Effects Of CCND1Amplification On Biological Behaviors And Responsiveness Of Endocrine Therapy In Primary Breast Cancer

BackgroundBreast cancer is the most common malignant tumor infemalesand the first cause of cancer death in women. Breast cancer is a complicated disease driven by multiple genes and has high heterogeneity, thus, only using the traditional histologic features could scarcely satisfy the clinical requirements. In recent years, great progress on correlating genes of breast cancerhas been achieved, and targeting HER-2is supposed to be a successful way in breast cancer treatment. A better understanding of the genetic changes in breast cancer provides favorable conditions for individual therapy. Our previous studies which used QM-FISH methods to detect the genetic instability of breast cancer, showed that CCND1amplification frequently occurred in breast cancer. CCND1plays an important role on tumorigenesis and tumor development, and is extremely closely related with breast cancer, the overexpression of its coding protein CyclinDl is more common. To date, the mechanism of highly expressed CyclinDl in different molecular classification of breast cancer remains unclear, and its correlations with ER positive breast cancer prognosis and the endocrine therapy effect are inconsistent. Moreover, the relationship between CCND1amplification and CyclinDl expression remains controversial.Our study is designed to determine the expressions of CCND1/CyclinDl in primary breast cancers, to explore the predicting function of expression of CyclinDl on CCDN1amplification, to find out the different Cut-off values of CyclinDl protein expression levels related with CCND1gene amplification, to analyze CCND1amplification/CyclinD1expression in different molecular classification of breast cancer and to explore the correlations among CCND1amplification and the prognosis of invasive breast cancer and the sensitivity to endocrine therapy.Our aim of this study is hoping that CCND1becomes a new molecular maker forgenotyping and therapeutic target in breast cancer.This study is divided into two parts as following:part I to study the relations between CCND1amplification and biological behaviors of invasive breast cancer; part IIto study the effects of CCND1on sensitivity to endocrine therapy in breast cancer.MethodsPart I:The clinical data of217primary breast cancer werereviewed retrospectively, Immunohistochemisty and FISH methods were used to detect the CyclinD1protein expression and CCND1gene statusand to determine their correlations in breast cancer. Then, we analyzed CyclinD1expressions in different molecular types of breast cancer, and explored its relationship with breast cancer prognosis and sensitivity of endocrine therapy.Part II:To silence CCND1gene expression, we performed cell transfection using expression plasmid into human breast cancer cell lines MCF-7and T47D. After transfection, RT-PCR and immunocytochemistry were used to detect CCND1gene silent effect. Cells were divided into three groups:the experiment group, the negative control group and the blank control group. Three different groups were co-cultivated with TAM and TOR in various concentrations for72h. Cell inhibition ratio was detected using MTS method, and IC50values were calculated to determine the effects of silencing CCND1expression on sensitivity to endocrine therapy.Results1. CCND1was amplified in41of the217(18.9%) primary breast cancer cases, and CyclinD1was overexpressed in132cases (60.8%). There was no statistic significant difference for CCND1amplification/CyclinD1overexpression in invasive ductal carcinoma and ductal carcinoma in situ.2. CyclinDl overexpression is associated with CCDN1amplification (P＜0.01), the expressions between them are consistent (Kappa=0.510±0.057). ROC curve analysis showed that the Cut-off value of CyclinDl+++was63%, and the Cut-off value of CCND1++was78%.3. The CCND1amplification/CyclinDl high expression rate was significantly higher in luminal type breast cancer than HER-2expression type, and there wasno CCND1amplification in triple negative breast cancer. CCND1amplification was positively associated with ER expression and was negatively associated with HER-2expression.4. The rate of CCND1amplification in ER positive breast cancer with lymph node metastasis (40.4%)was higher than that in those without lymph node metastasis (19.2%), even the difference was not significant (P=0.065).5. The highest interference efficiency achieved after cell transient transfection with liposome for72h in MCF-7/T47D (81%,86%).6. After down-regulating CCND1expression, the IC50value in MCF-7/T47D-shRNA group was obviously lower than that in MCF-7/T47D-NC group and blank control group (P＜0.01).Conclusions1. CyclinD1expression is found to exist extensively in primary breast cancer, it can appear in stage of DCIS, and survive in the later stages and metastatic tumor. Thus, it is an important factor in breast cancer occurrence and progression.2. The expressions of CyclinD1were different in different molecular types of breast cancer. CCND1amplification is positively associated with ER expression and is negatively associated with HER-2expression, thus, CCND1is a promising molecular makers for further breast cancer classification.3. Using immunohistochemical method to detect CyclinD1relative protein expression can predict CCND1gene amplification. Value of CyclinDl+++63%can presume CCND1amplification, and CyclinDl++78%can presume CCND1amplification positive.4. CCND1is the prediction marker for TAM drug resistance in breast cancer. In breast cancer wirh CCND1amplification, it is necessary to give anti-CyclinDl treatment.