Further studies will address roles of different genetic make (microsatellite instability higherin females). However, studies on experimental models remain a powerful means of investiga-ting these gender-related issues in colon carcinogenesis.

T1185

Effect of PTEN Downregulation on Invasion and Tumorigenic/MetastaticPotential of Human Colon Cancer CellsMarie-Josée Langlois, Francois Boudreau, Nathalie Perreault, Walid Chababi, CarolineSaucier, Julie Carrier, Nathalie Rivard

The PTEN phosphatase acts on phosphatidylinositols resulting from phosphatidylinositol3-kinase (PI3K) activation. Germinal inactive mutations of PTEN have been identified inCowden syndrome, a disease characterized by the development of hamartomatous polypsin the digestive tract and associated with an increased risk of cancer. However, little isknown about the specific cellular role of PTEN in human intestinal epithelial cells. Objective.To investigate the role of PTEN in human colorectal cancer cells. Methods. PTEN expressionwas analyzed by Western blot in human colorectal tumours, in normal epithelial crypt cells(HIEC) and in many colorectal cancer cell lines: Caco-2/15, DLD-1, HCT116, HT29, LoVo,Colo205 and SW480. Caco-2/15, HCT116 and CT-26 cells were infected with recombinantlentiviruses expressing a shRNA that specifically knocked-down PTEN. The impact of PTENdownregulation was analyzed on cell migration (wound assay), invasion (matrigel-coatedtranswells) and on tumour and metastasis formation in mice. Results. 1- PTEN expressionis decreased in approximately 60% of all colon cancer tumours of all grades. 2- PTEN isexpressed in all cancer cell lines but at a lower level than normal human intestinal epithelialcells. 3- The lentiviral infection of PTEN shRNA significantly increases spreading, migrationand invasion capacities of Caco-2/15 and HCT116 cells. 4- PTEN downregulation in bothCaco-2/15 and HCT116 cells increases tumour size following subcutaneous injection ofthese cells in nude mice. 5- Loss of PTEN expression in CT-26 (mouse colorectal cancercells) leads to an increase in their metastatic potential following tail-vein and intrasplenicinjections in BALB/c mice. Conclusions. Altogether, these results indicate that PTEN isinvolved in the control of migration/invasion capacity and tumorigenic/metastatic potentialof human colon cancer cells. Supported by CIHR Team grant.

T1186

Loss of RKIP Expression is Associated With Metastasis in EsophagealAdenocarcinomaAnand Gupte, Diana Cardona, Lisa R. Dixon, George A. Sarosi

BACKGROUND: The incidence of esophageal adenocarcinoma in North America and Europeis increasing at a rate faster than that for other solid tumors. Raf Kinase Inhibitor Protein(RKIP) has been shown to be a metastasis suppressor in many malignant tumors includingprostate, colon, liver and breast but its role in esophageal malignancies has not yet beenexplored. OBJECTIVE: To evaluate the role of RKIP in esophageal adenocarcinoma andmetastasis. METHODS: This was an IRB approved study. Patients were identified from aprospectively maintained cancer center database at University of Florida, Shands Hospital(SUF). Risk factors influencing adenocarcinoma and poor outcomes such as age, sex, priorGERD symptoms and BMI were evaluated. Paraffin embedded blocks were obtained frompatients that had previously undergone esophageal resection at SUF for known Barrett'sesophagus with high grade dysplasia (HGD) and esophageal adenocarcinoma or metastasis.RKIP expression in normal esophageal mucosa and primary and metastatic esophagealadenocarcinoma lesions was evaluated by immunohistochemistry (IHC). An independentGI pathologist graded RKIP expression which was graded on a scale (absent-0, weak-1,moderate-2 and strong-3). Categorical variables were expressed as proportions and comparedusing Fisher's exact test. RESULTS: Themean age of patients with esophageal adenocarcinomawas 67 years, 78% were males and 89% were overweight (BMI >25). 96% of patients withesophageal adenocarcinoma had prior symptoms of GERD. No differences in demographicswere noted between patients with and without metastasis. RKIP expression was significantlyhigher in normal esophageal mucosa and Barrett's metaplasia without adenocarcinoma (Meanexpression- 2.8). Adenocarcinoma tissue showed downregulation of RKIP (Mean expression-1.47). Metastatic esophageal tissue exhibited significantly decreased expression of RKIP(Mean expression-0.4, p= 0.001*). CONCLUSIONS: Decreased RKIP expression is associatedwith the development of metastatic disease in esophageal adenocarcinoma, and RKIP mayfunction as a metastasis suppressor protein in esophageal cancer. We speculate that RKIPmay be an important prognostic indicator in patients with esophageal adenocarcinoma andmay help us in individualizing treatment regimens based on its expression levels.Comparison of RKIP expression in normal esophageal mucosa, Barrett's metaplasia andesophageal adenocarcinoma with and without metastasis

