Colon Cancer Specimens Research Articles

Cortactin promotes colon cancer progression by regulating ERK pathway.

Cortactin is upregulated in various cancers including breast cancer, head and neck squamous cell carcinoma and gastric cancer. However, the role of cortactin in the pathogenesis of colon cancer remains unclear. mRNA expression of cortactin in colon cancer samples and cell lines was detected by quantitative real-time PCR (qRT-PCR), while protein expression of cortactin in colon cancer tissues and adjacent non-cancer tissues was assessed by immunohistochemistry. The role of cortactin in regulation of the proliferation of colon cancer derived cells were investigated both invitro and invivo. In the total of 60 paired colon cancer specimens, compared with the adjacent non-cancer tissues, the expression of cortactin mRNA was upregulated in 45 (75.0%). Immunohistochemical analysis showed significantly increased cortactin expression in colon cancer (42/60, 70.0%) compared to control tissues (18/60, 30.0%). Overexpression of cortactin promoted HCT116 cellular colony formation and tumor growth. Conversely, cortactin knockdown inhibited these effects in SW480 cells. Mechanistic analyses indicated that cortactin was able to activate the EGFR-ERK signaling pathway. Additionally, cortactin expression was associated with tumor size, tumor stages and lymphatic invasion, increased cortactin expression predicts poor prognosis in patients with colon cancer. In summary, cortactin demonstrated the promotive effect in human colon cancer cell growth and tumorigenicity. These results indicated that cortactin may serve as an effective target for gene therapy.

Investigation of methylation and protein expression of the Runx3 gene in colon carcinogenesis.

In the present study, the methylation and protein expression of the runt-related transcription factor 3 (Runx3) gene was detected in sporadic colorectal cancer, colonic adenoma and normal colon tissue to evaluate their clinical significance in colorectal carcinogenesis. A total of 34 colonic cancer specimens, 34 colonic adenoma specimens and 34 normal colonic tissue specimens were used in the study. The CpG island methylation status of the Runx3 gene was detected by methylation-specific polymerase chain reaction and the protein expression of Runx3 was detected by immunohistochemistry. The results showed that the rates of methylation of the Runx3 gene in colonic cancer and colonic adenomas were significantly higher than that in the normal colonic tissue (23.5, 20.6 vs. 0.0%; P<0.05). There was no significant difference in the percentage of methylation of the Runx3 gene between colonic adenoma and colonic cancer (P>0.05). The positive percentage of Runx3 protein expression was significantly lower in colonic cancer compared with colonic adenoma and normal tissue (17.7 vs. 61.8, 76.5%; P<0.05). Methylation of the promoter CpG islands of the Runx3 gene is an important genetic event of colon carcinogenesis and may be associated with an altered protein level of Runx3.

IL13 Receptor α2 Signaling Requires a Scaffold Protein, FAM120A, to Activate the FAK and PI3K Pathways in Colon Cancer Metastasis.

IL13 signaling through its receptor IL13Rα2 plays a critical role in colon cancer invasion and liver metastasis, but the mechanistic features of this process are obscure. In this study, we identified a scaffold protein, FAM120A (C9ORF10), as a signaling partner in this process. FAM120A was overexpressed in human colon cancer cell lines and 55% of human colon cancer specimens. IL13Rα2-FAM120A coimmunoprecipitation experiments revealed further signaling network associations that could regulate the activity of IL13Rα2, including FAK, SRC, PI3K, G-protein-coupled receptors, and TRAIL receptors. In addition, FAM120A associated with kinesins and motor proteins involved in cargo movement along microtubules. IL13Rα2-triggered activation of the FAK and PI3K/AKT/mTOR pathways was mediated by FAM120A, which also recruited PI3K and functioned as a scaffold protein to enable phosphorylation and activation of PI3K by Src family kinases. FAM120A silencing abolished IL13-induced cell migration, invasion, and survival. Finally, antibody blockade of IL13Rα2 or FAM120A silencing precluded liver colonization in nude mice or metastasis. In conclusion, we identified FAM120A in the IL13/IL13Rα2 signaling pathway as a key mediator of invasion and liver metastasis in colon cancer.

High-mobility group box 1 protein activating nuclear factor-κB to upregulate vascular endothelial growth factor C is involved in lymphangiogenesis and lymphatic node metastasis in colon cancer.

