Received: 15 March 2023

| Revised: 26 July 2023 | Accepted: 2 August 2023

DOI: 10.1111/cas.15934

ORIGINAL ARTICLE

Promotion of squamous cell carcinoma tumorigenesis byoncogene-­mediated THG-­1/TSC22D4 phosphorylation
Nohara Goto1,2 | Hiroyuki Suzuki1,3| Ling Zheng1 | Yasuhito Okano1 |Yukari Okita1| Yukihide Watanabe1| Yukinari Kato3| Mitsuyasu Kato1
1
Department of Experimental Pathology,
Faculty of Medicine, University ofTsukuba, Tsukuba, Japan2

Ph.D. Program in Human Biology,
School of Integrative and Global Majors,
University of Tsukuba, Tsukuba, Japan3

Department of Antibody Drug
Development, Tohoku University
Graduate School of Medicine, Sendai,Japan
Correspondence
Hiroyuki Suzuki, Department of Antibody
Drug Development, Tohoku University
Graduate School of Medicine, 2-­1 Seiryo-­machi, Aoba-­ku, Sendai, Miyagi 980-­8575,
Japan.
Email: hiroyuki.suzuki.b4@tohoku.ac.jp
Mitsuyasu Kato, Department ofExperimental Pathology, Faculty of
Medicine, University of Tsukuba, 1-­1-­1
Tennoudai, Tsukuba, Ibaraki 305-­8575,
Japan.
Email: mit-kato@md.tsukuba.ac.jp
Funding information
Japan Society for the Promotionof Science, Grant/Award Number:
17K19580, 25640059, 22K06995,
19K07473, 16K08706, 22K15596 and
20K16434

1|

Abstract
Carcinoma cells possess high proliferative and invasive potentials and exhibit a resilience against stresses, metabolic disorder, and therapeutic efforts. These properties
are mainly acquired by genetic alterations including driver gene mutations. However,the detailed molecular mechanisms have not been fully elucidated. Here, we provide
a novel mechanism connecting oncogenic signaling and the tumorigenic properties bya transforming growth factor-­β1-­stimulated clone 22 (TSC-­22) family protein, THG-­1(also called as TSC22D4). THG-­1 is localized at the basal layer of normal squamous epithelium and overexpressed in squamous cell carcinomas (SCCs). THG-­1 knockdown
suppressed SCC cell proliferation, invasiveness, and xenograft tumor formation. In
contrast, THG-­1 overexpression promoted the EGF-­induced proliferation and stratified epithelium formation. Furthermore, THG-­1 is phosphorylated by the receptor
tyrosine kinase (RTK)-­R AS-­ERK pathway, which promoted the oncogene-­mediatedtumorigenesis. Moreover, THG-­1 involves in the alternative splicing of CD44 variants,
a regulator of invasiveness, stemness, and oxidative stress resistance under the RTK
pathway. These findings highlight the pivotal roles of THG-­1 as a novel effector of
SCC tumorigenesis, and the detection of THG-­1 phosphorylation by our establishedspecific antibody could contribute to cancer diagnosis and therapy.
KEYWORDS

CD44, monoclonal antibody, phosphorylation, receptor tyrosine kinase, squamous cell
carcinoma, THG-­1, TSC22D4I NTRO D U C TI O N

cell carcinomas (SCCs) arise from epithelial tissues that can be classified as stratified squamous epithelium including skin,3 esophagus,4Protection against physical and chemical insults, infection, and watercervix,5 head and neck6 and nonsquamous epithelia including theloss is provided by the stratified squamous epithelium.1,2 Squamouslung airway epithelium.7 Despite advances of treatment in recent

Abbreviations: CD44s, CD44 standard; CD44v, CD44 variants; EGFR, epidermal growth factor receptor; FGFR, fibroblast growth factor receptor; GSH, reduced glutathione; HB-­EGF,
heparin-­binding epidermal growth factor-­like growth factor; IHC, immunohistochemistry; mAb, monoclonal antibody; NOD-­Scid, NOD.CB17-­Prkdc scid/NCrCrl; PI3K,
phosphatidylinositol-­3 kinase; ROS, reactive oxygen species; RTK, receptor tyrosine kinase; SAM68, Src-­associated in mitosis 68 kDa protein; SCC, squamous cell carcinoma; SD,
standard deviation; TSC-­22, transforming growth factor-­β1-­s timulated clone 22; xCT, cystine–­glutamate transporter.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction
in any medium, provided the original work is properly cited and is not used for commercial purposes.
© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.
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Cancer Science. 2023;114:3972–3983.

years, SCC survival has not been improved significantly.8 Further understanding of the molecular mechanisms underlying SCC progression is a prerequisite for improving patient therapy and prognosis.