T1187

Nanoscale Alterations in Early Colon Carcinogenesis are Determined byCytoskeletal Dysregulation in Microscopically Normal MucosaYolanda Stypula, Dhwanil Damania, Hariharan Subramanian, Suhasini Joshi, Ashish K.Tiwari, Tina P. Ward, Dhananjay Kunte, Seema R. Gandhi, Mart DeLaCruz, Ramesh K.Wali, Hemant K. Roy, Vadim Backman

Our group has developed a novel optical technology, partial wave spectroscopic microscopy(PWS) that enables unprecedented quantification of the nanoscale cellular architecture. Wehave shown that PWS marker, disorder strength (Ld), is exquisitely sensitive to subtle

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genetic/epigenetic alterations of colonic field carcinogenesis (PNAS 2008). Indeed, Ld frombrushing of the uninvolved rectum was able to predict neoplasia elsewhere in the colon(Cancer Res 2009). We hypothesized that cytoskeletal dysregulation may be responsible forthese PWS changes that are sensed below the level of resolution of conventional microscopyMethods: We had previously shown that CSK ShRNA knockdown HT29 had a markedinduction in Ld (PNAS 2008) so this served as our model for pharmacological disruptionof cytoskeleton studies. We also performed studies on colonic mucosal scrapings from 14week old (premalignant) rats treated with the carcinogen, azoxymethane (AOM). PWSanalysis was performed with our prototype instrument as previously described. Results:The rise in Ld in the cytoplasm of the CSK knockdowns was abolished by targeting actinfilaments (cytochalasin) or microtubules (colchicine). Indeed, there was almost a completenormalization to wildtype values suggesting that the majority of the Ld effect was mediatedthrough the cytoskeleton. We confirmed that the CSK construct had a three-fold increasein Ld when compared to untransfected HT29 (see Table 1). With regards to the proteinsinvolved, we demonstrated that in the premalignant azoxymethane-treated rat mucosa (previ-ously demonstrated to have increased Ld), we noted a marked induction in EB1 (200% ±18% of age-matched saline animals). However, other cytoskeletal proteins such as P-cadherinwere unaltered. Conclusions: We demonstrate, for the first time that during colonic fieldcarcinogenesis there is a profound alteration in the cytoskeleton which gives rise to nanoscalechanges. While the mechanisms remain unclear, a candidate approach implicates EB-1, theAPC binding protein. This suggests that cytoskeletal dysregulation may potentially impactupon signaling (APC, β-catenin etc) along with other cellular processes dysregulated in earlycarcinogenesis. Moreover, this underscores the power of PWS to probe the nano-architectureand give both clinical (screening through field effect detection) and biological (role of alteredcellular nanostructure) insights.Table 1: Effect size of CSK shRNA cells compared to HT29 cells

T1188

The Human Omentum Houses Distinct Populations of Immune Cells WithPotent Anti-Tumor Properties That are Depleted in Colorectal CancerDonal B. O' Connor, Donal O'Shea, Cliona O'Farrelly, Lydia Lynch, Des Winter

