increased angiogenesis, ColVII knockdown
resulted in amplied TGF-β1 signalling and upregulation of urokinase plasminogen activator (uPA), SERPINE1 and VEGFA
[86]. Clinically, RDEB tumors displayed increased EMT markers and increased levels of TGFβR1. Furthermore, loss of ColVII
directly correlated with decreased levels of involucrin, an epithelial dierentiation marker invivo [86]. Twaroski etal. [87]
conrmed ndings of increased TGF-β1 signalling and identied MEK/ERK, p38 and SMAD3 as downstream eectors and
mediators of an EMT phenotype. Together these studies provide an attractive model for the high aggressiveness and
high metastasis rates in RDEB cSCC.
3.3.3 Cancer‑associated fibroblasts induce EMT incSCC viaparacrine growth factor, cytokine, integrin andMMP signalling
Cancer-associated broblasts (CAFs) and other cancer-associated cell types in the tumor microenvironment are impli-
cated in several cancers to induce EMT via paracrine signalling (refer to Fig.2) [1, 15, 88]. Co-culture of broblast and
cSCC cancer cells has been shown to be paralleled by increased CAF and EMT marker expression [89]. In cSCC, broblast
subtype can also play a signicant role in cancer progression. For example, reticular broblasts favour EMT and invasion
compared to papillary broblasts [89]. On the same note, Bordignon etal. [90] reported dierent CAF subtypes with
diametric inuences on EMT markers and tumor invasiveness in cSCC. A TGF-β induced CAF population signicantly
increased invasiveness and tumorigenic expansion invivo but not FGF2-induced CAFs. The increased aggressiveness
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of TGF-β induced CAFs was associated with increased EMT markers (Vimentin, Snail, Slug and Twist) and altered TME
(e.g. COL1A1 secretion) in the tumor cells. Additional evidence, conrms that patient-derived broblasts from RDEB
patients secrete TGF-β to facilitate EMT in RDEB-cSCC cell lines [87]. However, broblasts contribute to EMT not only via
TGF-β secretion. For example, conditioned media derived from senescent broblasts induced EMT in post-senescent
keratinocytes via MMP-PAR-signalling. Matching clinical evidence suggest that EMT-associated traits and markers such
as gelatinolytic activity, PAR-1 expression, andTWIST expression were elevated in aged human skin samples [91]. This
might provide another mechanistic link between the risk factor age and cSCC progression aside from the continuous
accumulation of UV-induced mutations.
Clinically, Sasaki etal. [92] used independent clustering based on CAF and EMT-related markers to establish a signi-
cant correlation between clinic pathological subgroup and malignancy. Interestingly, the subgroups correlated with
clinical parameters such as lymph node metastasis, tumor thickness and tumor size. Ji etal. [15] showed that TSKs engage
in reciprocal signalling with CAFs, endothelial cells, macrophages, and myeloid-derived suppressor cells (MDSC). These
interactions form a complex network of signalling molecules, including ECM components (FN1, COL1A1), cytokines
(TGFB1, TGFB3, and CXCLs), growth factors (PGF, VEGFA), proteases (MMP9), and integrins (ITGA3, ITGB1). However, the
inuence of the individual factors on the EMT status remains subject to further investigation. For example, some evidence
suggests, that stromal macrophages do not play a major role in the induction of EMT in cSCC [59].The tumor microenvi-
ronment alters the EMT status of tumor cells not only through paracrine signalling but also through the properties and
composition of the ECM itself [93]. For example, HPV transformed-keratinocytes (N/TERT keratinocytes) elicit an EMT
response to bronectin via α3β1-integrins [94]. The inuence of matrix stiness and mechanotransduction pathways on
EMT has been investigated in other cancers but remains underexplored in cSCC [95, 96].