To investigate the roles of high-mobility group box 1 (HMGB1) protein in lymphangiogenesis and lymphatic node metastasis in colon cancer. Archival tumour specimens from patients with colon cancer were analysed in this retrospective immunohistochemical study. HMGB1, vascular endothelial growth factor C (VEGF-C) and podoplanin protein levels were analysed immunohistochemically. Invitro studies using the colon cancer cell line HCT116 were also undertaken to investigate the relationship between HMGB1, VEGF-C and nuclear factor (NF)-κB. Specimens from 70 patients with colon cancer were reviewed. The presence of positive HMGB1 immunohistochemical staining significantly correlated with lymphatic microvessel density, lymph node metastasis and VEGF-C immunohistochemical staining in colon cancer specimens. The presence of positive VEGF-C immunohistochemical staining significantly correlated with lymph node metastasis. The invitro studies demonstrated that HMGB1 upregulated VEGF-C mRNA and protein in a dose-dependent manner in HCT116 cells, and that this was mediated via NF-κB. HMGB1 immunohistochemical staining was significantly associated with lymphangiogenesis and lymphatic node metastasis in colon cancer. There was evidence that HMGB1 upregulates VEGF-C by activating NF-κB in a colon cancer cell line.

Chemokine (C-C Motif) Ligand 5 is Involved in Tumor-Associated Dendritic Cell-Mediated Colon Cancer Progression Through Non-Coding RNA MALAT-1.

Tumor micro-environment is a critical factor in the development of cancer. The aim of this study was to investigate the inflammatory cytokines secreted by tumor-associated dendritic cells (TADCs) that contribute to enhanced migration, invasion, and epithelial-to-mesenchymal transition (EMT) in colon cancer. The administration of recombinant human chemokine (C-C motif) ligand 5 (CCL5), which is largely expressed by colon cancer surrounding TADCs, mimicked the stimulation of TADC-conditioned medium on migration, invasion, and EMT in colon cancer cells. Blocking CCL5 by neutralizing antibodies or siRNA transfection diminished the promotion of cancer progression by TADCs. Tumor-infiltrating CD11c(+) DCs in human colon cancer specimens were shown to produce CCL5. The stimulation of colon cancer progression by TADC-derived CCL5 was associated with the up-regulation of non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1), which subsequently increased the expression of Snail. Blocking MALAT-1 significantly decreased the TADC-conditioned medium and CCL5-mediated migration and invasion by decreasing the enhancement of Snail, suggesting that the MALAT-1/Snail pathway plays a critical role in TADC-mediated cancer progression. In conclusion, the inhibition of CCL5 or CCL5-related signaling may be an attractive therapeutic target in colon cancer patients.

Tu1976 Statin Use After Diagnosis Improves Survival in Colorectal Cancer Patients

Background: The use of statins has been associated with a reduced incidence of colorectal cancer. Potentially, statins may also have an influence on survival of colon cancer patients when used after diagnosis. The primary aim of this observational study was to evaluate the influence of statin use after diagnosis of colon cancer on overall survival. The secondary aim was to investigate the molecular background in which statins are effective adjuvant therapeutic agents, specifically KRAS mutational status and Bone Morphogenetic Protein pathway functionality, as these are the two most promising and frequently mentioned potential molecular mechanisms. Methods: Data was derived from the PHARMO database network (PHARMO, Netherlands) and was linked to a colon cancer cohort (Eindhoven Cancer Registry). A Tissue Micro Array (TMA) consisting of 999 colon cancer specimens was constructed from patients who had a surgical resection between 2002 and 2008. Survival was analyzed with statin user status after diagnosis as a time-dependent covariate. Multivariate Poisson regression survival models were used to study the effect of statins on overall survival (OS). The tumors were analyzed for SMAD4, BMPR1a, BMPR1b and BMPR2 expression, and KRAS mutations to perform stratified analyses for intact BMP signaling status and KRAS mutation status. Results: In this cohort 21.0% (210/999) of the patients were defined as statin users after colon cancer diagnosis. Statin use after diagnosis was significantly associated with an improved OS with an adjusted Rate Ratio (RR) of 0.67 (95% CI 0.51-0.87, P= 0.003). When stratified for intact BMP signaling status, survival benefit of statin use after diagnosis was stronger in tumors with intact BMP signaling (adjusted RR 0.46 (95% CI 0.29-0.74, P=0.001) than in tumors with a non-intact BMP signaling pathway (adjusted RR 0.75, 95% CI 0.53-1.06, P=0.106). Statin use after diagnosis was not associated with an improved OS in KRAS wild-type tumors (Adjusted RR 0.81, 95% CI 0.56-1.18, P=0.273) or KRAS mutated-tumors (Adjusted RR 0.59, 95% CI 0.35-1.03, P=0.062). Conclusion: Statin use after diagnosis is associated with an improved overall survival in colon cancer patients. This survival benefit is more prominent in tumors with intact BMP signaling. The survival benefit is independent of KRAS mutation status.

Role of microRNA-30c targeting ADAM19 in colorectal cancer.

