Elevated expression of Tiam1 is associated with poor prognosis and promotes tumor progression in pancreatic cancer.

OBJECTIVE: T-cell lymphoma invasion and metastasis inducing factor 1 (Tiam1) is known to be involved in tumor progression. However, its molecular roles and mechanism in pancreatic ductal adenocarcinoma (PDAC) remain unclear. The purpose of this study is to determine Tiam1 expression levels and investigate its underlying molecular mechanism in PDAC.
MATERIALS AND METHODS: Tiam1 protein expression levels in PDAC tissues were examined using immunohistochemistry. Tiam1 expression was confirmed in pancreatic cancer (PC) cells by Western blot and immunofluorescence staining. Tiam1-silenced PC cells were created using short interfering RNA. Subsequently, colony formation, scratch, and migration and invasion assays were carried out to explore the molecular mechanisms of Tiam1 in PC cells.
RESULTS: The results indicated that Tiam1 expression was significantly higher in PDAC tissues than in paired non-tumor tissues, and overexpression of Tiam1 was significantly correlated with histological grade (P=0.040) and lymph node metastasis (P=0.031) in PDAC. The PDAC patients with high Tiam1 expression had significantly lower 5-year overall survival than patients with low Tiam1 expression. More importantly, univariate and multivariate analysis suggested that Tiam1 expression, along with lymph node metastasis, is a significant independent prognostic factor for patients with PDAC. Furthermore, we also demonstrated that the downregulation of Tiam1 was associated with decreased cell proliferation and reduced migratory and invasive capability.
CONCLUSION: High expression of Tiam1 plays a significant role in the progression of PDAC and may be a potential biomarker of poor prognosis as well as a therapeutic target.

Introduction

Pancreatic cancer (PC) is a highly lethal malignancy in which mortality closely parallels incidence. Pancreatic ductal adenocarcinoma (PDAC) is the most universal type of PC, and it has a 5-year survival rate of <5%.1 Currently, although great improvements in the diagnosis of PDAC have been made, the prognosis of PDAC patients is still generally poor. Tumor progression and metastasis are the major causes of mortality in PDAC patients.2,3 The high propensity of this cancer is included in the major causes due to the lack of progress in improving the prognosis for chemoresistance and early distant metastasis.4 Furthermore, the molecular mechanisms that underlie these events continue to be unclear.

T-cell lymphoma invasion and metastasis inducing factor 1 (Tiam1), a member of the guanine nucleotide exchange factors family that mediates the specific activation of Rac1, was first discovered by Habets et al as an invasion and metastasis-related gene in mice with aggressive T-cell lymphoma.5–8 The Tiam1 gene is located in the q22 band of chromosome 21 and the centromeric end of the Aml 21 gene, and it contains 2 exons separated by 1 intron; the 2 exons werê7.3 kb, and the intron was 14 kb. Previous studies have showed that Tiam1 is mainly expressed in normal brain and testis tissues and is minimally or not expressed in other normal tissues.9 In recent years, it has been confirmed that upregulation of Tiam1 expression is implicated in the formation of solid tumors, such as esophageal carcinoma,10,11 lung adenocarcinoma, and breast cancer.12,13 The potential prognostic relevance of Tiam1 expression in PDAC, however, has not been adequately explored.

In the present study, we concentrated on the analysis of alterations in Tiam1 expression in PDAC to clarify its associations with clinicopathological features and prognosis and identify the mechanism by which Tiam1 contributes to proliferation and epithelial–mesenchymal transition (EMT) events. These findings may provide a potential therapeutic target for the treatment of PDAC.

Materials and methods

Patients and specimens

Tissue samples from patients with PDAC tissues and paired adjacent paracancerous tissue samples were acquired from 81 patients. These patients were diagnosed at the Yanbian Hospital, China, and recruited in the present study. This study was approved by the Yanbian University’s ethics committee. Among the 81 PDAC patients, 33 were females and 48 males with age ranging from 38 to 85 years (median, 59 years). The average time of survival was 28.2 months for these patients, and it ranged from 1 to 81 months. The histological types were assigned according to the criteria of the World Health Organization Classification. No patients in this study received any adjuvant systemic treatment ahead of surgery. The subjects or their caregivers provided written informed consent.

Cell lines

The human PC cell BxPC-3, SW1990, Mia-paca-2, AsPC-1, and Capan-1 were supplied by the Cancer Research Center of Yanbian University, which were approved by Yanbian University’s ethics committee. The cells were seeded in high glucose DMEM (Gibco, Grand Island, NY, USA), containing 10% fetal bovine serum (FBS) (HyClone Laboratories Inc., Novato, CA, USA). And then, the cells were cultured at 37°C in 5% CO2 incubator.

