Abstract
The serine/threonine protein kinase B/Akt plays a central role in the coordination of multiple signal transduction processes involved in transcriptional regulation, cell survival, and apoptosis. Activation of Akt kinase is a prognostic factor in several types of cancers; however, its role in gastrointestinal cancers is not fully understood. Caspase-9 is an Akt substrate that belongs to the caspase family of proteases, which function as initiators of the mitochondrial apoptotic pathway. Little is known about the role of caspase-9 phosphorylation, which downregulates the apoptotic activity of the enzyme. In this study, we investigated the expression of phosphorylated (p)-Akt and phosphorylated (p)-caspase-9 in gastric and colorectal carcinoma and the relationship between p-Akt and p-caspase-9 expression and clinicopathological parameters of gastric and colorectal cancer patients. In total, 75 samples of advanced gastric adenocarcinoma (37 well-to-moderately differentiated and 38 poorly differentiated) and 76 samples of advanced colorectal adenocarcinoma (69 well-to-moderately differentiated and 7 poorly differentiated) were analyzed for p-Akt and p-caspase-9 expression by immunohistochemistry. Our results reveal a correlation between p-Akt and p-caspase-9 expression in gastric and colorectal cancers. Levels of p-caspase-9 were significantly higher in colorectal cancer than in gastric cancer, indicating tumor-specific regulation. Although the biological role of p-Akt/p-caspase-9 signaling remains unclear, we suggest that phosphorylation of caspase-9 may be a useful tool to assess the state of gastrointestinal cancer and the effects of anti-cancer therapy.
Keywords: Phosphorylated Akt, phosphorylated caspase-9, gastrointestinal carcinoma, apoptosis, cell death pathway
Introduction
The serine/threonine protein kinase B/Akt plays a central role in the coordination of multiple signal transduction processes involved in transcriptional regulation, cell survival, and apoptosis [1-3]. Akt is a major downstream effector of phosphatidylinositide-3-kinase (PI3K), which induces Akt activation via phosphorylation at key amino acid residues Thr-308 and Ser-473 [2]. In turn, phosphorylated Akt (p-Akt) targets a number of downstream substrates, including transcription factors, pro-apoptotic Bcl-2 family members, and caspases, promoting cell survival and blocking apoptosis. As a regulator of apoptosis and cell proliferation, Akt has been considered a key factor in many types of cancer [4-8]. Phosphorylation-dependent Akt activation has been shown to contribute to cancer resistance to chemotherapy and is associated with poor prognosis in many types of malignancies, including breast, prostate, and non-small cell lung cancers [9-13].
One of the Akt substrates is caspase-9, belonging to the caspase family of proteases, which function as initiators of the mitochondrial apoptotic pathway. Caspase-9 activity is important in the executing chemotherapy-induced apoptosis. The mechanism of caspase-9 activation involves the formation of a multimeric complex with Apaf-1 and cytochrome c, termed apoptosome; another level of regulation is phosphorylation at Ser 196 and Thr 125, which inhibits caspase-9 activation [1,14,15]. It has been shown that Akt targets caspase-9 at Ser196, downregulating its activity and decreasing apoptosis [1]; however, the exact mechanism is not fully elucidated.
It has been established that the expression of activated p-Akt may be used as a prognostic factor and therapy response predictor in patients with several types of cancer. However, the role of p-Akt in gastric and colorectal cancers is not fully understood. Even less is known about the significance of caspase-9 phosphorylation and the relationship between p-Akt and p-caspase-9 in carcinogenesis.
In this study, we examined the expression rates of p-Akt and p-caspase-9 in gastric and colorectal carcinomas by immunohistochemistry. We also investigated the relationship between p-Akt and p-caspase-9 expression and clinicopathological parameters of gastric cancer patients. To the best of our knowledge, this is the first report of the direct correlation between the expression of p-caspase-9 and p-Akt in surgically resected human gastric and colorectal carcinoma tissues.
We have previously analyzed the expression of cleaved (activated) caspase-8 and caspase-9, the autophagy marker microtubule-associated protein 1 light chain 3 (LC3), and survivin to elucidate the cell death pathways in gastric and colorectal carcinomas [16,17]. The purpose of this study was to analyze the phosphorylation status of Akt and caspase-9 in gastric and colorectal carcinoma and discuss the cell death pathway based on our previous and current results.
