The Investigation Of Correlation Between Gamma-Interferon-Inducible Lysosomal Thiol Reductase (GILT) And Breast Cancer

BACKGROUNDBreast cancer （BC） is the most frequent cancer among women all over the world. And it has become a public health event threatening the family of patients and the society. As the latest WHO statistics show that there are1.67million new cancer cases diagnosed in2012（25%of all cancers）, and there are180,000new cases in china （11.17%of cases all over the world）. Breast cancer is still the leading cause of cancer-related death in women, although preventive approaches and treatments have improved greatly in the past decades. It is now the second cause of cancer related death in more developed regions following lung cancer, while it is the most frequent cause of cancer related death in women in less developed regions （324,000deaths,14.3%of total）. Although the treatment of breast cancer was improved in the past decades and had come to standard strategies, there is still a range of patients who was resistant to the therapy. The heterogeneity of breast cancer and different host immune status may be the reasons for this phenomenon. Recently, the host immune status has been demonstrated to play an important role in malignant diseases, which causes interest from many investigators.Nowadays, study on tumor immune response has become a new hot spot in breast cancer research. By reviewing and analyzing our previous microarray results, comparing the gene expression profiles of breast cancer to normal breast tissue and benign breast lesion, we identified the immunology-related gene—IFI30as our gene of interest. IFI30was significantly up-regulated in breast cancer tissues, suggesting it may be involved in breast cancer pathogenesis. Human IFI30, also known as gamma-interferon-inducible lysosomal thiol reductase （GILT）, the only one localized in lysosomes and phagosomes, is a unique member of the thiol reductase family because its optimal pH is4.5-5.5. GILT is constitutively expressed in professional APC, but in other cell types it is induced by inflammatory cytokines such as IFN-γ, IL-1β, and TNF-α. GILT plays a pivotal role in exogenous antigen processing and presentation by catalyzing the reduction of the disulfide bounds of proteins. On one hand, GILT has been proved to promote Major Histocompatibility Complex （MHC） class Ⅱ-restricted antigen presentation, which is essential for CD4+T lymphocytes activation; On the other hand, GILT can facilitate the recognition of MHC class Ⅰ-restricted antigens by CD8+T lymphocytes. The expression level of GILT can definitely affect immune response to viral as well as tumor antigens. Previous studies have discovered that GILT was absent or expressed at greatly reduced levels in several human malignant diseases, such as melanomas, prostate cancer and glioblastoma, indicating a potential role of GILT in tumor immunology. Although accumulated evidence suggests that GILT plays important roles in tumor immunosurveillance, its role in breast cancer is still poorly understood.Based on the above findings, we hypothesized that GILT expression in breast cancer was involved in the pathogenesis, and had the potential to participate in immune escape. This study was designed to investigate the clinical significance of GILT in breast cancer and its potential roles in this disease, aimed at identifying novel prognostic factors and therapeutic targets for breast cancer, to provide a rationale and direction for study on anti-tumor immunology in breast cancer.PART Ⅰ:The correlation between GILT and breast cancerObjectiveTo identify the expression status of IFI30mRNA and protein in breast cancer tissues as well as adjacent normal breast tissues to verify the findings of cDNA microarray analysis. Furthermore, to make clear the difference of GILT protein level between primary as well as metastatic breast cancer tissues and noncancerous tissues to investigate the correlation between GILT and breast cancer and provide information for further study on its role in breast cancer.Methods 1. Analyze the result of cDNA microarrayCompare and analyze the differential gene expression profiles between breast cancer tissues （n=7） and normal breast tissues （n=7） as well as benign breast tumors （n=7）, to look for new promising oncogenic genes.2. Collect pure breast cancer cells and adjacent normal epithelial cells by Laser-capture microdissection （LCM）Breast cancer and its adjacent uninvolved frozen samples were obtained from breast cancer patients who underwent modified radical mastectomy in the department of breast diseases of Second Hospital of Shandong University, from December,2011to June,2012, were used for pure breast cancer cells and adjacent normal epithelial cells collection by LCM. The cells harvested were stored in200uL QIAzol Lysis Reagent at-80℃for further RNA extraction.3. RNA extraction and real-time RT-PCR assayTotal RNA was extracted from cells harvested by LCM and reverse-transcribed. Quantitative analysis of GILT mRNA expression was performed in paired breast cancer cells and normal epithelial cells by real-time PCR.4. Immunohistochemistry analysis of GILT expressionParaffin sections of19cases above were used for immunohistochemistry analyses to investigate the expression status of GILT in breast tissues. The protein level of GILT expression was evaluated by the density mean of the specific staining, which was calculated by ImagePro_Plus_IPP software. At the same time, the stained slides were reviewed and scored independently and blindly by two pathologists. And the expression