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AKT PLAYS A CRUCIAL ROLE IN GASTRIC CANCER

Yr LUO. RINA FU]IWARA. YUKIKO NISHIGUCHI and HIROKI KUNIYASU
Department of Molecular Pathology, Nara Medical University
Received April 4. 2016

Abbreviations : PI3K: phosphatidylinositol-3 kinase. VEGF: vascular endothelial growth factor,
pAKT: phosphorylated AKT. iNOS: inducible nitric oxide synthase. PTEN: tensin homolog
deleted on chromosome 10, NT: nitrotyrosine. hTERT: human telomerase r everse transcriptase,
CG: chronic gastritis without H. pylori, CAG: chronic active gastritis with H. pylori, CMG:
chronic metaplastic gastritis without H. pylori, CGA: chronic gastritis with atypia without H.
pylori. SHP2: src homology 2 domain-containing protein tyrosine phosphatase-2, hTR: humantelomerase RNA. EMT: epithelial-mesenchymal transition, NO: nitric oxide. ROS: reactive oxide
species
Abstract : The AKT protein is involved in the phosphatidylinositol-3 kinase signaling pathway
and is a vital regulator of survival. proliferation, and differentiation of various types of cells.
Helicobacter pylori induce epithelial cell proliferation and oxidative stress in chronic gastritis.
These alterations lead to telomere shortening and resultant telomerase activation. Specifically,
AKT is activated by H. pylori-induced inflammation; it subsequently promotes expression ofhuman telomerase r everse transcriptase, which encodes a catalytic subunit of telomerase; andinduces telomerase activity, which is an essential process of carcinogenesis. AKT activation
is increased in gastric mucosa with carcinogenic properties and is associated with low
survival of patients with gastric cancer. These findings suggest that AKT is pivotal in gastriccarcinogenesis and progression.
Key words: Helicobacter pylori, telomerase, hTERT, chronic gastritis

Introduction
Gastric cancer is the fourth most common cancer in the world ll_ Recent advances inmolecular analyses of preneoplastic and neoplastic lesions of the stomach have uncovereda large number of epigenetic and genetic alterations that determine a multistep processof stomach carcinogenesis 2l. Helicobacter pylori infection is inherently r esponsible for thepathogenesis of intestinal metaplasia, and its epidemiological contribution to gastric cancer issimilar in European and Japanese populations 3). In 1994, the International Agency for Research
on Cancer recognized H. pylori as a definite carcinogen of gastric cancer based on a strong

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epidemiological correlation between chronic colonization of H. pylori and gastric cancer 41 .
Moreover, a Mongolian gerbil model showed that H. pylori inoculation into the stomach is
closely associated with the occurrence of chronic gastritis, intestinal metastasis, and promotionof adenocarcinoma 51•
AKT is a pivotal regulator of cell survival, prolifer ation, and differentiation and a memberof the phosphatidylinositol-3 kinase (PI3K) signaling pathway. Stimulation of receptor tyrosine
kinases or G-proteins activates PI3K, which in turn activates AKT. AKT phosphorylation
is catalyzed by heat shock prot ein 90, and its dephosphor ylation is mediated via protein
phosphatase 2A. Subsequently, AKT regulates signaling via various growth factor s andcytokines. Upstream of AKT, activation of insulin-like growth factor-1 receptor, epidermal
growth factor receptor, and human epidermal growth factor receptor 2, which are important incancer progression, activates AKT 6' 71• Hence, AKT is a biomarker that predicts metastasis ofhuman gastrointestinal cancer 81 •Phosphorylation of AKT modulates signals from phosphatase and tensin homolog deleted
on chromosome 10 (PTEN) and from the mammalian target of rapamycin (mTOR), resulting
in diverse effects on cells 91. In this regard, AKT1 is recognized as an apoptotic inhibitor,
which contributes to cancer progression. Phosphorylation catalyzed by AKT inactivates Bcl2 antagonist of cell death, resulting in its dissociation from Bcl-2. In addition, AKT activates
nuclear factor KB, which in t urn upregulates transcription of many survival genes 101. AKT also
promotes angiogenesis through upregulation of vascular endothelial growth factor (VEGF) 111•
The AKT-microRNA regulatory network suggests that microRNA-mediated gene regulationinteracts with the AKT signal pathway 121• Thus, the expression/ activation of AKT promotes
tumorigenesis, and AKT represents a relevant molecular target for cancer treatment 71•

