cancers
ReviewAtypical Macropinocytosis Contributes to Malignant
Progression: A Review of Recent Evidence in Endometrioid
Endometrial Cancer Cells
Takayuki Kohno *and Takashi Kojima
Department of Cell Science, Research Institute for Frontier Medicine, Sapporo Medical University School of
Medicine, Sapporo 060-8556, Japan* Correspondence: kohno@sapmed.ac.jp

Simple Summary: A novel type of macropinocytosis has been identified as a trigger for the malignant progression of endometrial cancer. Transiently reducing epithelial barrier homeostasis
leads to macropinocytosis by splitting between adjacent cells in endometrioid endometrial cancer. Macropinocytosis causes morphological changes in well-differentiated to poorly differentiatedcancer cells. Inhibition of macropinocytosis promotes a persistent dormant state in the intrinsic KRASmutated cancer cell line Sawano. This review focuses on the mechanisms of atypical macropinocytosis
and its effects on cellular function, and it describes the physiological processes involved in inducing
resting conditions in endometrioid endometrial cancer cells.

Citation: Kohno, T.; Kojima, T.
Atypical Macropinocytosis
Contributes to Malignant
Progression: A Review of Recent
Evidence in Endometrioid
Endometrial Cancer Cells. Cancers
2022, 14, 5056. https://doi.org/
10.3390/cancers14205056

Abstract: Macropinocytosis is an essential mechanism for the non-specific uptake of extracellular
fluids and solutes. In recent years, additional functions have been identified in macropinocytosis,
such as the intracellular introduction pathway of drugs, bacterial and viral infection pathways, andnutritional supplement pathway of cancer cells. However, little is known about the changes in cellfunction after macropinocytosis. Recently, it has been reported that macropinocytosis is essential forendometrial cancer cells to initiate malignant progression in a dormant state. Macropinocytosis isformed by a temporary split of adjacent bicellular junctions of epithelial sheets, rather than from theapical surface or basal membrane, as a result of the transient reduction of tight junction homeostasis.
This novel type of macropinocytosis has been suggested to be associated with the malignant pathology
of endometriosis and endometrioid endometrial carcinoma. This review outlines the induction ofmalignant progression of endometrial cancer cells by macropinocytosis based on a new mechanism
and the potential preventive mechanism of its malignant progression.

Academic Editor: Andrzej Semczuk
Received: 5 September 2022
Accepted: 13 October 2022

Keywords: endometrioid-endometrial carcinoma; endometriosis; macropinocytosis; LSR; tight junctions;dormancy

Published: 15 October 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in

1. Introduction

published maps and institutional affil-

Macropinocytosis has long been known to be one of the mechanisms involved in the
uptake of extracellular components. This phenomenon was discovered in macrophages in1931 and is a widely confirmed biological phenomenon from amoebas and slime molds
to mammalian cells [1]. Endocytosis is the mechanism by which cells incorporate extracellular factors. Endocytosis is an essential mechanism for the uptake of the extracellularcomponents required for housekeeping tasks. According to the typical definition, they
are classified into two types: phagocytosis, which involves the uptake of solid substances,and pinocytosis, which involves the uptake of fluid components [2]. Macropinocytosis
is a process that nonspecifically incorporates extracellular components on a large scale.
Macropinocytosis is a physiologically important function of immune cells, endothelial cells,
and epithelial cells. In cancer cells, high macropinocytosis activity is used as a supply
pathway of amino acids required for cell proliferation [3]. A recent study identified a new

iations.

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pathway of amino acids required for cell proliferation [3]. A recent study identified a newroleforformacropinocytosis,
macropinocytosis,inducing
inducingthe
theinitiation
initiationof
ofmalignant
malignantprogression
progressionof
ofendometrial
endometrialrolecancercells[4].
Macropinocytosis
isattributed
tothedisruptionofepithelial
homeocancer cells [4]. Macropinocytosis is attributed to the disruption of epithelial cellcell
homeostastasis.
Evidencethatmacropinocytosis
contributes
tothemalignantprocessmaypoint
sis. Evidence that macropinocytosis contributes to the malignant process may point toward
towardnewfortargets
for the prevention
newtargets
the preventionof cancer.of cancer.
2.
2. UptakeUptake Mechanisms
Mechanismsof
ofExtracellular
ExtracellularComponents
Components
Most
incorporate
thethetargetfactors(Figure1) [5].1)Professional
Mostphagocytic
phagocyticcells
cellsactively
actively
incorporate
targetfactors(Figure[5]. Profesphagocytes,
such assuch
macrophages,
neutrophils,
and dendritic
cells, andsional phagocytes,
as macrophages,
neutrophils,
and dendritic
cells,non-professionaland non-profesphagocytes,
such assuch
fibroblasts
and epithelial
cells, incorporate
targets,
whichwhich
includesional phagocytes,
as fibroblasts
and epithelial
cells, incorporate
targets,
insolidcontaining
apoptotic
cells, throughreceptor-mediatedendocytosis[6].
cludecomponents
solid components
containing
apoptotic
cells, throughreceptor-mediatedendocytoProfessional
phagocytes
efficiently
engulf extracellularcomponents
through throughcomplementsis [6]. Professional
phagocytes
efficiently
engulf extracellularcomponents
commediatedopsonization.
Phagosomeformation
involves
membraneruffling
via PI3Kplement-mediated opsonization. Phagosome formation involves membrane rufflingvia
mediated
actin reorganization[7]. The[7].
coordinatedreconstructionof cell membranes
isPI3K-mediatedactin reorganization
The coordinatedreconstructionof cell memalso
required
Mycobacterium
tuberculosis
uses phagocytosis
as a routeasofaentry
thebranesis also[8].
required[8]. Mycobacterium
tuberculosis
uses phagocytosis
routeintoof entrycell,mechanism
is different
from thatfrom
of foreign
removalto prevent
own
into but
the its
cell,
but its mechanism
is different
that ofbody
foreignbody removal
to itsprevent
degradation[9]. Somecells express
CD47 CD47[10]. Theof CD47
withwiththe
its own degradation[9].cancer
Some cancer
cells express[10].interaction
The interactionof CD47
SIRPαofmacrophages
renders
cancercells
resistanttophagocytosis,
therebypromoting
the SIRPα of macrophages renders cancer cells resistant to phagocytosis, thereby promottumor
growthandandmetastasis[11].
Endometrial
canceralso
expresses
ing tumor
growthmetastasis[11].
Endometrial
canceralso
expressesCD47,
CD47,and
anditithas
has
been
pointed
out
that
its
inhibition
may
beatherapeutictarget[12].
been pointed out that its inhibition may be a therapeutic target [12].

Figure 1. Overview of the endocytic pathways. Phagocytosis incorporates extracellular components
Figure 1. Overview of the endocytic pathways. Phagocytosis incorporates extracellular components
through phagocytic receptors. Cells that phagocytose with high efficiency are termed professional
through phagocytic receptors. Cells that phagocytose with high efficiency are termed professional
cells,
Pinocytosis nonspecifinonspecifcells, whereas
whereas those
those with
with low
low efficiency
efficiency areare termed
termed non-professional
non-professional cells.
cells. Pinocytosis
icallyincorporates
solublecomponents,suchasproteins
andgrowthfactors.
Smallvesiclescally incorporates soluble components, such as proteins and growth factors. Small vesicles areareformedand caveolae-mediatedcaveolae-mediatedpathways
pathwaysduring
duringpinocytosis.
pinocytosis.
The
pathwayofformed throughthrough clathrin- andThepathwayof extensive incorporation,withmembrane
protrusion
fromthethecellcellsurface,is termed
macropinocytosis.
extensive
incorporation,withmembrane
protrusion
fromsurface,is termed
macropinocyThe endometrial
cancercancer
cell Sawano
exhibits
macropinocytosis
causedby a transient
split ofintertosis.
The endometrial
cell Sawano
exhibits
macropinocytosis
causedby a transientsplitofcellularspaces.
intercellular spaces.

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Pinocytosis can be classified according to the size of vesicles formed [13]. Small vesiclemediated pinocytosis is further classified into the following three types: clathrin-mediated,caveolae pathways and those mediated by neither. In the clathrin-mediated pathway,
clathrin-based triskelion vesicles facilitate intracellular transport [14]. Caveolae form vesicles associated with caveolin-1, which are smaller than clathrin-coated vesicles [15]. These
small vesicles originate from concavity of the cell membrane [16]. Pinocytosis, accompaniedby the formation of large vesicles, is called macropinocytosis. Macropinocytosis does not
require coat proteins such as clathrin for uptake. In addition, it is believed that there is nocommunication mechanism or interaction between the incorporated target and the cell [17].
In macropinocytosis, extracellular factors are incorporated into membrane protrusions via
a pathway that involves extensive actin rearrangement [18] and membrane ruffling [19]. A
mathematical model has been developed to explain the mechanism of formation of a large
macropinocytic cup during macropinocytosis [20].
The extracellular uptake mechanism is used as a route for viral infection [21]. Phagocytosis allows herpes simplex viruses (HSV-1) and acanthamoeba polyphaga mimivirus(AMPV) to enter cells, whereas in clathrin-mediated endocytosis, human immunodeficiency
virus type 1 (HIV-1) and influenza A virus (IAV) enter cells, while simian virus (SV40) andBK virus enter via caveolae. Adenovirus 3 (Ad3), coxsackievirus B (CVB), and respiratorysyncytial virus (RSV) enter cells through macropinocytosis [21–23].
3. A Universal Role of Macropinocytosis
Among immune system cells, macrophages and dendritic cells constantly perform
macropinocytosis to eliminate foreign substances and present antigens [24]. B cells, CD4+ T
cells, and CD8+ T cells utilize macropinocytosis for their physiological functions [25,26].
In fibroblasts, growth factors, such as PDGF and EGF, stimulate Ras and Src via
activation of RTKs, leading to macropinocytosis [27,28]. Circular dorsal ruffles (CDRs)
occur on the cell surface and are involved in the formation of macropinocytic cups [29].
The PI3K-Akt-TSC1-Rheb-mTORC1 pathway is associated with growth factor-dependent
macropinosome formation [30]. The macropinosome fuses with the lysosome, and its
contents are absorbed into the cell [3]. mTORC1/2 complexes regulate the recycling ofessential biomolecules in lysosomes through interactions with the nutrient source sensor
TSC1/2 [31,32].
In endothelial cells, macropinocytosis is a pathway for growth factor receptor internalization in a growth factor-dependent manner. Cell surface VEGFR2 is incorporated via
macropinocytosis by Cdc42 activation upon VEGF stimulation [33]. Cell surface FGFR1
is incorporated into Rab5-positive endosomes by macropinocytosis via RhoG activation
upon FGF2 stimulation [34]. Macropinocytosis is required for the activation of intracellularsignaling pathways that are dependent on growth factor stimulation.
Since collective epithelial cells have established cell polarity, they incorporate extracellular components from the apical and basal membranes. In polar MDCK cells, uptake
occurs via clathrin-mediated and clathrin-independent pathways on the apical surface,whereas caveolae-mediated uptake occurs on the basal membrane [13]. Two types ofplasma membrane ruffles precede macropinocytosis: circular dorsal ruffles (CDRs) arisingfrom the apical cell surface and planar ruffles arising from the cell periphery [3]. These
processes involve the activation of the RhoG-Rac pathway and reorganization of actin
filaments [35]. Recently, it was reported that macropinocytosis is necessary for breaking
out contact inhibition and initiating stratified cell growth in well-differentiated humanendometrial cancer cells (Figure 1) [4]. While macropinocytosis, which is Rac-dependent,
occurs through the temporary split of adjacent cell junctions; there is no protrusion of
the cell membrane prior to macropinocytosis. The triggering of this macropinocytosis isbased on a transient reduction in epithelial homeostasis by ligand stimulation of the tightjunction-associated protein lipolysis-stimulated lipoprotein receptor (LSR).