Immunohistochemistry and scoring

Immunohistochemistry staining was confirmed to study altered protein expression in PC and paracancerous tissues. Briefly, all paraffin sections were dewaxed, dehydrated, and rehydrated. Anti-Tiam1 antibody (1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was incubated with the sections and incubated at 4°C overnight. Following incubation with biotinylated secondary antisera, the streptavidin-biotin complex/horseradish peroxidase was employed. Finally, the sections were treated with diaminobenzidine and counterstained with hematoxylin. As depicted previously, staining for Tiam1 protein in tumor tissues was scored through a semiquantitative analysis. Staining intensity was graded as 0, negative; 1,weak; 2, medium; and 3, strong. Scores for the staining extent were as follows: 0 (0%), 1 (1%–25%), 2 (26%–50%), 3 (51%–75%), or 4 (76%–100%). Based on the semiquantitative grade estimated by multiplying these 2 values, we finally defined 3 intensity of the staining: negative (score=0); low expression (score=1–3), or high expression (≥4). Each section was captured with a microscope.

Immunofluorescence staining assay

Cells were cultured on coverslips overnight and fixed in 4% paraformaldehyde for 15 minutes. After fixation, the cells were permeabilized with 0.5% Triton X-100 for 10 minutes. And then incubated with rabbit anti-Tiam1 (1:200; Santa Cruz Biotechnology Inc.) at 4°C overnight followed by a fluorescein isothiocyanate-conjugated secondary anti-rabbit antibodies and a nuclear counterstain 4′,6-diamidino-2-phenylindole. Finally, the images were taken using immunofluorescence microscopy.

Western blot

The cells were collected in cell lysis buffer. Protein samples were separated on 6%–8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Immobilon P; EMD Millipore, Bedford, MA, USA). The membranes were incubated with primary antibodies at 4°C overnight, including Tiam1 (1:1,000; Santa Cruz Biotechnology Inc.), E-cadherin (1:1,000; CST, Bos-ton, MA, USA), Snail (1:1,000; CST), Slug (1:1,000; CST), Vimentin (1:1,000; CST), β-actin (1:1,000; Santa Cruz Biotechnology Inc.), followed by incubation with the corresponding secondary antibodies. Finally, the signals were visualized with the enhanced chemiluminescence substrate.

Cell transfection

The PC cells were plated in 6-well plates for transfection. Then special Tiam1 siRNA (5 µL) was transfected using Lipofectamine 3,000 (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. For Tiam1 knockdown, a control siRNA and 3 different siRNAs were purchased from RiboBio (Shanghai, China). The sequence was as follows: si-Tiam1-1:5′-CCTCCGTACAGTAATTATA-3′; si-Tiam1-2:5′-GGAGCTGATTTGCAAGACA-3′; and si-Tiam1-3:5′-GCAGTTTGCATG AGATGAA-3′. The effects were confirmed by Western blot analysis.

Colony formation assay

Cells were seeded in 6-well plates at a concentration of 5×103 cells/well and treated with siRNA. Cells were fixed in 4% paraformaldehyde (PFA) and stained with 0.1% crystal violet after culturing for 10 days. The images were observed by a camera.

Wound healing assay

For the wound healing assays, BxPC-3 and SW1990 cells were cultured in 6-well plates. The cells were scratched by scraping with a 10 µL tip after overnight incubation. The cells were incubated in low-serum (2%) medium with treatments, and images were obtained at 0, 24, and 48 hours using a microscope.

Migration and invasion assay

Cell migration and invasion ability were tested using a transwell assay. For migration assays, cells were plated and transfected with siRNA. The treated cells in 100 µL serum-free medium were placed in the top chamber and 500 µL of medium containing 20% FBS was added to the lower chamber. After incubation for 48 hours, the cells on the upper surface were removed and cells migrated to the lower chamber were fixed with 4% PFA and stained. The invasion assay was similar to that of the cell migration assay, except that the top chamber was pre-coated with Matrigel. The image of migrating and invading cells was captured under a microscope.

Statistical analysis

All data were expressed as mean ± SD and analyzed for statistical significance using GraphPad Prism 6 (Graph-Pad Software, Inc., San Diego, CA, USA) and the JMP software program (SAS Institute Inc, Cary, NC, USA). The χ2 test was used to calculate the correlations between the expression of Tiam1 protein and clinicopathological characteristics. The probability of survival was undertaken using the Kaplan–Meier method. The significance of various variables in the prognosis of the disease was assessed by the Cox proportional hazards regression model for univariate and multivariate analyses. Statistical analysis approaches mainly included Student’s t-test, log-rank test, and one-way analysis of variance test. A P-value <0.05 was considered to be significant in all cases.