of GILT was graded according to immunohistochemical scores, including intensity, proportion and total scores.5. Immunohistochemical staining of GILT in normal breast tissue--primary cancer--metastatic cancer44matched normal tissue--primary cancer--metastatic lymph nodes paraffin sections from the same patients with lymph node invasion, obtained from the department of pathology of the Second Hospital of Shandong University from Jan1,2005to Jun31,2010, were used for immunohistochemical staining of GILT. The immunohistochemistry result was evaluated the same way as described above.6. Statistical analysisData of real-time PCR as well as IPP was expressed as mean±SD. Statistical significance was evaluated with Student’s t test. Differences of GILT expression between breast cancer and noncancerous breast tissue were evaluated by Chi-square test. P<0.05was considered significant, and all tests were2-sided. All statistical analyses were performed by the statistical software package SAS9.1.3.Results1. Identify IFI30as gene of interestAmong the different expressed genes, we identified IFI30as our gene of interest, for it is the unique one up-regulated in cancer tissues both compared to normal breast tissues （fold change=5.4016, P<0.001） and benign breast tumors （fold change=6.6383, P<0.05）. And the role of IFI30in tumor immunology provides rationale for exploring its function in breast cancer, and there has been so far no detailed study on this gene in this disease.2. IFI30mRNA increased in breast cancer cellsReal-time PCR result confirmed IFI30mRNA increased （2.18-fold changes） in breast cancer cells compared with the corresponding adjacent normal epithelial cells （P=0.0427）.3. GILT expression were down-regulated significantly in cancerous tissuesThe immunohistochemical result showed that GILT was mainly expressed in cytoplasm of normal epithelial cells, breast cancer cells and breast stromal cells.To explore the changes of GILT expression during the progression of breast cancer, immunohistochemistry was performed on19paired formalin-fixed paraffin-embedded sections including breast cancer tissues and adjacent uninvolved epithelial tissues. In contrast to the up-regulation of the IFI30mRNA level, the protein level of GILT was down-regulated significantly in cancerous tissues compared with normal tissues.3.1The expression level of GILT evaluated by IPP softwareThe IPP analysis showed that the density mean of GILT expressed in breast cancer tissues was0.03659±0.0084, while which was higher in normal breast tissues, whose density mean was0.0512±0.0118. The result indicated that the GILT level was significantly down-regulated in breast cancer tissues compared with normal breast tissues （P=0.002）.3.2The expression level of GILT evaluated by immunohistochemical staining scoreAccording to the Harvey standard of immunohistochemical score evaluation, the intensity （P=0.006） were down-regulated significantly in cancerous tissues compared with normal tissues, while there was no significant difference found in proportion （P=0.062） and total score （P=0.062） of GILT expression between cancerous tissues and normal tissues.Taken together, GILT was significantly increased on mRNA level meanwhile decreased on the protein level in breast cancer cells compared with normal epithelial cells, indicating its potential role in tumorigenesis of breast cancer.4. GILT expression decreases with breast cancer development from normal to primary and metastatic cancersBoth primary breast cancer tissues and metastatic cancer tissues presented significant lower GILT expression than normal breast tissues in44matched normal—primary cancer--metastatic tissue samples, including intensity （P=0.002,0.031respectively） and proportion score （P=0.012,0.039respectively）.Conclusions1. IFI30mRNA increased in breast cancer cells compared with the corresponding adjacent normal epithelial cells.2. GILT expression was down-regulated significantly in transforming from normal epithelial cells to cancerous cells.3. GILT may be involved in the pathogenesis of breast cancer. PART Ⅱ:The clinical significance of GILT in breast cancer and its potential role in pathogenesis of the diseaseObjectiveTo make clear the influence of GILT on breast cancer, a large scale of immunohistochemistry analysis on breast cancer tissues as well as normal breast tissues was conducted. And the relationship between the expression of GILT in breast cancer tissues and clinicopathologic characteristics as well as prognosis of patients was studied, and the value of GILT as an independent prognostic factor was evaluated. Furthermore, to explore the potential role of GILT in the pathogenesis of breast cancer, we detected the expression of FOXP3in the interstitial tissues and investigated the possible correlation between GILT and FOXP3positive lymphocyte to explore the potential role of GILT in immune escape.Methods1. Immunohistochemistry analysis of GILT expression in218breast cancers and99noncancerous breast tissuesParaffin sections of218cases, newly diagnosed and surgically treated breast cancer and99cases of adjacent noncancerous breast tissues from the surgically treated breast cancer patients, which were histopathologically diagnosed at department of pathology of the Second Hospital of Shandong University, Linyi People’s Hospital, Linyi Tumor Hospital （China） from Jan1,2007to Dec31,2008, were used for immunohistochemistry analyses to investigate the expression status of GILT in breast tissues. The stained slides were reviewed and scored independently and blindly by two pathologists. And the