MicroRNAs (miRNAs) are deregulated in a number of cancers including colorectal cancer. MiR-30c belongs to miR-30 family, and is involved in a variety of malignant diseases. In this study, we detected the expression of miR-30c in colon cancer cell lines and clinical colon cancer specimens. MiR-30c was shown to be dramatically down-regulated both in cell lines and cancer tissues. Additionally, miR-30c could inhibit cancer cell growth, migration and invasion in vitro. Consistently, stable over-expression of miR-30c inhibited the growth and lung metastasis of colon cancer cell xenografts in vivo. Furthermore, bioinformatics algorithm and luciferase reporter assay indicated ADAM19 as a direct target of miR-30c. Of interest, further experiments demonstrated that inhibition of ADAM19 by miR-30c partially mediated the anti-tumor effect of miR-30c. Overall, our study provides the new insight that miR-30c inhibited colon cancer cells via targeting ADAM19. Thus, miR-30c might serve as a promising therapeutic strategy for colon cancer treatment.

Prognostic significance of autocrine motility factor receptor expression by colorectal cancer and lymph node metastases

BackgroundAutocrine motility factor receptor (AMFR) has been linked to metastasis and tumorigenicity. The aim of this study was to evaluate expression and prognostic significance of AMFR in colorectal carcinoma. MethodsAMFR expression was evaluated in 127 colon cancer specimens, 131 rectal cancer specimens, and 47 colonic and 25 rectal corresponding lymph node metastases. Clinicopathological correlates of prognostic significance were established by univariate and multivariate analysis. Spearman's correlation determined the association of expression between cancers and their metastases. ResultsAMFR was over-expressed by 22% of colon cancers and 18% of rectal cancers. AMFR over-expression correlated significantly with improved disease-free survival (DFS) (P < .05) in colon cancer and decreased DFS in corresponding nodal metastases. In rectal cancer, AMFR over-expression significantly correlated with decreased overall survival, DFS, and disease-specific survival (P < .001, P = .031, P = .005, respectively) and decreased overall survival in corresponding metastases. ConclusionAMFR may serve as a molecular prognosticator for colon cancer and rectal cancer.

An EGFR/PI3K/AKT axis promotes accumulation of the Rac1-GEF Tiam1 that is critical in EGFR-driven tumorigenesis.

Epidermal growth factor receptor (EGFR) signaling regulates cell growth and survival, and its overactivation drives cancer development. One important branch of EGFR signaling is through activation of GTPase Rac1, which further promotes cell proliferation, survival and cancer metastasis. Here, we show that EGFR activates Rac1 via inducing the accumulation of its specific guanine nucleotide exchange factor, T-cell lymphoma invasion and metastasis 1 (Tiam1) in non-small-cell lung cancer and colon cancer cells. Conversely, elevated Tiam1 is required for EGFR-induced tumorigenesis. In human lung adenocarcinoma and colon cancer specimens, Tiam1 expression strongly correlates with EGFR expression. We further reveal that AKT, a key downstream protein kinase of EGFR, phosphorylates Tiam1 at several consensus sites, facilitates the interaction of Tiam1 with scaffold proteins 14-3-3 and leads to an increase of Tiam1 stability. Subsequently, Tiam1 is dephosporylated and destabilized by PP2A. Together, our study identifies a bidirectional (phosphorylation and dephosphorylation) regulatory mechanism controlling Tiam1 stability and provides new insights on how EGFR signaling triggers Rac1 activation and cancer development.

Zileuton, 5-lipoxygenase inhibitor, acts as a chemopreventive agent in intestinal polyposis, by modulating polyp and systemic inflammation.

PurposeLeukotrienes and prostaglandins, products of arachidonic acid metabolism, sustain both systemic and lesion-localized inflammation. Tumor-associated Inflammation can also contribute to the pathogenesis of colon cancer. Patients with inflammatory bowel disease (IBD) have increased risk of developing colon cancer. The levels of 5-lipoxygenase (5-LO), the key enzyme for leukotrienes production, are increased in colon cancer specimens and colonic dysplastic lesions. Here we report that Zileuton, a specific 5-LO inhibitor, can prevent polyp formation by efficiently reducing the tumor-associated and systemic inflammation in APCΔ468 mice.Experimental DesignIn the current study, we inhibited 5-LO by dietary administration of Zileuton in the APCΔ468 mouse model of polyposis and analyzed the effect of in vivo 5-LO inhibition on tumor-associated and systemic inflammation.ResultsZileuton-fed mice developed fewer polyps and displayed marked reduction in systemic and polyp-associated inflammation. Pro-inflammatory cytokines and pro-inflammatory innate and adaptive immunity cells were reduced both in the lesions and systemically. As part of tumor-associated inflammation Leukotriene B4 (LTB4), product of 5-LO activity, is increased focally in human dysplastic lesions. The 5-LO enzymatic activity was reduced in the serum of Zileuton treated polyposis mice.ConclusionsThis study demonstrates that dietary administration of 5-LO specific inhibitor in the polyposis mouse model decreases polyp burden, and suggests that Zileuton may be a potential chemo-preventive agent in patients that are high-risk of developing colon cancer.