3.3.4 β‑catenin provides amechanistic explanation fortheclose link betweenstem‑like properties andEMT incSCC
Cancer stem cells have gained traction in recent years as they link major challenges modern cancer therapy faces includ-
ing recurrence, therapy resistance and metastasis [6, 97]. Even though stemness and EMT are separate phenomena, they
are closely linked[6]. Cells, staining positive for the stem cell markers CD44 and CD29, at the invasive front of tumors also
displayed characteristics consistent with EMT [98]. In three kidney organ transplant recipients, EMT markers (Vimentin,
Slug, and Snail) were co-expressed in CD133 expressing (stem) cells in invasive areas of skin SCCs but not concomitant
AKs or normal skin [60]. A mechanistic link is provided by the downregulation of E-cadherin, which releases sequestered
β-catenin from the cell membrane to the nucleus [69, 99]. Furthermore, this might work synergistically with dysregulated
Wnt/β-catenin signalling, an inducer of cancer stem cell properties [100102]. Clinical evidence shows the co-localization
of βcatenin and E-cadherin at cellular junctions [51, 69]. Loss of E-cadherin is associated with the disassembly of those
junctions, decreased adherence and increased nuclear β-catenin (Fig.2 and 4)[51, 69]. The transcriptional repression of
E-cadherin is mediated by canonical EMT-TFs (e.g. Snail) as well as other transcription factors such as Grhl3 (Fig.4)[61,
103]. Increased nuclear β-catenin has been observed in tumors with poor dierentiation and correlated to lymph node
metastasis [55, 58, 69].
The close link between stemness and EMT is supported by an increasing body of invitro evidence. Biddle etal. [104]
performed CD44/ EpCAM based sorting of the cell lines PM1, MET1 and MET2, derived from dysplastic skin, the primary
lesion, and a recurrence at the same anatomical site, respectively [105]. They observed an increase of in the Epcam low/
CD44 high population with increasing malignancy. Further, a more prominent EMT phenotype, sphere forming ability
but reduced proliferative ability distinguished the EpCAM low/CD44 high population from the EpCAM high/ CD44 high
population. CD44/ITGB1 based sorting of the A431 cell line also identied a subset within the cancer stem cells (CSC) that
display EMT characteristics. In murine xenograft models of A431 cells, the CD44 high/ ITGB1 high stem cells gave rise to
signicantly bigger and more aggressive tumors [98]. Additionally, the simultaneous regulation by common upstream
regulators including ARMC8, ΔNp63α, p38/NFκB, transglutaminase II (TGA2) and Axl [106111], as well as pharmaceuti-
cally active substances further tightens the link between of EMT and CSC properties [108, 112].
3.3.5 Proteases includingtheurokinase plasminogen activator system are underexplored markers andEMT effectors
Proteases such as matrix metalloproteases contribute to tumor cell invasion and EMT, via multiple mechanisms [113,
114]. Proteolytic cleavage of the ECM allows for increased migration and liberates latent signalling molecules such as
EGF, HGF, and TGF-β [115, 116]. In two-dimensional A431 cell culture, broad inhibition of MMPs and MMP-9 knockdown
reduced- EMT marker expression and motility. Additionally, the EMT-TF Snail induced expression of MMP-9 [117]. MMP-2
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contributes to the invasiveness and migratory abilities in TGF-β-induced EMT of RDEB cells [87]. TGF-β1 and EGF treatment
in Ras-transformed HaCaT-cells induced an EMT phenotype and increased collagen remodelling. The broad-spectrum inhi-
bition of MMPs using GM6001 increased cell attachment and abrogated collagen degradation [118]. An often-overlooked
contribution of MMPs to EMT signalling is mediated by G-protein-coupled cell surface receptors, transducing a signal
upon MMP cleavage [119]. MMPs, MMP-1, MMP-2, and MMP-3, secreted by senescent broblasts induced EMT markers
and migration via a PAR1-mediated mechanism [91].
Wilkins-Port etal. [118] identied the involvement of another protease system in TGF-β1 and EGF induced EMT of
ras-transformed HaCaT- cells, the urokinase plasminogen activator system (uPAS). Inhibition of the uPAS using amiloride
(uPA inhibitor) or plasminogen activator inhibitor 1 (PAI-1, encoded by SERPINE1), an endogenous uPA inhibitor, reduced
MMP-1 and MMP-10 levels as well as attenuated collagen. Additionally, PLAU (uPA) is part of the TSK-gene signature
identied by Ji etal. [15]. TGF-β1 and EGF induce the expression of PAI-1 and multiple studies report the transcriptional
regulation of SERPINE1 parallels the induction of EMT [47, 120]. Mizrahi etal. [47] found progressive down-regulation of
miR-497 through promotor methylation during the progression of SES to cSCC. Expression of miR-497 reduced SERPINE1
levels as well as levels of other EMT related genes.
