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BIOPHA-3384; No. of Pages 5 Biomedicine & Pharmacotherapy xxx (2014) xxx–xxx
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Original article
Clinicopathological significance of PI3K, Akt and survivin expression in gastric cancer Yongjuan Gu a,1, Shidai Jin a,1, Feng Wang b, Yibin Hua b, Li Yang b, Yongqian Shu a, Zhihong Zhang c,*,2, Renhua Guo a,*,3 a b c
Department of Medical Oncology, the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Rd, Nanjing, Jiangsu 210029, PR China Department of Surgery, the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Rd, Nanjing, Jiangsu 210029, PR China Department of Pathology, the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Rd, Nanjing, Jiangsu 210029, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 January 2014 Accepted 4 March 2014
Background: Gastric cancer is the second leading cause of cancer-related deaths worldwide, and is characterized by invasion and metastasis. Increasing attention is being focused on discovering molecular markers for diagnosis and prognosis. Our objective was to evaluate PI3K, Akt and survivin protein expression in gastric cancer, and their correlations with clinicohistological features and prognosis in patients with gastric cancer. Methods: Tissue samples were obtained from 70 patients with gastric cancer patients and 20 patients with normal gastric mucosa. The protein levels of PI3K, Akt and survivin were evaluated by immunohistochemistry. Statistical analyses were performed to establish the correlations between their expressions and patients’ clinicopathologic characteristics. Results: The positive expression rates of PI3K, Akt and survivin were significantly higher in the gastric cancer tissues compared to normal gastric mucosa (P < 0.05). Expression levels of PI3K, Akt and survivin proteins were significantly correlated with TNM stage, differentiation grade, lymph node metastasis and metastases to other organs (P < 0.05). Cooperative relationships were identified between PI3K and Akt, and PI3K and survivin (P < 0.01), suggesting the involvement of the PI3K/Akt/survivin signaling pathway in the tumorigenesis of gastric cancer. Conclusions: Protein expression of PI3K, Akt and survivin were significantly associated with the development, progression and metastasis of gastric cancer and may have value as diagnostic and prognostic markers in gastric cancer. ß 2014 Published by Elsevier Masson SAS.
Keywords: Gastric cancer PI3K/Akt/survivin Immunohistochemistry
1. Introduction Gastric cancer is one of the most common malignancies and the second leading cause of cancer-related deaths worldwide [1]. Over half of these cases occur in Eastern Asia, with the majority in China. Gastric cancer is characterized by invasion and metastasis which are the main factors contributing to its high mortality rate [2,3]. Despite improvements in palliative chemotherapy and
* Corresponding authors. E-mail addresses: [email protected] (Z. Zhang), [email protected], [email protected] (R. Guo). 1 Yongjuan Gu and Shidai Jin contributed equally to this work and share first authorship. 2 Tel.: +86 25 68 13 65 45; fax: +86 25 83 72 44 40. 3 Tel.: +86 25 68 13 67 14; fax: +86 25 83 71 00 40.
radiotherapy techniques, the survival rate of patients with advanced gastric cancer remains poor [4,5]. Considerable attention is being directed towards the discovery of molecular markers for diagnosis and prognosis, and to develop effective treatments for gastric cancer [6]. Currently, oncogenes and angiogenic and growth factors are most researched [7,8]. The phosphatidylinositol 3-kinase/protein kinase B (PI3K/ Akt) signaling pathway regulates many diverse cellular processes. Abnormal activation of this pathway is frequently observed in cancer, including lung, colon, pancreatic, prostate, breast, stomach and ovarian carcinomas [9]. Survivin is a member of the inhibitor of apoptosis protein (IAP) family, which is involved in the regulation of cell division and suppression of apoptosis [10]. The PI3K/Akt signaling pathway has been found to regulate survivin in human hepatocellular carcinoma HepG2 cells [11]; however, there are few reports on the effect of the
http://dx.doi.org/10.1016/j.biopha.2014.03.010 0753-3322/ß 2014 Published by Elsevier Masson SAS.
Please cite this article in press as: Gu Y, et al. Clinicopathological significance of PI3K, Akt and survivin expression in gastric cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.010
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PI3K/Akt/survivin signaling pathway in gastrointestinal cancer. In this study, we investigated the expressions of PI3K, Akt and survivin in gastric cancer tissues compared to normal gastric mucosa, and explored their relationships with clinicopathologic and prognostic characteristics in gastric cancer. Our aim was to determine their potential value as biological and prognostic markers in the occurrence and development of gastric cancer. 2. Materials and methods 2.1. Patients and tissue samples We selected 70 patients with pathologic (p-) stages I–IV gastric cancer who had undergone complete tumor resection and nodal dissection at the First Affiliated Hospital of Nanjing Medical University between February 2011 and July 2012. None of patients had received any preoperative therapy. Patients with a history of another malignancy or who died within 4 weeks of surgery were excluded from the study. In addition, 20 normal tissue samples were obtained as controls from patients with chronic gastritis who had undergone endoscopic biopsy. The study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University, and informed consent was obtained from each patient. The 70 patients with gastric cancer included 48 males and 22 females, with a median age of 57 years (range, 23–79 years). The histological classification, grading, and pathologic tumor-nodemetastasis (TNM) staging of the tumors were based on criteria defined by the 7th American Joint Committee on Cancer (AJCC) [12]. These showed that 27 patients had poorly differentiated tumors and 43 patients had moderately or well differentiated tumors; six patients were stage I, eight were stage II, 44 were stage III, and 12 were stage IV. Lymph node metastasis was found in 55 cases. 