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4. Disruption of Epithelial Homeostasis Triggers Cancer Malignancy
4.1. Regulation of Epithelial Homeostasis by Cell Polarity
To maintain homeostasis in epithelial cells, it is necessary to establish an adhesion
apparatus between adjacent cells and concomitantly establish cell polarity [36]. Adherence
junction formation precedes cell–cell junction formation [37]. Adherence junctions interact
with the actin cytoskeleton through the nectin-afadin and cadherin-α/β-catenin pathways [38]. The actin cytoskeleton forms circumferential actomyosin bundles and increases
the cell thickness [39]. Shroom3 and Lulu1/2 activate circumferential actomyosin bundles
via ROCK [40,41]. Tight junctions are established by polar proteins PAR3/PAR6/aPKC,
which interact with circumferential actomyosin bundles via ZO1/2/3 [42]. In matureepithelial cell sheets with sufficient epithelial barrier function, occludin (OCLN), claudins(CLDNs), and JAM-A are localized in the tight junction region between adjacent cells,
whereas tricellulin (Tric) accumulates in the tricellular contact region in an LSR dependent manner. The LSR is required for the localization of Tric to the tricellular contact
region [43], but the mechanism by which LSR accumulates in this region remains unclear.
OCLN, CLDNs, and Tric are transmembrane proteins with N- and C-termini in the cytoplasm, whereas JAM-A and LSR are single-transmembrane proteins with one or moreimmunoglobulin-like domains in the extracellular N-terminal region [44]. The LSR isa receptor for apolipoprotein B (ApoB) and ApoE [45], which are associated with lipid
metabolism in hepatocytes [46]. LSR localization is dependent on the cell density in epithelial cells. During the establishment of epithelial homeostasis, LSRs are distributed
broadly in the bicellular junction region and gradually accumulate in the tricellular contact
region [47]. This change in localization is reversible. LSR withdrawal from the tricellularcontact region impairs epithelial homeostasis [48]. Disruption of epithelial barrier function
abolishes cell polarity and promotes dedifferentiation [44,49].
4.2. Regulation of Epithelial Homeostasis by Cell Density
The cell adhesion apparatus senses cell density [50]. Epithelial cells have a contact
inhibition mechanism, which is one of the mechanisms that maintains cell density [51,52].
Contact inhibition involves two mechanisms: growth and motility inhibition [53,54]. These
contribute to the regulation of cell number during normal development and wound healing [55]. Physical cell loss and carcinogenesis disrupt contact inhibition [56,57]. Loss ofcontact inhibition and disruption of cell polarity are typical features of the malignant
transformation of cancer cells. These molecular mechanisms involve PAR3, TGF-β, andHippo pathways [53,58]. KRAS mutations are closely associated with the growth of malignant cells [59]. KRAS mutations are typical oncogenes found in one-quarter of adenocarcinomas [60]. KRAS mutations have been found in endometrial carcinomas andadenomyosis [61,62].
Sawano cell was established by endometrial adenocarcinoma of uterus [63]. Sawano
has a KRAS G13D heterozygous mutation and wild-type BRaf [4]. According to this study,
Sawano cells have high planar motility at low cell densities, whereas at high cell densities,
their growth and motility are temporarily arrested due to contact inhibition. After a certain
period of time under high cell density conditions, Sawano initiated multilayered cell growth.
In addition, in the presence of MAPK inhibitors, the cells maintained a highly differentiatedstate. Thus, Sawano cells are a good model for analyzing the malignant transformation
process of endometrial carcinoma.
5. LSR-Related Molecular Mechanisms Leading to Disruption ofEndometrial Homeostasis
5.1. Disruption of Endometrial Homeostasis by TGF-β Signaling
TGF-β is associated with increased migration and malignant transformation of endometrial carcinomas through activation of estrogen-related receptor alpha (ERRα); it isalso associated with invasion and epithelial-to-mesenchymal transition (EMT) [64]. TGF-β
increases HMGB1 expression and induces changes in LSR localization and downregulation,

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resulting in reduced epithelial barrier homeostasis [65–72]. TGF-β levels are also elevatedin high-grade stages of endometriosis [73]. TGF-β is released from endometrial epithelial
cells, endometrial stromal cells, endometriotic mesenchymal stem cells, and peritoneal
mesothelial cells and regulates endometriosis pathogenesis [74].
5.2. Disruption of Endometrial Homeostasis by Hippo-Yap Pathway
The Hippo-YAP pathway is involved in the regulation of cell number and apico-basal
polarity. This pathway is involved in tight junctions, adherens junctions, the planar cell
polarity (PCP) pathway, the mechanotransduction pathway, and growth factor signaling [52]. YAP also regulates the cell size [75]. YAP is localized in the nucleus, resulting in
the activation of transcription factors in various cell types, whereas YAP is degraded in
the cytoplasm, resulting in transcriptional repression in smaller and columnar cells [76].
In endometriotic tissue, YAP is localized in the cytoplasm, whereas in G1, G2, and G3stages of endometrioid carcinomas, YAP is localized in the nucleus [77,78]. LSR knockdown
enhanced cell motility and invasion in Sawano cells, and YAP knockdown resulted in cellquiescence [77]. Dobutamine, a β-adrenergic receptor agonist, decreases nuclear YAP levels
and increases cytoplasmic phosphorylated YAP levels in human U2OS cells [79]. Dobutamine has also been reported to suppress the high expression of YAP in gastric carcinoma,
resulting in suppression of cell motility and invasion [80]. In LSR-knockdown Sawano cells,
dobutamine treatment suppressed the enhancement of cell motility and invasion, associatedwith an increase in the amount of phosphorylated YAP [77]. Activation of the transcription
factor TEAD by YAP increased the expression of genes associated with cell proliferationand survival [81]. LSR-knockdown Sawano cells induce expression of the Hippo pathway
transcription factor TEAD and inducible gene amphiregulin (AREG) [77]. Immunohistochemical staining analysis using tissues from endometriosis and endometrial cancer
revealed that the expression of AREG, an EGFR ligand, is observed in the cytoplasm of cells
forming endometriotic tissue and that its expression level increases with increasing cancer
stage. In Sawano cells, cell motility and invasion, both of which were increased by LSR
knockdown, were significantly suppressed by AREG knockdown. Increased expression of
AREG has also been observed in the human pancreatic cancer cell line HPAC, wherein LSR
is knocked down, and in poorly differentiated pancreatic ductal adenocarcinomas (PDAC),
wherein no LSR is localized in the junctional region [66]. Thus, the downregulation ofepithelial barrier function caused by the suppression of LSR expression is involved in theregulation of cell motility and invasion through TEAD-dependent AREG expression via
the YAP-Hippo pathway.
6. Pathophysiological Changes in Endometriosis and Endometrial Carcinoma Are
Accompanied by Changes in LSR Localization
The endometrium is a regenerative tissue that undergoes periodic proliferation anddifferentiation under the influence of estrogen, progesterone, growth factors, and various
cytokines. Menstrual cycle influences the expression and localization of both bicellular
and tricellular tight junction proteins. Bicellular tight junction proteins such as CLDN-1,-3, -4, and -7 and OCLN accumulate in the subapical region during the proliferative phaseof the endometrium, whereas they are distributed in the basolateral region during thesecretory phase [82]. Levels of tricellular tight junction proteins also change dynamically.
Both Tric and LSR accumulate in the subapical region during the proliferative phase of theendometrium, wherein Tric is localized in the subapical region during the secretory phaseand LSR is widely distributed from the subapical region to the basolateral region [83].
A downregulation of expression of OCLN and CLDN-3, -4, and -7 has been observed inendometriosis [84], and an upregulation of expression of CLDN-3 and -4 has been reported
in endometrial cancer [85]. The expression of tricellular tight junction proteins is alteredduring endometrial pathogenesis [77,83]. These studies demonstrate that the localizationof LSR to tricellular contact regions is required for the maintenance of robust endometrial
epithelial barrier function. For example, in endometriosis, Tric accumulates in the subapicalCancers 2022, 14, 5056