Transforming growth factor-­
β1-­
stimulated clone 22 (TSC-­
22)

was identified as a TGF-­β1 target gene20 and possesses growth-­
suppressive function. 21 TSC-­
22 (TSC22D1) and related family

Genomic and molecular characterization of SCC including

members, including KIAA0669/TSC22D2, GILZ/TSC22D3, andlung, esophagus, head and neck, and skin SCCs have been stud-

THG-­1/TSC22D4 share a conserved Tsc-­box and leucine zipper do-

ied to understand the molecular characteristics and driver genemains. 22 Among them, THG-­1 was originally identified as a TSC-­22-­

9

mutations. Comprehensive genomic characterization of 178 lunginteracting protein23; however, the physiological and/or pathological

SCC revealed that broad range of receptor tyrosine kinase (RTK)

functions of THG-­1 have not been elucidated. In this study, we show

upregulation by the genomic alteration of epidermal growth fac-that THG-­1 is expressed in normal squamous epithelium and over-

tor receptor (EGFR) and fibroblast growth factor receptor (FGFR)

expressed in SCCs. THG-­1 is phosphorylated by receptor tyrosine

families were observed in 26% of tumors. The activation of thekinase pathways, which is important for the tumorigenic potential of

RAS-­BRAF pathway was observed in 24% of tumors. Furthermore,

THG-­1. Furthermore, THG-­1 and its phosphorylation promote theone of the components of the phosphatidylinositol 3-­kinase (PI3K)/

production of CD44v. These results indicated that THG-­1 is involved

AKT pathway was altered in 47% of tumors, and the alterations inin the tumorigenesis of SCC under the RTK signaling pathway.

the PI3K/AKT pathway genes were mutually exclusive with EGFR
alterations.10 The dependence of lung SCC on these individual alterations remains to be defined functionally, and the identificationof the substrate which determines the characteristics of SCC is also
important. Although molecular targeting therapies for lung adeno-

2|

M ATE R I A L S A N D M E TH O DS

2.1 | Cell lines

carcinoma against EGFR, BRAF, HER2, KRASG12C , and ALK fusions
have been successful,11 activating mutations in EGFR and ALK fu-

HEK293T and P3X63Ag8U.1 (P3U1) cells were purchased from thesions are typically not observed in lung SCC. Therefore, targeted

American Type Culture Collection (ATCC). HaCaT cells were provided

agents developed for lung adenocarcinoma are largely ineffective

by Dr. N.E. Fusenig (DKFZ). The TE series of esophageal tumor cellagainst lung SCC.

lines were obtained from the Cell Resource Center for Biomedical Re-

The outcome of esophageal SCC has remained unchanged during

search, Institute of Development, Aging and Cancer, Tohoku Univer-the last several decades, with a 5-­year survival rate ranging from 15%

sity. The KYSE series of esophageal tumor cell lines were purchased

to 20%.8 The driver gene mutations were also revealed by a large

from the Japanese Collection of Research Bioresources Cell Bank.

scale of whole-­exome or targeted deep sequencing. The mitogen-­

Human dermal fibroblasts were obtained from RIKEN BioResource

activated protein kinase and PI3K pathways are augmented by mul-

Research Center (BRC). These cells were cultured in Dulbecco's modi-

tiple mechanisms including the amplification and overexpression offied Eagle's medium (DMEM, Sigma-­Aldrich) supplemented with 10%

RTKs and the activating mutations KRAS and PIK3CA.12 Althoughfetal bovine serum (FBS) and 50 U/mL penicillin and 50 μg/mL strep-

the driver gene mutations were revealed in SCC, the substrates

tomycin (FUJIFILM Wako Pure Chemical). P3X63Ag8U.1 (P3U1) cells

which determine the property of SCC have not been fully elucidated.

were obtained from ATCC and cultured in RPMI1640 medium (Sigma-­

CD44 is a cell surface glycoprotein and known as a major re-

Aldrich) with 10% FBS. EGF was obtained from Sigma-­Aldrich.

ceptor for hyaluronic acid, which is involved in tumor cell adhesion,
migration, and invasiveness.13 CD44 is involved in cancer stem cellproperties, epithelial to mesenchymal transition, and a resistance14

to chemo-­and radiotherapy.

2.2 | Antibodies

The CD44 gene consists of multiple

exons in humans. The first five (1–­5) and the last five (16–­20) exons

THG-­1 (4G7, Abnova); FLAG epitope tag (M2, Sigma-­Aldrich); HA

produce CD44 standard form (CD44s). The variant exons can be al-

epitope tag (3F10, Roche Diagnostics); β-­actin (Sigma-­Aldrich);

ternatively spliced and assembled with exons contained in CD44s

E-­
C adherin (610,181, BD Bioscience); Phospho-­
EGFR (pTyr1068,

and are referred to as CD44 variants (CD44v).15 The CD44v pro-

C04301, Abcam); EGFR (sc-­101, Santa Cruz); Phospho-­ERK1/2

vides binding site for cytokines and growth factors. For instance,(pThr202/pTyr204, #9106, Cell Signaling); ERK1/2 (#9102, Cell Signal-

CD44v3–­10 functions as a coreceptor of RTKs by binding to FGFsing) or ERK2 (sc-­154, Santa Cruz); Phospho-­Akt (pS 473, #9271, Celland heparin-­binding epidermal growth factor-­
like growth factor

Signaling); Akt (#9272, Cell Signaling); c-­Myc (sc-­764, Santa Cruz);

16

and hepatocyte growth fac-

JunB (sc-­8 051, Santa Cruz); Ki67 (Leica); pan-­CD44-­PE (Sigma-­

tor receptor via the v6-­encoded region.17 Furthermore, CD44v in-(HB-­EGF) via the v3-­encoded region

Aldrich); CD44v3 (3G5, for immunohistochemistry [IHC], R&D Sys-

teracts with a subunit of cystine–­glutamate transporter (xCT) via

tems, Inc.); CD44v3 (C44Mab-­624 for flow cytometry); CD44v9 (RV3,

the v8–­10-­encoded region and confers oxidative stress resistancein several carcinomas.18 Therefore, CD44v is a promising target for

Cosmo Bio and C44Mab-­125).