Increased Sushi repeat-containing protein X-linked 2 is associated with progression of colorectal cancer.

Sushi repeat-containing protein X-linked 2 (SRPX2) is a novel chondroitin sulfate proteoglycan overexpressed in gastrointestinal cancer. Its role in tumor biology remains unknown. The aim of this study was to investigate the expression of SRPX2 in colorectal cancer and its potential association with cancer progression. The expression of SRPX2 and its clinicopathological significance was evaluated using immunohistochemistry in a tissue microarray including 88 colon cancer and pairing normal tissues. The impact of SRPX2 on behavior of colorectal cancer cells and possible mechanism was explored using gene transfection and silencing. Strong staining of SRPX2 was noted in 71 (80.7 %) of 88 colon cancer specimen and 30 (34.1 %) of 88 adjacent normal tissues (P < 0.001). The expression of SRPX2 was significantly correlated with histological differentiation grade (P = 0.003), infiltration depth (P = 0.003), and clinical stage (P = 0.006). The expression of SRPX2 was significantly higher in HCT116 than in HT29 and SW480 cells. Suppression of endogenous SRPX2 expression by small interfering ribonucleic acid (siRNA) in HCT116 cells resulted in significant reduction in the ability of cell proliferation, adhesion, migration, and invasion. Up-regulation of endogenous SRPX2 in SW480 cells significantly promoted the migration and invasion of SW480 cells. In addition, inhibition of SRPX2 by siRNA led to notable down-regulation of β-catenin, matrix metalloproteinase (MMP)-2, and MMP-9. These findings indicate that overexpressed SRPX2 exerts an oncogenic role in colorectal cancer. SRPX2 may promote the invasion of colorectal cancer through MMP-2 and MMP-9 modulated by Wnt/β-catenin pathway.

Bile acids regulate nuclear receptor (Nur77) expression and intracellular location to control proliferation and apoptosis.

Bile acids (BA) are endogenous agents capable of causing cancer throughout the gastrointestinal (GI) tract. To uncover the mechanism by which BAs exert carcinogenic effects, both human liver and colon cancer cells as well as mouse primary hepatocytes were treated with BAs and assayed for viability, genotoxic stress, and transcriptional response. BAs induced both Nur77 (NR4A1) and proinflammatory gene expression. The intracellular location of BA-induced Nur77 was time dependent; short-term (1-3 hours) exposure induced nuclear Nur77, whereas longer (1-2 days) exposure also increased cytosolic Nur77 expression and apoptosis. Inhibiting Nur77 nuclear export with leptomycin B decreased lithocholic acid (LCA)-induced apoptosis. Extended (7 days) treatment with BA generated resistance to BA with increased nuclear Nur77, viability, and mobility. While, knockdown of Nur77 in BA-resistant cells increased cellular susceptibility to LCA-induced apoptosis. Moreover, in vivo mouse xenograft experiments demonstrated that BA-resistant cells form larger tumors with elevated Nur77 expression compared with parental controls. DNA-binding and gene expression assays identified multiple survival genes (CDK4, CCND2, MAP4K5, STAT5A, and RBBP8) and a proapoptosis gene (BID) as Nur77 targets. Consistently, BA-induced upregulation of the aforementioned genes was abrogated by a lack of Nur77. Importantly, Nur77 was overexpressed in high percentage of human colon and liver cancer specimens, and the intracellular location of Nur77 correlated with elevated serum total BA levels in patients with colon cancer. These data show for the first time that BAs via Nur77 have a dual role in modulating cell survival and death. These findings establish a direct link between Nur77 and the carcinogenic effect of BAs.

Expanded KRAS and NRAS assays increase detection of mutations that predict for resistance to therapy in colorectal cancer patients.

519 Background: The selection of targeted therapies is guided by the analysis of somatic mutations. The identification of RAS activating mutations can be used to examine tyrosine kinase inhibitor therapeutic eligibility and prognosis. Beyond known mutations in RAS exon 2 (codons 12 and 13), the identification of additional mutations in RAS exons 3 and 4 (codons 61, 117 and 146) also predict for resistance to EGFR therapy in colorectal cancer (CRC). Meta-analysis supports screening for these additional mutations in any screening strategy prior to administration of EGFR mAb therapy in metastatic CRC patients (Sorich, MJ, et al., Ann Oncol, Aug 12, 2014). Methods: We expanded our existing allele-specific KRAS and sequencing-based NRAS assays to include codons 61, 117, and 146 and analytically validated these assays to CAP/CLIA standards. DNA, from microdissected colon tumor tissue that was wild-type for RAS exon 2, was tested for exon 3 and 4 mutations and included over 15 additional mutations. Results: Forty-two (9.1%) samples were identified as bearing either an exon 3 or 4 KRAS mutation amongst 461 colon cancer specimens. Exon 4 codon 146 mutations were more prevalent than three of the commonly screened exon 2 mutations: G12A, G12R, and G12S. Five (1.8%) of samples were identified as carrying exon 3 or 4 NRAS mutations amongst 272 colon cancer specimens. This included a single codon 146 mutation in exon 4. As is seen with exon 2, RAS mutations at exons 3 and 4 were mutually exclusive of activating BRAF mutations with ~10% of patients harboring V600E. The collection of additional data studying KRAS and NRAS mutation status is currently ongoing. Conclusions: The KRAS expanded coverage contributed an additional 5.5% to overall burden of specimens bearing mutations. The NRAS expanded coverage contributed an additional 1.8% to the mutational burden. These analyses in clinical cohorts support the observations made in a trial population (Douillard JV, et al. NEJM 369:1023-34, 2013). The expanded RAS coverage identifies additional patients unlikely to respond to EGFR-targeted therapies that would otherwise have been assessed as “no mutation detected” in using assays restricted to RAS exon 2.