expression of GILT was graded according to immunohistochemical scores, including intensity, proportion and total scores.2. Investigation of the relation between the GILT expressed in breast cancer tissues and certain clinicopathological characteristicsTo explore its potential role in breast cancer, analysis of the relation between GILT expressed in breast cancer tissues and certain clinicopathological characteristics as well as differential therapy strategies was conducted. The clinicopathological characteristics including age, body mass index （BMI）, menarche （year）, menopause, history of disease, disease side, peripheral blood leucocytes （×109/L）, peripheral blood neutrophils （×109/L）, peripheral blood lymphocytes （×109/L）, peripheral blood hemoglobin （g/L）, histology, tumor size, lymph nodes status, pTNM stage, hormone status, Her-2, Ki67index, molecular classification and TIL score of tumor.3. Survival analysisCollect prognosis information of218patients3years after diagnosis, and to investigate the correlation between GILT expression status and disease-free survival （DFS） and overall survival （OS） of patients. To evaluate the value of GILT as a breast cancer prognostic factor by multivariate analysis. To explore its potential mechanism influencing prognosis, analysis of the relation between GILT expressed in breast cancer tissues and certain clinicopathological characteristics was conducted.4. Immunohistochemical staining of FOXP344matched normal tissue--primary cancer--metastatic lymph nodes paraffin sections from the same patients with lymph node invasion were used for immunohistochemical staining of FOXP3in breast stromal cells. The immunohistochemistry result was evaluated by the same way above.5. Statistical analysisDifferences between lower and higher GILT expression group regarding clinicopathologic characteristics were evaluated by Chi-square test or Fisher’s exact test. Kaplan-Meier method and log-rank test were conducted to evaluate the influence of GILT expression on DFS as well as OS of patients. Furthermore, hazard ratios （HRs） and95%confidential intervals （CIs） were computed from multivariate COX regression models. Spearman analysis was used to investigate correlation between GILT expression status and the proportion of stromal FOXP3positive lymphocytes.Results1. Negative expression rate of GILT increased significantly in cancer tissues than noncancerous tissuesTo determine whether the expression level of GILT differs between breast cancer tissues and noncancerous breast tissues, immunohistochemistry analysis of218breast cancer tissue sections and99noncancerous breast tissue sections were conducted. The negative expression rate of GILT increased significantly from2.02%（2/99） in noncancerous breast tissues to15.6%（34/218） in breast cancer tissues （P<0.001）.2. Correlation between GILT expression and clinicopathologic characteristicsThe expression status of GILT in218cancer tissues was correlated with certain key clinicopathologic characteristics. The expression of GILT （including intensity, proportion and total score） was significantly correlated with Ki67index inversely and disease-free survival （DFS） and overall survival （OS） of3years after diagnosis positively.3. Loss of GILT in breast cancer was associated with disease-free survival and overall survivalThe expression patterns of GILT in breast cancer tissues were further correlated with the DFS and OS of the patients by Kaplan-Meier estimates. Adverse DFS and OS was observed in negative GILT expression patients, lower GILT range score of GILT expression group and lower total score of GILT expression group as determined by the log-rank test. And the multivariate analysis results showed that similarly to pTNM stage, lymph node status as well as tumor size, negative or reduced GILT expression was a significant independent adverse predictor for DFS and OS of the patients. To reveal the interaction between the prognostic value of the detection of GILT expression by immunohistochemistry and known clinicopathologic prognostic factors as well as treatment strategies, subgroup analysis for DFS as well as OS were conducted. DFS and OS favored higher GILT expression over decreased expression, including the intensity, range as well as total score of GILT staining, in all subgroups （HRs>1）.4. The proportion of FOXP3positive stromal lymphocytes increased with pathogenesis process of breast cancerThe intensity and proportion as well as total score of FOXP3expression in interstitial tissues increased with the process from normal breast to primary cancer and lymph node metastasis（P<0.001）. The expression status of GILT in cancer tissues in metastatic lymph nodes correlated with the proportion of FXOP3positive stromal lymphocytes, indicating the potential role of GILT loss in immune escape in breast cancer.Conclusions1. Loss of GILT expression in breast cancer was an independent prognostic factor for early recurrence and metastasis in breast cancer.2. GILT expression correlated with Ki67index inversely.3. The potential role of GILT loss in breast cancer pathogenesis may be involved in immune escape.Originality1. We demonstrate that GILT is downregulated in breast cancer, and absence of GILT is significantly associated with poor DFS as well as OS. GILT is a novel independent prognostic factor for early recurrence and metastasis as well as overall survival.2. The study investigates the correlation between GILT expressed in cancer tissues and proportion of Foxp3positive stromal lymphocytes to explore potential role of GILT loss in immune escape in breast cancer.