Colon Cancer Samples Research Articles

Abstract 2907: Protein Kinase D1 expression attenuates colon cancer progression

Abstract Objective: Protein Kinase D1 (PKD1) is an important modulator of several signal-transduction pathways in benign and malignant human diseases. Currently the role of PKD1 in colon cancer, which is the second-leading cause of cancer-related deaths, is not well established. We have found previously that advance stage prostate cancer has suppressed expression. This led us to analyze PKD1 expression patterns in various other cancer tissue samples including colon cancer. Materials and Methods: Tissue microarrays containing colon cancer samples with corresponding normal tissues were used to investigate the expression profile of PKD1 using immunohistochemistry. To determine PKD1 mediated effects on oncogenic β-catenin signaling in colon cancer, human colorectal adenocarcinoma cell line SW480. Stable transformants of SW480 were created with GFP and SW480-PKD1-GFP, PKD1 overexpression leads to attenuation of β-catenin/T cell factor (TCF) transcription activity measured by luciferase reporter assays. To determine the effect of PKD1 overexpression on other cancer associated genes, RT-PCR array (that had 84 cancer related genes) analysis was performed in SW480-GFP and SW480-PKD1-GFP cells. Results: Our expression analysis determined a significant decrease in PKD1 expression in advanced Dukes stage (II, III and IV) colon cancer samples as compared to non-neoplastic colon samples. We have also noticed downregulation and aberrant localization of β-catenin in colon cancer samples compared to non-neoplastic colon samples. Therefore, we also determined the function of PKD1 in oncogenic β-catenin signaling in colon cancer. Our confocal microscopy analyses demonstrates that PKD1 up-regulation caused translocation of β-catenin from the nucleus to the plasma membrane. PKD1 overexpression also reduced β-catenin/TCF interaction in the nucleus and suppressed the expression of its downstream signaling proteins. Phenotypic changes in the stable transformants were measured via proliferation assays, anchorage independent growth, anchorage dependent growth, cellular motility and invasion. The overexpression of PKD1 markedly suppressed cell proliferation in SW480 cells. The SW480-PKD1-GFP cells formed fewer colonies compared with SW480-GFP cells. The SW480-PKD-GFP cells showed very minimal or no motility and invasion compared to SW480-GFP cells. Our RT-PCR array data shows that PKD1 overexpression also modulates expression of tissue inhibitor of metalloproteinase 3 (TIMP3), interferon α, interferon β and tumor necrosis factor receptor (TNFR). Conclusion: In conclusion, PKD1 is down regulated in colon cancer and its downregulation is correlated with aberrant β-catenin subcellular localization. Our data suggest that the altered PKD1 expression may induce β-catenin dependent and independent signaling in colon cancer and suppression of PKD1 may play a critical role in colon carcinogenesis and colon cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2907. doi:10.1158/1538-7445.AM2011-2907

Abstract 4020: Characterization of novel BARD1 splice variants in colon cancer

Abstract BARD1 is a BRCA1 binding partner with putative tumor suppressive properties. Aberrant splice variants of BARD1 have been detected in various cancers, and it has been postulated that the presence of splice variants is cancer-specific. BARD1 expression patterns and their correlation with clinical outcome have not been assessed in colon cancer. In this study, we determined the occurrence and role of BARD1 splice variants in colon cancer. We characterized novel BARD1 splice variants and quantified the mRNA expression levels of BARD1 splice variants and full-length BARD1 in primary colon cancers and their corresponding normal tissue. Further, full-length BARD1 protein expression was correlated with clinical outcome in primary colon cancer samples. Methods: We performed PCR on cDNA from 15 colon cancer samples and matched normal colon tissues using primer targets in the first and last exon of BARD1 mRNA. The PCR produced several bands, that were submitted for direct sequencing. In a second approach the bands were subcloned and subsequently sequenced. The sequences were analyzed and splice variants were characterized. Splice variant specific qPCR primers were designed and expression levels were quantified using optimized SYBR Green qPCR assays. Control assays were performed to assure good RNA/cDNA quality and optimal reaction conditions. In addition, mRNA expression analysis was performed on a cDNA array consisting of pooled cDNA from 16 different human tissues (prostate, heart, brain, testis, placenta, leukocytes, thymus, lung, colon, pancreas, skeletal muscle, liver, kidney, spleen, small intestine, ovary). Full-length BARD1 protein expression was assessed via immunohistochemistry in a separate cohort of 82 primary colon cancer samples. The stained slides were scanned and visualized using the Aperio ImageScope system. Expression levels were assessed and correlated with clinical outcome data. Results: In addition to the full-length BARD1 mRNA, we now find 19 distinct BARD1 splice variants in colon cancer, the majority of which have not been previously described. 18 of the 19 splice variants lack one or more complete exons, thus creating new and specific exon-exon boundaries. Contrary to previous assumptions, these splice variants can also be found in the adjacent normal colon tissue as well as in various other normal human tissues. While BARD1 splice variants account for the majority of BARD1 mRNA, distinct variants show a cancer-specific regulation pattern. Further, loss of full-length BARD1 protein is associated with a poor prognosis (p=0.02). Conclusion: BARD1 splice variants commonly occur in colon cancer and normal colon tissue, with distinct variants showing a cancer-specific expression pattern. Loss of the full-length BARD1 protein correlates with a poor prognosis in colon cancer, consistent with a tumor suppressive function. Taken together, we suggest that splice variant regulation may affect BARD1 function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4020. doi:10.1158/1538-7445.AM2011-4020

