The Role Of TIP30and Egfr In HER-2-positive Advanced Breast Cancer And Its Clinical Significance

BackgroundBreast cancer is the most common malignancy in women. Its incidence increased year by year, accounting for female cancer mortality in second place, a serious threat to women’s health. In breast cancer patients, about25%to30%of breast cancer patients with HER-2overexpression, these patients are often expressed as prone to lymph node metastasis, relatively resistant to endocrine therapy,chemotherapy, remission shortened, poor prognosis than HER-2negative patients. Trastuzumab (Herceptin) is a humanized against HER-2monoclonal antibody portion of the outer membrane, which significantly improves therapeutic effect in HER-2-positive breast cancer, but not all patients with HER-2-positive used trastuzumab effectively. Patients in the course of neoadjuvant, adjuvant chemotherapy, or palliative care with HER-2-positive breast cancer are likely to appear resistant molecular target therapy. Therefore, looking for effective prognostic marker of HER-2-positive breast cancer with Herceptin therapy is of great significance.TIP30/CC3(Tat-interactive protein30) was a gene related to the inhibition of metastasis, which was found by Shtivelman etc. in1997by the techniques of differential display RNA. It did not express in high metastatic small cell lung cancer cell lines, while highly express in low metastatic small cell lung cancer cell line. It can bind to transcription cofactor Tat protein and regulate its transcription, and potentiate proliferation of human immunodeficiency virus (HIV), so also called HITAP2(HIV-1Tatinteraetive protein2). At home and abroad in recent years, several studies have found it in low expression or even missing in lung cancer, gastric cancer, liver cancer, colon cancer and other malignant tumors. Knock out TIP30gene, can make the mice spontaneous formation of liver cancer, bladder cancer and breast cancer and other tumors. These all indicated that TIP30was a tumor suppressor gene. There were studies show that TIP30can inhibit tumor occurrence and development by inhibiting tumor cell growth, promoting tumor cell apoptosis, inhibiting tumor angiogenesis, adjusting the DNA damage repair and increasing the sensitivity of tumor cells to chemotherapy. TIP30stimulated HIV-1together with Tat protein and RNA polymerase II and then activated the Tat protein transcription, took part in a variety of cell signaling pathways, adjusted the transcription of P53and inhibited the transcription of c-myc which was adjusted by Era in breast cancer cells. Most of the results in the studies of relationship between TIP30expression and the clinical outcomes of breast cancer patients showed that TIP30expression is negatively related to the axillary lymph node metastasis and vascular invasion, and down-regulation of TIP30is associated with poor prognosis of breast cancer patients. The study found that TIP30expression and HER-2expression were positively correlated, and the TIP30gene and HER-2were involved in MAPK pathway and PI3K/AKT signal cascade amplification pathway which regulate cell proliferation and differentiation. These showed that TIP30play an important role in the occurrence and development of breast cancer. Epidermal growth factor receptor (EGFR) was a proto-oncogene expression product of c-erbBl, which was one of epidermal growth factor receptor (HER) family members as HER-2. EGFR had a high expression or abnormal situation in lung cancer, colon cancer and other solid tumors. It can activate the downstream signaling pathways including MAPK/ERK and PI3K/AKT, combined with EGF and other ligands, and thus mediated tumor cell differentiation, growth, adhesion and cell migration, invasion and damage repair. Trastuzumab and EGFR tyrosine kinase inhibitors such as erlotinib (erlotinib) or gefitinib (gefitinib), were used together to inhibit growth of trastuzumab resistant cells, suggesting that EGFR upregulated was associated with trastuzumab resistance. Moreover, Xiao Hua etc. in the study of liver cancer and lung cancer found that TIP30can downregulate the EGFR signaling pathway and inhibit tumor occurrence and development by controlling endocytosis of the EGFR. However, so far there was no study about the relationship