Expression Of Tumor Suppressor Gene DBC2 In Breast Cancer And Its Function

ObjectiveBreast cancer is the most common malignancy in women and also is a genetic disease. Like other human cancers, it is thought to occur as the result of progressive accumulation of genetic aberrations. Familial breast cancer is characterized by an inherited susceptibility to breast cancer on basis of an identified germline mutation in one allele of a high penetrance susceptibility gene, such as BRCA1, BRCA2, CHEK2, TP53 or PTEN. But in more than 90% of cases, breast cancers result from a serial stepwise accumulation of acquired and uncorrected mutations in somatic genes, without any germline mutation playing a role. Up to now, the genetic alterations that precede development of sporadic breast cancer are poorly understood. Recently, Hamaguchi and coworkers have used representational difference analysis to identify a novel tumor suppressor gene DBC2 (Deleted in Breast Cancer 2), also named as RhoBTB2 gene, which belongs to the Rho GTPase family and is located at chromosome 8p21 deleted in some breast and lung tumor cell lines.DBC2 protein is an atypical member of Rho GTPase family. Unlike typical Rho GTPases, distinctive functions of DBC2 have been implied due to their unique structure such as transcriptional regulation and protein degradation. Studies indicated that DBC2 was homozygously deleted in 3.5% of primary breast cancers, gene expression was ablated in about 50% of breast and lung cancer cell lines, and several somatic missense mutations in DBC2 were isolated from primary tumors and cancer cell lines. Furthermore, reintroduction of DBC2 into a breast cancer cell line lacking endogenous DBC2 expression led to growth arrest. Although DBC2 was initially cloned as a tumor suppressor gene in breast cancer, its tumor suppression mechanism, the influence to tumor cells function and the status of expression in human sporadic breast cancer tissues remain unclear.In the present study, we will approach the antitumor effect of DBC2 at the following aspects:1. The expression status of DBC2 in sporadic breast cancer and its clinical significance.2. Construction of eukaryotic recombinant vector of human DBC2 and establishment of its stable expression cell line.3. Overexpression of exogenous DBC2 gene in breast tumor cells and the function study of RhoBTB2.Methods 1. The expression status of tumor suppressor gene DBC2 in sporadic breastcancer and its clinical significance(1) Patients and normal donors Sixty unrelated Chinese women with sporadic breast cancer, ranging in age from 28 to 70 years old (median 46) and 30 unrelated age matched control women with benign breast disease participated in this study. Normal control tissues were resected aside from benign breast disease tissues. Breast cancers were proved from surgical, pathological and clinical information obtained from their medical files. Any patient who had received chemotherapy or radiotherapy before obtaining specimens was excluded from this study. Survival was assessed in 56 patients as of August,2009 (4 Missing follow-up).(2) Cell lines and normal tissues samples HeLa:cervix carcinoma cell line; SKOV3,3AO:ovarian carcinoma cell line; BIU-87:bladder carcinoma cell line; MGC803:gastric carcinoma cell line; MCF-7, SKBR3:breast adenocarcinoma cell line; T-47D:breast ductal epithelial carcinoma cell line; HELF:human embryo lung fibroblasts cell line. Fetal brain tissue came from fetus of spontaneous abortion in Qi Lu Hospital, Shandong University.(3) The tissue samples from breast cancer patients and normal controls and cell samples from cell lines were collected and reverse transcription PCR (RT-PCR) was performed to detect the DBC2 mRNA. (4) The protein expression in 60 cases of breast cancer specimens and 30 cases of normal controls was detected using immunohistochemical staining (avidin-biotin-peroxidase complex method).