Results

Elevated expression of Tiam1 in PDAC

To analyze the clinical relevance of Tiam1 expression in human PDAC, first we obtained information about the alteration of Tiam1 protein expression in clinical specimens from the human protein atlas (www.proteinatlas.org). The results showed that expression of Tiam1 was higher in PC tissues than in normal tissues (Figure 1A). In addition, according to Oncomine data (www.oncomine.org), the Tiam1 mRNA levels were higher in PDAC tissues than in normal tissues (P<0.001; Figure 1B). These data indicated that Tiam1 is overexpressed in PDAC.

Figure 1

High expression of Tiam1 in pancreatic cancer tissues samples and pancreatic cancer cells.

Notes: (A) Immunohistochemistry of Tiam1 proteins in clinical specimens from the human protein atlas (www.proteinatlas.org). (B) Comparison of Tiam1 mRNA expression levels between the normal tissues and primary tumor or metastatic tumor tissues according to Oncomine data (www.oncomine.org) ***P<0.001. (C) The expression of Tiam1 in pancreatic tissue samples was detected by immunohistochemistry (DAB, ×200). a: Negative expression of Tiam1; b: weakly positive expression of Tiam1; c: strongly positive expression of Tiam1. (D) Western blot assays performed on pancreatic cancer cell lines as indicated. (E) Immunofluorescence staining of the Tiam1 protein in BxPC-3 and SW1990 pancreatic cancer cells; a nuclear staining pattern was also observed.

Abbreviations: Tiam1, T-cell lymphoma invasion and metastasis inducing factor 1; DAPI, 4′,6-diamidino-2-phenylindole; DAB, diaminobezidin.

Next, we examined the expression of Tiam1 in a cohort of 81 archived paraffin-embedded PDAC specimens and noncancerous specimens by immunohistochemistry (Figure 1C). Seventy-five of these tumor tissue samples showed high expression of Tiam1 (92.59%) and 30 had low expression (37.04%). The strongly positive expression rate in tumor tissues was higher than that of the matched non-cancerous tissues (41.98% vs 17.28%, P<0.001). There was a significant difference in the overexpression of Tiam1 between tumor and paracancerous tissue samples (Table 1).

Table 1

Tiam1 protein expression in different groups

Group	Case	Negative	Positive			Positive rate (%)	Strongly positive rate (%)
			+	++	+++
Pancreatic cancer tissues	81	6	41	29	5	92.59a	41.98a
Paracancerous tissues	81	51	16	14	0	37.04	17.28

Notes: Positive rate: percentage of positive cases with +, ++, and +++ staining scores. Strong positive rate: percentage of positive cases with ++ and +++ staining scores.

P<0.01.

Abbreviation: Tiam1, T-cell lymphoma invasion and metastasis inducing factor 1.

Subsequently, Tiam1 expression and subcellular localization in PC cell lines were explored using immunofluorescence staining. It was found that Tiam1 was also highly expressed in BxPC-3 and SW1990 cells (Figure 1D). Tiam1 protein was localized to the nucleus and the cytoplasm, mainly to the former (Figure 1E).

Tiam1 expression and clinicopathologic features

To determine the connection between PC progression and Tiam1 protein expression, we analyzed the correlations between Tiam1 expression and the clinicopathological attributes of PC. These data showed that the expression of Tiam1 was positively correlated with lymph node metastasis (P=0.031; Figure 2A) and histological grade (P=0.040; Figure 2B). However, Tiam1 overexpression was not associated with the sex, age, tumor location, or T classification of the patients with PDAC (Table 2).

Figure 2

Cancer patients with high Tiam1 expression are associated with poor prognosis.

Notes: (A and B) Correlations between Tiam1 expression and lymph node metastasis (A), pathological stage (B) of PDAC (P<0.05, P<0.05, respectively). (C) Kaplan–Meier survival curves in PDAC patients with high and low Tiam1 protein expression levels. (D) Patients with high or low Tiam1 mRNA levels obtained from the human protein atlas (www.proteinatlas.org).

Abbreviations: Tiam1, T-cell lymphoma invasion and metastasis inducing factor 1; PDAC, pancreatic ductal adenocarcinoma.

