Tumor Biol. DOI 10.1007/s13277-013-1499-z

RESEARCH ARTICLE

Downregulated expression of metastasis associated in colon cancer 1 (MACC1) reduces gallbladder cancer cell proliferation and invasion Yong Wang & Qiang Hong & Jianjun Wang & Yuejun Fang & Conggang Hu

Received: 15 October 2013 / Accepted: 29 November 2013 # International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract Metastasis associated in colon cancer 1 (MACC1), a key regulator of the hepatocyte growth factor (HGF)/MET signaling pathway, has been implicated in multiple human cancers. However, little is known regarding its expression and biological function in human gallbladder cancer (GBC). In this study, we focused on the clinical significance and biological functions of MACC1 in GBC and found that MACC1 protein overexpression was frequently detected in GBC tissues. Patients with MACC1-positive tumors had worse overall survival than patients with MACC1-negative tumors. Furthermore, treatment of GBC lines with MACC1-targeting small interfering RNA oligonucleotides (MACC1-siRNA) significantly reduced the proliferation of GBC-SD and OCUG-1 cell lines and diminished both anchorage-independent growth on soft agar and cell migration. These data indicate that MACC1 acts as a putative oncogene in GBC and could be a novel diagnostic and therapeutic target for GBC. Keywords Metastasis associated in colon cancer 1 . Cell proliferation . Gallbladder cancer . RNA interference

Background Gallbladder cancer (GBC) is the most common biliary tract malignancy and the seventh most common gastrointestinal Yong Wang and Qiang Hong contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s13277-013-1499-z) contains supplementary material, which is available to authorized users. Y. Wang (*) : Q. Hong : J. Wang : Y. Fang : C. Hu Department of General Surgery, Guangfu Hospital of Jinhua, 1296 Huancheng North Road, Jinhua City 321000, Zhejiang Province, People’s Republic of China e-mail: [email protected]

cancer, with an incidence of 2.5 per 100,000 persons according to the SEER program [1, 2]. Although the incidence is relatively low, the mortality rate is higher than that of other cancers [3]. Owing to the early infiltration of tumor cells through the lymphatic, perineural, and hematogenous routes as well as direct invasion into the liver, the median survival time of GBC patients is less than 1 year in spite of recent advances in surgical techniques and medical management [4, 5]. Therefore, it is of great clinical value to further identify effective early markers for the diagnosis and prognosis of the disease as well as novel therapeutic targets. Metastasis associated in colon cancer 1 (MACC1), which is located on chromosome 7, has been originally identified through genome-wide expression analyses, comparing primary and metastatic colon cancers [6]. MACC1 comprises 852 amino acids and contains multiple functional domains, including an Src homology 3 (SH3) domain together with a prolinerich (PXXP) motif, enabling MACC1 for protein-specific interactions [7]. Recent studies have shown that MACC1 acts as a key regulator of the hepatocyte growth factor (HGF)/ MET signaling pathway, which mediates various biological functions, including angiogenesis, growth, differentiation, and cellular motility and invasion [8]. Consistent with these critical roles, MACC1 has been characterized as an oncogene in HCC, lung adenocarcinoma, gastric carcinoma, and colorectal cancer [9–12]. The clinicopathological analysis showed that high MACC1 expression correlated with poor prognosis in a variety of solid cancers, and increased MACC1 expression was determined to be an independent prognostic biomarker [13]. In addition, MACC1 was also demonstrated to be of predictive value for therapy response [14]. However, the role of MACC1 in GBC remains elusive. The present study aims to elucidate the role of MACC1 in GBC. To this end, we examined MACC1 protein expression and localization in GBC tissues and investigated the phenotypic effects of MACC1 downregulation in cultured GBC cell lines.

Tumor Biol.

