Overexpression of TG-Interacting Factor Is Associated with Worse Prognosis in Upper Urinary Tract Urothelial Carcinoma

The American Journal of Pathology, Vol. 181, No. 3, September 2012 Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2012.05.024

Tumorigenesis and Neoplastic Progression

Overexpression of TG-Interacting Factor Is Associated with Worse Prognosis in Upper Urinary Tract Urothelial Carcinoma

Bi-Wen Yeh,*† Wen-Jeng Wu,‡§¶ Wei-Ming Li,‡§ Ching-Chia Li,‡§ Chun-Nung Huang,§ Wan-Yi Kang,储 Zi-Miao Liu,* and Huei-Sheng Huang储

prognostic target for selected patients with upper urinary tract UC. (Am J Pathol 2012, 181:1044–1055; http://dx.

From the Department of Medical Laboratory Science and Biotechnology* and the Institute of Basic Medical Sciences,† College of Medicine, National Cheng Kung University, Tainan; the Graduate Institute of Medicine,‡ College of Medicine, and the Department of Urology,§ Faculty of Medicine, College of Medicine, Kaohsiung Medical University Hospital, the Department of Urology,¶ Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University, Kaohsiung; and the Department of Pathology,储 Kuo General Hospital, Tainan, Taiwan

Urothelial carcinoma (UC), which tends to occur multifocally in the entire urothelium, including the renal pelvis, ureter, bladder, and proximal urethra, was the fourth most common cancer among men in the United States in 2010.1 Most UC (90% to 95%) occurs in the urinary bladder.2 On the basis of their histopathologic and clinical behavior, UCs can be classified into two main phenotypic variants: non–muscle invasive (Ta, T1, and Tis) and muscle invasive (T2, T3, and T4). The invasive and metastatic UC phenotype is a crucial event in tumor progression and the major cause of mortality in patients.3 Although aggressive UC is a major cause of morbidity and mortality, the molecular events associated with regulating its invasive phenotype are still unclear. Several clinicopathologic risk factors have been identified as clinical prognostic factors to predict tumor behaviors.4 However, a recent study has revealed that patient outcome after surgery for UC has not improved significantly in two decades, mainly because of these poor conventional prognostic factors.5 The incidence of renal pelvic and ureteral UC (upper urinary tract UC [UUT-UC]) is relatively low, accounting for only 5% to 10% of all UCs.6,7 Clinical stage and pathologic grade are the primary prognostic factors documented.8 However, differences in the biological potential are still observed, even in patients with the same

Prognostic outcome prediction would be useful for the treatment of patients with upper urinary tract urothelial carcinoma (UC). However, its prognostic biomarkers are not well established so far. According to the results of analysis of 168 human upper urinary tract UC specimens, overexpressed TG-interacting factor (TGIF) in nuclei of tumor tissues is significantly correlated with poor progression-free survival and higher cancer-related death. When both TGIF and p21 expression are altered, these patients had an even worse prognosis than those with one or no marker altered. Furthermore, to elucidate the role of TGIF in the progression of UC, overexpression of TGIF in RT4 or TSGH8301 cells was performed, and the results revealed that TGIF can significantly increase migration/invasion ability, matrix metalloproteinase expression, and invadopodia formation via the phosphatidylinositol 3-kinase–AKT pathway. In contrast, knockdown of TGIF with its specific short hairpin RNA inhibited the invasion ability of T24 cells. Besides, TGIF could inhibit p21WAF/CIP1 expression, upregulate cyclin D1 expression, and phosphorylate retinoblastoma to promote G1-S transition and cellular proliferation. In conclusion, we demonstrated that TGIF contributes to the progression of urothelial carcinoma via the phosphatidylinositol 3-kinase–AKT pathway. It may serve as an attractive therapeutic or

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Supported in part by grants from the National Science Council (Taipei, Taiwan; NSC95-2320-B-006-055-MY3, NSC98-2320-B-006-008-MY3, NSC98-2314-B-037-041-MY3) and Kaohsiung Medical University Hospital (Kaohsiung, Taiwan; KMUH95-5D36, KMUH98-8R37). Accepted for publication May 17, 2012. Supplemental material for this article can be found at http://ajp. amjpathol.org or at http://dx.doi.org/10.1016/j.ajpath.2012.05.024. Address reprint requests to Huei-Sheng Huang, Ph.D., Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan. E-mail: huanghs@ mail.ncku.edu.tw.

