Journal of Pathology
J Pathol June 2022; 257: 172–185
Published online 23 March 2022 in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/path.5876

ORIGINAL ARTICLE

Phosphorylation of hTERT at threonine 249 is a novel tumor
biomarker of aggressive cancer with poor prognosis in multiple
organsYoko Matsuda1* , Taro Yamashita2, Juanjuan Ye1 , Mami Yasukawa3, Keiko Yamakawa1, Yuri Mukai1,
Mitsuhiro Machitani3, Yataro Daigo4,5,6, Yohei Miyagi7, Tomoyuki Yokose8, Takashi Oshima9, Hiroyuki Ito10,
Soichiro Morinaga11, Takeshi Kishida12, Toshinari Minamoto13, Shinji Yamada14, Junko Takei14, Mika K Kaneko14,
Motohiro Kojima15, Shuichi Kaneko2, Tsutomu Masaki16, Masahiro Hirata16, Reiji Haba17, Keiichi Kontani18,
Nobuhiro Kanaji19, Nobuyuki Miyatake20, Keiichi Okano21, Yukinari Kato14,22 and Kenkichi Masutomi3*
1

Oncology Pathology, Department of Pathology and Host-Defense, Faculty of Medicine, Kagawa University, Kita-gun, Japan
Department of Gastroenterology, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Japan
3
Division of Cancer Stem Cell, National Cancer Center Research Institute, Tokyo, Japan
4
Department of Medical Oncology and Cancer Center, Shiga University of Medical Science, Otsu, Japan
5
Center for Advanced Medicine against Cancer, Shiga University of Medical Science, Otsu, Japan
6
Center for Antibody and Vaccine Therapy, Research Hospital, Institute of Medical Science Hospital, The University of Tokyo, Tokyo, Japan
7
Kanagawa Cancer Center Research Institute, Yokohama, Japan
8
Department of Pathology, Kanagawa Cancer Center, Yokohama, Japan
9
Department of Gastrointestinal Surgery, Kanagawa Cancer Center, Yokohama, Japan10
Department of Thoracic Surgery, Kanagawa Cancer Center, Yokohama, Japan
11
Department of Hepato-Biliary and Pancreatic Surgery, Kanagawa Cancer Center, Yokohama, Japan
12
Department of Urology, Kanagawa Cancer Center, Yokohama, Japan
13
Division of Translational and Clinical Oncology, Cancer Research Institute, Kanazawa University, Kanazawa, Japan
14
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan
15
Division of Pathology, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Chiba, Japan
16
Department of Gastroenterology and Neurology, Faculty of Medicine, Kagawa University, Kita-gun, Japan
17
Diagnostic Pathology, Faculty of Medicine, Kagawa University, Kita-gun, Japan18
Department of Thoracic, Breast and Endocrine Surgery, Faculty of Medicine, Kagawa University, Kita-gun, Japan
19
Department of Internal Medicine, Division of Hematology, Rheumatology and Respiratory Medicine, Faculty of Medicine, Kagawa University, Kitagun, Japan20
Department of Hygiene, Faculty of Medicine, Kagawa University, Kita-gun, Japan
21
Department of Gastroenterological Surgery, Faculty of Medicine, Kagawa University, Kita-gun, Japan
22
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan
2*Correspondence to: Y Matsuda, Oncology Pathology, Department of Pathology and Host-Defense, Faculty of Medicine, Kagawa University, 1750-1
Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan. E-mail: matsuda.yoko@kagawa-u.ac.jp or K Masutomi, Division of Cancer Stem Cell, National
Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. E-mail: kmasutom@ncc.go.jp

Abstract
Recent evidence indicates that RNA-dependent RNA polymerase (RdRP) activity of human telomerase reverse transcriptase (hTERT) regulates expression of target genes and is directly involved in tumor formation in a telomereindependent manner. Non-canonical function of hTERT has been considered as a therapeutic target for cancer therapy. We have previously shown that hTERT phosphorylation at threonine 249 (p-hTERT), which promotes RdRP activity, is an indicator of an aggressive phenotype and poor prognosis in liver and pancreatic cancers, using two cohorts
with small sample sizes with polyclonal p-hTERT antibody. To clarify the clinical relevance of p-hTERT, we developed
a speciﬁc monoclonal antibody and determined the diagnostic and prognostic value of p-hTERT in cancer specimens
using a large cohort. A monoclonal antibody for phosphorylated hTERT (p-hTERT) at threonine 249 was developed
and validated. The antibody was used for the immunohistochemical staining of formalin-ﬁxed, parafﬁn-embedded
specimens from 1523 cases of lung, colon, stomach, pancreatic, liver, breast, and kidney cancers. We detected elevated p-hTERT expression levels in cases with a high mitotic activity, high pathological grade, and high nuclear pleomorphism. Elevated p-hTERT expression was an independent prognostic factor for lung, pancreatic, and liver cancers.
Furthermore, p-hTERT expression was associated with immature and aggressive features, such as adenosquamous
carcinoma (lung and pancreas), invasive type of cancer (lung), high serum alpha-fetoprotein level (liver), andtriple-negative status (breast). In conclusion, RdRP activity indicated by p-hTERT expression predicts aggressive cancer phenotypes in various types of cancer. Thus, p-hTERT is a novel biomarker for the diagnosis of aggressive cancers
with a poor prognosis.
© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of GreatBritain and Ireland.
© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited.

Phosphorylation of hTERT in cancers

173

Keywords: hTERT; phosphorylation; RdRP; pathology; poor prognosis
Received 27 August 2021; Revised 21 January 2022; Accepted 25 January 2022

No conﬂicts of interest were declared.

