Yoshimi Ozaki-Ohgami et al.

Fig. 3. Postoperative survival curves of patients withpancreatic cancer according to expression of PTEN.
Patients with PTEN-negative tumors had significantly
worse prognoses than those with PTEN-positive tumors(P = 0.009). [ ], number of cases.

Fig. 4. Postoperative survival curves of patients withpancreatic cancer according to expression of p-Akt andPTEN. Patients with p-Akt-positive besides and PTENnegative tumors had signiﬁcantly worse prognoses thanthose with p-Akt-negative and PTEN-positive tumors (P= 0.0026). [ ], number of cases.

Patients with PTEN-positive tumors had signiﬁcantly better prognoses than those with PTENnegative tumors (P = 0.009; Fig. 3). Patients with
p-Akt-negative and PTEN-positive tumors had
signiﬁcantly better prognoses than those with thep-Akt-positive and PTEN-negative tumors (39.1%versus 0%, respectively; P = 0.0026; Fig. 4).
In 30 patients with pathological stage IVtumors, those with p-Akt-positive tumors hada significantly shorter survival time than thosewith p-Akt-negative tumors (P = 0.02; Fig. 5A),
and patients with PTEN-positive tumors had asigniﬁcantly longer survival time than those with
PTEN-negative tumors (P = 0.0386; Fig. 5B).
These results suggest that expression of p-Akt andPTEN can predict the prognoses of patients withpancreatic cancer.
Multivariate analysis by the Cox proportional hazards model and a stepwise procedure
was performed to determine the significance ofthe prognoses of patients with pancreatic cancer.
Fig. 5. Postoperative survival curves of patients with pancreatic cancer in stage IV. [ ], number of cases.
A: Patients with p-Akt-positive tumors had a signiﬁcantly shorter survival time than those with p-Akt-negativetumors (P = 0.02).
B: Patients with PTEN-positive tumors had a signiﬁcantly longer survival time than those with PTEN-negativetumors (P = 0.0386).

14

p-Akt and PTEN expression in pancreatic cancer

Table 3. Multivariate analysis of prognostic
factors by the Cox proportion hazard model

Covariates were p-Akt expression, PTEN expression, histology, venous invasion, arterial invasion,
lymphatic vessel invasion and pathological stage.
p-Akt expression, arterial invasion and lymph
node metastasis were found to be independent
prognostic factors in patients with pancreatic cancer (Table 3).

Hazard
ratio

95%
CIP
value

p-Akt expression†
2.294 1.078– 4.878 0.0201
Arterial invasion†
6.279 2.902–13.581 < 0.0001
Lymph node metastasis† 4.531 1.962–10.465 0.0005† Positive or negative.
CI, conﬁdence interval.
p-Akt Expression, arterial invasion and lymph nodemetastasis are independent prognostic factors in patients with pancreatic cancer.

Discussion
The PI3K-Akt signaling pathway is known totransmit survival signals and to protect a varietyof cells from apoptosis. Akt plays a critical rolein controlling the balance between cell survival
and apoptosis (Alessi et al., 1997). It has also
been reported that Akt plays an important role incell survival when cells are exposed to different
apoptotic stimuli, such as growth factor withdrawal, ultraviolet radiation, matrix detachment,
cell cycle discordance and DNA damage (Burnetet al., 1999; Camp et al., 2004). Phosphorylation of Akt is promoted by phosphatidylinositides
converted by PI3K products. Therefore, whenphosphorylation of Akt is inhibited, apoptosis
can be induced (Balendran et al., 1999; Brognardet al., 2001). Inactivation of PI3K using a speciﬁc inhibitor leads to dephosphorylation of Akt
at Ser473, consequently causing translocation ofAkt to the nucleus, where it is believed to regulate
the transcription of genes mediating cell survival
or apoptosis (Cardone et al., 1998). Therefore,
it is supposed that the PI3K-Akt pathway plays asignificant role in malignant cells. There is increasing evidence that Akt is activated in manydifferent cancers and that this signaling pathway confers a potent survival signal (West et al.,2002). The tumor suppression gene PTEN is anatural biological inhibitor of Akt in cell survival
signals. In the present study, we have demonstrated by immunohistochemistry the frequent
occurrence of p-AKT and PTEN expression in
surgically resected pancreatic cancers. In addition, our results reveal that p-Akt expression is asign of poor prognosis in patients with pancreaticcancer. Additional investigation is necessary toclarify the mechanism that controls p-Akt expression and activation. The inhibition of p-Akt may
be a molecular target for the treatment of pancreatic cancer.
PTEN negatively regulates the PI3-Akt
pathway and its mutation is associated with anaggressive tumor phenotype (Di Cristofano andPandolfi, 2000). The decreased expression ofPTEN in pancreatic cancer occurs at the mRNAand protein levels, and deletions or mutations are
not frequently observed (Ebert et al., 2002). Although it seems clear that the main role of PTEN
is to regulate the PI3K-Akt pathway, many important issues remain unresolved in pancreaticcancer. In the present study, we analyzed therelationship between p-Akt and PTEN expression and prognosis of patients with pancreaticcancer. The present immunohistochemical study
shows a significant association between positive
PTEN expression and good prognosis. Our dataalso demonstrate a tendency towards a close correlation between p-Akt and PTEN expression(P = 0.00549). Pancreatic cancer patients with
p-Akt-negative and PTEN-positive tumors had
significantly better prognoses than those with
p-Akt-positive and PTEN-negative tumors. Theseresults suggest that increased expression of PTEN
inhibits phosphorylation of Akt and subsequently
the ability of Akt to promote pancreatic cancer
invasion and resistance to apoptosis. Althoughadditional studies on the mechanism of PTEN are15