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region, as in the normal human endometrium, whereas LSR is widely distributed not
only in the subapical region, but also in the lateral region. In endometrial carcinoma G1,
in which most of the gland-like tissue remains, both Tric and LSR are heterogeneously
distributed from the subapical to the lateral regions. In stages G2 and G3, the expression
levels of both are decreased. Among endometrial cancer-derived cell lines, Sawano, HHUA,
and JHMUE-1 cells, which exhibit high to moderate differentiation, express LSR and Tric,
whereas spindle-shaped and fibroblast-like JHMUE-2 cells rarely express these markers.
Knockdown of LSR in Sawano cells resulted in decreased epithelial barrier function andincreased cell motility, invasion, and proliferation, as described above.
In the gland-like structure region of endometrial cancer tissue, upregulation of LSR
expression and downregulation of CLDN-1 expression have been observed by immunohistochemical staining analysis [86]. By contrast, in the invasive front area, LSR expression
is downregulated and CLDN-1 expression is markedly upregulated. The report has also
shown that, CLDN-1 expression upregulates in LSR knocked-down Sawano cells, while
the expression levels of OCLN, CLDN-3, -4, and -7 barely change. Prior to LSR knockdown, CLDN-1 is localized in the subapical region, but after the knockdown, it is widelydistributed from the subapical region to the basolateral region. Thus, there is a reciprocal
relationship between the expression of LSR and CLDN-1, yet almost no regulatory mechanism has been identified. Binding of Sp1 to the CLDN-1 promoter region is required for the
regulation of CLDN-1 expression in intestinal epithelium-derived cells [87]. Sp1-bindingsites exist in the promoter region of CLDN-1, -4, and -19 [88–90]. Sp1 is also involved in the
upregulation of CLDN-1 expression in Sawano cells [86]. Sp1 contributes to the expressionof matrix metalloproteinases (MMPs) in Sawano cells.
In oral squamous cell carcinoma (OSC) cells expressing CLDN-1, cell invasion isenhanced through cleavage of laminin-5γ2 chains by increased activity of MMP-14 (MTMMP-1) and MMP-2 [91]. Human colorectal cancer SW480 expressing CLDN-1 have beenreported to enhance cell invasion through the activation of MMP-2 and -9 [92]. The initialstage of cell invasion requires reconstitution of the extracellular matrix composition, in
addition to the depletion of intercellular junctions [93]. To date, 24 MMPs have been
identified [94]. MMP expression varies with the menstrual cycle [95]. MMP-3, -7, -9, and-13
are associated with disease susceptibility in endometriosis [96]. TGF-β is involved in theenhancement of MMP-2 and -3 secretion [97]. LSR knockdown increased the expression
levels of MMP-2, -9, -10, and -14 in Sawano cells [86]. MMP-14 is an initiator that activates
the MMP cascade by activation of proMMP-2 [98]. In Sawano cells, double knockdown of
LSR and CLDN-1 suppresses cell invasion enhanced by LSR knockdown [86]. While somestudies have linked increased expression of TGF-β with CLDN-1 expression [99], there
exist conflicting reports [100].
7. Internalization Pathways of Tight Junction Proteins
Tight junction complexes form static rigid seals, whereas their components exhibit
dynamic behavior. The exclusion of tight junction proteins from the cell surface membrane
is associated with internalization by clathrin-, caveolae-, and raft-mediated pathways, aswell as by macropinocytosis [101]. In intestinal epithelial cell T84, tight junction proteins
OCLN, CLDN-1, and JAM-A are internalized via the IFN-γ-induced macropinocytosislike pathway [102]. In contrast, in T84 cells, Escherichia coli cytotoxic necrotizing factor-1(CNF-1) induces caveolae-dependent intracellular translocation of OCLN without affecting
the localization of the AJ proteins E-cadherin and β-catenin [103]. In depolymerized MDCK
cells, in which actin filamentation is inhibited by latrunculin A, OCLN is internalized via
the caveolae [104]. GFP-CLDN-3 was incorporated from the lateral region by vesicles thatincorporated the plasma membrane of two adjacent cells [105]. In Caco-2 cells, Tric, OCLN,
CLDN-4, and ZO-1 are excluded from tight junctions in a poly L-arginine-dependent
manner. However, the underlying mechanism remains unknown [106]. In bovine retinal
endothelial cells (BRECs), OCLN is phosphorylated and ubiquitinated 30 min after VEGF
stimulation and translocates from tight junctions to early and late endosomes [107]. LSR isCancers 2022, 14, 5056

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excluded from tricellular tight junctions by LSR ligand stimulation, and LSR subsequently
colocalizes with Rab5-positive vesicles at bicellular junctions [4]. The pathway by which the
LSR translocates from the bicellular region to the intracellular region and the mechanism
by which the intracellularly translocated LSR is recycled to the junction region is unclear.
8. Regulation of Cell Motility by Macropinocytosis Induced by LSR Ligand
As mentioned above, decreased expression levels and altered localization of LSR,
which accumulates in tricellular tight junctions, alter the localization of Tric, leading to adecrease in the robustness of the epithelial barrier function. Many of these findings wereobtained through LSR knockdown experiments, in which it is difficult to temporally control
the suppression of gene expression. Hence, Kondoh et al. established an experimental
system that enables the simultaneous reduction of LSR expression in all areas of cultured
cells using LSR ligands [108]. Administration of the LSR ligand to Sawano cells induces LSR
localized in tricellular tight junctions into Rab5-positive intracellular vesicles via bicellularjunctions [4]. Under these conditions, the LSR ligand transiently reduced epithelial barrier
function, but it had little effect on OCLN expression and localization and did not completely
disrupt epithelial barrier function. TEM analysis revealed the presence of tight junctions,
adherens junctions, and desmosomes in bicellular junctions. Macropinocytosis does not
disrupt the tight junction barrier in a blood-tumor barrier model in rat gliomas [109].
The LSR ligand is a partial C-terminal peptide of the Clostridium perfringens iota-toxinb subunit. LSR bound to C. perfringens iota toxin translocates intracellularly throughmembrane rafts [110]. LSR expressed in mouse vascular endothelial cells is involved in
the establishment of barrier function [111]. Administration of the LSR ligand to micetransiently reduces the endothelial cell barrier function of the blood–brain barrier (BBB)
and enhances drug entry into the brain [112]. In the human cancer cell lines Sawanoand HPAC, administration of the LSR ligand decreased LSR expression and induced a
reduction in barrier function, resulting in enhanced malignant transformation of cancer
cells [66,113]. In contact-inhibited monolayers of Sawano cells under high cell density
conditions, administration of the LSR ligand resulted in transient and rapid enhancementof planar cell motility [4]. This process requires macropinocytosis, initiated by the cleavageof the gap between adjacent cells (Figure 2). LSR ligand-dependent pinocytosis does notoccur in apical or basal membranes. Additionally, no significant accumulation of actin
filaments was observed during macropinosome formation. In the presence of the Rac
inhibitor NSC23766 or in cells expressing a dominant negative Rac, LSR ligand-dependent
macropinocytosis is inhibited. Rac activity is required for JNK activation and is involved in
the subsequent enhancement of cell motility. Rac activates JNK [114]. JNK is associatedwith the regulation of cell motility in various cancer cells [115]. In Sawano cells, LSR
ligand stimulation enhances cell motility through JNK activation [113]. JNK inhibitors orknockdown of JNK not only suppresses cell motility but also inhibits macropinocytosis [4].
JNK-mTOR pathway is associated with macropinocytosis in HeLa cells [116].
The mechanism by which cell motility is enhanced following macropinocytosis in DCs
has been elucidated. In mature DCs that have undergone macropinocytosis, decreased
mTORC1 activity induces the expression of the lysosomal calcium channel TRPML1 andan increase in calcium ions, followed by enhanced cell motility via the activation of myosin
II [117]. Myosin II and ROCK play a central role in cell motility associated with actomyosin
reorganization [118]. Between neighboring adjacent junctions of Sawano cells, myosin
phosphatase MYPT1 accumulates during the contact inhibition phase and phosphorylated
myosin accumulates during the cell motility phase [47]. The myosin II inhibitor blebbistatin
and the ROCK inhibitor Y27632 did not affect the formation of macropinocytosis, suggestingthat LSR ligand-induced macropinocytosis triggers the initiation of planar cell motility.

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Figure2.2.Schematic
Schematicdiagram
diagramof
ofthe
theLSR
LSRligand-induced
ligand-inducedtransienttransientmacropinocytosis
macropinocytosisderived
derivedfrom
from
Figureintercellularsplit.
(Upperpanels)Inmatureepithelial
cells,
LSR
accumulates
inapicaltricellularintercellular split. (Upper panels) In mature epithelial cells, LSR accumulates in apical tricellujunctions.
Administration
of ofthetheLSR
spaces, leading
leading totolarjunctions.
Administration
LSRligandligandsplits
splitsthethe adjacentadjacent intercellularintercellular spaces,macropinocytosis. The intercellular adhesion apparatus was not disrupted by macropinocytosis. G,
macropinocytosis. The intercellular adhesion apparatus was not disrupted by macropinocytosis.
intercellular gap; TJ, tight junctions; AJ, adherens junctions; D, desmosomes; N, nucleus; Mt, mitoG, intercellular gap; TJ, tight junctions; AJ, adherens junctions; D, desmosomes; N, nucleus; Mt,
chondria. Bar, 1 μm. (Lower panels) The gap between adjacent cells expanded and filled with extramitochondria.
Bar,hour
1 µm.after(Lowerpanels) Theofgap
adjacent
cells expanded
and filledwithcellular fluid, one
administration
thebetween
LSR ligand.
Two hours
after addition,
numerous
extracellular
fluid,
one
hour
after
administration
of
the
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ligand.
Two
hours
after
addition,numermacropinosomes that incorporated the extracellular fluid appeared in the cytosol. The vesicles disous
macropinosomes
extracellular
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appearedcell
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the cytosol.
The vesicles
appeared
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afterincorporated
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cells
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hours
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Thecells then initiatedmalignantgrowth, resulting
in multilayered cellthree
growth.
Thispathwaywas
suppressedin the presenceof acellmacropinocytosis
inhibitor.
tilayered cell growth. This pathway was suppressed in the presence of a macropinocytosis inhibitor.

The mechanism by which cell motility is enhanced following macropinocytosis in
9.DCs
SLC9A1-MediatedMacropinocytosis
andthatIts AssociatedRegulationofhas been elucidated.
In mature DCs
have undergone
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deCancer Malignancy
creased mTORC1 activity induces the expression of the lysosomal calcium channel
The amiloride
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EIPA targets
macropinocytosis
TRPML1and an increasein calcium
ions, SLC9A1
followedandby suppresses
enhanced cell
motility via the[119].
actiSLC9A
familyofproteinsareinvolvedinmaintaining
intracellularpH
homeostasis[120].
vation of myosin II [117]. Myosin II and ROCK play a central role in cell
motility associThese
proteins
were formerly
named as[118].
sodiumhydrogenexchanger(NHE) junctions
family proated withactomyosin
reorganization
Betweenneighboring
adjacentof
teins[121].
EIPA
inhibits
theactivation
of
Rac
and
Cdc42
by
acidifying
theintracellular
Sawano cells, myosin phosphatase MYPT1 accumulates during the contact inhibition
pH and inhibiting macropinocytosis by preventing the translocation of Arp3 to the plasmaphase and phosphorylated myosin accumulates during the cell motility phase [47]. The
membrane [122]. The Arp2/3 complex is involved not only in actin filament branching andmyosin II inhibitor blebbistatin and the ROCK inhibitor Y27632 did not affect the forprotrusion but also in phagocytic cup formation [6,123]. During RSV infection, extracelmation of macropinocytosis, suggesting that LSR ligand-induced macropinocytosis triglular budding and spread of RSV are based on the regulation of the Arp2/3 complex bygers the initiation of planar cell motility.
transcribed viral proteins [124].
SLC9A1 is a ubiquitously expressed plasma membrane protein that directly interacts
9. SLC9A1-Mediated Macropinocytosis and Its Associated Regulation of Cancer Mawith ERM proteins [125] and ROCK [126] in its C-terminal region. SLC9A1 is a scaffold prolignancy
tein for Raf and ERK1/2 [127]. In triple-negative breast cancer MDA-MB-231 cells, SLC9A1
The amiloride
derivativetransformation
EIPA targets [128].
SLC9A1and suppresses
activation
triggers malignant
SLC9A1is involvedmacropinocytosis
in collective cell[119].
SLC9A
familyofproteinsareinvolvedinmaintaining
intracellularmigration in head and neck squamous cell carcinoma [129]. In A431 cells,pHanhomeostasis
increase in[120].
These
proteins
wereformerly
namedassodiumhydrogenexchanger(NHE) family
intracellular pH due to SLC9A1 activation is required for EGF-dependent macropinocytoproteins[121]. EIPA
theattention
activation
Rac andCdc42 bydrug
acidifying
intracellularsis[122]. SLC9A1
hasinhibits
attracted
asof
a novelanticancer
target the[130].
pH and inhibiting macropinocytosis by preventing the translocation of Arp3 to the plasma
membrane [122]. The Arp2/3 complex is involved not only in actin filament branching andCancers 2022, 14, 5056