AKT activation in the gastric mucosa
To establish the role of oxidative stress and AKT activation in gastric cancer development,
we examined the levels of phosphorylated AKT (pAKT), inducible nitric oxide synthase(iNOS), nitrotyrosine (NT), and human telomerase reverse transcriptase (hTER T , the catalytic
component of telomerase) by enzyme-linked immunosorbent assay in non-cancerous gastricmucosa and gastric cancers 131• We found that the levels of pAKT are directly correlatedwith the levels of iNOS, NT, and h TER T. Gastric mucosa is classified into 4 categories:
chronic gastritis without H. pylori (CG), chronic active gastritis with H. pylori (CAG), chronic
metaplastic gastritis without H. pylori (CMG), and chronic gastritis with atypia without H.
pylori (CGA). We found increasing levels of pAKT, iN OS, and NT in CG, CAG, CMG, andCGA. hTERT was detected only in CGA. These findings suggest that oxidative stress may be
implicated in AKT activation and hTER T induction and that CGA mucosa may represent ahigh-risk status for gastric carcinogenesis 131.
H. pylori infection is the major cause of chronic persistent inflammation of the gastric mucosa
5' 141• In gastric mucosal pathology, H. pylori induces chronic inflammation and increases the
production of reactive oxygen species. H. pylori stimulates proliferation of gastric mucosal cells
via type IV secretion of CagA followed by CagA phosphorylation by src homology 2 domain-

AKT PLAYS A CRUCIAL ROLE IN GASTRIC CANCER(3)

containing protein tyrosine phosphatase-2 (SHP2) 15' 16). CagA activates SHP2 phosphat ase, which
inhibits signal transducers and activators of tr anscription-mediated growth-suppressive signal
and activates extr acellular signal-r egulated kinase-mediated growth signal 17). The increased
growth activity may enhance the risk of gene alteration.
H. pylori-induced inflammation generates morphological changes, i.e., intestinal metaplasia,
which is transdifferentiation from gastric epithelium to the intestinal phenotype 18). In thatcondition, antr al or fundic mucosa of the stomach is replaced with mucosa that resembles
intestinal mucosa 19) through ectopic expr ession of CDX2 20). Intestinal metaplasia differs fromnormal mucosa in that cell division is accelerated 21' 22). Intestinal metaplasia shares several
genetic and epigenetic alterations with gastric cancer 3). We reported that mutated APC andabnormal CD44 transcripts are present in intestinal metaplasia as well as gastric cancer 23).
Furthermor e, we explained that microsatellite instability as well as p53 mutation 24) are detected
in 33% of intestinal metaplasias 25).

T elomer ase
The telomere is a repetitive "TT AGGG" sequence present at the ends of eukaryotic
chromosomes to maintain and pr otect their integrity 26). As cells divide, the t elomere isshortened in length; thus, the length of the telomere behaves like a marker of the division
limit for cells and/or for cell death 27). In stem cells, cancer cells, and cancer stem cells, thetelomere is elongated by t elomerase- a ribonucleopr otein enzyme- activity, which enables
cells to divide endlessly 28). Thus, through its reactivation, telomerase is a key enzyme to induce
immort alization and malignant properties in somatic cells. T elomerase activity is detect edin 85% of gastric cancers, regardless of tumor stage and histological types 29). In addition,
telomerase activity is detected in 23% of intestinal metaplasias and 50% of gastric adenomas,
whereas no activity in the corresponding gastric normal mucosa has been reported.

Telomerase RNA
Human telomerase RNA (hTR) is generated to function as the template RNA component oftelomerase, and h TR has 11 nucleotides complementary to the telomere sequence (TTAGGG)
30). h TR is expr essed in pre-crisis cell lines, non-neoplastic tissues, immortalized cell lines, andtumor specimens, but the expression level is not correlated with the level of telomer ase activity
31). Inter estingly, Blasco et al. reported that initial upr egulation of hTR is an early event andthat telomerase is activated only in end-stage tumors during multi-stage carcinogenesis 32).
We reported the expression of h T R and telomerase activity in gastric cancer and thecorresponding non-cancerous mucosa 33). Telomerase activity is detected in 88% of carcinoma
tissues. Although t umor specimens and non-cancerous mucosas express var ious levels of
h TR, 81% express h TR at higher levels in turnor specimens than that in the corresponding
mucosa. Gastric carcinoma cell lines also express h TR at high levels. Thirty-five percent of noncancerous mucosas show telomerase activity, and all non-cancerous mucosas contain intestinal
metaplasia. The incidence of telomerase-positive mucosa in moderate intestinal metaplasia is(4)Yi LUO eta!.