Abstract 1481: MUC13 expression enhances colon cancer progression

Abstract Background: Mucin, a family of high molecular weight glycoprotein has been implicated in a variety of cancers. MUC13 is a newly identified transmembrane mucin which has shown deregulated expression in gastric and ovarian cancers. However, limited information is available about the role of MUC13 in colon cancer progression. The present study investigated the expression profile, clinical relevance and functional significance of MUC13 expression in colon cancer progression. Materials and Methods: The MUC13 expression profile was determined by immunohistochemical (IHC) analysis using a novel MUC13 monoclonal antibody (PPZ0020) on a panel of colon cancer tissue microarrays containing non-malignant, colon cancer and liver metastasis tissue samples. Colon cancer cell lines which showed faint (SW480) and high (SW620) MUC13 expression were selected for over-expression and knockdown experiments, respectively, to determine the functional role of MUC13 in colon cancer progression. Functional studies, such as cell proliferation, colony formation, cell migration and cell invasion assays were performed in stable clones. The levels of Sonic hedehog (Shh), B cell moloney murine leukemia virus integration site 1 (Bmi-1) and Matrix metalloproteinase 1 (MMP1) were determined using real time PCR, Western blot and flow cytometry analyses. Results: Differential expression and sub-cellular localization of MUC13 was observed in cancerous and non-malignant tissues. MUC13 IHC analysis showed significantly higher membranous and cytoplasmic MUC13 expression in colon cancer and liver metastasis tissues compared to non-malignant tissue samples. Interestingly, liver metastasis tissue samples showed significantly higher nuclear MUC13 expression in addition to high membranous and cytoplasmic expression. The over-expression of MUC13 enhanced cell proliferation, colony formation, cell migration and cell invasion. In contrast, knockdown of MUC13 resulted in reduced cell proliferation, colony formation, cell migration and cell invasion. Additionally, over-expression of MUC13 increased the expression level of metastatic associated proteins such as Shh, Bmi-1 and MMP1 while knockdown of MUC13 decreased the expression level of these proteins. Conclusions: The present study suggests two important outcomes pertaining to MUC13 in colon cancer: (1) The aberrant expression of MUC13 could serve as a potential diagnostic/prognostic molecular tool for clinical use, and (2) MUC13 over-expression may enhance colon cancer carcinogenesis via modulation of metastatic proteins Bmi-1, Shh and MMP1. These data suggest a potential role of MUC13 in colon cancer progression and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1481. doi:10.1158/1538-7445.AM2011-1481

Protein Kinase A-mediated 14-3-3 Association Impedes Human Dapper1 to Promote Dishevelled Degradation

Wnt signaling regulates embryo development and tissue homeostasis, and its deregulation leads to an array of diseases, including cancer. Dapper1 has been shown to be a key negative regulator of Wnt signaling. However, its function and regulation remain poorly understood. In this study, we report that 14-3-3β interacts with human Dapper1 (hDpr1). The interaction is dependent on protein kinase A (PKA)-mediated phosphorylation of hDpr1 at Ser-237 and Ser-827. 14-3-3β binding attenuates the ability of hDpr1 to promote Dishevelled (Dvl) degradation, thus enhancing Wnt signaling. We further provide evidence that PKA-mediated Dpr1 phosphorylation may contribute to growth and tumor formation of colon cancer Caco2 cells. Finally, we show that cyclooxygenase-2 expression and PKA activation are positively correlated with Dvl protein levels in colon cancer samples. Together, our findings establish a novel layer of regulation of Wnt signaling by PKA via the 14-3-3-Dpr1-Dvl axis.

SNaPshot and StripAssay as Valuable Alternatives to Direct Sequencing for KRAS Mutation Detection in Colon Cancer Routine Diagnostics