Materials and methods
Tissue samples
In total, 75 samples of advanced gastric adenocarcinoma (37 well-to-moderately differentiated and 38 poorly differentiated) and 76 samples of advanced colorectal adenocarcinoma (69 well-to-moderately differentiated and 7 poorly differentiated) were analyzed for p-Akt and p-caspase-9 expression by immunohistochemistry. Formalin-fixed and paraffin-embedded surgically resected tumor tissues were obtained from the archives of the Department of Diagnostic Pathology of the Osaka Red Cross Hospital and the Kobe Central Hospital of Social Insurance. Three-micrometer-thick sections were cut consecutively and mounted on aminopropyltriethoxysilane-coated slides. The tumors were classified according to the TNM classification of malignant tumors [18]. The study was approved by the local ethics committee.
Immunohistochemistry
The sections were deparaffinized and subjected to heat-induced epitope retrieval using a pressure cooker (T-FAL; France) for 10 min; then, they were left at room temperature to cool in soaking solutions for 30 min. The optimal soaking solutions determined in preliminary experiments were 1 mM EDTA, pH 8.0, for phospho-Akt and 10 mM citrate buffer, pH 6.0, for phospho-caspase-9. The sections were washed in running tap water, followed by 10 mM phosphate buffered saline (PBS) pH 7.2, and incubated overnight at room temperature with a rabbit monoclonal antibody against phospho-Akt (Ser473) (1:100; Cell Signaling Technology, Danvers, MA, USA) or rabbit polyclonal antibody against phospho-caspase-9 (Ser196) (1:200; Abgent, San Diego, CA, USA). The sections were then rinsed in PBS and incubated with the secondary antibody-containing detection reagent Histofine Simple Stain MAX-PO (Nichirei, Tokyo, Japan) for 1 h at room temperature. The sections were then treated with diaminobenzidine to detect immunoreactivity and counterstained with Mayer’s hematoxylin. A section treated with PBS instead of primary antibody was used as negative control.
Immunostaining evaluation
The cytoplasmic or cell membrane staining for p-Akt and the cytoplasmic and nuclear staining for p-caspase-9 were considered positive. Each case was interpreted as positive by immunohistochemistry when the staining was observed in more than 10 of all neoplastic cells. The sections were examined for immunostaining by two independent investigators (A.S. and M. S) blinded to the experimental design.
Statistical analysis
The chi-squared test and Fisher’s exact test were used to analyze statistical differences in p-Akt and p-caspase-9 expression between gastric and colorectal adenocarcinomas, or between well-to-moderately and poorly differentiated adenocarcinomas, and the correlation of expression with patients’ age, sex, lymphatic or vascular invasion. The correlation between p-Akt and p-caspase-9 expression and tumor location, depth of invasion, lymph node metastasis, and pathological stage was analyzed using the Kruskal-Wallis test. The results were considered statistically significant when P < 0.05.
Results
P-Akt expression in gastric and colorectal carcinomas
As shown in Figure 1, p-Akt staining was localized to the cell membrane and cytoplasm, whereas p-caspase-9 was detected in the nuclei and cytoplasm. Positive immunostaining for p-Akt was observed in 30 of the 75 gastric adenocarcinoma cases (40%) and in 31 of the 76 colorectal carcinoma cases (41%); there was no significant difference in p-Akt expression between these tumor types. Positive staining for p-caspase-9 was observed in 20 of 75 (27%) and in 36 of 76 (47%) cases of gastric and colorectal carcinomas, respectively, and the difference between the tumor types was significant (P < 0.01). The expression of both p-Akt and p-caspase-9 was higher in well-to-moderately differentiated than in poorly differentiated gastric adenocarcinomas (P < 0.001 and P < 0.005, respectively), but no significant difference was observed for colorectal carcinomas (Table 1).
Figure 1.
Visualization of p-Akt and p-caspase-9 expression in gastrointestinal carcinomas by immunohistochemistry. A: Positive immunostaining for p-Akt in rectal carcinoma was localized to the cell membrane and cytoplasm of cancer cells. B: Positive immunostaining for p-caspase-9 in gastric carcinoma was localized to the nuclei and cytoplasm.
Table 1.