Introduction
Cancer causes about one in six deaths worldwide andits incidence is increasing [1]. The incidence, morbidity, and mortality of cancer vary substantially according to the sex of patients and the organ of origin [1].
Cancer is also heterogeneous in terms of biologicalbehavior, morphology, clinical responses to treatment, and prognosis, even for the same organ. Traditionally, this heterogeneity is considered to reﬂect
variation in accumulated somatic mutations that promote malignant transformation [2]. However, several
recent lines of experimental evidence indicate thatthe aged and cancer-prone phenotype might represent
the combined pathogenetic effects of the mutationload, epigenetic regulation, telomere dysfunction [3],altered stromal milieu [4], weakened stromal reaction,and decreased immune response against cancer
cells [5,6].
Telomeres are synthesized by telomerase, an enzyme
composed of the catalytic protein subunit human telomerase reverse transcriptase (hTERT) and an RNA component. hTERT activity is readily detected in normal
embryonic/pluripotent stem cells and in the majority ofhuman cancer cells, suggesting that it confers cell
immortality. However, the correlation between the enzymatic activity of hTERT and hTERT (TERT) expression
levels in cancer remains elusive [7]. Furthermore, there
is strong evidence for the importance of hTERT expression in cancer stem cells [8]. The cancer stem cellhypothesis predicts that a subset of tumor cells possesses
stem cell features in terms of self-renewal and differentiation capacity. Cancer stem cells are highly tumorigenic,
metastatic, resistant to treatment, and correlated withpoor prognosis in various solid tumors [9], and these features cannot be explained by the canonical function ofhTERT in the maintenance of telomeres. Speciﬁc antibodies against hTERT have been developed to evaluate
hTERT protein levels in clinical specimens [10,11].
However, no obvious correlation between hTERT expression levels and prognosis has been observed in immunohistochemical analyses of clinical samples [12,13].
We recently found that hTERT demonstrates RNAdependent RNA polymerase (RdRP) activity [14]. RdRP
is an enzyme that catalyzes the replication of RNA froman RNA template and is an essential protein of RNA
viruses. hTERT-RdRP activity generated dsDNAs thatare processed to siRNAs for the purpose of downregulating gene expression [14], and affected gene expression
by affecting RNA levels [15]. hTERT-RdRP is involvedin tumor formation in a telomere-independent manner[16]. The non-canonical function of hTERT has been
considered as an effective target for cancer therapy[17]. hTERT forms a complex with Brahma-related gene
1 (BRG1) and nucleostemin (NS) [15] and maintains
cancer stem cell properties via RdRP activity [18]. Furthermore, we recently demonstrated that hTERT is phosphorylated at threonine 249 (Thr249) by the serine/
threonine kinase CDK1, and this phosphorylation event
works as a molecular switch for the RdRP activity ofhTERT without affecting telomerase activity. hTERTRdRP activity was involved in the expression of various
genes such as Forkhead box O4 (FOXO4), a tumor suppressor gene, by preventing proper cell cycle. Most
importantly, the abrogation of hTERT phosphorylation
and RdRP activity signiﬁcantly inhibits tumorigenesis
in vivo by regulating the expression of FOXO4.
hTERT phosphorylation at Thr249 is correlated withpoor survival outcomes in pancreatic and liver cancers[16]. Moreover, eribulin mesylate, a speciﬁc inhibitorof hTERT-RdRP activity, inhibited ovarian cancer
cell growth in vitro [19] and glioblastoma cell growthin the subcutaneous and intracranial xenograft mouse[20]. The expression level of hTERT was correlated
to sensitivity for eribulin mesylate [19]. While eribulin was originally identiﬁed as an inhibitor of microtubular synthesis that is essential for cell division, andtherefore inhibition of hTERT-RdRP may affect the
microtubular network, these data reveal the value ofhTERT-RdRP for the diagnosis of cancer with a poor
prognosis and as a useful marker to predict responseto treatment.
Here, we successfully developed a mouse monoclonal
antibody that speciﬁcally recognizes hTERT phosphorylated at Thr249. This antibody provides the ﬁrst effective
tool for the visualization of hTERT-RdRP in cancer
using formalin-ﬁxed, parafﬁn-embedded tissues. Using
this antibody, we analyzed 1523 cancer specimens(including lung, colorectal, stomach, pancreatic, liver,breast, and kidney cancer specimens) to identify the clinicopathological characteristics of hTERT-RdRP-active
cancer.

Materials and methods
Generation of hybridoma-producing phosphospeciﬁc monoclonal antibody, TpMab-3
Details are provided in Supplementary materials andmethods. To generate a monoclonal antibody against
phosphorylated threonine 249 of hTERT (TpMab-3),© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org

J Pathol 2022; 257: 172–185
www.thejournalofpathology.com

Y Matsuda et al

174

hTERT phosphopeptide 244CEPERpTPVGQG254 wasused to immunize mice. Hybridoma was generated aspreviously reported [16]. Culture supernatants werescreened by enzyme-linked immunosorbent assay(ELISA) for the detection of the p-hTERT peptide andwild-type TERT peptide as previously reported [16].
Clone TpMab-3 (IgG1, kappa), which is speciﬁc for the
p-hTERT peptide, was ﬁnally established.

Immunoprecipitation–RdRP assay
Details are provided in Supplementary materials andmethods. hTERT was immunoprecipitated from humancell lines as described previously with an anti-hTERTmAb (clone 10E9-2) [15,16,21]. The sequence of the
RNA template was as follows: 5’-GGGAUCAUGUGGGUCCUAUUACAUUUUAAACCCA-3’.

Quantitative PCR
Cell culture and mitotic cell synchronization
The human cell lines used in the present study are listed
in supplementary material, Table S1. Cells were induced
to enter the mitotic phase for the enrichment of phosphohTERT and RdRP activity following a previously
described method [16,21]. In brief, cells were cultured
with standard medium, switched to medium containing
2.5 mM thymidine (Nacalai Tesque, Inc, Kyoto, Japan),and incubated for 24 h. Six hours after release, cells
were incubated in medium containing 0.1 μg/ml nocodazole (Sigma-Aldrich, St Louis, MO, USA) for 16 h.
Mitotic cells were retrieved by mitotic shake-off. Cells
arrested in mitosis with nocodazole were conﬁrmed byimmunoblotting using anti-phospho-histone H3(Ser10) antibodies.

siRNA transfection
HeLa cells were transfected with siRNAs using Lipofectamine 2000 (Thermo Fisher Scientiﬁc Inc, Waltham,
MA, USA) as previously reported [16]. After 48 h ofincubation, cells were treated with 0.1 μg/ml nocodazolefor 16 h. The sequences of siRNAs against hTERT wereas follows: TERT siRNA#1, GUGUCUGUGCCCGGGAGAATT; #2, GCAUUGGAAUCAGACAGCATT.
MISSION siRNA Universal Negative Control #1(Sigma-Aldrich) was used as a negative control.

Detection of phosphorylated hTERT by
immunoprecipitation
Details are provided in Supplementary materials andmethods. Anti-hTERT mouse monoclonal antibodies(clones 10E9-2 and 2E4-2) were generated, and thespeciﬁcity was evaluated as reported previously [15].
Anti-hTERT mouse mAb (clone 2E4-2) and Mouse
TrueBlot ULTRA: Anti-Mouse Ig HRP (Rockland, Gilbertsville, PA, USA) were used for immunoblotting todetect whole-hTERT proteins [21]. Anti-phosphohTERT mouse monoclonal Ab (clone TpMab-3) andMouse TrueBlot ULTRA: Anti-Mouse Ig HRP(Rockland) were used to detect phosphorylated hTERT.
For λ phosphatase treatment, the bead suspension withimmune complexes was treated with 2000 U of λ protein
phosphatase (λ-PPase) (Bio Academia, Osaka, Japan)
and 2 mM MnCl2 in λ-PPase reaction buffer [50 mM
Tris–HCl (pH 7.6), 100 mM NaCl, 2 mM DTT, 100 μM
EDTA, and 0.01% Brij 35] and incubated at 30  C for
30 min.

Total RNA was isolated from human cell lines using the
RNeasy Mini Kit (Qiagen, Hilden, Germany) and treatedwith the RNase-Free DNase Set (Qiagen). cDNAs weresynthesized from total RNA using SuperScript IV VILO
Master Mix (Thermo Fisher Scientiﬁc Inc), ampliﬁed by
PCR using the TaqMan Gene Expression Assay (TERT,
Hs00972650_ml; 18S rRNA, 4310893E, Thermo Fisher
Scientiﬁc), and analyzed with the StepOnePlus RealTime PCR System (Thermo Fisher Scientiﬁc).

Patients
The study was conducted in accordance with the principles embodied in the Declaration of Helsinki, 2013. All
experiments were approved by the ethics committeesof Kagawa University (permit number 2019-209), the
Human Genome/Gene Analysis Ethics Committee ofKanazawa University (approval No. 181), and Kanagawa Cancer Center (approval No. 177). Surgicallyresected tissues were obtained from consecutive caseseries that underwent surgical treatment at Kagawa
University Hospital (pancreas, between 2008 and2020; breast, 2005–2011) and Kanazawa University
Hospital (stomach, 2001–2009; liver, between 2005
and 2014). Tissue microarray (TMA) specimens wereobtained from the National Cancer Center Hospital
East (liver, between 2010 and 2019), Kanazawa University Hospital (colon, between 1997 and 2005),
and Platform of Supporting Cohort Study and Biospecimen Analysis (http://cohort.umin.jp/english/index.
html) (lung, stomach, pancreas, kidney). All tumors
were taken at diagnosis. We adhered to the REMARK
criteria [22].