TGIF Is a Prognostic Marker of UUT-UC 1045 AJP September 2012, Vol. 181, No. 3

stage or grade.9,10 Thus, it is important to seek potential prognostic markers to select a subset group of patients with risk of progression to facilitate more effective treatment. An unusually high incidence of UUT-UC has been documented in Taiwan and represents 20% to 25% of UC.4,11 Our previous investigation had found that in patients with UUT-UC, tumor stage is the only prognostic factor for both local recurrence and cancer-specific survival, after radical nephroureterectomy. In patients with ureteral UC who had undergone radical nephroureterectomy, advanced tumor stage and chronic renal disease stages were significantly associated with a worse prognosis.12,13 Although the exact causative reasons for the higher prevalence of UUT-UC is not known, environmental carcinogen exposure, such as ingested water contaminated with arsenic or herbal medicines that contain aristolochic acid, may explain some of these risks.14 –16 However, individual differences in the ability to counteract carcinogens may also influence the susceptibility and risk of developing UUT-UC. Because of the higher prevalence of UUT-UC in Taiwan, it is thus worthy to study this disease extensively to develop more effective therapeutic strategies. Previously, we found that 5=TG3=-interacting factor (TGIF) can inhibit p21WAF1/CIP1 (p21) expression and cell death.17,18 TGIF has been initially identified as a nuclear protein that functions as a transcriptional repressor within the transforming growth factor ␤ and retinoic acid signaling pathways.19,20 It belongs to the three–amino acid loop extension class of atypical homeodomain proteins.21 TGIF is implicated in diverse biological and pathologic functions, such as hematopoietic stem cell function, brain development,22 regulating vascularization of the embryonic placenta,23 differentiation of preadipocytes,24 and various types of human cancer.25–29 Epidermal growth factor signaling pathway can phosphorylate TGIF to prolong its half-life and increase its protein level, which results in the enhancement of TGIF function to acquire the resistance to transforming growth factor ␤–induced growth inhibition in cancer cells.30,31 Borlak et al32 found that TGIF might contribute to the initial malignant transformation of hepatocytes in an animal model of epidermal growth factor–induced hepatocellular carcinoma. These studies suggest that TGIF may play a role in tumorigenesis. The imbalance of the Tgiflocated chromosome (18p11) has also been found in UC of the bladder.28,33 The TGIF expression in microarray studies of UC patients from the ONCOMINE database (http://www.oncomine.org; free registration required for access) also shows 1.5- to 2.0-fold change of TGIF overexpression in UC to normal bladder tissues.34,35 However, its functional roles and the molecular mechanisms involved in UC and especially UUT-UC are still unclear. In this study, the prognostic significance of TGIF in UUT-UC patients who underwent radical nephroureterectomy was evaluated by using immunohistochemical staining. The results indicate that TGIF is important to the progression of UUT-UC. Therefore, we used the overexpression and short hairpin (shRNA) knockdown methods to elucidate the molecular mechanisms of TGIF in UC

cells and established that TGIF was involved in the cellular proliferation, migration/invasion, and invadopodia formation via the phosphatidylinositol 3-kinase (PI3K)– AKT pathway.

Materials and Methods Patients and Specimens The study was performed with the approval of the institutional review board (KMUH-IRB-970428) of the Department of Urology of Kaohsiung Medical University Hospital. The study cohort was composed of samples from 168 patients with UUT-UC, including 73 men and 95 women (mean age, 65.9 years), with a median follow-up of 40 months (range, 1–192 months), who had undergone radical nephroureterectomy at the Department of Urology of Kaohsiung Medical University Hospital between January 1991 and December 2005. Paraffin blocks that contained sufficient formalin-fixed tumor specimens were obtained for analysis. Ten normal urothelial tissue specimens serving as controls were sampled from radical nephrectomy for renal cell carcinoma and were pathologically confirmed to be free of cancer. The clinical and pathologic data were recorded retrospectively. The pathologic stage of the tumor (pT) was assessed according to the 2010 American Joint Committee on Cancer TNM classification. Tumor grade was assessed according to the grading system established by the World Health Organization in 2004. Postoperative follow-up consisted of interval history and physical examination, urinalysis, urine cytology, chest radiography, abdominal ultrasonography, intravenous urography, and abdominal computed tomography. Cystoscopy was performed every 3 months for the first 2 years, every 6 months for the next 2 years, and annually thereafter.

Reagents and Antibodies TRIzol RNA extraction kit, SuperScriptIII, RPMI 1640 medium, and Opti-MEM medium were obtained from Invitrogen (Carlsbad, CA). TurboFect transfection reagent was obtained from Fermentas (Glen Burnie, MD). Antibodies against matrix metalloproteinase (MMP)2, MMP9, and Ncadherin were obtained from Upstate Biotechnology (Lake Placid, NY), and antibodies against proliferating cell nuclear antigen (PCNA), TGIF, phospho-AKTSer473, retinoblastoma (Rb), cyclin D, and cortactin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against pRb807/811 and phosphatase and tensin homolog (PTEN) were obtained from Cell Signaling Technology (Boston, MA). Anti-␤-actin antibody and PI3K inhibitor (LY294002 or wortmannin) were obtained from Sigma-Aldrich (St. Louis, MO). Alexa Fluor 488 –phalloidin, Alexa 546 –labeled secondary antibodies, and ProLongR Gold antifade reagent with DAPI were obtained from Invitrogen. The pLKO.1-TGIF-shRNA, which targets the human TGIF gene sequence 5=-GCAAGAGATGAATTGCATTAT-3=, and a luciferase control (pLKO.1-shLuc) plasmid construct were obtained from the National RNAi Core Facility located at the Institute of

1046 Yeh et al AJP September 2012, Vol. 181, No. 3

Molecular Biology/Genomic Research Center, Academia Sinica (Taipei, Taiwan), supported by the National Research Program for Genomic Medicine Grants of the National Science Council (NSC97-3112-B-001-016).

