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Correlations of Foxo3 and Foxo4 expressions with clinicopathological features and prognosis of bladder cancer
Accepted Manuscript Title: Correlations of Foxo3 and Foxo4 expressions with clinicopathological features and prognosis of bladder cancer Authors: Yang Wang, Xin-Li Kang, Fan-Chang Zeng, Cong-Jie Xu, Jia-Quan Zhou, Dong-Ni Luo PII: DOI: Reference:
S0344-0338(16)30606-9 http://dx.doi.org/doi:10.1016/j.prp.2017.04.004 PRP 51782
To appear in: Received date: Revised date: Accepted date:
4-11-2016 13-3-2017 12-4-2017
Please cite this article as: Yang Wang, Xin-Li Kang, Fan-Chang Zeng, Cong-Jie Xu, Jia-Quan Zhou, Dong-Ni Luo, Correlations of Foxo3 and Foxo4 expressions with clinicopathological features and prognosis of bladder cancer, Pathology - Research and Practicehttp://dx.doi.org/10.1016/j.prp.2017.04.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Correlations of Foxo3 and Foxo4 expressions with clinicopathological features and prognosis of bladder cancer Running title: FoxO3 and FoxO4 expressions in bladder cancer
Yang Wang 1, *, Xin-Li Kang 1, Fan-Chang Zeng 1, Cong-Jie Xu 1, Jia-Quan Zhou 1, Dong-Ni Luo 2 1 Department
of Urology, Hainan General Hospital, Haikou 570311, P.R. China
2 Department
of Ultrasonography, Hainan General Hospital, Haikou 570311, P.R. China
*
Correspondence to: Dr. Yang Wang, Department of Urology, Hainan General Hospital, No.19
Xiuhua Road, Xiuying District, Haikou 570311, Hainan Province, P.R. China E-mail: [email protected] Tel/Fax: +86-0898-68642202
ABSTRACT Objective: The study is performed to explore the correlations of forkhead box O3 (FoxO3) and forkhead box O4 (FoxO4) expressions with clinicopathological features and prognosis of bladder cancer. Methods: Bladder cancer tissues and adjacent normal tissues from the recruited 222 patients were collected. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting and immunohistochemistry were applied to determine the expressions of FoxO3 and FoxO4. Spearman correlation analysis was conducted to examine the correlation between the expressions of FoxO3 and FoxO4. All patients were followed up and overall survival (OS) and disease-free survival (DFS) were recorded. Kaplan-Meier survival curve was drawn to determine the associations of FoxO3 and FoxO4 expressions and postoperative survival. Cox proportional hazards model was conducted to analyze the risk factors for poor prognosis of bladder cancer. Results: The mRNA and expressions of FoxO3 and FoxO4 proteins in the bladder cancer tissues were lower than that in the adjacent normal tissues (both P < 0.05). The positive rates of FoxO3 and FoxO4 were lower in the patients with lymph node metastasis than that in the patients without lymph node metastasis (P < 0.05), and significantly lower in the patients with non-muscle invasive bladder cancer (Tis-T1) than in those with non-muscle invasive bladder cancer (T2-T3) in TNM staging, and remarkably lower in the patients with high grade than in those with low grade in the histological type (P < 0.05). Furthermore, the expressions of FoxO3 and FoxO4 were positively correlated in the bladder cancer tissues (P < 0.05). Negative expressions of FoxO3 and FoxO4 and lymph node metastasis were the risk factors for the poor prognosis of bladder cancer. Conclusions: The FoxO3 and FoxO4 expressions may potentially associate with the clinicopathological features and prognosis of bladder cancer. Keywords: Forkhead box O3; Forkhead box O4; Bladder cancer; Clinicopathological features; Prognosis
1. Introduction Bladder cancer, the second malignancy of genitourinary tract only to prostate cancer (PC) and the 9th most common cancer worldwide, sees over 330,000 new cases were diagnosed and over 30,000 lives were lost per year [1]. Cigarette smoking and occupational exposure to aromatic amines have been found which were closely related to the risk of bladder cancer together with a family history of the carcinoma [2]. There are 80% of cases with non-muscle invasive bladder cancer when they were firstly diagnosed with a rather good prognosis, and they are likely to recur frequently and even progress to be muscle-invasive [3]. And the rest 20% of cases shows muscle invasion which are diagnosed with less favorable prognosis and their mortality rate accouts for about 50% [4]. For the treatment of invasive bladder cancer, the standard method remains radical surgical removal of the bladder within standard limits combined with additional neoadjuvant chemotherapy if necessary, which is still somewhat far from being satisfactory [5]. Although its exact mechanism remains unknown, tumorigenesis and progression of bladder cancer are closely related with chromosomal anomalies, epigenetic changes and genetic polymorphisms, and genetic changes are obviously involved in its treatment and prognosis [6]. Altered expression of oncogenes and tumor suppressors has been demonstrated the function in the initiation and progression of bladder cancer, making the discovery of new biomarkers of vital value in earlier detection and prognosis of bladder cancer [7]. Forkhead box class O (FoxO) transcription factors belong to the forkhead family of transcriptional regulators featured by a conserved DNA binding domain termed the ‘‘forkhead box’’, consisting of 4 members which are namely FoxO1, FoxO3, FoxO4 and FoxO6 [8, 9]. They are found to get involved in various cellular processes such as cell cycle regulation, apoptosis and resistance to reactive oxygen species [10]. It is also revealed that these transcription factors act as tumor suppressors, thus benefiting stem cell maintenance and lifespan extension [11]. Activated FoxO3 leads to remarkable atrophy of muscles and myotubes by stimulating overall protein
degradation and coordinately activating relevant pathways such as phosphoinositide-3 kinase (PI3K)-protein kinase B (PKB/c-Akt) signaling pathway, inducing expression of many autophagyrelated genes [12, 13]. FoxO4, the homolog of DAF-16 and also known as AFX, contributes to longevity and oxidative stress [14]. Previous studies have reported that FoxO3 and FoxO4 play a vitally important part in the development of many cancers such as breast cancer (BC) and PC [15, 16]. However, it still remains unveiled of their roles in the initiation, progression and prognosis of bladder cancer. Therefore, the study targets to investigate the correlations of the expressions of FoxO3 and FoxO4 with clinicopathological features and prognosis of bladder cancer.
