Preliminary Functional Studies Of Metastasis-associated Gene Tiam1 In Colorectal Cancer

BACKGROUND & OBJECTIVEColorectal cancer (CRC) is the second worldwide leading cause of cancer death in the world. The incidence rate of CRC in china is increasing fast during the past decades. Metastasis is one of the basic characteristic of malignant tumors and is the main cause which affects the therapeutic efficacy and leads to the death of cancer patients. It is an urgent task to work out the metastasis-associated factors and find out the preventive and therapeutic methods.In our previous studies, we prepared a cDNA microarray consisting of 447 tumor metastasis-associated candidate genes to screen metastasis-associated genes and obtained 51 candidate genes closely related to metastasis of colorectal cancer, four genes of which were selected as candidate metastatic genes of colorectal cancer by literature mining. Tiam1, one of the four candidate metastatic genes, was validated in the RNA level.Tiam1 is one of guanine nucleotide exchange factors (GEFs), which activate GTPases by promoting the exchange of their inactive GDP-bound forms to their active GTP-bound forms. Whereas Tiam1 displays GEF activity towards all three Rho-like GTPases Rac1, Cdc42 and RhoA in vitro, Tiam1 specifically activates Rac in vivo. Recent evidence suggests that Tiam1 could influence Rac GTPases signaling specificity in addition to promoting their activation. Tiam1 has been implicated to directly bind to many different cytoplasmic and membrane-associated proteins, which couples Tiam1-Rac activity to specific signaling pathways.Tiam1 was originally identified as the invasion- and metastasis-inducing gene by proviral tagging in combination with in vitro selection for invasiveness in T lymphoma cells. The role of Tiam 1 in cellular migration, invasion and metastasis may not be limited to T lymphoma. It was reported to be important in promoting the tumor progression in a variety of cancers such as breast cancer, lung cancer and Ras-induced skin tumors. The possible role of Tiam1 in the metastasis of colorectal cancer is unclear.In this study, we aim to clarify the possible role of Tiam1 gene in the proliferation, invasion and metastasis of CRC. It will be helpful to understand the molecular basis of CRC, and establish Tiam1 as a new target for early metastatic diagnostic markers and novel therapeutic strategies.METHODS1. Expression of Tiam1 in colorectal carcinomas and its clinical significanceThe expression of Tiam1 was examined in specimens of 157 CRC, 30 paired nomal colorectal mucosae, 35 regional lymphatic metastases and 32 adenoma by immunohistochemical (S-P) method.2. Effect of Tiam1 overexpression on the biological behaviors of human CRCThe expressions of Tiam1 gene in nine colorectal carcinoma cell lines were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Tiam1/C1199HA cDNA was transfected into HT29, which had no endogenous Tiam1 expression. The Tiam1 expression in transfectant was determined by RT-PCR, immunohistochemistry and Western blot. The biological behaviors of tranfectant were investigated by MTT assay, in vitro invasion assay, tumorigenesis, and in vivo metastasis assay by surgical orthotopic transplantation (SOI).3. Building the visualizing orthotopic animal model and observe real-time tumor growing and metastasis in colon cancerThe pEGFP-N1 plasmid was transfected into human colon carcinoma cell line SW480 to establish SW480/EGFP~+ cell line. MTT assay was engineered to detect the effect of EGFP on cell proliferation. The SW480/EGFP~+ cells were surgically orthotopically implanted as tissue fragments in the body of the colon of nude mice. Emitted fluorescence was collected and imaged through LT-9MACIMSYSPULS system and images were analyzed with IPP5.0 software. Whole-body optical images visualized real-time primary tumor growth and formation of metastatic lesions. The evaluation of orthotopic animal model was carried out with traditionally pathological methods.4. Lentivirus-mediated silencing of Tiam1 gene in human CRC cell lineWe transfected human CRC cell lines, SW480/EGFP~+ with Tiam1 specific RNAi lentiviral vectors as well as controls. Tiam1 silenced cells were screened by blasticidin. Quantitative RT-PCR and Western blot analysis were used to detect the expressions of Tiam1 mRNA and protein, respectively. MTT assay, plate colony formation assay, in vitro invasive assay, tumorigenesis, metastasis assay in vivo by whole-body visualizing orthotopic animal model of CRC were used to assess the functional effects of Tiam1 silencing on tumor cell proliferation, invasion and metastasis.5. Exploring the possible molecular mechanism underlying Tiara1-mediated multiple functions in CRC using gene microarray and comparative proteomics methodOligonucleotide microarray analysis was used to detect differences in gene expression between Tiam1-silencing SW480/EGFP~+ cells and the control.Comparative two-dimensional gel electrophoresis (2-DE) technology was performed to