NKD1 marks intestinal and liver tumors linked to aberrant Wnt signaling

The activity of the Wnt pathway undergoes complex regulation to ensure proper functioning of this principal signaling mechanism during development of adult tissues. The regulation may occur at several levels and includes both positive and negative feedback loops. In the present study we employed one of such negative feedback regulators, naked cuticle homolog 1 (Nkd1), to follow the Wnt pathway activity in the intestine and liver and in neoplasia originated in these organs. Using lineage tracing in transgenic mice we localized Nkd1 mRNA to the bottom parts of the small intestinal crypts and hepatocytes surrounding the central vein of the hepatic lobule. Furthermore, in two mouse models of intestinal tumorigenesis, Nkd1 expression levels were elevated in tumors when compared to healthy tissue. We utilized a collection of human intestinal polyps and carcinomas to confirm that NKD1 represents a robust marker of neoplastic growth. In addition, expression analysis of NKD1 in liver cancer showed that high expression levels of the gene distinguish a subclass of hepatocellular carcinomas related to aberrant Wnt signaling. Finally, our results were confirmed by bioinformatic analysis of large publicly available datasets that included gene expression profiling and high-throughput sequencing data of human colon and liver cancer specimens.

Abstract 3537: Suppression of miR-145 by long noncoding RNA RoR in colon cancer

Abstract Background: The human genome makes a large number of non-coding RNAs including long non-coding RNAs (lncRNAs) and microRNAs. Compared to well-characterized microRNAs, much less is known about lncRNAs. However, emerging evidence has implicated lncRNAs as master gene regulators; and they are often aberrantly expressed in a variety of human diseases including cancer. In particular, several studies suggest that lncRNAs can modulate the concentration and biological functions of microRNAs through a competing endogenous RNA (ceRNA) mechanism. We have previously demonstrated that lncRNA-RoR is a strong negative regulator of p53 in response to DNA damage and miR-145 is a direct transcription target for p53. Therefore, it is important to further characterize this p53 regulatory network involving lncRNA-RoR and miR-145, and to determine their role in tumorigenesis. Methods: To determine cell growth, we performed MTT assays. Protein levels from treated cells were analyzed by western blot. Real-time RT-PCR was used to determine relative expression of genes. Interaction between lncRNA-RoR and the key component (AGO2) of the RNA-induced silencing complex (RISC) was confirmed by RNA co-immunoprecipitation and RNA precipitation. Dual fluorescent in situ hybridization (FISH) was used to determine the relative levels of lncRNA-RoR and miR-145, and their co-localizations in colon cancer specimens. The effect of lncRNA-RoR on tumor growth was determined by a xenograft mouse model. Results: LncRNA-RoR suppressed miR-145 and upregulated c-Myc, one of major miR-145 target genes. In contrast, RoR-siRNA increased miR-145 expression and inhibited c-Myc. Of interest, miR-145 can also repress lncRNA-RoR expression. This reciprocal repression between lncRNA-RoR and miR-145 appeared to work through the CeRNA mechanism. Mechanistically, we showed that lncRNA-RoR directly interacted with AGO2, a key component of RISC complex, by RNA precipitation assays. This interaction was further confirmed by RNA immunoprecipitation with AGO2 antibody. In consistent with these finding, lncRNA-RoR stimulated proliferation in different cancer cells. Moreover, ectopic expression of lncRNA-RoR in HCT-116 cells significantly promoted tumor growth in a xenograft mouse model. Finally, experiments with dual FISH revealed an inverse correlation between lncRNA-RoR and miR-145 in colon cancer specimens. LncRNA-RoR was upregulated in colon cancer whereas miR-145 was downregulated in the same specimens. Conclusion: Taken together, these results suggest that lncRNA-RoR plays an oncogene role in colon cancer in part through suppression of the tumor suppressor miR-145. Thus, a better understanding of the p53-RoR-miR-145 regulatory system will provide new insight of colon cancer therapy. Citation Format: Jianguo Huang, Yin-Yuan Mo. Suppression of miR-145 by long noncoding RNA RoR in colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3537. doi:10.1158/1538-7445.AM2014-3537