Although direct sequencing is the gold standard for KRAS mutation detection in routine diagnostics, it remains laborious, time consuming, and not very sensitive. Our objective was to evaluate SNaPshot and the KRAS StripAssay as alternatives to sequencing for KRAS mutation detection in daily practice. KRAS exon 2-specific PCR followed by sequencing or by a SNaPshot reaction was performed. For the StripAssay, a mutant-enriched PCR was followed by hybridization to KRAS-specific probes bound to a nitrocellulose strip. To test sensitivities, dilution series of mutated DNA in wild-type DNA were made. Additionally, direct sequencing and SNaPshot were evaluated in 296 colon cancer samples. Detection limits of direct sequencing, SNaPshot, and StripAssay were 20%, 10%, and 1% tumor cells, respectively. Direct sequencing and SNaPshot can detect all 12 mutations in KRAS codons 12 and 13, whereas the StripAssay detects 10 of the most frequent ones. Workload and time to results are comparable for SNaPshot and direct sequencing. SNaPshot is flexible and easy to multiplex. The StripAssay is less time consuming for daily laboratory practice. SNaPshot is more flexible and slightly more sensitive than direct sequencing. The clinical evaluation showed comparable performances between direct sequencing and SNaPshot. The StripAssay is rapid and an extremely sensitive assay that could be considered when few tumor cells are available. However, found mutants should be confirmed to avoid risk of false positives.

MiniSOX9, a dominant-negative variant in colon cancer cells

Inherited and acquired changes in pre-mRNA processing have significant roles in human diseases, especially cancer. Characterization of aberrantly spliced mRNAs may thus contribute to understand malignant transformation. We recently reported an anti-oncogenic potential for the SOX9 transcription factor in the colon. For instance, the Sox9 gene knock out in the mouse intestine results in an excess of proliferation with appearance of hyperplasia. SOX9 is expressed in colon cancer cells but its endogenous activity is weak. We looked for SOX9 variants that may impair SOX9 activity in colon cancer cells and we discovered MiniSOX9, a truncated version of SOX9 devoid of transactivation domain as a result of retention of the second intron. A significant overexpression of MiniSOX9 mRNA in human tumor samples compared with their matched normal tissues was observed by real-time reverse transcriptase-PCR. Immunohistochemistry revealed that MiniSOX9 is expressed at high levels in human colon cancer samples whereas it is undetectable in the surrounding healthy tissues. Finally, we discovered that MiniSOX9 behaves as a SOX9 inhibitor, inhibits protein kinase Cα promoter activity and stimulates the canonical Wnt pathway. This potential oncogenic activity of the SOX9 locus gives new insights on its role in colon cancer.

Ubiquitination/deubiquitination and acetylation/deacetylation: Making DNMT1 stability more coordinated

In mammals, DNA methylation plays a crucial role in the regulation of gene expression, telomere length, cell differentiation, X chromosome inactivation, genomic imprinting and tumorigenesis1. DNA methylation patterns are established de novo by DNA methyltransferases (DNMTs) 3a and 3b, whereas DNMT1 maintains the parent-specific methylation from parental cells to their progeny2. After DNA replication, the new DNA strand is unmethylated. Thus with the mother methylated strand, the DNA is hemimethylated. The protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) recognizes and binds to the hemimethylated sites by its SRA domain. Then DNMT1 is recruited to the sites by the same domain, thereby it methylates the newly synthesized DNA strand3, 4. Increased DNMT1 abundance has been seen in many human cancers, and the roles of DNMT1 in tumorigenesis have been shown by some genetic knockout and RNA interference-mediated knockdown studies5, 6. Seeing that the mRNA abundance of DNMT1 contributes less to DNMT1 abundance, the stability of DNMT1 protein therefore plays an important role in human cancers7, 8. Ubiquitin-proteasome pathway is significant in the stability of DNMT18, but ubiquitin-mediated protein degradation can be enhanced or attenuated by some modifications like acetylation/deacetylation, protein methylation/demethylation, phosphorylation and S-nitrosylation9, 10, 11. Esteve et al demonstrated that SET7-mediated lysine methylation of DNMT1 decreased DNMT1 level by ubiquitin-mediated degradation10. Furthermore, an early study12 showed that HDAC inhibitors could induce degradation of DNMT1. These suggest that DNMT1-associated proteins may play key roles in DNMT1 stability. In a recent issue of Science Signaling, Du et al reported13 that some DNMT1-associated proteins can ubiquitinate/deubiquitinate and acetylate/deacetylate DNMT1. These modifications make the regulation of DNMT1 stability more coordinated (Figure 1). Figure 1 A model of posttranslational regulation of DNMT1 stability13. DNMT1 physically interacts with HAUSP, Tip60, UHRF1, HDAC1, and PCNA on chromatin. After the completion of DNA methylation in S phase, HAUSP dissociates from DNMT1 to enable DNMT1 degradation. ... They identified deubiquitinase HAUSP (herpesvirus-associated ubiquitin specific protease) as a DNMT1-associated protein. Knockout or knockdown of HAUSP increased DNMT1 ubiquitination and reduced its abundance, then they demonstrated the direct deubiquitination of DNMT1 in vitro by purified HAUSP recombinant proteins. They also found that overexpression of DNMT1-associated E3 ligase UHRF1 led to increased ubiquitination of DNMT1 and decreased abundance of DNMT1 mutant lacking the HAUSP interaction domain, but not the full-length protein. These results show the coordination between ubiquitination of DNMT1 by UHRF1 and deubiquitination by HAUSP. Furthermore, they found that knockdown of HDAC1 increased DNMT1 acetylation, and reduced DNMT1 abundance. Additionally, acetyltransferase Tip60 which was found to acetylate DNMT1 promoted its ubiquitination, then destabilized it. At last, Tip60 and HAUSP were found to regulate DNMT1 protein stability during the cell cycle. The following clinical samples of human colon cancer also revealed the correlation between the abundance of HAUSP and the abundance of DNMT1. Drugs targeting epigenetic modifications have been edging toward anticancer therapies. DNMT1 also provides a candidate anti-cancer target14. Although global genomic DNA hypomethylation and tumor suppressor genes hypermethylation have been frequently observed in different human cancers, this methylation change is not caused simply by increased levels of DNMT115. This paper shows that DNMT1, HAUSP, UHRF1, Tip60, HDAC1, and PCNA (proliferating cell nuclear antigen) can interact with each other, thus they regulate DNMT1 stability and activity by a more coordinated way. Consequently, the current or future epigenetic drugs such as 5-aza CdR (DNMT1 inhibitor), HDAC inhibitors, HAUSP inhibitors, and UHRF1 inhibitors may have a potential combination for effective cancer therapy. And equally, the drug interactions and side effects should be taken into account, as one of the inhibitors may affect the other targets.