separate the proteins between HT29/Tiam1 and HT29/mock cells and between SW480/EGFP~+/Tiam1~- and SW480/EGFP~+/mock cells respectively. Differentially expressed proteins were identified by Matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF-MS).RESULTSThe main results and findings are as follows:1. Expression of Tiam1 in CRCTiam1 was expressed heterogeneously among the normal colorectal mucosae, adenoma, carcinomas and lymphatic metastasis of colorectal carcinoma (x~2=23.561, P＜0.01). Tiam1 was higher expressed in adenoma compared with normal colorectal epithelium (Z=-2.423, P＜0.05). Tiam1 expression was significantly stronger in lymphatic metastasis in comparison with colorectal carcinoma (Z=-2.051, P＜0.05). Furthermore, the Tiam1 expression in colorectal carcinoma with lymph node metastasis is higher than that in colorectal carcinoma without lymph node metastasis (Z=-3.176, P＜0.01).2. Transfection of C1199Tiam1 cDNA and selection of stable clonesTo address the function of Tiam1 in CRC, we established stably transfected HT29 cells that overexpress Tiam1. As revealed by RT-PCR analysis, a higher level of Tiam1 expression was seen in the Tiam1 transfected clonel but not in the vector-only transfected clonel. The western blotting analysis also showed that Tiam1 expression in Tiam1 transfected clonel (designated HT29/Tiam1) was positive and vector-only transfected clonel (designated HT29/mock) was negative.3. Effect of Tiam1 overexpression on the biological behaviors of human CRCHT29/Tiam1 cells showed a significantly enhanced proliferation compared with the HT29/mock cells as determined by in vitro MTT assay. Next, the effect of Tiam1 on in vivo tumor growth was assessed by subcutaneous injection of HY29/Tiam1 and HT29/mock cells for twenty days. Compared with HT29/mock cells, the expression of Tiam1 led to a pronounced increase in HT29/Tiam1 cell growth starting from day 7, up to 2.5 fold increase in mice of tumor volume at day 20 after cell injection. Primary tumor growth after in vivo orthotopic implantation was also tested. In this assay, mice were sacrificed six weeks later, and tumors from surgical orthotopic implantation excised and weighed in HT29/Tiam1 group and HT29/mock group respectively. The result showed that Tiam1 expression led to a significant enhancement in orthotopic tumor growth.Invasion through the extracellular matrix (ECM) is an important step in tumor metastasis. The ECMatrix served as a reconstituted basement membrane matrix of proteins. The number of cells migrating the ECMatrix was counted and the result showed that the HT29/Tiam1 cells posses a remarkable increase in invasiveness as compared with HT29/mock cells (P＜0.01). A 1.9-fold increase in the number of HT29/Tiam1 cells with an invasive ability was observed. These data indicated that Tiam1 expression in colorectal cancer cells was associated with enhanced migratory and invasive ability.To unambiguously elucidate the enhanced effect of Tiam1 on colorectal cancer metastasis, we performed in vivo metastasis assay by SOI. Mice were sacrificed six weeks later because the mice of HT29/Tiam1 group were moribund. Autopsy was performed and the incidence of metastasis in the liver, lung and other organs were determined by macroscopic and histological examination. In HT29/Tiam1 group, 100% (7/7) of mice developed peritoneal metastasis that appeared as numerous white nodules on the peritoneum and the abdominal organs. In HT29/mock group, only 29% (2/7) of animal had peritoneal metastasis. The incidence of hepatic metastasis in mice of HT29/Tiam1 group was 57.1% (4/7), and two of them were easily detected by macroscopic observation and two were found by histological examination. HT29/mock group did not produce detectable tumors in livers. In addition, one of seven mice of HT29/Tiam1 group had tumor formation in the lung, speen, lymph node besides in the liver.4. Building of whole-body visualizing orthotopic animal model in colon cancerSW480/EGFP~+ cells stably expressed high-levels of enhanced green fluorescent protein (EGFP) and EGFP had no effect on cell proliferation. Visualizing orthotopic animal model developed with surgical orthotopic implantation (SOI) was built with 100% survival rate. After SOI, no animal had metastasis within 2 weeks, and subsequently, 6 weeks after SOI, 75% of animal had implantation metastasis and 50% of animal had liver metastasis. The whole-body visualizing orthotopic animal model was successfully validated by pathological detection.5. Down-regulation of Tiam1 expression by RNAiWe constructed plasmids that expressed short hairpin RNAs that were targeted against Tiam1 under the control of the U6 promoter by lentiviral vector. Four different sequences were originally selected for targeting the Tiam1 gene. We infected SW480/EGFP~+ cells with lentival vectors and examined Tiam1 mRNA and protein expression and found site B vector was the most effective at blocking Tiam1 expression (73%). Then, SW480/EGFP~+ cells were infected with pLenti6/siTiam1 (site B) or pLenti6 (control vector), and incubated with blasticidin to select blasticidin-resistant single clones. We found clone 2 exhibited a dramatic knock down of Tiam 1 protein expression (88%) (Designated SW480/EGFP~+/Tiam1~-).6. Effect of Tiam1 silencing on the biological behaviors of human CRCSW480/EGFP~+/Tiam1~- cells showed a significantly reduced proliferation compared with SW480/EGFP~+/mock and SW480/EGFP~+ cells as determined by in vitro MTT assay. In addition, SW480/EGFP~+/Tiam1~- cells had a significant reduction in their ability to form colonies in plate as compared with SW480/EGFP~+/mock and SW480/EGFP~+ cells.The result of in vitro invasion assay showed that SW480/EGFP~+/Tiam1~- cells had significantly reduced invasiveness as compared with SW480/EGFP~+/mock and SW480/EGFP~+ cells.The effect of Tiam1 on in vivo tumor growth was assessed by subcutaneous injection of SW480/EGFP~+/Tiam1~- and SW480/EGFP~+/mock cells for thirty days. Compared with SW480/EGFP~+/mock cells, the silencing of Tiam1 led to a pronounced decrease in SW480/EGFP~+/Tiam1~- cells growth starting from day 15, up to 1.3 fold decrease in mice of tumor volume at day 30 after cell injection.The metastatic ability evaluated by whole-body visualizing orthotopic animal model in SW480/EGFP~+/Tiam1~- cells was inhibited after Tiam1 silencing. In SW480/EGFP~+/mock group, 75% (6/8) of mice developed peritoneal metastasis, 37.5% (3/8) had hepatic metastasis, and 12.5% (1/8) had lung metastasis. In SW480/EGFP~+/Tiam1~- group, 37.5% (3/8) of mice had peritoneal metastasis and none of them had hepatic and lung metastasis.7. Exploring the molecular mechanism underlying Tiam1-mediated multiple functions in CRC using gene microarrayGene expression profiles of SW480/EGFP~+/Tiam1~- and SW480/EGFP~+/mock cells were obtained and the microarray findings were confirmed by Real Time PCR. We obtained 1026 of Tiam1-related genes which 647 genes were down-regulated and 379 gnes were up-regulated in SW480/EGFP~+/Tiam1~- cells. The function of these 647 differentially expressed genes involved in cell cycle, regulation of cell cycle, DNA replication, DNA metabolism and regulation of cellular physiological process. The pathway of these differentially expressed genes included wnt signalling pathway, p53 pathway, apoptosis signalling pathway, integrin signalling pathway, PI3 kanase pathway and Ras pathway.8. Exploring the molecular mechanism underlying Tiam1-mediated multiple functions in CRC using proteomics analysis and peptide mass fingerprintingPeoteomics represents a powerful approach to analyze alterations in protein expression and posttranslational modifications. To further understand the intracellular function of the Tiam1 in CRC, we implemented a proteomics-based approach to identify proteins associated with Tiam1 expression. The proteome of the HT29/Tiam1 and HT29/mock cells and of SW480/EGFP~+/Tiam1~- and SW480/EGFP~+/mock cells were compared using 2-D electrophoresis. Discrepancies in protein spots found and the significance of protein changes was evaluated using Student's t-tests. A number of spots were found to be significantly altered (P＜0.05) after Tiam1 transfection or Tiam1 silencing. Discrepancies in protein spots were excised from the 2-D gels and subjected to trypsin digestion and MALDI-TOF-MS. Twenty spots were identified successfully. Up-regulated proteins in HT29/Tiam1 cells include Fascin, heat shock protein 27 (HSP27), AnnexinⅡ, high-mobility group box1 (HMGB1), Glutathione S-transferase omega 1 (GSTO1) and IMP dehydrogenase. And down-regulated proteins in HT29/Tiam1 cells include Retinal dehydrogenase 1, annexinⅣand 11s regulator. Down-regulated proteins in SW480/EGFP~+/Tiam1~- cells include high mobility group box 1 (HMGB1), 26S proteasome non-ATPase regulatory subunit 7(PSD7), LysophospholipaseⅠ(LYPA1), Mitochondrial 28S ribosomal protein S22(MRP-S22), COPD(delta-COP), Phosphoglycerate mutase 1(PGAM1). These results suggested that these differentially expressed proteins were involved in cellular metabolism, cell proliferation, cytoskeletal network and other cell processes.9. transcriptome and integration with its proteome of Tiam1-related genesThe identity between transcriptome and proteome of Tiam1-related genes were systematically analysed and integrated. We obtained three genes, which HMGB1 and PGAM1 were up-regulated in HT29/Tiam1 cells and down-regulated in SW480/EGFP~+/Tiam1~- cells, while ANXA4 down-regulated in HT29/Tiam1 cells and up-regulated in SW480/EGFP~+/Tiam1~- cells.CONCLUSION1. Tiam1 gene plays an important role in proliferation, invasion and metastasis of CRC and is a metastasis-related gene. Clinically it may be a useful indicator of the tumor progression and metastasis in CRC.2. Tiam1 may involve in many pathways to mediate proliferation, invasion and metastasis of CRC. And we found three genes, HMGB1, PGAM1 and ANXA4, have close relationship with Tiam1. These data will be helpful to elucidate the molecular mechanism of Timn1 and provide new clues.