Tumor Biol. DOI 10.1007/s13277-015-4704-4

ORIGINAL ARTICLE

Upregulated LASP-1 correlates with a malignant phenotype and its potential therapeutic role in human cholangiocarcinoma Hongchen Zhang 1,2 & Zhizhen Li 1,2 & Bingfeng Chu 1,2 & Fei Zhang 1,2 & Yijian Zhang 1,2 & Fayong Ke 1,2 & Yuanyuan Chen 1,2 & Yi Xu 1,2 & Shibo Liu 1,2 & Shuai Zhao 1,2 & Haibin Liang 1,2 & Mingzhe Weng 1,2 & Xiangsong Wu 1,2 & Maolan Li 1,2 & Wenguang Wu 1,2 & Zhiwei Quan 1,2 & Yingbin Liu 1,2 & Yong Zhang 1,2 & Wei Gong 1,2

Received: 10 October 2015 / Accepted: 20 December 2015 # International Society of Oncology and BioMarkers (ISOBM) 2016

Abstract LIM and SH3 protein 1 (LASP-1) is demonstrated to play a key role in occurrence and development of tumors. However, the expression and function of LASP-1 in cholangiocarcinoma (CCA) remain largely unexplored. This study aimed to investigate the effect of regulated LASP-1 expression on migration, invasion, proliferation, and apoptosis of CCA cells and on tumorigenesis in vivo, and to examine clinico-oncological correlates of LASP-1 expression. Expression of LASP-1 by immunohistochemistry was evaluated in CCA tissue samples. HCCC-9810 and RBE cells were transfected with the LASP-1 small interfering RNA (siRNA), and the effect of knocking down LASP-1 gene expression on cell migration, invasion, proliferation, and apoptosis were examined by wound healing, transwell assays, CCK-8 assays, colony formation, and flow cytometry assays, respectively. Xenograft tumor model was used to validate the effect of downregulated LASP-1 in vivo. Our results demonstrated that LASP-1 was over-expressed in CCA tissues, positively correlating with larger tumors, poor histological differentiation, lymph node metastasis, advanced TNM stage, and poor prognosis in CCA patients (P < 0.05). Downregulation of LASP-1 in HCCC-9810 and RBE cell lines significantly increased cell Hongchen Zhang, Zhizhen Li and Bingfeng Chu contributed equally to this work. * Wei Gong [email protected]

1

Department of General Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, 1665 Kongjiang Road, Shanghai 200092, China

2

The Institute of Biliary Disease Research, School of Medicine, Shanghai Jiao Tong University, 1665 Kongjiang Road, Shanghai 200092, China

apoptosis and suppressed cell migration, invasion, and proliferation in vitro and tumorigenesis in vivo. Our results indicate that LASP-1 may essentially involve in the metastasis and growth of CCA and clinical significance of LASP-1 may reside in function as a biomarker to predict prognosis and as a promising therapeutic strategy for CCA patients by the inhibition of LASP-1 expression. Keywords LASP-1CCA . Prognosis . Cell migration . Cell invasion . Cell proliferation . Apoptosis

Introduction In recent years, the incidence of cholangiocarcinoma (CCA) has significantly increased, especially in South America and Asia, making it the second most common primary liver tumor and the most common biliary tract malignancy in the world [1]. Depending on its anatomic location, CCA is classified as either intrahepatic or extrahepatic. Although potentially radical surgery offers the only established treatment for CCA, the optimum time for surgical intervention is frequently precluded due to metastases or other comorbidities present at diagnosis [2]. Furthermore, the poor prognosis of CCA does not only result from high rates of unresectable tumors but also from high recurrences after resection. Currently, molecular studies have enhanced our understanding of CCA carcinogenesis, metastasis, and progression [3], which may enrich effective therapeutic strategies for CCA and improve patient survival. The LIM and SH3 protein 1(LASP-1), a focal adhesion protein, was originally identified two decades ago from a complementary DNA (cDNA) library of metastatic breast cancer [4]. The gene located in the chromosome 17q12 in human

Tumor Biol.