Expression of p-Akt and p-caspase-9 in gastric and colorectal adenocarcinoma samples
| p-Akt | p-caspase-9 | |
|---|---|---|
| Gastric adenocarcinoma (n = 75) | 30 (40%) | 20 (27%)c |
| Well-to-moderately differentiated (n = 37) | 23 (62%)a | 16 (43%)b |
| Poorly differentiated (n = 38) | 7 (18%)a | 4 (11%)b |
| Colorectal adenocarcinoma (n = 76) | 31 (41%) | 36 (47%)c |
| Well-to-moderately differentiated (n = 69) | 27 (39%) | 33 (48%) |
| Poorly differentiated (n = 7) | 4 (57%) | 3 (43%) |
P < 0.001;
P < 0.005;
P < 0.01.
Clinicopathological significance of p-Akt and p-caspase-9 expression in gastric and colorectal adenocarcinomas
The relationship between p-Akt and p-caspase-9 expression in gastric and colorectal carcinomas and clinicopathological factors (age, sex, tumor location, depth of invasion, lymph-node metastasis, lymphatic invasion, vascular invasion, or pathological stage) is presented in Table 2. In gastric carcinomas, the level of p-Akt expression was significantly positively associated with vascular invasion (P < 0.01), whereas no correlation was observed with the other clinicopathological parameters. No correlation was found between p-caspase-9 expression and clinicopathological factors in gastric carcinomas. In colorectal adenocarcinomas, the phosphorylation status of both proteins was not associated with any of the patients’ clinicopathological characteristics.
Table 2.
Correlation between p-Akt and p-caspase-9 expression and clinicopathological parameters in gastric and colorectal adenocarcinoma samples
| Gastric adenocarcinoma | Colorectal adenocarcinoma | ||||
|---|---|---|---|---|---|
|
|
|
||||
| p-Akt | p-cas 9 | p-Akt | p-cas 9 | ||
| Age (years) | Age (years) | ||||
| < 60 (n =10) | 3 (38) | 0 (0) | < 60 (n = 21) | 8 (38) | 12 (57) |
| ≥ 60 (n = 65) | 27 (41) | 20 (31) | ≥ 60 (n = 55) | 23 (42) | 24 (44) |
| Sex | Sex | ||||
| Male (n = 53) | 24 (45) | 16 (30) | Male (n = 44) | 15 (34) | 17 (39) |
| Female (n = 22) | 6 (27) | 4 (18) | Female (n = 32) | 16 (50) | 19 (59) |
| Location | Location | ||||
| Cardia (n = 18) | 8 (44) | 6 (33) | Right colon (n = 23) | 10 (43) | 11 (48) |
| Fundus (n = 32) | 12 (38) | 7 (22) | Left colon (n = 24) | 9 (38) | 14 (58) |
| Antrum (n =25) | 10 (40) | 7 (28) | Rectum (n = 29) | 12 (41) | 11 (38) |
| Depth of invasion* | Depth of invasion* | ||||
| pT2 (n = 17) | 5 (29) | 6 (35) | pT2 (n = 6) | 4 (67) | 4 (67) |
| pT3 (n = 21) | 12 (57) | 6 (29) | pT3 (n = 44) | 19 (43) | 20 (45) |
| pT4 (n = 37) | 13 (35) | 8 (22) | pT4 (n = 26) | 8 (31) | 12 (46) |
| Lymph node metastasis* | Lymph node metastasis* | ||||
| pN0 (n = 24) | 7 (29) | 6 (25) | pN0 (n = 35) | 15 (43) | 15 (43) |
| pN1 (n = 15) | 9 (60) | 6 (40) | pN1 (n = 28) | 11 (39) | 14 (50) |
| pN2 (n = 12) | 6 (50) | 3 (25) | pN2 (n = 13) | 5 (38) | 7 (54) |
| pN3 (n = 24) | 8 (33) | 5 (21) | |||
| Lymphatic invasion | Lymphatic invasion | ||||
| Ly (-) (n =16) | 6 (38) | 6 (38) | Ly (-) (n = 24) | 10 (42) | 14 (58) |
| Ly (+) (n = 59) | 6 (38) | 14(24) | Ly (+) (n = 52) | 21 (40) | 22 (42) |
| Vascular invasion | Vascular invasion | ||||
| V (-) (n = 40) | 10 (25)** | 9 (23) | V (-) (n = 38) | 17 (45) | 22 (58) |
| V (+) (n = 35) | 20 (57)** | 11 (31) | V (+) (n = 38) | 14 (37) | 14 (37) |
| UICC p-Stage* | UICC p-Stage* | ||||
| I B (n = 11) | 3 (27) | 4 (36) | IB (n = 6) | 4 (67) | 4 (67) |
| IIA and IIB (n = 23) | 11 (48) | 6 (26) | IIA and IIB (n = 28) | 10 (36) | 10 (36) |
| IIIA, IIIB and IIIC (n=41) | 6 (39) | 10 (24) | IIIA, IIIB and IIIC (n = 42) | 17 (40) | 22 (52) |
According to the TNM classification of malignant tumors, the numerals in parentheses indicate the percentage of positive tumors.
