International Journal of

Molecular Sciences

Review
Phenotypic Plasticity of Cancer Cells Based on Remodeling of
the Actin Cytoskeleton and Adhesive Structures
Svetlana N. Rubtsova , Irina Y. Zhitnyak and Natalya A. Gloushankova *

Institute of Carcinogenesis, N.N. Blokhin National Medical Research Center of Oncology, 24 Kashirskoye Shosse,
115478 Moscow, Russia; [email protected] (S.N.R.); [email protected] (I.Y.Z.)
* Correspondence: [email protected]; Tel.: +7-499-323-53-11

Abstract: There is ample evidence that, instead of a binary switch, epithelial-mesenchymal transition
(EMT) in cancer results in a flexible array of phenotypes, each one uniquely suited to a stage in
the invasion-metastasis cascade. The phenotypic plasticity of epithelium-derived cancer cells gives
them an edge in surviving and thriving in alien environments. This review describes in detail the
actin cytoskeleton and E-cadherin-based adherens junction rearrangements that cancer cells need to
implement in order to achieve the advantageous epithelial/mesenchymal phenotype and plasticity
of migratory phenotypes that can arise from partial EMT.

Keywords: cancer cells; EMT; plasticity; migration; actin cytoskeleton; E-cadherin; adherens junctions





 1. Introduction
Citation: Rubtsova, S.N.; Zhitnyak, Despite improvements in protocols of radio-, chemo-, and immunotherapy, distant
I.Y.; Gloushankova, N.A. Phenotypic metastases are still responsible for the great majority of cancer-related deaths. Detailed
Plasticity of Cancer Cells Based on studies of mechanisms of cancer cell dissemination are of great importance for understand-
Remodeling of the Actin ing tumor progression and developing new targeted drugs. In this Review, we present
Cytoskeleton and Adhesive current knowledge on how cancer cells acquire the ability to escape from primary tumor,
Structures. Int. J. Mol. Sci. 2021, 22, adapt their behavior to changes in their microenvironment during metastatic dissemination
1821. https://doi.org/10.3390/
and forming secondary (metastatic) tumors in distant organs or lymph nodes. Along with
ijms22041821
persistent cell proliferation and apoptosis suppression, one of the major characteristics
of tumor cells is their plasticity which allows them to switch between different modes of
Academic Editor: Jong Tai Chun
migration and support their survival, which results in successful metastatic colonization.
Recent data suggest that cancer cells expressing both epithelial and mesenchymal markers
Received: 22 December 2020
Accepted: 8 February 2021
maintain a high degree of plasticity, can survive in ectopic environments, exhibit a height-
Published: 12 February 2021
ened resistance to chemotherapy and have a high tumor initiating and metastatic potential.
Cells with a hybrid epithelial/mesenchymal phenotype are likely to be playing a major
Publisher’s Note: MDPI stays neutral
role in cancer progression.
with regard to jurisdictional claims in
2. Epithelial Cells
published maps and institutional affil-
iations. Most tumors in adults are carcinomas which arise from epithelial tissues. Epithelial tis-
sues are organized into layers composed of non-motile cells tightly connected by adhesive
structures (adherens junctions (AJs), tight junctions (TJs) and desmosomes) with adjacent
cells, and stably attached to the underlying basement membrane (BM) via hemidesmosomes
Copyright: © 2021 by the authors.
(Figure 1A–C) [1]. Epithelial cells exhibit apical–basal polarity of membrane domains, pro-
Licensee MDPI, Basel, Switzerland.
tein complexes, and cytoskeletal components. TJs and AJs form the apical junctional
This article is an open access article
complex [1]—a continuous belt around the apical part of cell—which is associated with
distributed under the terms and the circumferential actin bundle (Figure 1A,B). TJs define the boundary between the apical
conditions of the Creative Commons and basolateral domains in epithelial cells. TJs form a lateral diffusion barrier between
Attribution (CC BY) license (https:// the apical and basolateral domains. TJs are composed of occludin, claudins, and JAMs
creativecommons.org/licenses/by/ (Junctional Adhesion Molecules) that are linked to the actin cytoskeleton through ZO (zonu-
4.0/). lae occludens) proteins [2]. Apical-basal polarity is controlled by: (1) the apical complex—

Int. J. Mol. Sci. 2021, 22, 1821. https://doi.org/10.3390/ijms22041821 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 1821 2 of 23

the PAR proteins PAR3 and PAR6, aPKC, the CDC42 GTPase, the CRUMBS complex
(CRUMBS, PALS1, PATJ, and LIN-7); (2) the basolateral complex (SCRIB, DLG, LGL); and
(3) a cytoplasmic group of polarity proteins—PAR4/LKB1, PAR1/MARK, PAR5/14-3-3 [3].
Multi-level regulatory interactions between polarity proteins are essential for establishing
and maintaining cell polarity.

Figure 1. Organization of the actin cytoskeleton and adhesive structures in epithelial and mesenchymal cells. (A)—a
monolayer of epithelial cells. (B)—a close-up of an area in the dashed circle on A—stable cell-cell adhesion in epithelial cells
is provided by apical adhesion belts comprised by tight junctions (TJs) (red) and linear adherens junctions (AJs) (green),
both of which are closely associated with the circumferential actin bundle (yellow). (C)—a top view of a monolayer of
epithelial cells, connected by stable linear AJs. (D)—a mesenchymal cell exhibiting branched actin network (yellow) and
nascent focal adhesions (FAs) (purple) in lamellipodia at the leading edge. Closer to the center of the cell and in the rear
are mature FAs (purple) associated with straight actin bindles (yellow). Both nascent and mature FAs are connected to the
extracellular matrix (ECM) (pink). (E)—an area of cell-cell interaction between motile mesenchymal cells. (F)—a close-up of
the area in the dashed circle on E—overlapping lamellae containing branched actin network (yellow) point to the lack of
contact paralysis, unstable punctate AJs (green) are associated with straight actin bundles. Mature FAs (purple) connected
to the ECM (pink) are associated with straight actin bundles.

