miRNA-26b Overexpression in Ulcerative Colitis-associated Carcinogenesis

BACKGROUND Longstanding ulcerative colitis (UC) bears a high risk for development of UC-associated colorectal carcinoma (UCC). The inflammatory microenvironment influences microRNA expression, which in turn deregulates target gene expression. microRNA-26b (miR-26b) was shown to be instrumental in normal tissue growth and differentiation. Thus, we aimed to investigate the impact of miR-26b in inflammation-associated colorectal carcinogenesis. METHODS Two different cohorts of patients were investigated. In the retrospective group, a tissue microarray with 38 samples from 17 UC/UCC patients was used for miR-26b in situ hybridization and quantitative reverse transcription polymerase chain reaction analyses. In the prospective group, we investigated miR-26b expression in 25 fresh-frozen colon biopsies and corresponding serum samples of 6 UC and 15 non-UC patients, respectively. In silico analysis, Ago2-RNA immunoprecipitation, luciferase reporter assay, quantitative reverse transcription polymerase chain reaction examination, and miR-26b mimic overexpression were employed for target validation. RESULTS miR-26b expression was shown to be upregulated with disease progression in tissues and serum of UC and UCC patients. Using miR-26b and Ki-67 expression levels, an UCC was predicted with high accuracy. We identified 4 novel miR-26b targets (DIP1, MDM2, CREBBP, BRCA1). Among them, the downregulation of the E3 ubiquitin ligase DIP1 was closely related to death-associated protein kinase stabilization along the normal mucosa-UC-UCC sequence. In silico functional pathway analysis revealed that the common cellular pathways affected by miR-26b are highly related to cancerogenesis and the development of gastrointestinal diseases. CONCLUSIONS We suggest that miR-26b could serve as a biomarker for inflammation-associated processes in the gastrointestinal system. Because miR-26b expression is downregulated in sporadic colon cancer, it could discriminate between UCC and the sporadic cancer type.

Thiazolides promote apoptosis in colorectal tumor cells via MAP kinase-induced Bim and Puma activation

While many anticancer therapies aim to target the death of tumor cells, sophisticated resistance mechanisms in the tumor cells prevent cell death induction. In particular enzymes of the glutathion-S-transferase (GST) family represent a well-known detoxification mechanism, which limit the effect of chemotherapeutic drugs in tumor cells. Specifically, GST of the class P1 (GSTP1-1) is overexpressed in colorectal tumor cells and renders them resistant to various drugs. Thus, GSTP1-1 has become an important therapeutic target. We have recently shown that thiazolides, a novel class of anti-infectious drugs, induce apoptosis in colorectal tumor cells in a GSTP1-1-dependent manner, thereby bypassing this GSTP1-1-mediated drug resistance. In this study we investigated in detail the underlying mechanism of thiazolide-induced apoptosis induction in colorectal tumor cells. Thiazolides induce the activation of p38 and Jun kinase, which is required for thiazolide-induced cell death. Activation of these MAP kinases results in increased expression of the pro-apoptotic Bcl-2 homologs Bim and Puma, which inducibly bind and sequester Mcl-1 and Bcl-xL leading to the induction of the mitochondrial apoptosis pathway. Of interest, while an increase in intracellular glutathione levels resulted in increased resistance to cisplatin, it sensitized colorectal tumor cells to thiazolide-induced apoptosis by promoting increased Jun kinase activation and Bim induction. Thus, thiazolides may represent an interesting novel class of anti-tumor agents by specifically targeting tumor resistance mechanisms, such as GSTP1-1.

Soft tissue grafting to improve the attached mucosa at dental implants: A review of the literature and proposal of a decision tree.

BACKGROUND Scientific data and clinical observations appear to indicate that an adequate width of attached mucosa may facilitate oral hygiene procedures thus preventing peri-implant inflammation and tissue breakdown (eg, biologic complications). Consequently, in order to avoid biologic complications and improve long-term prognosis, soft tissue conditions should be carefully evaluated when implant therapy is planned. At present the necessity and time-point for soft tissue grafting (eg, prior to or during implant placement or after healing) is still controversially discussed while clinical recommendations are vague. OBJECTIVES To provide a review of the literature on the role of attached mucosa to maintain periimplant health, and to propose a decision tree which may help the clinician to select the appropriate surgical technique for increasing the width of attached mucosa. RESULTS The available data indicate that ideally, soft tissue conditions should be optimized by various grafting procedures either before or during implant placement or as part of stage-two surgery. In cases, where, despite insufficient peri-implant soft tissue condition (ie, lack of attached mucosa or movements caused by buccal frena), implants have been uncovered and/or loaded, or in cases where biologic complications are already present (eg, mucositis, peri-implantitis), the treatment appears to be more difficult and less predictable. CONCLUSION Soft tissue grafting may be important to prevent peri-implant tissue breakdown and should be considered when dental implants are placed. The presented decision tree may help the clinician to select the appropriate grafting technique.

