Effect Of Genomic Instability And Mutations On The Signaling Network In Colon Cancer Cells

BackgroundColorectal cancer (CRC) is one of the most common malignant tumors and its incidence be ranked in the No.5in the all of tumor incidence. In China, the morbidity and mortality of colorectal cancer is currently ranked in the No.4and annual increment rate of4%in some large cities. Moreover CRC also is the third most common cancer diagnosed and the second leading cause of cancer death in the United States. In the global, an annual new case for colorectal cancer is1.2million cases, about20%of patients with metastasis at the first vist in the hospital,5-year survival after full sure diagnosis is only55%.The progress for colorectal cancer is from the normal epithelium to atypical dysplasia, adenoma, cancer and cancer metastasis, which be called as classic "normal cell-adenoma-cancer" theory. A series of genes mutation, mismatch, the activation of oncegenes and the silence of tumor suppressor genes be enrolled in this complex multi-step process. However the etiology of colorectal cancer is not entirely clear yet. Some scientists point out that microsatellite instability (MSI) is one of the most important molecular mechanism which be involved in the process of CRC.Microsatellite instability is presented in about15%of colorectal cancers and plays critical roles in the development and progression of colorectal cancer. MSI is most likely multifactoral, including manifested by damaged DNA due to defects in the normal DNA repair process (Microsatellite Instability in Colorectal Cancer). Although clinical studies showed that MSI colon cancer has a more favorable prognosis and are less prone to lymph node and distance metastasis, the Stage II MSI patients can not benefit from5-fluorouracil (5-FU) adjuvant therapy according to NCCN Guidelines Version2.2012. Furthermore MSI phenotype may predict treatment response to the treatment of5-FU and irinotecan. The mechanism of MSI-induced carcinogenesis remains unclear.Compared with microsatellite instability genotyping, kras gene is also anthor potential predictive molecular markers of colorectal cancer which is same with the MSI. It not only can predict the treatment of patients with colorectal cancer but also speculated that the prognosis of patients with colorectal cancer. Activation of the kras gene is by point mutation. The vast majority of kras gene point mutations in colorectal cancer are codon12and13point mutation, which is about88%. kras gene mutations usually occur in the progression of small adenomas to large adenomas. kras gene mutations are the early molecular events in colorectal cancer which also play an important role in the process of development of colorectal. AimRecently, some studies have identified several regulatory pathways altered in colon cancer with MSI, including Mucin5(MUC5AC), apoptosis-related gene (CASP2) and cyclin D1. However recent application of microarray technology provides a great opportunity to assess mRNA expression alterations in distal colorectal cancers with microsatellite instability (MSI),the altered mRNA or DNA expression may not represent the protein interaction exactly and certainly does not reflect protein activation (i.e. phosphorylation) that drives cell transformation. However, there is no proteomic approaches be used for the colon cancer with MSI to detect the role of each pathway in MSI pathogenesis and the interaction of among these pathways remain to be undetermined. The goal of this study is to determine the global effect of microsatellite instability on the signaling pathways and network in colon cancer cells by protein pathway array (PPA). These altered proteins may be used to identify the patients with high risk for developing malignant CRC with MSI and as targets for preventing this deadly malignancy.Materials and methodsHuman colorectal adenocarcinoma and carcinoma cell lines HCT15, HCT116, HT29SW620, SW480and Caco-2which were purchased from the the American Type Culture Collection (USA) be selected in this study. HCT15and HCT116were instable for microsatellite (MSI), in contrast, HT29, SW620, SW480and Caco-2were stable for microsatellite (MSS). We profiled the expression and phosphorylation of110proteins in6well characterized colon cancer cell lines using Protein Pathway Array (PPA) to determine the global effect of microsatellite instability on the signaling pathways and network in colon cancer cells. The pathways and network involved by these proteins were determined using a knowledge-based computer program, Ingenuity Pathway Analysis (IPA) to figure the whole protein signal pathway network out. We explore the signaling pathway in kras mutant MSS CRC cells at the same time.Results1. We totally detected43proteins and phosphoproteins in110proteins we tested in colon caner cell lines with microsatellite instability. Our results showed that25proteins and phosphoproteins are more than1.5-fold change.8proteins are down-regulated:14-3-3β, p-p53(Ser392), Cdk2, p53, raf-1, Cdk6, PKCα and Hsp90and17proteins are up-regulated:p-Smadl/5(Ser463/465), Survivin, p-HGF R/c-MET (Y1234/1235), p-PKCa (Ser657), E-cadherin, PTEN, TNF-a, p-PTEN (Ser380), p-HGF R/c-MET (Y1003), p-p90RSK (Ser380), NF-κB p65, CyclinE, cdc2p34, p-β-Catenin (Ser33/37/Thr41), Bcl-6, p-p70S6Kinase (Thr389) and p-PKC a/βⅡ (Thr638/641) between microsatellite instability and microstatellite stable (MSS) cells.2. We totally detected64proteins and phosphoproteins in110proteins we tested in colon caner cell lines with microsatellite stability (HT29, SW480、SW620and Caco-2). Our results showed that35proteins and phosphoproteins are more than1.5-fold change.16proteins are down-regulated:NFkB50, p14, Cdk2, E-cadherin, PTEN, Vimentin, p-HGF R/c-MET (Y1003), p-PKC a/βⅡ, Cdc42, p-Stat3(Ser727), p-eIF4B (Ser422), p-PKC delta (Thr505), p-p38(Thr180/Tyr182), Notch4,ATF-1and Calretinin.3. Sixteen major pathways were affected in microsatellite instability cells including p53, PI3K/AKT,14-3-3β, ATM, HGF, etc. Different pathways were affected in MSI cells which may responsible for different functions like cell cycle, cell death cancer and so on.4. Fifteen major pathways were affected in kras mutant colon cancer cell lines with the microsatellite stability cells including HGF,14-3-3β, EGFR, PI3K/AKT, p53etc.5. Establish the interactive network in colon cancer cells with microsatellite instability.6. Effect of microsatellite instability and kras mutation on EGFR pathway. In MSI cells, Raf and14-3-3β were down-regulated, while p70S6K and p90RSK were activated by phosphorylation. However, the down-stream effectors, i.e. ERK1/2and CREB, of the EGFR pathway did not change significantly. Interestingly, PKCa pathway was activated by phosphorylation [p-PKCa (Ser657)] although total PKCa was reduced. In contrast, in kras mutant microsatellite stability cells, Ras and ERK as well as14-3-3β and p-CERB were up-regulated, while no change for Raf, p70S6k and p90RSk. In addition, PKCa was also up-regulated via phosphorylation as well as protein level.ConclusionsBased on the number of connections among the proteins, several key nodes were identified including p53, NF-kB p65, p-β-Catenin (Ser33/37/Thr41), E-cadherin. This complex network in MSI cells effects many cellular functions, including cell cycle progression, apoptosis, surviving, etc. Our conclusion is that different pathways were affected in MSI cells which may responsible for its carcinogenesis and p-p53(Ser392) and p-β-Catenin (Ser33/37/Thr41) may play critical roles in the CRC with MSI as a potential inhibitor for colon cancer with microsatellite instability. Addional, EGFR/RAF/MEK signaling pathway be specific activetied in kras mutant microsatellite stability cells which can be use to explore potential treatment options for microsatellite stable colon cancer with kras mutation.