Tissue processing
For preparing whole slides, formalin-ﬁxed and parafﬁnembedded (FFPE) tissues from the invasive andadvanced lesion which showed representative pathological histological features of tumors were used. TMA
specimens were made from the archives of FFPE tissues
used for routine histopathologic diagnosis at the pathology department of Kanagawa Cancer Center. In brief, an
experienced genitourinary pathologist centrally reviewed
the hematoxylin and eosin (H&E)-stained slides andmarked the areas to be punched out for a TMA. Subsequently, FFPE tissue cores (2 mm in diameter) corresponding to the marked areas on the H&E slides were obtainedusing manual tissue microarrayers (KIN-3 model;
AZUMAYA Inc, Tokyo, Japan/Mini Core; ALPHELYS,
Plaisir, France). For each case, two representative,© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org

J Pathol 2022; 257: 172–185
www.thejournalofpathology.com

Phosphorylation of hTERT in cancers

independent tumor areas and a non-neoplastic cortical area
were processed. Pathology specimens were used for lungcancer (TMA, n = 342), colon cancer (TMA, n = 117),
stomach cancer (whole slide, n = 80; TMA, n = 122),
pancreatic cancer (whole slide, n = 53; TMA, n = 199),
liver cancer (whole slide, n = 194; TMA, n = 199), breast
cancer (whole slide, n = 85), and kidney cancer (TMA,n = 132) (supplementary material, Table S2). [16]. Wholeslides of liver cancer (n = 194) were an expansion of thecohort published previously [16], and pancreatic cancer(n = 252) and TMA of liver cancer (n = 199) were an
independent group of previous cohorts.
The tissues were sliced serially into sections (3 μm
thick) for H&E and immunohistochemical staining andﬂuorescence in situ hybridization (FISH). Pathological
specimens were diagnosed by our pathologists basedon the World Health Organization Classiﬁcation of
Tumours [23–26]. Immunohistochemical staining was
performed for the detection of p40 (prediluted; Nichirei
Bioscience Inc, Tokyo, Japan), p63 (7JUL, prediluted;
Leica Biosystems, Wetzlar, Germany), cytokeratin 5/6(D5/16B4, prediluted; Agilent Technologies, Inc, Santa
Clara, CA, USA), napsin A (IP64, 1:100 dilution; Leica
Biosystems), and TTF1 (8G7G3/1, 1:50 dilution; Agilent Technologies, Inc) to distinguish adenocarcinoma
from squamous cell carcinoma for all lung cancer specimens. Immunohistochemical staining of estrogen receptor (6F11, prediluted; Leica Biosystems), progesterone
receptor (16, prediluted; Leica Biosystems), and HER2
(CB11, prediluted; Leica Biosystems) was performed
for all breast cancer cases to identify triple-negative
breast cancer. The pathological stage was diagnosed
based on the TNM Classiﬁcation of Malignant Tumors,
7th edition [27].

Immunohistochemical staining for phosphorylatedhTERT and Ki67
Tissue specimens were immunostained using the Ventana Discovery Staining System (Roche, Basel,
Switzerland) and the DISCOVERY ChromoMap DAB
Kit (Roche) according to the manufacturer’s instructions. The tissue sections were preheated with CC1(pH 9.0, Roche) for 30 min at 100  C. They were thenincubated with the mouse monoclonal anti-p-hTERT
antibody (1:500 dilution) or Ki67 (MIB1, 1:100 dilution;
Agilent Technologies, Inc) for 12 h at 25  C. Incubation
with OmniMap anti-mouse HRP-conjugated multimer
secondary antibody (Roche) was performed for 32 min
at 25  C. We performed immunohistochemical stainingof mouse IgG1 (isotype control; Abcam, Cambridge,UK) under the same conditions as p-hTERT and Ki67
staining, and conﬁrmed negative staining. The proportion of cancer cells with positively stained nuclei wasanalyzed at 200 by the authors (YMa, JY, KY, or
TY) [16]. The percentage of p-hTERT and Ki67 expression was scored every 10% as follows: 0–9%, 0; 10–19%, 10; 20–29%, 20; and so on.

175

Pathological assessment
The authors (YM, JY, or KY) reviewed H&E-stained
specimens for pathological assessments. Mitotic counts
in 10 high-power ﬁelds (HPF) were obtained using
H&E-stained specimens at a magniﬁcation of 400.
The mitotic count was scored based on the WHO classiﬁcation of breast tumors (ﬁeld diameter, 0.54 mm; score1, ≤8 mm; score 2, 9–16 mm; score 3, ≥17 mm) [23].
Ki67 immunostaining was performed to evaluate proliferative activity using breast cancer specimens to conﬁrm
the relationship between the mitotic count and Ki67
index.
Pathological grade was based on the TNM Classiﬁcation of Malignant Tumors, 7th edition (G1, well differentiated; G2, moderately differentiated; G3, poorly
differentiated; G4, undifferentiated; GX, cannot be
assessed) [27].
The nuclear score was assessed at 200 magniﬁcation
based on the WHO classiﬁcation of breast tumors (score
1, small, regular, uniform cells; score 2, moderateincrease in size and variability; score 3, marked variation) [23].

Quantitative ﬂuorescence in situ hybridization for
the analysis of telomere length
Slides were processed by FISH, as previously reported[28]. In brief, tissue sections were hybridized with
200 nM PNA probes for the telomere (telo C-Cy3 probe,5’-CCCTAACCCTAACCCTAA-3’; Panagene, Daejeon,
Korea) and the centromere (Cenp1-FITC probe, 5’CTTCGTTGGAAACGGGGT-3’; Panagene) for 3 min
at 80  C and then for 1 h at room temperature. The nuclei
were stained with DAPI (Molecular Probes, Eugene, OR,
USA). FISH images were captured using a ﬂuorescencemicroscope (FSX100; Olympus, Tokyo, Japan) at 800
magniﬁcation.
ImageJ (version 1.53a, Wayne Rasband, National
Institutes of Health, Bethesda, MD, USA; modiﬁed by
the plug-in AsKey, Kagawa, Japan) was used to estimate
the red, green, and blue intensities of individual nuclei.
As an entire nucleus will not necessarily be captured
within any given tissue section, the total corrected telomere signal for each nucleus was normalized by the corresponding integrated optimal density of the centromere
as the telomere/centromere ratio. Over 100 cells wereanalyzed for each sample. As a control for variation insample preparation, FISH was also performed on sections of a block preparation of a cultured cell strain,
HFL-1 (with a population doubling level of 20). The normalized telomere signals for each case were calculated asfollows: [median value of telomere/centromere ratio of target cells]/[median value of telomere/centromere ratio ofcontrol HFL-1 cells]. Patients were divided into two groups
based on [normalized telomere length of cancer cells]/[normalized telomere length of ﬁbroblasts], with a cut-off
value of 1.2 to obtain cases with short telomeres (under1.2) and cases with long telomere lengths (over 1.2).

© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org

J Pathol 2022; 257: 172–185
www.thejournalofpathology.com

Y Matsuda et al

176

Statistical analysis
Two groups were compared by the unpaired t-test, χ
test, and Fisher’s exact test. Three or more groups werecompared by an analysis of variance (ANOVA) andTukey’s test. Correlations were assessed by Pearson’sand Spearman correlation coefﬁcients. Survival was
analyzed by Kaplan–Meier curves and a Cox proportional hazard model. Univariate and multivariate analyses were performed using sex, age (cut-off value:
65 years old), p-hTERT expression (cut-off value:
median), and TNM stage. The level of signiﬁcance wasset at p < 0.05 for all analyses. Statistical analyses were
performed using JMP Pro 14 (SAS Institute Inc, Cary,
NC, USA) and Statistical Package for the Social Sciences version 22 (IBM Corp, New York, NY, USA).
2

Results
Characterization and validation of the mAb speciﬁc
for phosphothreonine 249 of hTERT
To investigate the clinical signiﬁcance of phosphothreonine 249 of hTERT, we generated a monoclonal antibody(TpMab-3) directed against hTERT phosphorylated at
Thr249. In combination with validated monoclonal antibodies speciﬁc for total hTERT proteins (clones 10E9-2
and 2E4-2) [15], we comprehensively validated the speciﬁcity of TpMab-3 by a standard validation method [16].
In brief, we performed immunoprecipitation followed byimmunoblotting (Figure 1A), treatment with λ-phosphatase
to conﬁrm that the signals are phosphothreonine-speciﬁc(Figure 1B), and an in vitro kinase assay reconstituted by
CDK1–cyclin B proteins with two different versions ofrecombinant hTERT proteins [hTERT amino acids
191–306 [16] (Figure 1C) and full-length hTERT protein[29] (Figure 1D)]. In addition, as a reciprocal experiment,
we further validated the speciﬁcity of TpMab-3 by treatment with the CDK-1 inhibitor RO-3306 (Figure 1E).
The suppression of hTERT by siRNAs speciﬁc for hTERT[18,29] further conﬁrmed that the signals are hTERT-speciﬁc(Figure 1F). More speciﬁcally, once we had suppressed
hTERT by siRNAs speciﬁc for hTERT [18,29] followed by
IP–IB, we were unable to detect hTERT signal by
TpMab-3, indicating that the signals are hTERT-speciﬁc.
Taken together, these data indicate that the signals are speciﬁc
for phosphothreonine 249 of hTERT protein. Given that thephosphorylation of Thr249 is necessary for hTERT-mediated
RdRP activity, we monitored whether TpMab-3 is able torecover RdRP activity in vitro (Figure 1G). In each case,
TpMab-3 identiﬁed phosphothreonine 249 (p-hTERT),
conﬁrming the sensitivity and speciﬁcity of the antibody. Thus, we concluded that TpMab-3 is effective
for immunohistochemical analyses. TpMab-3 revealed
expression in nuclei of HeLa cells but not in normalﬁbroblast BJ cells (Figure 1H). In addition, in several
human cell lines, we found that the level of p-hTERT
was associated with the mRNA level of hTERT (p =0.0063, R = 0.651; Figure 1I).

Phosphorylation of hTERT at threonine 249 occurs invarious cancers
The expression of p-hTERT (Thr249) was detected incancer cell nuclei (Figure 2A–G). The frequencies of
p-hTERT-positive cancer cells varied depending on cancer type and organ (Figure 2H). No positive associations
were found between the expression of p-hTERT andTNM stage (supplementary material, Figure S1), age(supplementary material, Figure S2), or sex (supplementary material, Figure S3).
Next, we analyzed the link between p-hTERT expression and pathological characteristics typically associatedwith aggressive phenotypes in cancer, including mitosis
score, pathological grade [30], and nuclear score[23,27,31] (Figure 3A–C and supplementary material,
Table S2). Populations of p-hTERT-positive cancer cells
exhibited a positive association with mitosis score incancers of the lung (p < 0.0001), colon (p = 0.0068),
stomach (p < 0.0001), pancreas (p = 0.0010), and liver(p < 0.0001) (Figure 3A). We conﬁrmed that the numberof mitosis per 10 HPF was positively correlated to theKi67 index (p < 0.0001; R = 0.373; supplementarymaterial, Figure S4).
p-hTERT expression increased as the pathologicalgrade increased in cancers of the lung (p < 0.0001), pancreas (p < 0.0001), and liver (p = 0.0005; Figure 3B).
We observed elevated p-hTERT expression in patientswith a high nuclear score in cancers of the lung(p < 0.0001) and liver (p < 0.0001; Figure 3C). These
results indicate that p-hTERT was highly expressed incancers with aggressive phenotypes, such as cancers
with high proliferative activity, a high pathologicalgrade, and severe nuclear atypia. The phosphorylation
event at threonine 249 is essential for RdRP activity
but does not affect telomerase activity, and we havereported that phosphorylation is not involved in telomere
maintenance in several cancer cell lines [16]. To validate
this result using clinical specimens, we evaluated thecorrelation between p-hTERT expression and telomere
length, as determined by FISH, in 1399 cancer specimens. In lung and liver cancers, p-hTERT expression
levels were higher in tissue sections with long telomeres
than in sections with short telomeres (lung, p = 0.0172;
liver, p = 0.0259; Figure 3D).
Univariate Cox regression analysis indicated that highp-hTERT expression was associated with short overall
survival in cancers of the lung [hazard ratio (HR) 0.58,
95% conﬁdence interval (CI) 0.36–0.93, p = 0.024],
pancreas (HR 0.69, 95% CI 0.5–0.94, p = 0.021), andliver (HR 0.49, 95% CI 0.27–0.85, p = 0.019;
Figure 4A and Table 1). Multivariate Cox regression
analysis indicated that high p-hTERT expression and
TNM stage are independent risk factors for short overall
survival in lung, pancreatic, and liver cancers (Table 1),
and disease-free survival in pancreatic and liver cancers(Table 2). Patients with a high level of p-hTERT showed
an association with pathological type in lung cancer;
high mitosis score in lung, colon, stomach, pancreas,
and liver cancers; high pathological grade in lung,© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org

J Pathol 2022; 257: 172–185
www.thejournalofpathology.com

Phosphorylation of hTERT in cancers

177

Figure 1. Validation of a monoclonal antibody (TpMab-3) speciﬁc for hTERT phosphorylated at Thr249. (A) Detection of endogenous p-hTERT fromHeLa cells treated with nocodazole to synchronize cells in mitosis. Endogenous hTERT was immunoprecipitated by an anti-hTERT mouse mAb(10E9-2) and detected by an anti-p-hTERT mouse mAb (TpMab-3) or an anti-hTERT mouse mAb (2E4-2). Mouse IgG was used as an isotype control
for immunoprecipitation. (B) hTERT immunoprecipitated from the cells was treated with λ-phosphatase and detected by an anti-p-hTERT mousemAb (TpMab-3) and an anti-hTERT mouse mAb (2E4-2). White arrowheads (in panels A and B) indicate hTERT signals and the band at 102 kDa (grayarrowhead) is a nonspeciﬁc signal from the secondary antibody [16]. (C) The recombinant hTERT fragment proteins (191–306 a.a.) [16] were phosphorylated by CDK1–cyclin B in vitro and phosphorylation of hTERT at threonine 249 by CDK1–cyclin B was conﬁrmed by TpMab-3. (D) Same as
panel C, in vitro kinase assay was performed in vitro using the recombinant hTERT full-length proteins [29]. Phosphorylation of hTERT at threonine
249 by CDK1–cyclin B was conﬁrmed by TpMab-3 and 2E4-2. (E) Cells were treated with a CDK1 inhibitor (RO-3306). hTERT was immunoprecipitated by 10E9-2 and detected by an anti-p-hTERT mouse mAb (TpMab-3, Thr249). (F) hTERT immunocomplex was immunoprecipitated (10E9-2)
from cells transfected with two different siRNAs speciﬁc for hTERT or siNC, followed by nocodazole treatment. The proteins were detected by
TpMab-3. (G) RdRP assay using hTERT immunoprecipitated with anti-hTERT mouse mAb (clone 10E9-2) or anti-phospho-hTERT (TpMab-3).
(H) Immunoﬂuorescence staining of p-hTERT in cell lines (BJ, human ﬁbroblast; HeLa, human cancer cells). Bar: 10 μm. (I) Immunohistochemical
staining of p-hTERT in cell lines (HFL-1, p-hTERT-negative human ﬁbroblast; 1301, p-hTERT-positive human leukemia cells). Bar: 20 μm. Correlations between TERT mRNA and TpMab-3 expression were determined using nine human cell lines. IB, immunoblotting; IP, immunoprecipitation.
© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org