3.3.6 miRNA andlncRNA expression profile changes duringEMT play acentral role inits regulation
During the progression from normal skin to cSCC, the expression of many non-coding RNAs such as miRNAs and lncRNA
are altered. miRNAs regulate the transcription of proteins by regulating the stability of their respective target mRNAs
[121]. Mizrahi etal. [47] investigated the dierential expression of miRNAs in a clinical progression of NSES to cSCC. The
changes in the miRNAs expression ranged from gradual increase/decrease to stepwise acquisition/loss creating a pro-
gression specic prole. For example, the loss of the tumor suppressor miR-497 increased SMAD signalling, EMT as well
as SERPINE1 expression. Other studies also found miRNAs dysregulating some of the signalling pathways central to EMT
including the PI3K/Akt pathway and the Wnt-pathway. The tumor suppressive miR-451a, a suppressor of PDK1, is down-
regulated in metastatic vs non-metastatic tumors [122]. The suppression of PTEN by miR-21 leads to increased pathway
activity and Akt activation [123, 124]. The oncogenic miR-22 is gradually upregulated with increasing grade of cSCC and
promotes stemness via Wnt- signalling [100]. A mechanistic study by Robinson etal. [125] identied miR-211 and miR-
205 as part of an iASPP/ p63 epigenetic feedback loop regulating EMT, with the latter directly targeting Zeb1 and p63.
LncRNA can act as molecular sponges for miRNAs by competing for binding with their cognate mRNAs [126]. How-
ever, other mechanism of action such as chromatin remodelling, transcriptional regulation or mRNA post-transcriptional
regulation are possible [121]. For example, the lncRNA HOTAIR sponges miR-326 leading to an increase of PRAF2 and a
more prominent EMT phenotype [126]. MALAT1 is upregulated in cSCC compared to normal skin and promotes EMT via
modulation of Wnt-signalling [127]. Clinically, Li etal. [128] found a correlation between the upregulation of LINC00319
and tumor size, lymphovascular invasion, and TNM stage. In cSCC cell lines, LINC00319 favoured migration, invasion,
and EMT marker expression [128]. LncRNA, H19, and miR-675 are upregulated in cSCC. Upregulation of H19 increased
miR-675 levels as well as the expression of EMT markers [129].
3.3.7 Environmental factors such asarsenite induce EMT andtransformation bywidespread alteration ofmiRNA
expression, mRNA expression andinduction ofIL‑6 signalling
After UV exposure, arsenic exposure is one of the biggest occupational hazards for developing NMSC [130]. The acute and
chronic toxicity of arsenite can be replicated invitro by exposing HaCaT cells to arsenite (0.1–1µM) for up to 28weeks
[124, 131]. Al-Eryani etal. [132] found a signicant dysregulation of EMT and cell cycle genes as early as 7 weeks after
exposure to arsenite. Banerjee etal. [131] reported decreases in ZO-1, a tight junction protein, and increased Slug after
19weeks. After 28weeks, the arsenic-exposed cells display an advanced EMT phenotype. Additionally, pathway analysis
of the dierentially expressed miRNAs and mRNAs showed inhibition of the ER stress pathway. Investigations into the
molecular mechanism implicate NFκB, PI3K and IL-6 signalling as the major contributors to the EMT phenotype (Fig.3).
IL-6 induces miR-21 via STAT3 cytokine signalling [123]. In return, miR-21 activates Akt via the inhibition of PTEN [124].
Furthermore, the increased CSC-like properties are ascribed to increase p38/ NFκB signalling as well as the induction of
IL-6 [107, 108]. Upregulation of the EMT-TF Snail provides a direct link to canonical EMT signalling [107, 108].
Other external factors such as UV and ROS also promote EMT in malignant keratinocytes. UV radiation induces the EMT
transcription factor Snail via an AP1-dependent mechanism and UV exposure is associated with decreased E-cadherin
levels clinically [46, 133]. Additionally, UV irradiation promotes cSCC via increased production of reactive oxygen species
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(ROS) [134]. Conversely, the tumor suppressor and negative modulator of oxidative stress, NAD(P)H dehydrogenase
(NQO1), is lost in cSCC. Adenoviral expression of NQO1 reduced ROS levels and attenuated EMT [135]. On the other hand,
cellular stress can induce autophagy by reducing the levels of the autophagy marker, p62 [136]. p62 directly binds and
stabilizes the EMT-TF Twist1 [137]. Additionally, p62 can induce NFκB signalling further linking inhibition of autophagy
to EMT [136].
