The Expression And Clinical Significance Of BCAT1 And CD147 In Esophageal Squamous Cell Carcinoma

Background:Esophageal cancer,derived from esophageal epithelium,is one of the most common upper digestive tract malignancies.Esophageal cancer includes two main histopathological subtypes--esophageal squamous-cell carcinoma(ESCC) and esophageal adenocarcinoma(EAC),and squamous-cell carcinoma accounts for about 90%.According to the report of International Agency for Research on Cancer(IARC),the incidence and mortality of the esophageal cancer rank eighth and sixth respectively in all the malignant tumors,with an estimated 456,000 new cases and an estimated 400,000 deaths in 2012 worldwide.China is one of the most high-risk regions,especially in Henan,Hebei,Jiangsu provinces.In China,,the incidence and mortality of the esophageal cancer rank fifth and fourth respectively, with an estimated 223,000 new cases and an estimated 197,000 deaths in 2012,approximately half of the total new cases and deaths of the esophageal cancer globally.However,the etiology of esophageal carcinoma is not yet fully illuminated,According to the reports of epidemiological investigation,Alcohol, tobacco,obesity,gastroesophageal reflux disease(GERD) and genetic predisposition are regarded as the main risk factors of esophageal carcinoma.the clinical symptoms of esophageal cancer are mild in the early stages, which are easy to be ignored by the patients,so it is unlikely to make a definite diagnosis and receive timely treatment at the disease’s early stages. Most patients are found advanced when come to see the doctor.While the treatment for esophageal cancer has made impressive progress,the survival of patients with advanced esophageal cancer is still poor,the five-year survival rate of patients diagnosed with esophageal squamous cell carcinoma is only about 15%.Nowadays,There are not molecular markers with high specificity and good utility available for ESCC,and the targeted therapy for ESCC is also very rare.therefor,we attempt to study the relationship between certain molecular markers and ESCC in this paper,which may be helpful to further understand the biological property of ESCC and provide more clues for the prevention and treatment of ESCC.BCAT1 (also known as ECA39)—branched-chain amino acid transaminase 1,located in human chromosome 12p12.1, encodes the cytosolic BCAT1.This enzyme catalyzes the reversible transamination reaction of the branched-chain amino acids(BCAAs) leucine,isoleucine and valine,resulting in transfer of the BCAA α-amino group toα-ketoglutarate to generate glutamate and the corresponding BCAA α-ketoacid(BCKA). Normally,BCAT1 is mainly expressed in the brain,spinal cord and peripheral neuron,in which BCAT1 may participate in the synthesis of neurotransmitter glutamate.BCAT1 is highly conserved during the evolution.Early studies have pointed out that BCAT1 is involved in cell cycle regulation in yeast and inducing mammalian cells apoptosis.Since then,a majority of research work have confirmed that the dysregulation of BCAT1 expression is associated with many human diseases.More importantly,BCAT1 is reported to be involved in a variety of human malignancies.BCAT1 was proven to be a direct target for MYC and originally isolated from Burkitt’s lymphoma in which MYC was activated by translocation.After that,a series of studies have shown that the transcription and/or translation of BCAT1 are up-regulated in certain malignancies,including breast carcinoma,non-seminoma, nasopharyngeal carcinoma,glioblastoma carrying wild-type IDH1, epithelial ovarian cancer and urothelial carcinoma.Combined with the in vivo and in vitro experiments,these studies have suggested that BCAT1 can promote cancer cell proliferation and metastasis and may be a molecular maker of poor prognosis.The specific mechanism underlying promoting progression of cancer of BCAT1 has not yet been fully elucidated.available evidences suggest it may be involved in boosting the metabolism,or synergy with other proteins to promote invasion and metastasis of cancer cells.Some researchers speculate that BCAT1 high expression allows cancer cells to catabolize BCAAs as an alternative source of carbon and nitrogen to support both ATP production and macromolecular synthesis,which are needed for rapid proliferation of cancer cells.This hypothesis is supported by a lot of researches directly or indirectly,For example, using positron emission tomography with 11C-leucine for in vivo tumor imaging has showed the high avidity of tumor amino acid uptake.in addition, as we know,branched-chain