Expression of the hyaluronan-mediated motility receptor RHAMM in tumor budding cells identifies aggressive colorectal cancers.

Expression of the hyaluronan-mediated motility receptor (RHAMM, CD168) predicts adverse clinicopathological features and decreased survival for colorectal cancer (CRC) patients. Using full tissue sections, we investigated the expression of RHAMM in tumor budding cells of 103 primary CRCs to characterize the biological processes driving single-cell invasion and early metastatic dissemination. RHAMM expression in tumor buds was analyzed with clinicopathological data, molecular features and survival. Tumor budding cells at the invasive front of CRC expressed RHAMM in 68% of cases. Detection of RHAMM-positive tumor budding cells was significantly associated with poor survival outcome (P = .0312), independent of TNM stage and adjuvant therapy in multivariate analysis (P = .0201). RHAMM-positive tumor buds were associated with frequent lymphatic invasion (P = .0007), higher tumor grade (P = .0296), and nodal metastasis (P = .0364). Importantly, the prognostic impact of RHAMM expression in tumor buds was maintained independently of the number of tumor buds found in an individual case (P = .0246). No impact of KRAS/BRAF mutation, mismatch repair deficiency and CpG island methylation was observed. RHAMM expression identifies an aggressive subpopulation of tumor budding cells and is an independent adverse prognostic factor for CRC patients. These data support ongoing efforts to develop RHAMM as a target for precision therapy.

Loss of tapasin correlates with diminished CD8(+) T-cell immunity and prognosis in colorectal cancer.

BACKGROUND Tapasin is a crucial component of the major histocompatibility (MHC) class I antigen presentation pathway. Defects in this pathway can lead to tumor immune evasion. The aim of this study was to test whether tapasin expression correlates with CD8(+) cytotoxic T lymphocyte (CTL) infiltration of colorectal cancer (CRC) and overall survival. METHODS A next-generation tissue microarray (ngTMA) of 198 CRC patients with full clinicopathological information was included in this study. TMA slides were immunostained for tapasin, MHC I and CD8. Marker expression was analyzed with immune-cell infiltration, patient survival and TNM-staging. RESULTS A reduction of tapasin expression strongly correlated with venous invasion (AUC 0.682, OR 2.7, p = 0.002; 95% CI 1.7-5.0), lymphatic invasion (AUC 0.620, OR 2.0, p = 0.005; 95 % CI 1.3-3.3), distant metastasis (AUC 0.727, OR 2.9, p = 0.004; 95% CI 1.4-5.9) and an infiltrative tumor border configuration (AUC 0.621, OR 2.2, p = 0.017; 95% CI 1.2-4.4). Further, tapasin expression was associated with CD8(+) CTL infiltration (AUC 0.729, OR 5.4, p < 0.001; 95% CI 2.6-11), and favorable overall survival (p = 0.004, HR 0.6, 95% CI 0.42-0.85). CONCLUSIONS Consistent with published functional data showing that tapasin promotes antigen presentation, as well as tumor immune recognition and destruction by CD8(+) CTLs, a reduction in tapasin expression is associated with tumor progression in CRC.