3.3.8 Thyroid hormone andEstrogen signalling modulates EMT incSCC
Male sex is a risk factor for cSCC. Male patients are often younger, present more frequently with metastatic disease and
have a worse prognosis [138141]. The increased incidence is often attributed life-style choices. However, more recent
research suggests an underlying biological cause [139, 142]. Some scarce evidence infers a role of hormonal signal-
ling in modulating EMT in cSCC. Chen etal. [42] reported estrogen-dependent activation of the FN1-STAT3 axis in the
vulva-derived A431 cell line. The subsequently induced EMT could be reversed with the inverse agonist XCT790. Nappi
etal. [143] reported on a link between thyroid hormones, EMT, and tumor stage. NANOG and Deiodinase 2 (D2) were
Fig. 3 Signalling for arsenite-induced EMT. Arsenite activates NFκB signalling pathways, which induce EMT via Snail as well as lead to the
secretion of IL-6. Autocrine IL-6 signalling induces EMT and activates PI3K signalling via miR-21 mediated repression of PTEN [76]
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proportionately increased signicantly with increased cSCC stage. D2 catalyzes the conversion of the thyroid hormone
(TH) T4 to T3. TH depletion of the growth medium and inhibition of D2 with rT3 both reverted the EMT phenotype in
SCC13 cells. A second study conrmed that T3-induced thyroid hormone receptor α (THR) directly binds the Zeb1 pro-
motor and induces transcription of the EMT-TF [144]. Additionally, high D2 levels correlated with more advanced stage,
a higher risk of relapse and lower overall survival in two independent datasets [144]. Together, these ndings warrant a
closer investigation of the hormonal inuence on EMT in cSCC.
3.3.9 EMT incSCC can be attenuated bytargeting MAPK, cytokine, growth factor, andNFkB signalling
Drug resistance and recurrence are two major challenges modern cancer therapy has to overcome. Due to its link to
both these phenomena, the modulation of EMT towards an epithelial phenotype has become a desirable therapeutic
avenue [6]. The development of therapy inducing MET would overcome some of these challenges. During past research
eorts, some drugs have elicited the desired reversion of an EMT phenotype in cSCC cell lines and xenografts (Table2).
Successful induction of MET was mostly achieved by targeting four major signalling pathways responsible for the induc-
tion EMT in cSCC: EGFR signalling, the PI3K/Akt/mTOR pathway, TGF-β signalling and NFκB signalling (refer to Fig.4 for
details). The kinase, Akt, takes a central role here with several drugs, reducing its activity also favourably modulating
EMT marker expression, reducing migratory and invasive properties. Direct pharmaceutical inhibition of Akt successfully
induced apoptosis, reduced tumor growth in xenografts whilst inducing MET [110, 145]. Inhibition of Cyclooxygenase 2
(COX-2) or Ornithine decarboxylase (ODC) with diclofenac and diuoromethylornithine (DFMO), respectively, reduced
p-Akt levels and in single agent and combination therapy [146]. Modulators of other upstream signalling of Akt like
EGFR and PI3K-signalling show promise as potential targets [124, 147149]. Inhibition of the eectors of EGF and TGF-β
induced signalling such as p38, JNK, AP1, MEK, and SMAD3 had a similar eect to inhibition of Akt [42, 87, 145, 150].
Tightly connected to p38 signalling is RelA, a component of the NFκB transcription factor. Induction of p-p38 lead to
decreases in p-RelA and abrogation of migration, spheroid formation and stemness [108]. Conversely, stabilization of
IκBα, a negative modulator of the NFκB pathway, increased cellular adhesion and lead to loss of migration, spheroid
formation and stemness [107].
