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Abstract

Our study mainly investigated ubiquitin-specific protease 4 (USP4) expression in pathogenesis of esophageal cancer. The data showed significantly increased expression of USP4 in cancer tissues compared to that in para-carcinoma tissues (68.38% $\pm$ 25.60% vs 13.04% $\pm$ 9.95%, $P=$ 0.000) and positive correlation between USP4 and pathology grade ( $r=$ 0.249, $P=$ 0.014), although survival analysis revealed that USP4 expression was positively associated with the prognosis (32.4% vs 10.9%, $P=$ 0.043). Grouped analysis revealed that the prognosis of patients with high USP4 expression were significantly better only in the small tumor subgroup (diameter $\leqslant$ 5 cm) (52.6% VS 8.6%, $P=$ 0.001) and the early stage subgroup (stages 1 and 2) (60.0% VS 16.7%, $P=$ 0.006). Moreover, in the subgroup of clinical stages 1 and 2 with tumor diameter $\leqslant$ 5 cm, high USP4 expression prolonged the survival time of esophageal cancer patients more significantly (75.5% VS 5.9%, $P=$ 0.000). Based on these results, we speculated that it was possible to significantly improve the prognosis of patients with low USP4 expression by targeted therapy in early esophageal cancer. Taken together, our study uncovered a previously unknown function of USP4 in esophageal cancer and more investigations would be carried out to further study its regulation gene network and molecular biological mechanism in esophageal cancer.

Keywords

Tissue microarray immunohistochemistry USP4 esophageal cancer prognosis

1. Introduction

Esophageal cancer is one of the four most common diagnosed cancers in China [1]. Despite the decreased incidence rate for esophageal cancer, China still is one of the areas with highest incidence rate of esophageal cancer [2]. There were approximately 500,000 newly diagnosed esophageal cancer cases in 2015 [1]. Better understanding of molecular aberrations associated with esophageal cancer might identify new diagnostic and therapeutic strategies.

Post-translational modifications by ubiquitin are emerging as major regulatory events in varies biological pathway. These include cell cycle progression, signal transduction, transcriptional regulation and apoptosis. The reverse process mediated by deubiquitinating enzymes (DUBs) has gained attention as an important regulatory mechanism in controlling protein turnover. To our knowledge, approximately 100 DUBs have been identified in human chromosomes, few of which have been functional characterized. The ubiquitin-specific proteases (USPs) occupy the largest subclass of DUBs. USP4 belonging to USPs is mapped on chromosome 3 (3p21.3), a region related to several human cancers including breast cancer, kidney cancer and lung cancer [3]. USP4 regulates a wide variety of normal and abnormal physiological functions, including innate immune activation, homologous recombination, pathological cardiac hypertrophy, neuronal apoptosis, exon skipping, osteoblast differentiation and oncogenesis [4, 5, 6, 7, 8, 9, 10]. USP4 shows ambivalent functions in different cancers with tissue- and tumor-specificity [10, 11, 12, 13, 14]. Currently, its role in esophageal cancer remains unclear.

In this present study, immunohistochemistry and tissue microarray were used to investigate the potential role of USP4 in tumor development and prognosis in esophageal cancer.

2. Materials and methods

2.1 Clinical materials

Esophageal cancer tissues were obtained from patients who accepted radical surgery for esophageal carcinoma in Huaian First People’s Hospital, Nanjing Medical University. Formalin-fixed paraffin embedded samples were used to construct tissue microarray (TMA) by Shanghai Outdo Biotech Co. Ltd. which including 100 esophageal cancer tissues and 80 adjacent para-carcinoma tissues. The tissue microarray was constructed by the methods used as previous reported.

The clinical information of esophageal cancer patients were showed in Table 3: 74 cases were male and 26 were female; the age distribution was from 48 to 82 years old; tumor size range was from 1.2 cm to 10 cm. The clinical grading distribution: 4 cases were stage 1, 42 cases were stage 2, 50 case was stage 3, 0 cases were stage 4, there were 4 cases missed clinical staging information.