(5) The relationship between the DBC2 expression and the pathological characteristics was analyzed by statistics. Cumulative survival time was calculated by the Kaplan-Meier method and curves were analyzed by the Log-rank and Gehan-Wilcoxon test. Multivariate survival analysis was performed using the Cox proportional hazard regression model.2. Construction of eukaryotic recombinant vector of human DBC2 and establishment of its stable expression cell line(1) Construction of recombinant vector of DBC2:Coding DNA sequence of human DBC2 was amplified by RT-PCR from HELF-6 cell line and was cloned into pMD19-T simple vector and then was transformed into competent E.coli JM109. The entire insert was sequenced from both directions by the method of Primer Walking. Full-length DNA of DBC2 was cloned into EcoRl-BamHl sites of pEGFP-N1 plasmid to get a recombinant eukaryotic expression vector named pEGFP-N1-DBC2.(2) Transformation:T-47D cells were transformed with recombinant plasmid by liposome LipofectAMINE 2000 Reagent following the manufacturer's instructions. Three groups were included in the transformation:①pEGFP-N1-DBC2 group;②pEGFP-N1 group;③liposome control group.(3) Screening of stable transformant clones:Stable transformants were selected by limiting dilution analysis and using G418 at a higher level than the minimum deadly concentration. The DBC2 expression of stable transformant clones was identified by RT-PCR and Western Blot.3. Overexpression of exogenous DBC2 gene in breast tumor cells and its function study of tumor suppressor gene DBC2To determine the effect of DBC2 on the growth, cell cycle, colony formation, apoptosis, invasion and metastasis of breast tumor cells, the recombinant plasmid pEGFP-N1-DBC2 carrying the full-length DBC2 cDNA was transfected into DBC2-negative breast tumor cells T-47D. (1) Cell proliferation was evaluated by MTT assayT-47D cells from different treated groups were incubated at 3×104/ml in 96-well flat bottom plates in DMEM medium supplemented with 10% FBS. Five identical plates were prepared for different time point assays (0,1,2,3,4 day, respectively). In each plate,5g/L MTT was added to cells for the last 4 h. Absorbance was determined with an enzyme-linked immunosorbent assay reader using 570 nm as test wavelength and 630 nm as reference wavelength.(2) Colony formation assayT-47D cells from different treated groups were plated in 6-well plates with a density of 200 cells per well in the presence of G418 for 2-3 weeks. The colonies were fixed in methanol and stained with Giemsa staining solution. The number of colonies with≥50 cells was counted and colony forming efficiency was calculated.(3) The cell cycle was detected by flow cytometric analysisT-47D cells from different treated groups (～106) were washed twice with cold PBS and were fixed in 70% cold ethanol for overnight at 4℃. Then, the cells were incubated with PI staining solution for 30 min in the dark. The stained cells were analyzed with a FACS Calibur flow cytometer using the Cell Quest software.(4) The apoptotic morphology detection by Hematoxylin-Eosin (HE) stainingT-47D cells were incubated in 6-well plate paved with grass slides at its bottom for 48 h at 37℃in 5% CO2. Then the slides were washed with PBS and were stained by HE method.(5) The apoptosis ratio was detected by flow cytometric analysisT-47D cells from different treated groups were collected and were washed with cold PBS. Five microliter Annexin V-APC and 10μl PI solution were added to cells suspension and incubated for 15 min in the dark. The stained cells were analyzed with flow cytometer.(6) Cell invasion assayCell invasion analysis was performed using a 24-well transwell chamber. Tumor cells were incubated in DMEM medium with serum free and were seeded in the upper chamber with an 8μm pore size insert precoated with ECMatrix in the 24-well plate and cultured for 72 h. Cells were allowed to migrate towards medium containing 10% FBS in the bottom chamber. The non-migratory cells on the upper membrane surface were removed with a cotton tip, and the migratory cells attached to the lower membrane surface were stained with 0.1% crystal violet. The crystal violet dye was eluted by 10% acetic acid and the absorbance was determined with an enzyme-linked immunosorbent assay reader using 570 nm as test wavelength.