Cell Culture and Transfection Assay Human UC cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 mg/mL of streptomycin, and 100 U/mL of penicillin and incubated at 37°C in a humidified 5% CO2 incubator. The transfection method was performed with TurboFect reagent according to the manufacturer’s instructions. Cell lysates were harvested and subjected to Western blot after incubation.

Cell Proliferation Assay UC cells or TGIF-transfected cells were subcultured to 6-cm dishes at a density of 2 ⫻ 105 cells per dish for various periods. Cell numbers were determined by manual counting with a counting chamber after staining with trypan blue (0.03%).

Colony Formation Assay Cells were seeded in dishes at a density of 2 ⫻ 105 cells per dish for approximately 2 to 3 weeks until the foci were visible. The colonies were washed twice with 1⫻ PBS and then stained with 0.5% crystal violet for 1 minute after fixation with 4% paraformaldehyde for 15 minutes. The colonies larger than 0.1 mm in diameter were counted with a microscope.

Western Blot Cell lysates were separated in an analytical 10% SDSPAGE gel and transferred onto the polyvinylidene difluoride membrane. Antibodies against human TGIF, p21, phospho-AKTSer473, PCNA, MMP2, MMP9, N-cadherin, cyclin D, pRb807/811, Rb, PTEN, and ␤-actin were used as primary antibodies. Rabbit or mouse IgG antibody coupled with horseradish peroxidase was used as a secondary antibody. The density of the immunoblots was determined by an image analysis system installed with a software BIO-ID (Vilber Lourmat, Marne-la-Vallée, France) after development with an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ).

Gelatin Zymography The activities of MMP2 were determined by gelatin zymography assay. Briefly, cells (2 ⫻ 105) were cultured in dishes with serum-free medium for 48 hours. The supernatant was collected and concentrated using Amicon Ultra-4 Centrifugal Filter Devices (Millipore, Billerca, MA). A total of 5 ␮g of pellets were resuspended in sample buffer containing 0.01% SDS, 0.12 M Tris-HCl, 20% glycerol, and 0.1% bromophenol blue and loaded on a 8% zymogram gelatin gels (Invitrogen). After electro-

phoresis, gels were washed twice with renaturing buffer containing 25 mL of Triton X-100 and 975 mL of H2O on a gyratory shaker for 30 minutes at room temperature to remove the SDS. The gel was then incubated for 24 hours at 37°C in reaction buffer (40 mmol/L Tris-HCl, pH 8.0, 10 mmol/L CaCl2, 0.01% NaN3). Gels were then stained with staining solution (1% Coomassie blue R-250, 0.25% Amido black, 250 mL of methanol, 50 mL of acetic acid, 200 mL of H2O) and destained in a destaining buffer (20% methanol, 10% acetic acid, and 70% water). Finally, gels were then submitted for image analysis.

Immunofluorescence and Confocal Microscopy Analysis Cells were plated on a dish containing glass coverslips coated with collagen IV for 12 hours. After transfection with plasmids for 24 hours, cells were washed and fixed with 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.2% Triton X-100, and then blocked with 10% fetal bovine serum of PBS. Cells were then stained with anti-cortactin antibody and/or Alexa Fluor 488 –phalloidin for detection of invadopodia formation. The images were captured on a Leica TCS SP2 confocal microscope and analyzed using a 63⫻ oil-immersion objective. Quantification of cells with invadopodia was performed in three independent experiments in which at least 200 cells were counted.

Immunohistochemistry Immunohistochemical staining of p21 and TGIF was performed on 4-␮m–thick paraffin-embedded tissue sections. After deparaffinization, rehydration, antigen retrieval, and nonspecific binding blocking, tissue sections were then incubated with an antibody against either p21 or TGIF at 37°C for 1 hour followed by incubation with horseradish peroxidase– conjugated antibody to develop staining with diaminobenzidine peroxidase substrate solution. Results were recorded with a magnifier digital camera (Olympus BX51 DP70, Tokyo, Japan).