AJs are particularly important for epithelial tissue integrity as they provide strong
calcium-dependent cell-cell adhesion. In non-tumorigenic epithelial cells and in carcinoma
cells that maintain the epithelial phenotype, AJs are organized linearly into zonulae ad-
herens (adhesion belt), located in the apical junctional complex just below TJs (Figure 1) [1].
These linear AJs are very stable and dissolve only during mitosis. Disruption of AJs re-
sults in loss of cell–cell adhesion and dissociation of the cells. In epithelial cells, AJs are
formed by transmembrane E-cadherin adhesion receptors whose cytoplasmic domains
bind to members of the catenin protein family, β-catenin and p120 (Figure 2) [4,5]. β-
catenin interacts with the N-terminal domain of α-catenin, the central part of the α-catenin
molecule contains the vinculin-binding domain, and α-catenin’s C-terminal domain di-
rectly binds actin filaments [6,7]. Contractile forces generated by actin-coupled myosin II
induce unfolding of the actin-binding domain of α-catenin, which enhances actin bind-
ing [8]. Force-dependent destabilization of the interactions between MI vinculin-binding
and MII and MIII inhibitory domains of α-catenin leads to opening of the MI domain [7,9]
that results in a significant increase in its affinity for vinculin, which, in turn, recruits
Int. J. Mol. Sci. 2021, 22, 1821 3 of 23

additional actin that stabilizes the cadherin/catenin complex. This junctional actin, tightly
associated with the circumferential actin bundle, is crucially important for the assembly
and maintenance of AJs [10,11].

Figure 2. Structure and composition of an epithelial adherens junction. E-cadherin molecules (green)
on the surface of adjacent cells connect with one another via their extracellular domains. Below
the cytoplasmic membrane (PM), the intracellular domain of E-cadherin interacts with p120 (pink)
and β-catenins (dull red). β-catenin binds to α-catenin (dark blue), which, in turn, interacts with
vinculin (blue) and directly with actin filaments (yellow). Vinculin binds to actin filaments to stabilize
the cadherin-catenin complex. Various actin-binding proteins such as VASP (bright red), EPLIN
(Epithelial Protein Lost in Neoplasm) (purple), α-actinin (orange), palladin (light green) and myosin
II (cyan-green) are associated with junctional actin.

Super resolution microscopy of Madine-Darby canine kidney (MDCK) epithelial

cells cultured on an E-cadherin-coated planar substrate demonstrated that E-cadherin-
based adhesions of the cells maintained a strict compartmentalized architecture. The
cadherin-catenin compartment containing E-cadherin and catenins and the actin cytoskele-
tal compartment containing actin cytoskeleton and actin-binding proteins (EPLIN (Ep-
ithelial Protein Lost in Neoplasm), myosin II, palladin, and α-actinin) sandwiched the
intermediate zone containing vinculin and VASP [12]. EPLIN additionally stabilizes the
circumferential actin bundle by inhibiting actin depolymerization and crosslinking actin
filaments [13]. SiRNA-mediated knockdown of EPLIN results in disappearance of circum-
ferential actin bundles and converting linear AJs into punctate AJs associated with radial
actin bundles [14]. Both myosin IIA and IIB isoforms are important for establishment and
maintenance of linear AJs. Actomyosin contractility defines the morphology of linear AJs,
drives compaction of epithelial cells and supports integrity of epithelial layers. Myosin IIA
is required for assembly and maintenance of junction complexes [15]. Myosin IIA depletion
disrupts formation of E-cadherin junctional belt in the apical part of epithelial cells. Myosin
IIB depletion decreases actin content in circumferential actin bundles associated with linear
AJs [16,17]. As was observed in 2D cultures of epithelial cells, AJ maturation required
activity of the Rho family of GTPases which recruited formins promoting elongation of
linear actin filaments [10]. mDIA1, recruited by active RAC1 [18], and FMNL2, recruited
by active RHOA [19], are necessary for E-cadherin stabilization in AJs.
Int. J. Mol. Sci. 2021, 22, 1821 4 of 23

Analyzing collisions between epithelial cells, we described the phenomenon of con-

tact paralysis of protrusive activity that was fundamentally different from the behavior
of mesenchymal cells in response to cell-cell interactions [20–22]. A collision between
epithelial cells resulted in dramatic cessation of protrusive activity in the zone of the
expanding cell-cell contact (contact paralysis) whereas after the initial cell-cell contact
mesenchymal cells continued formation of protrusions in the zone of cell-cell collision
(Figure 1E,F) [20,23]. Contact paralysis in epithelial cells is caused by tangential tension
at the cell-cell boundaries generated by actin-myosin bundles [24]. Live cell imaging and
analysis of the spatiotemporal regulation of RAC1 and RHOA activity and actomyosin
contractility during de novo formation of cell–cell adhesions detected high RAC1 activity
only at the early stages of formation of stable AJs during initial lamellipodia interactions,
but it was down-regulated during lateral expansion of linear AJs, while RHOA and ac-
tomyosin contractility were activated at the edges of expanding contact [25]. Contact
paralysis is essential for maintenance of stable cell-cell adhesion. Its loss during neoplastic
transformation may facilitate cancer cell dissemination during metastasis.

