The Mechanism Of Human Epidermal Growth Factor Receptor 2 Participates Proliferation Of Colorectal Adenocarcinoma

Part Ⅰ The expression of Her2 in cell membrane and cytoplasm in colorectal cancer cell line promotes its proliferationObjectives:The incidence of of colotectal cancer is at second place in female ptaients worldwide, and at third place in male patients. It is hot spot in sentific research to explore the initiation and development of colorectal cancer, and the machnism of related signaling pathway. It cannot be underestamited to identify novel therapy target and treatment to improve the life quality and prolong the survival of colorectal cancer.Human epidermal growth factor receptor 2 (Her2) is an important progonostic factor of breast cancer. Although Her2 pscitive (gene amplifaction or protein overexpression) is significantly correlate with prognosis of breast, the relationship between Her2 expression and biologic behavior of colorectal cancer is not elucidated. Although many studies have been undertaken for the relationship between Her2 amplification and human colorectal cancer, we fail to reach agreement on applicable conclusion. Lack of clinical guideline for treatment and prognosis of colorectal cancer shows much work need to be done for this field. It is important to further investigate the mechanism of how Her2 affects the development of colorectal cancer and if there any potential value to apply Her2 target therapy to colorectal cancer. The first step which is urgently needed is to explore Her2 gene amplification and protein over-expression in colorectal cancer, and do these amplification and over-expression influence the development of colorectal cancer.The present study investigate the role of Her2 in the development of colorectal cancer and its mechnasim. Firstly, we detected the expression of Her2 mRNA in human colorectal cancer cell lines, and developed a human colorectal cancer cell line which showed stable Her2 expression. A human colorectal cancer cell line with depressed Her2 expression was also established using Her2 siRNA. CCK8 (Cell Counting Kit-8), wound healing and Transwell migration assays were performed to evaluate the impact of Her2 on the in vitro proliferation, migration and aggressiveness of human colorectal cancer cell lines.Materials and Methods:Her2 Expression in human colorectal cancer cell linesCell linesHuman colorectal cancer cell lines DLD-1, HCT8, HCT116, SW480 and HT-29 were purchased from Chinese Academy of Science (Shanghai cell bank). Human breast cancer cell line SK-BR-3 was purchased from ACTT cell bank.Real time Quantitative PCRHer 2 expresssion of cell lines of human colorectal cancer was examined using Real-time PCR. Stable Her2 overexpression human colorectal cancer cell line (Sw480) and siNRA Her2 silence human colorectal cancer cell line (HT-29) were developed. Immunostaing of two types of monoclonal Her2 antobodies, detecting Her2 expression in cell membrane (CB11) and cytoplasm (Ab-3) respectively, was performed.To develop Her2 stable over expression in human colorectal cancer cell line (SW480) and siRNA silenced Her2 expression in human colorectal cancer cell line (HT-29)We developed Her2 stable over expression in human colorectal cancer cell line (SW480) using a sequence of Her2 gene which locates at extracellular membrane portion of colorectal cancer cell. The chosen sequence of Her2 gene was inserted into the genome of eukaryotic expression vector PIRES2-EGFP. A plasmid PIRES2-EGFP-HER2 was established. The cell line with siRNA silence Her2 gene was developed by transfecting Her2-RNAi-LV into human colorectal cancer cell HT-29.Detecting the location of Her2 protein by immunohistochemistryTwo types of Her2 monoclonal antibodies was applied to detect the extracellular portion (CB11) and intracellular portion (Ab-3) of Her2 protein in human colorectal cancer cell lines respectively. For the scoring of the results of immunohistochemistry, the scoring system of gastric cancer was used:1+ refers to weak or faint/barely perceptible membranous reactivity in≥10% of tumor cells; cells are reactive only in part of their membrane; 2+ refers to weak to moderate complete, basolateral or lateral membranous reactivity in≥10% of tumor cells; 3+ refers strong complete, basolateral or lateral membranous reactivity in≥10% of tumor cells.CCK8, wound healing and Transwell methodsThe impact of Her2 expression, including over expression and Her2 gene silence, to proliferation, emigration and aggressiveness of in vitro human colorectal cancer cell lines were measured using CCK8, wound healing and Transwell methods. Briefly, the transfected cells were cultured for 6 hours and stained by DAPI, then the cells which perforated the membrane of Transwell chambers were counted.StatisticsThe statistical softwear SPSS 19.0 was used to calculate the data which were obtained in the present study. The differences between two groups were calculated using Student’s T test. The differences among more than two groups were calculated using Oneway ANOVA test. The differences of Percentage between two or more groups were calculated using Chi square test. P< 0.05 was treated as with significantly difference.ResultsHer2 expression in 5 types of human colorectal cancer cell linesAmong the five types of human colorectal cancer cell lines studied in this study, HT-29 shows Her2 over-expression. The other four types of cell lines showed low or negative Her2 expression. Immunostaining showed that the Her2 monoclonal antibody Ab-3 expressed in the cytoplasm of SK-BR-3 and HT29, the scores of Her2 expression in them were recorded from weak positive to strong positive, while the expression of Ab-3 in DLD-1, HCT8, HCT116, SW480 was negative. The location at which Her monoclonal antibody CB11 reacted was cytoplasm of SK-BR-3 cell and cell membrane of HT-29, and the scores of Her2 expression in them were recorded from weak positive to strong positive, while the expression of Ab-3 in DLD-1, HCT8, HCT116, SW480 was negative.Her2 over expression improved the ability of proliferation of human colorectal cancer cell