between TIP30and EGFR expression in breast cancer with high expression of HER-2. The mechanism of TIP30in the development of breast cancer is still unclear and whether it makes a biological effect by influencing the expression of EGFR signaling pathway, which is needed to futher study.ObjectiveTo explore the expression of TIP30protein in HER-2-positive advanced breast cancer and HER-2-negative breast cancerTo discuss the relationship between the expression of TIP30and the characteristics (age、ECOG PS、Histology、ER/PR、PFS)To discuss the relationship between the expression of EGFR and the characteristics (age、ECOG PS、Histology、ER/PR、PFS)To discuss the relationship of TIP30and EGFR in breast cancerMaterials and methods1. Materials1.1SamplesThere were53breast cancer cases who underwent surgery from October2006to November2009in our hospital meeting the following conditions:(1) pathological diagnosis was primary invasive breast cancer;(2) HER-2immunohistochemical detection+++or FISH testing positive;(3) a complete clinical data;(4) measurable lesions;(5) to accept biochemotherapy (trastuzumab plus paclitaxel) for at least two cycles. There were another49cases of HER-2negative breast cancer tissues as the control group.1.2ReagentsRabbit anti-human EGFR monoclonal antibody, Rabbit anti-human TIP30polyclonal antibody, Horseradish peroxidase labeled second antibody, hematoxylin? Anhydrous ethanol, DAB chromogenic agent,95%alcohol,1%hydrochloric acid alcohol etc.2. Experimental procedure2.1TIP30protein expression in breast cancerDetected TIP30expression using immunohistochemistry in the paraffin sections of53cases of HER-2overexpressing breast cancer and49cases in the control group HER-2negative breast cancer. Paraffin tissue section, which were dewaxing hydration and repair with antigen, serum after closed drain, were added the TIP30antibody (1:50diluted concentration),4℃overnight. After45min and washed with PBS, then added horseradish peroxidase labeled secondary antibodies for1hour at room temperature. DAB color, wood grain redyeing, washing, dehydration, neutral rubber seal and the microscopic observation. Use semi-quantitative score double blind method, and randomly selecte5horizons from each section, count at least200cells respectively under optical microscope at high magnification (400times). TIP30appeared in the cytoplasm of brown granules for positive staining. Dyeing results with the microscopic observation of cell color degree and the product of two color scale score statistics. Cell color:no color is0points; light yellow for1point; tan for2points; brown for3points. Cell color scale:<10%were0,10%～30%for1point,31%～60%for2points,>60%for three points. The product of two score>2points was positive, and2points or less was negative expression.2.2Expression of TIP30and EGFR in breast cancer with high expression of HER-2The expressions of TIP30and EGFR in53breast cancer cases were detected by immunohistochemistry. Paraffin tissue section, which were dewaxing hydration and repair with antigen, serum after closed drain, were added the TIP30antibody (1:50diluted concentration),4℃overnight. After45min and washed with PBS, then added horseradish peroxidase labeled secondary antibodies for1hour at room temperature. DAB color, wood grain redyeing, washing, dehydration, neutral rubber seal and the microscopic observation. Use semi-quantitative score double blind method, and randomly selecte5horizons from each section, count at least200cells respectively under optical microscope at high magnification (400times). EGFR staining was predominantly located in the cell membrane, while TIP30staining was mainly located in cytoplasm. The method was same as that above.2.3The correlation of TIP30or EGFR and clinicopathologic, prognosis of HER-2-positive breast cancerWe studied the clinical data of53breast cancer patients, and analysis of TIP30and EGFR according to age (≥50years old or<50), histological type (invasive ductal carcinoma, other types), ECOG PS score (<2points or=2points), number of metastatic sites (<2,≥2), ER (negative, positive), PR (negative, positive) cases. Calculate the progression-free survival (PFS) combining with patient imaging data, according to RECIST criteria (2009).We discussed the relationship of TIP30expression and PFS