Deregulated expression of sprouty2 and microRNA-21 in human colon cancer: Correlation with the clinical stage of the disease

Sprouty protein is a novel feedback regulator involved in downstream inactivation of several growth factor receptor pathways. Sprouty2 (Spry2) protein was shown to be downregulated in human cancers. High levels of microRNA-21 (miRNA-21) expression have been associated with poor survival and poor response to adjuvant chemotherapy in cancer patients. But the effect of Spry2 in human colon cancer remained unknown. Paired tumor and normal mucosa samples from patients were examined for their expression of Spry2 mRNA and miRNA-21 by real-time quantitative RT-PCR analysis. Our results show that Spry2 was downregulated in human colon cancer, and its expression levels were lower in advanced-stage tumors than in early-stage tumors. There was a negative correlation between the expression levels of Spry2 and miRNA-21. Furthermore, overexpression of Spry2 suppressed the growth and migration of colon cancer cells with a concomitant increase in PTEN expression and reduction of Akt and MAPK phosphorylation. Spry2 inhibited the growth and tumorigenesis of colon cancer cells in vivo. Conclusively, we show for the first time that Spry2 expression is downregulated and miRNA-21 is upregulated in the clinical samples of colon cancer, which correlates with clinical stage of disease. Thus, Spry2 functions as a tumor suppressor in colon cancer.See commentary: Sprouty keeps bowel kinases regular in colon cancer, while miR-21 targets Sprouty

Distribution of trace elements like calcium, copper, iron and zinc in serum samples of colon cancer – A case control study

The pathogenesis of a number of diseases like cardiovascular diseases, diabetes and cancer has been associated with changes in the balance of certain trace elements. In the present study we aimed to investigate the levels of trace elements like calcium, copper, iron and zinc in colon cancer patients in comparison with healthy controls. Serum samples were collected from 256 colon cancer patients and 180 healthy age and sex matched controls. Trace element levels were detected using commercially available kits and an Auto-Analyzer (ChemWell 2910, Awareness Technology, and USA). The concentrations of calcium, copper and iron were not significantly different in patients in comparison with healthy controls. The concentration of zinc was significantly lower in colon cancer patients (p=0.001) as compared to normal subjects. Deficiency of zinc may play a role in the development of colon cancer or may contribute to damage already underway. Zn may represent an independent risk factor for colon cancer and therefore a possible target for prevention.

Candidate driver genes in focal chromosomal aberrations of stage II colon cancer

Chromosomal instable colorectal cancer is marked by specific large chromosomal copy number aberrations. Recently, focal aberrations of 3 Mb or smaller have been identified as a common phenomenon in cancer. Inherent to their limited size, these aberrations harbour one or few genes. The aim of this study was to identify recurrent focal chromosomal aberrations and their candidate driver genes in a well-defined series of stage II colon cancers and assess their potential clinical relevance. High-resolution DNA copy number profiles were obtained from 38 formalin-fixed, paraffin-embedded colon cancer samples with matched normal mucosa as a reference using array comparative genomic hybridization. In total, 81 focal chromosomal aberrations were identified that harboured 177 genes. Statistical validation of focal aberrations and identification of candidate driver genes were performed by enrichment analysis and mapping copy number and mutation data of colorectal, breast, and pancreatic cancer and glioblastomas to loci of focal aberrations in stage II colon cancer. This analysis demonstrated a significant overlap with previously identified focal amplifications in colorectal cancer, but not with cancers from other sites. In contrast, focal deletions seemed less tumour type-specific since they also showed significant overlap with focal deletions of other sites. Focal deletions detected were significantly enriched for cancer genes and genes frequently mutated in colorectal cancer. The mRNA expression of these genes was significantly correlated with DNA copy number status, supporting the relevance of focal aberrations. Loss of 5q34 and gain of 13q22.1 were identified as independent prognostic factors of survival in this series of patients. In conclusion, focal chromosomal copy number aberrations in stage II colon cancer are enriched in cancer genes that contribute to and drive the process of colorectal cancer development. DNA copy number status of these genes correlates with mRNA expression and some are associated with clinical outcome.