with ERM proteins [125] and ROCK [126] in its C-terminal region. SLC9A1 is a scaffold
protein for Raf and ERK1/2 [127]. In triple-negative breast cancer MDA-MB-231 cells,
SLC9A1 activation triggers malignant transformation [128]. SLC9A1 is involved in collective cell migration in head and neck squamous cell carcinoma [129]. In A431 cells, an increase in intracellular pH due to SLC9A1 activation is required for EGF-dependent
9 of 15
macropinocytosis [122]. SLC9A1 has attracted attention as a novel anticancer drug target[130].
In the presence of EIPA, similar to the knockdown of SLC9A1, few macropinosomes
In the presence of EIPA, similar to the knockdown of SLC9A1, few macropinosomes
were formed upon treatment with the LSR ligand; furthermore, no cell motility was conwere formed upon treatment with the LSR ligand; furthermore, no cell motility was confirmed under these conditions (Figure 3) [4]. Cryo-EM analysis showed that C. perfringens
firmed under these conditions (Figure 3) [4]. Cryo-EM analysis showed that C. perfringens
iota toxin, the origin of the LSR ligand, enters the cell under acidic conditions [131]. Traniota toxin, the origin of the LSR ligand, enters the cell under acidic conditions [131]. Transient acidification of the extracellular region by SLC9A1 activation may affect the infection
sient acidification of the extracellular region by SLC9A1 activation may affect the infec[132]. If the C. perfringens infection pathway is similar to the binding of the LSR ligand totion [132]. If the C. perfringens infection pathway is similar to the binding of the LSR ligand
the LSR, it is possible that the infection may be involved in the pathway by which genetto the LSR, it is possible that the infection may be involved in the pathway by which geically mutated KRAS cells awaken from dormancy. KRAS mutations are representative
netically mutated KRAS cells awaken from dormancy. KRAS mutations are representative
gene mutations in endometrial cancer [133]. As in endometriosis, lesions of adenomyosis
gene mutations in endometrial cancer [133]. As in endometriosis, lesions of adenomyosis
and their surrounding normal tissues have KRAS mutations, even though they are notand their surrounding normal tissues have KRAS mutations, even though they are notcancerous[62].
[62].LSR-targeted
LSR-targetedbacterial
bacterialinfections
infectionsand
andassociatedassociatedmacropinocytosis
macropinocytosismay
maybe
becancerous
involved
inthemalignanttransformationof
these
non-cancerous
cells.
It
has
been
reinvolved in the malignant transformation of these non-cancerous cells. It has been reported
portedthat bicellular
tight proteins,junction proteins,
such as
OCLN,and
CLDNs,
and
JAM-A,
areforrethatbicellular
tight junction
such as OCLN,
CLDNs,JAM-A,
arereceptors
ceptors for papillomavirus,
adenovirus, papillomavirus,
rotavirus,
influenzavirus,respiratory
severe acuterespiraadenovirus,
rotavirus, influenza
virus,
severe acutesyndrome
tory
syndrome(SARS),
andHIV-1[134].
Understandingthemechanisms
by
which
these
(SARS), and HIV-1 [134]. Understanding the mechanisms by which these exogenous factors
exogenous
factorsareincorporated
into
cells
may
help
avoid
infectionandcarcinogenesis.
are incorporated into cells may help avoid infection and carcinogenesis.

Figure3.3.Schematic
Schematicrepresentation
representationof
ofthe
themechanism
mechanismby
bywhichwhichendometrial
endometrialcancer
cancerSawano
Sawanoinitiates
initiates
Figuremalignantcell
cellgrowth.
growth. Mature
Mature epithelial
epithelial sheets,
sheets, where
where LSR
LSR localize
localize atat tricellular
tricellular tighttight junctions,junctions,malignantsuppress macropinocytosis. LSR ligand administration leads to the translocation of LSR from tricelsuppress macropinocytosis. LSR ligand administration leads to the translocation of LSR fromlular tight junctions. LSR interacts with Rab5 in the bicellular region, resulting in decreased LSR
tricellular tight junctions. LSR interacts with Rab5 in the bicellular region, resulting in decreased
expression. SLC9A1 is presumably activated through a signal that accompanies the alteration of LSR
LSR
expression.
SLC9A1
presumably
activateda signalthataccompanies
theof
alterationoflocalization,
followed
byisRacactivation.
SLC9A1throughscaffolds
Raf andERK
downstream
KRAS. AcLSR
localization,
followedbyRac
activation.
SLC9A1
scaffolds
Raf
and
ERK
downstreamofKRAS.
tivated Rac induces JNK activation, resulting in the formation of macropinocytosis, followed by an
ActivatedRac
induces
activation,in theformation ofmacropinocytosis,
followed by anincrease inplanarcellJNK
migration,
andresulting
eventually,
multilayered
cell
growth.
increase in planar cell migration, and eventually, multilayered cell growth.

Ras mutations, other than the KRAS G13D mutation, are also associated withRas mutations, other than the KRAS G13D mutation, are also associated with macromacropinocytosis. Analysis of human pancreatic adenocarcinoma-derived MIA PaCa-2
pinocytosis. Analysis of human pancreatic adenocarcinoma-derived MIA PaCa-2 cells withcells with KRAS G12C mutation, human bladder carcinoma-derived T24 cells with HRas
KRAS G12C mutation, human bladder carcinoma-derived T24 cells with HRas G12V, andG12V, and exogenous KRAS G12V-expressing NIH3T3 cells revealed that these cells obexogenous KRAS G12V-expressing NIH3T3 cells revealed that these cells obtain glutamine
tain glutamine needed for proliferation by uptake of albumin from the extracellular fluid
needed for proliferation by uptake of albumin from the extracellular fluid via macropinocyvia macropinocytosis [135]. In knock-in mice transfected with KRAS G12D into the pantosis [135]. In knock-in mice transfected with KRAS G12D into the pancreas, amino acids
creas, amino acids necessary for cancer cell growth are acquired through macropinocytonecessary for cancer cell growth are acquired through macropinocytosis, a mechanism thatsis, a mechanism that is rarely observed in normal cells [136]. These findings implicate
is rarely observed in normal cells [136]. These findings implicate macropinocytosis between
macropinocytosis between epithelial barrier homeostasis and initiation of malignant
epithelial barrier homeostasis and initiation of malignant transformation in KRAS-mutant
transformation
inisKRAS-mutant
cells.
Therefore,barrier
it is suggested
epithelial
barriercells.
Therefore, itsuggested thatthe epithelial
function thatmaythenegatively
regulate
the signaling pathways from mutant KRAS in the dormant state Sawano cells, in which
LSR is localized in tricellular tight junctions. It is speculated that LSR may be involved in
the initiation of the malignant transformation of endometriosis and endometrial cancer.
10. Conclusions
Atypical macropinocytosis, which is derived from the partial cleavage between adjacent cells, is essential for the initiation of malignant progression of endometrial cancer
cells. Aberrant cell proliferation, disruption of differentiation, and EMT are induced by theactivation of oncogenes, including Ras, and the inactivation of tumor suppressor genes,
including PTEN and p53. These processes involve reorganization of intercellular junctions.
LSR may be an initiating factor in the disruption of epithelial barrier homeostasis. Accu-

Cancers 2022, 14, 5056

10 of 15

mulation of LSR in tricellular tight junctions is necessary to maintain the robustness ofepithelial barrier function. As shown in Figure 3, changes in the localization of LSR intricellular tight junctions induce atypical macropinocytosis. Furthermore, macropinocytosis triggers a rapid increase in the motility of endometrial cancer cells and induction ofmultilayered cell growth. However, it is unclear when and where macropinocytosis occurs
in proliferating cancer cells. It is still unclear whether endogenous LSR ligands are involvedin the pathological process of endometriosis and the malignant progression of endometrial
cancer. The elucidation of these mechanisms may contribute to the development of new
therapeutic methods for these diseases. It is expected that mechanisms underlying theformation of atypical macropinocytosis will be discovered.
Author Contributions: Conceptualization, T.K. (Takayuki Kohno) and T.K. (Takashi Kojima);
writing—original draft preparation, T.K. (Takayuki Kohno); writing—review and editing, T.K.
(Takayuki Kohno) and T.K. (Takashi Kojima); visualization, T.K. (Takayuki Kohno); supervision;
T.K. (Takayuki Kohno). All authors have read and agreed to the published version of the manuscript.
Funding: This work was funded by JSPS KAKENHI Grant Number JP22K05357 and by The Akiyama
Life Science Foundation.
Conflicts of Interest: The authors declare no conflict of interest.

References
1.
2.
3.
4.
5.
6.
7.

8.
9.
10.
11.
12.

13.
14.
15.
16.
17.
18.