Most compounds tested targeted the aforementioned four major pillars of EMT signalling. However, there are a few
exceptions involving hormonal signal transduction and cytoskeletal signalling. XTC790, a reverse agonist for the nuclear
estrogen-related receptor α (ERRα), and rT3, a Dio2 inhibitor, elicited responses consistent with MET in A431 and SCC13
cell, respectively [42, 143]. Interestingly, inhibition of RhoA, a small GTPase involved in integrin and cell skeletal signal-
ling, had the opposite eect of all previously discussed drugs and in fact promoted EMT [151]. Additionally, proof of
concept studies using several natural compounds have reported modulation of EMT markers and properties in cSCC
cell lines [112, 149, 152, 153]. However, these studies lack proper validation of their targets, an in-depth assessment of
potential o-target activities, and consequently an understanding for the signalling pathways modulated. For example,
Wogonoside modulates p-PI3K, p-β-catenin and p-Wnt levels leaving serious doubts about its selectivity especially given
the lack of a specic target in the study [112]. Nevertheless, investigations into the suggested targets, EphB2 and HDAC3,
using selective inhibitors might prove fruitful given their implications in other cancers [154, 155].
4 Discussion
During the clinical progression from sun-exposed skin to metastatic cSCC, keratinocytes acquire an increasing number of
traits consistent with EMT [46, 51, 56] (refer to Fig.2). The increasing invasiveness paired with changes towards a spindle-
shape morphology and altered expression of markers are strong indicators for EMT. The evidence presented conrms
that in cSCC the acquisition of advanced EMT features represents the point of transition from benign to malignant, non-
invasive to invasive disease. A major dierence between dierentiated and classical pathway of cSCC progression is the
aggressiveness of the arising tumors as well as the point during the progression where tumor cells displaying advanced
EMT markers (Vimentin, Zeb1) can be observed [51, 52]. During the dierentiated pathway, the point of acquiring invasive
capabilities coincides with the expression of advanced EMT markers. During the classical pathway, the EMT transcription
factors Snail, Twist and Slug are readily expressed in the pre-neoplastic lesions [46]. Insitu cSCC (Bowens disease) display
signs of early EMT as well as stain positive for the expression of OVOL-transcription factors (OVOL-TFs), negative modu-
lators of EMT [50, 53]. Bowen’s disease acquiring de novo invasive capacity shows more advanced EMT markers at the
invasive front such as increased Vimentin [53]. The OVOL-TFs antagonize late-stage TFs such as ZEB1, hence suggesting
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Table 2 Drugs and their eects on EMT markers and properties in cSCC
Drug Target Markers and signalling Properties Model Refs.
SB431542 TGFBRI p-p38, pERK1/2 Migration, invasion, proliferation RDEB-cSCC [87]
SB203580 p38 Vim, FN1, MMP9, PAI-1 Migration, invasion RDEB-cSCC [87]
Trametinib MEK1/2 Vim, FN1, MMP9 Migration, invasion RDEB-cSCC [87]
PD169316 SMAD3 pSMAD3, PAI-1, MMP2, MMP9 Migration, invasion RDEB-cSCC [87]
ARP100 MMP2 MMP2, PAI-1 Migration, invasion RDEB-cSCC [87]
Avicularin n.s E-cad, N-cad, MMP9, Vim, pMEK, p-p65 Apoptosis SCC13 [152]
Aloeemodin, Kaempferitrin EphB2 E-cad, EphB2, MMP9, MMP2, Vim Proliferation, invasion, xenograft growth
apoptosis A431,
SCL-1 [154]
Wogonoside n.s E-cad, N-Cad, FN1, VEGF, MMP9, MMP14, p-PI3K,