encodes a protein containing an N-terminal LIM domain, followed by two internal nebulin repeats, and a C-terminal SRC homology 3 (SH3) domain [5]. Owing to its universal expression in many cell types and its unique composition allowing multiple protein–protein interactions, LASP-1 has been implicated in a wide array of biological functions, including cell morphology, signal transduction, and cell motility [6]. Furthermore, overexpression of LASP-1 has been found to correlate with a number of malignant phenotype in human, such as breast cancer [7], esophageal squamous cell carcinoma [8], gastric cancer [9], hepatocellular cancer [10], and pancreatic ductal adenocarcinoma [11], hinting a potential value of LASP-1 as a cancer biomarker. A growing body of evidence suggests that LASP-1 can promote cell migration, invasion, and proliferation in a wide variety of tumors both in vitro and in vivo [12]. However, the roles of LASP-1 as mediators of oncogenesis in CCA still remain elusive. In the current study, clinicopathological significance of LASP-1 was analyzed based on the expression of LASP-1 by immunohistochemistry (IHC) in CCA tissues and adjacent normal tissues. Silencing of the LASP-1 gene by small interfering RNA (siRNA) transfection significantly retarded cell migration, invasion and proliferation in vitro, as well as tumorigenesis in vivo. Our findings provide a novel insight for the diagnostic and therapeutic role of LASP-1 in the clinical practice of CCA.

Materials and methods Patient samples and cell lines Cancer tissue specimens and matched adjacent noncancerous tissues were obtained from 40 patients diagnosed with CCA who underwent a curative resection between January 2012 and January 2015 at the Department of General Surgery, Xinhua Hospital (Shanghai, China). These tissue specimens were formalin-fixed and paraffin-embedded and reviewed separately by two pathologists to confirm the diagnosis. No patients had received irradiation or chemotherapy prior to surgery. This study was performed with approval of the Ethics Committee of Xinhua Hospital and the ethical guidelines of the Declaration of Helsinki. In addition, written informed consent was obtained from all participants. Staging was performed according to the 7th AJCC-TNM Classification of Malignant Tumors. Follow-up data were collected until July 2015. The median follow-up was 11 months (range, 0.5–32 months). The human CCA cell lines, RBE and HCCC-9810 (provided by the Shanghai Cell Institute Country Cell Bank), were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10 % fetal bovine serum (Gibco, USA) at 37 °C in humidified atmosphere containing 5 % CO2.

Immunohistochemical staining analysis Immunohistochemistry (IHC) was performed to investigate the expression of LASP-1 protein in 40 pairs of CCA and adjacent non-cancer tissues. Briefly, after deparaffinization, antigen retrieval in 10 mmol/L sodium citrate, and inactivation of endogenous peroxidase activity in 3 % hydrogen peroxide for 15 min, the tissue sections were incubated overnight with polyclonal antibody against LASP-1 (1:200; Proteintech, Shanghai, China) at 4 °C. The IHC score was performed independently by two experienced pathologists, according to the extent and intensity of staining. The extent was scored as follows: 0 (less than 5 % positive tumor cells); 1 (5 to 25 %); 2 (25 to 50 %); and 3 (more than 50 %). The staining intensity was visually scored as follows: 0 (negative staining); 1 (weak staining); 2 (moderate staining); 3 and (strong staining). A final immunoreaction score was obtained by adding the extent score and the intensity score. A final score of 0–2 was defined as low expression, and 3–6 was defined as high expression. All discordant samples were reassessed and an agreement reached between both pathologists.

RNA extraction and quantitative real-time PCR analysis Total RNA was isolated from the cultured cells using the Trizol reagent (TaKaRa, Japan), and then converted into cDNA by using the PrimeScript Reverse Transcriptase (TaKaRa, Japan). LASP-1 messenger RNA (mRNA) expression was measured by quantitative realtime PCR (qRT-PCR) using the SYBR-Green method (TaKaRa, Japan) in accordance with user’s manual. PCR conditions were as follows: 95 °C for 30 s followed by 40 cycles at 95 °C for 5 s, and 60 °C for 34 s. Primers for detection of LASP-1 (Sangon, China) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows: LASP-1 (forward): 5′-CTGGAATGGGAGACCT GTTG-3′; LASP-1 (reverse): 5′-CCCTGGAATGTGTGGG TATG-3′; GAPDH (forward): 5′-CAACAGCCTCAAGAT CATCAGC-3′; GAPDH (reverse): 5′-TTCTAGACGGCAGGTC AGGTC-3′. The equation 2 − ΔCT (ΔCT = CT target − CT GAPDH ) was used to calculate the relative mRNA expression of LASP-1. The results were normalized to endogenous GAPDH expression.