King, J.S.; Kay, R.R. The Origins and Evolution of Macropinocytosis. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2019, 374, 20180158.
[CrossRef] [PubMed]
Hoeller, O.; Bolourani, P.; Clark, J.; Stephens, L.R.; Hawkins, P.T.; Weiner, O.D.; Weeks, G.; Kay, R.R. Two Distinct Functions for
PI3-Kinases in Macropinocytosis. J. Cell Sci. 2013, 126, 4296–4307. [CrossRef]
Recouvreux, M.V.; Commisso, C. Macropinocytosis: A Metabolic Adaptation to Nutrient Stress in Cancer. Front. Endocrinol. 2017,8, 261. [CrossRef] [PubMed]
Kohno, T.; Konno, T.; Kikuchi, S.; Kondoh, M.; Kojima, T. Translocation of LSR from Tricellular Corners Causes Macropinocytosis
at Cell-Cell Interface as a Trigger for Breaking out of Contact Inhibition. FASEB J. 2021, 35, e21742. [CrossRef] [PubMed]
Feng, M.; Jiang, W.; Kim, B.Y.S.; Zhang, C.C.; Fu, Y.-X.; Weissman, I.L. Phagocytosis Checkpoints as New Targets for Cancer
Immunotherapy. Nat. Rev. Cancer 2019, 19, 568–586. [CrossRef]
Uribe-Querol, E.; Rosales, C. Phagocytosis: Our Current Understanding of a Universal Biological Process. Front. Immunol. 2020,
11, 1066. [CrossRef] [PubMed]
Schlam, D.; Bagshaw, R.D.; Freeman, S.A.; Collins, R.F.; Pawson, T.; Fairn, G.D.; Grinstein, S. Phosphoinositide 3-Kinase Enables
Phagocytosis of Large Particles by Terminating Actin Assembly through Rac/Cdc42 GTPase-Activating Proteins. Nat. Commun.
2015, 6, 8623. [CrossRef]
Bohdanowicz, M.; Grinstein, S. Role of Phospholipids in Endocytosis, Phagocytosis, and Macropinocytosis. Physiol. Rev. 2013, 93,
69–106. [CrossRef] [PubMed]
Russell, D.G. Mycobacterium Tuberculosis: Here Today, and Here Tomorrow. Nat. Rev. Mol. Cell Biol. 2001, 2, 569–578. [CrossRef][PubMed]Matlung, H.L.; Szilagyi, K.; Barclay, N.A.; van den Berg, T.K. The CD47-SIRPα Signaling Axis as an Innate Immune Checkpoint inCancer. Immunol. Rev. 2017, 276, 145–164. [CrossRef] [PubMed]
Logtenberg, M.E.W.; Scheeren, F.A.; Schumacher, T.N. The CD47-SIRPα Immune Checkpoint. Immunity 2020, 52, 742–752.
[CrossRef] [PubMed]
Gu, S.; Ni, T.; Wang, J.; Liu, Y.; Fan, Q.; Wang, Y.; Huang, T.; Chu, Y.; Sun, X.; Wang, Y. CD47 Blockade Inhibits Tumor Progression
through Promoting Phagocytosis of Tumor Cells by M2 Polarized Macrophages in Endometrial Cancer. J. Immunol. Res. 2018,2018, 6156757. [CrossRef] [PubMed]
Sandvig, K.; Kavaliauskiene, S.; Skotland, T. Clathrin-Independent Endocytosis: An Increasing Degree of Complexity. Histochem.
Cell Biol. 2018, 150, 107–118. [CrossRef] [PubMed]
Young, A. Structural Insights into the Clathrin Coat. Semin. Cell Dev. Biol. 2007, 18, 448–458. [CrossRef] [PubMed]
Rothberg, K.G.; Heuser, J.E.; Donzell, W.C.; Ying, Y.-S.; Glenney, J.R.; Anderson, R.G.W. Caveolin, a Protein Component of
Caveolae Membrane Coats. Cell 1992, 68, 673–682. [CrossRef]
Rennick, J.J.; Johnston, A.P.R.; Parton, R.G. Key Principles and Methods for Studying the Endocytosis of Biological and Nanoparticle Therapeutics. Nat. Nanotechnol. 2021, 16, 266–276. [CrossRef] [PubMed]
Kerr, M.C.; Teasdale, R.D. Defining Macropinocytosis. Traffic 2009, 10, 364–371. [CrossRef] [PubMed]
Lin, X.P.; Mintern, J.D.; Gleeson, P.A. Macropinocytosis in Different Cell Types: Similarities and Differences. Membranes 2020,
10, 177. [CrossRef] [PubMed]Cancers 2022, 14, 5056

19.
20.
21.
22.
23.
24.
25.

26.
27.
28.

29.
30.
31.
32.
33.

34.
35.
36.
37.
38.
39.
40.
41.
42.
43.

44.
45.

46.

47.

11 of 15

Buckley, C.M.; King, J.S. Drinking Problems: Mechanisms of Macropinosome Formation and Maturation. FEBS J. 2017, 284,
3778–3790. [CrossRef] [PubMed]
Saito, N.; Sawai, S. Three-Dimensional Morphodynamic Simulations of Macropinocytic Cups. iScience 2021, 24, 103087. [CrossRef][PubMed]Mercer, J.; Helenius, A. Virus Entry by Macropinocytosis. Nat. Cell Biol. 2009, 11, 510–520. [CrossRef] [PubMed]
Lim, J.P.; Gleeson, P.A. Macropinocytosis: An Endocytic Pathway for Internalising Large Gulps. Immunol. Cell Biol. 2011, 89,
836–843. [CrossRef] [PubMed]
Paluck, A.; Osan, J.; Hollingsworth, L.; Talukdar, S.N.; Saegh, A.A.; Mehedi, M. Role of ARP2/3 Complex-Driven Actin
Polymerization in RSV Infection. Pathogens 2021, 11, 26. [CrossRef] [PubMed]
Norbury, C.C. Drinking a Lot Is Good for Dendritic Cells. Immunology 2006, 117, 443–451. [CrossRef] [PubMed]
García-Pérez, B.E.; de la Cruz-López, J.J.; Castañeda-Sánchez, J.I.; Muñóz-Duarte, A.R.; Hernández-Pérez, A.D.; Villegas-Castrejón,
H.; García-Latorre, E.; Caamal-Ley, A.; Luna-Herrera, J. Macropinocytosis Is Responsible for the Uptake of Pathogenic and
Non-Pathogenic Mycobacteria by B Lymphocytes (Raji Cells). BMC Microbiol. 2012, 12, 246. [CrossRef]
Charpentier, J.C.; Chen, D.; Lapinski, P.E.; Turner, J.; Grigorova, I.; Swanson, J.A.; King, P.D. Macropinocytosis Drives T Cell
Growth by Sustaining the Activation of MTORC1. Nat. Commun. 2020, 11, 180. [CrossRef] [PubMed]
Gu, Z.; Noss, E.H.; Hsu, V.W.; Brenner, M.B. Integrins Traffic Rapidly via Circular Dorsal Ruffles and Macropinocytosis during
Stimulated Cell Migration. J. Cell Biol. 2011, 193, 61–70. [CrossRef]
Mettlen, M.; Platek, A.; van der Smissen, P.; Carpentier, S.; Amyere, M.; Lanzetti, L.; de Diesbach, P.; Tyteca, D.; Courtoy, P.J.
Src Triggers Circular Ruffling and Macropinocytosis at the Apical Surface of Polarized MDCK Cells. Traffic 2006, 7, 589–603.
[CrossRef] [PubMed]
Yoshida, S.; Pacitto, R.; Sesi, C.; Kotula, L.; Swanson, J.A. Dorsal Ruffles Enhance Activation of Akt by Growth Factors. J. Cell Sci.
2018, 131, jcs220517. [CrossRef] [PubMed]
Yoshida, S.; Pacitto, R.; Inoki, K.; Swanson, J. Macropinocytosis, MTORC1 and Cellular Growth Control. Cell. Mol. Life Sci. 2018,75, 1227–1239. [CrossRef] [PubMed]
Inoki, K.; Zhu, T.; Guan, K.-L.L. TSC2 Mediates Cellular Energy Response to Control Cell Growth and Survival. Cell 2003, 115,
577–590. [CrossRef]
Condon, K.J.; Sabatini, D.M. Nutrient Regulation of MTORC1 at a Glance. J. Cell Sci. 2019, 132, jcs222570. [CrossRef] [PubMed]
Basagiannis, D.; Zografou, S.; Murphy, C.; Fotsis, T.; Morbidelli, L.; Ziche, M.; Bleck, C.; Mercer, J.; Christoforidis, S. VEGF Induces
Signalling and Angiogenesis by Directing VEGFR2 Internalisation through Macropinocytosis. J. Cell Sci. 2016, 129, 4091–4104.
[CrossRef]
Elfenbein, A.; Lanahan, A.; Zhou, T.X.; Yamasaki, A.; Tkachenko, E.; Matsuda, M.; Simons, M. Syndecan 4 Regulates FGFR1
Signaling in Endothelial Cells by Directing Macropinocytosis. Sci. Signal 2012, 5, ra36. [CrossRef]
Valdivia, A.; Goicoechea, S.M.; Awadia, S.; Zinn, A.; Garcia-Mata, R. Regulation of Circular Dorsal Ruffles, Macropinocytosis, andCell Migration by RhoG and Its Exchange Factor, Trio. Mol. Biol. Cell 2017, 28, 1768–1781. [CrossRef] [PubMed]
Zihni, C.; Balda, M.S.; Matter, K. Signalling at Tight Junctions during Epithelial Differentiation and Microbial Pathogenesis. J. Cell
Sci. 2014, 127, 3401–3413. [CrossRef] [PubMed]
Takai, Y.; Nakanishi, H. Nectin and Afadin: Novel Organizers of Intercellular Junctions. J. Cell Sci. 2003, 116, 17–27. [CrossRef]
Zihni, C.; Mills, C.; Matter, K.; Balda, M.S. Tight Junctions: From Simple Barriers to Multifunctional Molecular Gates. Nat. Rev.
Mol. Cell Biol. 2016, 17, 564–580. [CrossRef]
Citi, S. The Mechanobiology of Tight Junctions. Biophys. Rev. 2019, 11, 783–793. [CrossRef]
Nishimura, T.; Takeichi, M. Shroom3-Mediated Recruitment of Rho Kinases to the Apical Cell Junctions Regulates Epithelial and
Neuroepithelial Planar Remodeling. Development 2008, 135, 1493–1502. [CrossRef] [PubMed]
Chu, C.W.; Gerstenzang, E.; Ossipova, O.; Sokol, S.Y. Lulu Regulates Shroom-Induced Apical Constriction during Neural Tube
Closure. PLoS ONE 2013, 8, e81854. [CrossRef] [PubMed]
Hurd, T.W.; Gao, L.; Roh, M.H.; Macara, I.G.; Margolis, B. Direct Interaction of Two Polarity Complexes Implicated in Epthelial
Tight Junction Assembly. Nat. Cell Biol. 2003, 5, 137–142. [CrossRef] [PubMed]
Higashi, T.; Tokuda, S.; Kitajiri, S.; Masuda, S.; Nakamura, H.; Oda, Y.; Furuse, M. Analysis of the “angulin” Proteins LSR, ILDR1
and ILDR2–Tricellulin Recruitment, Epithelial Barrier Function and Implication in Deafness Pathogenesis. J. Cell Sci. 2013, 126,
966–977. [CrossRef] [PubMed]
Tsukita, S.; Yamazaki, Y.; Katsuno, T.; Tamura, A.; Tsukita, S. Tight Junction-Based Epithelial Microenvironment and Cell
Proliferation. Oncogene 2008, 27, 6930–6938. [CrossRef] [PubMed]
Yen, F.T.; Roitel, O.; Bonnard, L.; Notet, V.; Pratte, D.; Stenger, C.; Magueur, E.; Bihain, B.E. Lipolysis Stimulated Lipoprotein
Receptor: A Novel Molecular Link between Hyperlipidemia, Weight Gain, and Atherosclerosis in Mice. J. Biol. Chem. 2008, 283,
25650–25659. [CrossRef] [PubMed]
Stenger, C.; Hanse, M.; Pratte, D.; Mbala, M.L.; Akbar, S.; Koziel, V.; Escanyé, M.C.; Kriem, B.; Malaplate-Armand, C.; Olivier, J.L.;
et al. Up-Regulation of Hepatic Lipolysis Stimulated Lipoprotein Receptor by Leptin: A Potential Lever for Controlling Lipid
Clearance during the Postprandial Phase. FASEB J. 2010, 24, 4218–4228. [CrossRef] [PubMed]
Kohno, T.; Kikuchi, S.; Ninomiya, T.; Kojima, T. The Bicellular Tensile Force Sorts the Localization of LSRs in Bicellular andTricellular Junctions. Ann. N. Y. Acad. Sci. 2017, 1397, 185–194. [CrossRef]