The follow-up information of esophageal cancer patients were shown as bellows: the operation time of these patients was from January 2006 to October 2008 and the eventual follow-up time was September 2014, which followed 0–97 months. Among this follow-up time, 81 out of 100 patients were died of esophageal cancer with a median overall survival time of 14 months (0–60 months), 18 patients were still alive with a median overall survival time of 80 months (71–97 months), there was 1 patient lost to follow-up in September 2013 and was included in the survival group when statistical analysis. All patients were clinicopathologically diagnosed as esophageal squamous cell carcinoma and received no extra treatment before surgery. The patients’ detailed information can be found in Table 3.

2.2 Immunohistochemistry

To retrieval antigen and remove the endogenous peroxidase activity, sections were treated with citrate buffer and freshly prepared 0.3% hydrogen peroxide in methanol. Tissue sections were blocked with goat serum and subsequently incubated with primary antibody which anti-USP4 (1:1000, ab38510, Abcam) at 4 ${{}^{\circ}}$ C overnight, then incubated with secondary antibody (HRP-labeled anti-mouse antibody, DAKO), washed with PBS, visualized using diaminobenzidine (DAB) system and hematoxylin re-dying.

Three visual fields from different areas of each specimen were chosen at random with more than 100 cells in each area were calculated for immunohistochemistry evaluation. USP4 expression was scored according to the percentage of positive cells: $\leqslant$ 85% was regarded as low expression group and $>$ 85% as high expression group.

2.3 Statistical analysis

Paired T-test was used to analysis the difference between USP4 expression in esophageal cancer tissues and that in the adjacent tissues. The relationship between USP4 expression and clinical factors was calculated by Spearman’s correlation analysis. Kaplan-Meier method and log-rank test were used to analysis survival curves. Cox proportional-hazard regression model was developed to evaluate the association between the potential prognostic markers and overall survival at multivariate level. All statistical analyses were conducted using SPSS 17.0 software. $P<$ 0.05 was considered significantly.

Table 1
Difference of USP4 expression in esophageal cancer tissues and para-carcinoma tissues

Histology	No. patient	Expression score	P-value
Esophageal cancer	74	68.38% $\pm$ 25.60%	0.000
tissue
Adjacent normal	74	13.04% $\pm$ 9.95%
tissue

Mean $\pm$ Std. Deviation.

Figure 1.

Immunohistochemistry of USP4: USP4 was specifically expressed in cytoplasm of both esophageal cancer tissues and para-carcinoma tissues. The positive staining rate of USP4 in esophageal cancer tissues (A) was significantly higher than that in para-carcinoma tissues (B) (Magnification times: $\times$ 200).

3. Results

3.1 The expression difference of USP4 in esophageal cancer tissues and para-carcinoma tissues

The results of immunohistochemistry showed that USP4 protein was located in the cytoplasm of both esophageal cancer tissues and para-carcinoma tissues. USP4 expression was scored according to the percentage of positive cells and $>$ 85% was regarded as high expression group. High USP4 expression was noted in 34.0% cancer tissues and 0.0% para-carcinoma tissue. Paired T-test analysis revealed that ratio of USP4 positive staining was significantly higher in esophageal cancer tissues than in para-carcinoma tissues (68.38% $\pm$ 25.60% vs 13.04% $\pm$ 9.95%, $P=$ 0.000) (see Table 1, Fig. 1). And there is no correlation between USP4 expression in esophageal cancer tissues and para-carcinoma tissues ( $P=$ 0.216) (see Table 2).

