Lymphocyte-specific protein 1 inhibits the growth of hepatocellular carcinoma by suppressing ERK1/2 phosphorylation.

Lymphocyte-specific protein 1 (LSP1) has been reported to regulate cell biology in several human cancers including lymphoma and breast cancer. However, the functions of LSP1 in human hepatocellular carcinoma (HCC) are still unknown. In this study, we found that LSP1 expression was downregulated in HCC tissues and cell lines, and lower LSP1 expression was correlated with poor clinicopathological features including large tumor size, high Edmondson-Steiner grading and advanced tumor-node-metastasis (TNM) stage. Additionally, we demonstrated that patients with high LSP1 expression had significantly better overall survival and disease-free survival. Moreover, LSP1 was found to be an independent factor for predicting the prognosis of HCC patients. In vitro and in vivo assays showed that overexpressing LSP1 inhibited HCC growth by inducing both apoptosis and growth arrest. Mechanistically, we found that expression of phosphorylated extracellular regulated protein kinases 1 and 2 (ERK1/2) was downregulated after LSP1 overexpression, indicating LSP1 could suppress HCC growth by inhibiting the ERK pathway in HCC cells. Taken together, these results indicate that LSP1 may serve as a prognostic marker and a potential therapeutic target in human HCC.

B‐cell lymphoma 2

copy number variation

extracellular regulated protein kinases 1 and 2

hepatocellular carcinoma

kinase suppressor of Ras

lymphocyte‐specific protein 1

mitogen‐activated protein kinase

tumor–node–metastasis

Hepatocellullar carcinoma (HCC) is currently the fifth most frequently diagnosed malignant tumor and the third leading cause of cancer‐related deaths 1. Although therapeutic methods including surgical resection, liver transplantation and adjuvant therapy have been applied in the treatment of HCC, the overall 5‐year survival rate of HCC patients remains unsatisfactory 1, 2. Therefore, it is necessary to elucidate the molecular mechanisms of HCC pathogenesis, which may contribute to identifying novel therapeutic targets for HCC.

Lymphocyte‐specific protein 1 (LSP1), the gene for which is located in 11p15.5 and contains 20 exons, is a protein that consists of 339 amino acids 3. Previous studies have revealed that LSP1 was involved in multiple cancers including breast cancer 3, lymphomas 4, pancreatic cancer 5 and dermatofibroma 6. It has been reported that LSP1 was downregulated in all breast cancer patients, and LSP1 downregulation was a risk factor for breast cancer 3, 7 and non‐Hodgkin lymphoma 8. Results from a large pancreatic case–control study indicated that LSP1 may also be associated with pancreatic cancer susceptibility and survival 9. Additionally, LSP1 expression has been found to be useful in distinguishing dermatofibroma from dermatofibrosarcoma protuberans 6. Moreover, Nalesnik et al. 10 found through genome analysis that LSP1 had the most cases of copy number variation (CNV), including 46 deletions and 5 amplifications in 98 human HCC tissues. However, the expression and functions of LSP1 in human HCC remain largely unknown.

In this study, we demonstrated that LSP1 was downregulated in human HCC and was an independent prognostic factor for predicting both the overall and the disease‐free 5‐year survival of HCC patients. LSP1 inhibited the growth of HCC by inhibiting cell proliferation and promoting both apoptosis and growth arrest of HCC. Furthermore, LSP1 was inversely related to phosphorylated extracellular regulated protein kinases 1 and 2 (ERK1/2) protein expression in HCC cell lines. Our results suggested that LSP1 inhibited HCC growth by suppressing the ERK1/2 pathway.

Materials and methods

Human HCC clinical samples

Ninety HCC samples and paired adjacent benign tissues (>2 cm distance from the margin of the resection) were obtained from patients who had undergone curative surgery for HCC at the First Affiliated Hospital of Xi'an Jiaotong University from January 2008 to December 2010, with a median follow‐up time of 35 months. All tissues were stored at −80 °C in a deep freezer before protein extraction. The patients had not received any antitumor therapy before surgery. All HCC and normal liver tissue samples were pathologically confirmed. All samples were used after obtaining informed consent from patients. The Xi'an Jiaotong University Ethics Committee approved all protocols according to the Declaration of Helsinki (as revised in Tokyo 2004).