2. Materials and methods 2.1. Study Subjects From January 2010 to January 2012, 222 patients with bladder cancer in Hainan General Hospital were recruited for the study. Their clinical data and tumor tissues were collected. There were 158 males and 64 female patients with the age of 36 to 79 years and the mean age of 55.23 ± 8.72 years, which included a total of 141 patients with baldder cancer treated with radical cystectomy, 41 patients with patial cystectomy and 40 patients with electroresection. There were 126 patients with non-muscle invasive bladder cancer (Tis-T1) and 96 patients with muscle invasive bladder cancer (T2-T3) according to the 7th edition of the American Joint Committee on Cancer (AJCC) TNM staging in cancer manual [17]. According to the World Health Organization (WHO) histological classification of urinary bladder tumors [18], there were 154 patients in low grade and 68 patients in high grade according to histological classification. Patients with local lymph node metastasis were confirmed by pathologically diagnosing the excised tissue and dissected lymph nodes after bladder cancer surgery. There were 156 patients without local lymph node metastasis while 66 patients were diagnosed with lymph node metastasis. The bladder cancer tissues from all 222 cases were collected as the experiment group, and the adjacent normal tissues were collected
and confirmed by pathology as the control group (the tissues were located over 2 cm away from the edge of the tumor). The inclusion criteria: all patients were pathologically diagnosed as bladder cancer; each case was diagnosed with adequate imaging and clinical examination who received at least one follow-up or examination after discharge. The exclusion criteria: patients were treated with chemotherapy, radiotherapy or biotherapy; patients suffered with other tumor diseases; patients with distant metastasis before treatment. The present study with all the procedures was approved by the Ethic Committee of Hainan General Hospital and all patients were informed consent for the study and signed written informed consents. 2.2. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) The total RNA was extracted from the tumor tissues and adjacent normal tissues using the kit (TIANGEN Biotechnology Co., Ltd, Beijing, China). The kit for reverse transcription was purchased from Hangzhou Bioer Technology Co., Ltd. (Hangzhou, China), and the operating procedures were strictly conformed to the kit instructions. Ultraviolet spectrophotometer was used to detect the optical density (OD260/280) value of the extracted RNA samples and the RNA concentration was calculated and preserved at -80°C. The mRNA expressions of FoxO3 and FoxO4 were detected by qRT-PCR. Primers were designed using softwae Primer 5.0 on the basis of the gene sequences published in Genebank and miR BASE database, which were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). The primer sequences were shown in Table 1. The PCR kit was purchased from Bio-Rad, Inc., Hercules, CA, USA, and type 7500 qRT-PCR instrument was from ABI Company, Oyster Bay, NY, USA. The reaction condition of qRT-PCR was: pre-denaturation for 30 s at 95°C, and 40 cycles of denaturation for 5 s at 95°C, annealing for 30 s and extension for 30 s. With β-actin as an internal reference, Ct values were compared between target gene and β-actin to calculate the relative
expression of the mRNA of target genes: ΔCt target gene = Ct target gene - Ct β-actin, ΔΔCt = ΔCt experimental group
- ΔCt control group. The ratio of target gene expressions in two groups is represented as 2－ΔΔCt [19].
2.3. Western blotting Total protein of wax blocks was extracted by extraction reagent of paraffin wax, and bicinchoninic acid (BCA) assay was performed to determine the protein concentration. After denaturation, the total protein (50 μg) was conducted sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) at constant voltage. Semidry method was applied to transfer the electrophoresis product into polyvinylidene fluoride (PVDF) membrane sealed with 5% skimmed milk for 2 h at 4°C. The protein was added with the first antibodies (rabbit with FoxO3 polyclonal antibody and rabbit with FoxO4 polyclonal antibody from Abcam Inc., Cambridge, MA, USA) and preserved for 10 h at 4°C. Next, the protein was washed with Tris-buffered saline Tween-20 (TBST) for 4 times (10 min for each time), and then added with horse-radish peroxidase labeled second antibody (goat anti rabbit IgG; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Then, the protein was incubated for 2 h at 37°C, and washed for 4 times (10 min for each time) with TBST, finally chemi-Luminescence (ECL) kit was used for coloring. Gel electrophoresis imaging analysis system was performed for automatic imaging and the relative protein levels of FoxO3 and FoxO4 were analysed with β-actin as an internal reference. 2.4. Immunohistochemistcal (IHC) SP three-step method was applied for IHC staining to detect the protein expressions of FoxO3 and FoxO4 in the bladder cancer tissues and adjacent normal tissues. Normal bladder tissue sections were collected as positive control and phosphate buffer solution (PBS) instead of the first antibody as negative control, which were stained using HistostainTMSP-9000 staining kit (Zymed Laboratories Inc., South San Francisco, CA, USA). The paraffin sections underwent conventional dewaxing and then were heated by microwave to repair antigen. The heating was stopped for 5 min after boiling, and the tissue sections were heated again and then cooled to room temperature. After
washing with PBS, the sections were added with normal goat serum, and then with the first antibodies at 4°C overnight (rabbit with FoxO3 polyclonal antibody and rabbit with FoxO4 polyclonal antibody were all purchased from Abcam Inc., Cambridge, MA, USA. After rewarming and washing with PBS, the tissue sections were added with working fluid of second antibody (biotin labeled goat anti rabbit IgG) for 30 min at 37°C. After washing with PBS, the tissue sections were incubated with horseradish labeled working fluid, and 3, 3'-diaminobenzidine (DAB) was used for coloring for 5 ~ 10 min, which were ajusted the dying time under the microscope (×400) and sealed with gum after 1 min of hematoxylin staining, finally preserved after taking photo and printing. The proteins of FoxO3 and FoxO4 were mostly expressed in the nucleus with pale brown or brown particles and with weak expression in the cytoplasm. The brown particles in the nucleus were recognized as positive cells. Ten fields were randomly selected to observe and count the positive cells in each field. Positive cells < 10% was considered as negative expression. On the contrary, positive cells ≥ 10% was recognized as positive expression. 2.5. Follow-up Telephone follow-up was conducted to all patients, and outpatient follow-up was carried out to survival patients. The follow-up ended in March 2016. The overall survival (OS) was calculated, as well as the disease-free survival (DFS). The OS was the duration from the surgery to death or to the last follow-up and DFS was the duration from the surgery to tumor recurrence or metastasis. Kaplan-Meier curve was drawn in accordance with the results of follow-ups to compare OS and DFS. All patients were followed up completely. 2.6. Statistical analysis All the data were used statistical software SPSS for statistical analysis. Comparison between two groups was analyzed by student’ s t detection while comparison of measurement data between groups. The measurement data were presented as mean ± standard deviation, which were analyzed
by Least-Significant Difference Test (LSD-t) and paired-samples LSD-t between two groups and by one-way analysis of variance (ANOVA) among multiple groups. Baum-Welch Algorithm was conducted for heterogeneity of variance and comparisons between multiple groups were analysed by Dunnett’s T3 test. Enumeration data were measured by χ2 test. Univariate analysis of prognostic was performed for Kaplan-Meier curve and log-rank was used for detection. Multivariate analysis of prognostic was performed for Cox proportional hazards model, and Spearman was conducted for correlation analysis. P < 0.05 indicates that the difference is statistically significant.