Cys-141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines

Previously, we reported that human p53 is functionally inactivated by S-glutathionylation at Cys-141 during oxidative and DNA-damaging treatments. Here, we describe the presence of thiolated p53 and the dynamic nature of this modification in human tissues using unique and specific polyclonal antibodies raised against a 12-residue p53 peptide bearing a mixed disulfide at Cys-141. The affinity- purified antibodies (glut-p53) were sequence-specific in that they recognized the antigenic peptide but not the unthiolated peptide or a scrambled glutathionylated peptide in ELISAs. On immunoblots, the purified antibodies did not react with native p53 or recombinant p53 (rp53), but readily detected the glutathionylated or cysteinylated or ethanethiol-treated rp53 only under nonreducing conditions. Untreated HCT116 cells showed low levels of glut-p53, which increased markedly after H 2O 2, diamide, cisplatin, and doxorubicin treatments. Glut-p53 levels decreased sharply after cells were passed into oxidant-free medium, suggesting efficient dethiolation. The mutant p53 present in HT29 and T47D human cancer cells was also recognized. In vitro, the glut-p53 was rapidly degraded by rabbit reticulocyte lysates. Human prostate and prostate cancer tissues showed an abundant presence of glut-p53 in luminal epithelium, a site well known to generate ROS. Melanoma and colon cancer samples were also positive for glut-p53. The availability of the thiolation-specific antibodies should enhance our knowledge of p53 regulation in redox-perturbed states found in various diseases including cancer.

Aurora Kinase A Promotes Ovarian Tumorigenesis through Dysregulation of the Cell Cycle and Suppression of BRCA2

Aurora kinase A (Aurora-A) is known to regulate genomic instability and tumorigenesis in multiple human cancers. The underlying mechanism, however, is not fully understood. We examined the molecular mechanism of Aurora-A regulation in human ovarian cancer. Retrovirus-mediated small hairpin RNA (shRNA) was used to silence the expression of Aurora-A in the ovarian cancer cell lines SKOV3, OVCA432, and OVCA433. Immunofluorescence, Western blotting, flow cytometry, cytogenetic analysis, and animal assay were used to test centrosome amplification, cell cycle alteration, apoptosis, DNA damage response, tumor growth, and genomic instability. Immunostaining of BRCA2 and Aurora-A was done in ovarian, pancreatic, breast, and colon cancer samples. Knockdown of Aurora-A reduced centrosome amplification, malformation of mitotic spindles, and chromosome aberration, leading to decreased tumor growth. Silencing Aurora-A attenuated cell cycle progression and enhanced apoptosis and DNA damage response by restoring p21, pRb, and BRCA2 expression. Aurora-A was inversely correlated with BRCA2 in high-grade ovarian serous carcinoma, breast cancer, and pancreatic cancer. In high-grade ovarian serous carcinoma, positive expression of BRCA2 predicted increased overall and disease-free survival, whereas positive expression of Aurora-A predicted poor overall and disease-free survival (P < 0.05). Moreover, an increased Aurora-A to BRCA2 expression ratio predicted poor overall survival (P = 0.047) compared with a decreased Aurora-A to BRCA2 expression ratio. Aurora-A regulates genomic instability and tumorigenesis through cell cycle dysregulation and BRCA2 suppression. The negative correlation between Aurora-A and BRCA2 exists in multiple cancers, whereas the expression ratio of Aurora-A to BRCA2 predicts ovarian cancer patient outcome.

Circulating exosomes may provide a more sensitive platform to monitor disease progression compared to circulating tumor cells.