Tumor Biol.

Western blot analysis

In vitro migration and invasion assays

Protein samples were extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology, Beijing, China) containing protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). The concentration was measured using the bicinchoninic acid assay kit (Shanghai, Beyotime, China). Subsequently, equal amounts of proteins were separated using 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. After blocked with 10 % skim milk, the membranes were incubated with the primary antibody against LASP-1 (Proteintech, China). Anti-E-cadherin, anti-N-cadherin, anti-vimentin, anti-Bax, anti-Bad, anti-Bcl-2, and anti-GAPDH antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The membranes were then washed by TBST buffer and followed by incubation with peroxidase-conjugated secondary antibodies (goat antirabbit or goat anti-mouse IgG). At the end, the immunoreactive bands were visualized by using a Gel Doc 2000 (BioRad, USA).

In the wound-healing assay in vitro, cells were cultured as monolayer in 6-well plates. Artificial wounds were gently made by scratching with a 200-μL pipette tip. After washed with PBS to remove non-adherent cells and debris, the cells were then incubated at 37 °C with serum-free medium. The migration of cells toward the wound was detected under the microscope at time points of 0, 24, and 48 h, respectively, and expressed as the percentage of wound closure. Cell migration and invasion ability were examined at the time point of 24 h using 24-well Transwell chambers (BD Biosciences) according to the manufacturer’s protocol. Briefly, equal numbers of cells were seeded onto the upper chamber in 200 μL serum-free medium, and the lower chamber was filled with 500 μL medium containing 10 % FBS. After incubation at 37 °C for 24 h, the cells remaining on the upper chamber were scrubbed off with a cotton swab, and the migrated cells which attached to the lower surface were fixed with methanol and stained with 1 % crystal violet for 30 min. For quantification, the average number of migrated cells in five randomly chosen fields was reported. The invasion assay protocol was similar to that of the migration assay, with the exception that the upper chambers were coated with the Matrigel (BD Biosciences), which simulated the characteristics of the extracellular matrix.

Small interfering RNA transfection LASP-1 siRNA and its non-specific control siRNA were synthesized by Biomics Biotechnologies (Shanghai, China) and the siRNA sequence targeting LASP-1 was as follows: 5′CGCGCGGUGUAUGACUACAdTdT-3′. Transfections of siRNA were carried out using Lipofectamine 2000 (Invitrogen Life Technologies, CA, USA) according to the manufacturer’s protocol. The efficiency of the siRNA on knocking down LASP-1 expression was examined by qRT-PCR and Western blot 48 h after transfection.

Lentivirus-mediated siRNA construction and transfection To obtain cell lines stably silencing LASP-1 expression, short hairpin RNAs (shRNAs) were synthesized by Genomeditech (Shanghai, China) and inserted into pGMLV-SC5 RNAi vector containing a cytomegalovirus-driven enhanced green fluorescent protein (EGFP) reporter gene. Then, according to the manufacturer’s instruction, RBE cell lines were transfected with the recombinant lentivirus expressing LASP-1 siRNA or control siRNA (Lv-shLASP-1 or Lv-shCon) using Lipofectamine 2000 (Invitrogen Life Technologies CA, USA). After 24 h of incubation at 37 °C, transfection medium was replaced with complete medium containing 10 % FBS. Cells with lentivirus infection were subjected to qRT-PCR and Western blot analysis to verify the transfection efficiencies.