3. Results 3.1. Positive Rates of FoxO3 and FoxO4 Proteins in the Bladder Cancer and Adjacent Normal TissuesAs Fig. 1 shown, the proteins of FoxO3 and FoxO4 was mostly expressed in the cell nucleus but weakly expressed in the cell cytoplasm with positive reaction of pale brown or brown particles. As shown in Table 2, the positive rate of FoxO3 protein was lower in the bladder cancer tissues (32.43%) than that in the adjacent normal tissues (71.62%) (P < 0.05), and the positive rate of FoxO4 protein was lower in the bladder cancer tissues (34.23%) than that in the adjacent normal tissues (62.16%) (P < 0.05). 3.2. MRNA and Protein Expression of FoxO3 and FoxO4 in the Bladder Cancer and Adjacent Normal Tissues As shown in Fig. 2, mRNA and the protein expressions of FoxO3 and FoxO4 in the bladder cancer tissues were significantly decreased as compared with the adjacent normal tissues (all P < 0.05). 3.3. Correlation between the Expressions of FoxO3 and FoxO4 in the Bladder Cancer Tissues Spearman correlation analysis was performed to detecte the correlation with the expressions of FoxO3 and FoxO4 in the bladder cancer tissues, which showed the positive cerrelation between the expressions of FoxO3 and FoxO4 in the bladder cancer tissues (r = 0.522, P < 0.001, Fig. 3).
3.4. Association of FoxO3 and FoxO4 Protein with the Clinicopathological Features in the Bladder Cancer tissues As shown in Table 3, the positive rates of FoxO3 and FoxO4 protein were significantly lower in the patients with lymph node metastasis than that in those without lymph node metastasis (P < 0.05), also significantly lower in those with invasive bladder cancer (T2-T3) than that in those with superficial bladder cancer (Tis-T1) (P < 0.05). According to WHO histological classification, the positive rates of FoxO3 and FoxO4 protein were significantly lower in the patients with high grade than that in those with low grade (P < 0.05). However, there was no significant difference in gender or age, with regard to the positive rates of FoxO3 and FoxO4 protein (both P > 0.05). 3.5. Association of FoxO3 and FoxO4 Expressions with Postoperative Survival All 222 bladder cancer patients were followed up for 60 months but out of 24 cases, and 118 cases died of this cancer. The OS was 46.85%. As Kaplan-Meier curve showed (Fig. 4), the OS of patients with positive expressions of FoxO3 and FoxO4 in the bladder cancer tissues was higher than those with negative expressions (P = 0.005 and P = 0.031, respectively). There were 140 patients with local recurrence and/or metastasis (In the follow-up, patients with local recurrence and/or metastasis, margin-positive bladder cancer, vascular and/or neural invasion were treated with adjuvant systemic chemothera.) and the DFS was 36.94%. As Kaplan-Meier curve showed (Fig. 5), the DFS of patients with positive expressions of FoxO3 and FoxO4 in the bladder cancer tissues was higher than those with negative expressions (P < 0.001 and P < 0.001, respectively). 3.6. Prognostic Factors for Bladder Cancer As shown in Table 4, Cox proportional hazard model analysis indicated that negative expressions of FoxO3 and FoxO4, TNM stage, lymph node metastasis and WHO histological classification were the prognostic factors for bladder cancer (all P < 0.05), while the age and gender showed no association with the prognosis of bladder cancer (both P > 0.05).
4. Discussion Generally, there were no major advances in the treatment of metastatic bladder cancer in the last 3 decades [20]. Although cystectomy combined with neoadjuvant chemotherapy is associated with improved outcomes among patients with bladder cancer, the survival rate for those in later stages remains poor [21]. Therefore, it is of vital importance to address the disease from the perspective of molecular levels. It was found out in the study, that the expressions of FoxO3 and FoxO4 decreased in the bladder cancer tissues as compared to the adjacent normal tissues, which were positively correlated with each other. As has been previously pointed out, FoxO family is regulated by multiple signaling pathways, most notably by PI3K-PKB/c-Akt signaling pathway [13]. The pathway and its related kinase can get FoxO protein phosphorylated at the first and second phosphorylation sites which creates a binding site for the chaperone protein 14-3-3 and furthermore binds to FoxO factors in the nucleus, leading to its nuclear exclusion and ensuing inability to bind DNA [16]. Direct phosphorylation by the pathway leads to nuclear exclusion and FoxO inactivation [8, 22]. As a result, bladder cancer develops with the gradual phosphorylation by the pathway. It is probably in such a way that FoxO3 and FoxO4 showed a decrease in bladder cancer tissues. Paik et al. [23] reported in their study that broad somatic deletion of all FoxO family members created a condition which is progressive cancer-prone and characterized by hemangiomas and thymic lymphomas, demonstrating that FoxOs are tumor suppressors, which further backs our results. The study also revealed that patients with invasive bladder cancer showed lower positive rates of FoxO3 and FoxO4 protein than those with superficial bladder cancer, and the patients with lymph node metastasis showed lower positive rates of FoxO3 and FoxO4 protein than the ones without. About 90% of all primary bladder tumors are transitional-cell carcinomas, which originate in the bladder mucosa, then progressively invade lamina propria, and move into muscularis propria, perivesical fat and contiguous pelvic structures, and the progression is companied with increasing
incidence of lymph node metastasis [24]. As the carcinoma deteriorates into later stages concerning invasion and lymph node metastasis, constant Akt activation in bladder cancer cells then promotes cellular differentiation and survival by down-regulating a large quantity of transcription factors, FoxO3 and FoxO4 included [25]. In addition, we also found that positive expressions of FoxO3 and FoxO4 in bladder cancer tissues were associated with higher OS rate among patients. There is a high probability that activated FoxO3 and FoxO4 proteins enhance apoptosis through the induction of the expressions of death receptor ligands which include Fas ligand, Bcl-2 family members and tumor necrosis factor-related apoptosis-inducing ligand [26], thus preventing the progression of bladder cancer. Furthermore, functions of FoxO family also lie in its ability to act as tumor suppressors, benefiting stem cell maintenance and promote lifespan extension [11]. Ursula et al [15] have reported that in a complicated way related with FoxO3, breast cancer can be repressed to a certain degree, which is in consistent with the finding here. To sum up, we can come to the conclusion that FoxO3 and FoxO4 expressions may be associated with the clinicopathological features and prognosis of bladder cancer. Therefore, FoxO3 and FoxO4 expressions may act as prognostic indicators for bladder cancer. It is our sincere hope that this study can help promote individually-designed therapies in clinical treatment of bladder cancer so as to increase survival rate for the patients with bladder cancer. However, this study was based on not large enough samples to substantiate our conclusion. Therefore, more studies with larger samples need to be launched to verify the result.
Competing Interests The authors have declared that no competing interests exist
Acknowledgements This study was supported by the project of Natural Science Foundation of Hainan Province (No. 310128). All the authors are grateful to reviewers for all the critical comments to the manuscript and for those who made additional data available from their published papers.