Introduction: Tumor-infiltrating cytotoxic T cells are critical indicators for an effective anti-tumor immune response to colorectal cancer. The greater omentum has recently beenidentified as a site of accumulation of unique T lymphocyte subsets. Its role in anti-tumorimmunity has not been investigated. Aim: This study aimed to determine if the immunerepertoire of the omentum is distinct from other adipose tissue depots and to evaluate itspotential anti-tumor role against colorectal cancer. Methods: Following ethical approval andinformed consent, omental samples from healthy patients (n=8) and patients with colorectalcancer (n=8), were analysed by Flow Cytometry to characterise lymphocyte populations.Adipose tissue samples from subcutaneous tissue, mesenteric and pericolic fat (appendicesepiploicae) and matched peripheral blood were also examined. NKR+T cells were isolatedfrom omentum and blood of colorectal cancer patients by magnetic bead separation andtheir cytotoxicity against autologos cancer cells was measured using a flow based cytoxicityassay. Results: The omentum has a higher concentration of lymphocytes compared to otheradipose tissue depots. (Omentum: 2.46x104/g, Mesenteric fat: 1.27x104/g, Pericolic fat4.7x103/g, Subcutaneous fat: 8.73 x103/g). In particular, iNKT's (invariant natural killer Tcells) and NKR+T's(natural killer receptor positive T cells) were found at 14-fold and 5-foldhigher concentrations respectively, in omentum compared to other fat depots. iNKT's andNKR+T's were found at much higher concentrations than in peripheral blood.(100-fold and20-fold respectively) Omental iNKT's and NKR+ T's were significantly depleted in colorectalcancer patients. (p=0.004, p=0.001)No significant difference was observed in overall lympho-cyte numbers or proportions of other T cells. Despite their depletion in cancer, omentalNKR+ T cells effectively killed autologos tumor cells, but not uninvolved colon, even at lowratios. Conclusion: This study identifies the omentum as a distinct site of accumulation ofcytotoxic immune cells which are specifically depleted in CRC. We have also shown thatomental NKR+T cells kill autologos tumor effectively In Vitro. These findings imply a rolefor the omentum in colorectal tumor immunity.

T1189

Alcohol Promotes EMT in Colon Cancer Cells via an iNOS and MembraneRaft-Mediated MechanismChristopher B. Forsyth, Fredric S. Cohen, Artem Ayuyan, Lijuan Zhang, Maliha Shaikh

Alcohol consumption has been linked to an increased risk of colon, breast, and several otherhuman cancers yet the mechanisms are not established. Epithelial mesenchymal transition(EMT) is an established program in embryogenesis. Recent studies from many labs haveshown EMT may be reactivated in cancer cells and promote cancer cell metastases andinvasion. We have recently shown that alcohol promotes EMT characteristic changes incolon and breast cancer cells as well as normal intestinal epithelial cells through activationof the transcription factor Snail. The AIM of this study was to identify mechanisms throughwhich alcohol activates the key EMT transcription factor Snail and could therefore promotecancer through activation of EMT. Our HYPOTHESIS was that alcohol stimulation of indu-cible nitric oxide synthase (iNOS) activity in plasma membrane rafts was a key mechanismfor alcohol activation of Snail and EMT in cancer cells. Methods. Caco-2 human coloncancer cells were stimulated with physiological levels of alcohol (43mM, 0.2%).Snail activa-tion analysis was conducted using the Snail phosphoserine-246 (pS246) Ab and Ab tototal Snail. Activated Snail was assessed by western blotting of nuclear extracts as well asdeconvoluted z-stack immunofluorescent microscopy on whole cells. In addition, we usedour recently described novel methods for isolation of physiological membrane rafts usingno detergents and at 37°C followed by western blotting analysis of sucrose density gradientfractions. Results. Our data show that alcohol stimulates nuclear localization of activated p-S246 Snail by 30 min. We also show that siRNA knockdown of the PAK1 kinase prevents