lineCCK8 method was applied in the stable Her2 overexpression human colorectal cancer cell line (Sw480) for consective 5 days to detect the ability of proliferation of the tranfected SW480 PIRES2-EGFP-Her2 cells which was significantly higher than those of non-transfected cells (p<0.05). All of the experiment were undertook triplicate.Her2 over expression improved the ability of migration of human colorectal cancer cell lineTanswell migration assay was performed to detect the ability of aggressiveness of stable Her2 overexpression human colorectal cancer cell line (SW480). The cells were cultured on the upper chamber with 8.0 um holes for 36 hours. The the cells were stained by crystal violet and observed by inverted microscope. Compared with non-transfected SW480 cells, the Her2 overexpression SW480 was with significantly increased cells which can perforate the basal membrane and entered into the lower chamber of the transwell. All of the experiment were undertook triplicate.RNAi silenced Her2 repressed the aggressiveness of HT-29 cell in vitroFor the Her2 siRNA HT-29 cells, their ability of migration through the basal membrane was signifancantly higher the control cells (p=0.003 t test), while there were no significantly difference between the the blank control cells of upper chamber and lower chamber. So to the negative control cells. All of the experiment were undertook triplicate.ConclusionsThere was Her2 overexpression in human colorectal cell line HT-29, while DLD-1, HCT8, HCT116, and SW480 showed lower or negative expression. These results means the existence of Her2 amplification and overexpression in human colorectal cancer cells. Our results also showed that the gene amplification or protein overexpression in colorectal cells made them with higher ability of proliferation and aggressiveness.Part II Immunohistochemical results of HER2/neu protein expression assessed by rabbit monoclonal antibodies SP3 and 4B5 in colorectal carcinomasObjectives:Colorectal carcinoma is a leading cause of cancer-related deaths worldwide. Although chemotherapy has shown to be an efficient management, ongoing improvement is needed, especially for advanced stage. Targeted cancer therapy provide a promising way to tailor cancer treatment with more selective for cancer cells than normal cells. Rabbit monoclonal antibodies represent a novel type of immunoreagents that may combine the best properties of both mouse monoclonal antibodies and of rabbit antisera. Recently developed rabbit monoclonal Her2 antibodies have higher affinity and specificity. This study aims to investigate Her2 expression in colorectal carcinomas using these two rabbit monoclonal Her2 antibodies, and to clarify the relationship between protein overexpression and gene amplification of Her2 and their clinicopathologic importance.Materials and methodsWe examined 106 cases colorectal carcinomas obtained from 2003 to 2007 from the surgical pathological database of the First Affiliated Hospital of Wenzhou Medical University. The patients was composed of 39 men and 52 women with a median age of 60.09 (34-81 years). In all cases colectomy was performed and their clinical data, including gender, age, stage, recurrence, lymph node metastasis, and follow-ups were collected. Tissue microarray (TMA) was constructed from formalin-fixed and paraffin embedded blocks. One tissue section was chosen for each case on which three random representative locations of cancer foci and one location of normal mucosa were marked. Having matched the marked foci with the tissue paraffin block,4 cores of tissue per case were embedded into the recipient paraffin blocks using a tissue arrayerv. Sections were then cut for immunostaining. Rabbit Monoclonal antibodies for Her2 was stained according to manufacturer recommendation. The IHC positive cases and 10 negative cases were chosen to preform FISH. FISH was performed using the Pathvysion HER-2 DNA probe kit. Both the immunostaining of membrane and cytoplasm were scored. The membrane staining was evaluated on a score of 0 to 3 using the score system suggested by Hoffman et al. Scoring of membrane was as follows:0, no reactivity or FISH analysis shows homogenous (A) and heterogenous (B) amplification of HER-2 (clusters of red signals) in colorectal cancer.RseultsImmunostaining results and clinicopathologic data of related cases are listed in Table-1. In cases with immunoscores of 1+ and 2+ (both of immunoreactivity of SP3 and 4B5), most cancer cells showed week lateral or basolateral (U-shaped) membranous staining. All 3 cases with immunoscores of 3+ showed intensive complete membranous staining. The staining intensity of 4B5 was stronger than that of SP3. In 5 cases scored 2+for 4B5 staining, only 1 scored 1+ and 1 scored 2+ for SP3 staining. There were no notable background staining noted for both antibodies. In positive cases for 4B5 antibody,89%(16/18) showed cytoplasmic staining coexist with membranous staining, and all of the positive cells showed weak immunoreactivity (Figure 1). Of 7 SP3 cytoplasmic positive cases,2 cases showed strong staining.There were 3 signet ring cell carcinomas and 4 mucinous adenocarcinomas in our series of cases. None of them overexpressed 4B5 and SP3 in either membrane or cytoplasm. Three cases of surrounding normal glands showed 4B5 weak membranous staining (1+). FISH was undertaken for all TMA slides, and all HER2/neu 2+ and 3+ staining sections were analyzed. All 3 cases of HER2/neu 3+ also exhibited Her2 amplification. For the cases scored 2+, only 1 case with 4B5 immunoreactivity was amplified. None of the 2 cases of strong cytoplasmic staining showed amplification. We defined Her2 2+ and 3+ as overexpression, and there was no correlation between HER2/neu membranous and cytoplasmic overexpression and gender, age, tumor size, TNM stage (p>0.05). There was no correlation between Her2 (for 4B5 staining) overexpression and overall survival (p=0.33).ConclusionsIn our study,4B5 was more sensitive to detect HER2/neu of colorectal carcinoma than SP3.2.8% patients with colorectal patients might benefit from anti-HER2/neu therapy.