Table 2
Correlation between USP4 expression in esophageal cancer tissues and para-carcinoma tissues

		USP4 expression
		in para-carcinoma
USP4 expression in	Correlation Coefficient	0.145
cancer tissues	P-value	0.216
	N	74

Table 3

Correlation between clinical factors and USP4 expression in esophageal cancer tissues

Clinical factors	USP4 expression in esophageal cancer		P value
	Low	High
Gender			0.346
Male	49	23
Female	15	11
Age			0.912
$\leqslant$ 60	20	11
$>$ 60	44	23
Tumor size			0.707
$\leqslant$ 5 cm	35	19
$>$ 5 cm	19	10
Lost	10	5
Grade			0.014
I	4	1
II	47	18
III	13	15
T stage			0.084
T1	1	3
T2	6	4
T3	52	26
T4	3	0
Lost	2	1
N stage			0.436
N0	31	13
N1	19	12
N2	10	7
N3	3	1
Lost	1	1
cTNM stage			0.891
1 $+$ 2	30	15
3	32	17
Lost	2	2

Table 4

Cox regression analysis independent factor of esophageal cancer

	Overall survival
	HR (95% CI)	$P$
USP4 expression	0.874 (0.489–1.562)	0.649
Gender	0.570 (0.272–1.193)	0.136
Tumor size	1.480 (0.861–2.544)	0.156
T	1.856 (0.982–3.508)	0.057
N	1.145 (0.734–1.786)	0.550
cTNM	1.254 (0.823–1.911)	0.293

3.2 The relationship between USP4 expression and clinical factors in esophageal cancer

Patients were stratified into high and low USP4 expression groups according to the percentage of positive cells. Spearman’s correlation analysis showed that USP4 expression was not associated with any clinical factors significantly except pathological grade ( $r=$ 0.249, $P=$ 0.014). The detailed information was shown in Table 3.

3.3 The correlation between USP4 expression and prognosis of esophageal cancer

Kaplan-Meier analysis and log-rank test were applied to determine the correlation between USP4 expression and prognosis of all esophageal cancer patients or grouped patients.

The results showed that USP4 expression in cancer tissues was significantly positively correlated with the overall survival rate of all esophageal cancer patients (32.4% vs 10.9%, $P=$ 0.043). Detailed analysis was shown in Fig. 2. At the same time, gender ( $P=$ 0.024), tumor size ( $P=$ 0.042), T staging ( $P=$ 0.026), N staging ( $P=$ 0.001) and clinical staging ( $P=$ 0.000) were all significantly correlated with the prognosis. But Cox Multivariate analysis revealed that none of these indexes was independent prognostic factor ( $P>$ 0.050). Detailed analysis was shown in Table 4.

Figure 2.

Kaplan-Meier survival curves dependent on USP4 expression were calculated with log-rank test.

Subsequently, we grouped esophageal cancer patients according to the different clinical factors and further analyzed the relationship between USP4 and the prognosis in each group. Results revealed that: the survival rates of patients with high USP4 expression were significantly better than those with low USP4 expression only in the small tumor subgroup (diameter $\leqslant$ 5 cm) (52.6% VS 8.6%, $P=$ 0.001) (see Fig. 3A) and the early stage subgroup (stages 1 and 2) (60.0% VS 16.7%, $P=$ 0.006) (see Fig. 3C). However, among the big tumor subgroup (diameter $>$ 5 cm) and the late stage esophageal cancer patients (stage 3 and no stage 4), USP4 expression did not significantly affect the prognosis ( $P=$ 0.073, $P=$ 0.298) (see Fig. 3B and D). Moreover, in the subgroup of clinical stage 1 and 2 with tumor diameter $\leqslant$ 5 cm, high USP4 expression prolonged the prognosis more significantly (75.5% VS 5.9%, $P=$ 0.000) (see Fig. 4).

Figure 3.

Kaplan-Meier survival curves dependent on USP4 expression in the subgroup of (A) tumor size $\leqslant$ 5 cm, (B) tumor size $>$ 5 cm, (C) cTMA stage 1 $+$ 2, (D) cTMA stage 3 were calculated with log-rank test.

4. Discussion

USP is a large family that contains a lot of proteins with different functions. As a member of USP, USP4 has been reported correlating with cancers in a few reports, but its function in different cancers is conflicting. To our knowledge, there was no report about USP4 expression in esophageal cancer. Here, our studies focused on the correlation between USP4 expression and occurrence, development as well as the prognosis of esophageal cancer.