amino acids play a key role in the maintenance of lean body mass and regulation of skeletal muscle protein metabolism.According to the clinical observation, many patients with advanced cancer will suffer from involuntary weight loss or even cachexia-anorexia syndrome, which may be contributed to alterations in amino acid metabolism in cancer,including reduced dietary intake,increased oxidation of branched-chain amino acids.Although BCAT1 has been reported to have an impact on promoting cancer progression in many common human malignancies so far,however,Methylated BCAT1 has been detected in most colorectal cancer and early-stage non-small cell lung cancer tissues by some researchers.As we know,gene methylation is usually associated with expression suppression,Thus,it seems to imply that BCAT1 is not expressed or at a low level in most of colorectal cancer and early-stage non-small cell lung cancer cases.these studies make the relationship between BCAT1 and human malignancies become complicated and confusing.therefore,it is necessary to carry out more extensive researches to reveal its role in human malignancies.Whole genome sequencing analysis of esophageal squamous cell carcinoma tissues has revealed that proto-oncogene MYC is amplified in most cases,Moreover,a majority of researches have showed that MYC may facilitate the progress of esophageal squamous cell carcinoma,and overexpression of MYC is often associated with poor prognosis.Based on the above mentioned reports,It is reasonable to speculate whether BCAT1,as a downstream target gene of MYC,may also be involved in the progression of esophageal squamous cell carcinoma? While the expression and clinical significance of BCAT1 in esophageal squamous cell carcinoma has not yet been studied so far,so we attempt to figure it out in this paper.CD147,also termed as extracellular matrix metalloproteinase inducer (EMMPRIN),is a transmembrane glycoprotein,belonging to the immunoglobulin superfamily.CD147 plays a vital role in the promotion of tumor invasion and metastasis by up-regulating matrix metalloproteinases(MMPs) secreted from adjacent fibroblasts.Besides, CD 147 is an indispensable assembly factor for monocarboxylate transporters(MCTs),aerobic glycolysis(Warburg effect),a hallmark of cancer,will lead to high lactate production in the tumor cells.MCTs can transport excessive lactate out of the cells to facilitate tumor survival and growth.Meanwhile,it has also been reported that CD 147 can mediate the chemotherapy resistance through other mechanisms.It has been demonstrated that CD 147 is overexpressed in a variety of cancers,such as breast cancer,cervical cancer,endometrial cancer,lung cancer, hepatocellular carcinoma,ovarian cancer and so on. CD 147 promotes cancer cell proliferation,invasion and metastasis through its various functions.Overexpression of CD 147 usually indicates poor prognosis.whereas researches on the expression and clinical significance of CD 147 in esophageal squamous cell carcinoma remain comparatively rare,and results vary from each other.Metastasis is the most important characteristic of the malignant tumor.the poor prognosis of esophageal squamous carcinoma is mainly due to its metastasis and recurrence. As mentioned above,BCAT1 and CD 147 can both promote cancer cell proliferation,invasion and metastasis,in addition, there seems to be a correlation between BCAT1 and CD 147 within the intricate molecular network of breast cancer cell, in which MYC can regulate both BCAT1 and CD 147 expression,and Silencing of BCAT1 gene or inhibition of BCAT1 by specific inhibitor will both downregulate the expression of CD 147 and reduce cancer metastasis, we image if these two moloculars can co-express in other types of cancer,like esophageal squamous cell carcinoma.Objective:1.To study the expression and clinica1 significance of BCAT1 and CD 147 in esophageal squmous cell carcinoma;2.To preliminary explore the relationship between the expression of BCAT1 and CD 147 in esophageal squmous cell carcinoma;Material and Method:50 cases formalin-fixed paraffin-embedded specimen of the patients with primary esophageal squamous cell carcinoma who underwent radical surgery barely from January 2010 to December 2012 in the Department of thoracic surgery were collected.29 adjacent normal esophageal epithelium specimen were also collected as control group.The clinicopathologic data and follow-up information of these patients were obtained at the same time.The expression of BCAT1 and CD147 were detected in 50 cases of esophageal squamous cell carcinoma tissues and 29 cases of