Cell culture

The human immortalized normal hepatic cell line LO2 and six HCC cell lines (HepG2, MHCC‐97L, Hep3B, SMMC‐7721, MHCC‐97H and Huh7) were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Life Technologies, Carlsbad, CA, USA), 100 units·mL−1 penicillin and 100 μg·mL−1 streptomycin (Sigma‐Aldrich, St. Louis, MO, USA), aerated with air containing 5% CO2 in an incubator at 37 °C.

Immunohistochemistry

Ninety paraformaldehyde‐fixed, paraffin‐embedded HCC tissue sections and corresponding adjacent benign tissue sections were used to perform the immunohistochemistry. The sections were heated at 60 °C for 2 h and then deparaffinized in xylene and rehydrated through descending alcohol concentrations. They were washed in PBS (pH 7.4) before antigen retrieval in citrate buffer. Endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide for 15 min at room temperature. The sections were blocked for 30 min using 10% goat plasma and then incubated at 4 °C overnight with the primary antibodies directed against LSP1 (Cat. No. ab133506, Abcam, Cambridge, MA, USA) or Ki‐67 (9027; Cell Signaling Technology, Danvers, MA, USA). After washing in PBS, they were incubated with horseradish peroxidase‐conjugated secondary antibody (Goldenbridge Biotechnology, Zhongshan, China) according to the manufacturer's recommendations. A negative control was prepared by replacing the primary antibody with PBS. Finally, the sections were visualized with diaminobenzidine and counterstained with hematoxylin, then dehydrated in alcohol and xylene and mounted onto glass slides.

Western blotting

Western blotting analysis was performed using standard techniques. The following antibodies were used: LSP1 (ab133506, Abcam), ERK1/2 (sc‐292838, Santa Cruz Biotechnology, Dallas, TX, USA), phosphorylated ERK1/2 (p‐ERK1/2; sc‐16982, Santa Cruz Biotechnology), cyclin D1 (ab137875, Abcam), B‐cell lymphoma 2 (Bcl2; ab32124, Abcam) and β‐actin (sc‐4778, Santa Cruz Biotechnology).

MTT assay

Hepatocellular carcinoma cells transfected with LAP1 expressing vector or negative control vector were seeded in 96‐well plates in triplicate at densities of approximately 5000 cells per well and cultured in DMEM containing 10% FBS. Cell viability was analyzed using thiazolyl blue,3‐[4,5‐dimethylthylthiazol‐2‐yl]‐2,5 diphenyltetrazolium broide (MTT; Sigma‐Aldrich) assay at the indicated time points.

BrdU assay

Cells grown on coverslips (Fisher, Pittsburgh, PA, USA) were incubated with bromodeoxyuridine (BrdU) for 1 h and stained with anti‐BrdU antibody (Sigma‐Aldrich) according to the manufacturer's instructions. Gray level images were acquired under a laser scanning microscope (Axioskop 2 plus, Carl Zeiss Co. Ltd, Jena, Germany).

Cell cycle assay and cell apoptosis detection

The cells were collected and washed with PBS. The washed cells were resuspended in PBS and fixed in 75% ethanol. Then, the fixed cells were stained with propidium iodide (PI) supplemented with RNase A (Sigma‐Aldrich) for cell cycle analysis with a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA). An Annexin‐V‐FLUOS Staining Kit (Roche Diagnostics, Indianapolis, IN, USA) was used to analyze the level of apoptosis. Data were collected and analyzed with modfit software (BD Biosciences).

Cell transfection

The pcDNA/LSP1 and Control were purchased from Origene Biotechnology (Beijing, China; sc118682). The day before transfection, 5 × 106 Hep3B or HepG2 cells were seeded in 100 mm dishes. Cells were transfected using Lipofectamine 2000 Reagent (Invitrogen/Life Technologies) following the manufacturer's instructions. The medium containing the retroviruses was collected 48 and 72 h after transfection. Viral transduction was performed by incubating cells with the viral supernatant (25%) supplemented with polybrene (8 μg·mL−1) overnight at 37 °C. Further experiments were performed 48–96 h after viral transduction.