In vitro tumorigenesis assays Cell viability and proliferation was measured using the Cell Counting Kit-8 assay (CCK-8; Dojindo). Cells were plated at a density of 1500 cells per well in 96-well plates and were allowed to adhere overnight. The CCK8 counting reagent was added to each well at different time points (0, 24, 48, 72, and 96 h) and the cells were then incubated at 37 °C for 3 h. The absorbance value (OD) at 450 nm was measured for each sample using an Automated Microplate Reader (Bio-Tek, USA). All the experiments were repeated three times, and the mean was calculated. Anchorage-independent growth was assessed by colony formation assay. Cells were plated in a 6-well plate with a concentration of 500 cells per well and allowed to grow for 8 days to form natural colonies. When the cell numbers in most single colonies were greater than 50, the colonies were washed twice with phosphate buffered saline (PBS), fixed with 4 % methanol, and stained with 0.5 % crystal violet solution (Sigma-Aldrich). The number of colonies were then counted and photographed under a microscope (Leica, Germany).

Tumor Biol.

Analysis of cell apoptosis An Annexin-V-FITC apoptosis detection kit (BD Biosciences, USA) was used to measure cell apoptosis. HCCC-9810 and RBE cell lines were harvested and washed twice with cold PBS after transfected with LASP-1 siRNA and control siRNA for 48 h. Subsequently, 100 μL of 1× binding buffer containing 2.5 μL of FITC-conjugated annexin-V and 1 μL of PI (100 μg/mL) was added to each sample and incubated at room temperature in the dark for 30 min. Finally, the samples were detected using the flow cytometry (BD, San Diego, USA). In vivo tumorigenesis assays Nude mice, aged 4–6 weeks, were obtained from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China). They were maintained in pathogenfree conditions according to the experimental animal guidelines and all experimental procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University (Shanghai, China). To evaluate tumor formation, 1 × 106 of cells (Lv-shCon or Lv-shLASP-1) in a volume of 100 uL were injected subcutaneously into the left axilla of the mice. Tumor size was measured every week, and tumor volume was calculated using the following formula: tumor volume (mm3) = (L × W2)/2 (L = length and W = width). After 23 days, the mice were sacrificed and the tumors were weighed and photographed.

markedly higher in the CCA tissues than that in adjacent noncancerous tissues (P < 0.001), which was confirmed by Western blot analysis (Fig. 1b, c). Furthermore, higher LASP-1 staining scores were positively correlated with larger tumor volume (≥5 cm), poor histological differentiation, lymph node metastasis, as well as advanced TNM stage (P < 0.05); while age, gender, tumor location, nerve invasion, and vascular invasion did not significantly correlate with LASP-1 expression (Table 2). Among 14 patients in tumor size above 5 with high expression LASP-1, 11 showed low differentiation of tumor at the same time. Upregulated LASP-1 expression is associated with poor prognosis in CCA LASP-1 expression was correlated with overall survival in CCA patients using the Kaplan–Meier method and the log-rank test. The overall survival of those patients with low expression of LASP-1 was significantly higher than that of patients with high expression (log rank, 8.557; P = 0.003) (Fig. 1d). The multivariate Cox regression analysis further revealed that LASP-1 expression was an independent prognostic factor for patients with CCA (hazard ratio, 2.689; 95 % confidence interval, 1.042–6.942; P = 0.041) (Table 3). Knockdown of LASP-1 inhibited migration and invasion of CCA cells by inducing EMT

Statistical analysis Statistical analysis was conducted using the SPSS software (version 19.0, USA). Student’s t test was used to determine the statistical significance of differences between comparison groups. The Pearson χ2 test was used in immunohistochemical analysis and clinicopathological associations of LASP-1 expression. The survival curve was plotted using the Kaplan– Meier method and multivariate statistical analysis was performed using a Cox proportional hazard regression model. Results of all assays were presented as mean ± standard deviation (SD). P < 0.05 was considered to be statistically significant in this study. All assays were performed independently in triplicate.