Materials and methods Patients and clinicopathological data The study was approved by the ethics committee of Guangfu Hospital of Jinhua, and all patients provided informed consent. Cancer tissue specimens were obtained from 40 GBC patients who underwent radical cholecystectomy (without prior radiotherapy or chemotherapy) between 2008 and 2012 at the Department of General Surgery, Guangfu Hospital of Jinhua, China. In addition, 40 patients with cholelithiasis who underwent simple cholecystectomy were included as controls. All diagnoses of GBC, cholelithiasis, and lymph node metastasis were confirmed by histopathological examination, and all tissue samples were fixed in 4 % formalin immediately after removal and embedded in paraffin for immunohistochemical staining. Immunohistochemical analysis and evaluation of MACC1 expression Immunohistochemical staining was performed using the standard immunoperoxidase staining procedure, and MACC1 expression in the benign and malignant specimens was evaluated according to the methods described by Pinheiro et al. [15]. The sections were semi-quantitatively scored according to the extent of immunoreactivity as follows: 0, 0 % immunoreactive cells; 1, <5 % immunoreactive cells; 2, 5–50 % immunoreactive cells; and 3, >50 % immunoreactive cells. Additionally, the staining intensity was scored semiquantitatively as follows: 0, negative; 1, weak; 2, intermediate; and 3, strong. The final immunoreaction score was defined as the sum of both parameters (extension and intensity), and the samples were classified as negative (0), weakly stained (1–2), moderately stained (3), and strongly stained (4–6). For statistical purposes, only moderate and strong final immunoreaction scores were considered positive; the other final scores were considered negative. Cell culture The GBC-SD cell line was purchased from Shanghai Institutes for Biological Sciences (Shanghai, China). The OCUG-1 cell line was obtained from the Health Science Research Resources Bank (Osaka, Japan). All cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 10 % (v/v) fetal bovine serum and routinely cultivated in a humidified incubator at 37 °C with 5 % CO2. Lentivirus-mediated RNA interference For pFH1UGW lentivirus-mediated silencing of MACC1, one effective sequence of human MACC1 short hairpin RNAs

(shRNAs) was used: 5′-GAGTTAGTCGCACGTCTCA-3′. Non-silencing shRNA, with the scrambled sequence 5′GCCAGCTTAGCACTGACTC-3′, was used as a control. These shRNAs were synthesized and inserted into the pFH1UGW lentivirus core vector containing a cytomegalovirus (CMV)-driven enhanced green fluorescent protein (EGFP) reporter gene and driven by the H1 promoter. Recombinant lentivirus expressing MACC1 small interfering RNA (siRNA) or control siRNA (si-MACC1 or siCTRL) was produced by HEK293 cells cotransfected with the core vector and pVSVG and pCMV D R 8.92 vectors using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The virus was harvested and purified by centrifugation and used for subsequent studies. Quantitative real-time PCR Total RNA was extracted from cultured cells with Trizol reagent (Invitrogen). The following primers were used for detecting the expression of MACC1 and β-actin (internal control): MACC1 (sense), 5′-TTCTTTTGATTCCTCCGG TGA-3′; MACC1 (antisense), 5′-TTCTTTTGATTCCTCC GGTGA-3′; β-actin (sense), 5′-GTGGACATCCGCAAAG AC-3′; and β-actin (antisense), 5′-AAAGGGTGTAACGC AACTA-3′. The protocol was performed according to the manufacturer's instructions. Results were normalized to the expression of β-actin. Western blotting analysis Total cell lysates were separated on a 10 % SDS-PAGE gel and then transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Membranes were blocked and then probed with primary antibody against MACC1 (1:400 dilution, Abcam, Cambridge, UK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz) and visualized using the enhanced chemiluminescent detection reagent. Cell proliferation assay Cell proliferation was assessed with the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay following the manufacturer's instructions. In addition, the absorbance at 490 nm was detected using a microplate reader (Bio-Rad, Hercules, CA, USA). A growth curve was prepared according to the absorbance values at 490 nm. The results reflect the average of three replicates under the same conditions.

Tumor Biol.

Fig. 1 The enhanced expression of MACC1 in GBC and negative association with prognosis. Immunohistochemistry of MACC1 expression (scale bar, 100 μm). The positive reaction was predominantly localized to the cell cytoplasm. a, b Negative staining in normal glandular epithelium (red arrow). c, d Moderate to strong staining in an

adenocarcinoma (black arrow). e The average staining scores of MACC1 expression in cholelithiasis and GBC tissues. f Kaplan–Meier plots of overall survival in GBC patients with positive and negative MACC1 expression scores

Colony formation assay

colonies were counted by light microscopy. Experiments were performed in triplicate.

Both non-transfected and transfected GBC-SD and OCUG-1 cells (200 cells/well) were seeded in six-well plates. The culture medium was changed at regular time intervals. After 14 days of culture, adherent cells were washed twice with PBS and fixed with 4 % paraformaldehyde for 30 min at room temperature. Colonies were stained with Giemsa solution for 15 min and then washed with water and air-dried. Cell

Table 1 Association of MACC1 expression with the clinicopathological characteristics of GBC

Parameter

Age (years) Sex Histopathological subtypes

TNM stage Lymph node metastasis Perineural invasion Gallbladder stones a

Statistically significant

In vitro cell invasion assay Cell invasion was assessed using 6.5-mm transwell chambers with a pore size of 8 μm (Corning). Transwell upper chambers were precoated with 50 μg/chamber of solubilized basement membrane in the form of Matrigel (BD Biosciences, Franklin