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[17] S.B. Edge, C.C. Compton, The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM, Ann Surg Oncol. 17 (2010) 1471-1474. [18] L.H. Sobin, The WHO histological classification of urinary bladder tumours, Urol Res. 6 (1978) 193-195. [19] K.P. Lu, N.F. Alejandro, K.M. Taylor, M.M. Joyce, T.E. Spencer, K.S. Ramos, Differential expression of ribosomal L31, Zis, gas-5 and mitochondrial mRNAs following oxidant induction of proliferative vascular smooth muscle cell phenotypes, Atherosclerosis. 160 (2002) 273-280. [20] T. Powles, J.P. Eder, G.D. Fine, et al., MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer, Nature. 515 (2014) 558-562. [21] H.B. Grossman, R.B. Natale, C.M. Tangen, et al., Neoadjuvant chemotherapy plus cystectomy compared with cystectomy alone for locally advanced bladder cancer, N Engl J Med. 349 (2003) 859-866. [22] C. Mammucari, G. Milan, V. Romanello, et al., FoxO3 controls autophagy in skeletal muscle in vivo, Cell Metab. 6 (2007) 458-471. [23] J.H. Paik, R. Kollipara, G. Chu, et al., FoxOs are lineage-restricted redundant tumor suppressors and regulate endothelial cell homeostasis, Cell. 128 (2007) 309-323. [24] L.A. Kiemeney, S. Thorlacius, P. Sulem, et al., Sequence variant on 8q24 confers susceptibility to urinary bladder cancer, Nat Genet. 40 (2008) 1307-1312. [25] L.C. Trotman, A. Alimonti, P.P. Scaglioni, J.A. Koutcher, C. Cordon-Cardo, P.P. Pandolfi, Identification of a tumour suppressor network opposing nuclear Akt function, Nature. 441 (2006) 523-527. [26] Z. Fu, D.J. Tindall, FOXOs, cancer and regulation of apoptosis, Oncogene. 27 (2008) 2312-2319.
Legends Fig. 1. Expressions of FoxO3 and FoxO4 proteins in the bladder cancer and adjacent normal tissues by immunohistochemical staining (× 400). Note: A, the expression of FoxO3 in the adjacent normal tissues; B, the expression of FoxO3 in the bladder cancer tissues; C, the expression of FoxO4 in the adjacent normal tissues; D, the expression of FoxO4 in the bladder cancer tissues. Fig. 2. MRNA and protein expressions of FoxO3 and FoxO4 in the bladder cancer and adjacent normal tissues using qRT-PCR and western blotting. Note: A, relative expressions of FoxO3 and FoxO4 mRNA by qRT-PCR (with the mRNA expression in the adjacent normal tissues as 1); B, the expressions of FoxO3 and FoxO4 proteins by Western blotting; C, relative expressions of FoxO3 and FoxO4 protein; *, P < 0.05 compared with the adjacent normal tissues; qRT-PCR, quantitatve real- time polymerace chain reaction. Fig. 3. Correlation between the expressions of FoxO3 and FoxO4 proteins in the bladder cancer tissues. Fig. 4. Association of FoxO3 (A) and FoxO4 (B) expressions with OS. Note: OS, the overall survival. Fig. 5. Association of FoxO3 (A) and FoxO4 (B) expressions with DFS. Note: DFS, disease-free survival.
Figr-1
Figr-2
Figr-3
Figr-4
Figr-5
Table 1 The primer sequences of FoxO3 and FoxO4 for qRT-PCR. Gene
Primer sequence
FoxO3
F: 5'-CGTGGGTAAAAAGGTGTTCC-3'
Temperature (°C) 60
R: 5'-CAAGCCTCCAAACTCAGGAC-3' FoxO4
F: 5'-CCGAAGGTGCCACTCCAAAC-3'
60
R: 5'-GAATAAGGCCGGTGTGCGTT -3 β-actin
F: 5'-GGACCTGACTACCTCATGAA-3'
50
R:5'-CTTAATGTCACGCACGATTTCC-3' Note: qRT-PCR, quantitative real-time polymerase chain reaction; F, forward; R, reverse; FoxO3, forkhead box O3; FoxO4, forkhead box O4.
Table 2 The expressions of FoxO3 and FoxO4 proteins in the bladder cancer and adjacent normal tissues
FoxO3
FoxO4
Bladder cancer tissues
Adjacent normal tissues
(n = 222)
(n = 222)
-
150
63
+
72
159
Positive rate
32.43%
71.62%
-
146
84
+
76
138
Positive rate
34.23%
62.16%
Note: FoxO3, forkhead box O3; FoxO4, forkhead box O4.
P
< 0.001
< 0.001
Table 3 Association of FoxO3 and FoxO4 proteins with the clinicopathological features of patients with bladder cancer FoxO3 Cas es
Feature
Gender
-
+
15 0
7 2
Positive rate (%)
FoxO4 P
0.87 5
-
+
14 6
7 6
Positive rate (%)
0.085
Male
158
10 6
5 2
32.91%
98
6 0
39.24%
Female
64
44
2 0
31.25%
48
1 6
21.88%
0.10 0
Age
0.051
< 50 years
56
43
1 3
23.21%
43
1 3
23.21%
≥ 50 years
166
10 7
5 9
35.54%
10 3
6 3
37.95%
0.00 3
Lymph node metastasis
0.001
No
156
96
6 0
38.46%
92
6 4
41.03%
Yes
66
54
1 2
18.18%
54
1 2
18.18%
WHO histological classification
0.00 2
0.014
Low grade
154
94
6 0
38.96%
93
6 1
39.61%
High grade
68
56
1 2
17.65%
53
1 5
22.06%
0.00 1
TNM stage Tis-T1
126
74
5 2
41.27%
23
P
< 0.001 66
6 0
47.62%
T2-T3
96
76
2 0
20.83%
80
1 6
16.67%
Note: WHO, World Health Organization; TNM, tumor node metastasis; FoxO3, forkhead box O3; FoxO4, forkhead box O4.
24
Table 4 Risk factors for poor prognosis of patients with bladder cancer by Cox proportional hazard model analysis with OS. Factor
B S.E. Wald χ2
df
P
RR
95% CI
Gender
-0.422 0.447 0.892 1.000 0.345
0.656 0.273 1.574
Age
-0.076 0.475 0.026 1.000 0.873
0.927 0.366 2.351
FoxO3
0.554 0.229 5.822 1.000 0.016
1.740 1.110 2.728
FoxO4
1.162 0.252 21.256 1.000 < 0.001 3.195 1.950 5.236
TNM stage
2.974 0.391 57.789 1.000 < 0.001 19.563 9.088 42.112
Lymph node metastasis
1.664 0.353 22.196 1.000 < 0.001 5.279 2.642 10.547
WHO histological classification
0.607 0.271 5.021 1.000 0.025
1.835 1.079 3.121
Note: WHO, World Health Organization; TNM, tumor node metastasis; RR, relative risk; CI, confidence interval; OS, overall survival; FoxO3, forkhead box O3; FoxO4, forkhead box O4.