For generation of anti-­phospho-­THG-­1(pS264) antibody, BALB/ccancer diagnosis and therapy. Therefore, understanding the molecu-

mice (CLEA) were immunized with injections of synthetic peptide

lar mechanism of CD44v processing is important for targeting CD44

PLDSRPS(pS)PALYFT (pTHG-­1 peptide) plus C-­terminal cysteine

in cancer therapy.19

conjugated with the keyhole limpet hemocyanin at the C-­terminus

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GOTO et al.

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GOTO et al.

(Eurofins Genomics KK). The spleen cells were fused with mouse

mice (Charles River) with Matrigel (BD Biosciences). For the xeno-

myeloma P3U1 cells using a Sendai virus envelope, GenomONE-­CF

grafts of the HaCaT-­HRasG12V cells, 5 × 10 6 cells were subcutane-(Ishihara Sangyo).

26

Supernatants were subsequently screened using

ously inoculated into female BALB/cAJcl-­n u/nu mice (nude mice)

enzyme-­linked immunosorbent assay which reacted with pTHG-­1(CLEA). After 60 days (TE13 cells) and 45 days (HaCaT-­HRasG12V

peptide but not non-­phospho-­THG-­1 peptide (PLDSRPSSPALYFT).

cells), the tumor volumes were approximated using the following

From the positive wells, hybridomas were cloned by limiting dilution.

equation: volume = (a × b2) π/6, where a and b are the lengths ofClone 18 could be applied to the immunoblot analysis, and clone 21

the major and the minor axes, respectively. Tumors were fixed andcould be applied to immunofluorescence and IHC. Anti-­THG-­1 rabbit

embedded in paraffin, and the sections were stained with indi-

serum was obtained by the immunization of peptide (human THG-­1

cated antibodies.

amino acid 176–­193: PTSPG​PQ A​RSF​TGG​LGQ L plus C-­terminal cysteine) (Sigma-­Aldrich). The rabbit serum was affinity-­purified using
the SulfoLink immobilization kit (Thermo Fisher Scientific).

2.6 | Immunohistochemistry

2.3 | DNA constructs and transfection

ness, deparaffinized in xylene, rehydrated in graded ethanol solu-

The paraffin-­embedded tissues were sectioned at 2-­to 5-­μm thicktion, and immersed in citrate-­NaOH buffer (10 mM sodium citrate,

cDNAs of TSC-­22, GILZ, THG-­1, HRas, and CD44v3–­10 werepH 6.0) for 20 min at 115°C, 121 kPa. After blocking with Super-cloned by polymerase chain reaction (PCR) and cloned into pcDNA,

Block T20 (Thermo Fisher Scientific, Inc.), sections were incubated

pcDEF3, pPyCAG-­IB (provided by Dr. Niwa), and pCAG-­IP27 vectors.

with anti-­THG-­1 (4G7), phospho-­THG-­1(pS264) (clone 21), Ki67, and

The THG-­1 and HRas mutants were generated using a PCR-­based

CD44v9 (RV3) overnight at 4°C and then treated with the EnVision+

method. cDNA of KIAA0669 was obtained from RIKEN BRC. cDNAs

kit for mouse (Agilent Technologies Inc.) or Histofine reagent for ratof constitutively active form of ERK2 (L73P/S151D) were provided

IgG (Nichirei biosciences, Inc.) for 30 min. Color was developed using

by Dr. Fukamizu. 28 These constructs were transfected into cells by

3,3′-­diaminobenzidine tetrahydrochloride (DAB; Agilent Technolo-

FuGENE6 (Roche Diagnostics). HaCaT cells stably expressing FLAG-­

gies Inc.). Counterstaining was performed with hematoxylin (FUJI-

THG-­1 (WT and mutants) and HA-­HRasG12V were selected and main-

FILM Wako Pure Chemical Corporation).

tained in the presence of 1 μg/mL of puromycin (Sigma-­Aldrich) and4 μg/mL of Blasticidin S (FUJIFILM Wako Pure Chemical).

2.4 | RNAi

2.7 | Specimens and tissue microarrays
Tissue microarrays of tumors of the esophagus (124 cases: 121 SCCs
and 3 adenocarcinomas), the lung (114 cases: 34 SCCs, 58 adenocar-

RNAi-­mediated knockdown of THG-­1 was performed by using thecinomas, 10 large cell carcinomas, and 12 small cell carcinomas), thepSUPER RNAi system (OligoEngine) and pLKO.1 (for lentivirus, Ad-

cervix uteri (120 SCCs, 8 adenosquamous carcinomas and 10 adeno-

dgene). The 19-­bp targeting sequence was 5’-­GGACG​TGT​GTG​GAT​

carcinomas), and the stomach (20 cases: 1 SCC and 19 adenocarci-

GTTTA-­3′ for shTHG-­1#2,

nomas) were obtained from Cybrdi.