P < 0.01.
Relationship between p-Akt and p-caspase-9 expression in gastric and colorectal carcinomas
To investigate whether p-caspase-9 levels were affected by p-Akt expression, we examined a possible correlation between Akt and caspase-9 phosphorylation in gastric and colorectal carcinomas and found a significant positive correlation between p-Akt and p-caspase-9 in both types (r = 0.47, P < 0.0001; r = 0.59, P < 0.0001, respectively).
Discussion
In the present study, we examined the phosphorylation status of Akt kinase and caspase-9 in gastric and colorectal carcinomas by immunohistochemistry. Among gastric and colorectal samples, 40 and 41% stained positive for pAkt, while 27 and 47% were positive for p-caspase-9, respectively. We found that the expression of p-caspase-9 was significantly higher in colorectal than in gastric carcinomas (P < 0.01), although there was no significant difference in p-Akt staining. In our previous studies, we attempted to elucidate the cell death pathways in gastric and colorectal carcinoma by analyzing the expression of cleaved caspase-8 and caspase-9, the autophagy marker microtubule-associated protein 1 light chain 3 (LC3), and survivin [16,17]. We found that the expression of cleaved caspase-9 was significantly lower in colorectal than in gastric carcinoma (P < 0.0001) [16]. On the contrary, LC3 and survivin expression was significantly higher in colorectal than in gastric carcinoma (P < 0.0001 and P < 0.01, respectively) [16,17], suggesting that different cell-death pathways may be activated in these types of tumors. Caspase-9, an initiator of the mitochondrial apoptotic pathway, is activated upon complexing with cytochrome c and Apaf-1, which cleaves the pro-enzyme of caspase-9 into the active form. Once initiated, caspase-9 cleaves procaspase-3 and procaspase-7, which then execute the apoptotic program. In contrast, caspase-9 activity can be downregulated via phosphorylation by pro-apoptotic protein kinases, including Akt. The results of the present study showing that colorectal carcinomas had much higher levels of phosphorylated caspase-9 than gastric tumors are consistent with lower expression of activated cleaved caspase-9, suggesting that the high expression of p-caspase-9 in colorectal carcinoma may be part of the mechanism to suppress the mitochondrial apoptotic pathway via caspase-9 inhibition.
We also showed that the phosphorylation of both Akt and caspase-9 was enhanced in well-to-moderately differentiated gastric adenocarcinomas compared to that in poorly differentiated tumors (P < 0.001 and P < 0.005, respectively). Other researchers reported that p-caspase-9 (Thr 125) expression was higher in intestinal than in diffuse gastric carcinomas, although there was no significant difference in caspase-9 or Akt phosphorylation among histological subtypes [19,20]. In contrast, Murakami et al. reported that p-caspase-9 expression was lower in differentiated than in undifferentiated tumors but, similar to the other study, there was no difference in Akt phosphorylation among histological subtypes [21]. Further investigations are needed to clarify the relationship between p-Akt or p-caspase-9 levels and tumor differentiation.
The analysis of the relationship between phosphorylation status and clinicopathological parameters showed that in gastric carcinomas, the level of p-Akt was significantly higher in the cases with vascular invasion than in those without invasion (P < 0.01), whereas other clinicopathological factors were not associated with Akt or caspase-9 phosphorylation. Similarly, Murakami et al. reported that p-Akt expression in gastric carcinoma showed no significant correlation with tumor size, pathology, or invasion [21]; however, they found significant positive association of Akt phosphorylation with poor prognosis, especially for T3/T4 gastric cancer. Other studies have also indicated that p-Akt expression may help to predict the clinical outcome for gastric cancer patients. Nam et al. in a study of 311 gastric carcinoma cases showed that p-Akt was highly expressed in the early stage of pTNM and inversely correlated with lymphatic invasion or lymph node metastasis [20], while Henderson-Jackson et al. showed p-Akt upregulation during the transition from normal colorectal mucosa to carcinoma, supporting the role of Akt activation in colon carcinogenesis [22]. In contrast, a large cohort study by Baba et al. showed that p-Akt expression in colorectal cancer was associated with low disease stage and favorable prognosis [23].