2.2. Immunohistochemistry The surgical samples and gastric biopsies were fixed in 10% formalin and embedded in paraffin (FFPE). Four serial sections, 4 mm thick, were cut from representative blocks from each case. One section was subjected to conventional hematoxylin and eosin (H&E) staining for standard pathologic examination, and the other three sections were used for immunohistochemical staining. Immunohistochemical staining was performed according to the EnVision two-step technique. FFPE sections were mounted on superfrost microscope slides, deparaffinized and rehydrated in a graded ethanol series. They were exposed to 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity, and heat-induced antigen retrieval was performed by pretreating the slides in 0.01 M citrate buffer (pH 6.0) at 98 8C for 15 min in a microwave oven. The sections were incubated for 10 min at room temperature with normal serum before being incubated with primary antibody (1:100) at 4 8C overnight. The slides were then rinsed in Tris-buffered saline (TBS) for 10 min and incubated with secondary antibody for 30 min at room temperature. The following primary antibodies were used: PI3K rabbit polyclonal anti-human antibody; Akt rabbit polyclonal anti-human antibody; survivin goat polyclonal anti-human antibody (Cell Signaling Technology; Beverly, MA, USA). The corresponding secondary antibodies were obtained from Zhongshan Golden Bridge Biotechnology (Beijing, China). The slides were developed with diaminobenzidine (DAB) for 5 min, counterstained with hematoxylin, dehydrated, cleared and mounted. Sections treated with phosphate buffered saline (PBS) instead of primary antibody were used as negative controls; slides mounted with known gastric cancer samples were used as positive controls. All the
slides were processed by the same technician under the same laboratory conditions. The immunohistochemical results were evaluated simultaneously by two independent observers who had no prior knowledge of the patients. PI3K and survivin proteins in the cytoplasm were stained brown-yellow, while Akt protein in the nuclei was stained brown-yellow or brown. The percentages of positively stained cells were calculated by counting 200 cells from four different fields under high magnification, with. The staining intensities were scored as follows: colorless (0); buffy (1); lightbrown (2); or brown-yellow (3). The extent of staining was scored as the percentage of positive cells as follows: negative (0); 10% (1); 11%–50% (2); 51%–75% (3); > 75% (4). The product of the two scores was calculated. A protein was considered to be overexpressed if the final score > 2. 2.3. Statistical analysis All statistical analyses were performed using SPSS v. 16.0 software (SPSS; Chicago, IL, USA). The x2-test and Fisher’s exacttest were used to identify correlations between the expression levels of PI3K, Akt and survivin with patients’ clinicopathologic characteristics. Spearman’s rank correlation-test was used to determine the relationships between PI3K, Akt and survivin protein expressions. P-values were based on two-tailed statistical analysis. A P-value < 0.05 was considered statistically significant. 3. Results 3.1. Expression rates of PI3K, Akt and survivin in gastric cancer tissue in relation to normal gastric mucosa The positive expression rates for PI3K, Akt and survivin proteins in the 70 gastric cancer tissue samples were 74.3% (52/70), 37.1% (26/70) and 62.9% (44/70), respectively. The corresponding rates in the 20 normal gastric mucosa samples were 30% (6/20), 0% (0/20) and 0% (0/20), respectively (Fig. 1). These results demonstrated that expression of PI3K, Akt and survivin were significantly higher in gastric cancer tissues compared to normal gastric mucosa (P < 0.05; Table 1). 3.2. The relationships between protein expressions of PI3K, Akt and survivin and clinicopathologic features in gastric cancer The expressions of PI3K, Akt and survivin proteins in the 70 gastric cancer tissue samples were significantly correlated with differentiation grade, TNM stage, lymph node metastasis and metastases to other organs (P < 0.05). In contrast, no significant associations were observed with gender, age, carcinoembryonic antigen (CEA) and tumor size (P > 0.05; Table 2). 3.3. The relationships between the expression of PI3K, Akt and survivin proteins in gastric cancer tissue Analysis of PI3K, Akt and survivin immunohistochemical staining patterns showed that PI3K and Akt were positively coexpressed in 24/70 (34.3%) gastric cancer tissue samples and negatively co-expressed 16/70 (22.9%) samples; PI3K and survivin were positively co-expressed in 40/70 (57.1%) samples and negatively co-expressed in 14/70 (20.0%) samples; Akt and survivin were positively co-expressed in 19/70 (27.1%) samples and negatively co-expressed in 19/70 (27.1%) samples (Table 3). Correlation analyses identified positive associations between PI3K and Akt (r = 0.336; P < 0.001); and between PI3K and survivin (r = 0.528; P < 0.001), where r is the correlation coefficient.
Please cite this article in press as: Gu Y, et al. Clinicopathological significance of PI3K, Akt and survivin expression in gastric cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.010
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Fig. 1. Immunohistochemical staining of PI3K, Akt and survivin proteins in gastric cancer tissues. Light-brown or brown-yellow staining indicates positive expression of the proteins: 1: negative protein expression; 2: positive protein expression (score: 1+); 3: positive protein expression (score: 2+). Magnification 200.
Table 1 Positive expression rates of PI3K, Akt and survivin proteins in gastric cancer tissues and normal gastric mucosa samples. Protein and pathology type
n
Positive n (%)
P value
PI3K Gastric cancer Normal gastric mucosa
70 20
52 (74.3) 6 (30.0)
0.014
Akt Gastric cancer Normal gastric mucosa
70 20
26 (37.1) 0 (0.0)
0.047
Survivin Gastric cancer Normal gastric mucosa
70 20
44 (62.9) 0 (0.0)
0.001
However, no association was found between Akt and survivin (P > 0.05) in gastric cancer tissue. 4. Discussion PI3K is a heterodimer with p85 regulatory and p110 catalytic subunits [13]. The p110 subunit has protein kinase homology, and activities in common with protein kinases and phospholipases. These enzymes catalyze phosphorylation at the D3 position of PI(4)P and PI(4,5)P2 to produce PI(3,4,5)P3. PIP3 is generated by activation of PI3K in response to growth factors and other stimuli.