Dual role of tumour-infiltrating T helper 17 cells in human colorectal cancer

BACKGROUND The immune contexture predicts prognosis in human colorectal cancer (CRC). Whereas tumour-infiltrating CD8+ T cells and myeloid CD16+ myeloperoxidase (MPO)+ cells are associated with favourable clinical outcome, interleukin (IL)-17-producing cells have been reported to correlate with severe prognosis. However, their phenotypes and functions continue to be debated. OBJECTIVE To investigate clinical relevance, phenotypes and functional features of CRC-infiltrating, IL-17-producing cells. METHODS IL-17 staining was performed by immunohistochemistry on a tissue microarray including 1148 CRCs. Phenotypes of IL-17-producing cells were evaluated by flow cytometry on cell suspensions obtained by enzymatic digestion of clinical specimens. Functions of CRC-isolated, IL-17-producing cells were assessed by in vitro and in vivo experiments. RESULTS IL-17+ infiltrates were not themselves predictive of an unfavourable clinical outcome, but correlated with infiltration by CD8+ T cells and CD16+ MPO+ neutrophils. Ex vivo analysis showed that tumour-infiltrating IL-17+ cells mostly consist of CD4+ T helper 17 (Th17) cells with multifaceted properties. Indeed, owing to IL-17 secretion, CRC-derived Th17 triggered the release of protumorigenic factors by tumour and tumour-associated stroma. However, on the other hand, they favoured recruitment of beneficial neutrophils through IL-8 secretion and, most importantly, they drove highly cytotoxic CCR5+CCR6+CD8+ T cells into tumour tissue, through CCL5 and CCL20 release. Consistent with these findings, the presence of intraepithelial, but not of stromal Th17 cells, positively correlated with improved survival. CONCLUSIONS Our study shows the dual role played by tumour-infiltrating Th17 in CRC, thus advising caution when developing new IL-17/Th17 targeted treatments.

VE1 immunohistochemistry predicts BRAF V600E mutation status and clinical outcome in colorectal cancer

AIM VE1 is a monoclonal antibody detecting mutant BRAFV600E protein by immunohistochemistry. Here we aim to determine the inter-observer agreement and concordance of VE1 with mutational status, investigate heterogeneity in colorectal cancers and metastases and determine the prognostic effect of VE1 in colorectal cancer patients. METHODS Concordance of VE1 with mutational status and inter-observer agreement were tested on a pilot cohort of colorectal cancers (n = 34), melanomas (n = 23) and thyroid cancers (n = 8). Two prognostic cohorts were evaluated (n = 259, Cohort 1 and n = 226, Cohort 2) by multiple-punch tissue microarrays. VE1 staining on preoperative biopsies (n = 118 patients) was compared to expression in resections. Primary tumors and metastases from 13 patients were tested for VE1 heterogeneity using a tissue microarray generated from all available blocks (n = 100 blocks). RESULTS Inter-observer agreement was 100% (kappa = 1.0). Concordance between VE1 and V600E mutation was 98.5%. Cohort 1: VE1 positivity (seen in 13.5%) was associated with older age (p = 0.0175) and MLH1 deficiency (p < 0.0001). Cohort 2: VE1 positivity (seen in 12.8%) was associated with female gender (p = 0.0016), right-sided tumor location (p < 0.0001), higher tumor grade (p < 0.0001) and mismatch repair (MMR)-deficiency (p < 0.0001). In survival analysis, MMR status and postoperative therapy were identified as possible confounding factors. Adjusting for these features, VE1 was an unfavorable prognostic factor. Preoperative biopsy staining matched resections in all cases except one. No heterogeneity was found across any primary/metastatic tumor blocks. CONCLUSION VE1 is highly concordant for V600E and homogeneously expressed suggesting staining can be analysed on resection specimens, preoperative biopsies, metastatic lesions and tissue microarrays.

Binding studies on isolated porcine small intestinal mucosa and in vitro toxicity studies reveal lack of effect of C. perfringens beta-toxin on the porcine intestinal epithelium

Beta-toxin (CPB) is the essential virulence factor of C. perfringens type C causing necrotizing enteritis (NE) in different hosts. Using a pig infection model, we showed that CPB targets small intestinal endothelial cells. Its effect on the porcine intestinal epithelium, however, could not be adequately investigated by this approach. Using porcine neonatal jejunal explants and cryosections, we performed in situ binding studies with CPB. We confirmed binding of CPB to endothelial but could not detect binding to epithelial cells. In contrast, the intact epithelial layer inhibited CPB penetration into deeper intestinal layers. CPB failed to induce cytopathic effects in cultured polarized porcine intestinal epithelial cells (IPEC-J2) and primary jejunal epithelial cells. C. perfringens type C culture supernatants were toxic for cell cultures. This, however, was not inhibited by CPB neutralization. Our results show that, in the porcine small intestine, CPB primarily targets endothelial cells and does not bind to epithelial cells. An intact intestinal epithelial layer prevents CPB diffusion into underlying tissue and CPB alone does not cause direct damage to intestinal epithelial cells. Additional factors might be involved in the early epithelial damage which is needed for CPB diffusion towards its endothelial targets in the small intestine.

