High Expression of Asparagine Synthetase Is Associated with Poor Prognosis of Breast Cancer in Chinese Population

Introduction

Breast cancer (BC) is one of the most frequent cancers among women of more than 35 years of age in both developing and developed countries.^1,2 The occurrence of BC is associated with heredity, and this disease often attacks women before and after menopause. BC usually derives from glandular epithelium of the breast and severely threatens physical and mental health of the cases. BC is highly heterogeneous at molecular and clinical levels.³ The characteristics of BC mainly include breast lump, dysgalactia, thoracalgia, and distant metastasis. It has been estimated that about 235,000 women were diagnosed with BC in 2014, and the incidence rate of BC has been increased in recent years.^4,5 Treatments for BC mainly depend on surgical resection and radiotherapy. However, about 5%–45% of cases would face recurrence, producing a great challenge.⁶ What is more, although overall survival of BC has been greatly improved in the past decade with the advancements in adjuvant therapy and early detection, this malignancy is still one of the leading causes for cancer-related deaths.^7,8 The 5-year survival rate of BC patients in advanced stage is only 20%.⁹ Therefore, it is needed to find novel and efficient ways to better prognosis prediction and treatment of BC.

Asparagine is a nonessential amino acid, and widely required for cellular growth and functions.^10,11 Cells receive asparagine physiologically through two major ways: circulating ASNS or plasmatic asparagine.¹²ASNS consists of two domains: the N-terminal catalyzing the hydrolysis of glutamine, and the C-terminal catalyzing the carboxylate of aspartate. Asparagine synthetase (ASNS) gene encodes ASNS protein, which belongs to the aminotransferase family and catalyzes the 1-glutamine and 1-aspartate into 1-glutamate and 1-asparagine in an ATP-dependent manner.^13–15 Previous studies have claimed that ASNS played important roles in cancer cell growth. According to Li et al., the knockdown of ASNS could inhibit the growth of melanoma and epidermoid carcinoma cells, indicating it was a candidate target in melanoma treatment.¹⁶ Yang et al. proposed that ASNS might contribute to the tumorigenesis of BC, and that decreased expression of ASNS inhibited the proliferation of BC cells and induced cell cycle arrest.¹⁷

In the present study, the authors measured the expression of ASNS in BC using quantitative real-time PCR and estimated the role of ASNS in the proliferation of BC cells with MTT assay. Furthermore, the prognostic value of ASNS in BC patients was also evaluated through Kaplan–Meier method and Cox regression analysis.

Materials and Methods

Patients and specimens

In this study, 135 BC patients who underwent surgical resection in Weihai Municipal Hospital were included. None of them had received any radio/chemotherapy before surgery. Clinical parameters, including age, HER2 status, endocrine therapy, family history, histological grade, vascular invasion, and PR status were collected from electronic record system. Patients who were lost in follow-up were excluded from this study. BC tissue samples were obtained from all recruited participants. In addition, 56 adjacent normal tissue samples were also resected from the patients as controls. This study was supported by the Ethics Committee of Weihai Municipal Hospital. Besides, all participants signed the written informed consents.

Cell lines and cell culture

BC cell line MCF7 was provided by Prof. Moon Hyeong-Gon (Seoul National University, Seoul, Republic of Korea). All cells were cultured in DMEM, which was supplemented with 1 × antibiotic/antimycotic and 10% fetal bovine serum (Gibco) in a humidified atmosphere with 5% CO₂ at 37°C.

siRNA and transient transfection

Human ASNS siRNA (si-ASNS) and negative control siRNA were obtained from Dharmacon (Chicago, IL). Transient transfection was conducted using Lipofectamine RNAiMax Reagent (Invitrogen) based on corresponding protocols. Then cells were cultured under normal conditions.

MTT assay

MTT assay was performed to detect the proliferation ability of transfected MCF7 cells. First, the transfected cells were incubated in 96-well plates at a density of 2000 cells/well for 0, 12, 24, 36, 48, and 60 h, respectively. Then the cells were coincubated with MTT for 4 h. Finally, 200 μL DMSO was added to each well to terminate reactions. The proliferation ability of MCF7 cells was measured with spectrophotometer at a wavelength of 490 nm.

RNA extraction and quantitative real-time PCR

Total RNA was isolated from all BC tissue specimens and normal controls using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Then the isolated RNA was used to synthesize cDNA with the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Finally, real-time PCR was conducted with the Light-Cycler 480 Real-Time PCR System (Roche Diagnostic GmbH, Manheim, Germany) based on the manufacturer's instructions. GAPDH was used as internal reference. The relative expression of ASNS mRNA was analyzed through 2^−ΔΔCT method. Every sample was measured at least three times.

Statistical analyses

All statistical analyses were carried out in software SPSS 18.0 and Sigmaplot 12.5. All data were presented as mean ± SD. Student's t-test was used to determine difference in ASNS expression between BC tissues and normal controls. Chi-square test was adopted to analyze the relationship between ASNS expression and clinical features of the patients. Overall survival curves were estimated by Kaplan–Meier method. Finally, the prognostic performance of ASNS in BC was evaluated by Cox regression analysis. Two-side p value was used in this study, and results were considered to be statistically significant when p was less than 0.05.