PIP3 then activates downstream signaling pathways by phosphorylating several kinases which are involved in the regulation of a variety of cellular processes, including 3-phosphoinositide-dependent kinase-1 (PDK1) and Akt [14]. PIP3 binds to the PH domains of Akt, leading to Akt membrane recruitment, which alters the structure of Akt to allow subsequent phosphorylation by PDK1. Phosphorylated Akt (pAkt) is the active form of Akt. This is released from the cellular membrane to the cytoplasm, where it participates in molecular processes which promote cell growth, proliferation, such as glycometabolism, protein synthesis, and antiapoptotic activities [15,16]. Overactivation of PI3K has been associated with oncogenesis; conversely, the PI3K inhibitor LY294002 was found to block the PI3K signaling pathway to inhibit cell proliferation and induce apoptosis [17]. Antagonism to PI3K is often absent in mutated or deficient tumor suppressor genes, such as PTEN. This often results in disorders to physiological functions and normal metabolic activities and overexpression of Akt, which can lead to excessive cell proliferation and malignant transformation [18]. The role of Akt in tumorigenesis was confirmed in a model that showed infection with retroviral vectors expressing activated forms of Akt led to the formation of malignant tumors [19]. The central role played by the PI3K/Akt signaling pathway in the regulation of normal cell growth and apoptosis has made this pathway a target in cancer research. Survivin is a recently discovered IAP that regulates cell division and suppresses apoptosis. Abnormal activation of survivin can lead
Please cite this article in press as: Gu Y, et al. Clinicopathological significance of PI3K, Akt and survivin expression in gastric cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.010
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Table 2 Relationships between the expressions of PI3K, Akt and survivin and the clinicopathologic characteristics in patients with gastric cancer. Clinicopathologic parameters
n
PI3K
Akt
Survivin
Positive n (%)
P value
Positive n (%)
P value
Positive n (%)
P value
30 40
23 (76.7) 29 (72.5)
0.693
8 (26.7) 18 (45.0)
0.116
21 (70.0) 23 (57.5)
0.284
Gender Male Female
48 22
34 (70.8) 18 (81.8)
0.329
18 (37.5) 8 (36.4)
0.927
32 (66.7) 12 (54.5)
0.330
Tumor size 5 cm < 5 cm
42 28
32 (76.2) 20 (71.4)
0.655
17 (40.5) 9 (32.1)
0.480
28 (66.7) 16 (57.1)
0.419
CEA High Normal
11 59
7 (63.6) 45 (76.3)
0.614
4 (36.4) 22 (37.3)
1.000
7 (63.6) 37 (62.7)
1.000
Differentiation Moderately/well Poorly
43 27
28 (65.1) 24 (88.9)
0.027
7 (16.3) 19 (70.4)
0.000
22 (51.2) 22 (81.5)
0.011
TNM stage I–II III–IV
14 56
6 (42.9) 46 (82.1)
0.008
2 (14.3) 24 (42.9)
0.048
5 (35.7) 39 (69.6)
0.019
LN metastasis 1 0
55 15
47 (85.5) 5 (33.3)
0.000
25 (45.5) 1 (6.7)
0.006
42 (76.4) 2 (13.3)
0.000
Metastasis Yes No
12 58
12 (100.0) 40 (69.0)
0.025
8 (66.7) 18 (31.0)
0.046
12 (100.0) 32 (55.2)
0.009
Age 60 < 60
CEA: carcinoembryonic antigen; TNM: tumor-node-metastasis; LN: lymph node.
Table 3 The relationships between PI3K, Akt and survivin protein expression in gastric cancer. Survivin
Akt +
+
PI3K + Survivin +
16 28
2 24
14 12
4 40
19 25
7 19
– –
– –
to aberrant cell cycle progression, inhibition of apoptosis and induction of carcinogenesis. Survivin is abundantly expressed in fetal tissues and is overexpressed in many human cancers [20]. Several studies have demonstrated that overexpression of survivin is correlated with poor prognosis in patients with gastric cancer, and contributes to chemoresistance [21]. However, a study by Kania et al. found that survivin protein was undetectable in normal gastric mucosa, and survivin mRNA was only weakly detectable. In contrast, both survivin mRNA and protein were significantly increased in biopsy specimens of gastric cancer, suggesting that changes in survivin expression play an important role in the transformation from normal gastric mucosa to gastric malignancy [22]. Consistent with this report, we found no positive expression of survivin protein in the 20 normal gastric mucosa samples. However, we detected significantly higher levels survivin protein in the 70 gastric cancer tissues (P > 0.05). These results indicated that survivin is specifically expressed in malignant gastric tumors, and may be a potential independent prognostic factor in gastric cancer. Expressions of PI3K and Akt proteins were also significantly increased in gastric cancer tissues (P > 0.05). Furthermore, expressions of PI3K, Akt and survivin were significantly correlated
with tumor differentiation grade, TNM stage, lymph node metastasis and metastasis to other organs (P < 0.05), but were not correlated with gender, age, tumor size or CEA (P > 0.05). These findings suggested that PI3K, Akt and survivin play important roles in the invasion, progression and metastasis of gastric cancer, and may be valuable molecular markers and therapeutic targets in gastric cancer. We also investigated the relationships between PI3K/Akt and survivin in the carcinogenesis of gastric cancer. Previous reports have shown that the PI3K/Akt signaling pathway is involved in tumor growth and angiogenesis and can reflect the grade of malignancy in human gastric adenocarcinomas [23]. In addition, abnormal activation of PI3K/Akt signaling pathway has been implicated in regulating the expression of survivin in tumor cells. Studies have shown that LY294002, a PI3K inhibitor, not only blocked the PI3K signaling pathway and Akt phosphorylation, but also significantly decreased survivin expression [15,24]. Guo et al. found that downregulation of survivin expression by tamoxifen in HepG2 cells was mediated at transcriptional and posttranscriptional levels by the PI3K/Akt/mTOR signaling pathway to induce apoptosis [25]. Zhao et al. showed that overexpression of PI3K and Akt increased expression of survivin mRNA in chicken embryonic fibroblast cells, demonstrating that PI3K regulated survivin expression through Akt activation [26]. These reports indicate that PI3K regulates the expression of Akt; the PI3K/Akt pathway then regulates the expression of survivin in response to cytokines and growth factors; and the PI3K/Akt/survivin pathway plays a central role in cell proliferation. In agreement with these reports, we identified significant associations between PI3K and Akt (P < 0.001), and between PI3K and survivin (P < 0.001). This suggested that PI3K and Akt, and PI3K and survivin, cooperated to promote the formation, progression and metastasis of gastric cancer. No direct association was found between the expressions of Akt and survivin. This may have been a consequence of too few samples and not including pAkt in