25
S0344-0338(16)30606-9 http://dx.doi.org/doi:10.1016/j.prp.2017.04.004 PRP 51782
To appear in: Received date: Revised date: Accepted date:
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Please cite this article as: Yang Wang, Xin-Li Kang, Fan-Chang Zeng, Cong-Jie Xu, Jia-Quan Zhou, Dong-Ni Luo, Correlations of Foxo3 and Foxo4 expressions with clinicopathological features and prognosis of bladder cancer, Pathology - Research and Practicehttp://dx.doi.org/10.1016/j.prp.2017.04.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Correlations of Foxo3 and Foxo4 expressions with clinicopathological features and prognosis of bladder cancer Running title: FoxO3 and FoxO4 expressions in bladder cancer
Yang Wang 1, *, Xin-Li Kang 1, Fan-Chang Zeng 1, Cong-Jie Xu 1, Jia-Quan Zhou 1, Dong-Ni Luo 2 1 Department
of Urology, Hainan General Hospital, Haikou 570311, P.R. China
2 Department
of Ultrasonography, Hainan General Hospital, Haikou 570311, P.R. China
*
Correspondence to: Dr. Yang Wang, Department of Urology, Hainan General Hospital, No.19
Xiuhua Road, Xiuying District, Haikou 570311, Hainan Province, P.R. China E-mail: [email protected] Tel/Fax: +86-0898-68642202
ABSTRACT Objective: The study is performed to explore the correlations of forkhead box O3 (FoxO3) and forkhead box O4 (FoxO4) expressions with clinicopathological features and prognosis of bladder cancer. Methods: Bladder cancer tissues and adjacent normal tissues from the recruited 222 patients were collected. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting and immunohistochemistry were applied to determine the expressions of FoxO3 and FoxO4. Spearman correlation analysis was conducted to examine the correlation between the expressions of FoxO3 and FoxO4. All patients were followed up and overall survival (OS) and disease-free survival (DFS) were recorded. Kaplan-Meier survival curve was drawn to determine the associations of FoxO3 and FoxO4 expressions and postoperative survival. Cox proportional hazards model was conducted to analyze the risk factors for poor prognosis of bladder cancer. Results: The mRNA and expressions of FoxO3 and FoxO4 proteins in the bladder cancer tissues were lower than that in the adjacent normal tissues (both P < 0.05). The positive rates of FoxO3 and FoxO4 were lower in the patients with lymph node metastasis than that in the patients without lymph node metastasis (P < 0.05), and significantly lower in the patients with non-muscle invasive bladder cancer (Tis-T1) than in those with non-muscle invasive bladder cancer (T2-T3) in TNM staging, and remarkably lower in the patients with high grade than in those with low grade in the histological type (P < 0.05). Furthermore, the expressions of FoxO3 and FoxO4 were positively correlated in the bladder cancer tissues (P < 0.05). Negative expressions of FoxO3 and FoxO4 and lymph node metastasis were the risk factors for the poor prognosis of bladder cancer. Conclusions: The FoxO3 and FoxO4 expressions may potentially associate with the clinicopathological features and prognosis of bladder cancer. Keywords: Forkhead box O3; Forkhead box O4; Bladder cancer; Clinicopathological features; Prognosis
1. Introduction Bladder cancer, the second malignancy of genitourinary tract only to prostate cancer (PC) and the 9th most common cancer worldwide, sees over 330,000 new cases were diagnosed and over 30,000 lives were lost per year [1]. Cigarette smoking and occupational exposure to aromatic amines have been found which were closely related to the risk of bladder cancer together with a family history of the carcinoma [2]. There are 80% of cases with non-muscle invasive bladder cancer when they were firstly diagnosed with a rather good prognosis, and they are likely to recur frequently and even progress to be muscle-invasive [3]. And the rest 20% of cases shows muscle invasion which are diagnosed with less favorable prognosis and their mortality rate accouts for about 50% [4]. For the treatment of invasive bladder cancer, the standard method remains radical surgical removal of the bladder within standard limits combined with additional neoadjuvant chemotherapy if necessary, which is still somewhat far from being satisfactory [5]. Although its exact mechanism remains unknown, tumorigenesis and progression of bladder cancer are closely related with chromosomal anomalies, epigenetic changes and genetic polymorphisms, and genetic changes are obviously involved in its treatment and prognosis [6]. Altered expression of oncogenes and tumor suppressors has been demonstrated the function in the initiation and progression of bladder cancer, making the discovery of new biomarkers of vital value in earlier detection and prognosis of bladder cancer [7]. Forkhead box class O (FoxO) transcription factors belong to the forkhead family of transcriptional regulators featured by a conserved DNA binding domain termed the ‘‘forkhead box’’, consisting of 4 members which are namely FoxO1, FoxO3, FoxO4 and FoxO6 [8, 9]. They are found to get involved in various cellular processes such as cell cycle regulation, apoptosis and resistance to reactive oxygen species [10]. It is also revealed that these transcription factors act as tumor suppressors, thus benefiting stem cell maintenance and lifespan extension [11]. Activated FoxO3 leads to remarkable atrophy of muscles and myotubes by stimulating overall protein
degradation and coordinately activating relevant pathways such as phosphoinositide-3 kinase (PI3K)-protein kinase B (PKB/c-Akt) signaling pathway, inducing expression of many autophagyrelated genes [12, 13]. FoxO4, the homolog of DAF-16 and also known as AFX, contributes to longevity and oxidative stress [14]. Previous studies have reported that FoxO3 and FoxO4 play a vitally important part in the development of many cancers such as breast cancer (BC) and PC [15, 16]. However, it still remains unveiled of their roles in the initiation, progression and prognosis of bladder cancer. Therefore, the study targets to investigate the correlations of the expressions of FoxO3 and FoxO4 with clinicopathological features and prognosis of bladder cancer.