3. Epithelial-Mesenchymal Transition (EMT)

Epithelial cells can acquire mesenchymal phenotypes using a program known as
the epithelial–mesenchymal transition (EMT). While undergoing EMT, epithelial cells
lose apical-basal polarity and stable cell-cell adhesion and acquire migratory activity.
EMT is implicated in various biological processes such as embryonic development, tissue
repair, wound healing and pathological conditions such as tissue/organ fibrosis and
cancer progression. The bulk of carcinoma cells in primary tumors exhibit epithelial
characteristics but in order to metastasize, carcinoma cells undergo EMT to acquire a more
mesenchymal phenotype that will allow them to detach from neighboring cells, overcome
tissue barriers and migrate through tissues. During EMT, carcinoma cells lose their apical-
basal polarity; stable E-cadherin-based AJs and the curcumferential actin bundle supporting
their integrity become disrupted; de novo actin polymerization leads to appearance of
dynamic lamellipodia which are the engine of cancer cell migratory and invasive activity.
Cells acquire front-rear polarity and become capable of directional migration.
EMT is the most widely studied example of phenotypic plasticity, and its contribution
to promoting cancer cell invasion and metastasis has been established for many types
of carcinomas [26–29]. The most common sequence of events nesessary to successfully
colonize distant organs is termed “the invasion-metastasis cascade” and includes the
following steps: (1) invasion at the local site through BM; (2) migration in surrounding
tissues; (3) entrance into a circulatory system (blood or lymph vessel) and traveling through
circulation; (4) arrest at a distant site and exit from the circulatory system; (5) survival and
proliferation in a distant organ, resulting in formation of micro- and macrometastases [30].
In cancer, EMT is activated by signaling pathways from TGFβ (Transforming Growth
Factor Beta), EGF (Epidermal Growth Factor), HGF (Hepatocyte Growth Factor), Notch,
FGF (Fibroblast Growth Factor), Wnt, and IGF (Insulin-Like Growth Factor), signals from
tumor microenvironment (e.g., cancer-associated macrophages or fibroblasts), hypoxia and
increased matrix stiffness [29,31–34].
In many cancer types these signals activate core EMT-inducing transcription fac-
tors (EMT-TFs)—SNAIL, SLUG, TWIST1, ZEB1, and ZEB2 via transcriptional and post-
transcriptional mechanisms [35]. These EMT-TFs are considered the key drivers of can-
cer progression, and their high expression has been detected in many invasive carcino-
mas [36–40]. EMT-TFs down-regulate the expression of genes associated with the epithelial
phenotype (e.g., E-cadherin, occludin, cytokeratins, polarity genes) and induce the expres-
sion of genes that sustain the mesenchymal phenotype (N-cadherin, vimentin, fibronectin,
and certain integrins) and matrix metalloproteinases (MMPs) (Table 1) [31,41]. Expression
of mesenchymal markers in cells of epithelial origin may be advantageous for cell migration.
N-cadherin increased migration and invasion of breast cancer cells regardless of E-cadherin
expression [42,43]. Vimentin, through its interaction with RhoGEFs, could promote cell
Int. J. Mol. Sci. 2021, 22, 1821 5 of 23

migration by influencing structure and dynamics of the actin cytoskeleton [44]. Vimentin
binding to the cytoplasmic tail of β3 integrin could directly affect integrin-mediated sig-
naling in the cell [45]. However, the mechanisms regulating vimentin involvement in
controlling cell migration require further studies.

Table 1. EMT transcription factors and their targets.

Down-Regulated Up-Regulated
EMT-TF References References
Expression Expression
E-cadherin
[46,47] Vimentin [55,56]
Plakophilin-2
[48] Claudin-11 [57]
Claudin-4
[48] MMP1 [56]
Cytokeratins 17, 18,
[48] MMP2 [55,56,58]
SNAIL 19, 20
[48] MMP7 [56]
Gelsolin
[48–51] MT1-MMP [56]
Occludin
[52,53] Fibronectin [56]
Integrins α3, α6, β4
[54] Integrins α2, β1, β3 [31,41,53]
Crumbs3
E-cadherin [59,60] Vimentin [60]
SLUG
Occludin [50,61] Fibronectin [60]
N-cadherin [64,65]
E-cadherin [62–64] Vimentin [63,64,66]
TWIST1 α-catenin [62,63] Smooth muscle actin [62,63]
γ-catenin [63] Fibronectin [62,63]
Integrin α5 [67]
E-cadherin [68,69] N-cadherin [71]
ZEB1 Occludin [70] Fibronectin [71]
Crumbs3 [70] Vimentin [72]
N-cadherin [73]
Vimentin [56,74]
E-cadherin [56,73] MMP1 [56]
ZEB2
α-catenin [73] MMP2 [56]
MT1-MMP [56]
Fibronectin [56]

EMT-TFs play essential roles in cancer cell migration, invasion and metastasis. Studies
in vitro demonstrated that exogenous SNAIL and SLUG increased migratory and invasive
capacity of cancer cells [47,60,75]. SNAIL promoted collective migration in squamous
carcinoma cells by inducing the expression of claudin-11 [57]. ZEB1 and ZEB2 induced
EMT in epithelial cells and promoted cell migration and invasion in breast and colorectal
cancer cells [38,73,76]. ZEB1 promoted metastasis in the KPC mouse model of pancreatic
cancer [77]. TWIST1 expression enhanced cell motility in hepatocellular carcinoma cells [64].
Expression of TWIST1 correlated with lymph node metastasis of breast cancer [37].
Besides promoting metastatic dissemination, the EMT program and EMT-TFs appear
to serve as major drivers of cancer progression. EMT-TFs such as SNAIL and SLUG can
activate and maintain stemness traits in carcinoma cells as was shown for mammary and
thyroid carcinoma respectively [78,79]. EMT-TFs have been shown to promote DNA dam-
age repair and radioresistance [80,81]. Emerging evidence suggests that EMT contributes
to increased cell survival, suppression of apoptosis and resistance to chemotherapy and
immunotherapy [31,35,82,83].

4. Hybrid Epithelial-Mesenchymal Phenotype

During the last decade it has been revealed that in cancer, EMT is not a binary switch
between epithelial and mesenchymal states, but a process which, depending on particular
combinations of intrinsic and extrinsic factors, generates subpopulations of cells in various
intermediate states between the epithelial and mesenchymal phenotypes [26,27,84]. Carci-
Int. J. Mol. Sci. 2021, 22, 1821 6 of 23