p-WNT, p-β-cat, p-AKT Viability, colony formation, stemness, prolifera-
tion, invasion, microtubule formation, xenograft
growth, apoptosis
SCL-1, SCC12 [112]
rT3 Dio2 E-Cad, N-Cad, Vim, Zeb1 Migration SCC13 [43]
Ginsenoside®- Rg3 HDAC3 E-Cad, N-Cad, Vim, Snail, HDAC3, c-Jun Migration, invasion A431, SCC12 [155]
LY2109761 TGFBRI/II E-Cad, pSMAD2/3, Vim, FN1, Slug Migration, invasion SCL-1 [156]
XTC790 ERRα E-Cad, p53, FN1, Vim, pSTAT3, pATR, pAMPKα Proliferation, migration, apoptosis A431 [42]
Niclosamide STAT3 E-Cad, pSTAT3, FN1, Vim n.s A431 [42]
Lapatinib HER2/ EGFR PTEN, p-PTEN, E-Cad, pAKT, p-mTOR, Wnt,
β-catenin, N-cad, Vim, Slug Apoptosis, autophagy, viability A431 [147]
LY294002 PI3K E-Cad, pAKT, Vim Migration, invasion As-transformed HaCaT [124]
NC9 TG2 E-Cad, Twist, Snail, Slug, Vim, FN1, N-Cad, HIF1α Spheroid formation, migration, invasion A431, SCC13 [110]
Akt inhibitor VIII Akt E-Cad, Vim, Slug, pAkt Adhesion, migration PM1, MET1, MET4 [56]
Caeic Acid Fyn Kinase E-Cad, N-Cad, Vim, Snail, p-p38, p-RelA Migration, stemness, spheroid formation As-transformed HaCaT [108]
SB203580 p38 E-Cad, N-Cad, Vim, Snail, p-p38, p-RelA Migration, stemness, spheroid formation As-transformed HaCaT [108]
BAY 11–7082 p-IκBα E-Cad, N-Cad, Vim, Snail Adhesion, stemness, spheroid formation, tumor
formation As-transformed HaCaT [107]
Y27632 RhoA E-Cad, Vim, N-Cad, Snail, Slug n.s A5RT3 [151]
Diclofenac COX-2 p-AKT, p-ERK1/2, p-MAPKAP2, Snail, Twist, MMP-2,
COX-2 Tumor growth, migration, colony formation,
apoptosis,A431 (Xenograft) [146]
DMFO OCD p-AKT, p-ERK1/2, p-MAPKAP2, ODC Tumor growth, colony formation, apoptosis A431 (Xenograft) [146]
Diclofenac and DMFO COX-2/ OCD p-AKT, p-ERK1/2, p-MAPKAP2, Slug, Twist, MMP-9,
MMP-2, COX-2, ODC Tumor growth, migration, colony formation,
apoptosis A431 (Xenograft) [146]
API-59CJ-Ome Akt p-AKT, Slug, N-Cad, FN1 Tumor growth A431 [146]
Triciribine p38 p-p38, p-MAPKAP2, MMP-2, MMP-9, N-Cad, E-Cad Tumor growth, proliferation, apoptosis CsA-treated A431 (Xenograft) [145]
SB-203580 Akt p-AKT, pmTOR, p-p38, p-MAPKAP2, MMP-2, MMP-9,
N-Cad, E-Cad Tumor growth, proliferation, apoptosis CsA-treated A431 (Xenograft) [145]
Triciribine/ SB-203580 p38/ Akt p-AKT, pmTOR, p-p38, p-MAPKAP2, MMP-2, MMP-9,
N-Cad, E-Cad Tumor growth, proliferation, apoptosis CsA-treated A431 (Xenograft) [145]
luteolin n.s FN1, Vim, Twist, Snail, N-Cad, MMP-9, p-Akt,
p-GSK3β, E-Cad Migration, invasion A431 [153]
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Table 2 (continued)
Drug Target Markers and signalling Properties Model Refs.
Quercetin n.s FN1, Vim, Twist, Snail, N-Cad, MMP-9, p-Akt,
p-GSK3β, E-Cad Migration, invasion A431 [153]
Wortmannin PI3K p-Akt, Vim, E-Cad n.s A431 [148]
GSP n.s EGFR, p-ERK1/2, N-Cad, FN1, Vim, E-Cad Migration, invasion SCC13 [149]
Erlotinib EGFR N-Cad, FN1, Vim, E-Cad Invasion SCC13 [149]
UO126 MEK p-p38, p-ERK, Vim, E-Cad n.s Transformed HaCaT (II-3andH375) [150]
SP600125 JNK Vim, E-Cad n.s Transformed HaCaT (II-3andH375) [150]
[6]-Gingerol AP1 Vim, E-Cad n.s Transformed HaCaT (II-3andH375) [150]
Italics : attenuated; bold : induced; n.s.: not specied; GSP: grape seed proanthocyanidins
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that insitu cSCC is conned to the epidermal layer due to the inhibition of late stage EMT by the OVOL-TFs [49, 50, 71].
Finally, the clinical extreme of cSCC, sc-SCC, can also be explained by EMT involvement and is plausible given that EMT
involvement in the formation of scSCC has been shown for a closely related cancer of cSCC, scSCC- of the head and neck
[74, 157159].