2. Materials and methods 2.1. Study Subjects From January 2010 to January 2012, 222 patients with bladder cancer in Hainan General Hospital were recruited for the study. Their clinical data and tumor tissues were collected. There were 158 males and 64 female patients with the age of 36 to 79 years and the mean age of 55.23 ± 8.72 years, which included a total of 141 patients with baldder cancer treated with radical cystectomy, 41 patients with patial cystectomy and 40 patients with electroresection. There were 126 patients with non-muscle invasive bladder cancer (Tis-T1) and 96 patients with muscle invasive bladder cancer (T2-T3) according to the 7th edition of the American Joint Committee on Cancer (AJCC) TNM staging in cancer manual [17]. According to the World Health Organization (WHO) histological classification of urinary bladder tumors [18], there were 154 patients in low grade and 68 patients in high grade according to histological classification. Patients with local lymph node metastasis were confirmed by pathologically diagnosing the excised tissue and dissected lymph nodes after bladder cancer surgery. There were 156 patients without local lymph node metastasis while 66 patients were diagnosed with lymph node metastasis. The bladder cancer tissues from all 222 cases were collected as the experiment group, and the adjacent normal tissues were collected
and confirmed by pathology as the control group (the tissues were located over 2 cm away from the edge of the tumor). The inclusion criteria: all patients were pathologically diagnosed as bladder cancer; each case was diagnosed with adequate imaging and clinical examination who received at least one follow-up or examination after discharge. The exclusion criteria: patients were treated with chemotherapy, radiotherapy or biotherapy; patients suffered with other tumor diseases; patients with distant metastasis before treatment. The present study with all the procedures was approved by the Ethic Committee of Hainan General Hospital and all patients were informed consent for the study and signed written informed consents. 2.2. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) The total RNA was extracted from the tumor tissues and adjacent normal tissues using the kit (TIANGEN Biotechnology Co., Ltd, Beijing, China). The kit for reverse transcription was purchased from Hangzhou Bioer Technology Co., Ltd. (Hangzhou, China), and the operating procedures were strictly conformed to the kit instructions. Ultraviolet spectrophotometer was used to detect the optical density (OD260/280) value of the extracted RNA samples and the RNA concentration was calculated and preserved at -80°C. The mRNA expressions of FoxO3 and FoxO4 were detected by qRT-PCR. Primers were designed using softwae Primer 5.0 on the basis of the gene sequences published in Genebank and miR BASE database, which were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). The primer sequences were shown in Table 1. The PCR kit was purchased from Bio-Rad, Inc., Hercules, CA, USA, and type 7500 qRT-PCR instrument was from ABI Company, Oyster Bay, NY, USA. The reaction condition of qRT-PCR was: pre-denaturation for 30 s at 95°C, and 40 cycles of denaturation for 5 s at 95°C, annealing for 30 s and extension for 30 s. With β-actin as an internal reference, Ct values were compared between target gene and β-actin to calculate the relative
expression of the mRNA of target genes: ΔCt target gene = Ct target gene - Ct β-actin, ΔΔCt = ΔCt experimental group
- ΔCt control group. The ratio of target gene expressions in two groups is represented as 2－ΔΔCt [19].
2.3. Western blotting Total protein of wax blocks was extracted by extraction reagent of paraffin wax, and bicinchoninic acid (BCA) assay was performed to determine the protein concentration. After denaturation, the total protein (50 μg) was conducted sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) at constant voltage. Semidry method was applied to transfer the electrophoresis product into polyvinylidene fluoride (PVDF) membrane sealed with 5% skimmed milk for 2 h at 4°C. The protein was added with the first antibodies (rabbit with FoxO3 polyclonal antibody and rabbit with FoxO4 polyclonal antibody from Abcam Inc., Cambridge, MA, USA) and preserved for 10 h at 4°C. Next, the protein was washed with Tris-buffered saline Tween-20 (TBST) for 4 times (10 min for each time), and then added with horse-radish peroxidase labeled second antibody (goat anti rabbit IgG; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Then, the protein was incubated for 2 h at 37°C, and washed for 4 times (10 min for each time) with TBST, finally chemi-Luminescence (ECL) kit was used for coloring. Gel electrophoresis imaging analysis system was performed for automatic imaging and the relative protein levels of FoxO3 and FoxO4 were analysed with β-actin as an internal reference. 2.4. Immunohistochemistcal (IHC) SP three-step method was applied for IHC staining to detect the protein expressions of FoxO3 and FoxO4 in the bladder cancer tissues and adjacent normal tissues. Normal bladder tissue sections were collected as positive control and phosphate buffer solution (PBS) instead of the first antibody as negative control, which were stained using HistostainTMSP-9000 staining kit (Zymed Laboratories Inc., South San Francisco, CA, USA). The paraffin sections underwent conventional dewaxing and then were heated by microwave to repair antigen. The heating was stopped for 5 min after boiling, and the tissue sections were heated again and then cooled to room temperature. After
washing with PBS, the sections were added with normal goat serum, and then with the first antibodies at 4°C overnight (rabbit with FoxO3 polyclonal antibody and rabbit with FoxO4 polyclonal antibody were all purchased from Abcam Inc., Cambridge, MA, USA. After rewarming and washing with PBS, the tissue sections were added with working fluid of second antibody (biotin labeled goat anti rabbit IgG) for 30 min at 37°C. After washing with PBS, the tissue sections were incubated with horseradish labeled working fluid, and 3, 3'-diaminobenzidine (DAB) was used for coloring for 5 ~ 10 min, which were ajusted the dying time under the microscope (×400) and sealed with gum after 1 min of hematoxylin staining, finally preserved after taking photo and printing. The proteins of FoxO3 and FoxO4 were mostly expressed in the nucleus with pale brown or brown particles and with weak expression in the cytoplasm. The brown particles in the nucleus were recognized as positive cells. Ten fields were randomly selected to observe and count the positive cells in each field. Positive cells < 10% was considered as negative expression. On the contrary, positive cells ≥ 10% was recognized as positive expression. 2.5. Follow-up Telephone follow-up was conducted to all patients, and outpatient follow-up was carried out to survival patients. The follow-up ended in March 2016. The overall survival (OS) was calculated, as well as the disease-free survival (DFS). The OS was the duration from the surgery to death or to the last follow-up and DFS was the duration from the surgery to tumor recurrence or metastasis. Kaplan-Meier curve was drawn in accordance with the results of follow-ups to compare OS and DFS. All patients were followed up completely. 2.6. Statistical analysis All the data were used statistical software SPSS for statistical analysis. Comparison between two groups was analyzed by student’ s t detection while comparison of measurement data between groups. The measurement data were presented as mean ± standard deviation, which were analyzed
by Least-Significant Difference Test (LSD-t) and paired-samples LSD-t between two groups and by one-way analysis of variance (ANOVA) among multiple groups. Baum-Welch Algorithm was conducted for heterogeneity of variance and comparisons between multiple groups were analysed by Dunnett’s T3 test. Enumeration data were measured by χ2 test. Univariate analysis of prognostic was performed for Kaplan-Meier curve and log-rank was used for detection. Multivariate analysis of prognostic was performed for Cox proportional hazards model, and Spearman was conducted for correlation analysis. P < 0.05 indicates that the difference is statistically significant.