noma cells frequently undergo partial EMT (pEMT) by acquiring mesenchymal traits while
retaining epithelial markers. Cells possessing the hybrid epithelial/mesenchymal pheno-
type retain expression of cytokeratins or E-cadherin or EpCAM (Epithelial Cell Adhesion
Molecule) while inducing a mesenchymal marker vimentin and, in a substantial number of
cases, N-cadherin [26,29,78,83,85–88]. Cells undergoing pEMT switch from stable linear
AJs to unstable punctate AJs. The robust circumferential actin bundle is replaced with
dynamic lamellipodia in the front and straight actin bundles assosiated with AJs and focal
adhesions. Cells with the hybrid epithelial/mesenchymal phenotype acquire migratory
activity [22].
EMT in cancer exhibits great diversity and is a local and dynamic process [78,89]. It is
considered that the hybrid epithelial/mesenchymal phenotype is a plastic state, prone to
changes depending on the cell’s microenvironment. This phenotype, which allows the cell
to quickly adapt and change its morphology and migratory properties accordingly is espe-
cially advantageous during metastatic colonization [84,86,90,91]. Single-cell transcriptomic
analysis of genes associated with stemness (OCT4 and SOX2) and EMT (SNAI2, SKP2 and
TWIST1) in mouse models of human triple negative breast cancer demonstrated higher
expression of these genes in early stage metastatic disease than either in primary tumors or
in advanced stage metastatic disease [92]. Using multicolour fluorescence-activated cell
sorting and single cell RNA sequencing, various subpopulations of hybrid phenotype cells
expressing different combinations of epithelial and mesenchymal markers were identified
in mouse models of skin and breast carcinoma [84]. Intravital microscopy of a breast
cancer model harboring an EMT-driven color switch revealed a population of tumor cells
undergoing EMT at the boundaries of tumor lobules adjacent to the blood vessel-enriched
stroma [93]. In a lineage-labelled mouse model of pancreatic ductal adenocarcinoma, it
was observed that tumor cells undergoing the pEMT program migrated as clusters and
exhibited epithelial-mesenchymal plasticity [94]. Single-cell transcriptomic analysis of
cells from head and neck squamous cell carcinoma patients showed that cells exhibiting
the pEMT program spatially localized at the leading edges of primary tumors in contact
with cancer-associated fibroblasts (CAFs) [95]. EMT may be regulated by growth factors
secreted by the cells from the tumor microenvronment. It was shown that CAFs secreting
HGF and TGFβ promoted activation of carcinoma cells migration [96,97]. In another study,
migration and intravasation of tumor cells was induced by tumor-associated macrophages
producing EGF [98,99].
A significant amount of data accumulated in recent years shows that cells with the hy-
brid epithelial/mesenchymal phenotype exhibit high metastatic potential [27,83,84,100,101].
Intravenous injection of different subpopulations of squamous cell carcinoma cells derived
from hair follicles or prostate carcinoma cells demonstrated increased lung metastasis of
pEMT tumor cells as compared to cells with mesenchymal phenotype [85,102]. Analysis of
circulating tumor cells (CTCs) originated from the primary tumor and travelling through
the bloodstream has provided important insights into phenotypic traits of disseminating
cancer cells. CTCs displaying both epithelial (e.g., E-cadherin, cytokeratins) and mesenchy-
mal markers (e.g., vimentin) have been found in the blood of patients with breast, lung,
colon, prostate, and liver cancers [90,103–105]. RNA profiling of breast cancer CTCs has
revealed the existence of different EMT states [90]. The presence of expression of epithe-
lial markers in CTCs is associated with better survival of cancer cells and poorer clinical
prognosis. In prostate cancer patients, detection of CTCs expressing high levels of EpCAM,
correlated with poor survival. CTCs with low levels of EpCAM did not affect survival of
the patients [106]. In another study, the presence of CTCs with the epithelial/mesenchymal
phenotype, co-expressing cytokeratin, high levels of ALDH1, and nuclear TWIST1, in the
blood of metastatic breast cancer patients had a significant negative prognostic value [107].
In the blood of patients with breast, lung and head and neck cancer along with single
CTCs, CTC clusters were also found [90,108–110]. CTCs obtained from patients with
various types of cancers were often joined into clusters by E-cadherin-based AJs. These
clusters were more resistant to anoikis in the bloodstream and more effective at metastatic
Int. J. Mol. Sci. 2021, 22, 1821 7 of 23

outgrowth in distant organs [111,112]. To transit through capillary vessels, CTC clusters of
≤20 cells could reversibly stretch into single-file chains [113]. Association of clusters with
platelets could protect cancer cells from elimination by immune system [27]. Studies of
patients with lung, breast and head and neck cancer demonstrated that CTC clusters were
associated with poorer prognosis as compared to single CTCs [108–110].

5. Role of E-Cadherin in Carcinoma Cell Dissemination

For a long time, it was an accepted fact that down-regulation of E-cadherin expression
plays a key role in carcinoma progression by promoting invasion and metastasis [114,115].
Loss of E-cadherin expression has been found in esophageal, gastric, breast, colon, prostate,
and liver cancer [116–121]. It is clearly apparent that down-regulation of E-cadherin expres-
sion leads to destabilization of AJs and facilitates the initial dissociation of cells from the
primary tumor. Tumor-suppressive role of E-cadherin may also be explained by its negative
role in regulation of the canonical WNT/β-catenin pathway through sequestering β-catenin
at the cell membrane [122]. Additionally, E-cadherin localized at the membrane in AJs can
negatively regulate the ligand-dependent activation of EGFR, IGF-1R, and c-Met [123,124].
E-cadherin in the apical zonulae adherens has been found to recruit DROSHA and DGCR8,
the core components of the RNA-induced silencing complex (RISC) and various mRNAs
and miRNAs including miRNA-24 and miRNA-200c, via PLEKHA7. MiRNA-24 and
miRNA-200c associated with zonulae adherens down-regulate expression of the oncogenes
MYC and JUN, and a pluripotency factor SOX2 by silencing their mRNAs [125].
Although E-cadherin has been considered a tumor suppressor, accumulating evidence
suggests a more complicated role of E-cadherin in cancer cell biology. A wealth of histo-
chemical data points to retention of E-cadherin expression in many invasive carcinomas
and their metastases (e.g., ductal breast, colorectal, prostate, pancreas carcinoma and
oral squamous cell carcinoma) [126–131]. Well-differentiated cells that maintain epithelial
morphology and apico-basal polarity form the bulk of many early stage carcinomas [31].
In vitro, cells of various carcinomas (e.g., MCF-7 and T-47D breast carcinoma, A-549 lung
carcinoma, HT-29, Caco-2 and T84 colon carcinoma cell lines) are capable of maintaining
stable linear AJs associated with the circumferential actin bundles. Many types of car-
cinomas (e.g., breast, colorectal, prostate, oral squamous cell carcinoma) can invade as
multicellular groups in which cells remain attached to the neighbors by E-cadherin-based
AJs (collective invasion) (Figure 3A) [132–135]. Collective invasion may be facilitated by
CAFs connected with cancer cells by heterophilic E-N-cadherin AJs. CAFs may act as
leader cells and remodel ECM creating migration tracks for follower cancer cells [136,137].

Figure 3. E-cadherin-based adherens junctions (AJs) facilitate cancer cell dissemination. (A)—collective invasion. (B)—
migration over a monolayer of normal epithelial cells. (C)—invasion of the monolayer of normal epithelial cells. (D)—a
circulating tumor cell (CTC) cluster traveling through circulation. Orange—cancer cells, grey—normal epithelial cells,
purple—endothelial cells. Green—E-cadherin-based AJs, red arrows—direction of cell migration.