(7) Cell migration assayThe test method was the same as cell invasion assay except that there was not ECMatrix in the upper chamber and incubation time was 8 h.Results1. The expression status of tumor suppressor gene DBC2 in sporadic breast cancer and its clinical significance(1) Expression of DBC2 in embryon and different cancer cell linesDBC2 was expressed in human fetal brain tissue, human embryonic lung fibroblasts cells, uterine cervical carcinoma, ovarian carcinoma, bladder carcinoma, gastric carcinoma, breast adenocarcinoma cells MCF-7 and SKBR3, was not expressed in T-47D, a breast ductal epithelial carcinoma cell line.(2) Detection of DBC2 mRNA in breast cancer tissues and normal controlsThe DBC2 expression at RNA level in 60 breast cancer tissues and 30 normal breast samples was analyzed by RT-PCR. DBC2 mRNA expression was amplified in 28 of 30 normal breast samples, in contrast, most of breast cancer lacked DBC2. In 60 breast cancer samples,36 lost DBC2 mRNA expression. There was a significant difference of negative ratio between these two groups (6.7% versus 60.0%, P<0.001).(3) Expression analysis of DBC2 in breast cancer tissue and normal controlsWe further detected the expression of DBC2 at protein level by immunohistochemistry (IHC) in breast cancer tissue samples and normal breast tissue. Peroxidase staining revealed cytoplasmic expression in almost all of breast ductal epithelial cells samples from normal breast tissue (28/30). Whereas DBC2 was only weakly expressed in part of breast cancer samples or was not expressed in the most of breast cancer samples (36/60). These results were consistent with the RT-PCR analysis.(4) Association of loss of DBC2 expression in breast cancer tissues and clinicopathologic informationResults revealed an association between age of onset, progesterone receptor (PR) expression, tumor type and loss expression of DBC2. There was significantly higher negative ratio of DBC2 in patients of age≥50 years old than that of in patients of age <50 years old (90.0% versus 45.0%, P=0.000). Meanwhile, there was an increase of DBC2 loss expression ratio among patients with positive PR compared to those with negative PR (72.2% versus 41.7%,P=0.018). In this study, we also found that frequent loss of DBC2 occurred easily in ductal carcinoma as opposed to lobular carcinoma (71.4% versus 28.6%, P=0.000). This result agreed with the expression analysis of DBC2 in different cancer cell lines. But there was no correlation between loss of DBC2 expression and clinical stages, axillary lymph nodes metastasis, estrogen receptor (ER) and HER2 expression.(5) Prognostic value of DBC2 expression for patients with breast cancerTo investigate the association of DBC2 expression with patient survival, the survival data from 56 patients with breast cancer (4 missing follow-up) were assessed. In univariate analysis, the expression ratio of DBC2 significantly correlated with the long-term survival rate of patients. The survival rate of patients with DBC2-positive breast cancer was significantly higher than that of patients with DBC2-negative breast cancer (P=0.02). Results from the multivariate analysis showed that DBC2 expression was a significant prognostic factor and correlated with a better clinical outcome (relative risk,0.090; 95% confidence interval,0.010-0.806; P= 0.03).2. Construction of eukaryotic expression vector of DBC2 and establishment of its stable expression cell lineFull-length human DBC2 was obtained from human fetal lung cell line HELF-6 with the addition of EcoRl restriction endonuclease recognition sites at its 5'end and BamHl site at the 3'end. The recombinant eukaryotic expression vector with green fluorescent protein, pEGFP-N1-DBC2, was successfully constructed and identified by digestion with restriction enzyme. DBC2 gene was introduced into T-47D cells and stable transformants were obtained by screening with G418 and were identified by RT-PCR and Western blot. This provided a perfect cell model for the functional analysis of DBC2.3. Overexpression of exogenous DBC2 gene in breast tumor cells and its functional study(1) The morphological changes of T-47D cells with DBC2 expressionThe overexpression of DBC2 caused the morphological changes of T-47D cells compared with T-47D negative controls and empty vector transfected (Mock) group. There was decreased numbers of cells, abundant cytoplasm, increased cytoplasmic particles and cells arranged in groups in breast tumor cells transfected with DBC2.