Yoshimi Ozaki-Ohgami et al.

needed, increasing the expression of PTEN protein may contribute to a new, effective treatment
for pancreatic cancer.
In conclusion, we have found that a high expression level of p-Akt protein is signiﬁcantly correlated with poor prognosis, and that high expression of PTEN protein is significantly correlatedwith good prognoses in patients with pancreaticcancer. Furthermore, it has been suggested thatpatients with negative expression of p-Akt andpositive expression of PTEN have good prognoses. Therefore, the expression of p-Akt andPTEN protein determined by immunohistochemistry may be a new method for predicting prognoses in patients with pancreatic cancer. It may bethat p-Akt and PTEN expression are novel targets
in the search for effective therapeutic options forpancreatic cancer.

LS, et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor.
Cell 1999:96;857–868.
7 Cardone MH, Roy N, Stennicke HR, Salvesen GS,Franke TF, Stanbridge E, et al. Regulation of cell
death protease caspase-9 by phosphorylation. Science 1998;282:1318–1321.
8 Camp ER, Li J, Minnich DJ, Brank A, Moldawer LL,
MacKay SL, et al. Inducible nuclear factor-kap-paB
activation contributes to chemotherapy resistance ingastric cancer. J Am Coll Surg 2004:199;249–258.
9 Cheng JQ, Ruggeri B, Klein WM, Sonoda G, Altomare DA, Watson DK, et al. Ampliﬁcationof AKT2in human pancreatic cells and inhibition of AKT2
expression and tumorigenicity by antisense RNA.
Proc Natl Acad Sci 1996:93;3636–3641.
10 Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y,et al. Akt phosphorylation of BAD couples survival
signals to the cell-intrinsic death machinery. Cell
1997;91:231–241.
11 Di Cristofano A, Pandolﬁ PP. The multiple roles ofPTEN in tumor suppression. Cell 2000:100;387–390.
12 Ebert MP, Fei G, Schandl L, Mawin C, Dietzmann K,Herrera P, et al. Reduced PTEN expression in thepancreas overexpressing transforming growth factorbeta 1. Br J Cancer 2002:86;257–262.
13 Franke TF, Yang SI, Chan TO, Datta K, Kazlzuskas A, Morrison DK, et al. The protein kinase encoded by the Akt proto-oncogene is a target of thePDGF-activated phosphatidylinositol 3-kinase. Cell
1995:81;727–736.
14 Hinemann V. Present and future treatment of pancreatic cancer. SeminOncol 2002:29;23–31.
15 Itoh N, Semba S, Itoh M, Takeda H, Kawata S, Yamakawa M. Phoshorylation of Akt/PKB is requiredfor suppression of cancer cell apoptosis and tumor
progression in human colorectal carcinoma. Cancer
2002:94;3127–3134.
16 Leslie NR, Downes CP. PTEN: The down side ofPI3-kinase signaling. Cell signal 2002:14;285–295.
17 Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang
SI, et al. PTEN, aputative proten tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 1997:275;1943–1947.
18 Malik SN, Brattain M, Ghosh PM, Troyer DA, Prihoda T, Bedolla R, et al. Immunohistochemical
demonstration of Phospho-Akt in high gleason grade
prostate cancer. Clin Cancer Res 2002:8;1168–1171.
19 McMenamin ME, Soung P, Perera S, Kaplan I, Loda
M, Selers WR. Loss of PTEN expression in paraffin-embedded primary prostate cancer correlateswith high Gleason score and advanced stage. Cancer
1999:59;4291–4296.
20 Mutter GL, Lin MC, Fitzgerald JT, Kum JB, Baak
JP, Lees JA, et al. Altered PTEN expression as a di-Acknowledgments: We thank Dr. Kiyoshi Kishi (Division of Surgery, Tottori Prefectural Central Hospital,Japan) for providing surgically resected specimens frompatients with pancreatic cancer.