Table 2

The relationship between high expression of Tiam1 protein and the clinicopathological parameters of patients with PDAC

Characteristic	Case	Tiam1 expression		Chi-squared	P-value
		Low	High
Sex				0.005	0.946
Male	48	28	20
Female	33	19	14
Age (years)				0.650	0.420
<60	40	25	15
≥60	41	22	19
Tumor location				0.406	0.524
Head	54	30	24
Body/tail	27	17	10
Tumor size				0.101	0.750
<4 cm	54	32	22
≥4 cm	27	15	12
Lymph node status				4.653	0.031a
Yes	34	15	19
No	47	32	15
Pathological status				6.448	0.040a
I	1	1	0
II	76	46	30
III	4	0	4
T stage				5.011	0.082
T1	3	2	1
T2	68	36	32
T3	10	9	1

Note:

P<0.05.

Abbreviations: Tiam1, T-cell lymphoma invasion and metastasis inducing factor 1; PDAC, pancreatic ductal adenocarcinoma.

Tiam1 expression is inversely related to prognosis in PDAC patients

To further substantiate the significance of Tiam1 expression in PC progression, the association between the survival period and Tiam1 expression was evaluated by applying Kaplan–Meier analysis using the log-rank test. Total survival analysis using the Kaplan–Meier approach indicated that patients with high Tiam1 expression had lower 5-year survival rates than patients without Tiam1 overexpression (log-rank value=25.93, P=0.000; Figure 2C). However, the data from the human protein atlas (www.proteinatlas.org) until now showed that PC patients with low Tiam1 mRNA had lower 5-year survival rates (Figure 2D). Further investigation is required to determine the relationship between expression of Tiam1 mRNA and protein and prognosis of PC.

Univariate analysis using the Cox proportional hazards model revealed that lymph node metastasis (P=0.023), pathological grade (P=0.006), and Tiam1 expression (P=0.000) were associated with overall survival. Moreover, multivariate analysis showed that the high expression level of Tiam1 was a strong predictor of a negative prognosis (hazard ratio, 2.753; 95% CI: 1.670–4.536; P=0.000; Table 3). Consequently, we demonstrated that Tiam1 expression was significantly associated with prognosis and was also an independent risk factor for PDAC patients.

Table 3

Univariate and multivariate analyses of prognosis in patients with pancreatic cancer

Variable	HR (95% CI)	P-value
Univariate
Gender	1.260 (0.800–1.984)	0.319
Age, years	0.918 (0.591–1.426)	0.703
Tumor location	0.950 (0.594–1.518)	0.829
Tumor size, d/cm	0.926 (0.581–1.473)	0.744
Lymph node status	1.697 (1.077–2.674)	0.023a
Pathological status	4.478 (1.540–13.018)	0.006a
T classification	0.689 (0.415–1.144)	0.150
Tiam1 expression	2.861 (1.765–4.636)	0.000b
Multivariate
Lymph node status	1.672 (1.051–2.660)	0.030a
Pathological status	2.336 (0.802–6.802)	0.120
Tiam1 expression	2.753 (1.670–4.536)	0.000b

Notes:

P<0.05.

P<0.01.

Abbreviations: HR, hazard ratio; Tiam1, T-cell lymphoma invasion and metastasis inducing factor 1.

Alteration of Tiam1 expression affects proliferation, migration, and invasion of PC cells

To further elucidate the functional significance of Tiam1 in the growth and progression of PC, loss-of-function experiments were first performed to knockdown the expression of Tiam1 in SW1990 cells and BxPC-3 using a specific plasmid infection. The efficiency of the knockdown of Tiam1 expression was confirmed by Western blot (Figure 3A). A colony formation assay showed that suppressing Tiam1 expression significantly inhibited the proliferation of siTiam1-BxPC-3 and siTiam1-SW1990 cells compared with the proliferation of the corresponding group of mock cells (Figure 3B).

Figure 3

Tiam1 knockdown inhibited the proliferation, migration, and invasion of pancreatic cancer cells.

Notes: (A) Western blot showing Tiam1 expression in BxPC-3 and SW1990 cells upon Tiam1 knockdown. (B) Proliferating capability of transfected cells was evaluated using colony formation assays. (C) A scratch wound healing assay was used to determine the effects of si-Tiam1 on pancreatic cancer cells motility. (D) Migration and invasion of Tiam1 knockdown cells was measured by transwell assay. **P<0.01.

Abbreviations: con, control; Tiam1, T-cell lymphoma invasion and metastasis inducing factor 1.

Additionally, we examined the influential role of Tiam1 in the migration rates of PC cells using wound healing assays. The results showed that BxPC-3 and SW1990 cells with decreased Tiam1 had delayed wound closure compared with that in control cells (Figure 3C). Matrigel transwell assays showed a similar phenomenon, in which decreased Tiam1 levels inhibited BxPC-3 and SW1990 cell invasion (Figure 3D). Altogether, these findings suggest that decreased expression of Tiam1 could significantly suppress PC cell proliferation, migration, and invasion.

