Yonago Acta medica 2006;49:19–27

Expression of Phospho-Akt and PTEN Proteins in Human
Breast Cancer in Relation to Tumor Progression andPatient Survival
Osamu Yamamoto, Yoshihito Gomyo, Yasuaki Hirooka, Shunichi Tsujitani andMasahide Ikeguchi
Division of Surgical Oncology, Department of Surgery, School of Medicine, Tottori University
Faculty of Medicine, Yonago 683-8504 Japan

Phosphatidylinositol 3-kinase (PI3-kinase) controls mitogenesis, cellular growth andtransformation in a variety of cancers. The serine-threonine kinase Akt is a downstream target of PI3-kinase, and phosphorylated Akt (Phospho-Akt) inhibits apoptosis.
Phosphatase and tensin homolog detected on chromosome ten (PTEN) is a tumor suppressor that antagonizes PI3-kinase activity, negatively regulates its downstream-target,
Akt, inhibits phosphorylation of Akt, and medicates cell-cycle arrest and apoptosis. To
clarify whether the PI3-kinase/Akt pathway and PTEN relate to breast cancer, we examined the expression of pathway-related proteins such as Phospho-Akt and PTEN in clinical specimens. Immunohistochemical analysis was performed on tissue specimens surgically obtained from 221 patients with breast cancer. The association of Phospho-Aktand PTEN expression with clinicopathological variables and the prognosis of patientswere analyzed. Of 221 breast carcinomas, positive Phospho-Akt expression was observed in 91 (41.1%) and positive PTEN expression in 119 (53.8%). Phospho-Akt expression and loss of PTEN expression signiﬁcantly correlated with tumor staging, tumor sizeand lymph node metastasis. Patients with Phospho-Akt-positive tumors had signiﬁcantly inferior disease-free survival or over-all survival to those with Phospho-Akt-negativetumors, while those with PTEN positive tumors were better than those with PTEN negative tumors. Moreover, patients with Phospho-Akt-positive and PTEN-negative tumors
had a signiﬁcantly inferior disease-free survival and over-all survival compared to thosewith Phospho-Akt-negative and PTEN-positive tumors. Multivariate analysis revealed
that expression of Phospho-Akt and tumor size were the independent factors (P = 0.024).
We demonstrated that the expression of Phospho-Akt signiﬁcantly correlated with tumor
progression and patients survival with breast cancer. Phospho-Akt/PTEN expression
status is possibly a deﬁnitive prognostic factor in clinical breast cancer.
Key words: breast cancer; immunohistochemistry; phosphatase and tensin homolog detectedon chromosome ten; phosphorylated-Akt; prognosis
Aber rant functions of phosphatidylinositol
3-kinase (PI3-kinase) contribute to mitogenesis,
cellular growth and transformation in a varietyof cancers including breast cancer (Carpenter etal., 1990; Varticovski et al., 1994). PI3-kinaseis activated by insulin, various growth factors

Abbreviations: ER, estrogen receptor; PBS, phosphate-buffered saline; PgR, progesterone receptor; PI3-kinase, phosphatidylinositol 3-kinase; Phospho-Akt, phosphorylated-Akt; PTEN, phosphatase and tensin homolog detected on chromosome ten19

O. Yamamoto et al.

lates cell cycle progression and survival.
Breast cancer is the most common cancer inwomen, accounting for about 30% of all female
malignancies, and is a major cause of morbidity
and mortality in the female population (Landis etal., 1998). In this report, to clarify whether thePI3-kinasse/Akt pathway and PTEN are related
to breast cancer, we examined the expression ofpathway-related proteins such as Phospho-Aktand PTEN in clinical specimens of breast cancer.

and cytokines (Burgering and Coffer, 1995), andcatalyzes the phosphorylation of inositol phospholipids at the 3 position to generate phosphatidylinositol 3,4,5-trisphosphate and phosphatidyliositol 3,4-bisphosphate. Serine-threonine kinase
Akt is a downstream target of PI3-kinase. Akt is
phosphorylated at Thr-308 and Ser-473 by PI3kinase, thus Akt is converted to phosphorylated
Akt (Phospho-Akt) (Franke et al., 1997), which
phosphorylates BAD, caspase-9, Forkhead transcription factors and IκB kinase α which correlatewith apoptosis (Datta et al., 1997; Ozes et al.,1999). Akt has also been shown to inhibit theRaf-MEK-ERK pathway through phosphorylationof Raf-1 in myotubes, and to overcome constitutively activated MAPK-induced cell-cycle arrest
in MCF7 cells (Rommel et al., 1999). Akt is thus
an important regulator of cell proliferation andsurvival. Amplification of genes encoding Aktisoforms has been found in several types of human cancers. Phospho-Akt plays an importantrole in the development and/or progression of asubset of human cancers.
In addition, mutations in the phosphataseand tensin homolog deleted on chromosome ten(PTEN), one of the most frequently mutatedtumor suppressor genes on 10q23.3, results inelevated Akt activity. Germline mutations ofPTEN/MMAC1/TEP1 has been reported to correlate with Cowden syndrome, Bannayan-RileyRuvalcade, proteus and proteus-like syndromes(Marsh et al., 1999; Zhou et al., 2001). PTEN
antagonizes PI3-kinase activity, negatively regulates its downstream-target Akt and mediates cellcycle arrest and apoptosis. Speciﬁcally, cell line
analyses have shown that PTEN appears to suppress breast cancer growth by down-regulation ofPI3-kinase, with resultant G1 arrest and cell death(Weng et al., 1999). Studies of embryonic stem
cells have shown that cells featuring mutationsof the PTEN gene exhibited an increased growthrate and an advanced entry into S-phase (Sun etal., 1999). The accelerated G1/S transition wasaccompanied by down-regulation of p27, a major
inhibitor of G1 cyclin-dependent kinases. Overall, results have demonstrated that PTEN regu-Materials and Methods
Specimen collection
Specimens, embedded in parafﬁn, which had beencollected from patients with breast cancer who
underwent surgery at Tottori University Hospital,
Yonago, and Tottori Red Cross Hospital, Tottori,
between 1987 and 2002, were examined. There
were 221 post-operated patients, aged from 25 to87 years (mean, 55.5), with a mean follow-up of
67.8 months (range, 7–180). Fifty-three (23.9%)
patients had a relapse and 35 (15.8%) died of thecancer, while 176 (79.6%) were alive at the time ofthe completion of the study (August 2003). Cancer was residual in 8 patients and the disease freesurvival of 13 patients was unknown. Eighty-one(41.2%) patients were at stage I, 59 (26.7%) stageIIA, 34 (15.4%) stage IIB, 27 (12.2%) stage IIIA,1 (0.5%) stage IIIB and 9 (4.1%) stage IV. The
clinicopathological ﬁndings were determined according to tumor-node-metastasis (Stmad, 1999).
The patients were given adjuvant chemotherapy
according to St. Gallen recommendations from1998.
Histopathological examination
Resected specimens were fixed in 10% bufferedformalin for 48 h, and then were cut into 5-mmslices. Slices were embedded in parafﬁn, and thetissue parafﬁn blocks were cut into 4-µm-thick sections for hematoxylin-eosin and immunostaining.
20