References
1 Alessi DR, James SR, Downes CP, Holmes AB,Gaffney PR, Reese CB, et al. Characterization of a3-phosphoinositide-dependent protein kinase which
phosphorylates and activates protein kinase Balpha.
Curr Biol 1997:7;261–269.
2 Balendran A, Casamayor A, Deak M, Paterson A,Gaffney P, Currie R, et al. PDK1 acuires PDK2 activity in the presence of a synthetic peptide derived
from the carboxyl terminus of PRK2. Curr Biol
1999:9;393–404.
3 Bardeesy N, DePinho RA. Pancreatic cancer biology and genetics. Nat Rev Cancer 2002:2;897–909.
4 Brognard J, Clark AS, Ni Y, Dennis PA. Akt/protein
kinase B is constitutively active in non-small celllung cancer cells and promotes cellular survival andresistance to chemotherapy and radiation. Cancer
Res 2001:61;3986–3997.
5 Bondar VM, Sweeney-Gotsch B, Andreeff M, MillsGB, NcConkey DJ. Inhibition of the phoshatidylinositol 3’-kinase-AKT pathway induces apoptosis inpancreatic carcinoma cells in vivo and in vitro. MolCancer Ther 2002:12;989–997.
6 Burnet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu16

p-Akt and PTEN expression in pancreatic cancer

suppressor PTEN. Cell 1998:95;29–39.
26 Tang D, Okada H, Ruland J, Liu L, Stambolic V,Mark TW, et al. Akt is activated in response to an
apoptotic signal. J Biol Chem 2001;276:30461–30466.
27 West KA, Sianna-Castillo S, Dennis PA. Activationof the PI3K/Akt pathway and chemotherapeutic resistance. Drug Resist Update 2002:5;234–248.
28 Zhong H, Chiles K, Feldser D, Laughner E, Hanrahan C, Georgescu MM, et al. Modulation of hypoxiainducible factor 1 alpha expression by the epidermal
growth factor/phosphatidylinositol 3-kinase/PTEN/
AKT/FRAP pathway in human prostate cancer cells:
implications for tumor angiogenesis and therapeutics. Cancer Res 2000:60;1541–1545.

agnostic marker for the earliest endometrial precancers. J Natl Cancer Inst 2000:92;924–930.
21 Perez-Tenorio G, Stal O. Activation of Akt/PKB in
breast cancer predicts a worse outcome among endocrine treated patients. Br J Cancer 2002:86;540–545.
22 Perren A, Wenig LP, Boag AH, Ziebold U, Thakore K, Dahia PL, et al. Immunohistochemicalevidence of loss of PTEN expression in primary
ductal adenocarcinomas of the breast. Am J Pathol
1999:155;1253–1260.
23 Shi X, Liu S, Kleeff J, Friess H, Buchler MW. Acquired resistance of pancreatic cancer cell towards
5-Fluorouracil and gemcitabine is associated withexpression of apoptosis-regulation genes. Oncology
2002:62;354–362.
24 Sobin LH, Wittekind Ch. TNM classification ofmalignant tumuors (6ed.). New York: John Wiley &Sons; 2002. p. 93–96.
25 Stambolic V, Suzuki A, de la Pompa JL, Brothers
GM, Mirtsos C, Sasaki T, et al. Negative regulationof PKB/Akt-dpendent cell survival by the tumor

Received December 12, 2005;
accepted December 22, 2005
Corresponding author: Yoshimi Ozaki-Ohgami

17