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sthis Snail phosphorylation and nuclear localization. In addition, we show that a chemicalinhibitor of iNOS as well as siRNA iNOS knockdown prevent activation of Snail by alcohol.Finally, we show that at rest, Snail is associated with Flotillin-2 membrane rafts while iNOSis associated with Caveolin-1 rafts. PAK1 is not associated with the membrane rafts. Uponstimulation PAK1 is translocated to Flotillin-2 rafts along with iNOS and Snail and activatedSnail is translocated to the nucleus. Conclusions. Our data support a mechanism is whichFlotillin-2 membrane rafts form a signaling platform for alcohol-induced activation of Snailvia iNOS and PAK1.These are the first data to show iNOS activation of PAK1 and Snailand Snail association with membrane rafts by any stimulus and provide new insight intomechanisms regulating EMT as well as mechanisms through which alcohol may promotecolon cancer progression through EMT that could be targeted for therapy.

T1190

MicroRNA Modulation is Involved in the Progression Phase of ColonCarcinogenesis in the Azoxymethane-Treated Rat Model: Implications forNSAID ChemopreventionDhananjay Kunte, Ramesh K. Wali, Yolanda Stypula, Ashish K. Tiwari, Mart DeLaCruz,Tina P. Ward, Seema R. Gandhi, Hemant K. Roy

Background: Epigenetic events are critical in driving the progression from histologicallynormalmucosa to invasive colorectal cancers. Althoughmethylation has received considerableattention, recently interest is focused on microRNAs. While miRNAs have been shown tobe overexpressed or downregulated in CRCs, their global role, however, remains unclear.Moreover, no compelling data exists on the impact of miRNAs with well-established chemop-reventive agents such as NSAIDS. We, therefore, systematically investigated this at variousphases or carcinogenesis and chemoprevention with the well validated azoxymethane (AOM)rat model of colon carcinogenesis Methods: Male Fisher 344 rats were given two weeklyi.p. injections of either saline or azoxymethane (15 mg/kg; Sigma). AOM- rats were thenrandomized to either AIN76a diet alone or supplemented with 320 ppm of sulindac for 40weeks, after which they were euthanized. Total RNA was isolated from the uninvolvedcolonic tissue and tumors and the RNA was processed for miRNA microarray analysis usingAgilent arrays which analyzed >300 miRNAs. The arrays were scanned on an Agilent G2565Microarray Scanner and data analysis was done with Agilent Feature Extraction and GeneSpr-ing GX v7.3.1 software packages. Results: Comparative analysis of the differential miRNAexpression during colon carcinogenesis and during NSAID-mediated chemoprevention isgiven in table 1 with increase and decrease defined as >1.5 or <0.67 fold). We noted thatalteration of miRNAs levels was more evident in the tumor than the ininvolved mucosa,indicating that miRNA modulation is a marker of progression phase of carcinogenesis. TheNSAID sulindac modulated 16 of the miRNAs, although, in general, not the same ones thatwere involved in tumorigenesis. Conclusion: Our work provides the first global demonstra-tion of miRNA role in the AOM-treated rat model of colon carcinogenesis. Taken together,it suggests that miRNA dysregulation is involved in the “progression” rather than the initiationphase of colon carcinogenesis.We also demonstrate, for the first time, that thewell-establishedchemopreventive agent sulindac had a marked impact on miRNA expression but at an earlystage (uninvolved mucosa) indicating that chemoprevention is not simply a reversal of thecarcinogenic process but rather activating new molecular pathways.Table1: Number of miRNAs differentially expressed during CRC progression andchemoprevention

T1191

Intra-Tumoral Variability in Prostaglandin E2 Levels in Human ColorectalCancer Liver Metastases is Associated With Differences in NAD+ Levels andExpression of NAD+-Dependent 15-Prostaglandin DehydrogenaseAlastair Young, Gillian Hawcroft, Ritchie Chalmers, Giles Toogood, Mark Hull