5’-­GAAGC​C TG​GTT​GGC​ATTGA3’ for shTHG-­1#3,
5’-­CCATC​AGT​GGT​C AC​C TAGA-­3′ for shTHG-­1#4,

2.8 | Semiquantitative RT-­PCR

and 5’-­GCGCG​CTT​TGT​AGG​ATTCG-­3′ for the nontargeting control.
TE13 cells transfected with pSUPER.puro-­shTHG-­1#2, pSUPER.puro-­

Total RNA was isolated using Isogen (Nippon Gene), and cDNA wasshTHG-­1#3, and pSUPER.puro-­control were selected and maintained insynthesized from PrimeScript RT Master Mix (TaKaRa). Semiquanti-

the presence of 1 μg/mL of puromycin. For lentivirus-­mediated THG-­1tative PCR was performed using TaKaRa Ex taq (TaKaRa). The PCRknockdown, control, #2, and #4 sequences were cloned into pLKO.1,

primers to amplify both CD44s and CD44v,18 and β-­actin21 were de-and the lentiviruses were generated in HEK293T cells.

scribed previously.

TE13 shTHG-­1#2 cells were transfected with pcDNA3 or
pcDNA3-­CD44v3–­10 vectors, and stable cell lines were generated
in the presence of 0.8 mg/mL of G418 (Nacalai Tesque, Inc.).

2.5 | Tumor xenograft formation in mice

2.9 | Statistical analysis
All data are expressed as mean ± standard deviation (SD). Statisticalanalysis was conducted with ANOVA and Sidak's multiple comparisons test. All calculations were performed using GraphPad Prism 8

For the xenografts of TE13 cells, 1 × 107 cells were subcutane-

ously inoculated into NOD.CB17-­Prkdc scid/NCrCrl (NOD-­S cid)

(GraphPad Software, Inc.). A p-­value of <0.05 was considered statistically significant.

13497006, 2023, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.15934 by Cochrane Japan, Wiley Online Library on [05/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License3974

Other methods of protein-­based assay (immunoblot analysis

THG-­1 was observed in esophageal SCC (positive THG-­1 expres-and dephosphorylation assay), cell-­based assays (cell proliferation

sion in 112 of 121 specimens [92.6%]) and cervical SCC (positive

assay, three-­dimensional [3D] culture assay, sphere formation assay,

THG-­1 expression in 96 of 120 specimens [80%]) (Figure 1A).

immunofluorescence, and flow cytometry), and the supplemental

Moreover, THG-­1 expression was observed in lung SCC (23 of

figures are available in the supplemental materials and methods in

34 specimens [67.6%]), but not in normal lung epithelium, ad-Appendix S1.

enocarcinoma, and small cell carcinoma (Figure 1B). THG-­1 wassporadically expressed in large cell lung carcinoma (Figure 1B).

3|R E S U LT S

3.1 | Distribution of THG-­1 in normal andcarcinoma tissues
To investigate the roles and functions of THG-­1 , we examined the

Furthermore, THG-­1 expression was observed in gastric SCC,
but not adenocarcinoma (Figure 1C). THG-­1 was mainly distributed in the cytoplasm and/or accumulated on cell–­c ell contact
sites.

3.2 | Tumorigenic functions of THG-­1

tissue distribution by IHC. Using a specific anti-­T HG-­1 antibody(Figure S1A), we found that THG-­1 was localized in the basal

To investigate the function of THG-­1 in SCC, we established THG-­

layer of stratified squamous epithelium including the forestom-

1-­k nockdown cells using two independent shRNAs (#2 and #3) inach, skin, and hair follicle epithelium of mice (Figure S1B). We

esophageal SCC cell line TE13 cells (Figure 2A). We first foundnext investigated THG-­1 expression in human squamous epithe-that cell proliferation was reduced in THG-­
1-­
k nockdown cells

lium and SCCs. THG-­1 is localized in the basal layer of normal(Figure 2B). THG-­1 knockdown also inhibited cell proliferation inesophagus and uterine cervix mucosa, and diffuse expression offive out of six human esophageal SCC cell lines (Figure S2). We

F I G U R E 1 Distribution of THG-­1
in normal stratified epithelium andsquamous cell carcinomas (SCCs).
(A) Immunohistochemistry of THG-­
1 in normal esophagus, esophageal
SCC, normal cervix uteri, and cervical
SCC. (B) Immunohistochemistryof THG-­1 in normal lung and lung
SCC, adenocarcinoma, large cell
carcinoma, and small cell carcinoma.
(C) Immunohistochemistry of THG-­1 ingastric SCC and adenocarcinoma. Scalebars: 100 μm.

13497006, 2023, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.15934 by Cochrane Japan, Wiley Online Library on [05/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License| 3975

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F I G U R E 2 Functions of THG-­1 incell invasiveness and tumorigenicity.
(A) Anti-­THG-­1, E-­C adherin, and β-­
Actin immunoblot analyses of TE13
cells expressing shRNAs targeting
THG-­1 (shTHG-­1#2 and shTHG-­1#3)
or control shRNA. (B) Effect of THG-­1knockdown on cell proliferation. Error
bars represent means ± SDs. (C, D) Effectof THG-­1 knockdown on cell invasion
into the collagen gels in 3D culture assay.
Scale bars: 50 μm. Error bars representmeans ± SDs. **p < 0.01. (E, F) Effect of
THG-­1 knockdown on tumor formationin NOD-­Scid mice (scale bar: 1 cm). Error
bars represent means ± SDs. **p < 0.01.
Immunohistochemistry of THG-­1 in
xenograft tumors of TE13 cells (control,
shTHG-­1#2, and shTHG-­1#3) (scale bars:
50 μm).