Cancers 2022, 14, 5056

48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.

62.
63.
64.
65.

66.

67.

68.

69.

70.

71.

72.

12 of 15

Kohno, T.; Konno, T.; Kojima, T. Role of Tricellular Tight Junction Protein Lipolysis-Stimulated Lipoprotein Receptor (LSR) in
Cancer Cells. Int. J. Mol. Sci. 2019, 20, 3555. [CrossRef] [PubMed]
Martin, T.A.; Jiang, W.G. Loss of Tight Junction Barrier Function and Its Role in Cancer Metastasis. Biochim. Biophys. Acta
Biomembr. 2009, 1788, 872–891. [CrossRef] [PubMed]
Guillot, C.; Lecuit, T. Mechanics of Epithelial Tissue Homeostasis and Morphogenesis. Science (1979) 2013, 340, 1185–1189.
[CrossRef]
Loza, A.J.; Koride, S.; Schimizzi, G.V.; Li, B.; Sun, S.X.; Longmore, G.D. Cell Density and Actomyosin Contractility Control theOrganization of Migrating Collectives within an Epithelium. Mol. Biol. Cell 2016, 27, 3459–3470. [CrossRef] [PubMed]
Gumbiner, B.M.; Kim, N.G. The Hippo-YAP Signaling Pathway and Contact Inhibition of Growth. J. Cell Sci. 2014, 127, 709–717.
[CrossRef]
Mendonsa, A.M.; Na, T.-Y.; Gumbiner, B.M. E-Cadherin in Contact Inhibition and Cancer. Oncogene 2018, 37, 4769–4780.
[CrossRef] [PubMed]
McClatchey, A.I.; Yap, A.S. Contact Inhibition (of Proliferation) Redux. Curr. Opin. Cell Biol. 2012, 24, 685–694. [CrossRef][PubMed]
Garcia, M.A.; Nelson, W.J.; Chavez, N. Cell–Cell Junctions Organize Structural and Signaling Networks. Cold Spring Harb. Perspect.
Biol. 2018, 10, a029181. [CrossRef]
Eisenhoffer, G.T.; Rosenblatt, J. Bringing Balance by Force: Live Cell Extrusion Controls Epithelial Cell Numbers. Trends Cell Biol.
2013, 23, 185–192. [CrossRef] [PubMed]
Stramer, B.; Mayor, R. Mechanisms and in Vivo Functions of Contact Inhibition of Locomotion. Nat. Rev. Mol. Cell Biol. 2017, 18,
43–55. [CrossRef] [PubMed]
Buckley, C.E.; St Johnston, D. Apical–Basal Polarity and the Control of Epithelial Form and Function. Nat. Rev. Mol. Cell Biol.
2022, 23, 559–577. [CrossRef] [PubMed]
Haigis, K.M. KRAS Alleles: The Devil Is in the Detail. Trends Cancer 2017, 3, 686–697. [CrossRef]
Li, S.; Balmain, A.; Counter, C.M. A Model for RAS Mutation Patterns in Cancers: Finding the Sweet Spot. Nat. Rev. Cancer 2018,18, 767–777. [CrossRef]
Byron, S.A.; Gartside, M.; Powell, M.A.; Wellens, C.L.; Gao, F.; Mutch, D.G.; Goodfellow, P.J.; Pollock, P.M. FGFR2 Point Mutationsin 466 Endometrioid Endometrial Tumors: Relationship with MSI, KRAS, PIK3CA, CTNNB1 Mutations and Clinicopathological
Features. PLoS ONE 2012, 7, e30801. [CrossRef]
Inoue, S.; Hirota, Y.; Ueno, T.; Fukui, Y.; Yoshida, E.; Hayashi, T.; Kojima, S.; Takeyama, R.; Hashimoto, T.; Kiyono, T.; et al. Uterine
Adenomyosis Is an Oligoclonal Disorder Associated with KRAS Mutations. Nat. Commun. 2019, 10, 5785. [CrossRef] [PubMed]
Satoh, T.; Nishida, M.; Miyazaki, Y.; Sugita, M.; Arai, Y.; Oki, A.; Kono, K.; Tsunoda, H.; Kasahara, K.; Kubo, T. Establishment of a
Cisplatin-Resistant New Human Endometrial Adenocarcinoma Cell Line, Sawano Cells. Hum. Cell 1995, 8, 67–72. [PubMed]
Ray, I.; Meira, L.B.; Michael, A.; Ellis, P.E. Adipocytokines and Disease Progression in Endometrial Cancer: A Systematic Review.
Cancer Metastasis Rev. 2022, 41, 211–242. [CrossRef]
Konno, T.; Takano, K.; Kaneko, Y.; Kakuki, T.; Nomura, K.; Yajima, R.; Kakiuchi, A.; Kohno, T.; Himi, T.; Kojima, T. Guanylate
Binding Protein-1-Mediated Epithelial Barrier in Human Salivary Gland Duct Epithelium. Exp. Cell Res. 2018, 371, 31–41.
[CrossRef]
Kyuno, T.; Kyuno, D.; Kohno, T.; Konno, T.; Kikuchi, S.; Arimoto, C.; Yamaguchi, H.; Imamura, M.; Kimura, Y.; Kondoh, M.; et al.
Tricellular Tight Junction Protein LSR/Angulin-1 Contributes to the Epithelial Barrier and Malignancy in Human Pancreatic
Cancer Cell Line. Histochem. Cell Biol. 2020, 153, 5–16. [CrossRef]
Kodera, Y.; Kohno, T.; Konno, T.; Arai, W.; Tsujiwaki, M.; Shindo, Y.; Chiba, H.; Miyakawa, M.; Tanaka, H.; Sakuma, Y.; et al.
HMGB1 Enhances Epithelial Permeability via P63/TGF-β Signaling in Lung and Terminal Bronchial Epithelial Cells. Tissue
Barriers 2020, 8, 1805997. [CrossRef]
Kodera, Y.; Chiba, H.; Konno, T.; Kohno, T.; Takahashi, H.; Kojima, T. HMGB1-Downregulated Angulin-1/LSR Induces Epithelial
Barrier Disruption via Claudin-2 and Cellular Metabolism via AMPK in Airway Epithelial Calu-3 Cells. Biochem. Biophys. Res.
Commun. 2020, 527, 553–560. [CrossRef]
Miyakawa, M.; Konno, T.; Kohno, T.; Kikuchi, S.; Tanaka, H.; Kojima, T. Increase in Epithelial Permeability and Cell Metabolism
by High Mobility Group Box 1, Inflammatory Cytokines and TPEN in Caco-2 Cells as a Novel Model of Inflammatory Bowel
Disease. Int. J. Mol. Sci. 2020, 21, 8434. [CrossRef]
Shindo, Y.; Arai, W.; Konno, T.; Kohno, T.; Kodera, Y.; Chiba, H.; Miyajima, M.; Sakuma, Y.; Watanabe, A.; Kojima, T. Effects ofHistone Deacetylase Inhibitors Tricostatin A and Quisinostat on Tight Junction Proteins of Human Lung Adenocarcinoma A549
Cells and Normal Lung Epithelial Cells. Histochem. Cell Biol. 2021, 155, 637–653. [CrossRef]
Ohwada, K.; Konno, T.; Kohno, T.; Nakano, M.; Ohkuni, T.; Miyata, R.; Kakuki, T.; Kondoh, M.; Takano, K.; Kojima, T. Effectsof HMGB1 on Tricellular Tight Junctions via TGF-β Signaling in Human Nasal Epithelial Cells. Int. J. Mol. Sci. 2021, 22, 8390.
[CrossRef] [PubMed]
Kojima, T.; Shindo, Y.; Konno, T.; Kodera, Y.; Arai, W.; Miyakawa, M.; Ohwada, K.; Tanaka, H.; Tsujiwaki, M.; Sakuma, Y.; et al.
Dysfunction of Epithelial Permeability Barrier Induced by HMGB1 in 2.5D Cultures of Human Epithelial Cells. Tissue Barriers
2022, 10, 1972760. [CrossRef]

Cancers 2022, 14, 5056

73.
74.
75.
76.
77.

78.
79.

80.
81.
82.

83.

84.
85.
86.

87.
88.
89.
90.
91.

92.

93.
94.
95.

96.