Introduction Cyclooxygenase (COX)-2 (synthesis) and NAD+-dependent 15-prostaglandin(PG) dehydrogenase (15-PGDH; catabolism) are rate-limiting enzymes in control of tissuePGE2 levels. Their function may be affected by the tumour micro-environment (eg. hypoxia)leading to changes in local PGE2 concentration and altered cancer cell behaviour. Epithelial-mesenchymal transition (EMT) is thought to be crucial for tumour cell invasion and meta-stasis. EMT is promoted by PGE2 in several cell types In Vitro. We investigated the relationshipbetween PGE2 levels, COX-2 and 15-PGDH expression, and NAD+ levels in human colorectalcancer liver metastases (CRCLM), and the role of hypoxia and PGE2 in CRC cell EMT InVitro. Methods Paired central and peripheral tissue from 20 CRCLM were obtained tomeasure tissue PGE2 and NAD+ levels, as well as immunoreactive 15-PGDH and COX-2protein content. 15-PGDH activity was measured by a [3H]-PGE2 degradation assay in thepresence (1μM) or absence of exogenous NAD+. Parallel In Vitro studies used a LIM1863human CRC cell model of transforming growth factor (TGF)β-induced EMT and MCF-7human breast cancer cells. Results PGE2 levels were increased by a mean (95%CI) of 26%(1%-52%) in the centre of CRCLM relative to peripheral tissue (mean 762 vs 604 pg/mgprotein). Counter-intuitively, immunoreactive 15-PGDH protein levels were also increased(14% [3%-27%]) in the centre of tumours, as was 15-PGDH activity in the presence ofadded NAD+ (16% [3%-29%]). However, NAD+ levels were 59% (25%-72%) lower in thecentre of CRCLM than periphery (mean 192 vs 546 pmol/mg) suggesting inefficient 15-PGDH-dependent catabolism may explain raised PGE2 levels. By contrast, there were nosignificant regional differences in immunoreactive COX-2 protein levels. In central areas ofCRCLM, cancer cells exhibiting low E-cadherin expression (EMT marker) had higher expres-sion of 15-PGDH than neighbouring E-cadherin-positive cells. Our CRCLM tissue observa-tions were mirrored by LIM1863 cells undergoing EMT such that in hypoxic (1% O2)conditions LIM1863 cells contained significantly less NAD+ and exhibited higher 15-PGDH

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protein levels compared with normoxic cells. 15-PGDH activity was significantly decreasedin hypoxic MCF-7 cells compared with normoxic counterparts when NAD+ was present ata limiting concentration (no exogenous added). Furthermore, PGE2 (0.1-10 μM) promotedTGFβ-induced EMT in LIM 1863 cells in a concentration-dependent manner. ConclusionRegional differences in PGE2 levels and 15-PGDH expression/activity are apparent in humanCRCLM (possibly linked to a hypoxic micro-environment). PGE2 promotes TGFβ-inducedEMT in human CRC cells.

T1192

The Role of Transketolase-Like Proteins During Early Stages of ColorectalCarcinogenesisJeff Brasky, Ashish K. Tiwari, Seema R. Gandhi, Dhananjay Kunte, Yolanda Stypula,Ramesh K. Wali, Tina P. Ward, Mart DeLaCruz, Hemant K. Roy