J Pathol 2022; 257: 172–185
www.thejournalofpathology.com

Y Matsuda et al

178

Figure 2. Variation in p-hTERT expression among cancers. (A) Lung adenocarcinoma, (B) colon adenocarcinoma, (C) gastric adenocarcinoma,(D) pancreatic adenocarcinoma, (E) hepatocellular carcinoma, (F) breast adenocarcinoma, and (G) renal cell carcinoma. Original magniﬁcation: 400. Bar: 50 μm. (H) Box plot of p-hTERT expression in cancers in various organs.

pancreas, and liver cancers; and high nuclear score inlung cancers (supplementary material, Table S2), suggesting that high p-hTERT expression contributes tothe proliferation and highly aggressive morphologicalfeatures of those cancers.
Overall survival and disease-free survival were longer
for patients whose tumors showed low p-hTERT expression than for patients whose cancers showed high p-

hTERT expression in cancers of the lung (p = 0.0223;
p = 0.0866), pancreas (p = 0.0199; p = 0.0494), andliver (p = 0.0168; p = 0.0128; p = 0.0008; p = 0.0006;
Figure 4B and supplementary material, Figures S5 andS6). These data indicate that high p-hTERT expression isclosely associated with poorer prognosis, independent of
TNM stage, supporting its prognostic value in lung,pancreatic, and liver cancers.

© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org

J Pathol 2022; 257: 172–185
www.thejournalofpathology.com

Phosphorylation of hTERT in cancers

179

Figure 3. Associations of p-hTERT expression with mitosis score, pathological grade, nuclear grade, and telomere length. (A) p-hTERT expression in
mitotic cells (arrows). The plot shows expression of p-hTERT for each mitosis score in cancers from various organs. (B) Upper panels: lung adenocarcinoma case classiﬁed as pathological grade 1, well-differentiated adenocarcinoma. Lower panels: lung adenocarcinoma case classiﬁed as pathological grade 3, poorly differentiated adenocarcinoma. The plot shows expression of p-hTERT for each pathological grade in various organs.
(C) Upper panels: hepatocellular carcinoma case with nuclear score 1; small, regular, uniform cells. Lower panels: hepatocellular carcinoma casewith nuclear score 3, marked variation. The plot shows p-hTERT for each nuclear score in various organs. (A–C) Original magniﬁcation: 400.
Bar: 50 μm. Spearman’s rank correlation coefﬁcient. (D) Left panel: lung adenocarcinoma case with a long telomere length. Right panel: lung adenocarcinoma case with a short telomere length. FISH images of telomeres (red), centromeres (green), and DAPI (blue); original magniﬁcation: 800.
Bar: 20 μm. Patients were divided into two groups based on [normalized telomere length of cancer cells]/[normalized telomere length of ﬁbroblasts]
with a cut-off value of 1.2 to obtain cases with short telomeres (under 1.2) and cases with long telomere lengths (over 1.2). The plot shows expression of p-hTERT for Short and Long telomere length groups in various organs. Student’s t-test.
© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org

J Pathol 2022; 257: 172–185
www.thejournalofpathology.com

Y Matsuda et al

180

Figure 4. p-hTERT expression is a potential biomarker for poor prognosis in lung, pancreatic, and liver cancers. (A) Hazard ratio for overall
survival. (B) Kaplan–Meier analysis for overall survival. Cut-off value for p-hTERT expression was 40% for lung, 20% for pancreatic, and livercancer according to the median expression. The number of patients at risk is shown below the graph. *p < 0.05 and **p < 0.01 by log-rank test.
(C) p-hTERT expression of adenocarcinoma (ADC), adenosquamous carcinoma (ASC), and squamous cell carcinoma (SCC). (D) Lung squamouscell carcinoma case. Original magniﬁcation: 400. Bar: 200 μm. (E) Expression levels of ΔNp63, p63, and cytokeratin 5/6 were associatedwith p-TERT. Red box, positive for ΔNp63, p63, or cytokeratin 5/6; gray box, negative. Student’s t-test.

hTERT phosphorylation is associated with aggressive
features
Based on our ﬁndings that the phosphorylation ofhTERT at T249 correlates with poor prognosis in lung,pancreatic, and liver cancers, we further analyzed theclinicopathological characteristics of p-hTERT in thesecancers. Lung and pancreatic cancers with squamous differentiation (adenosquamous carcinoma, an aggressive
and immature phenotype [32,33], and squamous cell carcinoma) demonstrated higher p-hTERT expression levels
than those of adenocarcinoma (p = 0.0473; p < 0.0001,
Figure 4C). Furthermore, the squamous cell makers
ΔNp63 and cytokeratin 5/6 (Figure 4D) were associatedwith p-hTERT (p < 0.01; p = 0.02, Figure 4E). A previous
report has shown that ΔNp63α induces TERT promoter
activation and RNA splicing in mice [34]; therefore, phTERT expression in cancers might be closely associatedwith squamous differentiation.
Furthermore, p-hTERT expression was correlated with
the invasive type of lung adenocarcinoma (supplementarymaterial, Figure S7A); serum levels of AFP, which is a representative oncofetal protein activated in liver cancer withstem cell properties (supplementary material, Figure S7B);
and triple-negative breast cancer, which is a highly aggressive form of breast cancer with stem cell properties(supplementary material, Figure S7C). In the same cohort,mitosis score (supplementary material, Figure S8) and telomere length were not correlated with overall survival anddisease-free survival (supplementary material, Figure S9).
Previously, we have reported that CDK1 phosphorylates
hTERT at threonine 249, which represents RdRP activity [16]. hTERT-RdRP activity involves tumor formation
via regulating the expression of target genes. In the present
study, we have shown that h-TERT-RdRP activity, which isa non-telomelic function of hTERT, is closely associatedwith aggressiveness and poorer prognosis via the regulationof proliferation and differentiation in cancer (supplementarymaterial, Figure S10).