Please cite this article in press as: Gu Y, et al. Clinicopathological significance of PI3K, Akt and survivin expression in gastric cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.010
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our analyses. Therefore, further investigations are required to verify these findings. In summary, our results have demonstrated that expression of PI3K, Akt and survivin is closely related to the occurrence, development and prognosis of gastric cancer. Furthermore, we identified cooperative relationships between these three proteins, which may be implicated in the transformation from normal gastric mucosa to malignant tumors. However, the details of the regulatory mechanisms in the PI3K/Akt/survivin signaling pathway remain controversial and there is a need for further research. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (81172217), the Medical Important Talents of Jiangsu Province (RC201157), and the Project of Oncology Translational Medicine Central of Jiangsu Province (BL2012008). The authors thank Weimin Zhang for excellent technical help. References [1] Roder DM. The epidemiology of gastric cancer. Gastric Cancer 2002;5(Suppl. 1): 5–11. [2] Steeg PS. Tumor metastasis: mechanistic insights and clinical challenges. Nat Med 2006;12:895–904. [3] Smith BR, Stabile BE. Extreme aggressiveness and lethality of gastric adenocarcinoma in the very young. Arch Surg 2009;144(6):506–10. [4] De Vita F, Giuliani F, Galizia G, Belli C, Aurilio G, Santabarbara G, et al. Neoadjuvant and adjuvant chemotherapy of gastric cancer. Ann Oncol 2007;18(Suppl. 6):120–3. [5] Orditura M, Martinelli E, Galizia G, Vitiello F, Fasano M, Muto P, et al. Chemoradiotherapy as adjuvant treatment of gastric cancer. Ann Oncol 2007;18 (Suppl. 6):133–5. [6] Yasui W, Oue N, Aung PP, Matsumura S, Shutoh M, Nakayama H. Molecular pathological prognostic factors of gastric cancer: a review. Gastric Cancer 2005;8:86–94. [7] Ye B, Jiang LL, Xu HT, Zhou DW, Li ZS. Expression of PI3K/AKT pathway in gastric cancer and its blockade suppresses tumor growth and metastasis. Int J Immunopathol Pharmacol 2012;25(3):627–36.
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[8] Tian WY, Chen WC, Li R, Liu L. Markers CD40, VEGF, AKT, PI3K, and S100 correlate with tumor stage in gastric cancer. Onkologie 2013;36(1–2):26–31. [9] Vivanco I, Sawyers CL. The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2002;2(7):489–501. [10] Cheng Z, Hu L, Fu W, Zhang Q, Liao X. Expression of survivin and its splice variants in gastric cancer. J Huazhong Univ Sci Technolog Med Sci 2007;27(4): 393–8. [11] Guo RH, Jin SD, Cai J, Huang ZH, Shu YQ. Insulin/PI3K signalling pathway regulates the expression of survivin in liver cancer HepG2 cells. Zhonghua Zhong Liu Za Zhi 2008;30(10):745–8. [12] Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A. AJCC cancer staging manual, 7th ed., New York: Springer; 2010. [13] Chu Y, Liu W, Cui Q, Feng G, Wang Y, Jiang X. Analysis of phosphatidylinositol 3-kinase activation in the adipose tissue of gestational diabetes mellitus patients and insulin resistance. J Huazhong Univ Sci Technolog Med Sci 2010;30(4):505–8. [14] Higa M, Shimabukuro M, Shimajiri Y, Takasu N, Shinjyo T, Inaba T. Protein kinase B/Akt signalling is required for palmitate-induced beta-cell lipotoxicity. Diabetes Obes Metab 2006;8(2):228–33. [15] Nicholson KM, Anderson NG. The protein kinase B/Akt signalling pathway in human malignancy. Cell Signal 2002;14(5):381–95. [16] Dudek H, Datta SR, Franke TF, Birnbaum MJ, Yao R, Cooper GM, et al. Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science 1997;275(5300):661–5. [17] Kim S, Kang J, Qiao J, Thomas RP, Evers BM, Chung DH. Phosphatidyliosiyol 3kinase iinhibition down-regulatates surviving and facilitates TRAIL-mediated apoptosis in neuroblastoma. J Pediatr Surg 2004;39(4):516–21. [18] Samuels Y, Wang Z, Bardelli A, Silliman N, Ptak J, Szabo S, et al. High frequency of mutations of the PIK3CA gene in human cancers. Science 2004;304(5670): 554. [19] Mende I, Malstrom S, Tsichlis PN, Vogt PK, Aoki M. Oncogenic transformation induced by membrane-targeted Akt2 and Akt3. Oncogene 2001;20(32): 4419–23. [20] Lee GH, Joo YE, Koh YS, Chung IJ, Park YK, Lee JH, et al. Expression of survivin in gastric cancer and its relationship with tumor angiogenesis. Eur J Gastroenterol Hepatol 2006;18(9):957–63. [21] Bertazza L, Mocellin S, Marchet A, Pilati P, Gabrieli J, Scalerta R, et al. Survivin gene levels in the peripheral blood of patients with gastric cancer independently predict survival. J Transl Med 2009;22(7):111. [22] Kania J, Konturek SJ, Marlicz K, Hahn EG, Konturek PC. Expression of survivin and caspase-3 in gastric cancer. Dig Dis Sci 2003;48(2):266–71. [23] 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. [24] Shi M, Zhang H, Li M, Xue J, Fu Y, Yan L, et al. Normal endometrial stromal cells regulate survival and apoptosis signaling through PI3K/AKt/Survivin pathway in endometrial adenocarcinoma cells in vitro. Gynecol Oncol 2011;123(2): 387–92. [25] Guo R, Wang T, Shen H, Ge HM, Sun J, Huang ZH. Involvement of mTOR and survivin inhibition in tamoxifen-induced apoptosis in human hepatoblastoma cell line HepG2. Biomed Pharmacother 2010;64(4):249–53. [26] Zhao P, Meng Q, Liu LZ, You YP, Liu N, Jiang BH. Regulation of survivin by PI3K/ Akt/p70S6K1 pathway. Biochem Biophys Res Commun 2010;395(2):219–24.