4.1 Akt regulation paralleled byactivated TGFβ signalling iscentral intheregulation ofEMT incSCC
Clinical studies identied the increased activation of Akt, overexpression of EGFR and the presence of active nuclear IκKβ
as predictors of aggressive and metastatic disease [56, 160, 161]. Conrming these ndings, invitro studies identify the
canonical EGFR/MAPK-pathway, the PI3K/Akt-pathway, and the NFκB-pathway as the central pillars of EMT regulation in
cSCC (Fig.4). Akt can be activated via PI3K and PDK1 by mitogen receptors such as EGFR [162]. Another important hub,
GSK3-β, connects Akt signalling with NFκB signalling as well as stemness signalling through stabilization of β-catenin
[163, 164].
The fourth pillar of EMT (Fig.4), canonical TGF-β signalling, was described in an early study by Davies etal. [150]. The
minimal requirement of the immortal keratinocyte cell line, HaCaT, to undergo EMT is a combination treatment of EGF
and TGF-β. The activation of EGFR signalling can be mimicked by transfection with mutant Ras. Constitutively active
Ras was able to activate MAPK signalling which is required to potentiate SMAD-dependent activation of AP1 family TFs.
AP1-mediated EMT was an observation that Ji etal. [15] should repeat. Since then TGF-β has been shown to contribute
Fig. 4 Synthesis of EMT signalling in cSCC based on clinical and experimental studies evaluated in this review (Supplementary Table1 and
2). The signalling investigated by cell line-based studies was merged in this EMT pathway map under the assumption of transferability
between the dierent aetiologies and models. Multiple changes can induce EMT in cSCC. Hormonal, cytokine, growth factor, ECM signalling
all contribute cooperatively to the extent and nature of the EMT programme. Central signalling hubs such as Akt, MKKs or NFκB are attrac-
tive drug targets that could be used to attenuate EMT in cSCC [76]
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to the induction of EMT via autocrine signalling, paracrine signalling, as well as the induction of an EMT-favouring CAF
phenotype. Additionally, suppressors of aberrant canonical SMAD-dependent TGF-β signalling such as SMAD4 and KLF4
are lost during cSCC pathogenesis [64, 66]. TGF-β and underlying SMAD, ERK and P38 signalling induces the transcrip-
tion of multiple EMT related proteins such as COX-2, Snail, Slug, proteases (MMP-2, MMP-9) and SERPINE1 [47, 156, 165].
An overview over the complex signalling circuity underlying EMT and the crosstalk between the four pillars is provided
in Fig.4.
4.2 EMT‑targeting therapeutics might be valuable inadjuvant therapies forhigh‑risk tumors
andimmunotherapy
Immunosuppression is a major risk factor for cSCC [23]. In murine models of cSCC, cyclosporineA-mediated repression
of immunosurveillance gave rise to aggressive tumors- bearing a strong EMT signature [166]. Similar observations in
humans also point towards a potential application of EMT-targeting therapeutics to mitigate malignant transformation in
immunosuppressed patients [167]. Additionally, EMT markers have been linked to resistance against immunotherapy, an
emerging treatment for inoperable advanced and metastatic cSCC [7, 15, 167]. EMT itself and the closely linked stem-like
properties have been linked to drug resistance and recurrence in multiple cancers [6]. Consequently, reducing EMT and
associated stemness might prove useful in assisting current and future therapeutics to overcome these challenges. As
shown in Table2, targeting canonical EGFR, canonical TGF-βR, NFκB, and PI3K-signalling successfully reverse EMT. Within
in these pathways, connecting hubs such as AKT pose interesting drug targets (Fig.4). Downstream eector kinases such
as ERK or p38 can also be used to attenuate EMT and prevent the transcriptional induction of EMT-TFs.
However, the targeted treatments used in the past are merely valuable proof-of-concept studies due to their insuf-
cient drug properties for clinical translation. Lacking selectivity, unclear mechanism of action, or a poor side eect
prole are only a few of the problems that need to be addressed. However, these studies can serve as part of the target
validation process or as lead for the development of more selective compounds. Replacing compounds with current
alternatives that have possibly undergone pre-clinical and clinical testing or are approved by the respective governing
body can be a valid strategy to assess their ecacy against cSCC.