Cellular Migration/Invasion Assay Cell migration/invasion assay was performed by a modified Boyden chamber assay (48-well micro-Boyden chamber; Neuroprobe, Gaithersburg, MD). After transfection, the cells were harvested and counted with a counting chamber after staining with trypan blue (0.03%) to measure the cell viability. Viable cells (1.5 ⫻ 104) were added to the upper chamber of uncoated polycarbonate membrane (8-␮m pore size) for migration assay or membrane coated with Matrigel (BD Bioscience, Bedford, MA) for invasion assay, respectively. RPMI 1640 medium supplemented with 10% fetal bovine serum was placed into each well of the bottom chamber to act as a chemoattractant. After an additional incubation for 12 hours at 37°C, cells that migrated to or invaded the bottom surface of the membrane were fixed with 100% methanol for 10 minutes, followed by staining with 10%

TGIF Is a Prognostic Marker of UUT-UC 1047 AJP September 2012, Vol. 181, No. 3

Normal urothelia (UUT)

B

p21

TGIF (+)

Log rank p=0.002

Follow-up (months)

p21

Case 1

Case 1

Case 2

Case 2

F None marker altered

Disease-specific survival

TGIF (-)

Advanced (UUT-UC) TGIF

E

Progression-free survival

D

C

Superficial (UUT-UC) TGIF

TGIF

TGIF (-)

TGIF (+)

Log rank p<0.001

Follow-up (months)

Cancer-specific survival

A

One marker altered Two marker altered

Log rank p<0.001

Follow-up (months)

Figure 1. Patients with both prognostic factor alterations (TGIF and p21) had a worse cancer-specific survival than those with one or no adverse prognostic factors. Immunohistochemical staining was performed to detect endogenous TGIF in normal UUT epithelial (A) and TGIF and p21 protein expression (B and C) in 168 specimens of UUT-UC patients as described in Materials and Methods. Original magnification, ⫻200. Progression-free (D) and cancer-specific (E) survival with regard to TGIF expression were analyzed by Kaplan-Meier curves. F: The specimens were classified into three groups: no marker altered, one marker altered (⬎50% of positive cells in nuclei for TGIF or ⬍10% of positive cells in nuclei for p21), and two markers altered (⬎50% of positive cells in nuclei for TGIF and ⬍10% of positive cells in nuclei for p21). The 5-year cancer-specific survival rates of the three groups were analyzed by the Kaplan-Meier curves as indicated.

Giemsa for 30 minutes. The cell numbers were counted under a microscope in 5 random fields (100⫻). Means were based on the numbers from the triplicate wells for each reaction.

Statistical Analysis A representative result is presented; all experiments were performed at least three times. The association between p21 and TGIF expression and clinicopathologic features was assessed using the ␹2 test. Cancer-specific survival was determined using the Kaplan-Meier method, followed by the log-rank test. Statistical analyses were performed using SPSS statistical software version 17.0 (SPSS Inc, Chicago, IL). For all in vitro experiments, differences between the groups were statistically evaluated using the unpaired Student’s t-test. All values were displayed as mean ⫾ SD for three determinations.

Results Alteration of TGIF and p21 Correlate with Poor Prognosis of UUT-UC Patients Previous studies have addressed the role of p21 as an independent prognostic marker, which evidenced that altered p21 expression is associated with an increased

risk of UC disease progression and decreased patients’ overall survival.36 –38 Therefore, the correlation of the expression of TGIF/p21 with the clinical behaviors of UUT-UC patients was evaluated immunohistochemically. Ten normal urothelial specimens from patients with UUT were analyzed. A representative result is showed in Figure 1A. It indicates that TGIF expression in nuclei is ⬍40% (Figure 1A). A normal urothelial specimen of the bladder also shows a similar result (see Supplemental Figure S1A at http://ajp.amjpathol.org). Therefore, tumors with ⬎50% TGIF expression in nuclei were categorized as having altered TGIF status. In tumor tissues of UUT, highly expressed TGIF in nuclei (⬎50% of positive cells in the nuclei) (Figure 1, B and C) was found in 55 specimens (32.7%), whereas low expression of p21 in nuclei (⬍10% of positive cells in the nuclei) (Figure 1, B and C) was found in 66 specimens (39.2%). Tumor tissues of UC of the bladder also show similar results (see Supplemental Figure S1, B and C, at http://ajp.amjpathol.org). In addition, p21 immunoreactivity was also found in the cytoplasm of specimens (Figure 1, B and C). The association of TGIF expression with clinicopathologic characteristics of 168 patients with UUT-UC is listed in Table 1. No significant correlation was found between TGIF expression status and pT stage (P ⫽ 0.712) or tumor grade (P ⫽ 0.168). However, a positive TGIF expression was significantly associated with higher

1048 Yeh et al AJP September 2012, Vol. 181, No. 3

Table 1.