Abstract 533: MicroRNA486-5p is a KRas target involved in promoting cell proliferation in lung cancer

Abstract KRAS-induced lung cancer is a very common disease, for which there are currently no effective therapies. Direct targeting of KRAS has failed in clinical trials and intense efforts are underway to identify KRAS targets that play a crucial role in oncogenesis. One promising KRAS-regulated pathway that has so far been overlooked is the microRNA pathway. Even though many microRNAs that regulate expression of KRAS are known, microRNAs regulated by oncogenic KRAS remain largely unknown. Our goal was to identify microRNAs regulated by oncogenic KRAS in lung cells that could contribute to the oncogenic phenotype. Due to a reported positive correlation between microRNA486-5p (miR-486-5p) expression and the presence of KRAS mutations in colon cancer specimens, we decided to investigate in lung cells whether KRAS regulates miR-486-5p. For that purpose we used an immortalized human primary lung epithelial cell line (SALEB) and its isogenic KRAS-transformed counterpart (SAKRAS), as well as lung cancer cell lines with either gain-of-function or loss-of-function of KRAS. We found, in all models tested, a positive correlation between expression of oncogenic KRAS and expression of miR-486-5p. We also found a negative correlation between expression of oncogenic KRAS and expression of miR-486-5p targets, including FoxO1, a tumor suppressor. In order to evaluate how miR-486-5p affects Ras-induced oncogenic properties, we transfected miR-486-5p inhibitor oligonucleotides into KRAS-positive lung cancer cell lines H358 and A549. Inhibition of miR-486-5p expression leads to reduced growth in clonogenic assays and reduced MTT viability. This reduction is not associated with increased cell death, but, as measured by 3H-Thymidine incorporation, it is associated with decreased cell proliferation. Interestingly, transfection of miR-486-5p double-stranded RNA mimic oligonucleotides into KRAS negative lung cancer cell line H1703 or into H358 cells with loss of function of KRAS by RNA interference leads to enhanced proliferation and clonogenicity. Taken together, these results indicate, not only that miR-486-5p is a KRAS target in lung cancer, but also that miR-486-5p acts as an oncomiR contributing to KRAS-induced cell proliferation. Further understanding of miR-486-5p targets could uncover novel pathways for KRAS-induced lung cancer therapy design. Citation Format: Mateus N. Aoki, Amanda C. P. Salviatto, Tatiana C. C. Lobo, Ana Cláudia O. Carreira, Mari C. Sogayar, Daniela S. Basseres. MicroRNA486-5p is a KRas target involved in promoting cell proliferation in lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 533. doi:10.1158/1538-7445.AM2014-533

Abstract 180: Interaction between bone marrow-derived mesenchymal stem cells and tumor cells induces expression of fibronectin in human colon cancer cells

Abstract Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to contribute to the formation of tumor stroma. We reported recently that in an orthotopic nude mouse model of human colon cancer, MSCs migrated to tumor stroma, where they differentiated into carcinoma-associated fibroblast (CAF)-like cells and promoted tumor growth and metastasis. However, the molecular mechanisms of this phenomenon have not been elucidated. We performed microarray analysis of gene expression in KM12SM cells and found that expression of fibronectin (FN) mRNA was increased markedly by direct co-culture of KM12SM cells with MSCs. FN, a glycoprotein in extracellular matrix and also a maker of epithelial-mesenchymal transition (EMT), has been implicated in the development of several types of human cancer. Many reports indicate that EMT is an important step during the cascade of tumor progression. However, the potential role of FN in carcinogenesis is not fully elucidated. We examined FN expression in surgical specimens of human colon cancer and colon adenoma by immunohistochemistry. A total of 87 tumors were analyzed. Levels of FN expression in tumor cells correlated with disease stage, and FN immunoreactivity was detected in invasive cancers, especially at the invasive edge, but less in mucosal cancers or adenomas. Furthermore, in an orthotopic nude mouse model of colon cancer, mRNA expression levels of FN were shown to be higher in the tumors produced by KM12SM cells mixed with MSCs than in the tumors produced by KM12SM cells alone.These findings suggest that the interaction between cancer cells and MSCs recruited into tumor stroma may play a role in tumor growth and metastasis via induction of FN expression in invasive colon cancer. Citation Format: Kei Shinagawa, Yasuhiko Kitadai, Yuichiro Tanaka, Ryo Yuge, Mieko Onoyama, Shinji Tanaka, Wataru Yasui, Kazuaki Chayama. Interaction between bone marrow-derived mesenchymal stem cells and tumor cells induces expression of fibronectin in human colon cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 180. doi:10.1158/1538-7445.AM2014-180