In vivo experiments

Female BALB/c nude mice (Centre of Laboratory Animals, The Medical College of Xi'an Jiaotong University, Xi'an, China), 4–6 weeks old, were used to establish a nude mouse xenograft model. Hep3B cells (5 × 106) transfected with pcDNA/LSP1 or Control vectors were mixed in 100 μL of Matrigel and were inoculated subcutaneously into the flank of the nude mouse. Tumor volume was determined by measuring two of its dimensions with calipers every 7 days, and then calculated as tumor volume = length × width × width/2. All mice were sacrificed at 4 weeks after the injection of HCC cells. The xenograft tumor tissues were explanted for pathological examination. All in vivo protocols were approved by the Institutional Animal Care and Use Committee of Xi'an Jiaotong University.

Statistical analysis

All statistical analyses were performed using the statistical package spss for Windows Version 20.0 (IBM Corp., Armonk, NY, USA) or prism 5 software (GraphPad Software, Inc., San Diego, CA, USA). The quantitative data were compared between groups using Student's t test and Chi squared test was used to analyse the clinical characteristics of the patients. The Kaplan–Meier method and log‐rank test were used to compare the cumulative recurrence and survival rates. The independent factors influencing the survival and recurrence of HCC patients were determined using the Cox proportional hazards model. P < 0.05 was considered to be statistically significant.

Results

LSP1 was significantly downregulated in human HCC

To identify the expression level of LSP1 in human HCC, firstly, we used immunohistochemistry and western blot to detect LSP1 expression in HCC tissues and adjacent tissues. The results showed that LSP1 was mainly expressed in the cytoplasm (Fig. 1A), and LSP1 was significantly downregulated in HCC tissues compared with adjacent tissues (Fig. 1A, P < 0.001; Fig. 1B, P < 0.05). Then, we further examined the LSP1 expression in HCC cell lines by western blot. This consistently revealed that LSP1 protein expression was downregulated in six HCC cell lines (HepG2, MHCC‐97L, Hep3B, SMMC‐7721, MHCC‐97H and Huh7) compared with the human immortalized normal hepatic cell line LO2 (Fig. 1C). Among them, the relative expression levels were lowest in HepG2 and Hep3B cell lines. These data demonstrated that LSP1 was obviously downregulated in human HCC.

Figure 1

The expression levels of LSP1 in HCC. (A) Immunohistochemical analyses of LSP1 protein in HCC tissues (b,d) and matched noncancerous tissues (a,c). (a,b: ×100; c,d: 400). Scale bar: 100 m. IHC score, immunohistochemistry score. (B) Representative western blot analysis of LSP1 expression in HCC tissues (T) and matched noncancerous tissues (NT) was shown. (C) Representative western blot analysis of LSP1 expression in HCC cell lines and the immortalized hepatic cell line LO2 was shown. Values are depicted as mean ± SEM,*P < 0.05, **P < 0.01, by t test.

Clinical significance of LSP1 expression in HCC

To determine whether LSP1 expression is associate with clinical features in HCC patients, we divided 90 patients into two different groups according to the median level of LSP1 expression. Statistical analysis indicated that the expression of LSP1 was significantly correlated with tumor size (P = 0.003), Edmondson–Steiner grading (P = 0.049) and TNM tumor stage (P = 0.001) (Table 1). Kaplan–Meier survival curves showed that low LSP1 expression in HCC was significantly correlated with worse overall survival (P < 0.01) and disease‐free survival (P < 0.001) (Fig. 2). Moreover, multivariate Cox regression analysis indicated that LSP1 expression and tumor size were independent prognostic factors for predicting both 5‐year overall survival and disease‐free survival in HCC patients (P < 0.01, respectively, Table 2). These data suggested that downregulated LSP1 was correlated with poor prognosis of HCC patients and LSP1 may be a promising prognostic marker for HCC.