adjacent normal esophageal tissues by immunohistochemistry technique respectively.The correlations of BCAT1 and CD 147 expression with clinicopathologic features(including gender,age,degree of differentiation,tumor size,lymph node metastasis and clinical stage) and prognosis were analyzed,at last, the relationship between the expression of BCAT1 and CD147 in esophageal squmous cell carcinoma was also analyzed.Results:1. the expression of BCATI,CD 147 in esophageal squmous cell carcinoma and adjacent normal esophageal tissuesBCAT1 was mainly expressed in the cytoplasm of cancer cells presenting brown particles,while CD 147 was mainly expressed in cancer cells membrane,partially expressed in the cytoplasm of cancer cells presenting yellowish-brown particles.In 50 specimens,staining with the BCAT1 antibody was positive in 44 cases,the expression rate of BCAT1 was 88%(44 out of 50 cases),among which,10 cases (20%) showed low expression,27 cases (54%) showed intermediate expression,and 7 cases (14%) showed high expression.In a11,38 out of 50 cases (76%) express CD147,8 cases (16%) showed low expression,21 cases (42%) showed intermediate expression,9 cases (18%) showed high expression.BCATl expression tended to be higher in poorly differentiated ESCC,but in some well or moderately-differentiated ESCC,BCAT1 was mainly expressed in cancer nest peripheral cells. CD 147 was widely expressed in highly invasive ESCC and with the higher expression in invasive frontier cells.Both BCAT1 and CD 147 were mainly expressed as basal staining in normal esophageal squamous epithelia.2.The association between BCAT1,CD147 expression and the clinic-pathological characteristics of ESCC patientsStatistical results of the IHC showed that the expression of BCAT1 within cancerous lesions was correlated with the following variables:histologic grades of differentiation,tumor size(T) and pTNM stage.the average BCAT1 intensity was 3.85±1.69 in well or moderately-differentiated ESCC group,while the average BCAT1 intensity was 5.00±0.91in poorly differentiated ESCC group,Higher BCAT1 expression was significantly correlated with poorly differentiated ESCC(p<0.05). the average BCAT1 intensity was 2.57±1.90,4.10±1.42 and 4.54±1.61 in T1-T2/T3 and T4 group respectively.the expression of BCAT1 was higher in T3 and T4 group,and the difference was significant(p<0.05). the average BCAT1 intensity was 3.33±1.68 in early stage of ESCC patients whose postoperative pathological stage were I and Ⅱ,while the average BCAT1 intensity was 4.38±1.50 in advanced ESCC patients, Higher BCAT1 expression was significantly correlated with advanced pTNM stage(p<0.05).On the other hand,the expression of CD 147 within cancerous lesions was only correlated with tumor size(T),However,there was no association found between the expression of CD 147 and other clinic-pathological characteristics, the average CD 147 intensity was 1.86±2.55,3.84±1.83in T1-T2,T3-T4 group respectively, the expression of CD 147 was higher in T3-T4 group,and the difference was significant(p<0.05).3.prognostic implications of the expression of BCAT1 and CD 147 in esophageal squmous cell carcinomaAfter analyzing the survival information,we found that the average survival time of BCAT1-negative patients was 45.83 months,while the average survival time of BCAT1-positive patients was 17.49 months.the expression of BCAT1 was significantly correlated with poor outcome of ESCC patients(p<0.05).Meanwhile, the average survival time of CD147-negative patients was 40.83 months, whereas the average survival time of CD147-positive patients was 14.43 months, the expression of CD 147 was also significantly correlated with poor outcome of ESCC patients(p<0.05). Cox regression analysis showed postoperative pathological TNM stage and expression of CD 147 were independent prognostic indicators(p=0.008 and p=0.003,respectively).4.the relationship between the expression of BCAT1 and CD 147 in ESCC patientsIn this study,we did not find any correlation between the expression of BCAT1 and CD 147 in ESCC patients by IHC(p>0.05).Conclusions:1. BCAT1 and CD147 expressed in most ESCC patients, suggesting that they may play an important role in the development of esophageal squamous cell carcinomas;2. BCAT1 expression was positively correlated with poorer differentiation,larger tumor and advanced pTNM stage in ESCC patients;BCATl expression was negatively correlated with patients’survival time;3. the expression of CD 147 was also negatively correlated with patients’survival time, the expression of CD 147 was an independent prognostic indicator.