As was mentioned earlier, E-cadherin may contribute to dissemination of cancer

cells through circulation by helping form CTC clusters (Figure 3D). Using mouse and
human models of luminal and basal invasive ductal breast carcinomas, it has been recently
demonstrated that E-cadherin contributes to metastasis by acting as a survival factor for
cancer cells [111,112]. Loss of E-cadherin reduced proliferation and survival of CTCs and
Int. J. Mol. Sci. 2021, 22, 1821 8 of 23

their dissemination via TGFβ-stimulated accumulation of reactive oxygen species and

induction of apoptosis [138].
Although cancer cells initiating the invasion-metastasis cascade acquire a more mes-
enchymal phenotype, reversal to the epithelial phenotype (mesenchymal-epithelial tran-
sition, MET) and re-expression of E-cadherin are important for metastatic colonization.
Earlier, immunohistochemical studies of invasive breast cancer have detected higher levels
of E-cadherin expression in metastases than in the primary tumor [139]. Direct intravital
microscopy in mice demonstrated that carcinoma cells spontaneously undergoing EMT and
acquiring migratory activity reverted to epithelial phenotype in growing metastases [85,89].
It was shown that EMT-TFs TWIST1 and PRRX1 (Paired Related Homeobox 1) that were
necessary for initiation of metastasis, needed to be repressed for successful metastatic
outgrowth [140–142]. In a mouse breast cancer model it was recently shown that metastatic
colonization by cancer cells required a metastatic niche activation in distant organs that
promoted the shift toward a more epithelial phenotype and up-regulation of E cadherin
expression [143,144]. Taken together, these data point at extreme importance of E-cadherin-
based AJs in cancer cell dissemination.
We were first to show that E-cadherin-based AJs in neoplastically transformed epithe-
lial cells undergoing pEMT were different from the linear AJs found in normal epithelial
cells. Using a panel of chemically or oncogenically transformed IAR rat liver epithelial
cells, we observed radial (punctate) AJs, which behaved radically differently from the
stable linear AJs in normal epithelial cells [22]. These punctate AJs were associated with
straight actin bundles and were very dynamic and unstable much like the N-cadherin-
based AJs of bona fide mesenchymal cells. Formation and maintenance of punctate AJs
depended on myosin II-mediated contractility, as both the ROCK inhibitor Y-27632 and
the myosin II ATPase inhibitor blebbistatin reduced mature punctate AJs to nascent dot-
like AJs. Transformed epithelial cells with these punctate AJs were capable of effective
collective migration on 2D adhesive substrates and in migration chambers [145]. We also
found that neoplastically transformed epithelial cells retaining E-cadherin expression could
form E-cadherin-based AJs with normal epithelial cells. Expression of E-cadherin and its
assembly into dynamic punctate AJs allowed transformed cells to migrate over epithelial
monolayer and to invade the monolayer (Figure 3B,C) [146]. When the formation of punc-
tate AJs in transformed cells was abolished by expression of either a dominant negative
E-cadherin construct or an anti-E-cadherin siRNA, migration over and invasion of the
epithelial monolayer considerably decreased. Depletion of N-cadherin did not have any
effect on invasive behavior of transformed cells. Thus, cancer cell dissemination may be
dependent on formation of E-cadherin-based cell-cell contacts between cancer cells and the
surrounding normal cells.
In a recent work by Indra et al. spatial organization of punctate AJs in A-431 car-
cinoma cells was studied in great detail. It was shown that punctate AJs consisted of
dense, paracrystalline nanoclusters formed through cis and trans interactions of cadherin
ectodomains, interspersed with less dense cadherin regions [147]. It was also shown that
F-actin at punctate AJs consisted of two different structurally distinct regions—stable
bundle stalk enriched with calponin and highly dynamic AJ proximal region of branched
F-actin at the tip of the bundle enriched with VASP and F-actin depolymerization factor,
cofilin-1. The assembly and disassembly of both F-actin and cadherin clusters were tightly
coupled [148].

6. Rearrangement of the Cytoskeleton and Adhesive Structures in Cells Undergoing EMT

While undergoing EMT, cells have to reorganize their cytoskeleton to weaken cell-cell
adhesion and acquire efficient directional motility. As mentioned earlier, weakening of
cell-cell adhesion may be connected with transcriptional repression of the CDH1 gene that
encodes E-cadherin or loss of surface E-cadherin through protein endocytosis [91,149]. The
earliest stages of EMT leading to disruption of cell-cell contacts that allows a cell to escape
from neighboring cells remained unexplored until now. Recently, using live cell imaging,
Int. J. Mol. Sci. 2021, 22, 1821 9 of 23