10580 Background: Circulating tumor cells (CTCs) have been used to monitor disease progression in patients with different types of metastatic cancer. However only 50% of metastatic breast, 57% of metastatic prostate, and 18% of metastatic colon cancer blood specimens have adequate levels of CTCs for clinical laboratory analysis. Levels of exosomes have recently been shown to correlate with tumor progression. Methods: Exosomes from 1 ml of plasma were isolated by ultracentrifugation. CD-81 antibodies were used to capture and measure the exosome level of breast cancer samples (n=14) and healthy controls (n=4). CTCs were measured for all samples using the Cell Search CTC test protocol. Subsequently, EpCam positive exosomes were captured from metastatic breast (n=10), prostate (n=2), and colon cancer (n=3) samples, and compared to healthy controls (n=7). RNA was extracted from these EpCam positive exosomes and microRNA-21 (miR-21) expression was quantified by a qRT-PCR. Results: The preliminary study of breast cancer samples established 11 of the 14 samples (78.6%) had CD-81 specific exosome levels significantly above the level found in the 4 healthy samples (p=0.002). Only 7 of the 14 (50%) specimens analyzed had more than 5 CTCs, the clinical threshold for metastatic breast cancer. Three cancer samples had CD-81 measured exosome levels below the average of normal samples, one of these had >5 CTCs. miR- 21 analysis of 15 additional metastatic cancer specimens, 5 of which had >5 CTCs, found miR-21 averaged 4.2 × 106, 4.82 × 106 and 5.05 × 106 copies in the breast, prostate, and colon cancer samples respectively. Conversely, the plasma specimens from healthy donors collected in EDTA tubes averaged 1.8 × 104 copies of miR21. Conclusions: This study suggests that exosome analysis from plasma samples may offer a greater opportunity to monitor and track disease than CTC analysis, while acknowledging that further study will be necessary to establish clinical definitions of exosome load. Furthermore, tumor-derived exosomes provide the ability to characterize tumor of origin miR content, which demonstrates additional opportunities for tumor-specific exosome-based biomarker analysis from a blood sample. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Caris MPI

Incidence and initial characterization of circulating tumor cells in different cancer entities.

e21083 Background: Circulating tumor cell (CTC) quantification has been approved for monitoring of blood from metastatic breast, colon and prostatic cancer patients. In other cancers little is known about the presence of CTCs. CTCs might represent a potential alternative to invasive biopsies and assessment of their receptor profile may be helpful in planning and monitoring treatment strategies. This study was undertaken to investigate prevalence and characteristics of CTCs in a variety of solid tumors. Methods: After informed consent, fifty consecutive 20 mL-peripheral blood samples were collected from 38 patients with different metastatic epithelial cancers (n = 30) or metastatic melanoma (n = 8). After lysis of erythrocytes, cells were enriched for tumor cells by CD45 depletion. Cells were stained for CD45 to exclude leukocytes from the analysis and for the epithelial markers EpCAM and Cytokeratin 7, 8 or the melanoma marker HMW-MAA/MCSP, respectively. In 16 samples from patients affected by epithelial cancers a co-staining for EGFR was carried out. Control tubes including appropriate isotypic controls were added. Cells were analysed on a FACSCanto II. CTCs were defined EpCAM+Cytokeratin+CD45- in case of epithelial cancers or as HMW-MAA/MCSP+CD45- in case of melanoma. Results: In blood from patients with epithelial cancers CTCs were detected in 20/40 samples (50%). The absolute number of CTCs ranged from 2 to 37 (median 3) per 10 mL of blood. Besides breast cancer (5/9) and colon cancer (3/11) samples, CTCs were also detected in ovarian (4/8), cervix (2/4), head and neck (3/3), gastric (2/3) and non-small cell lung cancer (1/2) samples. In ten out of 16 samples (62.5%) CTCs were positive for EGFR (4 breast cancer, 2 head and neck, 2 ovarian, 1 gastric and 1 cervix cancer sample). In blood from melanoma patients CTCs were detected in 6/10 samples (60%). The absolute number of CTCs ranged from 5 to 551 (median 19) per 10 mL of blood. Conclusions: CTCs can often be detected in blood from patients with metastatic epithelial cancer or melanoma. CTCs were more abundant in melanoma compared to epithelial cancers. Phenotypic characterization identified EGFR as potential target to eradicate the EGFR-positive pool of CTCs. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Merck Serono Merck Serono Merck Serono

Abstract 5342: Phage display selelction on tumor histological specimens

Abstract A method was developed to obtain phage displayed ligands that bind to a select population of cells in histological specimens of freshly harvested solid human cancers. It combines phage display panning with laser capture microdissection (LCM). This method allows selection of phage ligands bound to subpopulations of specific cells contained in tumor tissue on histological sections. Naïve phage scFv library was incubated directly on a histological section of human breast cancer that was snap frozen immediately after surgical resection. Tumor stromal cells were captured by LCM and bound phages were recovered by bacterial infection. Individual phage clones selected after three rounds of panning were evaluated for their binding ability by immunofluorescence staining on tumor tissue from the same patient. Based on DNA analysis, 20% of 3rd output clones represented a single clone. This clone selectively bound tumor stroma in the breast cancer specimen and did not bind the malignant cell population. This clone, expressed as a soluble scFv, showed no significant binding to normal tissues, 13 other breast cancers or 4 colon cancer samples. This method is applicable for selection of ligands to virtually any portion of a histological specimen amenable to LCM. This may speed the process of generating ligands to any subset of cells or noncellular feature present on histological specimens. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5342.