Discussion
Our results clearly indicated that p-hTERT expression isa strong risk factor independent of TNM stages in lungcancer, pancreatic cancer, and liver cancer, three highly
aggressive cancer types with poor prognosis. Moreover,
p-hTERT expression was strongly associated withmarkers of squamous cell differentiation and aggressive
features, suggesting that p-hTERT expression is a common molecular event in these aggressive cancers. Using© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org

J Pathol 2022; 257: 172–185
www.thejournalofpathology.com

Lung© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org

ND

0.1215<0.0001**

0.0376*0.0781

0.0441*

P value<0.0001**

0.08370.0240*0.14070.0243*

P valueColon

0.09 (0.04–0.22)

1
0.32 (0.13–0.76)

ND

0.91 (0.44–1.92)

0.42 (0.19–0.93)
11

1.09 (0.52–2.30)

1

HR

Colon

0.11 (0.05–0.28)

0.39 (0.17–0.92)

ND
11.03 (0.50–2.13)

1<0.0001**0.0101*0.81410.0321*

0.3388

P value<0.0001**

0.0315*0.94530.19500.08 (0.02–0.38)

1
0.35 (0.18–0.66)

ND

1.49 (0.87–2.56)

0.83 (0.50–1.39)
1

10.77 (0.42–1.38)

1HR

Stomach0.11 (0.02–0.49)

0.41 (0.23–0.75)

ND
11.12 (0.67–1.87)

110.93 (0.56–1.55)

0.97 (0.55–1.69)

10.60 (0.28–1.30)

10.7767

StomachHR

1.11 (0.55–2.21)

P value1HRHR, hazard ratio; TNM stage, UICC 7th edition; ND, not determined.
*p < 0.05;**p < 0.01.

IV

0.61 (0.33–1.14)
0.20 (0.12–0.35)

0.61 (0.37–0.97)

High

II
III

0.65 (0.41–1.05)
1

>65
Low

hTERT

1

1

≤65

Age (years)

I1.64 (1.01–2.66)

TNM stage

1

Female

HR

Lung

ND

Male

Sex

Multivariate

IV

0.20 (0.12–0.35)

0.58 (0.36–0.93)

High

III

1

Low

hTERT

1
0.58 (0.31–1.08)

10.70 (0.44–1.12)

≤65
>65

Age (years)

III

1.73 (1.07–2.79)

TNM stage

1

Female

HR

Male

Sex

Univariate

Table 1. Univariate and multivariate analyses of overall survival

0.0017**0.0013**0.1461

0.48640.3753

P value

0.0040**0.0039**0.65480.78940.9021

P value

Pancreas

ND

0.48 (0.20–1.17)
0.14 (0.02–1.26)

10.70 (0.51–0.97)

1.03 (0.76–1.41)
110.88 (0.65–1.20)

1HR

PancreasND

0.13 (0.02–1.16)

10.48 (0.20–1.16)

0.69 (0.50–0.94)

11
1.02 (0.75–1.38)

0.82 (0.61–1.10)

1HR

0.10760.08000.0311*0.83700.4215

P value

0.06800.10270.0207*0.91000.1887

P valueLiver

0.15 (0.02–1.12)

0.24 (0.14–0.44)
0.08 (0.04–0.17)

1

0.48 (0.26–0.87)

1.05 (0.63–1.74)
110.84 (0.43–1.63)

1

HR

Liver

0.17 (0.02–1.31)

0.08 (0.04–0.17)

1
0.25 (0.14–0.45)

0.49 (0.27–0.89)

11
1.12 (0.68–1.84)

0.99 (0.52–1.91)

1HR

0.0644<0.0001**<0.0001**

0.0148*0.85950.6050P value0.0892<0.0001**<0.0001**

0.0191*0.66630.9816

P value

BreastND

0.77 (0.20–3.03)
0.10 (0.03–0.39)

1

0.93 (0.37–2.45)

0.77 (0.27–2.17)
1

1

ND

HR

BreastND

0.14 (0.04–0.47)

1
0.97 (0.29–3.23)

1.15 (0.46–2.98)

1

10.87 (0.34–2.24)

NDHR

0.71640.0009**0.88420.6148

P value0.0016**

0.96410.75620.7778

P value

Kidney0.03 (0.01–0.09)

0.12 (0.02–062)
0.14 (0.06–0.35)

10.82 (0.39–1.73)

0.65 (0.33–1.28)
11

1.65 (0.72–3.81)

1HR

Kidney0.04 (0.01–0.09)

0.14 (0.06–0.35)

1
0.14 (0.03–0.66)

1.02 (0.53–1.96)

1

10.78 (0.41–1.49)

1.46 (0.69–3.08)

1HR<0.0001**

0.0110*<0.0001**

0.59840.20990.2403

P value<0.0001**<0.0001**

0.0130*0.96070.45410.3201

P value

Phosphorylation of hTERT in cancers
181

J Pathol 2022; 257: 172–185
www.thejournalofpathology.com

0.11 (0.07–0.19)

III© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org

ND<0.0001**

0.0001**0.16630.95240.6470P value<0.0001**<0.0001**

0.08870.73990.4084

P value0.02 (0.003–0.07)

0.08 (0.02–0.34)

ND
1

1.43 (0.65–3.12)

110.85 (0.39–1.84)

1.39 (0.64–3.05)

1

HR

Colon

0.02 (0.004–0.08)

0.08 (0.02–0.36)

11.40 (0.66–2.95)
ND

1

1.13 (0.53–2.34)

0.88 (0.43–1.83)
1

1

HR

Colon

HR, hazard ratio; TNM stage, UICC 7th edition; ND, not determined.
*p < 0.05;**p < 0.01.

IV

0.12 (0.07–0.20)

III

High

1
0.35 (0.20–0.59)

0.73 (0.47–1.13)

Low

hTERT

I
II

1

≤65
>65

TNM stage

1.11 (0.71–1.73)

10.99 (0.94–1.53)

Female

1

Male

HR

Lung

Age

Sex

Multivariate

0.34 (0.20–0.58)

II

ND

0.68 (0.44–1.06)
1

High
I

TNM stage

IV1

1.08 (0.70–1.65)

>65

Low

1.20 (0.78–1.86)
1Female
≤65

Age

hTERT

1

Male

HR

Lung

Sex

Univariate<0.0001**

0.0006**0.37440.67140.4051

P value<0.0001**

0.0009**0.38250.74550.7423

P value

Table 2. Univariate and multivariate analyses of disease-free survival

0.17 (0.03–0.84)

0.32 (0.16–0.65)

10.82 (0.48–1.42)

110.75 (0.44–1.29)

0.85 (0.46–1.57)

1HR

Stomach0.17 (0.04–0.78)

0.32 (0.16–0.64)

10.67 (0.39–1.14)ND

10.81 (0.48–1.37)

1.04 (0.58–1.89)
1

1

HR

Stomach0.0297*0.0017*

0.48150.29680.5997

P value

0.0228*0.0013**0.13590.43370.8929P valueND

0.17 (0.02–1.56)

10.33 (0.12–0.89)

0.73 (0.54–0.99)

111.13 (0.84–1.51)

0.87 (0.33–0.65)

1HR

Pancreas0.02 (0.002–0.16)

0.16 (0.02–1.44)

0.33 (0.12–0.89)

0.75 (0.55–1.00)
11

1.11 (0.83–1.49)

0.82 (0.62–1.09)
1

1

HR

Pancreas0.11730.0293*0.0442*0.42260.3331

P value

0.0004**0.10220.0287*0.05290.47430.1678

P valueND

0.23 (0.13–0.39)

1
0.55 (0.40–0.75)

0.67 (0.49–0.92)

110.81 (0.60–1.09)

0.94 (0.64–1.38)

1

HR

Liver

0.09 (0.06–0.45)

0.24 (0.14–0.40)

0.57 (0.41–0.78)

0.67 (0.49–0.92)
110.80 (0.59–1.08)

0.98 (0.68–1.43)
1

1

HR

Liver

<0.0001**

0.0002**0.0135*0.17000.7561

P value<0.0001**<0.0001**

0.0004**0.0137*0.14290.9323

P valueND

0.15 (0.06–0.39)