Table 1

Correlation between the clinicopathological characteristics and expression of LSP1 in human HCC. AFP, α‐fetoprotein; HBV, hepatitis B virus; LSP1Low and LSP1High, low and high expression, respectively, of LSP1 in HCC

Clinicopathological feature		Total no. of patients (n = 90)	No. of patients		P
			LSP1Low	LSP1High
Age (years)	<50	24	14	10	0.847
	≥50	66	37	29
Sex	Male	65	33	32	0.069
	Female	25	18	7
HBV	Absent	21	13	8	0.580
	Present	69	38	31
Serum AFP level (ng·mL−1)	<400	35	20	15	0.942
	≥400	55	31	24
Tumor size (cm)	<5	36	13	21	0.003**
	≥5	54	38	16
No. of tumor nodules	1	69	37	32	0.291
	≥2	21	14	7
Cirrhosis	Absent	39	22	17	0.966
	Present	51	29	22
Vennous infiltration	Absent	57	31	26	0.566
	Present	33	20	13
Edmondson–steiner grading	I + II	67	42	25	0.049*
	III + IV	23	9	14
TNM tumor stage	I + II	62	28	34	0.001**
	III + IV	28	23	5

*P < 0.05, **P < 0.01.

Figure 2

Prognostic significance of LSP1 in HCC cases. Kaplan‐Meier 5‐year (A, P < 0.01) overall and (B, P < 0.001) disease‐free survival curves of HCC patients according to the level of LSP1 expression revealed that the high LSP1 group patients had a better prognosis than low LSP1 group patients.

Table 2

Multivariate Cox regression analysis of 5‐year overall and disease‐free survival of 90 HCC patients. The data show the hazard ratio (HR) and 95% confidence interval (CI)

Variables	Overall survival			Disease‐free survival
	HR	95% CI	P	HR	95% CI	P
LSP1 expression	3.774	1.429, 9.964	0.007**	6.562	2.260, 19.050	0.002**
Tumor size	3.249	1.230, 8.586	0.017*	5.532	1.692, 18.087	0.005**
Edmondson–Steiner grading	2.777	1.089, 7.081	0.132	2.962	1.126, 7.791	0.228
TNM tumor stage	6.511	1.784, 23.770	0.205	6.746	2.181, 20.869	0.103

*P < 0.05, **P < 0.01.

LSP1 inhibited HCC cell proliferation and promoted apoptosis

In order to explore the biological functions of LSP1 in HCC progression, we transfected pcDNA/LSP1 plasmid into HepG2 and Hep3B cell lines. Western blot revealed LSP1 was upregulated when transfected with pcDNA/LSP1 (Fig. 3A). Then we carried out the MTT and Brdu assays to explore the changes of HCC proliferation. The results showed overexpressing LSP1 resulted in decreased proliferation of HepG2 and Hep3B (Fig. 3B–E). Then, flow cytometry analysis was utilized to detect changes of cell apoptosis (Fig. 3F,G). The results revealed that overexpressed LSP1 induced cell apoptosis both in HepG2 and Hep3B cells. Therefore, these results suggested that LSP1 exerted a tumor suppressive role in HCC by inhibiting proliferation and promoting both apoptosis and growth arrest.

Figure 3

LSP1 regulates proliferation and apoptosis in HCC cells. (A) HepG2 and Hep3B cells that had been transfected with pcDNA/LSP1 were subjected to western blot for LSP1. (B,C) Cell proliferation was measured by MTT assay; overexpressed LSP1 was found to reduce the viability of HepG2 and Hep3B cells. (D,E). Representative quantification of BrdU positive cells. (F,G) Quantification of the apoptotic cell population by flow cytometry. Both HepG2 and Hep3B cells with overexpressed LSP1 were composed of a larger subset of apoptotic cells compared with the control cells. PI‐A: the apoptosis cell stained by propidium iodide(PI). FITC‐A: the apoptosis cell stained by fluorescein isothiocyanate (FITC). *P < 0.05, **P < 0.01, by t test; n = 3 repeats with similar results.

To further confirm LSP1 could inhibit HCC proliferation and promote apoptosis, we conducted animal experiments. Hep3B cells that had been infected with retroviruses were implanted into nude mice via subcutaneous injection. Tumor growth curves, generated over 28 days, revealed that overexpressed LSP1 slowed down Hep3B tumor growth in mice (Fig. 4A–C). We performed immunohistochemistry for Ki‐67 in the xenografted tissues. As expected, LSP1 overexpression inhibited proliferation in vivo (Fig. 4D). Taken together, these data indicated that LSP1 inhibited tumor proliferation.