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BIOPHA-3384; No. of Pages 5 Biomedicine & Pharmacotherapy xxx (2014) xxx–xxx
Available online at
ScienceDirect www.sciencedirect.com
Original article
Clinicopathological significance of PI3K, Akt and survivin expression in gastric cancer Yongjuan Gu a,1, Shidai Jin a,1, Feng Wang b, Yibin Hua b, Li Yang b, Yongqian Shu a, Zhihong Zhang c,*,2, Renhua Guo a,*,3 a b c
Department of Medical Oncology, the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Rd, Nanjing, Jiangsu 210029, PR China Department of Surgery, the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Rd, Nanjing, Jiangsu 210029, PR China Department of Pathology, the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Rd, Nanjing, Jiangsu 210029, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 January 2014 Accepted 4 March 2014
Background: Gastric cancer is the second leading cause of cancer-related deaths worldwide, and is characterized by invasion and metastasis. Increasing attention is being focused on discovering molecular markers for diagnosis and prognosis. Our objective was to evaluate PI3K, Akt and survivin protein expression in gastric cancer, and their correlations with clinicohistological features and prognosis in patients with gastric cancer. Methods: Tissue samples were obtained from 70 patients with gastric cancer patients and 20 patients with normal gastric mucosa. The protein levels of PI3K, Akt and survivin were evaluated by immunohistochemistry. Statistical analyses were performed to establish the correlations between their expressions and patients’ clinicopathologic characteristics. Results: The positive expression rates of PI3K, Akt and survivin were significantly higher in the gastric cancer tissues compared to normal gastric mucosa (P < 0.05). Expression levels of PI3K, Akt and survivin proteins were significantly correlated with TNM stage, differentiation grade, lymph node metastasis and metastases to other organs (P < 0.05). Cooperative relationships were identified between PI3K and Akt, and PI3K and survivin (P < 0.01), suggesting the involvement of the PI3K/Akt/survivin signaling pathway in the tumorigenesis of gastric cancer. Conclusions: Protein expression of PI3K, Akt and survivin were significantly associated with the development, progression and metastasis of gastric cancer and may have value as diagnostic and prognostic markers in gastric cancer. ß 2014 Published by Elsevier Masson SAS.
Keywords: Gastric cancer PI3K/Akt/survivin Immunohistochemistry
1. Introduction Gastric cancer is one of the most common malignancies and the second leading cause of cancer-related deaths worldwide [1]. Over half of these cases occur in Eastern Asia, with the majority in China. Gastric cancer is characterized by invasion and metastasis which are the main factors contributing to its high mortality rate [2,3]. Despite improvements in palliative chemotherapy and
* Corresponding authors. E-mail addresses: [email protected] (Z. Zhang), [email protected], [email protected] (R. Guo). 1 Yongjuan Gu and Shidai Jin contributed equally to this work and share first authorship. 2 Tel.: +86 25 68 13 65 45; fax: +86 25 83 72 44 40. 3 Tel.: +86 25 68 13 67 14; fax: +86 25 83 71 00 40.
radiotherapy techniques, the survival rate of patients with advanced gastric cancer remains poor [4,5]. Considerable attention is being directed towards the discovery of molecular markers for diagnosis and prognosis, and to develop effective treatments for gastric cancer [6]. Currently, oncogenes and angiogenic and growth factors are most researched [7,8]. The phosphatidylinositol 3-kinase/protein kinase B (PI3K/ Akt) signaling pathway regulates many diverse cellular processes. Abnormal activation of this pathway is frequently observed in cancer, including lung, colon, pancreatic, prostate, breast, stomach and ovarian carcinomas [9]. Survivin is a member of the inhibitor of apoptosis protein (IAP) family, which is involved in the regulation of cell division and suppression of apoptosis [10]. The PI3K/Akt signaling pathway has been found to regulate survivin in human hepatocellular carcinoma HepG2 cells [11]; however, there are few reports on the effect of the
http://dx.doi.org/10.1016/j.biopha.2014.03.010 0753-3322/ß 2014 Published by Elsevier Masson SAS.
Please cite this article in press as: Gu Y, et al. Clinicopathological significance of PI3K, Akt and survivin expression in gastric cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.010
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PI3K/Akt/survivin signaling pathway in gastrointestinal cancer. In this study, we investigated the expressions of PI3K, Akt and survivin in gastric cancer tissues compared to normal gastric mucosa, and explored their relationships with clinicopathologic and prognostic characteristics in gastric cancer. Our aim was to determine their potential value as biological and prognostic markers in the occurrence and development of gastric cancer. 2. Materials and methods 2.1. Patients and tissue samples We selected 70 patients with pathologic (p-) stages I–IV gastric cancer who had undergone complete tumor resection and nodal dissection at the First Affiliated Hospital of Nanjing Medical University between February 2011 and July 2012. None of patients had received any preoperative therapy. Patients with a history of another malignancy or who died within 4 weeks of surgery were excluded from the study. In addition, 20 normal tissue samples were obtained as controls from patients with chronic gastritis who had undergone endoscopic biopsy. The study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University, and informed consent was obtained from each patient. The 70 patients with gastric cancer included 48 males and 22 females, with a median age of 57 years (range, 23–79 years). The histological classification, grading, and pathologic tumor-nodemetastasis (TNM) staging of the tumors were based on criteria defined by the 7th American Joint Committee on Cancer (AJCC) [12]. These showed that 27 patients had poorly differentiated tumors and 43 patients had moderately or well differentiated tumors; six patients were stage I, eight were stage II, 44 were stage III, and 12 were stage IV. Lymph node metastasis was found in 55 cases. 