Bevacizumab continuation versus no continuation after first-line chemotherapy plus bevacizumab in patients with metastatic colorectal cancer: a randomized phase III non-inferiority trial (SAKK 41/06)

BACKGROUND Chemotherapy plus bevacizumab is a standard option for first-line treatment in metastatic colorectal cancer (mCRC) patients. We assessed whether no continuation is non-inferior to continuation of bevacizumab after completing first-line chemotherapy. PATIENTS AND METHODS In an open-label, phase III multicentre trial, patients with mCRC without disease progression after 4-6 months of standard first-line chemotherapy plus bevacizumab were randomly assigned to continuing bevacizumab at a standard dose or no treatment. CT scans were done every 6 weeks until disease progression. The primary end point was time to progression (TTP). A non-inferiority limit for hazard ratio (HR) of 0.727 was chosen to detect a difference in TTP of 6 weeks or less, with a one-sided significance level of 10% and a statistical power of 85%. RESULTS The intention-to-treat population comprised 262 patients: median follow-up was 36.7 months. The median TTP was 4.1 [95% confidence interval (CI) 3.1-5.4] months for bevacizumab continuation versus 2.9 (95% CI 2.8-3.8) months for no continuation; HR 0.74 (95% CI 0.58-0.96). Non-inferiority could not be demonstrated. The median overall survival was 25.4 months for bevacizumab continuation versus 23.8 months (HR 0.83; 95% CI 0.63-1.1; P = 0.2) for no continuation. Severe adverse events were uncommon in the bevacizumab continuation arm. Costs for bevacizumab continuation were estimated to be ∼30,000 USD per patient. CONCLUSIONS Non-inferiority could not be demonstrated for treatment holidays versus continuing bevacizumab monotheray, after 4-6 months of standard first-line chemotherapy plus bevacizumab. Based on no impact on overall survival and increased treatment costs, bevacizumab as a single agent is of no meaningful therapeutic value. More efficient treatment approaches are needed to maintain control of stabilized disease following induction therapy. CLINICAL TRIAL REGISTRATION ClinicalTrials.gov, number NCT00544700.

Prognostic Relevance of Palliative Primary Tumor Removal in 37,793 Metastatic Colorectal Cancer Patients: A Population-Based, Propensity Score-Adjusted Trend Analysis

OBJECTIVE To assess whether palliative primary tumor resection in colorectal cancer patients with incurable stage IV disease is associated with improved survival. BACKGROUND There is a heated debate regarding whether or not an asymptomatic primary tumor should be removed in patients with incurable stage IV colorectal disease. METHODS Stage IV colorectal cancer patients were identified in the Surveillance, Epidemiology, and End Results database between 1998 and 2009. Patients undergoing surgery to metastatic sites were excluded. Overall survival and cancer-specific survival were compared between patients with and without palliative primary tumor resection using risk-adjusted Cox proportional hazard regression models and stratified propensity score methods. RESULTS Overall, 37,793 stage IV colorectal cancer patients were identified. Of those, 23,004 (60.9%) underwent palliative primary tumor resection. The rate of patients undergoing palliative primary cancer resection decreased from 68.4% in 1998 to 50.7% in 2009 (P < 0.001). In Cox regression analysis after propensity score matching primary cancer resection was associated with a significantly improved overall survival [hazard ratio (HR) of death = 0.40, 95% confidence interval (CI) = 0.39-0.42, P < 0.001] and cancer-specific survival (HR of death = 0.39, 95% CI = 0.38-0.40, P < 0.001). The benefit of palliative primary cancer resection persisted during the time period 1998 to 2009 with HRs equal to or less than 0.47 for both overall and cancer-specific survival. CONCLUSIONS On the basis of this population-based cohort of stage IV colorectal cancer patients, palliative primary tumor resection was associated with improved overall and cancer-specific survival. Therefore, the dogma that an asymptomatic primary tumor never should be resected in patients with unresectable colorectal cancer metastases must be questioned.