next investigated the THG-­1 function using a 3D culture assay,

epithelium as shown in Figure 2C. Control cells formed stratified epi-

in which air exposure promotes the stratification of SCC cells on

dermoid structure, and differentiated flattened cells were observed

the collagen gel as well as the invasion into the gels in the pres-on the surface. However, the differentiated cells were not observed

ence of EGF. 29 The invasion of TE13 cells into the gels was signifi-

on the surface of the HaCaT-­THG-­1 cells (Figure 3C). Furthermore,cantly reduced in THG-­1-­k nockdown cells (Figure 2C,D). Finally,

the proliferation marker Ki67-­positive cells was increased from 13%

we investigated the role of THG-­1 in tumor formation in NOD-­

to 60% with spreading over two-­thirds of the layers of the THG-­

Scid mice. As shown in Figure 2E,F, the tumor-­forming ability was1-­transfected epithelium (Figure 3D).EGF-­
induced cell migration

significantly reduced by THG-­1 knockdown. These results indi-

and invasion were observed in TE13 cells, which were suppressedcated that THG-­1 is overexpressed in SCC and has critical roles in

by THG-­1 knockdown (Figure S3A,B). These results indicated thattumorigenic ability.

overexpression of THG-­1 promotes EGF-­induced proliferation andinhibits differentiation during the stratified epithelium formation.

3.3 | Effect of THG-­1 in EGF-­mediatedproliferation and stratified epithelium formation

3.4 | Phosphorylation of THG-­1 by the
RTK pathway

We examined the effect of overexpression of THG-­1 in human keratinocyte HaCaT cells (Figure 3A). When the cells were cultured in

We next investigated the effect of THG-­1 on EGF signaling using HaCaT-­

the presence of EGF in 0.1% FBS condition, the HaCaT-­THG-­1 cells

THG-­1 cells. As shown in Figure S4A, THG-­1 overexpression did not(clones 3 and 7) formed foci and showed increased cell proliferation

affect EGF signaling including EGF-­induced phosphorylation of EGFR,

compared with the mock-­transfected control cells (Figure 3B). We

ERK, and Akt and the induction of JunB and c-­Myc. However, slowly

next investigated the effect of THG-­1 on the formation of stratified

migrated bands of THG-­1 were induced by EGF, suggesting that EGF

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3976

F I G U R E 3 Effect of THG-­1 inepidermal growth factor (EGF)-­induced
proliferation and stratified epithelium
formation. (A) Anti-­THG-­1, FLAG, andβ-­Actin immunoblot analyses of HaCaT
cells expressing FLAG-­THG-­1 (clone 3
and clone 7) or control (mock). (B) Effectof THG-­1 on cellular proliferation andmorphology in the presence of 50 ng/mL
of EGF (in 0.1% FBS). Error bars representmeans ± SDs. (C) Effect of THG-­1 on
the formation of stratified epitheliumon collagen gels in 3D culture assay.
Morphology of stratified epithelium incontrol and THG-­1-­transfected HaCaT
cells. Sections were stained with anti-­
Ki67 antibody, and Ki67-­positive cells
were counted (D). **p < 0.01. Error bars
represent means ± SDs. Scale bars: 50 μm.

signaling modifies THG-­1 in a post-­translational manner. To confirm thewith constitutively active HRasG12V and ERK2 (L73P/S151D) (Fig-nature of the slowly shifted THG-­1, we treated the immunoprecipitatedure S5B,C), suggesting that THG-­1 S264 is one of the important amino

THG-­1 with lambda protein phosphatase. As a result, the EGF-­induced

acids to be phosphorylated by the EGF-­Ras-­ERK pathway. We next

slowly shifted THG-­1 was completely eliminated (Figure 4A), indicating

established monoclonal antibodies (mAbs) which recognize phos-that THG-­1 is phosphorylated by EGF. The phosphorylation of THG-­1phorylated THG-­
1 (pS264). An anti-­phospho-­THG-­1(pS264) mAb

was inhibited by AG1478 (an EGFR inhibitor) and PD98059 (a MEK(clone 18) recognized only the slowly migrated form of THG-­1 in theinhibitor), but not by LY294002 (a PI3 kinase inhibitor) (Figure S4B).

presence of HRasG12V and ERK2 (L73P/S151D) (Figure S6A), and in

THG-­1 phosphorylation was also induced in THG-­1 cotransfected withEGF-­treated HaCaT-­THG-­1 cells (Figure S6B). Furthermore, the an-constitutively active HRasG12V (Figure 4B). Furthermore, THG-­1 phos-

tibody also recognized the endogenously phosphorylated THG-­1 inphorylation was also induced by the constitutively active form of ERK2

the presence of EGF (Figure 4D). In immunofluorescence analysis(L73P/S151D), but not by that of Akt (myr Akt) (Figure S4C).