13 of 15

Nezhat, F.; Datta, M.S.; Hanson, V.; Pejovic, T.; Nezhat, C.; Nezhat, C. The Relationship of Endometriosis and Ovarian Malignancy:
A Review. Fertil. Steril. 2008, 90, 1559–1570. [CrossRef] [PubMed]
Zhou, W.-J.; Yang, H.-L.; Shao, J.; Mei, J.; Chang, K.-K.; Zhu, R.; Li, M.-Q. Anti-Inflammatory Cytokines in Endometriosis. Cell.
Mol. Life Sci. 2019, 76, 2111–2132. [CrossRef] [PubMed]
Zeng, Q.; Hong, W. The Emerging Role of the Hippo Pathway in Cell Contact Inhibition, Organ Size Control, and Cancer
Development in Mammals. Cancer Cell 2008, 13, 188–192. [CrossRef]
Halder, G.; Dupont, S.; Piccolo, S. Transduction of Mechanical and Cytoskeletal Cues by YAP and TAZ. Nat. Rev. Mol. Cell Biol.
2012, 13, 591–600. [CrossRef]
Shimada, H.; Abe, S.; Kohno, T.; Satohisa, S.; Konno, T.; Takahashi, S.; Hatakeyama, T.; Arimoto, C.; Kakuki, T.; Kaneko, Y.;
et al. Loss of Tricellular Tight Junction Protein LSR Promotes Cell Invasion and Migration via Upregulation of TEAD1/AREG in
Human Endometrial Cancer. Sci. Rep. 2017, 7, 37049. [CrossRef]
Konno, T.; Kohno, T.; Okada, T.; Shimada, H.; Satohisa, S.; Kikuchi, S.; Saito, T.; Kojima, T. ASPP2 Suppression Promotes
Malignancy via LSR and YAP in Human Endometrial Cancer. Histochem. Cell Biol. 2020, 154, 197–213. [CrossRef]
Bao, Y.; Nakagawa, K.; Yang, Z.; Ikeda, M.; Withanage, K.; Ishigami-Yuasa, M.; Okuno, Y.; Hata, S.; Nishina, H.; Hata, Y. A
Cell-Based Assay to Screen Stimulators of the Hippo Pathway Reveals the Inhibitory Effect of Dobutamine on the YAP-Dependent
Gene Transcription. J. Biochem. 2011, 150, 199–208. [CrossRef]
Zheng, H.X.; Wu, L.N.; Xiao, H.; Du, Q.; Liang, J.F. Inhibitory Effects of Dobutamine on Human Gastric Adenocarcinoma. World J.
Gastroenterol. 2014, 20, 17092–17099. [CrossRef]
Paul, S.; Xie, S.; Yao, X.; Dey, A. Transcriptional Regulation of the Hippo Pathway: Current Understanding and Insights fromSingle-Cell Technologies. Cells 2022, 11, 2225. [CrossRef] [PubMed]
Someya, M.; Kojima, T.; Ogawa, M.; Ninomiya, T.; Nomura, K.; Takasawa, A.; Murata, M.; Tanaka, S.; Saito, T.; Sawada, N.
Regulation of Tight Junctions by Sex Hormones in Normal Human Endometrial Epithelial Cells and Uterus Cancer Cell Line
Sawano. Cell Tissue Res. 2013, 354, 481–494. [CrossRef] [PubMed]
Shimada, H.; Satohisa, S.; Kohno, T.; Takahashi, S.; Hatakeyama, T.; Konno, T.; Tsujiwaki, M.; Saito, T.; Kojima, T. The Roles ofTricellular Tight Junction Protein Lipolysis-Stimulated Lipoprotein Receptor in Malignancy of Human Endometrial Cancer Cells.
Oncotarget 2016, 7, 27735–27752. [CrossRef] [PubMed]
Grund, S.; Grümmer, R. Direct Cell-Cell Interactions in the Endometrium and in Endometrial Pathophysiology. Int. J. Mol. Sci.
2018, 19, 2227. [CrossRef] [PubMed]
Leech, A.O.; Cruz, R.G.B.; Hill, A.D.K.; Hopkins, A.M. Paradigms Lost—An Emerging Role for over-Expression of Tight Junction
Adhesion Proteins in Cancer Pathogenesis. Ann. Transl. Med. 2015, 3, 184. [CrossRef] [PubMed]
Shimada, H.; Satohisa, S.; Kohno, T.; Konno, T.; Takano, K.; Takahashi, S.; Hatakeyama, T.; Arimoto, C.; Saito, T.; Kojima, T.
Downregulation of Lipolysis-Stimulated Lipoprotein Receptor Promotes Cell Invasion via Claudin-1-Mediated Matrix Metalloproteinases in Human Endometrial Cancer. Oncol. Lett. 2017, 14, 6776–6782. [CrossRef]
Wang, H.B.; Wang, P.Y.; Wang, X.; Wan, Y.L.; Liu, Y.C. Butyrate Enhances Intestinal Epithelial Barrier Function via Up-Regulationof Tight Junction Protein Claudin-1 Transcription. Dig. Dis. Sci. 2012, 57, 3126–3135. [CrossRef]
Dufresne, J.; Cyr, D.G. Activation of an SP Binding Site Is Crucial for the Expression of Claudin 1 in Rat Epididymal Principal
Cells. Biol. Reprod. 2007, 76, 825–832. [CrossRef]
Honda, H.; Pazin, M.J.; Ji, H.; Wernyj, R.P.; Morin, P.J. Crucial Roles of Sp1 and Epigenetic Modifications in the Regulation of the
Cldn4 Promoter in Ovarian Cancer Cells. J. Biol. Chem. 2006, 281, 21433–21444. [CrossRef]
Luk, J.M.; Tong, M.K.; Mok, B.W.; Tam, P.C.; Yeung, W.S.B.; Lee, K.F. Sp1 Site Is Crucial for the Mouse Claudin-19 Gene Expression
in the Kidney Cells. FEBS Lett. 2004, 578, 251–256. [CrossRef]
Oku, N.; Sasabe, E.; Ueta, E.; Yamamoto, T.; Osaki, T. Tight Junction Protein Claudin-1 Enhances the Invasive Activity of Oral
Squamous Cell Carcinoma Cells by Promoting Cleavage of Laminin-5 Γ2 Chain via Matrix Metalloproteinase (MMP)-2 andMembrane-Type MMP-1. Cancer Res. 2006, 66, 5251–5257. [CrossRef] [PubMed]
van Itallie, C.M.; Fanning, A.S.; Anderson, J.M.; Boireau, S.; Samuel, M.S.; Pannequin, J.; Ryan, J.L.; Choquet, A.; Chapuis, H.;
Rebillard, X.; et al. Claudin-1 Regulates Cellular Transformation and Metastatic Behavior in Colon Cancer. J. Clin. Investig. 2005,
115, 1765–1776. [CrossRef]
Castro-Castro, A.; Marchesin, V.; Monteiro, P.; Lodillinsky, C.; Rossé, C.; Chavrier, P. Cellular and Molecular Mechanisms of
MT1-MMP-Dependent Cancer Cell Invasion. Annu. Rev. Cell Dev. Biol. 2016, 32, 555–576. [CrossRef]
Brown, G.T.; Murray, G.I. Current Mechanistic Insights into the Roles of Matrix Metalloproteinases in Tumour Invasion andMetastasis. J. Pathol. 2015, 237, 273–281. [CrossRef] [PubMed]
Gaide Chevronnay, H.P.; Selvais, C.; Emonard, H.; Galant, C.; Marbaix, E.; Henriet, P. Regulation of Matrix Metalloproteinases
Activity Studied in Human Endometrium as a Paradigm of Cyclic Tissue Breakdown and Regeneration. Biochim. Biophys. Acta
(BBA)-Proteins Proteom. 2012, 1824, 146–156. [CrossRef] [PubMed]
Pandit, A.; das Mahapatra, P.; Saha, P.; Srivastava, A.K.; Swarnakar, S. Interleukin-1β Activated C-FOS Transcription Factor Binds
Preferentially to a Specific Allele of the Matrix Metalloproteinase-13 Promoter and Increases Susceptibility to Endometriosis. J.
Cell Physiol. 2022, 237, 3095–3108. [CrossRef]

Cancers 2022, 14, 5056

97.