3. Results 3.1. Positive Rates of FoxO3 and FoxO4 Proteins in the Bladder Cancer and Adjacent Normal TissuesAs Fig. 1 shown, the proteins of FoxO3 and FoxO4 was mostly expressed in the cell nucleus but weakly expressed in the cell cytoplasm with positive reaction of pale brown or brown particles. As shown in Table 2, the positive rate of FoxO3 protein was lower in the bladder cancer tissues (32.43%) than that in the adjacent normal tissues (71.62%) (P < 0.05), and the positive rate of FoxO4 protein was lower in the bladder cancer tissues (34.23%) than that in the adjacent normal tissues (62.16%) (P < 0.05). 3.2. MRNA and Protein Expression of FoxO3 and FoxO4 in the Bladder Cancer and Adjacent Normal Tissues As shown in Fig. 2, mRNA and the protein expressions of FoxO3 and FoxO4 in the bladder cancer tissues were significantly decreased as compared with the adjacent normal tissues (all P < 0.05). 3.3. Correlation between the Expressions of FoxO3 and FoxO4 in the Bladder Cancer Tissues Spearman correlation analysis was performed to detecte the correlation with the expressions of FoxO3 and FoxO4 in the bladder cancer tissues, which showed the positive cerrelation between the expressions of FoxO3 and FoxO4 in the bladder cancer tissues (r = 0.522, P < 0.001, Fig. 3).
3.4. Association of FoxO3 and FoxO4 Protein with the Clinicopathological Features in the Bladder Cancer tissues As shown in Table 3, the positive rates of FoxO3 and FoxO4 protein were significantly lower in the patients with lymph node metastasis than that in those without lymph node metastasis (P < 0.05), also significantly lower in those with invasive bladder cancer (T2-T3) than that in those with superficial bladder cancer (Tis-T1) (P < 0.05). According to WHO histological classification, the positive rates of FoxO3 and FoxO4 protein were significantly lower in the patients with high grade than that in those with low grade (P < 0.05). However, there was no significant difference in gender or age, with regard to the positive rates of FoxO3 and FoxO4 protein (both P > 0.05). 3.5. Association of FoxO3 and FoxO4 Expressions with Postoperative Survival All 222 bladder cancer patients were followed up for 60 months but out of 24 cases, and 118 cases died of this cancer. The OS was 46.85%. As Kaplan-Meier curve showed (Fig. 4), the OS of patients with positive expressions of FoxO3 and FoxO4 in the bladder cancer tissues was higher than those with negative expressions (P = 0.005 and P = 0.031, respectively). There were 140 patients with local recurrence and/or metastasis (In the follow-up, patients with local recurrence and/or metastasis, margin-positive bladder cancer, vascular and/or neural invasion were treated with adjuvant systemic chemothera.) and the DFS was 36.94%. As Kaplan-Meier curve showed (Fig. 5), the DFS of patients with positive expressions of FoxO3 and FoxO4 in the bladder cancer tissues was higher than those with negative expressions (P < 0.001 and P < 0.001, respectively). 3.6. Prognostic Factors for Bladder Cancer As shown in Table 4, Cox proportional hazard model analysis indicated that negative expressions of FoxO3 and FoxO4, TNM stage, lymph node metastasis and WHO histological classification were the prognostic factors for bladder cancer (all P < 0.05), while the age and gender showed no association with the prognosis of bladder cancer (both P > 0.05).
4. Discussion Generally, there were no major advances in the treatment of metastatic bladder cancer in the last 3 decades [20]. Although cystectomy combined with neoadjuvant chemotherapy is associated with improved outcomes among patients with bladder cancer, the survival rate for those in later stages remains poor [21]. Therefore, it is of vital importance to address the disease from the perspective of molecular levels. It was found out in the study, that the expressions of FoxO3 and FoxO4 decreased in the bladder cancer tissues as compared to the adjacent normal tissues, which were positively correlated with each other. As has been previously pointed out, FoxO family is regulated by multiple signaling pathways, most notably by PI3K-PKB/c-Akt signaling pathway [13]. The pathway and its related kinase can get FoxO protein phosphorylated at the first and second phosphorylation sites which creates a binding site for the chaperone protein 14-3-3 and furthermore binds to FoxO factors in the nucleus, leading to its nuclear exclusion and ensuing inability to bind DNA [16]. Direct phosphorylation by the pathway leads to nuclear exclusion and FoxO inactivation [8, 22]. As a result, bladder cancer develops with the gradual phosphorylation by the pathway. It is probably in such a way that FoxO3 and FoxO4 showed a decrease in bladder cancer tissues. Paik et al. [23] reported in their study that broad somatic deletion of all FoxO family members created a condition which is progressive cancer-prone and characterized by hemangiomas and thymic lymphomas, demonstrating that FoxOs are tumor suppressors, which further backs our results. The study also revealed that patients with invasive bladder cancer showed lower positive rates of FoxO3 and FoxO4 protein than those with superficial bladder cancer, and the patients with lymph node metastasis showed lower positive rates of FoxO3 and FoxO4 protein than the ones without. About 90% of all primary bladder tumors are transitional-cell carcinomas, which originate in the bladder mucosa, then progressively invade lamina propria, and move into muscularis propria, perivesical fat and contiguous pelvic structures, and the progression is companied with increasing
incidence of lymph node metastasis [24]. As the carcinoma deteriorates into later stages concerning invasion and lymph node metastasis, constant Akt activation in bladder cancer cells then promotes cellular differentiation and survival by down-regulating a large quantity of transcription factors, FoxO3 and FoxO4 included [25]. In addition, we also found that positive expressions of FoxO3 and FoxO4 in bladder cancer tissues were associated with higher OS rate among patients. There is a high probability that activated FoxO3 and FoxO4 proteins enhance apoptosis through the induction of the expressions of death receptor ligands which include Fas ligand, Bcl-2 family members and tumor necrosis factor-related apoptosis-inducing ligand [26], thus preventing the progression of bladder cancer. Furthermore, functions of FoxO family also lie in its ability to act as tumor suppressors, benefiting stem cell maintenance and promote lifespan extension [11]. Ursula et al [15] have reported that in a complicated way related with FoxO3, breast cancer can be repressed to a certain degree, which is in consistent with the finding here. To sum up, we can come to the conclusion that FoxO3 and FoxO4 expressions may be associated with the clinicopathological features and prognosis of bladder cancer. Therefore, FoxO3 and FoxO4 expressions may act as prognostic indicators for bladder cancer. It is our sincere hope that this study can help promote individually-designed therapies in clinical treatment of bladder cancer so as to increase survival rate for the patients with bladder cancer. However, this study was based on not large enough samples to substantiate our conclusion. Therefore, more studies with larger samples need to be launched to verify the result.
Competing Interests The authors have declared that no competing interests exist
Acknowledgements This study was supported by the project of Natural Science Foundation of Hainan Province (No. 310128). All the authors are grateful to reviewers for all the critical comments to the manuscript and for those who made additional data available from their published papers.