Association of TGIF Expression with Clinicopathologic Characteristics of 168 Patients with UUT-UC No. (%) of patients Variable

Total (N ⫽ 168)

TGIF negative (n ⫽ 113)

TGIF positive (n ⫽ 55)

P value

73 (43.5) 95 (56.5)

47 (41.6) 66 (58.4)

26 (47.3) 29 (52.7)

0.486

73 (43.5) 95 (56.5)

49 (43.4) 64 (56.6)

24 (43.6) 31 (56.4)

0.973

78 (46.4) 74 (44.0) 16 (9.6)

56 (49.6) 48 (42.5) 9 (7.9)

22 (40.0) 26 (47.3) 7 (12.7)

0.406

71 (42.3) 97 (57.7)

51 (45.1) 62 (54.9)

20 (36.4) 35 (63.6)

0.280

71 (42.2) 47 (28.0) 41 (24.4) 9 (5.4)

51 (45.1) 29 (25.7) 27 (23.9) 6 (5.3)

20 (36.4) 18 (32.7) 14 (25.4) 3 (5.5)

0.712

58 (34.5) 110 (65.5)

43 (38.1) 70 (61.9)

15 (27.3) 40 (72.7)

0.168

45 (26.8) 34 (20.2)

21 (18.6) 12 (10.6)

24 (43.6) 22 (40.0)

0.001* ⬍0.001*

Age, years ⱕ65 ⬎65 Sex Male Female Tumor location Renal pelvis Ureter Multiple pT stage Non–muscle invasive Muscle invasive pT stage Ta/T1 T2 T3 T4 Tumor grade Low High Oncologic outcomes Disease progression Cancer death *Statistically significant (␹2 test).

disease progression (P ⫽ 0.001) and cancer-related death rates (P ⬍ 0.001). To elucidate the prognostic value of TGIF expression, univariate and multivariate Cox regression analyses were performed. Univariate analysis revealed that TGIF status, pT stage, and tumor grade were significantly associated with progression-free and cancerspecific survival (Table 2). Multivariate Cox regression analysis revealed that only TGIF status and pT stage were independent prognostic indicators of progression-free and cancer-specific survival (Table 3). Kaplan-Meier curves also demonstrated significant differences in progression-free (P ⫽ 0.002, Figure 1D) and cancer-specific survival (P ⬍ 0.001, Figure 1E) in regard to TGIF expression. The 5-year progression-free and cancer-specific survival rates were 52.0% and 58.5% for patients with positive TGIF expression, re-

Table 2.

Univariate Analysis of the Prognostic Factors for Progression-Free and Cancer-Specific Survival in UUT-UC Patients

Variable Age Sex Tumor location pT stage (muscle) pT stage Tumor grade TGIF status

Progression-free survival

Cancer-specific survival

␹2 test

␹2 test

P value

1.408 0.235 0.417 0.518 3.638 0.162 13.461 ⬍0.001* 37.697 ⬍0.001* 5.269 0.022* 9.334 0.002*

*Statistically significant (log rank test).

P value

0.296 0.586 0.143 0.705 3.925 0.104 13.699 ⬍0.001* 46.081 ⬍0.001* 4.879 0.027* 15.388 ⬍0.001*

spectively, compared with 74.1% and 86.5% for patients with negative TGIF expression. According to the status of p21 and TGIF expression, three groups were classified: no marker altered, one marker altered (⬎50% of positive cells in nuclei in TGIF or ⬍10% of positive cells in nuclei for p21), and two markers altered (⬎50% of positive cells in nuclei for TGIF and ⬍10% of positive cells in nuclei for p21), respectively. The association among marker expression and clinicopathologic characteristics of patients is given in Table 4. Ninety-eight cases (58.3%) had at least one marker altered. Among the features, patients with two markers altered had a higher tumor stage (P ⫽ 0.018), a higher disease progression rate (P ⫽ 0.006), and a higher cancer-related death rate (P ⬍ 0.001) (Table 4). The KaplanMeier curves indicated that there were significant differences in the cancer-specific survival rate (P ⬍ 0.001) according to the numbers of markers expression (Figure 1F). Accordingly, UUT-UC patients with both TGIF and p21 markers altered had the worst prognosis. Significant differences were also seen in the 5-year cancerspecific survival rate among patients with no marker alerted (90.7%), with only one marker altered (72.9%), and with two markers altered (44.7%) (P ⬍ 0.001; Figure 1F). These results suggest that the altered expression of TGIF and p21 may be critical to the progression of UUT-UC.

Expression of TGIF and p21 in Human UC Cells Our results indicate that TGIF overexpression is significantly associated with the progression of UUT-UC.

TGIF Is a Prognostic Marker of UUT-UC 1049 AJP September 2012, Vol. 181, No. 3

Table 3.

Multivariate Analysis of the Prognostic Factors for Progression-Free and Cancer-Specific Survival in UUT-UC Patients

Variable Progression-free survival pT stage Ta/T1 T2 T3 T4 Trend TGIF status Negative Positive Tumor grade Low High Cancer-specific survival pT stage Ta/T1 T2 T3 T4 Trend TGIF status Negative Positive Tumor grade Low High

Hazard ratio

95% CI

P value

1 1.799 5.906 17.690

0.727–4.455 2.602–13.407 4.880–64.128

0.204 ⬍0.001* ⬍0.001* ⬍0.001*

1 2.507

1.380–4.552

0.003*

1 1.195

0.563–2.537

0.642

1 3.249 7.258 20.936

1.101–9.586 2.525–20.864 4.441–98.712

0.033* ⬍0.001* ⬍0.001* ⬍0.001*

1 3.536

1.742–7.254

0.001*

1 1.203

0.492–2.941

0.686

*Statistically significant.