1
0.50 (0.21–1.19)

0.75 (0.40–1.42)

110.80 (0.40–1.63)

ND

HR

BreastND

0.17 (0.07–0.43)

0.60 (0.27–1.34)

0.84 (0.45–1.56)
110.75 (0.39–1.45)

1

ND

HR

Breast<0.0001**

0.11820.37450.5463

P value

0.0002**0.21240.57030.3833

P valueND

0.07 (0.02–0.21)

1
0.07 (0.01–0.42)

0.94 (0.36–2.44)

11
1.47 (0.53–4.11)

0.88 (0.35–2.26)

1HR

Kidney0.001 (0–0.04)

0.07 (0.02–0.21)

0.08 (0.01–0.42)

1.14 (0.46–2.83)
1

1

1.27 (0.49–3.28)

0.92 (0.37–2.29)
1

1

HR

Kidney<0.0001**

0.0033**0.89150.46200.7983

P value<0.0001**<0.0001**

0.0031*0.7756

0.62120.8601

P value

182
Y Matsuda et al

J Pathol 2022; 257: 172–185
www.thejournalofpathology.com

Phosphorylation of hTERT in cancers

a large number of cohorts, we also found that p-hTERT
is correlated with the following parameters: (1) mitotic
activity in lung, colon, stomach, pancreatic, and livercancers; (2) pathological grade (differentiation) in lung,pancreatic, and liver cancers; (3) nuclear score (nuclear
pleomorphism) in lung and liver cancers; (4) squamous
differentiation in lung and pancreas cancers; and(5) aggressive immature features in lung, pancreatic,
liver, and breast cancers, as summarized in supplementary material, Table S3. Our ﬁndings suggest the utilityof histological evaluations of p-hTERT expression for
prognostic stratiﬁcation in these deadly cancers.
hTERT canonically regulates telomere lengthening via
hTERT recruitment to telomeres at the S-phase. However,
we have previously demonstrated that it exhibits RdRP
activity at the M-phase [14], and this non-canonical hTERT
function is acquired by CDK1-mediated phosphorylation[16]. In this study, we identiﬁed a positive correlation
between p-hTERT expression and the incidence of mitosis
in our cancer panels, indicating that hTERT-RdRP is activated in various human cancers with high frequencies ofmitotic cells. Furthermore, p-hTERT expression did notshow a clear association with telomere length, suggestingthat immunoreactivity of the newly developed monoclonal
antibody reﬂects RdRP activity rather than telomerase
activity.
We found that p-hTERT expression has prognostic value
in lung, pancreatic, and liver cancers but not in colon andstomach cancers. Interestingly, although p-hTERT expression increased as the mitosis score increased in all of thesecancers, p-hTERT expression decreased as the pathologicalgrade increased in colon and stomach cancers. It is unclear
why the relationships between p-hTERT expression, mitosis scores, and pathological scores differed according to
the cancer origin. We found that cancer cells in the mucosal
layer showed higher expression levels of p-hTERT thanthose in the muscular or subserosal layer in colon and stomach cancers (unpublished data). It is possible that additional
mechanisms regulate p-hTERT expression, potentiallyrelated to the tumor microenvironment, in the mucosal layerof cancer originating in luminal organs.
Although p-hTERT expression is associated withimmature and aggressive pathological/clinical featuresin several cancers, correlation with poor prognostic outcome was only shown in lung, pancreatic, and liver cancers. One possibility to explain the different clinicalimpacts of p-hTERT expression on prognosis in distinct
cancer subtypes would be the surgical procedures andcurability. Curative surgical resection is generallyachieved in limited cases of lung, liver, and pancreaticcancers, potentially due to the microscopic tumor dissemination at the time of surgery, resulting in earlyrecurrence with poor prognosis. In contrast, radical resection could be technically achieved irrespective ofimmature and aggressive features in some colon and stomach cancers. In these cancers, the value of p-hTERT
expression evaluation might be limited. Another possibility
is that more impactful genetic alteration or molecularabnormality, in addition to p-hTERT phosphorylation,
might exist and determine the patients’ prognosis in colon,

183

stomach, and breast cancers. To clarify the prognostic utility of p-hTERT in low-risk patients, therefore, we haveanalyzed p-hTERT expression in pancreatic neuroendocrine tumor and found an association between p-hTERT
expression and large tumor size and high mitotic activity,
suggesting that p-hTERT might be a marker of cancers
with aggressive features even in low-risk localized disease(unpublished data).
We have also analyzed the intensity and H-score ofphosphorylation of hTERT 249T of most of the cases(83 hepatocellular carcinoma cases were not analyzed).
However, only high H-score was correlated with poor
prognosis in lung cancer but not in other cancers. The
percentage of phosphorylation of hTERT showed themost signiﬁcant correlation with prognosis in lung, pancreas, and liver cancers as compared with the intensity
and H-score. Evaluation of the percentage of p-hTERT
might be the best approach in a clinical setting.
Our results indicate that the evaluation of p-hTERT
could be utilized for prognostic stratiﬁcation in clinical settings. Furthermore, our monoclonal antibody can detect phTERT in formalin-ﬁxed, parafﬁn-embedded samples
using an automatic immunostaining system, thus providing
a basis for the development of a novel clinical diagnostic
tool to identify patients with aggressive cancer.

Acknowledgements
We would like to express our gratitude to Yuko
Narusawa, Misa Tanimoto, Masumi Ito, and Dr Futoshi
Suizu (Oncology Pathology, Kagawa University) for
technical support and Maiko Tada and Etsuyo Matsubara(Oncology Pathology, Kagawa University) for assistancein preparing the manuscript. This work was supported inpart by a grant-in-aid from AMED under Grant
No. 20cm0106473h0001 and an Extramural Collaborative Research Grant of the Cancer Research Institute,
Kanazawa University to YM, a grant-in-aid from theJapan Society for the Promotion of Science (JSPS)
Kakenhi Grant No. JP21H02900 to TY, Platform Projectfor Supporting Drug Discovery and Life Science
Research [Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)] from AMED
under Grant No. JP20am0101078 to YK, a Grant-in-Aid
from AMED under Grant No. JP21fk0210049 andJP20ck0106403, and the National Cancer Center
Research and Development Fund (30-A-4) to KM. This
study was supported by a Grant-in-Aid for ScientiﬁcResearch on Innovative Areas from JSPS KAKENHI
Grant No. JP: 16H06277.

Author contributions statement
YMa, JY and NM curated the data. YMa, YK and KM
acquired funding. JY, MY, KY, YMu, MM, SY, JT
and MKa carried out the investigation. YMa, TYa, YK
and KM were responsible for methodology, and YD,
YMi, TYo, TO, HI, SM, TK, TM, MKo, SK, TM, MH,© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org

J Pathol 2022; 257: 172–185
www.thejournalofpathology.com

Y Matsuda et al

184

RH, KK, NK and KO for resources. YMa was responsible for software. YMa, TYa and KM supervised thestudy. YMa wrote the original draft, and YMa, TYa
and KM reviewed and edited the ﬁnal manuscript.