we analyzed early events during EMT induced by EGF in IAR-20 rat liver epithelial cells
(Figure 4) [150]. We detected rapid (within 5–10 min) fragmentation and dissolution
of circumferential actin bundle, a structure crucial for maintenance of stable linear AJs.
Simultaneously, we observed formation of dynamic lamellipodia containing branched actin
network at the cell-cell boundaries and appearance of retrograde actin-myosin flow. We
detected increased phosphorylation of the actin-binding protein EPLIN within minutes
of the addition of EGF. It had been shown earlier that phosphorylation of EPLIN had
resulted in its degradation through ubiquitin-proteasome-dependent mechanism [151].
It is known that EPLIN stabilizes circumferential actin bundle [13]. Thus, EGF-induced
phosphorylation and degradation of EPLIN may lead to disruption of the circumferential
actin bundle at the earliest stages of EMT. We also found that the early EMT-induced
disruption of circumferential actin bundle was followed by transformation of the stable
linear E-cadherin-based AJs into dynamic punctate AJs which were associated with straight
actin bundles and co-localized with a tension-sensitive protein zyxin. The presence of zyxin
indicated generation of centripetal forces at the cell-cell boundaries. Taken together, these
observations reveal that early EMT promotes increased dynamics in the cell-cell contact
areas, particularly the transformation of stable AJs and actin structures into dynamic ones.
Contact paralysis—an essential property of the stable control AJs—disappeared, leading
to weakening of cell-cell adhesion and disruption of cell-cell contacts. Cells released
from the stable cell-cell contacts could then acquire front-rear polarity and eventually, a
migratory phenotype.
Front-rear polarity is an important characteristic of mesenchymal cells that defines
directionality of migration. Front-rear polarity is a major actin cytoskeleton reorganisa-
tion; however, other cytoskeletal systems as well as polarity proteins contribute to its
establishment. Generation of front-rear polarity is largely controlled by CDC42. Using
stably expressed fluorescence resonance energy transfer (FRET) biosensors, it was found
that in morphologically non-polarized cells, local activation of CDC42 and its spatial
gradient drove the formation of initial protrusive fronts upon uniform chemotactic stimu-
lation [152]. When the apical junctional complex was disrupted, CDC42 and the polarity
protein complex PAR6-aPKC re-localized from the TJ region to the leading edge and in-
duced re-localization of the centrosome and the Golgi apparatus to the front of the cell.
Microtubule-organizing centers (MTOCs) associated with the centrosome and the Golgi
apparatus promote microtubule (MT) growth towards the cell front, and subsequent cell
migration and scattering [153,154]. MTs play a key role in the establishment and mainte-
nance of front–rear polarity organizing MT-mediated intracellular transport, delivering of
kinases and guanine nucleotide exchange factors (GEFs) for Rho GTPases to the leading
edge [44,155]. MT-dependent delivery of mRNAs of the proteins that regulate the actin
cytoskeleton and local translation also controls protrusion persistence in mesenchymal-
like cells [156]. The EB1 protein localized at the plus ends of growing MTs recruits the
CLIP-170-mDIA1 complex to accelerate actin filament elongation during lamellipodia
formation [157].
In an in vitro model of EMT leading to epithelial cell scattering, it was found that
protrusive activity at the free cell edges contributed to cell scattering via formation and
attachment of integrin-mediated focal adhesions (FAs) to substrate and actomyosin contrac-
tility that transmitted to the rear cell-cell boundaries and caused disruption of the cell-cell
contacts [158]. Integrins play an essential role in front-rear polarity and cell migration by en-
gaging the Rho family of small GTPases (Rac, Rho and Cdc42) that coordinate cytoskeletal
dynamics. Rho-dependent localization of myosin IIB at the cell rear is required for front-
back polarity maintenance and tail retraction during mesenchymal migration [159,160].
Int. J. Mol. Sci. 2021, 22, 1821 10 of 23

Figure 4. Dissolution of the circumferential actin bundle, degradation of EPLIN and rearrangement of E-cadherin-based
adherens junctions (AJs) during the early stages of epithelial-mesenchymal transition (EMT). Adapted from Zhitnyak
et al., Cells 2020, 9, 578 [150]. Top row: progression of EMT induced by Epidermal Growth Factor (EGF). 1—epithelial
cells before treatment with EGF. An island of non-motile cells tightly connected by linear AJs. Circled area in higher
magnification below: a stable linear AJ between epithelial cells, associated with a robust circumferential actin bundle.
EPLIN supports the bundle integrity by cross-linking actin filaments. 2—early stages of EGF-induced EMT (5–10 min).
Protrusive activity increases at the cell edges and protrusions begin to form at the cell-cell boundaries. Contact paralysis at
the cell-cell boundaries disappears. Linear AJs are partially disassembled and replaced by punctate AJs. Circled area in
higher magnification below: Dissolution of the circumferential actin bundle and reorganization of linear AJs. Part of the
AJ still maintains its original linear configuration and is associated with the remnants of the circumferential actin bundle.
Phosphorylation of EPLIN results in its detachment from the circumferential actin bundle which leads to disintegration of
the bundle. Bottom part—formation of small punctate AJs associated with nascent straight actin bundles. 3—later stages of
EGF-induced EMT (15–60 min). Cells acquire migratory properties and detach from each other, breaking cell-cell adhesion.
The new punctate AJs formed by migratory cells are dynamic and unstable. Circled area in higher magnification below:
mature punctate AJs during later stages of EMT. The AJs are longer than in 2 and are associated with thicker straight actin
bundles again fortified by EPLIN.

7. Mesenchymal Migration
During metastasis, as was shown by intravital microscopy, cancer cells use different
modes of migration such as mesenchymal migration of individual cells or groups of cells
or amoeboid migration. This choice is dependent on substrate adhesiveness, composition
of the extracellular matrix (ECM), activity of Rac and Rho GTPases regulating cytoskeletal
dynamics, and MMP activity [161–163]. Among the MMPs, MT1-MMP plays a central role
in pericellular matrix degradation [164]. Actin cytoskeleton dynamics is the basis for cell
migration. In order to migrate, cells can use two properties of actin filaments: the ability to
push by polymerization and the ability to contract by interacting with myosin (Figure 5).
Carcinoma cells use mesenchymal mode of migration moving along the BM, invad-
ing surrounding tissues or migrating in distant organs during metastatic colonization.
Mesenchymal migration is characterized by repeating cycles that include extension of
protrusions at the leading cell edge, their attachment to the substratum via FAs followed
by detachment and retraction of the rear as a result of the cell contractility, which results
in cell translocation. Migrating cells form flat protrusions (lamellipodia) and finger-like
protrusions (filopodia). Extension of lamellipodia is driven by pushing forces generated
Int. J. Mol. Sci. 2021, 22, 1821 11 of 23

by coordinated polymerization of a branched network of actin filaments oriented with

their barbed ends towards the leading edge (Figure 1D, Figure 5B) [44,165,166]. This
branched actin network is highly dynamic and is regulated by: (1) actin nucleators; (2)
proteins promoting actin filament elongation; (3) capping proteins; (4) filament severing
proteins [165,167]. The Arp2/3 complex consisting of seven subunits (Arp2, Arp3, ArpC1-
C5) which nucleates a new actin filament off the side of a pre-existing filament plays the
key role in formation of the branched actin network in lamellipodia [168]. The Arp2/3
complex requires activation by a nucleation-promoting factor WAVE that exists as a part
of a pentameric protein complex [167,169]. The WAVE regulatory complex is recruited to
lamellipodia by RAC1-GTP [170]. Ena/VASP proteins and formin family proteins binding
with barbed ends of actin filaments promote their elongation [171–173]. Lamellipodin,
which has been shown to interact both with the WAVE regulatory complex [174] and
Ena/VASP proteins [175] in lamellipodia, can regulate lamellipodia dynamics and cell
adhesion to ECM [176]. Actin filament debranching and severing is mediated by the actin
depolymerizing factor (ADF)/cofilin family proteins [177].