Activated Akt may phosphorylate caspase-9 and inhibit its pro-apoptotic activity. In this study, we show a correlation between Akt and caspase-9 phosphorylation both in gastric and colorectal carcinoma. The levels of p-caspase-9 were significantly higher in colorectal than in gastric carcinoma, indicating tumor-specific regulation. Although the biological role of p-Akt/p-caspase-9 signaling remains unclear, we believe that the phosphorylation of caspase-9 may be a useful tool to assess the state of cancer development and cancer therapy.
In conclusion, our investigation suggests that phosphorylation-dependent activation of Akt and inhibition of caspase-9 may be inter-related and that the p-Akt/p-caspase-9 pathway may constitute a possible mechanism of apoptotic suppression specific for colorectal and gastric carcinomas. Further elucidation of tumor-specific apoptotic pathways will provide a basis for the development of individualized approaches to cancer therapy.
Acknowledgements
The authors thank Dr. Masayuki Shintaku and Dr. Toshihiko Miyake for their kind support. This study was supported by a Grant-in-Aid for Scientific Research (No. 23590396) from the Japan Society for the Promotion of Science.
Disclosure of conflict of interest
None.
References
- 1.Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S, Reed JC. Regulation of cell death protease caspase-9 by posphorylation. Science. 1998;282:1318–1321. doi: 10.1126/science.282.5392.1318. [DOI] [PubMed] [Google Scholar]
- 2.Downward J. Mechanisms and consequences of activation of protein kinase B/Akt. Curr Opin Cell Biol. 1998;10:262–7. doi: 10.1016/s0955-0674(98)80149-x. [DOI] [PubMed] [Google Scholar]
- 3.Shtilbans V, Wu M, Burstein DE. Current overview of the role of Akt in cancer studies via applied mmunohistochemistry. Ann Diagn Pathol. 2008;12:153–60. doi: 10.1016/j.anndiagpath.2007.12.001. [DOI] [PubMed] [Google Scholar]
- 4.Robey RB, Hay N. Is Akt the “Warburg kinase”? Akt-energy metabolism interactions and oncogenesis. Semin Cancer Biol. 2009;19:25–31. doi: 10.1016/j.semcancer.2008.11.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Cicenas J. The potential role of Akt phosphorylation in human cancers. Int J Biol Markers. 2008;23:1–9. doi: 10.1177/172460080802300101. [DOI] [PubMed] [Google Scholar]
- 6.Michl P, Downward J. Mechanisms of disease: PI3K/AKT signaling in gastrointestinal cancers. Z Gastroenterol. 2005;43:1133–9. doi: 10.1055/s-2005-858638. [DOI] [PubMed] [Google Scholar]
- 7.Kobayashi I, Semba S, Matsuda Y, Kuroda Y, Yokozaki H. Significance of Akt phosphorylation on tumor growth and vascular endothelial growth factor expression in human gastric carcinoma. Pathobiology. 2006;73:8–17. doi: 10.1159/000093087. [DOI] [PubMed] [Google Scholar]
- 8.Roy HK, Olusola BF, Clemens DL, Karolski WJ, Ratashak A, Lynch HT, Smyrk TC. AKT protooncogene overexpression is an early event during sporadic colon carcinogenesis. Carcinogenesis. 2002;23:201–5. doi: 10.1093/carcin/23.1.201. [DOI] [PubMed] [Google Scholar]
- 9.Kirkegaard T, Witton CJ, McGlynn LM, Tovey SM, Dunne B, Lyon A, Bartlett JM. AKT activation predicts outcome in breast cancer patients treated with tamoxifen. J Pathol. 2005;207:139–46. doi: 10.1002/path.1829. [DOI] [PubMed] [Google Scholar]