Results

Upregulated expression of ASNS mRNA in BC tissues

The relative expression of ASNS mRNA in BC tissues and normal controls was determined by qRT-PCR. The results showed that the relative expression of ASNS was significantly higher in BC tissues than in normal controls (Fig. 1, p < 0.05).

Relationship between ASNS expression and clinical features

Chi-square test was conducted to analyze the association between ASNS expression and clinical features of BC patients. According to the median value of ASNS expression, the authors divided cancer tissues into high and low expression groups. As shown in Table 1, ASNS expression was affected by histological grade (p = 0.017), vascular invasion (p = 0.009), and PR status (p = 0.014). However, there was no significant association for ASNS expression with age (p = 0.361), HER2 status (p = 0.222), endocrine therapy (p = 0.112), or family history (p = 0.191).

ASNS knockdown inhibited the proliferation of MCF7 cells

To explore the role of ASNS in the development of MCF7 cells, MTT assay was conducted. All MCF7 cells were transfected by si-ASNS and negative control siRNA, and decreased expression of ASNS was observed in si-ASNS group. As shown in Figure 2, ASNS downregulation significantly suppressed the proliferation ability of MCF7 cells (p < 0.05).

The prognostic value of ASNS for BC patients

The Kaplan–Meier method and Cox regression analyses were carried out to determine the prognostic role of ASNS in BC patients. All patients were followed up every 3 months in the first 2 years and then every 6 months in the last 3 years. During the follow-up, 79 patients died, including 66 (70.2%) with high ASNS expressions and 13 (31.7%) with low expressions. According to Kaplan–Meier curves, patients with high ASNS expressions had shorter survival time than those with low expressions (Fig. 3, p < 0.05). Besides, as listed in Table 2, histological grade (p = 0.003, HR = 1.954, 95% CI = 1.254–3.046), HER2 status (p = 0.035, HR = 1.623, 95% CI = 1.035–2.546), PR status (p = 0.001, HR = 2.194, 95% CI = 1.380–3.487), vascular invasion (p = 0.019, HR = 1.714, 95% CI = 1.093–2.689), and ASNS expression (p < 0.001, HR = 3.919, 95% CI = 2.151–7.142) were related to the prognosis of BC patients. What is more, multivariate analysis suggested that histological grade (p = 0.019, HR = 1.709, 95% CI = 1.092–2.674), PR status (p = 0.007, HR = 1.897, 95% CI = 1.188–3.028), and ASNS expression (p < 0.001, HR = 3.293, 95% CI = 1.790–6.058) were prognostic factors for BC patients.

Discussion

BC is one of the most common invasive malignancies in the world.¹⁸ A variety of biomarkers have been investigated for their roles in the development and progression of BC. Chen et al. revealed that miR-22 targeted glucose transporter protein type 1 and was a promising marker for the prognosis of BC patients.¹⁹ Sun et al. demonstrated that miR-200c enhanced radiosensitivity and suppressed the autophagy of BC cells through regulating UBQLN1.²⁰ In the study of Wu et al., serum levels of CEA and CA15–3 before surgery could act as prognostic biomarkers in BC.²¹This study was the first one to explore the relationship of ASNS expression with the prognosis of BC patients.

It is reported that the silencing of ASNS in human sarcoma cell lines reduced the percentage of cells at S phase and inhibited the synthesis of new polypeptide.²² Moreover, there is evidence supporting that ASNS is overexpressed in various cancers and related with the development and progression of the cancers, such as in prostate cancer, brain tumors, and lymphoblastic leukemia.^23–25 In the present study, they also assessed the influence of ASNS expression on the growth of BC cells. MTT assay showed that cells, after knocking down ASNS, proliferated slower than those expressing ASNS. The results of this study were consistent with those in previous reports.

In addition, in this study, they first examined the expression of ASNS mRNA in BC tissues and healthy controls, and ASNS mRNA was positively expressed in BC tissues, which was in accordance with findings in previous reports. The following Chi-square test demonstrated that high ASNS expression was related to PR status, histological grade, and vascular invasion. Based on the above results, they speculated that ASNS expression might be associated with the occurrence and development of BC. Finally, survival curves suggested that patients with high ASNS expressions had poor prognosis compared with those with low expressions. Furthermore, Cox regression analysis highlighted that ASNS could be a prognostic biomarker for BC patients. The findings of this study were in accordance with those by Zhang et al., who demonstrated that the expression of ASNS was higher in HCC tissues and that patients with lower ASNS expression levels had poor prognosis.²⁶ Therefore, ASNS may be a promising therapeutic target for malignant tumors.

Conclusion

In summary, the overexpression of ASNS mRNA was observed in BC tissues compared with adjacent normal ones. Chi-square test unveiled that high ASNS expression was correlated with certain clinical factors. In addition, the knockdown of ASNS by siRNA could significantly inhibit the proliferation of BC cells. Both survival curves and Cox regression analyses proved that ASNS was related to the prognosis of BC patients and its upregulation predicted poor prognosis. However, molecular mechanism through which ASNS worked in BC has not yet been precisely illustrated, which needs more and further research in the future.

Author Contributions

Conceived and designed the experiments: C.Q.; Performed the experiments: X.Y.; Analyzed the data: Z.Z.; Contributed reagents/materials/analysis tools: C.Q.; Wrote the article: X.Y.

Author Confirmation Statement

All coauthors have reviewed and approved the article before submission.

Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.