Figure 5. Plasticity of cancer cell migration. (A)—A primary epithelial tumor through partial epithelial- mesenchymal
transition (pEMT) or epithelial-mesenchymal transtion (EMT) gives rise to motile cells capable of invasion via individ-
ual or collective mesenchymal migration. Specific microenvironment conditions govern reversible transitions between
mesenchymal and amoeboid migration modes (collective-amoeboid transition (CAT), mesenchymal-amoeboid transition
(MAT), amoeboid-mesenchymal transition (AMT)). In a distant metastasis, cells cease to migrate and revert to the original
epithelial phenotype (mesenchymal-epithelial transition (MET)). (B–D)—Modes of cancer cell migration. Reorganized actin
cytoskeleton and relative activity of Rho and Rac in the front and rear of the cells during individual (B), collective (C), or
amoeboid (D) migration.

Arp2/3 subunits overexpression signals poor prognosis for patients with lung, breast,
and colorectal cancers. It is correlated with cancer progression, invasion and metastasis.
3D migration of cancer cells in vitro or in vivo has been shown to require Arp2/3 activity.
Overexpression of the WAVE regulatory complex is observed in various carcinomas (breast,
colon, liver, lung, ovary and prostate). Generally, overexpression of WAVE complex compo-
nents is associated with reduced survival and lymph node invasion and metastasis. [178].
Lamellipodin has been shown to promote metastasis (specifically, tumor invasion and
intravasation) in an orthotopic mouse breast cancer model, possibly through its interactions
with Ena/VASP and WAVE. In breast cancer patients, moderate increase in Lamellipodin
levels correlated with poorer prognosis [179]. Mena/VASP expression in breast, cervical,
colorectal and pancreatis cancers correlated with high risk of metastases [180–183]. Mena
invasion isoform promoted effective single-cell chain migration in mouse model of breast
cancer [184].
Int. J. Mol. Sci. 2021, 22, 1821 12 of 23

Mesenchymal cells’ attachement to ECM via FAs is mediated by transmembrane

heterodimeric receptors integrins. Small nascent adhesions constantly assemble at the edge
of lamellipodia. Binding to ECM induces recruitment to FAs of paxillin, vinculin, and talin,
which link integrins to actin filaments, leading to integrin activation and clustering [185].
Contractile forces generated by mutual sliding of actin and bipolar myosin II filaments
induce growth and maturation of nascent adhesions into FAs. Cross-linking of actin
filaments by α-actinin bundles the actin filaments associated with FAs into stress fibers and
increases contractile forces (Figure 1D, Figure 5B) [186]. Actomyosin-mediated contraction
of the cell body promotes disassembly of FAs at the cell rear, retraction of the rear and cell
displacement [44,166]. FAs, being key components of cell migration machinery, play an
important role in cancer cell dissemination. Integrins are involved in mechanotransduction
through sensing the stiffness of the underlying surfaces and transmitting that information
inside the cells, allowing them to adapt to their microenvironment. FAs contain multiple
signaling molecules including FAK and Src family kinases, tyrosine phosphatases, and
adaptor proteins [185]. It has also been found that integrin-mediated FAs accumulate
regulators of RhoGTPase activity. It is known that the activity of Rho GTPases is regulated
by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and
guanine nucleotide dissociation inhibitors (GDIs) [161–187]. Localization of GEFs and
GAPs in different zones within cells results in local activation of Rho GTPases. Recently
it has been found that integrin-based adhesions play a key role as multiprotein scaffolds
spatially segregating GEFs and GAPs to promote RAC1 activation [188] which induces
Arp2/3-dependent polymerization of the actin network and lamellipodia formation at the
cell edge.
Changes in expression patterns of integrins on cancer cells and in the production,
secretion and remodeling of ECM by cancer cells and CAFs leading to increased tumor
ECM stiffness, contribute to migratory behavior of cancer cells [189]. ECM stiffness affects
localization and transcriptional activity of the mechanosencitive transcriptional co-factors
YAP and TAZ [190,191]. ECM stiffness also promotes migration via FAK-dependent acti-
vation of RAC1 [192]. Integrin-specific signaling is essential for cancer progression. α6β4
intergin cooperates with oncogenic RTKs EGFR, ErbB2 while αvβ3 integrin cooperates with
c-Met to amplify oncogenic signaling in tumors [193–195]. In cancer cells, integrin-specific
signaling is also involved in pro-mitogenic and pro-survival signaling pathways such as
the RAS/ERK, PI3K/AKT, and NF–kB (nuclear factor–kappa B) as has been thoroughly
described in several reviews [196,197]. Integrin-dependent phosphorylation by Src and
FAK of β-catenin and subsequent disruption of AJs contributes to weakening of cell-cell
adhesion of pancreatic carcinoma cells growing on collagen type I [198]. Integrin-specific
signaling is not restricted by focal adhesions. Upon endocytosis of integrins, FAK binds to
and becomes activated on integrin-containing endosomes. Integrin endosomal signalling
plays a key role in supporting anchorage-independent growth and anoikis resistance
nessesary for survival in vasculature and formation of metastasis by cancer cells [199].
Using mesenchymal mode of migration, cells can move as single cells or as groups
of cells, a process known as collective migration (Figure 5A–C). Cells of many types of
carcinomas (e.g., ductal breast carcinoma, squamous cell carcinoma, colon carcinoma
and others) can employ collective migration to invade adjacent tissues [200]. Collective
migration is driven by front-rear polarization of leading cells that move using mesenchymal
mode of migration and coordinate migration of the follower cells via signaling molecules
and cadherin-based AJs [201]. At the invasion front, the lamellipodia of the leading cells
generate traction forces on the substrate via integrins, while the cell–cell junctions transmit
these forces from the front to the rear of the migrating group, which enables migration
of follower cells [202]. The choice of a leader cell and establishment of its polarity is far
from clear. The asymmetric distribution of cadherin-based adhesions may contribute to
coordination of collective migration by restricting lamellipodial activity in the zones of
cell-cell contacts and recruiting Rac GEFs at the leading cell edge [203]. Activation of
leader cells depends on extracellular stimuli, such as ECM ligands or growth factors from
Int. J. Mol. Sci. 2021, 22, 1821 13 of 23

microenvironment [201]. It was also demonstrated that cells behind the prospective leaders
could locally increase traction forces to facilitate leader cell formation [204].