Background: There is increasing evidence that metabolic activity modulates colorectal cancerrisk. Diabetes is a well known risk factor for colonic neoplasia. Intriguingly, a recent reportsuggested that glucose deprivation fostered development of K-ras mutational pathway (Yunet al., Science 2009) suggesting that metabolic hemostasis may be tightly regulated toevade colon cancer phenotype. However, the molecular features involved in these metabolicpathways remain incompletely elucidated. Few candidate proteins like transketolase likegenes (TKTL1&2) have been implicated in a variety of malignancies (bladder, head andneck etc). These regulate not only gycolysis but also ribose formation and hence relevantfor the DNA backbone, gene make up and repair. Since little is known about this criticalpathway in early colon carcinogenesis, we explored TKTL expression in a well-validatedazoxymethane (AOM)-treated rat model. Materials and Methods: Twenty Fisher 344 ratswere randomized to AOM (2 weekly injections of 15 mg/kg IP) or saline. Animals wereeuthanized at 14 weeks, a stage prior to the development of adenomas (~20 weeks). Beforescrapping, all colons were examined and demonstrated to be neoplasia free. RNA wasextracted using TRI reagent and protein lysates were collected using 1x cell lysis buffer fromthe colonic mucosal scrapings. TKTL2 expression was studied by real time PCR using TKTL2Taqman probe while TKTL1/2 expression was demonstrated on western blots using standardprotocols. Results: The real time PCR showed drastic reduction in TKTL2 expression (75%decrease) in AOM rats as compared to their age matched controls. Additionally, 30%reduction in TKTL1/2 expression was noted in protein expression in AOM rats on westernblot, suggesting that TKTL2 is the major TKTL isoform to be down regulated during colorectalcarcinogenesis.Conclusions:We demonstrate herein, for the first time, that TKLT expressionis lost early in colon carcinogenesis. The implications are multifold including the inductionof oxidative stress, decreased DNA base synthesis and glycolysis. These may contribute tothe increased mutagenesis that is characteristic of field carcinogenesis in both AOM ratmodel and human colorectal carcinogenesis. Further assessment of the role of TKTL isoformsas early events in colon carcinogenesis and potential targets for chemoprevention ishighly needed.

T1193

MicroRNAs Regulate 5-Fluorouracil Resistance in Human Colon Cancer CellsKen Kurokawa, Toshihito Tanahashi, Tutomu Iima, Yuta Yamamoto, Kensei Nishida,Kiyoshi Masuda, Yuki Kuwano, Shigetada Teshima-kondo, Masakazu Fukushima,Kazuhito Rokutan

Background and Aims: MicroRNAs (miRNAs) are one of the non-coding functional RNAsthat play a potential role in post transcriptional regulation of cellular proliferation, differenti-ation, and cell death. Given these features of miRNAs, several lines of evidence have suggesteda crucial role of distinct miRNAs in determining the drug sensitivity and resistance. Recently,several genes have been proposed to be related with 5-FU resistance, while there is noconsistent finding in the transcriptome analyses. In line with these novel pharmacologicalinsights, this study was designed to identify miRNAs involved in the resistance against 5-fluorouracil (5-FU). Methods and Results: Two human colon cancer cells (DLD-1/S andKM12C/S) and their 5-FU resistant lines (DLD-1/R and KM12C/R) were used. Cells weretreated with 1, 5, 10, 20, 30, and 60 μM 5-FU, and the live cells were counted up to 72hour with MTT assay. Although the parental cells were suppressed with 5 μM 5-FU, theresistant cells showed normal growth even in the presence of 60 μM 5-FU. Cell cycle analysiswith FACS showed that after the 5-FU treatment, the majority of DLD-1/S and KM12C/Scells moved to the S phase, and the G1 phase was decreased. However, 5-FU did not affectcell cycle pattern in DLD-1/R cells. KM12C/R responded to 10 μM or higher concentrationsof 5-FU and increased the cell numbers in S phase. To analyze the expression profiles ofmiRNAs, we used a miRNA microarray (Agilent) that covered a total of 723 human miRNAs.The duplicate results with the same RNA were highly correlated (0.999 - 0.993). Shown inthe hierarchical clustering, 57miRNAswere differentially expressed under the 5-FU treatmentin each cell. In particular, 30 miRNAs were over-expressed in the resistant cells (8 miRNAsfor DLD-1/R and 22 miRNAs for KM12C/R), but no common miRNA was found in bothcell lines. In DLD-1/R, the maximum change (1.80-fold) was observed in hsa-miR-19bderived from miR-17-92 cluster. In KM12C/R, the top change (2.04-fold) was hsa-miR-21.The expressions of two miRNAs were accurately validated by qRT-PCR. Interestingly, oneof the target genes of hsa-miR-19b was E2F7, a transcriptional factor regulating the cellcycle. Conclusions: Our results suggest that miR-19b and miR-21 may play an importantrole in the post-transcriptional regulation of cell cycle progression in the presence of 5-FU.This novel function may be related to the drug resistance.