Figure 4

LSP1 inhibited tumor growth in vivo. (A) Representative pictures of HCC xenografts from both control Hep3B cells (top) and overexpressed LSP1 Hep3B cells (bottom). Tumor volume (B) and weight (C) revealed that LSP1 significantly inhibited tumor growth. (D) Tumor nodules were subjected to immunohistochemical staining for Ki‐67 and quantitative analysis. Representative immunostaining revealed that LSP1 significantly reduced the number of Ki‐67 positive cells. Values are depicted as the mean relative level of Ki‐67. P < 0.05, **P < 0.01, by t test. Scale bar: 100 μm.

LSP1 suppressed HCC cell cycle progression in vitro

We have confirmed that LSP1 could inhibit HCC cell proliferation. To further confirm the results, flow cytometric analysis of cell cycle distribution was performed to detect the cell cycle of HepG2 and Hep3B cell lines. The results revealed that overexpressed LSP1 led to cell cycle arrest at G0/G1 phase and reduced the percentages of cells at S phase in both cell lines compared with the corresponding percentages in negative control cells (Fig. 5). Therefore, LSP1 could suppress HCC cell cycle progression in vitro.

Figure 5

LSP1 suppressed HCC cell cycle progression. Effects of overexpressed LSP1 on the cell cycle progression of Hep3B cells (A) and HepG2 cells (B) measured by flow cytometry analysis. The proportion of S‐phase cells was significantly reduced in pcDNA/LSP1 transfected cell lines compared with the control group. Overexpressed LSP1 induced G0/G1 arrest of HCC cells. FL2‐A: the area of second fluorescence channel.

LSP1 inhibited the ERK1/2 pathway

As we have mentioned before, LSP1, as a tumor suppressor, could inhibit the ERK pathway to exert its role 11. Also, deletion of LSP1 expression results in increased p‐ERK2 in rat HCC 11. So we needed to know whether LSP1 could regulate ERK1/2 activation in human HCC. It has been confirmed that the gene for cyclin D1, a cell cycle regulator, could promote cancer cell proliferation and could be induced by the mitogen‐activated protein kinase (MAPK) /ERK pathway 12, 13, 14, 15. And Bcl2, an important anti‐apoptotic protein, could be regulated by the MAPK/ERK pathway 16. Western blot was performed to analyze the expression level of ERK1/2, p‐ERK1/2, Bcl2 and cyclin D1. We found that overexpressed LSP1 could decrease the expressions of p‐ERK1/2, Bcl2 and cyclin D1 in both Hep3B and HepG2 cell lines (Fig. 6). The total expression of ERK1/2 showed little change. However, p‐ERK1/2 level was downregulated by Flag‐LSP1, which meant the ERK1/2 pathway was inhibited (Fig. 6B,C). Also, the change of cyclin D1 expression indicated that the cell cycle in cells with pcDNA/LSP1 was restrained compared with the control cells (Fig. 6D). The result that protein expression of Bcl2 was also downregulated (Fig. 6E) revealed LSP1 could accelerate HCC cell apoptosis. These data consistently indicated that LSP1 could inhibit cell proliferation and promote cell apoptosis, through inhibiting the ERK1/2 pathway in HCC cells.

Figure 6

Overexpressed of LSP1 inhibited the ERK1/2 pathway. (A) HepG2 and Hep3B cells were transfected with pcDNA/LSP1, and subjected to western blot for LSP1, ERK1/2, p‐ERK1/2, Bcl2 and cyclin D1. (B–E) LSP1 overexpression decreased p‐ERK1/2 (B,C), cyclin D1 (D) and Bcl2 (E) protein levels in HepG2 and Hep3B cells. Data are representative of multiple repeats with similar results. *P < 0.05, **P < 0.01.