(Figure 4E), THG-­1 was detected diffusely in the cytoplasm in the

To identify the phosphorylation site(s) of THG-­1, we focused on

absence of EGF. EGF stimulation changed the localization of THG-­1

the conserved P-­X-­S/T-­P sequence, which is known to be phosphor-at the ruffling membranes. Another anti-­phospho-­THG-­1(pS264)

ylated by ERK,

30

264

and found that S

corresponds to the ERK con-mAb (clone 21) could detect phosphorylated THG-­1 in both cyto-

sensus sequence (Figure S5A). Therefore, we transfected the THG-­1

plasm and ruffling membranes only in the presence of EGF. Finally,(WT) and THG-­1 (S264A) constructs into cells and stimulated them

we stained the esophageal SCC sections with the anti-­phospho-­with EGF. As shown in Figure 4C, THG-­1 (S264A) was not phos-

THG-­1 (pS264) antibody (clone 21) and found that the THG-­1 phos-

phorylated by EGF. Similar results were obtained by cotransfection

phorylation was detected in those tumors (Figure 4F).

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F I G U R E 4 Phosphorylation of THG-­1 by the epidermal growth factor (EGF)-­R AS-­ERK pathway. (A) Phosphorylation of THG-­1 by EGF.
HaCaT-­FLAG-­THG-­1 cells were treated with EGF. Immunoprecipitated THG-­1 was incubated with or without lambda protein phosphatase.
Immunoblot analysis was performed using anti-­FLAG antibody. The arrow indicates the phosphorylated THG-­1. (B) THG-­1 is phosphorylated
by constitutively active HRasG12V, but not by wild type (WT). HEK293T cells were transfected with FLAG-­THG-­1 with HRasWT and HRasG12V.
Immunoblot analysis was performed using anti-­FLAG and HA antibodies. (C) S264 is an essential phosphorylation site of THG-­1. HEK293T
cells were transfected with FLAG-­THG-­1 (WT and S264A). The cells were treated with 50 ng/mL of EGF, and immunoblot analysis was
performed using anti-­FLAG, pERK, and ERK1/2 antibodies. (D) Using anti-­phospho-­THG-­1(pS264) mAb (clone 18), phosphorylated THG-­1 wasdetected by immunoblot analysis in TE13 cells. (E) The phosphorylation of THG-­1 was detected by immunofluorescence in HaCaT-­HA-­THG-­1cells (scale bars: 20 μm) using anti-­phospho-­THG-­1(pS264) mAb (clone 21). Anti-­HA antibody detected HA-­THG-­1. (F) The phosphorylationof THG-­1 was detected by immunohistochemistry in esophageal SCC using anti-­phospho-­THG-­1(pS264) mAb (clone 21) and anti-­THG-­1antibody. Scale bars: 100 μm.

3.5 | Requirement of THG-­1 phosphorylation inRas-­mediated tumorigenesis(WT) compared with HRasG12V. In contrast, THG-­1 (S264A) dominantly suppressed HRasG12V-­m ediated tumor formation. Furthermore, the stratified Ki67-­p ositive regions were observed at the

To investigate the requirement of THG-­
1 phosphorylation foredge of HRasG12V-­THG-­1 (WT) tumors. In contrast, a few layered

G12V

Ki67-­p ositive regions were detected in HRasG12V-­THG-­1 (S264A)

tumorigenesis, we established HaCaT cells expressing HRas,

HRasG12V-­THG-­1 (WT), and HRasG12V -­THG-­1 (S264A) (Figure 5A).

tumors (Figure 5C). These results indicated that THG-­
1 phos-We inoculated them into nude mice. As shown in Figure 5B, thephorylation is essential for the enhancement of Ras-­m ediatedtumor growth was significantly promoted in HRasG12V-­THG-­1tumorigenesis.

13497006, 2023, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.15934 by Cochrane Japan, Wiley Online Library on [05/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

3978

F I G U R E 5 Requirement of THG-­
1 phosphorylation in Ras-­mediatedtumorigenesis. (A) Anti-­phospho-­THG-­
1(pS264) mAb (clone 18), FLAG, HA, and β-­
Actin immunoblot analyses of HaCaT cells
expressing HRasG12V, HRasG12V-­THG-­1(WT), and HRasG12V-­THG-­1 (S264A) (B)
Tumor formation in nude mice. Error bars
represent means ± SDs. **p < 0.01. (C) The
sections were stained with anti-­THG-­1
and Ki67 antibodies. Scale bars: 100 μm.

3.6 | Regulation of EGF-­induced CD44 variant
inclusion by THG-­1

Figure 6D and found that they were identified as v3–­10, v6–­10, andv8–­10 isoforms. We next used anti-­CD44v9 antibody, which recognizes the variant exon 9-­encoded sequence, and confirmed that the

To investigate the THG-­1 functions in SCC development in detail, we

induction of CD44v9 by EGF was reduced in THG-­1-­knockdown cells

performed RNA-­sequence analysis and found that CD44, an impor-in flow cytometry (Figure 6E) and immunoblot analysis (Figure S7B).

tant marker for cancer stem cell and tumor invasiveness, is down-

Furthermore, the recovery of CD44v3–­10 cDNA into the THG-­1-­

regulated by THG-­1 knockdown (Figure S7A). We confirmed thatknockdown cell line (TE13 shTHG-­1 #2) significantly promoted thethe expression of CD44 was decreased in THG-­1-­knockdown cells

sphere-­forming ability (Figure 6F,G). These results indicated that theusing an anti-­pan-­CD44 antibody, which recognizes both CD44s and