Phospho-Akt and PTEN in breast cancer

Fig. 1. Immunohistochemical staining patterns for Phospho-Akt expression in breast cancer.
Phospho-Akt antigen exists in both the cytoplasm and nucleus (× 100).

Fig. 2. PTEN expression in breast cancer. PTEN expression is found in the cytoplasm (× 100).

tissue sections underwent antigen retrieval withthe target retrieval solution at pH 6.1 (Dako,Grostrup, Denmark) in an autoclave at 120˚C for
15 min. The slides for immunohistochemistry for
PTEN were placed in citric acid buffer (10 mM)

Immunohistochemistry for Phospho-Aktand PTEN
The 4 µm-thick sections were dewaxed using xylene and transferred to alcohol. Deparaffinized21

O. Yamamoto et al.

and heated in an autoclave at 120˚C for 15 min.
Endogenous peroxidase activity was blocked byincubating sections with 0.3% hydrogen peroxide in methanol for 10 min at room temperature.
Slides were then washed 3 times in phosphatebuffered saline (PBS) and incubated in 10%
normal goat serum for 1 h to reduce nonspeciﬁc
antibody binding. Then slides were incubatedovernight at 4˚C with the following primary antibodies: polyclonal antibodies raised against
Phospho-Akt (Ser 473) (diluted 1:50; Cell Signaling Technology, Danvers, MA) and PTEN (diluted1:100; Upstate Biotechnology, Lake Placid, NY).
After washing the slides with PBS, biotinylated
antibodies were applied as second antibody: antirabbit immunoglobulin (IgG + IgA + IgM) conjugated with biotin for 1 h , followed by incubation
with a sterptvidin-peroxidase complex (HistoﬁneABC kit; Nichirei, Tokyo, Japan) for 1 h at room
temperature. The reaction products were visualized with diaminobenzidine as the chromogen andthe slides were counterstained with hematoxylin.
All immunostained sections were evaluated in ablinded manner with no knowledge of the clinicaland pathological factors. Intensity is designatedas negative when less than 5% of tumor cells arestained and positive when over 5% of tumor cells
are stained.

Statistical analysis
The χ2 test was used to determine the signiﬁcanceof the association between different variables.
The comparison of Phospho-Akt and PTEN expression with disease-free survival and over-allsurvival were performed by univariate and multivariate analysis using the Kaplan-Meier test andCox proportion hazard model. The level of signiﬁcance was set at P < 0.05.

Results
Phospho-Akt and PTEN expression
The immunoreactivity of Phospho-Akt was predominantly observed in the cytoplasm and thenucleus (Fig. 1). The expression of Phospho-Aktprotein was seen in 91 (41.1%) of the breast tumors. Phospho-Akt expression signiﬁcantly correlated with staging, tumor size and lymph nodemetastasis (Table 1). PTEN expression was foundin the cytoplasm of breast cancer cells; shownin Fig. 2. Of the 221 breast tumors, 119 (53.8%)
showed PTEN protein expression, which signiﬁcantly correlated inversely with tumor stage, tu-Table 1. Immunohistochemical staining for Phospho-Akt and PTEN in relation to the clinicalstage in the current study
VariableAgeSize
2 cm ≥2 cm <Lymph node metastasis
AbsentPresentHistological stageIIIAIIBIIIAIIIBIV

Negative

Phospho-Akt
Positive

P value

Negative

PTEN
Positive

P value

60.7 ± 10.6

48.6 ± 10.4

< 0.0556.0 ± 14.0

54.7 ± 10.3

0.96

8050

3160

< 0.053270

7940< 0.05102283259< 0.05

4755

87
32

< 0.0571
35121002

2024
22
717

< 0.052427
25210567329614< 0.0522