Tiam1 promoted PC cell metastasis via activating EMT

An increasing number of reports have revealed that cell invasion and migration are associated with altered levels of EMT biomarkers.14 To ascertain Tiam1’s underlying mechanism in EMT, Western blot analysis was used to observe the expression of epithelial and mesenchymal protein markers. The results indicated that the silencing of Tiam1 expression in BxPC-3 and SW1990 cells increased the expression of E-cadherin while reducing the expression of mesenchymal markers, such as vimentin, slug, and snail (Figure 4). These results indicate that Tiam1 is a necessary mediator for promotion of PC EMT processes.

Figure 4

Tiam1 promoted pancreatic cancer cell migration and invasion by promoting EMT.

Note: The changes in the protein expression levels of the epithelial marker E-cadherin and the mesenchymal markers vimentin, snail, and slug in si-Tiam1-transfected BxPC-3 and SW1990 cells as detected by Western blot.

Abbreviations: con, control; Tiam1, T-cell lymphoma invasion and metastasis inducing factor 1; EMT, epithelial–mesenchymal transition.

Discussion

PC is one of the most lethal malignancies in the world, and each year ~40,000 patients die from this cancer. In contrast to the steady decrease in mortality for most cancers, the incidence and death rates of PC are increasing. Therefore, there is an urgent need to examine the molecular mechanisms that are relevant to the progression of PDAC and search for novel therapeutic strategies.

A number of roles in the regulation of cellular functions are exhibited by Tiam1, and these roles depend on the substratum, the specific cell type, and other factors.15 Li et al and Ding et al confirmed that the overexpression of Tiam1 predicts poor prognosis in ovarian carcinoma and hepatocellular carcinomas,16,17 and Yang et al indicated that upregulation of Tiam1 correlates with poor prognosis in head and neck squamous cell carcinoma.18 In line with these results, in this study, we assessed the expression level of Tiam1 in PDAC and found for the first time that Tiam1 was upregulated significantly in PDAC compared with that in non-cancerous tissues. Furthermore, elevated Tiam1 expression was positively correlated with lymph node metastasis and pathological status in PDAC patients, which indicated that Tiam1 potentially played an important role in the progression of PDAC.

Few studies to date have reported an association between Tiam1 expression and prognosis in PDAC. To determine whether the levels of Tiam1 expression might function as a prognostic factor for human PDAC, we analyzed Tiam1 expression and patient survival rates. We discovered that compared with patients with high Tiam1 expression, those with low Tiam1 expression had a longer 5-year overall survival. Consistent with our findings, the data from the human protein atlas showed that high levels of Tiam1 mRNA in PC were negatively correlated with overall patient survival. Most importantly, both the univariate and multivariate analyses confirmed that Tiam1 expression was an independent risk factor for poor prognosis, along with lymph node status.

Uncontrolled growth and metastasis, which are important features of malignant tumors, are the main causes of cancer-related death. Michiels et al considered that Tiam1 overexpression may contribute to oncogenic transformation.7 Moreover, Tiam1 was found to play an important role in regulating the tumor microenvironment,19 as well as modification of cell polarity and the actin cytoskeleton,20 all of which are thought to be associated with tumor progression. Here, we knocked down the expression of Tiam1 in BxPC-3 and SW1990 cells via siRNA transfection and found that cell growth was significantly inhibited. Downregulation of Tiam1 also significantly inhibited cell invasion and migration. These findings demonstrated that Tiam1 may function as an oncogene in PC cells and that its expression may facilitate PC development and progression.

EMT is a process whereby cancer cells develop migration capability and invasiveness, which leads to poor prognosis and cancer metastasis.21,22 Consistent with the studies by Liu et al, in which Tiam1 overexpression was associated with thyroid carcinoma metastasis,23 in this study, we discovered that Tiam1 overexpression promoted EMT by decreasing the expression of the mesenchymal markers vimentin and snail and by elevating the expression of the epithelial marker E-cadherin. These results confirmed that Tiam1 promoted cell migration and invasion by promoting the process of EMT in PC. However, the role of Tiam1 in regulating PC progression and metastasis requires further in-depth studies in the future.

This study demonstrated that Tiam1 overexpression is strongly related to PDAC progression and is an independent prognostic factor. Moreover, the suppression of Tiam1 in PC cells significantly inhibited cell proliferation, migration, and invasion. Our findings indicated that Tiam1 may also represent a new molecular biomarker for human PDAC.