Therefore, we further confirmed and elucidated the phenomena in a UC cell model. UC cell lines (RT4, TSGH8301, J82, and T24) were selected to analyze their expression of TGIF, PTEN, phosphorylation of AKTSer473,

Table 4.

and p21 by using Western blot. As shown in Figure 2A, J82 and T24 cells had higher expression of TGIF and phosphorylation of AKTSer473 but lower expression of p21 than RT4 and TSGH8301 cells. Moreover, the cellular migration activity was also measured in these UC cells. It has been reported that highly invasive cancer cells tend to escape from the primary tumor and metastases by forming actin-rich protrusions, invadopodia, to degrade the basement membrane and invade the stroma compartments.39 – 41 The actin-associated proteins (cortactin, Arp2/3 complex, Wiskott-Aldrich syndrome protein, neuronal Wiskott-Aldrich syndrome protein, gelsolin, and cofilin), and MMPs (membrane type 1 MMP, MMP2, and MMP9) are also involved in the regulation of invadopodia.39,40 As shown in Figure 2, T24 cells with higher TGIF expression exhibited a more increased migration ability (Figure 2B), invadopodia formation (Figure 2, C and D), N-cadherin (Figure 2E), and MMP2/MMP9 activity (Figure 2F). Accordingly, these results indicate that the invasive UC cells have a higher migration ability and invadopodia formation, which may be related to their high TGIF expression and AKT phosphorylation.

TGIF Is Involved in the Promotion of Migration/ Invasion Ability and Invadopodia Formation of UC Cells We further explored whether the TGIF was involved in the promotion of migration/invasion ability by using gene overexpression and knockdown methods. Chemotaxis chamber analysis was performed to evaluate the effects of TGIF on the ability of migration/invasion. Overexpression of TGIF in RT4 cells could markedly increase their ability of migration (Figure 3A) and invasion (Figure 3B) in

Association of Alterations in TGIF and p21 Expression with Clinicopathologic Characteristics of Patients with UUT-UC No. (%) of patients Variable

Age, years ⱕ65 ⬎65 Sex Male Female Tumor location Renal pelvis Ureter Multiple pT stage Non–muscle invasive Muscle invasive Tumor grade Low High Oncologic outcomes Disease progression Cancer death *Statistically significant (␹2 test).

None altered (n ⫽ 70)

One altered (n ⫽ 75)

Two altered (n ⫽ 23)

P value

31 (44.3) 39 (55.7)

31 (41.3) 44 (57.7)

11 (47.8) 12 (52.2)

0.845

34 (48.6) 36 (51.4)

28 (37.3) 47 (62.7)

11 (47.8) 12 (52.2)

0.355

33 (47.1) 30 (42.9) 7 (10.0)

37 (49.3) 34 (45.3) 4 (5.4)

8 (34.8) 10 (43.5) 5 (21.7)

0.209

29 (41.4) 41 (58.6)

38 (50.7) 37 (49.3)

4 (17.4) 19 (82.6)

0.018*

28 (40.0) 42 (60.0)

25 (33.3) 50 (66.7)

5 (21.7) 18 (78.3)

0.267

11 (15.7) 6 (8.6)

23 (30.7) 17 (22.7)

11 (47.8) 11 (47.8)

0.006* ⬍0.001*

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RT4 TSGH J82

A

a dose-dependent manner, respectively. In addition, overexpression of TGIF could significantly increase formation of F-actin and cortactin-positive invadopodia in RT4 (Figure 3C) and TSGH8301 cells (see Supplemental Figure S2 at http://ajp.amjpathol.org), respectively. The expression of TGIF was also confirmed by Western blot (data not shown). In addition, overexpression of TGIF in RT4 cells also increased MMP2 protein expression and its enzyme activities by using gelatin zymography assay (Figure 3D). In contrast, knockdown of TGIF by its specific shRNA could decrease phosphorylation of AKTSer473, MMP9 expression (Figure 3E), and invasive ability of T24 cells (Figure 3F). Collectively, the results indicate that TGIF contributes the ability of migration/invasion and invadopodia formation of UCs, which is consistent with the important roles of TGIF in the progression of UUT-UC patients (Figure 1 and Tables 1– 4).

T24

TGIF 1

1.8

3.2

4.2

1

0.8

0

1

1

1.3

5.8

2.9

1

1

0.8

0.03

PTEN

p-AKTSer473

p21 β -actin

TSGH

RT4

T24

Migration (cell number per field)

B 350

***

300

The PI3K/AKT Pathway Is Involved in the TGIFInduced Migration/Invasion Phenotypes

250 200

Aberrant activation of the PI3K/AKT pathway in the invasive and metastatic phenotype of UC progression has been demonstrated.42– 44 As shown in Figure 2A, the J82 and T24 cells had high TGIF expression and AKTSer473 phosphorylation. We, therefore, explored the possibility of whether TGIF-induced migration/invasion effects were mediated through the PI3K/AKT pathway. Figure 4, A and B, show that pretreatment with PI3K inhibitor LY294002 and wortmannin could attenuate TGIF-induced migration activity of the RT4 cells. Furthermore, overexpression of TGIF effectively induced phosphorylation of AKTSer473 in RT4 and TSGH8301 cells, respectively (Figure 4, C and D). In addition, knockdown of TGIF by its specific shRNA in T24 cells could decrease phosphorylation of AKTSer473 (Figure 3E). Therefore, we suggest that TGIF promotes the progression of UC, including the PI3K/ AKT pathway.