2.2. Immunohistochemistry The surgical samples and gastric biopsies were fixed in 10% formalin and embedded in paraffin (FFPE). Four serial sections, 4 mm thick, were cut from representative blocks from each case. One section was subjected to conventional hematoxylin and eosin (H&E) staining for standard pathologic examination, and the other three sections were used for immunohistochemical staining. Immunohistochemical staining was performed according to the EnVision two-step technique. FFPE sections were mounted on superfrost microscope slides, deparaffinized and rehydrated in a graded ethanol series. They were exposed to 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity, and heat-induced antigen retrieval was performed by pretreating the slides in 0.01 M citrate buffer (pH 6.0) at 98 8C for 15 min in a microwave oven. The sections were incubated for 10 min at room temperature with normal serum before being incubated with primary antibody (1:100) at 4 8C overnight. The slides were then rinsed in Tris-buffered saline (TBS) for 10 min and incubated with secondary antibody for 30 min at room temperature. The following primary antibodies were used: PI3K rabbit polyclonal anti-human antibody; Akt rabbit polyclonal anti-human antibody; survivin goat polyclonal anti-human antibody (Cell Signaling Technology; Beverly, MA, USA). The corresponding secondary antibodies were obtained from Zhongshan Golden Bridge Biotechnology (Beijing, China). The slides were developed with diaminobenzidine (DAB) for 5 min, counterstained with hematoxylin, dehydrated, cleared and mounted. Sections treated with phosphate buffered saline (PBS) instead of primary antibody were used as negative controls; slides mounted with known gastric cancer samples were used as positive controls. All the
slides were processed by the same technician under the same laboratory conditions. The immunohistochemical results were evaluated simultaneously by two independent observers who had no prior knowledge of the patients. PI3K and survivin proteins in the cytoplasm were stained brown-yellow, while Akt protein in the nuclei was stained brown-yellow or brown. The percentages of positively stained cells were calculated by counting 200 cells from four different fields under high magnification, with. The staining intensities were scored as follows: colorless (0); buffy (1); lightbrown (2); or brown-yellow (3). The extent of staining was scored as the percentage of positive cells as follows: negative (0); 10% (1); 11%–50% (2); 51%–75% (3); > 75% (4). The product of the two scores was calculated. A protein was considered to be overexpressed if the final score > 2. 2.3. Statistical analysis All statistical analyses were performed using SPSS v. 16.0 software (SPSS; Chicago, IL, USA). The x2-test and Fisher’s exacttest were used to identify correlations between the expression levels of PI3K, Akt and survivin with patients’ clinicopathologic characteristics. Spearman’s rank correlation-test was used to determine the relationships between PI3K, Akt and survivin protein expressions. P-values were based on two-tailed statistical analysis. A P-value < 0.05 was considered statistically significant. 3. Results 3.1. Expression rates of PI3K, Akt and survivin in gastric cancer tissue in relation to normal gastric mucosa The positive expression rates for PI3K, Akt and survivin proteins in the 70 gastric cancer tissue samples were 74.3% (52/70), 37.1% (26/70) and 62.9% (44/70), respectively. The corresponding rates in the 20 normal gastric mucosa samples were 30% (6/20), 0% (0/20) and 0% (0/20), respectively (Fig. 1). These results demonstrated that expression of PI3K, Akt and survivin were significantly higher in gastric cancer tissues compared to normal gastric mucosa (P < 0.05; Table 1). 3.2. The relationships between protein expressions of PI3K, Akt and survivin and clinicopathologic features in gastric cancer The expressions of PI3K, Akt and survivin proteins in the 70 gastric cancer tissue samples were significantly correlated with differentiation grade, TNM stage, lymph node metastasis and metastases to other organs (P < 0.05). In contrast, no significant associations were observed with gender, age, carcinoembryonic antigen (CEA) and tumor size (P > 0.05; Table 2). 3.3. The relationships between the expression of PI3K, Akt and survivin proteins in gastric cancer tissue Analysis of PI3K, Akt and survivin immunohistochemical staining patterns showed that PI3K and Akt were positively coexpressed in 24/70 (34.3%) gastric cancer tissue samples and negatively co-expressed 16/70 (22.9%) samples; PI3K and survivin were positively co-expressed in 40/70 (57.1%) samples and negatively co-expressed in 14/70 (20.0%) samples; Akt and survivin were positively co-expressed in 19/70 (27.1%) samples and negatively co-expressed in 19/70 (27.1%) samples (Table 3). Correlation analyses identified positive associations between PI3K and Akt (r = 0.336; P < 0.001); and between PI3K and survivin (r = 0.528; P < 0.001), where r is the correlation coefficient.
Please cite this article in press as: Gu Y, et al. Clinicopathological significance of PI3K, Akt and survivin expression in gastric cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.010
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Fig. 1. Immunohistochemical staining of PI3K, Akt and survivin proteins in gastric cancer tissues. Light-brown or brown-yellow staining indicates positive expression of the proteins: 1: negative protein expression; 2: positive protein expression (score: 1+); 3: positive protein expression (score: 2+). Magnification 200.
Table 1 Positive expression rates of PI3K, Akt and survivin proteins in gastric cancer tissues and normal gastric mucosa samples. Protein and pathology type
n
Positive n (%)
P value
PI3K Gastric cancer Normal gastric mucosa
70 20
52 (74.3) 6 (30.0)
0.014
Akt Gastric cancer Normal gastric mucosa
70 20
26 (37.1) 0 (0.0)
0.047
Survivin Gastric cancer Normal gastric mucosa
70 20
44 (62.9) 0 (0.0)
0.001
However, no association was found between Akt and survivin (P > 0.05) in gastric cancer tissue. 4. Discussion PI3K is a heterodimer with p85 regulatory and p110 catalytic subunits [13]. The p110 subunit has protein kinase homology, and activities in common with protein kinases and phospholipases. These enzymes catalyze phosphorylation at the D3 position of PI(4)P and PI(4,5)P2 to produce PI(3,4,5)P3. PIP3 is generated by activation of PI3K in response to growth factors and other stimuli.