THG-­1-­CD44v axis is essential to confer the tumorigenic potential

CD44v (Figure 6A). As shown in Figure 6B, CD44s and CD44v are

to SCC cells.

produced by alternative splicing. Using a primer set which amplifies
both CD44s and CD44v isoforms (Figure 6B), we performed semiquantitative RT-­PCR to determine the difference of CD44 isoforms
in control and the THG-­1-­knockdown cells. As shown in Figure 6C,

3.7 | Regulation of CD44 variant splicing by
THG-­1-­knockdown and -­overexpressed tumors

the CD44v transcript (~1000 bp) was reduced in THG-­1-­knockdown
cells. Since ERK signaling is known as an inducer of CD44v transcription,

31

We next investigated the CD44 splicing in tumor sphere-­forming

we investigated the CD44v transcription in the presence ofcondition. In THG-­1-­knockdown cells, we could observe the reduced

EGF. EGF stimulation promoted the CD44v transcriptions in con-sphere forming ability in the presence of EGF and bFGF (Figure S8A).

trol cells, which was not observed in THG-­1-­knockdown cells (Fig-In this condition, the CD44v transcription was also reduced in THG-­

ure 6D). Furthermore, we sequenced three major bands as shown in1-­knockdown cells (Figure 7A). Furthermore, IHC showed that both

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GOTO et al.

F I G U R E 6 Regulation of epidermal
growth factor (EGF)-­induced CD44
variant splicing by THG-­1. (A) Flowcytometry using anti-­Pan-­CD44
antibody conjugated with PE in TE13
cells expressing shRNAs targeting THG-­1(shTHG-­1#2 and shTHG-­1#3) or control
shRNA. (B) Schematic illustration of
CD44s and CD44v. Primers to amplify
CD44s and CD44v by semiquantitative
RT-­PCR are indicated (arrows). (C)Semiquantitative RT-­PCR analysis of
CD44s and CD44v in control and THG-­
1-­knockdown cells in 10% FBS condition.
(D) Semiquantitative RT-­PCR analysis
of CD44s and CD44v in the presenceor absence of 50 ng/mL of EGF (in 0.1%
FBS) for 48 h. The indicated bands wereidentified as CD44v3–­10, v6–­10, andv8–­10 by sequence analysis. (E) Cells
were cultured in the presence or absenceof 50 ng/mL of EGF (in 0.1% FBS) for
48 h. Flow cytometry was performedby anti-­CD44v9 (RV3) antibody. (F) The
mock and CD44v3–­10-­trasfected THG-­1-­
knockdown cells (TE13 shTHG-­1 #2) weretreated with anti-­CD44v3 (C44Mab-­6) andanti-­CD44v9 (C44Mab-­1) antibodies andanalyzed using flow cytometry. (G) The
sphere number (left) was counted, and theviability (right) was determined using the
CellTiter-­Glo® 3D Cell Viability Assay inmock and CD44v3–­10-­trasfected THG-­
1-­knockdown cells. **p < 0.01. Error bars
represent means ± SDs.

CD44v9 (Figure 7B) and CD44v3 (Figure S8B) were hardly detected

and have common histological features such as keratin pearls. SCC

in THG-­1-­knockdown xenograft tumors. As shown in Figure S8C, theexhibited resistance to therapy and high invasiveness by potentiat-

THG-­1 (WT) could potentiate Ras-­mediated tumor sphere formation,

ing the property of normal squamous epithelium including strati-

but the THG-­1 (S264A) could not. In this condition, the transcription

fied epithelium formation to protect from stresses1 and migration

G12V-­THG-­1 (WT)-­expressing HaCaT

during wound closure, 32,33 respectively. In this study, we showed

cells (Figure 7C). Furthermore, HaCaT HRasG12V-­THG-­1 (WT) tu-

a novel mechanism connecting oncogenic RTK signaling and themors showed an enlarged CD44v9-­positive region compared with

SCC tumorigenesis by THG-­1. We also showed the involvementof CD44v was elevated in HRas

HRasG12V and HRasG12V-­THG-­1 (S264A) tumors (Figure 7D). Theseof the RTK-­THG-­1 axis to promote the inclusion of variant exons

results indicated that the RTK-­THG-­1 axis is involved in the inclusion

in CD44 mRNA.

of CD44 variant exons.

We first revealed the tumor-­promoting functions of THG-­1.
Its knockdown in SCC cells reduced the proliferation, invasive-

4|

DISCUSSION

ness, and tumor growth in immunodeficient mice (Figure 2). THG-­1
overexpression promoted EGF-­
induced proliferation, inhibited
differentiation during stratified epithelium formation (Figure 3),

In this study, we provided a novel mechanism of oncogene-­induced

and potentiated Ras-­
mediated tumorigenesis through phosphor-

SCC development through THG-­
1 phosphorylation. SCCs arise

ylation (Figures 4 and 5). We had also performed the knockout offrom stratified squamous epithelium and nonsquamous epithelia