Results Relationship between LASP-1 expression and clinical characteristics of CCA patients The paraffin-embedded specimens of CCA tissues and adjacent noncancerous tissues from 40 cases were examined by IHC (Fig. 1a). As shown in Table 1, LASP-1 expression was

The finding of significantly upregulated expression of LASP1 in CCA tissues intrigued us to look into LASP-1 biological behaviors in CCA cells in vitro. We downregulated the expression of LASP-1 by siRNA transfection in HCCC-9810 and RBE cell lines. The efficacy of siRNA to reduce LASP1 mRNA and protein expression was measured by qRT-PCR and Western blot, respectively. It showed that mRNA and protein levels of LASP-1 were significantly downregulated in LASP-1 silencing group compared to the control siRNA group (p < 0.05) (Fig. 2a, b). The wound healing assay indicated that downregulation of LASP-1 markedly inhibited the relative migration distance in HCCC-9810 and RBE cells (Fig. 2c). The transwell assays verified that the number of penetrating cells in the LASP-1 siRNA group decreased significantly compared with the negative control group (p < 0.05) (Fig. 2d, e). Given that epithelial-to-mesenchymal transition (EMT) is a process which occurs in the initiation of metastasis for cancer progression, we evaluated the expression changes of several EMT biomarkers by Western blotting to identify the involvement LASP-1 in the invasiveness of CCA cells. Upon LASP-1 silencing, the exp r e s s i o n o f e p i t h e l i a l m a r k e r, E - c a d h e r i n , w a s

Tumor Biol.

Fig. 1 a Expression level of LASP-1 protein in CCA and adjacent noncancer tissues measured by immunohistochemistry (×200 magnification). Non-cancer tissues: negative LASP-1 expression in adjacent non-cancer tissues. Cholangiocarcinoma tissues: (i) weak LASP-1 expression in well differentiated CCA tissues; (ii) moderate LASP-1 expression in moderately differentiated CCA tissues; (iii) strong LASP-1 expression in poorly differentiated CCA tissues. b, c Western blot analysis of

LASP-1 expression level in paired CCA tissues and adjacent noncancerous tissues. Expression levels were normalized for GAPDH. (T tumor tissues; N noncancerous tissues). d Kaplan–Meier overall survival curve of CCA patients correlated with LASP-1 expression. Patients with tumors of high LASP-1 expression had significantly poorer prognosis than those with the low expression (P = 0.003)

upregulated, while the expression of the mesenchymal markers, N-cadherin and vimentin were downregulated (Fig. 2f).

Knockdown of LASP-1 promoted CCA cells apoptosis by disrupting the balance of Bcl-2 family proteins

Knockdown of LASP-1 inhibited proliferation and colony forming of CCA cells The cell viability assay showed that after 24 h of incubation, the cell number started to increase less in the HCCC-9810 and RBE cell lines with LASP-1 silencing than control groups (Fig. 3a). Moreover, the colony formation capacity showed that the number and size of the colonies were remarkably decreased in both HCCC-9810 and RBE cell lines with silencing LASP-1 (P < 0.05), compared to negative controls (Fig. 3b, c). Table 1 Immunohistochemical analysis of LASP-1 expression in CCA

Group

CCA tissues Non-cancer tissues a b

The results of flow cytometry showed that a majority of cells in the control group were alive and intact, but in the cells with LASP-1 siRNA transfection, apoptotic and dead cells increased significantly (Fig. 4a). The percentage of apoptotic cells of control group and LASP-1 siRNA group were 3.5 and 38.0 % in the HCCC-9810 cells (P < 0.05), and 3.7 and 24.7 % in the RBE cells (P < 0.05), respectively. It is well known that the Bcl-2 family play a fundamental role in regulation of the mitochondrial pathway of apoptosis and the balance between anti- and proapoptotic Bcl-2 family members determines the fate of cells to either live or die [13]. Therefore, we assessed the expression of several Bcl-2 family proteins by

No. of cases

40 40

Expression of LASP-1b Low

High

18 38

22 2

χ2

P

23.8

<0.001a

Statistically significant; P values were calculated using the Kappa test

Expression of LASP-1 was evaluated by the immunoreaction scores based on the extent and intensity of IHC staining. The score of 0–2 was defined as low expression, and 3–6 was defined as high expression