In the future, therapeutic strategies could be amended by new targets and drug classes such as miRNA therapeu-
tics that replace tumor suppressor miRNAs lost during progression or targeting EMT-TF directly [168]. With increasing
advancements in RNA-based technologies, ectopic OVOL-TF induction might become feasible [169]. Modulation of p62
levels to attenuate EMT and subsequent induction of apoptosis via an autophagy-dependent mechanism is an interesting
mechanism [136]. Inhibitors of NFκB or Wnt/βcatenin signalling could attenuate the EMT-linked stemness and EMT and
hence help mitigate -associated complications such as therapy resistance. Hence, increasing research in small molecule
and peptide inhibitors makes targeting key protein–protein such as SMAD interactions or the dimeric NFκB transcription
factor become more attainable [170].
The uPA system is a regulator of MMPs and hence might be an attractive therapeutic target to reduce invasion and
metastasis. The upregulation of uPA/ uPAR and SERPINE1 is associated with poor prognosis in multiple cancers including
HNSCC and many other solid tumors [171174]. uPA-uPAR complexes can interact with integrins, vitronectin and LDLR
endocytosis receptors as well as induce plasmin-mediated ECM degradation [171174]. PAI-1 modulates cell adhesion
and migration via competing with integrins and uPAR for vitronectin binding sites and can act as co-receptor for LDLRs
and convey mitogenic signalling [172, 174176] High levels of both uPAS components and PAI-1 might also be required
for the precise spatial and temporal regulation of tumor cell invasion and focalized ECM remodelling [177, 178]. Addition-
ally, amiloride derivatives, a class of uPA inhibitors, have shown some success in murine models completely preventing
metastasis in an aggressive pancreatic cancer model [179, 180]. The induction of uPA activity by specic combination of
EGF and TGF-β1 treatment in HaCaT derivatives alongside of induction of EMT implies a strong link between the regula-
tion of uPAS and the regulation of EMT in keratinocytes [15, 118]. Some clinical evidence supports the exploration of uPA
as a therapeutic target. Minaei etal. [181] report the signicant upregulation of PLAU (uPA) and PLAUR (uPA receptor) in
metastatic tumors vs. non-metastatic primary cSCCs.
4.3 EMT markers can aid intumor risk stratification andare linked functionally toinvasion andmetastasis
In multiple cancers, EMT markers are found on cells with invasive capacities and on circulating tumor cells [10, 14, 15].
This links EMT conceptually to metastasis. In cSCC, EMT markers also correlate with increased metastatic risk, increased
progression, decreased recurrence and increased disease-specic death [54, 56, 151]. Barrette etal. [56] and Toll etal.
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1 3
[58] linked EMT markers to invasion and metastasis in cSCC. Additional biomarkers for aggressive and metastatic disease
include the indicators of increased signalling of two additional pillars, the overexpression of EGFR and the presence of
active nuclear IκKβ [160, 161]. This warrants further investigation into an EMT-marker based risk stratication of primary
tumors aiming to predict metastasis and guide clinical interventions. This would put cSCC in the line of cancers that push
for clinical adaptation of EMT markers for risk stratication as well as meeting a desperate need for metastasis markers
in cSCC [18, 182184].
5 Conclusion
EMT plays a pivotal role in the clinical progression of cSCC. EMT-markers are associated with worse patient outcome and
recurrence. This makes EMT a desirable process to target using small molecule inhibitors and other therapeutics. Poten-
tial therapeutic targets include members of the driving signalling pathways (EGFR, NFκB, TGF-β, PI3K) and eectors that
execute changes such as ECM degradation (uPA). Modulating EMT or mitigating its eects has the potential to become
a powerful therapeutic approach to assist current therapies such as immunotherapy as well as targeted therapies in
development.
Author contributions Search, selection, and extraction of information were executed by BG under the supervision and control of MR. BG, BA,
JP and MR contributed to the development of the concept and editing. All authors have read and approved the nal manuscript. All authors
have agreed on the journal to which the article will be submitted. All authors agree to take responsibility and be accountable for the contents
of the article. All authors read and approved the nal manuscript.
Funding BG was funded by a scholarship through a NHMRC Ideas Grant (APP1181179) to MR and BA.
Declarations
Competing interests The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article
are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. or g/ licen ses/ by/4. 0/.
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