Category

No. of cases

MACC1 No. of positive cases (%)

χ2

P value

0.227

0.634

0.755

0.385

8.027

0.018a

7.349

0.007a

0.029

0.865

9.858

0.002a

0.152

0.697

<60 ≥60 Male Female High Middle Low 0–I II–IV

9 31 11 29 8 14 18 5 35

8 (88.9) 23 (74.2) 7 (63.6) 24 (82.8) 3 (37.5) 12 (85.7) 16 (88.9) 1 (20.0) 30 (85.7)

Negative Positive Absent Present Negative Positive

19 21 12 28 17 23

14 (73.7) 17 (83.0) 5 (41.7) 26 (92.9) 14 (82.4) 17 (77.3)

Tumor Biol. Table 2 Correlation between patient survival time and MACC1 expression Survival time

<1 year ≥1 year a

No. of cases

MACC1

28 12

No. of positive cases (%)

χ2

P value

Result

25 (89.3) 6 (50.0)

5.352

0.021a

MACC1 expression is associated with a poor prognosis in patients with GBC

Statistically significant

Lakes, NJ, USA). DMEM/10 % FBS was added to the bottom chamber. GBC cells (3×104 per chamber) were seeded in serum-containing media in the upper well of the transwell chambers and incubated for 48 h at 37 °C and 5 % CO2. The invasive cells in the lower chamber (below the filter surface) were fixed in 70 % ethanol, stained with 0.1 mg/ml crystal violet solution, and counted under a microscope (×20 magnification). Five random visual fields were counted for each well, and the average was determined. Statistical analysis Statistical analysis was conducted with SPSS software, version 18.0 (SPSS Inc., Chicago, IL, USA). The data are expressed as the means±SD. Independent Student's t test and χ 2 test were used to assess the effects of MACC1 in the cell lines. The Kaplan–Meier test was used for univariate survival analysis. The Cox proportional hazard model was used for multivariate analysis and for determining the 95 %

Table 3 Univariate log-rank analysis of disease-specific survival (DSS) Parameter

Category Case Median survival time P value number (months) (95 % CI) (n)

Age (years)

<60 ≥60 Sex Male Female Histopathological High Middle subtypes Low TNM stage 0–I II–IV Lymph node Negative metastasis Positive Perineural Absent invasion Present MACC1 Negative Positive a

Statistically significant

confidence interval (CI). Results were considered to be statistically different at P <0.05.

8 32 11 29 8 14 18 5 35 19 21 12 28 9 31

8.0 (5.8–10.2) 8.0 (2.2–13.8) 11.0 6.5 (3.7–9.3) 36.0 (26.3–45.7) 8.0 (5.9–10.1) 6.0 (3.6–8.4) 36.0 6.5 (4.4–8.6) 15.0 (9.5–20.5) 5.0 (2.9–7.1) 36.0 6.5 (4.2–8.8) 36.0 (0–79.7) 6.0 (4.0–8.0)

0.930

To determine the potential role of MACC1 in GBC progression, we evaluated MACC1 expression in GBC and cholelithiasis tissues by IHC (Fig. 1a–e). MACC1 was mainly located in the cytoplasm. Approximately 77.5 % (31/40) of the GBC cases had positive MACC1 staining in the tumor cells. In contrast, only 21.5 % (11/40) of the cases had positive staining in the cholelithiasis tissues (Fig. 1e, P <0.001). Next, we assessed the correlation between MACC1 expression levels and histopathological parameters in patients with GBC. As shown in Table 1, the MACC1 overexpression was found to be significantly correlated with the histopathological subtypes (P <0.05); the TNM stage (P <0.01); and the perineural invasion (P <0.01), but not with sex, age, history of gallstones, or lymph node metastasis, indicating a potential role of MACC1 expression in promoting aggressive phenotypes in GBC. Among the 40 GBC patients, the survival information was obtained through phone calls. Twelve patients survived over 1 year and 28 patients survived less than 1 year, with an average survival time of 9.7 months. The patients who survived less than 1 year were found to have significantly higher MACC1 expression than the patients who survived over 1 year (Table 2). The Kaplan–Meier survival analysis revealed that the histopathological subtypes (P <0.01), the TNM stage (P <0.05), the presence of lymph node metastasis (P <0.01), and the perineural invasion (P <0.05) were significantly associated with the average survival time. The average survival Table 4 Multivariate analysis of disease-specific survival (DSS) Parameter