significantly higher than that in mild intestinal metaplasia. whereas hTR overexpression isdetected in mild intestinal metaplasia as well as moderate intestinal metaplasia. Interestingly,
the degree of metaplasia of H. pylori-infected mucosa increased concordantly with the level ofhTR expression and telomerase activity. These results indicated that hTR overexpression iscaused by H. pylori, which plays a role upstream of telomerase activity as an early event instomach carcinogenesis 33l.
In the stomach, various kinds of inflammation affect the growth of the mucosa. Specifically,
intestinal metaplasia and chronic atrophic gastritis exhibit accelerated cell growth and active
cell cycling 22' 34l. Although factors that upregulate hTR expression are unknown. continuous
inflammatory and regenerative processes may stimulate hTR expression by affecting stem
cells, and, subsequently, these processes may enhance the activity of telomerase in noncancerous mucosa of the stomach. Therefore, it is likely that hTR overexpression in noncancerous mucosa may reflect an earlier process of multistep carcinogenesis of the stomach.

Telomere shortening
Telomere shortening of non-cancerous tissues is determined by regenerative processes andis specific to the individual organs or tissue components, since regeneration of tissues requires
various degrees of cell division, which also occur in chronic gastritis 21' 22l. We examined thedifference in telomere shortening of the gastric mucosal epithelium using an in situ telomere
quantification technique. Then, we investigated the relationship between telomere length andH. pylori infection, and we also assessed the difference between telomere length characterizing
intestinal metaplasia of cancer patients and that of non-cancer patients 35l.
In healthy subjects without H. pylori infection, the telomere volumes of gastric epithelial
tissues are 70- 79% that of intramucosal lymphocytes (internal control). In patients without
gastric cancer, the telomere volume of H. pylori-infecte d mucosa is significantly less than thatof H. pylori-negative mucosa in both metaplastic and non-metaplastic tissues (P < 0.0001). In
gastric cancer patients, telomere volumes in intestinal metaplasias adjacent to cancer are 75%
that of intestinal metaplasias in non-cancer patients (P = 0.0001) 35l. H. pylori infection is closely
associated with shortening of telomeres, which subsequently stimulates necessary elongation of
the telomeres by telomerase activity, especially in the gastric epithelium.

Telomere and AKT
The catalytic subunit of hTERT is responsible for telomerase activity and telomer eelongation and is suppressed in differentiated cells 36l. We reported that telomere shortening
is a significant factor for the induction of hTERT expression in gastric mucosa 35l. hTERT
expression is detected in 7% of the corresponding mucosas of cancer-associated int estinal
metaplasias and 3% of cancer-negative intestinal metaplasias, in which telomere volume ismarkedly reduced 35l. We classified the status of gastric mucosa according to inflammationand immortalization by h TER T expression. AKT is a key protein that links inflammationand tumorigenesis; hence. it became the focus of our studies. iNOS is a significant mediator

AKT PLAYS A CRUCIAL ROLE IN GASTRIC CANCER(5)

of inflammation in the gastric mucosa. Increased expression of iNOS is epigenetically induced
upon H. pylori infection as a host defense 37). NT is a marker of nitric oxide (NO)-induced
protein degradation. In a previous study, we examined the expression of iNOS and NT toevaluate inflammation and the expression of hTERT as a marker of immortality. Our dat ashowed that the levels of pAKT are associated with hTERT expression in CGA 13l. In addition,in another study we showed that pAKT levels are associated with hTERT expression in
gastric adenocarcinomas 13l. Taken together, these findings suggest that the immortality ofgastric mucosal cells is associated with inflammation-induced AKT activation.
It is notable that pAKT levels are elevated in certain gastric mucosa; specifically CAG, CMG,
and CGA. Interestingly, high pAKT levels in CAG are associated with H. pylori-induced active
inflammation. In contrast, increased pAKT in CMG suggests that persistent iNOS upregulation
develops during the metaplastic process. The highest pAKT levels in CGA may develop
during carcinogenic processes 38l. In vitro analysis showed that only CGA and MKN28 cancer
cells showed upregulation of pAKT in response to brief NO exposure. In Barrett esophagus,
activation of AKT is associated with dysplasia-carcinoma sequence 39). Moreover, only CGA
mucosa expressed hTERT. These findings suggest that CGA is likely a distinct category in thegastric carcinogenesis process.
hTERT activity is regulated by phosphorylation and expression of hTERT, and protein
kinase C and AKT phosphorylate hTERT 40' 41l. AKT-catalyzed phosphorylation of hTERT
induces intranuclear translocation of hTERT and, subsequently, activates hTERT. In contrast.
ring finger protein 1. an E3 ubiquitin ligase, decreases the activity of hTERT by ubiquitination
42)