(2) Overexpression of DBC2 in breast tumor cells inhibites proliferationThe wild type DBC2 expression in T-47D cells significantly inhibited their growth compared with untreated negative cell control and mock group (P<0.01).(3) Overexpression of DBC2 in breast tumor cells prevents colony formationBreast tumor cells, unlike normal breast cells, have the tendency to form colonies in the culture. The overexpression of DBC2 significantly diminished the capacity of breast tumor cells to form colonies. Compared with mock group, the expression of DBC2 caused reduction in the number of colonies formed by T-47D cells (26%±4.24%, P<0.01).(4) Overexpression of DBC2 in breast tumor cells arrestes the cell cycleCell cycle analysis showed that there were significant differences in proportions of G0/G1 and S phase between T-47D cells transfected with DBC2 and their control groups. Expression of DBC2 in breast tumor cells increased the proportion of G0/G1 phase (80.23% vs 46.32% T-47D controls and 53.92%mock controls) and reduced the proportion of S phase (11.36% vs 46.91% T-47D controls and 35.54% mock controls) and thereby prevented tumor cells entering DNA synthesis phase. (5) Overexpression of DBC2 in breast tumor cells promotes the apoptosisResults from Annexin V-APC/PI analysis revealed that T-47D cells transfected with DBC2 underwent obvious apoptosis (22.07%±1.67%) than negative cell controls (8.31%±1.10%) and mock (11.25%±2.59%) (P< 0.01). HE morphological staining also showed that there were clearly apoptosis morphological changes in breast tumor cancer cells of DBC2 overexpression, such as cells becoming round, cytoplasmic density increasing, cell nucleus shrinking, concentrated dye and typical apoptotic bodies.(6) Effect of DBC2 on invasion and migration ability of breast tumor cellsTo investigate the effect of DBC2 on breast tumor cells invasion and metastasis ability, the transwell system precoated with ECMatrix was used in this study. Our results showed that overexpression of DBC2 did not affect the invasion ability of breast tumor cells compared with negative cell controls and mock controls, (0.50±0.02 and 0.56±0.06,0.47±0.04, respectively, P>0.05). The effect of DBC2 on breast tumor cells migration ability also be evaluated by transwell assay. The overexpression of DBC2 did not affect the migration ability of breast tumor cells compared with negative cell controls and mock controls, (0.51±0.05 and 0.53±0.20, 0.51±0.10, respectively, P>0.05).Conclusion1. High frequently loss of DBC2 generally exist in breast cancer tissues and the inactivation of DBC2 may occur at the transcription level. The loss of DBC2 expression more frequently occur in postmenopausal patients with age≥50 and in patients with PR-positive expression. Infiltrative ductal carcinoma subtype has higher negative ratio of DBC2 than that of infiltrative lobular carcinoma subtype. Furthermore, DBC2 expression may be controlled by female hormones and it functions as tumor suppressor in a tissue specific manner.2. DBC2 expression can serve as a considerable factor for prognosis of sporadic breast cancer.3. DBC2 plays antitumor roles by inhibiting proliferation, preventing colony formation, arresting cell cycle and promoting the apoptosis of tumor cells. But DBC2 does not affect the invasion and migration ability of breast tumor cells. Originality1. We demonstrate, for the first time, that loss of DBC2 expression is a frequently event in human breast cancer and that frequently loss of DBC2 significantly correlates with the clinicopathology, outcome and prognosis of breast cancer patients.2. It is the first time, we thoroughly reveal the function of DBC2 as a tumor suppressor by proliferation, apoptosis, invasion and metastasis in cell functional study.3. Our finding raises new issues for the follow-up study of the DBC2 gene and put forward new ideas for gene targeted therapy of DBC2.