Category HR

95 % CI

P value

0.142 MACC1 expression

0.003a

0.015a 0.005a 0.013a 0.001a

Negative Positive TNM stage 0–I II–IV Lymph node metastasis Negative Positive Histopathological subtypes High Middle Low Perineural invasion Absent Present HR hazard ratio a

Statistically significant

4.298 1.134–88.887 0.038a 0.008 0.084–15.014 0.930 4.368 1.060–6.134

0.037a

2.061 0.828–3.402

0.151

0.073 0.218–3.170

0.787

Tumor Biol.

time for MACC1-negative patients was significantly higher than that for patients with positive MACC1 expression (P <0.01; Table 3, Fig. 1f). To obtain a more precise estimate about prognosis, the Cox proportional hazard regression model was applied. Results confirmed that the MACC1 expression, as well as the presence of lymph node metastasis, was negatively correlated with postoperative survival, suggesting that high MACC1 expression is a risk factor (Table 4). Lentiviral siRNA effectively and specifically inhibited MACC1 mRNA and protein expression in GBC-SD and OCUG-1 cells Since MACC1 was overexpressed in GBC and linked with poor prognosis of patients aforementioned, the oncogenic function of MACC1 was determined by lentivirus shRNAmediated disruption of MACC1 expression in two GBC cell lines (GBC-SD and OCUG-1), which showed high expression of MACC1 (Fig. 2a). To determine the lentiviral transfection efficiency in GBC-SD and OCUG-1 cells, GFP expression was examined by microscopy at a multiple oblique illumination (MOI) of 40 at 3 days after transfection (Fig. 2b). The

Fig. 2 Lentivirus-mediated siRNA decreased the expression of MACC1 in GBC-SD and OCUG-1 cells. a MACC1 protein was highly expressed in GBC cell lines (GBC-SD and OCUG-1) as suggested by Western blotting. b The transfection efficiency was determined 3 days after incubation with lentivirus at an MOI of 40. Transfected cells labeled with GFP were observed under a light microscope and fluorescence microscope. Light micrograph (upper); fluorescent micrograph (lower) (×100). c Total RNA was extracted at 5 days after infection, and relative MACC1 mRNA expression was determined using quantitative real-time PCR. β-

efficiency of lentiviral transduction in GBC-SD and OCUG-1 cells was more than 90 %. After being transfected with scramble or MACC1 shRNA as indicated, the mRNA and protein expression of MACC1 in GBC-SD and OCUG-1 cells were analyzed by real-time PCR and Western blot assay. As shown in Fig. 2c, the levels of MACC1 mRNA in GBC-SD and OCUG-1 cells transduced with MACC1 shRNA (si-MACC1) were significantly decreased by 71 % (P <0.001) and 64 % (P <0.001), respectively. In agreement with the real-time PCR analysis, Western blot analysis demonstrated a significant reduction in MACC1 protein level in the si-MACC1 group (Fig. 2d). These results indicated that lentivirus-mediated shRNA effectively and specifically inhibited MACC1 mRNA and protein expression in GBC-SD and OCUG-1 cells. Effects of MACC1 downregulation on GBC-SD and OCUG-1 cell proliferation To further evaluate whether MACC1 gene silencing in GBCSD and OCUG-1 cells inhibits cell growth and proliferation, the MTT assay and colony formation assay were performed.

actin was used as an internal control. Data represent the mean±SD of three independent experiments. ***P <0.001, compared with si-CTRL. d Total cellular proteins were extracted at 5 days after infection and determined by Western blotting analysis using antibodies against MACC1 and GAPDH as an internal control. Data represent one out of three separate experiments. CTRL black control, si-CTRL cells transduced with lentivirus-mediated scr-siRNA, si-MACC1 cells transduced with lentivirusmediated MACC1-siRNA

Tumor Biol.

As shown in Fig. 3a, b, the cell viability of the transfected cells was detected using an MTT assay daily for 5 days, and downregulation of MACC1 expression markedly inhibited the cell viability of GBC-SD and OCUG-1 in a timedependent manner. After 5 days of infection, the percentage of viable cells in GBC-SD and OCUG-1 cells transfected with MACC1 shRNA (si-MACC1) significantly decreased, as compared with the negative controls (CTRL and si-CTRL) (P <0.001). Furthermore, the colony formation evaluation also showed that silencing MACC1 significantly decreased the number of colonies in GBC-SD and OCUG-1 (Fig. 3c, d) cells compared with the CTRL and si-CTRL groups (P <0.001, respectively). These results suggested that MACC1 played a vital role in GBC cell growth and proliferation. Effect of MACC1 downregulation on the invasiveness of GBC-SD and OCUG-1 cells Metastasis is frequently observed in patients with gallbladder cancer, and therefore, we wondered whether MACC1 might