Time-dependent supraimplant mucosa changes: short communication

PURPOSE The aim of this short communication was to analyze time-dependent changes of the supraimplant mucosa architecture in the esthetic zone. MATERIALS AND METHODS Five patients underwent single-tooth replacement with implant crowns in the anterior maxilla. The supraimplant soft tissue was conditioned with fixed provisional crowns. Quadrantlike digital impressions were taken with an intraoral optical scanning device at three time points: t0, immediately after removal of the provisional (baseline); t1, after 5 minutes; and t2, after 10 minutes. To analyze time-dependent mucosal changes, the corresponding digital files were superimposed for each patient, and baseline (t0) scans were compared with t1 and t2 scans, respectively. Wilcoxon rank sum tests were used for statistical calculations with a strict level of significance at P < .01. RESULTS Mean values for supraimplant soft tissue changes were statistically significantly different after 5 minutes (5.5%; standard deviation ± 0.3%) in comparison to the results after 10 minutes (21.7%; standard deviation ± 1.8%). The direction of mucosa shrinkage showed a trend toward palatal sites. CONCLUSION Based on the findings of this analysis, changes in supraimplant mucosa architecture seem to be affected only slightly during the first 5 minutes after removal of soft tissue support.

Training primary care physicians to offer their patients faecal occult blood testing and colonoscopy for colorectal cancer screening on an equal basis: a pilot intervention with before-after and parallel group surveys.

OBJECTIVES Primary care physicians (PCPs) should prescribe faecal immunochemical testing (FIT) or colonoscopy for colorectal cancer screening based on their patient's values and preferences. However, there are wide variations between PCPs in the screening method prescribed. The objective was to assess the impact of an educational intervention on PCPs' intent to offer FIT or colonoscopy on an equal basis. DESIGN Survey before and after training seminars, with a parallel comparison through a mailed survey to PCPs not attending the training seminars. SETTING All PCPs in the canton of Vaud, Switzerland. PARTICIPANTS Of 592 eligible PCPs, 133 (22%) attended a seminar and 106 (80%) filled both surveys. 109 (24%) PCPs who did not attend the seminars returned the mailed survey. INTERVENTION A 2 h-long interactive seminar targeting PCP knowledge, skills and attitudes regarding offering a choice of colorectal cancer (CRC) screening options. OUTCOME MEASURES The primary outcome was PCP intention of having their patients screened with FIT and colonoscopy in equal proportions (between 40% and 60% each). Secondary outcomes were the perceived role of PCPs in screening decisions (from paternalistic to informed decision-making) and correct answer to a clinical vignette. RESULTS Before the seminars, 8% of PCPs reported that they had equal proportions of their patients screened for CRC by FIT and colonoscopy; after the seminar, 33% foresaw having their patients screened in equal proportions (p<0.001). Among those not attending, there was no change (13% vs 14%, p=0.8). Of those attending, there was no change in their perceived role in screening decisions, while the proportion responding correctly to a clinical vignette increased (88-99%, p<0.001). CONCLUSIONS An interactive training seminar increased the proportion of physicians with the intention to prescribe FIT and colonoscopy in equal proportions.

CHARACTERIZATION OF CYTOCHROME P-450 MIXED FUNCTION OXIDASE OF RAT COLON MUCOSA

Non-pregnant, female adult rats pretreated with either phenobarbital (PB) or (beta)-naphthoflavone ((beta)NF) through short-course intraperitoneal injections were shown by sodium dithionite-reduced carbon monoxide difference spectroscopy and NADPH-cytochrome c in vitro assay to contain cytochrome P-450 and NADPH-dependent reductase associated with the microsomal fraction of colon mucosa. These two protein components of the mixed function oxidase system were released from the microsomal membrane, resolved from each other, and partially purified by using a combination of techniques including solubilization in nonionic detergent followed by ultracentrifugation, anion exchange and adsorption column chromatographies, native gel electrophoresis, polyethylene glycol fractionation and ultrafiltration.^ In vitro reconstitution assays demonstrated the cytochrome P-450 fraction as the site of substrate and molecular oxygen binding. By the use of immunochemical techniques including radial immunodiffusion, Ouchterlony double diffusion and protein electroblotting, the cytochrome P-450 fraction was shown to contain at least 5 forms of the protein, having molecular weights as determined by SDS gel electrophoresis identical to the corresponding hepatic cytochrome P-450. Estimation of total cytochrome P-450 content confirmed the preferential induction of particular forms in response to the appropriate drug pretreatment.^ The colonic NADPH-dependent reductase was isolated from native gel electrophoresis and second dimensional SDS gel electrophoresis was performed in parallel to that for purified reductase from liver. Comparative electrophoretic mobilities together with immunochemical analysis, as with the cytochrome P-450s, reconstitution assays, and kinetic characterization using artificial electron acceptors, gave conclusive proof of the structural and functional homology between the colon and liver sources of the enzyme.^ Drug metabolism was performed in the reconstituted mixed function oxidase system containing a particular purified liver cytochrome P-450 form or partially pure colon cytochrome P-450 fraction plus colon or liver reductase and synthetic lipid vesicles. The two drugs, benzo{(alpha)}pyrene and benzphetamine, which are most representative of the action of system in liver, lung and kidney, were tested to determine the specificity of the reconstituted system. The kinetics of benzo{(alpha)}pyrene hydroxylation were followed fluorimetrically for 3-hydroxybenzo{(alpha)}pyrene production. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Development of colorectal cancer gene therapy