Abstract 2961: Expression of ZEB1, an E-cadherin repressor correlates with poor patient survival in colon cancer and mediates claudin-1 dependent repression of E-cadherin

Abstract Claudin family of transmembrane proteins are expressed in tissue specific manner and are regulated differentially in different cancer types. In colon cancer, we have reported dysregulated claudin-1 expression and cellular distribution which correlates with cancer progression especially metastases. However, the mechanism/s underlying claudin-1 dependent regulation of colon cancer remains largely unexplored. Initially, manipulation of claudin-1 in colon cancer cells resulted in reciprocal changes in E-cadherin expression, at transcriptional level. In the current studies, using a tightly regulated microarray analyses, we show that ZEB-1, an E-cadherin repressor changes upon manipulation of claudin-1 expression. However, the claudin-1 dependent changes in ZEB-1 expression were parallel to claudin-1 expression and reciprocal to E-cadherin expression. We have further confirmed this finding at the RNA and protein levels using semi-quantitative RT-PCR and immunoblot analyses. A causal role of ZEB-1 in the claudin-1 mediated E-cadherin regulation was determined by overexpressing full-length ZEB1 cDNA construct in SW620siRNA cells (where claudin-1 expression is stably inhibited and E-cadherin is expressed). ZEB-1 over-expression decreased E-cadherin expression in these cells. In parallel, we inhibited ZEB1 expression in SW480Claudin-1 cells (stably overexpressing claudin-1). Effective inhibition of ZEB1 expression (80-90%) resulted in increased E-cadherin expression even in presence of overexpressed claudin-1 and thus supported a causal role for changes in ZEB1/E-cadherin axis in Claudin-1 dependent changes. We further determined the expression of Claudin-1, E-cadherin and ZEB1 in matched colon cancer samples, using immunoblot analyses, and observed a parallel correlation between the expressions of Claudin-1 and ZEB1 and inverse correlation with E-cadherin expression. We further confirmed our findings using two large clinicogenomic databases representing patients from the Moffitt Cancer Center in Tampa, Florida and the Vanderbilt Medical Center in Nashville, TN (MCC and VMC). Claudin-1 and ZEB1 mRNA expression was noted to be higher in colorectal cancer versus adenomas, normal colon and carcinoma in situ in the VMC training and MCC test sets. Most strikingly, patients with higher than median ZEB1 expression showed significantly worse overall and disease-specific survival in 195 patients with colorectal cancer from MCC dataset compared to those who express lower than median levels of ZEB1. Further analysis is being conducted to determine if the levels of E-cadherin and ZEB1 are inversely proportional on a patient-patient basis. Taken together, our data supports that in colon cancer claudin-1, a tight junction protein, regulates E-cadherin expression through direct/indirect modulation of ZEB-1 expression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2961.

Abstract B29: Expression of BARD1 predicts outcome in colon cancer

Abstract Background: The BARD1/BRCA1 heterodimer has been implicated in several different pathways crucial for maintaining genomic integrity, DNA repair and cell survival. Complex functions have been ascribed to BARD1: It acts as a chaperone that recruits BRCA1 to the nucleus to form an ubiquitin E3 ligase complex involved in DNA repair and chromosomal stability. On the other hand, BARD1 may also promote cell survival by inhibiting BRCA1-dependent apoptosis through nuclear retention of BRCA1. Mutations in the BARD1 gene, as well as aberrant expression of BARD1 splice isoforms, have been associated with poor prognosis in breast and ovarian cancer. Here, we assess the occurrence and role of BARD1 splice isoforms in colon cancer formation. Methods: Total RNA from primary human colon cancer samples and matched normal colon tissue was extracted and reverse transcribed. Gene specific PCR primers were designed to analyze the occurrence and pattern of BARD1 splice variants in colon cancer. Splice variants were subcloned and sequenced. Protein expression of BRCA1 and BARD1 in paraffin-embedded cancer tissues was detected by immunohistochemistry and correlated with clinicopathologic parameters. Results: mRNA analysis of primary colon cancers confirmed a specific pattern of BARD1 splice isoforms distinct from normal controls. Certain isoforms appear to be tumor specific. Full length BARD1 is upregulated in non-metastatic cancer samples compared to matched normal tissue, which is in line with its anti-apoptotic function. Interestingly, full length BARD1 is dramatically downregulated in metastatic cancer tissue supporting its role as a tumor suppressor. Immunohistochemical analysis of colon cancer samples confirmed the loss of full length BARD1 in metastatic tissues on the protein level and revealed a significant correlation (p&amp;lt;0.05) between expression levels of BARD1 and outcome, in which low expression of BARD1 is associated with a worse prognosis. Conclusion: Our results suggest critical roles for the BRCA1 chaperone BARD1 in colon cancer formation. Alternative splice variants of BARD1 commonly occur in colon cancer tissue and expression of full-length BARD1 is differentially regulated along the cancer-metastasis axis affecting cancer outcome. We hypothesize that in colon cancer tissue, BRCA1 function critically depends on BARD1 expression. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B29

Optional splicing of T‐Cell Intercellular antigen‐1 (TIA‐1), a RNA‐binding Protein Regulates VEGF Expression in Colorectal Cancer