150 100

*

50 0

RT4

C

TSGH

T24

Cortactin

F-actin

Merge

RT4 8μm

8μm

8μm

8μm

8μm

8μm

TSGH

T24

8μm

8μm

D

E

100

Cell with Invadopodia (%)

8μm

***

RT4 TSGH T24

N-cadherin

80

β-actin

F

60

pro-MMP9 MMP9 pro-MMP2

40

**

20

MMP2

0

RT4

TSGH

T24

1

53.8

149.3

Figure 2. Comparison of the expression status of TGIF, phosphorylated AKT, p21, and phenotypes of UC cells. UC cell lines (RT4, TSGH8301, J82, and T24) were used to detect their protein expression of TGIF, PTEN, phosphorylated AKT, and p21 (A). The expression of ␤-actin was used as an internal control for Western blot. B: The ability of cellular migration was measured by a modified Boyden chamber assay as described in Materials and Methods (original magnification ⫻100). Data represent mean ⫾ SD of triplicate experiments. C: Invadopodia formation ability of UC cell lines on fixation and staining with Alexa Fluor 488 –phalloidin (green) and cortactin (red). Representative cells of RT4 (upper), TSGH 8301 (middle), and T24 (bottom) were presented. Arrows indicate the invadopodia structure. Scale bar ⫽ 8 ␮m. D: Cells with invadopodia were scored by percentage in at least 200 cells. Data represent mean ⫾ SD of triplicate samples in three separate experiments. N-cadherin expression (E) and MMP2/MMP9 activities (F) were also detected by Western blot and gelatin zymography assay, respectively. The MMP2 activities were quantified by densitometer analysis. **P ⬍ 0.01, ***P ⬍ 0.001.

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Overexpression of TGIF Promotes G1-S Transition to Increase Growth of UC Cells p21 plays an important role as a cyclin-dependent kinase inhibitor to arrest the cell cycle at the G1 phase.45 The interplay of cyclin D1 and cyclin-dependent kinase can phosphorylate Rb to release E2F to promote S-phase entry.46 As shown in Figure 5A, T24 cells with high TGIF expression and low p21 expression exhibited a more rapid proliferative rate. Furthermore, cell cycle–related molecules were measured to clarify whether the TGIF was involved in the regulation of G1-S transition. We found that overexpression of TGIF in RT4 and TSGH8301 cells could inhibit p21 promoter activation (Figure 5B) and protein expression but increase cyclin D1 expression, phosphorylation of RbS807/811, and PCNA protein expression (Figure 5, C

and D). In addition, TGIF could increase cell numbers and markedly promote the colonies formation in RT4 (Figure 5, E and G) and TSGH8301 cells (Figure 5, F and H). According to these results, we suggest that TGIF can promote G1-S transition of cell cycle to increase cellular growth.

Discussion Spread of cancer cells during metastasis to distant organs in the body is a critical step in cancer progression to be a major cause of cancer patient mortality.47 For the prevention of disease progression and improvement of the prognosis of UC after surgery, adjuvant or neoadjuvant cisplatin-based systemic chemotherapy has been adapted in combination with radical surgery. However,

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even doing so, disease progression and tumor metastasis are commonly found postoperatively. Therefore, to establish effective therapeutic strategies, exact assessment of high-risk factors that affect tumor invasion/progression and finding new prognostic biomarkers are important. In our clinical study, we found that overexpression of TGIF is significantly associated with worse progressionfree and cancer-specific survival in patients treated by radical nephroureterectomy for UUT-UC. The 5-year progression-free and cancer-specific survival rates were 52.0% and 58.5% for patients with positive TGIF expression, respectively, compared with 74.1% and 86.5% for patients with negative TGIF expression. Our data suggest that aside from pT stage, TGIF overexpression is an independent prognostic factor for poor survival in UUT-UC. Looking for TGIF expression status in the surgical pathologic specimens may assist urologists to select patients at high risk of progression and poor survival who need more aggressive therapy and close postoperative monitoring to improve their outcome. The present study is the first clinical article to report the prognostic effect of TGIF on UC. Furthermore, we demonstrated that there was a link between altered p21 expression and poorer prognosis in UUT-UC patients after radical nephroureterectomy