8. Amoeboid Migration
Amoeboid migration does not depend on integrin-based adhesions to the substratum
and is driven by bleb-like protrusions sustained by high levels of Rho-mediated actomyosin
contractility (Figure 5D). During amoeboid migration blebs grow as a result of intracellular
pressure generated by actomyosin contraction pushing the membrane out in the regions
where the cell membrane detaches from the cortex or where cortical actin exhibits local
weaknesses [204–207]. Using amoeboid mode of migration, cancer cells can squeeze
through pores in the ECM without requiring pericellular proteolysis [162,208]. Amoeboid
migration is often faster than mesenchymal migration [207].
The molecular mechanisms controlling rearrangement of actin structures during
active blebbing are not entirely clear. In expanding blebs of rounded cells membrane-
associated proteins spectrin, adducin, ankyrin B1, myosins 1C and 1E have been found.
Ezrin and moesin accumulate at the bleb membrane when expansion ceases and the
actin cytoskeleton begins to assemble within the bleb by elongating cortical filaments
and new filament polymerization driven by Arp2/3 and mDIA1 nucleation followed by
α-actinin, tropomyosin, and myosin II accumulation [209,210]. In confined carcinoma cells
that use amoeboid leader bleb-based migration, ERK-mediated EPS8 bundling activity
modulates actin cortex and promotes cortex tension and intracellular pressure to drive
bleb-based migration and leader bleb formation [211]. It remains to be elucidated how
blebbing cells gain traction forces to promote forward cell translocation in the absence
of adhesions at the leading edge. A study of migration of mammary carcinoma cells in
Matrigel demonstrated that actin and myosin II could accumulate at the cell rear in a
uropod-like structure and that β1 integrin was required for contraction of this uropod-
like structure, which promoted cell migration [212]. It has been proposed that migrating
cells can also employ non-specific interactions with surrounding substrate when rearward
flowing actin cytoskeleton generates friction between cell and the substrate [213]. It is also
suggested that in liquid, constant rearward plasma membrane flow generates tangential
viscous forces that can propel the cell forward in the absense of any adhesion. RHOA-
dependent increased endocytosis at the rear end of the cells and forward trafficking of
membrane vesicles maintain the continuity of the membrane flow [214]. It has also been
shown that melanoma cells migrating through compliant matrices form large blebs at the
front which destroy the collagen matrix through mechanical interaction. The collagen is
then internalized by the cells using macropinocytosis [215].

9. Mesenchymal-Ameboid Transition (MAT)

Phenotypic plasticity allows migrating tumor cells to pass through tissues with differ-
ent molecular, structural and mechanical characteristics. Variations in substrate properties
influence modes of cell migration (Figure 5A) [200,207,210]. Reduced cell–substrate adhe-
sion favors the transition from mesenchymal to amoeboid phenotype. Studies of Walker
256 breast carcinosarcoma cell migration on micropatterned surfaces demonstrated that
when cells moved from an adhesive to a non-adhesive substrate, lamellipodia could change
to blebs and vice versa [216]. HT1080 fibrosarcoma cells cultured on a 2D adhesive sub-
strate preferentially exhibited mesenchymal phenotype but on a substrate with reduced
adhesiveness, a part of the cell population switched to forming blebs [217]. Cells switch
to amoeboid migration under conditions of physical confinement and decreased adhe-
sion [218]. Cells of breast and colon cancer cell lines migrating in 10-µm channels could
switch between mesenchymal and amoeboid modes of migration. During migration in
narrow 3-µm channels the same cells only exhibited blebbing at the leading edge. The
transition from mesenchymal to amoeboid migration was accompanied by a loss of F-actin
stress fibers. Cells passing through a narrow microchannel did not assemble FAs even
Int. J. Mol. Sci. 2021, 22, 1821 14 of 23

on an adhesive substrate. A marked increase in cell velocity in narrow 3-µm channels

compared to wider 10-µm channels was observed [219].
Plasticity of cell migration can be regulated via different signaling pathways. For
example, MTLn3 mammary tumor cells overexpressing EGFR that possessed mesenchymal
phenotype on adhesive substrates, exhibited amoeboid invasion both in vitro (3D collagen
matrix) and in vivo (orthotopic injection into mice mammary fat pad) [220]. Met may
also be involved in amoeboid cell motility. It has been demonstrated that MDA-MB-
231 breast cancer cells expressing high levels of activated Met formed membrane blebs
independently of HGF/SF and invaded three-dimensional matrix [221]. TGFβ signaling
which has been shown to activate RHOA-dependent contractility [222] may contribute to
MAT. In hepatocellular carcinoma cells, down-regulation of EGFR promoted TGFβ-induced
transition to amoeboid invasion [223]. TGFβ also increased the number of MDA-MB-231
cells migrating in collagen matrices using amoeboid mode [224]. In some cases, hypoxia
may be an important MAT-inducing factor. For example, 4T1 murine breast carcinoma cells
that exhibited collective mesenchymal migration in collagen matrix in normoxia, in hypoxia
switched to amoeboid migration. This switch may be connected to the downregulation of
MMP observed in hypoxic conditions [225].

10. Conclusions
The studies summarized in this review demonstrate how pEMT may result in the
cell phenotype diversity observed in cancers. Reversible changes in cytoskeleton and
adhesive structure organization allow cancer cells to fine-tune their reaction to the microen-
vironment and change their phenotype and migratory properties to realize their invasive
and metastatic potential. Further research is needed to shed more light on the molecular
mechanisms that regulate these rearrangements. A deeper understanding of the EMT and
plasticity of cell migration will help develop new therapeutic strategies to prevent cancer
cell dissemination.

Author Contributions: Conceptualization, N.A.G.; writing—original draft preparation, S.N.R.,

N.A.G.; writing—review and editing, S.N.R., I.Y.Z., N.A.G.; visualization, I.Y.Z.; project admin-
istration, N.A.G.; funding acquisition, N.A.G. All authors have read and agreed to the published
version of the manuscript.
Funding: The research was funded by the Russian Science Foundation, grant number 16-15-10288.
The work in the laboratory was also funded by Russian Foundation for Basic Research grant number
18-54-16005.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: No new data were created or analyzed in this study. Data sharing is
not applicable to this article.
Acknowledgments: The author thank Reviewer 3 for their incredible dedication and constructive
criticism which helped improve the manuscript during submission.
Conflicts of Interest: The authors declare no conflict of interest.

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