Tumor Biol. Table 2 Association of LASP-1 expression with the clinicopathological characteristics of CCA

Variable

Category

Expression of LASP-1 Low

High

χ2

P

Age

<60

6 12

10 12

0.606

0.436

Gender

≥60 Male Female ≥5 cm

14 4 5 13

12 10 14 8

2.349

0.125

5.105

0.024

Location

<5 cm

7 11

13 9

1.616

0.204

Histology differentiation

Intra hepatic Extra hepatic

13 5

6 16

8.021

0.005

Nerve invasion

Well/moderate Poor Absent Present

8 10 9 9

10 12 10 12

0.004

0.949

0.082

0.775

Absent Present Absent Present 0-I II–IV

13 5 6 12

9 13 16 6

3.922

0.048

6.208

0.013

Tumor size (cm)

Vascular invasion Lymph node metastasis TNM stage

Values set in italics indicate statistical significance, P < 0.05

Western blot analysis. In Fig. 4b, the increased expression of Bad and Bax protein and the decreased expression of Bcl-2 protein were both observed in LASP-1 siRNA treated CCA cells. Knockdown of LASP-1 inhibited tumor growth in vivo Since LASP-1 conferred a growth advantage to CCA cells in vitro, its effect in vivo was investigated. Both qRT-PCR

Table 3

and Western-blot analysis demonstrated that the lentivirusmediated infection effectively suppressed the expression level of LASP-1 (Fig. 5a, b). Then, we established the xenograft tumor model in the nude mice by subcutaneously injecting LASP-1-depleted or control RBE cells. The tumors removed from these animals were shown in Fig. 5c. It was observed that LASP-1 knockdown significantly inhibited tumor growth suggested by the reduced weight and size of the xenografted tumors (Fig. 5d, e).

Univariate and multivariate analysis of the association of prognosis with clinicopathological characteristics and LASP-1 expression in CCA

Characteristics

Age (<60/≥60) Gender (male/female) Tumor size (<5/≥5 cm) Location (intra hepatic/extra hepatic) Histology differentiation (well or moderate/poor) Nerve invasion (present/absent) Vascular invasion (present/absent) Lymph node metastasis (present/absent) TNM stage (0-I/II-IV) LASP-1 expression (low/high) Values set in italics indicate statistical significance, P < 0.05 HR hazard ratio; CI confidence interval

Univariable analysis

Multivariable analysis

HR (95% CI)

P

HR (95% CI)

P

1.431 (0.647–3.167) 1.373 (0.614–3.072) 1.410 (0.642–3.098) 0.654 (0.293–1.460) 0.753 (0.340–1.671) 1.043 (0.472–2.303) 2.185 (0.959–4.979) 2.870 (1.271–6.480) 2.669 (1.137–6.269) 3.301 (1.405–7.757)

0.376 0.440 0.392 0.300 0.486 0.917 0.063 0.011 0.024 0.006

– – – – – – – 0.360 (0.150–0.869) 0.561 (0.218–1.441) 2.689 (1.042–6.942)

– – – – – – – 0.023 0.230 0.041

Tumor Biol.

Fig. 2 Inhibition of LASP-1 reduced migration and invasion of HCCC9810 and RBE cells. a, b Knockdown of LASP-1 in HCCC-9810 and RBE cells revealed by qRT-PCR and Western blotting, GAPDH was used as an internal control. c Wound-healing assays for HCCC-9810 and RBE cells with or without knockdown of LASP-1. Cell monolayers were wounded by scratching with a 200-μL pipette tip. The wound closure in LASP-1 siRNA group was significantly delayed. d Transwell migration assays showed that HCCC-9810 and RBE cells transfected with LASP-1 siRNA had reduced number of migrated cells as

compared to the control group. e Transwell invasion assays showed that HCCC-9810 and RBE cells transfected with LASP-1 siRNA exhibited reduced invasion abilities compared to the control group. f LASP-1 silencing modulated the expression of EMT-related proteins in CCA cells. The expression levels of E-cadherin, N-cadherin, and vimentin were detected by Western blot analysis. GAPDH was used as the loading control. Data were represented as the mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001