References
1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J
Clin 2019; 69: 7–34.
2. Arai T, Takubo K. Clinicopathological and molecular characteristics
of gastric and colorectal carcinomas in the elderly. Pathol Int 2007;
57: 303–314.
3. Ishikawa N, Nakamura K, Izumiyama-Shimomura N, et al. Changesof telomere status with aging: an update. Geriatr Gerontol Int 2016;
16((Suppl 1)): 30–42.
4. DePinho RA. The age of cancer. Nature 2000; 408: 248–254.
5. Arai T, Sakurai U, Sawabe M, et al. [Pathologic characteristics ofmalignant neoplasms occurring in the elderly]. Gan To Kagaku
Ryoho 2011; 38: 1571–1576 [in Japanese].
6. DeGregori J. Challenging the axiom: does the occurrence of oncogenic mutations truly limit cancer development with age? Oncogene
2013; 32: 1869–1875.
7. Noureen N, Wu S, Lv Y, et al. Integrated analysis of telomerase enzymatic activity unravels an association with cancer stemness and proliferation. Nat Commun 2021; 12: 139.
8. Hannen R, Bartsch JW. Essential roles of telomerase reverse transcriptase hTERT in cancer stemness and metastasis. FEBS Lett
2018; 592: 2023–2031.
9. Batlle E, Clevers H. Cancer stem cells revisited. Nat Med 2017; 23:1124–1134.
10. Horvai AE, Kramer MJ, Garcia JJ, et al. Distribution and prognostic
signiﬁcance of human telomerase reverse transcriptase (hTERT)
expression in giant-cell tumor of bone. Mod Pathol 2008; 21:423–430.
11. Kumaki F, Kawai T, Churg A, et al. Expression of telomerase reverse
transcriptase (TERT) in malignant mesotheliomas. Am J Surg Pathol
2002; 26: 365–370.
12. Kumaki F, Kawai T, Hiroi S, et al. Telomerase activity and expressionof human telomerase RNA component and human telomerase reverse
transcriptase in lung carcinomas. Hum Pathol 2001; 32: 188–195.
13. Gonzalo V, Petit A, Castellví-Bel S, et al. Telomerase mRNA expression and immunohistochemical detection as a biomarker of malignant
transformation in patients with inﬂammatory bowel disease.
Gastroenterol Hepatol 2010; 33: 288–296.
14. Maida Y, Yasukawa M, Furuuchi M, et al. An RNA-dependent RNA
polymerase formed by TERT and the RMRP RNA. Nature 2009; 461:
230–235.
15. Maida Y, Yasukawa M, Okamoto N, et al. Involvement of telomerase
reverse transcriptase in heterochromatin maintenance. Mol Cell Biol
2014; 34: 1576–1593.
16. Yasukawa M, Ando Y, Yamashita T, et al. CDK1 dependent phosphorylation of hTERT contributes to cancer progression. Nat
Commun 2020; 11: 1557.

17. Thompson CAH, Wong JMY. Non-canonical functions of telomerase
reverse transcriptase: emerging roles and biological relevance. Curr
Top Med Chem 2020; 20: 498–507.
18. Okamoto N, Yasukawa M, Nguyen C, et al. Maintenance of tumor
initiating cells of deﬁned genetic composition by nucleostemin.
Proc Natl Acad Sci U S A 2011; 108: 20388–20393.
19. Yamaguchi S, Maida Y, Yasukawa M, et al. Eribulin mesylate targets
human telomerase reverse transcriptase in ovarian cancer cells. PLoS
One 2014; 9: e112438.
20. Takahashi M, Miki S, Fujimoto K, et al. Eribulin penetrates brain
tumor tissue and prolongs survival of mice harboring intracerebral
glioblastoma xenografts. Cancer Sci 2019; 110: 2247–2257.
21. Maida Y, Yasukawa M, Ghilotti M, et al. Semi-quantitative detectionof RNA-dependent RNA polymerase activity of human telomerase
reverse transcriptase protein. J Vis Exp 2018; 136: e57021.
22. McShane LM, Altman DG, Sauerbrei W, et al. Reporting recommendations for tumor marker prognostic studies (REMARK). J NatlCancer Inst 2005; 97: 1180–1184.
23. WHO Classiﬁcation of Tumours Editorial Board. WHO Classiﬁcationof Tumours, Breast Tumours (Vol. 2, 5th edn). World Health Organization: Lyon, 2019.
24. Moch H, Humphrey PA, Ulbright TM, et al. (eds). WHO Classiﬁcation of Tumours of the Urinary System and Male Genital Organs(4th edn). World Health Organization: Lyon, 2016.
25. Travis WD, Brambilla E, Burke AP, et al. WHO Classiﬁcation of
Tumours of the Lung, Pleura, Thymus and Heart (Vol. 7, 4th edn).
World Health Organization: Lyon, 2015.
26. WHO Classiﬁcation of Tumours Editorial Board. WHO Classiﬁcationof Tumours, Digestive System Tumours (Vol. 1, 5th edn). World
Health Organization: Lyon, 2019.
27. Sobin LH, Gospodarowicz MK, Wittekind C (eds). TNM Classiﬁcation of Malignant Tumours (7th edn). Wiley-Blackwell: Hoboken,
2010.
28. Matsuda Y, Ishiwata T, Izumiyama-Shimomura N, et al. Gradual telomere shortening and increasing chromosomal instability among
PanIN grades and normal ductal epithelia with and without cancer
in the pancreas. PLoS One 2015; 10: e0117575.
29. Maida Y, Yasukawa M, Masutomi K. De novo RNA synthesis by
RNA-dependent RNA polymerase activity of telomerase reverse transcriptase. Mol Cell Biol 2016; 36: 1248–1259.
30. Edge SB, Compton CC. The American Joint Committee on Cancer:
the 7th edition of the AJCC Cancer Staging Manual and the futureof TNM. Ann Surg Oncol 2010; 17: 1471–1474.
31. Ramanathan SP, Krajnc M, Gibson MC. Cell-size pleomorphism
drives aberrant clone dispersal in proliferating epithelia. Dev Cell
2019; 51: 49–61.e44.
32. Mordant P, Grand B, Cazes A, et al. Adenosquamous carcinoma of
the lung: surgical management, pathologic characteristics, and prognostic implications. Ann Thorac Surg 2013; 95: 1189–1195.
33. Fang Y, Su Z, Xie J, et al. Genomic signatures of pancreatic adenosquamous carcinoma (PASC). J Pathol 2017; 243: 155–159.
34. Vorovich E, Ratovitski EA. Dual regulation of TERT activity throughtranscription and splicing by ΔNP63α. Aging (Albany NY) 2008; 1:
58–67.

SUPPLEMENTARY MATERIAL ONLINE
Supplementary materials and methods
Figure S1. p-hTERT expression is not associated with TNM stage
Figure S2. p-hTERT expression is not associated with age
Figure S3. p-hTERT expression is not associated with sex
Figure S4. Positivity of Ki67 was associated with mitosis© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org

J Pathol 2022; 257: 172–185
www.thejournalofpathology.com

Phosphorylation of hTERT in cancers

185

Figure S5. p-hTERT and survival
Figure S6. p-hTERT and survival of liver cancer cohorts
Figure S7. (A) Noguchi classiﬁcation and p-hTERT in lung cancer. (B) Serum AFP levels and p-hTERT in liver cancer. (C) Triple-negative breast
cancer and p-hTERT
Figure S8. Mitosis score and survival
Figure S9. Telomere length and survival
Figure S10. Non-telomeric and telomeric function of hTERT
Table S1. Human cell lines used
Table S2. Clinicopathological characteristics of patients with high and low hTERT phosphorylation
Table S3. Clinicopathological factors related to high levels of p-hTERT© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org

J Pathol 2022; 257: 172–185
www.thejournalofpathology.com