PIP3 then activates downstream signaling pathways by phosphorylating several kinases which are involved in the regulation of a variety of cellular processes, including 3-phosphoinositide-dependent kinase-1 (PDK1) and Akt [14]. PIP3 binds to the PH domains of Akt, leading to Akt membrane recruitment, which alters the structure of Akt to allow subsequent phosphorylation by PDK1. Phosphorylated Akt (pAkt) is the active form of Akt. This is released from the cellular membrane to the cytoplasm, where it participates in molecular processes which promote cell growth, proliferation, such as glycometabolism, protein synthesis, and antiapoptotic activities [15,16]. Overactivation of PI3K has been associated with oncogenesis; conversely, the PI3K inhibitor LY294002 was found to block the PI3K signaling pathway to inhibit cell proliferation and induce apoptosis [17]. Antagonism to PI3K is often absent in mutated or deficient tumor suppressor genes, such as PTEN. This often results in disorders to physiological functions and normal metabolic activities and overexpression of Akt, which can lead to excessive cell proliferation and malignant transformation [18]. The role of Akt in tumorigenesis was confirmed in a model that showed infection with retroviral vectors expressing activated forms of Akt led to the formation of malignant tumors [19]. The central role played by the PI3K/Akt signaling pathway in the regulation of normal cell growth and apoptosis has made this pathway a target in cancer research. Survivin is a recently discovered IAP that regulates cell division and suppresses apoptosis. Abnormal activation of survivin can lead
Please cite this article in press as: Gu Y, et al. Clinicopathological significance of PI3K, Akt and survivin expression in gastric cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.010
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Table 2 Relationships between the expressions of PI3K, Akt and survivin and the clinicopathologic characteristics in patients with gastric cancer. Clinicopathologic parameters
n
PI3K
Akt
Survivin
Positive n (%)
P value
Positive n (%)
P value
Positive n (%)
P value
30 40
23 (76.7) 29 (72.5)
0.693
8 (26.7) 18 (45.0)
0.116
21 (70.0) 23 (57.5)
0.284
Gender Male Female
48 22
34 (70.8) 18 (81.8)
0.329
18 (37.5) 8 (36.4)
0.927
32 (66.7) 12 (54.5)
0.330
Tumor size 5 cm < 5 cm
42 28
32 (76.2) 20 (71.4)
0.655
17 (40.5) 9 (32.1)
0.480
28 (66.7) 16 (57.1)
0.419
CEA High Normal
11 59
7 (63.6) 45 (76.3)
0.614
4 (36.4) 22 (37.3)
1.000
7 (63.6) 37 (62.7)
1.000
Differentiation Moderately/well Poorly
43 27
28 (65.1) 24 (88.9)
0.027
7 (16.3) 19 (70.4)
0.000
22 (51.2) 22 (81.5)
0.011
TNM stage I–II III–IV
14 56
6 (42.9) 46 (82.1)
0.008
2 (14.3) 24 (42.9)
0.048
5 (35.7) 39 (69.6)
0.019
LN metastasis 1 0
55 15
47 (85.5) 5 (33.3)
0.000
25 (45.5) 1 (6.7)
0.006
42 (76.4) 2 (13.3)
0.000
Metastasis Yes No
12 58
12 (100.0) 40 (69.0)
0.025
8 (66.7) 18 (31.0)
0.046
12 (100.0) 32 (55.2)
0.009
Age 60 < 60
CEA: carcinoembryonic antigen; TNM: tumor-node-metastasis; LN: lymph node.
Table 3 The relationships between PI3K, Akt and survivin protein expression in gastric cancer. Survivin
Akt +
+
PI3K + Survivin +
16 28
2 24
14 12
4 40
19 25
7 19
– –
– –
to aberrant cell cycle progression, inhibition of apoptosis and induction of carcinogenesis. Survivin is abundantly expressed in fetal tissues and is overexpressed in many human cancers [20]. Several studies have demonstrated that overexpression of survivin is correlated with poor prognosis in patients with gastric cancer, and contributes to chemoresistance [21]. However, a study by Kania et al. found that survivin protein was undetectable in normal gastric mucosa, and survivin mRNA was only weakly detectable. In contrast, both survivin mRNA and protein were significantly increased in biopsy specimens of gastric cancer, suggesting that changes in survivin expression play an important role in the transformation from normal gastric mucosa to gastric malignancy [22]. Consistent with this report, we found no positive expression of survivin protein in the 20 normal gastric mucosa samples. However, we detected significantly higher levels survivin protein in the 70 gastric cancer tissues (P > 0.05). These results indicated that survivin is specifically expressed in malignant gastric tumors, and may be a potential independent prognostic factor in gastric cancer. Expressions of PI3K and Akt proteins were also significantly increased in gastric cancer tissues (P > 0.05). Furthermore, expressions of PI3K, Akt and survivin were significantly correlated
with tumor differentiation grade, TNM stage, lymph node metastasis and metastasis to other organs (P < 0.05), but were not correlated with gender, age, tumor size or CEA (P > 0.05). These findings suggested that PI3K, Akt and survivin play important roles in the invasion, progression and metastasis of gastric cancer, and may be valuable molecular markers and therapeutic targets in gastric cancer. We also investigated the relationships between PI3K/Akt and survivin in the carcinogenesis of gastric cancer. Previous reports have shown that the PI3K/Akt signaling pathway is involved in tumor growth and angiogenesis and can reflect the grade of malignancy in human gastric adenocarcinomas [23]. In addition, abnormal activation of PI3K/Akt signaling pathway has been implicated in regulating the expression of survivin in tumor cells. Studies have shown that LY294002, a PI3K inhibitor, not only blocked the PI3K signaling pathway and Akt phosphorylation, but also significantly decreased survivin expression [15,24]. Guo et al. found that downregulation of survivin expression by tamoxifen in HepG2 cells was mediated at transcriptional and posttranscriptional levels by the PI3K/Akt/mTOR signaling pathway to induce apoptosis [25]. Zhao et al. showed that overexpression of PI3K and Akt increased expression of survivin mRNA in chicken embryonic fibroblast cells, demonstrating that PI3K regulated survivin expression through Akt activation [26]. These reports indicate that PI3K regulates the expression of Akt; the PI3K/Akt pathway then regulates the expression of survivin in response to cytokines and growth factors; and the PI3K/Akt/survivin pathway plays a central role in cell proliferation. In agreement with these reports, we identified significant associations between PI3K and Akt (P < 0.001), and between PI3K and survivin (P < 0.001). This suggested that PI3K and Akt, and PI3K and survivin, cooperated to promote the formation, progression and metastasis of gastric cancer. No direct association was found between the expressions of Akt and survivin. This may have been a consequence of too few samples and not including pAkt in