Colorectal cancer is the number two cancer killer in the United States. Although primary colorectal cancer can be resected by surgery, patients often die from metastatic disease. Liver is the most common site of metastasis for colorectal cancer. It is difficult to selectively kill metastatic colon cancer cells without damaging normal liver functions. Thus it becomes a high priority to develop a selective targeting system for the treatment of colorectal cancer liver metastasis. ^ In the current study, a gene therapy strategy that allows a therapeutic gene to selectively destroy metastatic colon cancer cells without affecting normal liver cells is developed. The APC gene is frequently mutated in colorectal cancers. These mutations activate β-catenin responsive promoters. An optimized β-catenin responsive promoter, containing TCF consensus binding sites, was engineered for this study. This TCF promoter was found to express preferentially in APC mutated/β-catenin activated colorectal cancers while maintaining a low expression level in cell lines of liver origin. A recombinant adenoviral vector AdTCF-TK, in which the TCF promoter controls expression of the herpes simplex virus thymidine kinase gene, selectively destroyed colorectal cancer cells in vitro. AdTCF-TK virus and ganciclovir treatment also inhibited the growth of solid tumour derived from the colon cancer cell line DLD-1 in nude mice. In a control experiment, the growth inhibition effect of the same virus was attenuated in a liver cancer cell line. ^ In the present study, a novel method was developed to target therapeutic gene expression to colon cancer cells at reduced liver toxicity to the patients. The same gene therapy design may also be applied to treat tumours carrying mutations in the β-catenin gene, which is a central component of the APC signal transduction pathway. In summary, the principle for a rational design of a cancer specific treatment approach is demonstrated in this study. In the future, mutations in cancer patients will be more easily identified. Using the same principle developed in this study, specific regimen can be designed to treat these patients based on the specific genetic changes found in the tumour. ^

The biologic and signaling effects of Tiam1 overexpression in colorectal carcinoma

Cellular migration is essential to many normal cellular processes. In tumor cells, aberrant activation of the normal pathways regulating migration is one of the critical steps in the development of metastasis. Previously, I demonstrated for the first time that overexpression of Tiam1, a guanine nucleotide exchange factor (GNEF) for small G proteins in the Rho family, could alter migration in colorectal tumor cells. ^ This dissertation focuses on the roles of Tiam1 in promoting cell migration, survival, and metastasis of colorectal carcinoma cells, utilizing the model system I developed. To determine the in vivo phenotype of the migratory cell lines, athymic nude mice were injected with cells into the orthotopic site. Several of the mice injected with cells of increased migratory potential had metastases. Thus, the in vitro selection for increased migration resulted in increased metastatic potential in vivo, and therefore, the Tiam1-overexpressing cells provide a model to examine signal transduction pathways important to this process. ^ To examine effects of Tiam1 signaling on small G proteins critical to cellular functions associated with migration, I examined the activation status of the small G proteins Rac, Rho, and Cdc42. The cells of increased migratory potential have increased GTP-bound Rac and Rho, compared to control SW480 cells. Cells that overexpress Tiam1 are more migratory and are resistant to detachment-induced death, or anoikis. To determine which effects and phenotypes were Tiam1-specific, we utilized siRNA to downregulate Tiam1 expression. These results demonstrate that Tiam1 is sufficient but not required for the migration of colorectal carcinoma cells in our model system, and that the biologically selected cells have additional changes that promote migration besides the increase in Tiam1. I also show that Tiam1 protects colorectal carcinoma cells from detachment-induced death, but is not required for anoikis resistance in the biologically selected migratory cells. ^ In summary, my studies demonstrate a heretofore-unknown regulator of phenotypes critical to the development of colorectal carcinoma metastases, overexpression of Tiam1. Understanding the mechanism by which Tiam1 contributes to cellular migration and metastasis is crucial to developing desperately needed new therapies for colorectal carcinoma. ^