Abstract 587: Loss of colonic claudin-3 expression characterize colon cancer patients and predicts poor patient survival

Abstract The loss of cell-cell adhesion plays important role in promoting epithelial transformation and malignant cancer phenotype. In this regard, expression of claudin-3, a tight junction-integral protein, is reported to increase in prostate and ovarian cancers which contrast the paradigm. Notably, claudin-3 is highly expressed in the colon however its role in the regulation of colonic epithelial homeostasis or colon cancer remains unclear. In current studies, we have confirmed that claudin-3 is the most abundant claudin family member in the colon, and is present throughout the colonic crypt though it is more concentrated at the crypt top among the terminally differentiated colonocytes. We further found that in spontaneously differentiating Caco-2 cells, claudin-3 expression is markedly increased in terminally differentiated cells and correlates negatively with Vimentin and Smooth muscle actin (SMA) expressions. Dedifferentiation of the epithelial cells underlies colon carcinogenesis. In accordance, in a panel of 14 colon cancer cell lines, claudin-3 expression was either undetectable or low in 10 cell lines. In further analysis, using microarray data from a cohort of 268 {colorectal carcinoma (250) and normal (10)} colon cancer (CRC) specimens combined from the Vanderbilt Medical Center and the Moffitt Cancer Center, we found that claudin-3 expression level is significantly decreased in both colorectal adenoma (p&amp;lt;0.05) and carcinoma (p&amp;lt;0.001) versus normal mucosa. Further, IHC analysis of a colon cancer tissue microarray {TMA; colon tumors (67) and normal (10)} showed 23% of the tumors to be claudin-3 negative. In tumors, when claudin-3 was expressed, it was localized primarily in the cytoplasm compared to the surface expression in normal mucosa and its expression decreased with the increase in tumor grade. Overall, we observed a negative intensity score for claudin-3 with higher tumor grade. Further, based on the median expression levels, we found a significant and positive correlation of the greater levels of claudin-3 expression with patient survival (disease specific, disease free or overall survival; p&amp;lt;0.0008). We found a similar and significant suppression of claudin-3 expression in colon tumors induced in mice either spontaneously (by inactivation of the APC gene; APCmin mice) or pharmacological means [Azoxymethane (AOM) and Dextran sodium sulfate (DSS) administration). Notably, claudin-3 negative tumors retained E-cadherin expression suggesting that claudin-3 loss may be a rather sensitive marker for neoplastic colonic epithelial cells. Taken together, our data support strong correlation between claudin-3 expression, colonic epithelial differentiation and patient survival and highlights the tissue-specific regulation of claudin-3 and its efficacy as a potential colorectal tumor marker. Citation Format: Rizwan Ahmad, Zhimin Chen, Jian Wang, xi chen, Robert D. Beauchamp, Mary Kay Washington, Punita Dhawan, Amar B. Singh. Loss of colonic claudin-3 expression characterize colon cancer patients and predicts poor patient survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 587. doi:10.1158/1538-7445.AM2014-587

Abstract 283: Biomarker search using gene expression databases in a phase III, controlled clinical trial of postoperative adjuvant chemotherapy for stage III colon cancer (acts-cc): Correlation between gene expression and DNA copy number