Fig. 3 MACC1 exhibiting oncogenic property in GBC cell lines. a, b Cellular proliferation of untransfected or transfected GBC-SD cells (a) and OCUG-1 cells (b) were measured using an MTT assay daily for 5 days. c, d GBC-SD cells and OCUG-1 cells were seeded at 200 cells/ well, and the cells were allowed to form colonies. Colony numbers were counted and recorded. e Representative photos of crystal violet-stained GBC-SD and OCUG-1 cells that migrated through polycarbonate membranes. f A statistical plot of the average number of migrated GBC-SD and OCUG-1 cells in each group. Graph shows mean± SD; ***P <0.001. Data represent one of three separate experiments. CTRL black control, si-CTRL cells transduced with lentivirusmediated scr-siRNA, si-MACC1 cells transduced with lentivirusmediated MACC1-siRNA

be involved in the migration of GBC-SD and OCUG-1 cells. Matrigel invasion transwell assay was conducted to confirm the effect of MACC1 on the invasion properties of GBC-SD and OCUG-1 cells. As indicated, knockdown of MACC1 significantly decreased the invasive ability of both GBC-SD and OCUG-1 cells (Fig. 3e, f; P <0.001 and P <0.01, respectively, for GBC-SD and OCUG-1), which suggested that MACC1 might be involved in the enhanced metastasis property of GBC. These results further explained the aforementioned correlation of MACC1 with the poor prognosis of GBC patients.

Discussion GBC is a highly invasive and rapidly proliferating cancer, and the prognosis for this type of cancer remains poor even with the most aggressive conventional treatments [16]. Therefore, it is important to find novel methods that can effectively inhibit GBC cell growth and metastasis. Targeting therapies have shown promise in achieving these goals [17]. MACC1

Tumor Biol.

acts as a transcription activator for c-MET. Thereby, aberrant activation of c-MET could cause oncogenesis and cancer metastasis by activating the HGF/MET signaling axis [6, 18]. MACC1 was first identified as a colon cancer oncogene that promotes metastasis and proliferation and associates with peritoneal dissemination and a higher stage of TNM classification in colorectal carcinomas [6]. Recent studies have also linked MACC1 upregulation to cancer development and progression in other solid tumors. In hepatocellular cancer, MACC1 was validated as a prognostic biomarker for tumor progression and survival, including early-stage and AFP-normal patients, and also as a predictive biomarker for therapy response [9]. In gastric cancer, MACC1 contributed to a poor prognosis by promoting tumor cell proliferation and invasion as well as the EMT [11]. On the basis of these findings, it is implicated that MACC1 plays an important role in carcinogenesis processes [19]. However, to date, MACC1 has never been linked to GBC. In the present study, we found for the first time that significantly increased MACC1 expression was detected in the vast majority of GBC tissues when compared with cholecystitis tissues. Moreover, increased MACC1 protein expression correlated not only with advanced tumor status but also with poor patient survival, suggesting the significance of MACC1 in GBC advancement. Notably, Cox multivariate analysis indicated that a high nuclear MACC1 expression level was an independent factor for the indication of poor prognosis in GBC patients. Thus, MACC1 could be a potential therapeutic target in GBC. To further evaluate the biological significance of MACC1 in GBC pathogenesis, we employed lentivirally transduced siRNA to effectively and specifically reduce endogenous MACC1 expression in the GBC cell lines. We observed that, in GBC-SD and OCUG-1 cells, MACC1 knockdown markedly decreased cell proliferation, migration, and invasiveness by MTT, colony formation, and transwell assays, respectively. These findings are consistent with an earlier report by Meng et al., in which the downregulation of MACC1 inhibited the invasiveness of nasopharyngeal carcinoma cells [20]. These qualities might contribute to MACC1-associated aggressive biological behaviors of GBC. Further studies are required to elucidate the mechanism by which MACC1 mediates aggressiveness in GBC cells. In summary, the present study showed that MACC1 might be involved in GBC progression and aggressiveness. The classification of patients according to MACC1 expression levels provides a valuable tool with which to identify GBC patients with poor prognoses. Our findings might also extend our knowledge of the biological progression of GBC and could provide a new therapeutic target for GBC.

Acknowledgments This work was supported by the Jinhua Committee of Science and Technology (No. 2012-3-018). We thank the pathology department of the Guangfu Hospital of Jinhua for the experiment equipment supply. Conflicts of interest None

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