THG-­1 in TE13 cells, which exhibited a severe growth retardation

13497006, 2023, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.15934 by Cochrane Japan, Wiley Online Library on [05/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

|

3980

F I G U R E 7 Regulation of CD44
variant splicing in THG-­1-­knockdown and-­overexpressed tumors. (A) Decreased
CD44v transcription in tumor sphere
formation of THG-­1-­knockdown cells.
(B) The xenograft tumor sections inFigure 2E were stained by anti-­CD44v9(RV3) antibody. Scale bars: 100 μm. (C)Increased CD44v transcription in HaCaT-­
HRasG12V-­THG-­1 (WT) cells comparedwith HRasG12V-­THG-­1 (S264A). (D) The
xenograft tumor sections in Figure 5B
were stained by anti-­CD44v9 (RV3)
antibody. Scale bars: 100 μm.

and senescent phenotypes,34 indicating that THG-­1 is essential for

not investigated the expression and phosphorylation of THG-­1 intumor proliferation and protects cellular senescence.

the abovementioned tumors. There is a possibility that THG-­1 medi-

Therapeutic strategies against the RTK pathway including anti-­
RTK mAbs35 and RTK36,37/RAS38,39/RAF40/MEK41 inhibitors haveates the malignant property of those carcinomas. Therefore, it could
be a future diagnostic marker to be considered.

been progressively developed, and their clinical trials for SCC have

We showed that THG-­1 is a regulator of CD44 splicing (Figure 6)

been conducted. We need reliable diagnostic biomarkers to predictand promotes cell proliferation in the presence of EGF (Figure 3). We

the efficacy and prognosis of patients. THG-­1 S264 is one of the amino

found that CD44v expression was decreased in THG-­1-­knockdown

acids that is phosphorylated by the RTK-­R AS-­ERK pathway, whichcells (Figure 6) and xenograft tumors (Figure 7). Previously, studies

plays crucial roles in RAS-­mediated tumorigenesis (Figure 5). The

showed that the inclusion of CD44 variant exon is activated by the264

can be detected by established

RAS-­R AF-­MEK-­ERK signaling cascade.31,44 The Src-­associated in mi-phosphorylation of THG-­1 at S
264

) mAb in IHC (Figure 4F). Therefore, thetosis 68 kDa protein (SAM68) enhanced ERK-­mediated inclusion ofphosphorylation of THG-­1 would be a useful biomarker for diag-anti-­phospho-­THG-­1(pS

the variant exon 5.31 Phosphorylated SAM68 bound to the variant

nosis to determine the sensitivity to RTK-­t argeted therapy in SCC.

exon 5 elements and interacted with SRm160, a splicing activator

Moreover, THG-­1 was also detected in gastric SCC (Figure 1C), a rareof pre-­mRNAs containing GAA repeats, which was necessary for

carcinoma type in stomach. Furthermore, squamous carcinoma or avariant exon 5 inclusion upon Ras activation.45 We also showed that42

squamous subtype is also observed in breastand pancreatic car-

THG-­1 is phosphorylated by the RTK-­Ras-­ERK pathway, and THG-­1

cinomas,43 which are rare but the most malignant tumors. We have

(S264A) mutant could promote neither Ras-­mediated tumorigenesis

13497006, 2023, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.15934 by Cochrane Japan, Wiley Online Library on [05/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License| 3981GOTO et al.

|

GOTO et al.

(Figure 5B) nor CD44v expression (Figure 7C,D). Therefore, phosphorylation of THG-­1 is important to induce CD44v expression.
CD44v potentiates RTK signaling as a coreceptor by recruiting
FGFs and HB-­EGF, which plays critical roles in tumor invasion andYukari Okita

https://orcid.org/0000-0002-7279-4634

Yukihide Watanabe
Yukinari Kato
Mitsuyasu Kato

https://orcid.org/0000-0003-1014-2910

https://orcid.org/0000-0001-5385-8201
https://orcid.org/0000-0001-9905-2473

stemness.46 CD44v also plays a critical role in regulation of reactive
oxygen species (ROS) defense and tumor growth.18 CD44v interacts

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AU T H O R C O N T R I B U T I O N S
All authors have contributed significantly and are in agreement withthe content of the manuscript.
AC K N O​W L E D​ G E ​M E N T S
We thank Ms. Yuko Nakayama and Ms. Yoko Miyake for technicalassistance.
F U N D I N G I N FO R M AT I O NThis work was supported by a JSPS KAKENHI Grant Number
22K06995, 19K07473, and 16K08706 (to H. S.), 22K15596 and
20K16434 (to L. Z), and 17K19580 and 25640059 (to M. K.).
C O N FL I C T O F I N T E R E S T S TAT E M E N T
The authors have no conflict of interest. Dr. Mitsuyasu Kato is anassociate editor of Cancer Science.
E T H I C S S TAT E M E N T
Approval of the research protocol by an Institutional Review Board:
N/A.
Informed consent: N/A.
Registry and the Registration No. of the study/trial: N/A.
Animal studies: Animal experiments were performed with the approval of the Animal Ethics Committee of the University of Tsukuba.
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https://orcid.org/0000-0003-2646-5132

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S U P P O R T I N G I N FO R M AT I O N
Additional supporting information can be found online in the
Supporting Information section at the end of this article.

How to cite this article: Goto N, Suzuki H, Zheng L, et al.
Promotion of squamous cell carcinoma tumorigenesis byoncogene-­mediated THG-­1/TSC22D4 phosphorylation.
Cancer Sci. 2023;114:3972-3983. doi:10.1111/cas.15934

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GOTO et al.