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[17] S.B. Edge, C.C. Compton, The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM, Ann Surg Oncol. 17 (2010) 1471-1474. [18] L.H. Sobin, The WHO histological classification of urinary bladder tumours, Urol Res. 6 (1978) 193-195. [19] K.P. Lu, N.F. Alejandro, K.M. Taylor, M.M. Joyce, T.E. Spencer, K.S. Ramos, Differential expression of ribosomal L31, Zis, gas-5 and mitochondrial mRNAs following oxidant induction of proliferative vascular smooth muscle cell phenotypes, Atherosclerosis. 160 (2002) 273-280. [20] T. Powles, J.P. Eder, G.D. Fine, et al., MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer, Nature. 515 (2014) 558-562. [21] H.B. Grossman, R.B. Natale, C.M. Tangen, et al., Neoadjuvant chemotherapy plus cystectomy compared with cystectomy alone for locally advanced bladder cancer, N Engl J Med. 349 (2003) 859-866. [22] C. Mammucari, G. Milan, V. Romanello, et al., FoxO3 controls autophagy in skeletal muscle in vivo, Cell Metab. 6 (2007) 458-471. [23] J.H. Paik, R. Kollipara, G. Chu, et al., FoxOs are lineage-restricted redundant tumor suppressors and regulate endothelial cell homeostasis, Cell. 128 (2007) 309-323. [24] L.A. Kiemeney, S. Thorlacius, P. Sulem, et al., Sequence variant on 8q24 confers susceptibility to urinary bladder cancer, Nat Genet. 40 (2008) 1307-1312. [25] L.C. Trotman, A. Alimonti, P.P. Scaglioni, J.A. Koutcher, C. Cordon-Cardo, P.P. Pandolfi, Identification of a tumour suppressor network opposing nuclear Akt function, Nature. 441 (2006) 523-527. [26] Z. Fu, D.J. Tindall, FOXOs, cancer and regulation of apoptosis, Oncogene. 27 (2008) 2312-2319.
Legends Fig. 1. Expressions of FoxO3 and FoxO4 proteins in the bladder cancer and adjacent normal tissues by immunohistochemical staining (× 400). Note: A, the expression of FoxO3 in the adjacent normal tissues; B, the expression of FoxO3 in the bladder cancer tissues; C, the expression of FoxO4 in the adjacent normal tissues; D, the expression of FoxO4 in the bladder cancer tissues. Fig. 2. MRNA and protein expressions of FoxO3 and FoxO4 in the bladder cancer and adjacent normal tissues using qRT-PCR and western blotting. Note: A, relative expressions of FoxO3 and FoxO4 mRNA by qRT-PCR (with the mRNA expression in the adjacent normal tissues as 1); B, the expressions of FoxO3 and FoxO4 proteins by Western blotting; C, relative expressions of FoxO3 and FoxO4 protein; *, P < 0.05 compared with the adjacent normal tissues; qRT-PCR, quantitatve real- time polymerace chain reaction. Fig. 3. Correlation between the expressions of FoxO3 and FoxO4 proteins in the bladder cancer tissues. Fig. 4. Association of FoxO3 (A) and FoxO4 (B) expressions with OS. Note: OS, the overall survival. Fig. 5. Association of FoxO3 (A) and FoxO4 (B) expressions with DFS. Note: DFS, disease-free survival.
Figr-1
Figr-2
Figr-3
Figr-4
Figr-5
Table 1 The primer sequences of FoxO3 and FoxO4 for qRT-PCR. Gene
Primer sequence
FoxO3
F: 5'-CGTGGGTAAAAAGGTGTTCC-3'
Temperature (°C) 60
R: 5'-CAAGCCTCCAAACTCAGGAC-3' FoxO4
F: 5'-CCGAAGGTGCCACTCCAAAC-3'
60
R: 5'-GAATAAGGCCGGTGTGCGTT -3 β-actin
F: 5'-GGACCTGACTACCTCATGAA-3'
50
R:5'-CTTAATGTCACGCACGATTTCC-3' Note: qRT-PCR, quantitative real-time polymerase chain reaction; F, forward; R, reverse; FoxO3, forkhead box O3; FoxO4, forkhead box O4.
Table 2 The expressions of FoxO3 and FoxO4 proteins in the bladder cancer and adjacent normal tissues
FoxO3
FoxO4
Bladder cancer tissues
Adjacent normal tissues
(n = 222)
(n = 222)
-
150
63
+
72
159
Positive rate
32.43%
71.62%
-
146
84
+
76
138
Positive rate
34.23%
62.16%
Note: FoxO3, forkhead box O3; FoxO4, forkhead box O4.
P
< 0.001
< 0.001
Table 3 Association of FoxO3 and FoxO4 proteins with the clinicopathological features of patients with bladder cancer FoxO3 Cas es
Feature
Gender
-
+
15 0
7 2
Positive rate (%)
FoxO4 P
0.87 5
-
+
14 6
7 6
Positive rate (%)
0.085
Male
158
10 6
5 2
32.91%
98
6 0
39.24%
Female
64
44
2 0
31.25%
48
1 6
21.88%
0.10 0
Age
0.051
< 50 years
56
43
1 3
23.21%
43
1 3
23.21%
≥ 50 years
166
10 7
5 9
35.54%
10 3
6 3
37.95%
0.00 3
Lymph node metastasis
0.001
No
156
96
6 0
38.46%
92
6 4
41.03%
Yes
66
54
1 2
18.18%
54
1 2
18.18%
WHO histological classification
0.00 2
0.014
Low grade
154
94
6 0
38.96%
93
6 1
39.61%
High grade
68
56
1 2
17.65%
53
1 5
22.06%
0.00 1
TNM stage Tis-T1
126
74
5 2
41.27%
23
P
< 0.001 66
6 0
47.62%
T2-T3
96
76
2 0
20.83%
80
1 6
16.67%
Note: WHO, World Health Organization; TNM, tumor node metastasis; FoxO3, forkhead box O3; FoxO4, forkhead box O4.
24
Table 4 Risk factors for poor prognosis of patients with bladder cancer by Cox proportional hazard model analysis with OS. Factor
B S.E. Wald χ2
df
P
RR
95% CI
Gender
-0.422 0.447 0.892 1.000 0.345
0.656 0.273 1.574
Age
-0.076 0.475 0.026 1.000 0.873
0.927 0.366 2.351
FoxO3
0.554 0.229 5.822 1.000 0.016
1.740 1.110 2.728
FoxO4
1.162 0.252 21.256 1.000 < 0.001 3.195 1.950 5.236
TNM stage
2.974 0.391 57.789 1.000 < 0.001 19.563 9.088 42.112
Lymph node metastasis
1.664 0.353 22.196 1.000 < 0.001 5.279 2.642 10.547
WHO histological classification
0.607 0.271 5.021 1.000 0.025
1.835 1.079 3.121
Note: WHO, World Health Organization; TNM, tumor node metastasis; RR, relative risk; CI, confidence interval; OS, overall survival; FoxO3, forkhead box O3; FoxO4, forkhead box O4.
25