To determine the role of post‐transcriptional regulation of Vascular Endothelial Growth Factor (VEGF) in colorectal cancer we investigated the relationship between VEGF and the RNA binding protein TIA‐1. An endogenous splice variant of TIA‐1 was expressed in 66% of colon cancer samples and in cancer cells with high angiogenic VEGFxxx expression. This splice variant resulted in a truncated protein termed short TIA‐1 (sTIA‐1). Whereas full length TIA‐1 bound to the VEGFxxx isoform (RNA immunoprecipitation, MS2‐Maltose Binding Protein assay) there was no TIA‐1 binding in cells expressing sTIA‐1. siRNA knockdown of TIA‐1 resulted in higher expression of total VEGF. Using VEGF terminal exon luciferase reporter constructs, TIA‐1 reduced translation of the angiogenic (exon 8a) but not the anti‐angiogenic (exon 8b) isoforms.In summary, we show that over expression of the wild type TIA‐1 in adenocarcinoma cells expressing sTIA‐1 results in restoration of the anti‐angiogenic VEGF isoforms. This suggest that TIA‐1 can act as both a splicing and splice specific translational regulator of VEGF and alternative splicing of TIA‐1 could lead to enhanced angiogenesis in colorectal cancer. Supported by AICR.

A rapid, sensitive, reproducible and cost-effective method for mutation profiling of colon cancer and metastatic lymph nodes

BackgroundAn increasing number of studies show that genetic markers can aid in refining prognostic information and predicting the benefit from systemic therapy. Our goal was to develop a high throughput, cost-effective and simple methodology for the detection of clinically relevant hot spot mutations in colon cancer.MethodsThe Maldi-Tof mass spectrometry platform and OncoCarta panel from Sequenom were used to profile 239 colon cancers and 39 metastatic lymph nodes from NSABP clinical trial C-07 utilizing routinely processed FFPET (formalin-fixed paraffin-embedded tissue).ResultsAmong the 238 common hot-spot cancer mutations in 19 genes interrogated by the OncoCarta panel, mutations were detected in 7 different genes at 26 different nucleotide positions in our colon cancer samples. Twenty-four assays that detected mutations in more than 1% of the samples were reconfigured into a new multiplexed panel, termed here as ColoCarta. Mutation profiling was repeated on 32 mutant samples using ColoCarta and the results were identical to results with OncoCarta, demonstrating that this methodology was reproducible. Further evidence demonstrating the validity of the data was the fact that the mutation frequencies of the most common colon cancer mutations were similar to the COSMIC (Catalog of Somatic Mutations in Cancer) database. The frequencies were 43.5% for KRAS, 20.1% for PIK3CA, and 12.1% for BRAF. In addition, infrequent mutations in NRAS, AKT1, ABL1, and MET were detected. Mutation profiling of metastatic lymph nodes and their corresponding primary tumors showed that they were 89.7% concordant. All mutations found in the lymph nodes were also found in the corresponding primary tumors, but in 4 cases a mutation was present in the primary tumor only.ConclusionsThis study describes a high throughput technology that can be used to interrogate DNAs isolated from routinely processed FFPET and identifies the specific mutations that are common to colon cancer. The development of this technology and the ColoCarta panel may provide a mechanism for rapid screening of mutations in clinically relevant genes like KRAS, PIK3CA, and BRAF.Trial RegistrationClinicalTrials.gov: NSABP C-07: NCT00004931

RTSβ as a Novel 5-fluorouracil Resistance Marker of Colorectal Cancer: A Preliminary Study

Introduction: Colorectal cancer is the most common form of malignancy in Taiwan and the third leading cause of death from cancer, preceded only by lung and hepatic cancers. Colorectal cancer is typically treated by surgical intervention and/or chemotherapy and radiotherapy, if necessary. To date, 5-fluorouracil (5-FU) is the most commonly used anti-cancer chemotherapy drug. However, patients commonly experience resistance to the drug therefore limiting its efficiency. In this study, we measured the expression of rTSβ in human colon cancer as a novel 5-FU resistance marker. Materials and Methods: We collected 172 colon cancer samples from 4 different hospitals (including 21 pairs of colon cancer biopsies and 151 pathologic slides of colon cancer). In vitro, we measured the cytotoxicity of 5-FU and 5-FU plus leucovorin in H630 and H630-1 colon cancer cell lines. Results: The results revealed that rTSβ was expressed in 115 (66.9 %) pathology samples and that tumour expression was higher than in corresponding normal tissue. Survival rates of up to 5 years following treatment was significantly higher for patients without rTSβ expression than for those with rTSβ expression (P = 0.0023). In vitro, H630-1 (with rTSβ overexpression) had significantly higher IC50 of 5-FU than did H630. IC50of 5-FU decreased when leucovorin was added. Conclusions: Results indicate a close relationship between rTSβ expression and resistance to the drug 5-FU in human colorectal cancer. These results provide further evidence for rTSβ expression as a novel 5-FU resistance marker of colorectal cancer. Key words: Chemotherapy, Immunohistochemistry, Prognosis