(Figure 1 and Tables 1– 4). This observation in UUT-UC is concordant with reports concerning bladder UC and other human cancers.36 –38,48,49 Moreover, when both TGIF and p21 gene expression are altered, these patients have worst clinical outcome. We found that combining evaluation of the status of TGIF and p21 in UUT-UC specimens by immunostaining can further improve the ability to select patients with a risk of disease progression. Accordingly, following up and treating these patients with more intensive/aggressive therapeutics after surgery are beneficial to improving outcome. In the in vitro study, we demonstrated that overexpression of TGIF enhanced the migration/invasion ability, formation of invadopodia, and MMP expression in RT4 (Figure 3) and TSGH8301 cells (see Supplemental Figure S2 at http://ajp.amjpathol.org), which exhibited low TGIF expression and phosphorylation of AKTSer473 (Figure 2). F-actin–associated proteins, cortactin, and MMP2 (data not shown) were also detected at the invadopodia (Figure 2C). Invadopodia formation has been observed in various cancer cells, such as mammary adenocarcinoma, melanoma, glioblastoma, colon cancer, prostate cancer, and bladder cancer.50 –53 Therefore, it will be urgent to elucidate the role of TGIF in the regulation of invadopodia

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Figure 5. TGIF could promote G1-S transition to increase cellular proliferation. A: Proliferative rates of the UC cells were measured by counting cells manually for various periods with a counting chamber after staining with trypan blue. The values were expressed as mean ⫾ SD of triplicate samples at each time point in four independent experiments. B: The p21-luc plasmids were co-transfected with various doses of pcDNA3.1-TGIF into RT4 cells. After incubation for 24 hours, cells were harvested for measurement of p21 luciferase activity. RT4 cells (C) and TSGH8301 cells (D) were transiently transfected with various doses of TGIF plasmids to detect p21, cyclin D, pRb807/811, Rb, and PCNA protein expression by Western blot as described in Materials and Methods section. RT4 cells (E and G) or TSGH8301 cells (F and H) were transiently transfected with various doses of TGIF plasmids to detect cellular proliferation by counting cells manually for various periods or performing a colony formation assay, respectively. The numbers of colonies in each well were counted and are displayed in the bar chart. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001.

formation to predict cancer progression for the improvement of therapeutic efficacy. Besides, we demonstrated that TGIF facilitates cellular migration/invasion of UC via activation of PI3K/AKT pathway. The PI3K/AKT pathway plays a critical role in cellular proliferation and survival.54 Aberrant activation of this pathway in the UC cells with invasive and metastatic phenotype, such as J82 and T24 cells, has also been identified.42– 44 We also found high phosphorylation of AKTSer473 and elevated TGIF expression in J82 and T24 cells (Figure 2). Phosphorylation of AKTS473 was involved in the TGIF-induced cellular migration/invasion of RT4 and TSGH8301 cells (Figure 4, C and D). However, the cross talk between TGIF and PI3K/AKT pathway is warranted for further elucidated. Previous studies found that in cancer cells with overexpression of the HER2/neu receptor, the PI3K/AKT pathway can be activated to phosphorylate p21 protein, leading to its translocation to the cytoplasm to play an antiapoptotic role.55–57 In this study, we found that high percentages of p21 were expressed in the cytoplasm of UC specimens (Figure 1). Whether the activation of the PI3K/AKT pathway also contributes to the p21 translocation to the cytoplasm of UC is yet to be determined. On the other hand, our previous studies have proved that TGIF can interact with histone deacetylase 1 to bind to the promoter of p21 to inhibit its expression in keratinocytes.17,18 In UC cells, overexpression of TGIF could also down-regulate p21 promoter and protein expression and up-regulate expression of cell-cycle molecules to

promote G1-S transition to increase cellular proliferation (Figure 5). This is in concordance with a previous report that cellular analysis of mouse embryonic fibroblasts of Tgif-null mice also demonstrated the involvement of TGIF in the regulation of proliferation and progression through the G1 cell cycle phase.58 However, in our clinical study, UUT-UC patients with both TGIF and p21 markers altered (⬎50% of positive cells in nuclei for TGIF and ⬍10% of positive cells in nuclei for p21) had the worst prognosis, higher tumor stage, disease progression rate, cancerrelated death rate (Table 4), and 5-year cancer-specific survival rate (Figure 1) when compared with those who manifested only one marker or no marker altered. It seems that TGIF and p21 are not entirely biologically dependent. We suppose the cancer biology is complicated and might involve multiple signal pathways. Although TGIF can inhibit p21 expression at its transcriptional level to promote cell cycle, TGIF can also activate PI3K/AKT pathway to enhance progression of UC cells as described. In conclusion, we demonstrate that the alteration of TGIF is critical to the progression of UUT-UC patients after radical nephroureterectomy, which can promote cellular migration/invasion, invadopodia formation, and proliferation of UC cells via PI3K/AKT pathway. It may serve as a therapeutic target and a promising biomarker for the prognosis evaluation of UUT-UC patients. Assessment of TGIF status may provide additional prognostic information and help identify individuals in need of adjuvant treatments and close follow-up postoperatively.

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