Discussion

Currently, HCC patients still have a poor prognosis and early diagnosis plays a pivotal role in treatment and clinical outcomes 17. Elucidating the underlying molecular mechanisms of HCC progression is critical for identifying novel therapeutic targets for HCC. Recent years, more and more cell elements, such as microRNAs (miRNAs) 18, 19, 20, long non‐coding RNAs (lncRNAs) 21, 22 and proteins 20, 23, have been identified as potential therapeutic targets for HCC. For example, miR‐185 suppresses the epithelial‐mesenchymal transition progression via the up‐regulation of E‐cadherin and down‐regulation of vimentin in epithelial and mesenchymal HCC cells 24. Also, the lncRNA HOTAIR, overexpressed in HCC and associated with tumor size, could activate autophagy by increasing ATG3 and ATG7 expression, promoting HCC cell proliferation 25. Protein NUPR1 has been identified as a potential therapeutic target in hepatocarcinogenesis, which may contribute to the progression of HCC 26. LSP1, which is mainly expressed in lymphocytes 27, neutrophils 28, macrophages 29 and endothelium 30, has been reported to be correlated with several kinds of cancers, such as breast cancer 31, lymphomas 8, pancreatic cancer 9 and dermatofibroma 6. Polymorphisms of LSP1 are associated with high risk of breast cancer in women who have an existing mutation of the BRCA1 gene 32. More importantly, in rat HCC, the LSP1 gene was reported to be a tumor suppressor, existing at high CNV, which can regulate hepatocellular proliferation and migration in rat via inhibiting the MAPK/ERK pathway 10, 11. However, there are no previous reports about LSP1 expression and function in human HCC.

In order to explore the expression of LSP1 in human HCC and understand its role in human HCC, we initially investigated the expression of LSP1 protein in 90 HCC patients using immunohistochemistry and western blot. Our data showed that the expression of LSP1 was significantly lower in HCC compared with matched normal, tumor‐adjacent tissues. Furthermore, LSP1 expression was significantly correlated with tumor size, Edmondson–Steiner grading and TNM tumor stage. Importantly, our data indicated that higher LSP1 expression is significantly correlated with a better 5‐year patient survival for all HCC patients. Multivariate Cox regression analysis found that LSP1 was an independent factor in predicting both overall 5‐year survival and disease‐free survival in HCC patients. These results suggested that LSP1 may be a potential prognosis marker in HCC patients. Furthermore, we explored the functions of LSP1 in HCC cell biology both in vivo and in vitro and found that LSP1 could suppress HCC growth by regulating cell proliferation, apoptosis and cell cycle progression.

It has been reported that LSP1 can target the scaffold kinase suppressor of Ras (KSR) to F‐actin filaments and regulate cell growth and migration 33. KSR was described as a major regulator of cell growth, because it acts as a scaffold for Raf and mitogen activated protein kinase (MAPK) 34. Previous studies have found the MAPK/ERK pathway was excessively activated in many cancer such as bladder cancer 35, lung cancer 36 and HCC 37. The MAPK/ERK pathway is a positive regulator of biological features of carcinoma, including proliferation 38, apoptosis 39 and differentiation 40. Many compounds can inhibit steps in the MAPK/ERK pathway, and therefore are potential drugs for treating cancer. The first drug licensed to act on this pathway is sorafenib, a Raf kinase inhibitor 41. In this study, we found LSP1 could inhibit ERK1/2 phosphorylation, and thus inhibit its downstream proteins, such as Bcl2 and cyclin D1. Thus, we demonstrated that LSP1 could inhibit the ERK1/2 pathway to suppress cell proliferation and the cell cycle, and promote cell apoptosis.

In summary, this study indicated that LSP1 is a tumor suppressor in human HCC. LSP1 could suppress cell proliferation and the cell cycle and promote cell apoptosis through inhibiting the ERK1/2 pathway. Thus, LSP1 may potentially act as a clinical biomarker and may also be a therapeutic target in HCC.

Author contributions

HZ, YW, ZL, BY, CD, MX, YJ and QL carried out the cell biology and molecular biology experiments and drafted the manuscript. QL, KT, SW, HZ, YW, ZL, BY, CD, MX, YJ and QL participated in the design of the study and performed the statistical analysis. All authors read and approved the final manuscript.