AKT is associated with diverse pro-tumoral responses, e.g., hTERT activation. In our study,
the significance of AKT phosphorylation and hTERT on the prognosis of gastric cancer was
examined. AKT activation by epidermal growth factor increased hTERT expression andtelomer ase activity. In contrast. AKT inactivation by inhibitors and knockdown decr eased
hTERT expression and telomerase activity in MKN28 gastric cancer cells. In 40 gastric cancer
tissues, significant correlations were found among the levels of pAKT, hTERT expr ession,and telomere length. The pAKT levels or the levels of pAKT/hTERT are not associated withclinicopathological parameters, including stage and nodal metastasis. However, survival ratesof the high-level pAKT patients or the high-level pAKT and high-level hTERT patients are
significantly poorer than those of other patients. These findings suggest that AKT and hTERT
ar e good molecular targets for the treatment of gastric cancer.
AKT is associated with cancer cell survival via alteration of Bcl-2 antagonist of cell death,p53, forkhead, nuclear factor KB, mTOR. and PTEN JO) 43l. Moreover, dysregulated PTEN/ PI3K/
AKT signaling interacts with the Wingless-INT pathway to induce epithelial-mesenchymaltransition (EMT), which is usually associated with cancer stem cell-phenotype and poor
prognosis 44l. A recent report states that hTERT promotes transforming growth factor- /3-induced and /3 -catenin-induced EMT by inducing /3 -catenin nuclear translocation and its
transcriptional activity for vimentin, a mesenchymal factor, expression 45l. Therefore, PTEN/
PI3K/ AKT signaling enhances EMT and stem cell phenotypes. In our study, the associationof AKT phosphorylation, TER T expression, and telomer ase activity was confirmed in MKN28

(6)Yi LUO et al.

gastric cancer cells and gastric cancer tissues. These associations could result in poor prognoses
in patients with high pAKT levels or high pAKT/ hTERT levels; further. a multivariate
analysis revealed that pAKT levels or pAKTI h TERT levels are reliable prognostic factor s.
The examination of more gastric cancer cases is required to confirm the hypothesis that the
EMT/ stem cell phenotype affects disease progression.
Angiogenesis is an essential phenotype for cancer progression 46), and VEGF expression isassociated closely with neovascularization and cancer progression in many malignancies. The
PI3K/ AKT pathway induces VEGF response, which includes other downstream inducer ssuch as mitogen-activated protein kinase (extracellular signal-r egulated kinases or p38), Sr c,
focal adhesion kinase, Rho family GTPases, and endothelial NO 47). The PI3K/ AKT pathway
increases the secretion of VEGF from cancer cells by hypoxia-inducible factor !-dependentand independent mechanisms 48); therefore, AKT suppression could result in an anti-angiogenic
effect on gastric cancer.
Our data showed that AKT and hTERT are present in high levels in gastric cancer, andthe concurrent synthesis of these 2 proteins at high levels is associated with a poor prognosis.
These results suggest that AKT and hTERT are plausible molecular targets for the treatmentof gastric cancer.

Conclusions
In gastric carcinogenesis. H. pylori is an essential factor stimulating the transformationof gastric epithelial cells. H. pylori induces iNOS, synthesis of reactive oxide species (ROS).
and telomere shortening as a r esult of repetitive destruction/ regener ation cycles in chronic
active gastritis (Fig. 1). The inflammatory changes activate AKT, which promotes hTERT
expression and telomer ase activation. Telomerase activity immortalizes epithelial cells to enableuncontrolled cancer cell division and instigates malignant phenotypes advancing cancer. Thesefindings demonstrated that AKT plays a pivotal role in gastric cancer development. Thus, thestudy of AKT as a promising molecular target to prevent gastric cancer development and/ or
progression should be reconsidered.

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Fig. 1. Central role of AKT in H. pylori-induced gastric carcinogenesis
hTERT: human telomerase reverse transcriptase. ROS: r eactiveoxide s pecies, iN OS: in ducible nitric ox ide synth ase, PI3K:
phosphatidylinositol-3 kinase, and H. pylori: Helicobacter pylori

AKT PLAYS A CRUCIAL ROLE IN GASTRIC CANCER(7)

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