14 of 15

Mwaura, A.N.; Riaz, M.A.; Maoga, J.B.; Mecha, E.; Omwandho, C.O.A.; Scheiner-Bobis, G.; Meinhold-Heerlein, I.; Konrad, L. Roleof Betaglycan in TGF-β Signaling and Wound Healing in Human Endometriotic Epithelial Cells and in Endometriosis. Biology
2022, 11, 513. [CrossRef]
98. Paz, H.; Pathak, N.; Yang, J. Invading One Step at a Time: The Role of Invadopodia in Tumor Metastasis. Oncogene 2014, 33,
4193–4202. [CrossRef] [PubMed]
99. Chang, J.W.; SEO, S.T.; IM, M.A.; WON, H.-R.; LIU, L.; OH, C.; JIN, Y.L.; PIAO, Y.; KIM, H.J.; KIM, J.T.; et al. Claudin-1 Mediates
Progression by Regulating EMT through AMPK/TGF-β Signaling in Head and Neck Squamous Cell Carcinoma. Transl. Res.
2022, 247, 58–78. [CrossRef] [PubMed]
100. Wang, K.; Pascal, L.E.; Li, F.; Chen, W.; Dhir, R.; Balasubramani, G.K.; DeFranco, D.B.; Yoshimura, N.; He, D.; Wang, Z. Tight
Junction Protein Claudin-1 Is Downregulated by TGF-β1 via MEK Signaling in Benign Prostatic Epithelial Cells. Prostate 2020, 80,
1203–1215. [CrossRef] [PubMed]
101. Stamatovic, S.M.; Johnson, A.M.; Sladojevic, N.; Keep, R.F.; Andjelkovic, A.V. Endocytosis of Tight Junction Proteins and theRegulation of Degradation and Recycling. Ann. N. Y. Acad. Sci. 2017, 1397, 54–65. [CrossRef]
102. Bruewer, M.; Utech, M.; Ivanov, A.I.; Hopkins, A.M.; Parkos, C.A.; Nusrat, A. Interferon-γ Induces Internalization of Epithelial
Tight Junction Proteins via a Macropinocytosis-like Process. FASEB J. 2005, 19, 923–933. [CrossRef] [PubMed]
103. Hopkins, A.M.; Walsh, S.V.; Verkade, P.; Boquet, P.; Nusrat, A. Constitutive Activation of Rho Proteins by CNF-1 Influences Tight
Junction Structure and Epithelial Barrier Function. J. Cell Sci. 2003, 116, 725–742. [CrossRef]
104. Shen, L.; Turner, J.R. Actin Depolymerization Disrupts Tight Junctions via Caveolae-Mediated Endocytosis. Mol. Biol. Cell 2005,
16, 3919–3936. [CrossRef] [PubMed]
105. Matsuda, M.; Kubo, A.; Furuse, M.; Tsukita, S. A Peculiar Internalization of Claudins, Tight Junction-Specific Adhesion Molecules,
during the Intercellular Movement of Epithelial Cells. J. Cell Sci. 2004, 117, 1247–1257. [CrossRef]
106. Yamaki, T.; Ohtake, K.; Ichikawa, K.; Uchida, M.; Uchida, H.; Ohshima, S.; Juni, K.; Kobayashi, J.; Morimoto, Y.; Natsume, H.
Poly-l-Arginine-Induced Internalization of Tight Junction Proteins Increases the Paracellular Permeability of the Caco-2 Cell
Monolayer to Hydrophilic Macromolecules. Biol. Pharm. Bull. 2013, 36, 432–441. [CrossRef] [PubMed]
107. Murakami, T.; Felinski, E.A.; Antonetti, D.A. Occludin Phosphorylation and Ubiquitination Regulate Tight Junction Trafficking
and Vascular Endothelial Growth Factor-Induced Permeability. J. Biol. Chem. 2009, 284, 21036–21046. [CrossRef] [PubMed]
108. Krug, S.M.; Hayaishi, T.; Iguchi, D.; Watari, A.; Takahashi, A.; Fromm, M.; Nagahama, M.; Takeda, H.; Okada, Y.; Sawasaki, T.;et al. Angubindin-1, a Novel Paracellular Absorption Enhancer Acting at the Tricellular Tight Junction. J. Control. Release 2017,
260, 1–11. [CrossRef] [PubMed]
109. Pernet-Gallay, K.; Jouneau, P.H.; Bertrand, A.; Delaroche, J.; Farion, R.; Rémy, C.; Barbier, E.L. Vascular Permeability in the RG2Glioma Model Can Be Mediated by Macropinocytosis and Be Independent of the Opening of the Tight Junction. J. Cereb. Blood
Flow Metab. 2017, 37, 1264–1275. [CrossRef] [PubMed]
110. Nagahama, M.; Takehara, M.; Kobayashi, K. Interaction of Clostridium Perfringens Iota Toxin and Lipolysis-Stimulated Lipoprotein Receptor (LSR). Toxins 2018, 10, 405. [CrossRef] [PubMed]
111. Sohet, F.; Lin, C.; Munji, R.N.; Lee, S.Y.; Ruderisch, N.; Soung, A.; Arnold, T.D.; Derugin, N.; Vexler, Z.S.; Yen, F.T.; et al.
LSR/Angulin-1 Is a Tricellular Tight Junction Protein Involved in Blood-Brain Barrier Formation. J. Cell Biol. 2015, 208, 703–711.
[CrossRef]
112. Zeniya, S.; Kuwahara, H.; Daizo, K.; Watari, A.; Kondoh, M.; Yoshida-Tanaka, K.; Kaburagi, H.; Asada, K.; Nagata, T.; Nagahama,M.; et al. Angubindin-1 Opens the Blood–Brain Barrier in Vivo for Delivery of Antisense Oligonucleotide to the Central Nervous
System. J. Control. Release 2018, 283, 126–134. [CrossRef] [PubMed]
113. Konno, T.; Kohno, T.; Kikuchi, S.; Shimada, H.; Satohisa, S.; Saito, T.; Kondoh, M.; Kojima, T. Epithelial Barrier Dysfunction andCell Migration Induction via JNK/Cofilin/Actin by Angubindin-1. Tissue Barriers 2020, 8, 1695475. [CrossRef] [PubMed]
114. Huang, C.; Jacobson, K.; Schaller, M.D. MAP Kinases and Cell Migration. J. Cell Sci. 2004, 117, 4619–4628. [CrossRef]
115. Magi, S.; Tashiro, E.; Imoto, M. A Chemical Genomic Study Identifying Diversity in Cell Migration Signaling in Cancer Cells. Sci.
Rep. 2012, 2, 823. [CrossRef]
116. Ichimizu, S.; Watanabe, H.; Maeda, H.; Hamasaki, K.; Ikegami, K.; Chuang, V.T.G.; Kinoshita, R.; Nishida, K.; Shimizu, T.; Ishima,Y.; et al. Cell-Penetrating Mechanism of Intracellular Targeting Albumin: Contribution of Macropinocytosis Induction andEndosomal Escape. J. Control. Release 2019, 304, 156–163. [CrossRef] [PubMed]
117. Bretou, M.; Sáez, P.J.; Sanséau, D.; Maurin, M.; Lankar, D.; Chabaud, M.; Spampanato, C.; Malbec, O.; Barbier, L.; Muallem, S.;et al. Lysosome Signaling Controls the Migration of Dendritic Cells. Sci. Immunol. 2017, 2, eaak9573. [CrossRef] [PubMed]
118. Vicente-Manzanares, M.; Ma, X.; Adelstein, R.S.; Horwitz, A.R. Non-Muscle Myosin II Takes Centre Stage in Cell Adhesion and
Migration. Nat. Rev. Mol. Cell Biol. 2009, 10, 778–790. [CrossRef] [PubMed]
119. Masereel, B. An Overview of Inhibitors of Na+/H+ Exchanger. Eur. J. Med. Chem. 2003, 38, 547–554. [CrossRef]
120. Fliegel, L. Structural and Functional Changes in the Na+ /H+ Exchanger Isoform 1, Induced by Erk1/2 Phosphorylation. Int. J.
Mol. Sci. 2019, 20, 2378. [CrossRef] [PubMed]
121. Donowitz, M.; Ming Tse, C.; Fuster, D. SLC9/NHE Gene Family, a Plasma Membrane and Organellar Family of Na+ /H+
Exchangers. Mol. Aspects Med. 2013, 34, 236–251. [CrossRef] [PubMed]Cancers 2022, 14, 5056

15 of 15

122. Koivusalo, M.; Welch, C.; Hayashi, H.; Scott, C.C.; Kim, M.; Alexander, T.; Touret, N.; Hahn, K.M.; Grinstein, S. Amiloride Inhibits
Macropinocytosis by Lowering Submembranous PH and Preventing Rac1 and Cdc42 Signaling. J. Cell Biol. 2010, 188, 547–563.
[CrossRef]
123. Rotty, J.D.; Wu, C.; Bear, J.E. New Insights into the Regulation and Cellular Functions of the ARP2/3 Complex. Nat. Rev. Mol. Cell
Biol. 2013, 14, 7–12. [CrossRef] [PubMed]
124. Mehedi, M.; McCarty, T.; Martin, S.E.; le Nouën, C.; Buehler, E.; Chen, Y.-C.; Smelkinson, M.; Ganesan, S.; Fischer, E.R.; Brock,L.G.; et al. Actin-Related Protein 2 (ARP2) and Virus-Induced Filopodia Facilitate Human Respiratory Syncytial Virus Spread.
PLoS Pathog. 2016, 12, e1006062. [CrossRef] [PubMed]
125. Denker, S.P.; Huang, D.C.; Orlowski, J.; Furthmayr, H.; Barber, D.L. Direct Binding of the Na-H Exchanger NHE1 to ERM Proteins
Regulates the Cortical Cytoskeleton and Cell Shape Independently of H+ Translocation. Mol. Cell 2000, 6, 1425–1436. [CrossRef]
126. Tominaga, T.; Ishizaki, T.; Narumiya, S.; Barber, D.L. P160ROCK Mediates RhoA Activation of Na-H Exchange. EMBO J. 1998, 17,4712–4722. [CrossRef]
127. Hendus-Altenburger, R.; Pedraz-Cuesta, E.; Olesen, C.W.; Papaleo, E.; Schnell, J.A.; Hopper, J.T.S.; Robinson, C.V.; Pedersen, S.F.;
Kragelund, B.B. The Human Na+/H+ Exchanger 1 Is a Membrane Scaffold Protein for Extracellular Signal-Regulated Kinase 2.
BMC Biol. 2016, 14, 31. [CrossRef]
128. Amith, S.R.; Wilkinson, J.M.; Baksh, S.; Fliegel, L. The Na+/H+ Exchanger (NHE1) as a Novel Co-Adjuvant Target in Paclitaxel
Therapy of Triple-Negative Breast Cancer Cells. Oncotarget 2015, 6, 1262–1275. [CrossRef]
129. Kaminota, T.; Yano, H.; Shiota, K.; Nomura, N.; Yaguchi, H.; Kirino, Y.; Ohara, K.; Tetsumura, I.; Sanada, T.; Ugumori, T.; et al.
Elevated Na+/H+ Exchanger-1 Expression Enhances the Metastatic Collective Migration of Head and Neck Squamous Cell
Carcinoma Cells. Biochem. Biophys. Res. Commun. 2017, 486, 101–107. [CrossRef]
130. Guan, X.; Luo, L.; Begum, G.; Kohanbash, G.; Song, Q.; Rao, A.; Amankulor, N.; Sun, B.; Sun, D.; Jia, W. Elevated Na/H Exchanger
1 (SLC9A1) Emerges as a Marker for Tumorigenesis and Prognosis in Gliomas. J. Exp. Clin. Cancer Res. 2018, 37, 255. [CrossRef]
131. Yamada, T.; Yoshida, T.; Kawamoto, A.; Mitsuoka, K.; Iwasaki, K.; Tsuge, H. Cryo-EM Structures Reveal Translocational Unfoldingin the Clostridial Binary Iota Toxin Complex. Nat. Struct. Mol. Biol. 2020, 27, 288–296. [CrossRef] [PubMed]
132. Amith, S.R.; Fong, S.; Baksh, S.; Fliegel, L. Na+/H+ Exchange in the Tumour Microenvironment: Does NHE1 Drive Breast Cancer
Carcinogenesis? Int. J. Dev. Biol. 2015, 59, 367–377. [CrossRef] [PubMed]
133. Levine, D.A. Integrated Genomic Characterization of Endometrial Carcinoma. Nature 2013, 497, 67–73. [CrossRef] [PubMed]
134. Torres-Flores, J.M.; Arias, C.F. Tight Junctions Go Viral! Viruses 2015, 7, 5145–5154. [CrossRef]
135. Commisso, C.; Davidson, S.M.; Soydaner-Azeloglu, R.G.; Parker, S.J.; Kamphorst, J.J.; Hackett, S.; Grabocka, E.; Nofal, M.; Drebin,J.A.; Thompson, C.B.; et al. Macropinocytosis of Protein Is an Amino Acid Supply Route in Ras-Transformed Cells. Nature 2013,
497, 633–637. [CrossRef]
136. Davidson, S.M.; Jonas, O.; Keibler, M.A.; Hou, H.W.; Luengo, A.; Mayers, J.R.; Wyckoff, J.; del Rosario, A.M.; Whitman, M.; Chin,
C.R.; et al. Direct Evidence for Cancer-Cell-Autonomous Extracellular Protein Catabolism in Pancreatic Tumors. Nat. Med. 2017,23, 235–241. [CrossRef]