- 10.Perez-Tenorio G, Stal O. Activation of AKT/PKB in breast cancer predicts a worse outcome among endocrine treated patients. Br J Cancer. 2002;86:540–5. doi: 10.1038/sj.bjc.6600126. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Tokunaga E, Kimura Y, Oki E, Ueda N, Futatsugi M, Mashino K, Yamamoto M, Ikebe M, Kakeji Y, Baba H, Maehara Y. Akt is frequently activated in HER2/neu-positive breast cancers and associated with poor prognosis among hormone-treated patients. Int J Cancer. 2006;118:284–9. doi: 10.1002/ijc.21358. [DOI] [PubMed] [Google Scholar]
- 12.Schlieman MG, Fahy BN, Ramsamooj R, Beckett L, Bold RJ. Incidence, mechanism and prognostic value of activated AKT in pancreas cancer. Br J Cancer. 2003;89:2110–5. doi: 10.1038/sj.bjc.6601396. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Tang JM, He QY, Guo RX, Chang XJ. Phosphorylated Akt overexpression and loss of PTEN expression in non-small cell lung cancer confers poor prognosis. Lung Cancer. 2006;51:181–91. doi: 10.1016/j.lungcan.2005.10.003. [DOI] [PubMed] [Google Scholar]
- 14.Shi Y. Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell. 2002;9:459–70. doi: 10.1016/s1097-2765(02)00482-3. [DOI] [PubMed] [Google Scholar]
- 15.Allan LA, Clarke PR. Apoptosis and autophagy: Regulation of caspase-9 by phosphorylation. FEBS J. 2009;276:6063–73. doi: 10.1111/j.1742-4658.2009.07330.x. [DOI] [PubMed] [Google Scholar]
- 16.Shintani M, Sangawa A, Yamao N, Miyake T, Kamoshida S. Immunohistochemical analysis of cell death pathways in gastrointestinal adenocarcinoma. Biomed Res. 2011;32:379–86. doi: 10.2220/biomedres.32.379. [DOI] [PubMed] [Google Scholar]
- 17.Shintani M, Sangawa A, Yamao N, Kamoshida S. Immunohistochemical expression of nuclear and cytoplasmic survivin in gastrointestinal carcinoma. Int J Clin Exp Pathol. 2013;6:2919–27. [PMC free article] [PubMed] [Google Scholar]
- 18.Sobin LH, Gospodarowicz MK, Wittekind C eds. TNM Classification of Malignant Tumours. 7th edition. New Jersey: Wiley-Blackwell; 2009 [Google Scholar]
- 19.Yoo NJ, Lee SH, Jeong EG, Lee SH. Expression of phosphorylated caspase-9 in gastric carcinomas. APMIS. 2007;115:354–9. doi: 10.1111/j.1600-0463.2007.apm_632.x. [DOI] [PubMed] [Google Scholar]
- 20.Nam SY, Lee HS, Jung GA, Choi J, Cho SJ, Kim MK, Kim WH, Lee BL. Akt/PKB activation in gastric carcinomas correlates with clinicopathologic variables and prognosis. APMIS. 2003;111:1105–13. doi: 10.1111/j.1600-0463.2003.apm1111205.x. [DOI] [PubMed] [Google Scholar]
- 21.Murakami D, Tsujitani S, Osaki T, Saito H, Katano K, Tatebe S, Ikeguchi M. Expression of phosphorylated Akt (pAkt) in gastric carcinoma predicts prognosis and efficacy of chemotherapy. Gastric Cancer. 2007;10:45–51. doi: 10.1007/s10120-006-0410-7. [DOI] [PubMed] [Google Scholar]
- 22.Henderson-Jackson EB, Helm J, Ghayouri M, Hakam A, Nasir A, Leon M, Bui M, Yeatman T, Coppola D. Correlation between Mcl-1 and pAKT protein expression in colorectal cancer. Int J Clin Exp Pathol. 2010;3:768–74. [PMC free article] [PubMed] [Google Scholar]
- 23.Baba Y, Nosho K, Shima K, Hayashi M, Meyerhardt JA, Chan AT, Giovannucci E, Fuchs CS, Ogino S. Phosphorylated AKT expression is associated with PIK3CA mutation, low stage, and favorable outcome in 717 colorectal cancers. Cancer. 2011;117:1399–408. doi: 10.1002/cncr.25630. [DOI] [PMC free article] [PubMed] [Google Scholar]