Discussion

new diagnostic and prognostic biomarkers and identification of specific therapeutic targets are therefore become urgent in the clinical practice of CCA. LASP-1 is an actin-binding protein regulating cytoskeleton dynamics and taking part in cell migration, proliferation, and survival in numerous human cell

CCA is an aggressive malignancy of the biliary tract that carries high mortality typically due to its late presentation and limited curative medical therapies [14]. Detection of

Tumor Biol. Fig. 3 LASP-1 silencing inhibited proliferation and colony formation of CCA cells. a Cell viability of CCA cells was measured by the CCK-8 assay. Knockdown of LASP-1 reduced cell proliferation in HCCC-9810 and RBE cells. b, c Anchorageindependent growth was examined by colony formation assay. Knockdown of LASP-1 suppressed colony forming ability of CCA cells compared with controls. The number of colonies were calculated and depicted by the bar graph. Data were represented as the mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001

lines [7–11], and the upregulation of LASP-1 is observed in a range of malignant tumors, which strongly suggests the oncological and clinical significance of this protein. Our study, for the first time, revealed that LASP-1 protein essentially contributes to the oncogenesis and development of CCA. We also demonstrated the significant suppression of CCA by downregulating the expression of LASP-1 both in vitro and in vivo. IHC staining showed that LASP-1 was highly expressed in CCA tissues compared with that in their adjacent noncancerous tissues. Moreover, the clinicopathological data indicated that LASP-1 expression was significantly associated with larger tumors, poor histological differentiation, lymph node metastasis, and advanced TNM stage. In particular, high LASP-1 expression in cancer tissues indicated a shorter overall survival time of CCA patients. Multivariate analysis further identified LASP-1 as an independent prognostic factor for poor prognosis in CCA patients, which is consistent with the previous studies on gastric cancer [9], colorectal cancer [15], bladder cancer [16], and hepatocellular cancer [17]. The

positive correlation of the expression of LASP-1 with the progression of CCA suggested that detection of LASP-1 expression in resected CCA samples could not only serve as a postoperative prognostic factor for CCA patients, but also imply a possible therapeutic target for CCA treatment. The effect of LASP-1 on CCA pathogenesis was further evaluated in vitro with HCCC-9810 and RBE cell lines. Silencing LASP-1 gene by siRNA transfection significantly downregulated the levels of LASP-1 mRNA and protein in both cell lines, suggesting that LASP-1 siRNA effectively inhibited endogenous expression of LASP-1. Wound healing and transwell migration assays showed that LASP-1 silencing suppressed CCA cells migration and invasion. Meanwhile, the results of cell viability and colony formation revealed that CCA cell proliferation after LASP-1 silencing was significantly inhibited. Other researchers also reported the similar results in some other tumors, including gastric cancer [9], hepatocellular cancer [17], and breast cancer [18].

Tumor Biol.

Fig. 4 LASP-1 silencing induced apoptosis in CCA cells. a Apoptosis was determined by flow cytometry. Cells in the lower right quadrant represent early apoptotic cells, and those in the upper right quadrant represent late apoptotic cells. The percentage of apoptotic cells is presented as the mean ± SD (n = 3). b LASP-1 silencing modulated the

expression of Bcl-2 family proteins in CCA cells. The expression levels of Bad, Bax, and Bcl-2 were detected by Western blot analysis. GAPDH was used as the loading control. Significant differences from the control are indicated by *p < 0.05; **p < 0.01; ***p < 0.001