Please cite this article in press as: Gu Y, et al. Clinicopathological significance of PI3K, Akt and survivin expression in gastric cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.010
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our analyses. Therefore, further investigations are required to verify these findings. In summary, our results have demonstrated that expression of PI3K, Akt and survivin is closely related to the occurrence, development and prognosis of gastric cancer. Furthermore, we identified cooperative relationships between these three proteins, which may be implicated in the transformation from normal gastric mucosa to malignant tumors. However, the details of the regulatory mechanisms in the PI3K/Akt/survivin signaling pathway remain controversial and there is a need for further research. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (81172217), the Medical Important Talents of Jiangsu Province (RC201157), and the Project of Oncology Translational Medicine Central of Jiangsu Province (BL2012008). The authors thank Weimin Zhang for excellent technical help. References [1] Roder DM. The epidemiology of gastric cancer. Gastric Cancer 2002;5(Suppl. 1): 5–11. [2] Steeg PS. Tumor metastasis: mechanistic insights and clinical challenges. Nat Med 2006;12:895–904. [3] Smith BR, Stabile BE. Extreme aggressiveness and lethality of gastric adenocarcinoma in the very young. Arch Surg 2009;144(6):506–10. [4] De Vita F, Giuliani F, Galizia G, Belli C, Aurilio G, Santabarbara G, et al. Neoadjuvant and adjuvant chemotherapy of gastric cancer. Ann Oncol 2007;18(Suppl. 6):120–3. [5] Orditura M, Martinelli E, Galizia G, Vitiello F, Fasano M, Muto P, et al. Chemoradiotherapy as adjuvant treatment of gastric cancer. Ann Oncol 2007;18 (Suppl. 6):133–5. [6] Yasui W, Oue N, Aung PP, Matsumura S, Shutoh M, Nakayama H. Molecular pathological prognostic factors of gastric cancer: a review. Gastric Cancer 2005;8:86–94. [7] Ye B, Jiang LL, Xu HT, Zhou DW, Li ZS. Expression of PI3K/AKT pathway in gastric cancer and its blockade suppresses tumor growth and metastasis. Int J Immunopathol Pharmacol 2012;25(3):627–36.
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[8] Tian WY, Chen WC, Li R, Liu L. Markers CD40, VEGF, AKT, PI3K, and S100 correlate with tumor stage in gastric cancer. Onkologie 2013;36(1–2):26–31. [9] Vivanco I, Sawyers CL. The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2002;2(7):489–501. [10] Cheng Z, Hu L, Fu W, Zhang Q, Liao X. Expression of survivin and its splice variants in gastric cancer. J Huazhong Univ Sci Technolog Med Sci 2007;27(4): 393–8. [11] Guo RH, Jin SD, Cai J, Huang ZH, Shu YQ. Insulin/PI3K signalling pathway regulates the expression of survivin in liver cancer HepG2 cells. Zhonghua Zhong Liu Za Zhi 2008;30(10):745–8. [12] Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A. AJCC cancer staging manual, 7th ed., New York: Springer; 2010. [13] Chu Y, Liu W, Cui Q, Feng G, Wang Y, Jiang X. Analysis of phosphatidylinositol 3-kinase activation in the adipose tissue of gestational diabetes mellitus patients and insulin resistance. J Huazhong Univ Sci Technolog Med Sci 2010;30(4):505–8. [14] Higa M, Shimabukuro M, Shimajiri Y, Takasu N, Shinjyo T, Inaba T. Protein kinase B/Akt signalling is required for palmitate-induced beta-cell lipotoxicity. Diabetes Obes Metab 2006;8(2):228–33. [15] Nicholson KM, Anderson NG. The protein kinase B/Akt signalling pathway in human malignancy. Cell Signal 2002;14(5):381–95. [16] Dudek H, Datta SR, Franke TF, Birnbaum MJ, Yao R, Cooper GM, et al. Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science 1997;275(5300):661–5. [17] Kim S, Kang J, Qiao J, Thomas RP, Evers BM, Chung DH. Phosphatidyliosiyol 3kinase iinhibition down-regulatates surviving and facilitates TRAIL-mediated apoptosis in neuroblastoma. J Pediatr Surg 2004;39(4):516–21. [18] Samuels Y, Wang Z, Bardelli A, Silliman N, Ptak J, Szabo S, et al. High frequency of mutations of the PIK3CA gene in human cancers. Science 2004;304(5670): 554. [19] Mende I, Malstrom S, Tsichlis PN, Vogt PK, Aoki M. Oncogenic transformation induced by membrane-targeted Akt2 and Akt3. Oncogene 2001;20(32): 4419–23. [20] Lee GH, Joo YE, Koh YS, Chung IJ, Park YK, Lee JH, et al. Expression of survivin in gastric cancer and its relationship with tumor angiogenesis. Eur J Gastroenterol Hepatol 2006;18(9):957–63. [21] Bertazza L, Mocellin S, Marchet A, Pilati P, Gabrieli J, Scalerta R, et al. Survivin gene levels in the peripheral blood of patients with gastric cancer independently predict survival. J Transl Med 2009;22(7):111. [22] Kania J, Konturek SJ, Marlicz K, Hahn EG, Konturek PC. Expression of survivin and caspase-3 in gastric cancer. Dig Dis Sci 2003;48(2):266–71. [23] 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. [24] Shi M, Zhang H, Li M, Xue J, Fu Y, Yan L, et al. Normal endometrial stromal cells regulate survival and apoptosis signaling through PI3K/AKt/Survivin pathway in endometrial adenocarcinoma cells in vitro. Gynecol Oncol 2011;123(2): 387–92. [25] Guo R, Wang T, Shen H, Ge HM, Sun J, Huang ZH. Involvement of mTOR and survivin inhibition in tamoxifen-induced apoptosis in human hepatoblastoma cell line HepG2. Biomed Pharmacother 2010;64(4):249–53. [26] Zhao P, Meng Q, Liu LZ, You YP, Liu N, Jiang BH. Regulation of survivin by PI3K/ Akt/p70S6K1 pathway. Biochem Biophys Res Commun 2010;395(2):219–24.
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