The results indicated that USP4 protein was localized in the cytoplasm and the positive staining rate was significantly higher in esophageal cancer tissues than that in para-carcinoma tissues (68.38% $\pm$ 25.60% VS 13.04% $\pm$ 9.95%, $P=$ 0.000). However, subsequent survival correlation analysis revealed that the expression of USP4 in cancer tissues was significantly positively related to the prognosis of patients with esophageal cancer (32.4% VS 10.9%, $P=$ 0.043). Similar conclusions could be found in the study of Xiaozhi Hou et al. They found that USP4 could activate NF- $\kappa$ B and promote TNF- $\alpha$ induced apoptosis of cancer cells by deubiquitination of RIP1, thus inhibiting the progression of head and neck squamous cell carcinoma (SCCHH), although significantly increased USP4 expression was observed in cancer tissues compared to adjacent tissues [11]. Then they speculated that USP4 would be upregulated by immune cells in the course of host’s immune response to cancer, thus promoted the apoptosis of cancer cells. Above all, we hypothesized that the molecular mechanism and biological function of USP4 in esophageal cancer may be similar with that in SCCHH. At the same time, esophageal cancer cells with higher histological grade were more malignant, thus may stimulate more intense immune response and higher USP4 expression.

In order to further understand the biological function of USP4, we divided esophageal cancer patients into different subgroups according to the clinical indicators and analyzed the correlation between USP4 expression and the prognosis in each subgroup, respectively. Analysis results showed that USP4 expression in cancer tissues was significantly positively correlated with the prognosis in the small tumor subgroup (diameter $\leqslant$ 5 cm) (52.6% VS 8.6%, $P=$ 0.001) and the early stage subgroup (stages 1 and 2) (60.0% VS 16.7%, $P=$ 0.006), but did not significantly affect the prognosis of other subgroups. In addition, we found that in the subgroup of clinical stages 1 and 2 with tumor diameter $\leqslant$ 5 cm, patients with high USP4 expression had more higher survival rate up to 75.5%, while the survival rate of patients with low USP4 was only 5.9%, closing to that of the advanced stage patients. Based on these results, we suggested that in early stage of esophageal cancer patients with smaller tumor size, USP4 might play significant cancer inhibition role, and it’s absent in cancer tissues could be a dangerous signal that associated with quite poor prognosis. According to the previous research, the mortality of esophageal cancer was extremely high, only less than 50% survival rate of patients in stages 1 and 2 consisted with the data in our research (32.6%) [15]. Therefore, it was possible to significantly improve the survival time by targeted therapy in early esophageal cancer patients with low USP4 expression. That was to say: USP4 may be an important marker for esophageal cancer targeted therapy.

Figure 4.

Kaplan-Meier survival curves dependent on USP4 expression in the subgroup of cTMA stage 1 $+$ 2 with tumor size $\leqslant$ 5 cm were calculated with log-rank test.

As the previous literatures showed, USP4 overexpressed in many types of cancers and its increased levels appeared to promote the development of colorectal cancer and lung cancer [10, 13]. On the other hand, some reports predicted that USP4 also acted as a tumor suppressor. A previous study demonstrated that USP4 could negatively regulate TNF-induced cell migration [16], which is important to cancer metastasis. Moreover, USP4 could also decrease Wnt signal pathway, which could promote tumorigenic process [17]. Based on these results, we assumed that USP4 might suppress the tumorigenic process in esophageal cancer by negatively regulating TNF-induced cell migration or Wnt signal pathway. The mechanism of USP4 suppressing the esophageal cancer needs to be further investigated.

Obviously, the function of USP4 was complex and various indicting its tissue- and tumor-specificity, and was different even in the different subgroup of the same type of cancer. Next step, more samples would be required to further investigate the relationship between USP4 expression and the different esophageal cancer subtypes. At the same time, different tumor cell lines with USP4 knockdown or overexpression would be constructed to investigate regulation gene network and molecular biological mechanism of USP4 in esophageal cancer in the future.

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Ubiquitin-specific protease 4 improves the prognosis of the patients in esophageal cancer