LASP-1 silencing inhibiting migration and invasion of CCA cells may be triggered by an EMT-like phenotypic transition. EMT is a reversible process by which cancer cells can switch from a sessile epithelial phenotype to an invasive mesenchymal state [19]. The hallmarks of EMT include loss of contact inhibition, remodeling of extracellular matrix, and reorganization of cytoskeleton, along with expression of mesenchymal markers and reduction of epithelial markers [20]. In this study, we found that knockdown of LASP-1 induced EMT by elevating protein level of the epithelial marker Ecadherin and reducing protein levels of the mesenchymal

markers N-cadherin and vimentin. Wang et al. has shown that LASP-1 played a critical role in the TGF-β mediated EMT process in colorectal cancer metastasis [21]. It was confirmed in this study that knockdown of LASP-1 contributed to the inhibition of migration and invasion of CCA cells by inducing EMT. Our experiments in vitro and in vivo also revealed that proapoptotic effect could give rise to significant inhibition in proliferation of CCA cells with LASP-1 silencing. Apoptosis, or programmed cell death, is a process that results in the controlled elimination of unhealthy or damaged cells and maintains the healthy survival/death balance [22]. Notably, the

Tumor Biol.

Fig. 5 LASP-1 silencing suppressed the growth of tumor in nude mice. a The lentiviral transduction efficiency was tested after infection. Relative LASP-1 mRNA expression was determined using qRT-PCR. GAPDH was used as an internal control. b The inhibited effect of Lv-shLASP-1 on LASP-1 protein levels in RBE cell lines by Western blot; GAPDH was used as an internal control. c In vivo tumor formation assay was carried

out by subcutaneously injecting RBE cells into nude mice. Tumors were excised from the animals and evaluated. d Average weights of LvshLASP-1 nude tumors and control tumors. e Tumor volume measured periodically in Lv-shLASP-1 tumors and control tumors. Data points were presented as mean volume ± SD. *p < 0.05; **p < 0.01; ***p < 0.001

impairment of apoptosis is a critical step in tumor pathogenesis. Bcl-2 family proteins can act as either cellular bodyguards or assassins by which positively or negatively control apoptosis. The Bcl-2-associated death promoter (Bad) acts as pro-apoptotic activator, Bcl-2-associated X protein (Bax) acts as pro-apoptotic effector, whereas Bcl-2 suppresses the pro-apoptotic role of Bad and Bax [23]. Our data showed that LASP-1 knockdown induced apoptosis of CCA cells by reducing the expression of Bcl-2 and increasing the expressions of Bad and Bax. Thus, the regulative role of LASP-1 in tumor growth and progression of CCA may be linked to the finding that knockdown of LASP-1 promoted apoptosis of CCA cells by disrupting the balance of Bcl-2 family proteins. Zyxin, localized primarily at the sites of cell adhesion, is a binding partner of LASP-1 and a key player for correct assembly and disassembly of actin filaments [24]. It has been reported that the decreased cell migration ability after LASP-1 silencing can be explained by the functional loss of zyxin which affects the actin cytoskeleton required for cell motility [25]. Furthermore, recent studies have shown that zyxin also shuttles between the cytoplasm and the nucleus, regulating the signal transduction [26]. Therefore, it is conceivable that after LASP-1 silencing, zyxin moved from focal adhesions and accumulated in the nucleus, leading to apoptosis, decreased cell motility, and inhibition of proliferation by regulating gene expression. However, the mechanisms underlying changes in the clinico-oncological behaviors of CCA by downregulated expression of LASP-1, particularly the downstream signaling pathways, need further investigations.

In summary, our study has shown that LASP-1 was overexpressed in CCA tissues. Moreover, expression of LASP-1 is significantly related to tumor size, histology differentiation, lymph node metastasis, TNM tumor stage, and poor prognosis in patients with CCA. In addition, knockdown of LASP-1 could successfully affect cell migration, invasion, proliferation, and apoptosis in CCA cell lines and inhibit tumorigenesis in vivo. Therefore, LASP-1 has potential as a novel prognostic biomarker and a promising anti-tumor therapeutic target in the treatment of CCA.

Acknowledgments This study was supported by the Introductory Funding project from Shanghai Science and Technology Bureau (124119a0600) to WG and Science and Technology funding project of Shanghai Jiao Tong University, School of Medicine (13xj22003) to BFC.

Compliance with ethical standards This study was performed with approval of the Ethics Committee of Xinhua Hospital and the ethical guidelines of the Declaration of Helsinki. In addition, written informed consent was obtained from all participants. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University (Shanghai, China). Conflicts of interest None

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