Abstract Background: The ACTS-CC trial is a randomized, controlled phase III study designed to validate the noninferiority of S-1 to UFT/LV as adjuvant chemotherapy for stage III colon cancer and rectosigmoid cancer. A prospective biomarker search was performed as an auxiliary study of the ACTS-CC trial, using formalin-fixed, paraffin-embedded (FFPE) specimens obtained from 892 patients. The gene expression levels of 5-FU metabolizing enzymes and folate metabolizing enzymes and alterations of genome-wide copy numbers in tumor have been reported (2012 and 2013 Annual Meetings of AACR). In the present study, we performed correlation analyses of gene expression and DNA copy number data to characterize colorectal cancer and to prepare for a pre-planned analysis of predictive or prognostic biomarkers. Methods: FFPE specimens for this biomarker study were obtained after receiving informed consent from 892 (gene expression analysis) or 795 (DNA copy number analysis) of 1535 patients enrolled between April 2009 and January 2010. Macro-dissections were performed to extract total RNA and genomic DNA from 10-μm-thick FFPE specimens of colon cancer. Gene expression levels of 11 enzymes (TS, DPD, TP, OPRT, FPGS, GGH, DHFR, MTHFR, MTHFD, FOLRA, GART) related to 5-FU and folic acid metabolism were studied according to the Danenberg Tumor ProfileTM method (Shirota, Y. JCO 2001). Somatic copy-number alterations in cancer were analyzed by high-density SNP array analysis (Human 250K StyI array, Affymetrix, Santa Clara, CA). To detect copy-number alterations, the scanned image CEL files were imported into Partek Genomics Suite v6.6 (Partek Inc., St. Louis, MO, USA). We compared mRNA level, gene copy number and genome-wide segment copy number across a total of 161 colorectal cancers. Results: Gene expression levels were successfully estimated in 521-808 samples (84.0-90.0%, median 89.4%). Genome-wide DNA copy numbers profiles of 161 samples were obtained by GeneChip. Extracted genomic DNA from 616 of 777 samples that were not subjected to microarray analysis served as validation samples. Frequencies of copy number gain (&amp;gt;2.3) ranged from 0% to 57% for 11 genes, whereas frequencies of copy number loss (&amp;lt;1.7) ranged from 24.8% to 87%. Among 11 genes, the DNA copy numbers of GART, GGH, MTHFD1, and FPGS most strongly correlated with mRNA expression: correlation coefficients of these genes were 0.48, 0.45, 0.42, and 0.41, respectively. Gene expressions of OPRT or TP were influenced not only by their own DNA copy numbers but also by other genomic segments of 20q or 18q on genome-wide analysis. Conclusions: Gene expression of folate metabolizing enzymes positively correlated with DNA copy numbers in patients with stage III colorectal cancer. Such correlations may merit further evaluation to identify potential biomarkers. Citation Format: Hiroyuki Uetake, Toshiaki Ishikawa, Megimi Ishiguro, Shigeyuki Matsui, Kenichi Sugihara, ACTS-CC Study Group. Biomarker search using gene expression databases in a phase III, controlled clinical trial of postoperative adjuvant chemotherapy for stage III colon cancer (acts-cc): Correlation between gene expression and DNA copy number. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 283. doi:10.1158/1538-7445.AM2014-283

Abstract 4808: Aflibercept is highly active in clinically relevant, patient derived xenografts of colorectal cancer

Abstract Aflibercept (ziv-aflibercept in USA), a new fusion protein, targets vascular endothelial growth factor (VEGF)-A, VEGF-B, and placental growth factor (PlGF). Aflibercept + FOLFIRI is approved for treating metastatic colorectal cancer (mCRC) that is resistant to or has progressed following an oxaliplatin containing regimen. We investigated the pharmacological activity of aflibercept in CRC patient derived xenografts (PDX), most metastases-derived, and characterized PDX molecular and phenotypic properties. In 48 CRC PDX, Whole Exome Sequencing revealed: 85% APC, 79% TP53, 51% KRAS, 3% NRAS, 25% PIK3CA, 11% PTEN and 8% BRAF mutants. There was no significant association between angiogenic factors, tumor mutation profiles, and response to aflibercept. In tumors, protein levels of human (h) VEGF-A were significantly higher than mouse (m) VEGF-A, hPlGF, and mPlGF (Chiron et al, ESMO, 2013). Affymetrix probes detected VEGF-A and PIGF but not VEGF-B mRNA in most PDX. There was no significant association between expression of angiogenic factors and response to aflibercept. Histological sections of CRC tumors from PDX, xenografts created from cell lines, and patients’ tumor specimens indicated that stromal involvement in human tumors was recapitulated in PDX but not in tumors from cell lines. PlGF expression was analyzed by immunohistochemistry on colon cancer tissue arrays. A significant number of colon cancer specimens were positive for PlGF, expressed in tumor cells and stroma. Aflibercept resulted in anti-tumor activity in 47/48 CRC PDX models and complete tumor stasis in 31/48 models. Efficacy data from 5 PDX models responsive to aflibercept and CRC standard of care (SOC) drugs, all administered as monotherapies, are below. The robust activity of aflibercept in CRC PDX models that are sensitive to SOC drugs suggest that CRC patients would benefit from aflibercept treatment, independently of mutational status or expression of angiogenic factors. In Vivo Sensitivity Profile (optimal Tumor/Control (%))CRC PDX ModelPrior TreatmentsAflibercept 25 mg/kg, SC, 2x/wk5-FU100 mg/kg, IP, 1x/wkIrinotecan 50 mg/kg, IV, 1x/wkOxaliplatin 10 mg/kg, IP, Q2WCetuximab30 mg/kg, IV,1x/wkKRAS StatusCFX 1991Not available-2.531.51.856.342.1MutantCFX 2032Not available3.147.76.760.535.7MutantCFX 2048Not available-10.326.221.350.12.6Wild-typeCFX 20655-FU, leucovorin-11.920.614.471.82.2Wild-typeCFX 2067None6.253.045.158.914.2Wild-type Citation Format: Rebecca G. Bagley, Zakia Amalou, Marielle Chiron, Vincent Vuaroqueaux, Anne Caron, Nina Baltes, Heinz H. Fiebig. Aflibercept is highly active in clinically relevant, patient derived xenografts of colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4808. doi:10.1158/1538-7445.AM2014-4808