by univariate and multivariate analysis, and evaluate whether TIP30is an independent prognostic indicator for breast cancer patients with biochemotherapy (trastuzumab in combination with paclitaxel).2.4Correlation between TIP30and EGFR in breast cancerUse immunohistochemical staining method to detect the expression of TIP30and EGFR in HER-2-positive breast cancer and preliminary study the relationship between TIP30and EGFR by spearman correlation analysis.3. Statistical analysisAll the statistical calculations used the SPSS16.0software. The relationship between the expression of EGFR or TIP30and clinicopathologic variables, as well as the TIP30expression between HER-2-positive breast cancer and HER-2-negative evaluated by Pearsonχ2tests. The relationship between TIP30and EGFR was analyzed by Spearman correlation analysis. Univariate analysis of PFS was used Kaplan-Meier method, and Cox proportional hazards model was applied for multivariate analysis of individual parameters for correlations with PFS rate. P value <0.05was considered the significant statistical.Results1. The expression of TIP30in HER-2overexpressed breast cancer and HER-2negative breast cancer:TIP30positive immunostaining was found in56.6%(30/50) of HER-2overexpressed breast cancer, while44.9%(22/49) of HER-2negative breast cancer had positive stained, but the difference was not significantly(P=0.237).2. The relationship between EGFR or TIP30and clinicopathologic features: There was no correlation between the expression of TIP30protein and age, pathological pattern, metastasis, ER and PR. While the expression of TIP30was inversely associated with ECOG PS (P=0.003). EGFR protein expression and the patients’age, ECOG PS, pathological type, the number of transfer parts, and ER, PR expression had no correlation.3.The relationship between biochemotherapy effect in HER-2overexpressed breast cancer and clinicopathologic features:Use RECIST Version1.1for efficacy evaluation of53patients. The research results showed that there was no correlation between progression-free survival (PFS) and age, pathological pattern, metastasis, EGFR,ER and PR. While there was a significant correlation between PFS and ECOG PS,TIP30(P<0.05).After two cycles of biochemotherapy of the30patients with positive TIP30expression, the clinical benefit rate (CBR) was73.3%(22/30), with a non-response rate of26.7%(8/30); by contrast, the CBR was only30.4%in23patients with negative TIP30expression, with a non-response rate of69.6%, showing a significant difference between two groups (P=0.02). Survival analysis showed that HER-2-positive advanced breast cancer patients with positive TIP30expression had a significantly longer progression-free Survival time than those who with negative TIP30expression (P<0.001). Multivariate analysis showed that TIP30is an independent predictor of PFS of HER-2-positive breast cancer (P<0.001).4. The relationship between TIP30and EGFR in HER-2positive advanced breast cancer:In53cases of HER-2positive advanced breast tissues, TIP30positive rate was56.6%(30/53), and EGFR overexpression rate was26.4%(14/53). Spearman correlation analysis showed that TIP30and EGFR protein in HER-2positive advanced breast tissues had a significant negative correlation (r=-0.339, P=0.013).Conclusions1. The difference of the expression of TIP30was not significantly between HER-2overexpressed breast cancer and HER-2negative breast cancer.2.There was no correlation between the expression of TIP30protein and age, pathological pattern, metastasis, ER and PR. While the expression of TIP30was inversely associated with ECOG PS.3.After the biochemotherapy,the clinical benefit rate (CBR)and Progression-free Survival (PFS)of TIP30positive expression group compared with negative group had a significant advantage. Prompt TIP30protein in HER-2positive advanced breast tissues up-regulated, It could be an important biomarker in predicting the prognosis of HER-2-positive advanced breast cancer patients. TIP30could be used to predict the efficacy before the biochemotherapy in patients with HER-2-positive advanced breast cancer.4.The expression of TIP30and EGFR is negative correlation in HER-2positive advanced breast cancer. The expression of TIP30may be related to the regulation of EGFR signaling pathway.