Colon Cancer Samples Research Articles

Abstract 1198: Presence of non-canonical tumorigenesis pathways in early-onset sporadic rectal cancer.

Abstract Chromosomal instability (CIN) caused by β-Catenin dependent aberrant activation of canonical Wnt signaling or Microsatellite instability (MSI) triggered by inactivation of mismatch repair (MMR) pathway, are the two major genetic instability pathways that drive classical age-related sporadic colorectal cancer (CRC). Activation of canonical Wnt signaling and MSI are primary tumor initiating events in about 80% and 15% of late-onset CRC cases, respectively. Canonical Wnt signaling is also reported as a secondary event in tumors primarily driven by MSI. These inferences are based on seminal studies that included a disproportionately greater number of colon tumors (as compared to rectal tumors). Recent genome-wide studies have suggested colon and rectal cancer to be a single entity, though several other studies appear to indicate otherwise. Exclusive studies on rectal cancer (RC), especially the early-onset sporadic subtype, have been fewer. Despite a recent trend of increased worldwide incidence, early-onset sporadic rectal cancer (EOSRC) is not well understood. We profiled canonical Wnt, KRAS and p53 (components of the classical colorectal carcinoma progression model) and MSI status in a panel of 298 colorectal cancer samples. 41% of EOSRC samples did not harbor Wnt or MSI pathways; the high proportion of a ‘double negative’ entity was neither identified in late-onset RC samples nor in colon cancer samples. KRAS mutation frequency was also significantly lower in EOSRC (24%). Since CIN is a hallmark of canonical Wnt activation driven CRC, we profiled genome-wide DNA copy number alterations (CNAs) in microsatellite stable EOSRC samples and surprisingly identified extensive chromosomal aberrations in both Wnt active and Wnt inactive subtypes suggesting the interesting possibility of presence of CIN in the absence of canonical Wnt activation. Several CNAs were detected exclusively in Wnt inactive samples (being absent in Wnt active samples) and were validated by quantitative PCR. As expected, a few CNAs, such as an amplification detected at 17q12 (ERBB2/GRB7), were present in both subtypes. Genome-wide transcript profiling performed in parallel revealed the elevated expression of genes located within the amplifications, validated by quantitative reverse transcription PCR (Q-RT-PCR). More importantly, aberrant activation of non-canonical signaling pathways was identified in a subset of the ‘double negative’ EOSRC samples, based on unsupervised (hierarchical clustering) and supervised (significance analysis of microarrays and gene set enrichment analysis) analyses of the transcriptome data. Elevation of non-canonical pathway gene transcripts was confirmed by Q-RT-PCR. Our study has therefore revealed presence of unique tumorigenesis pathways in EOSRC samples distinct from canonical pathways that drive late-onset CRC. Citation Format: Ratheesh Raman, Viswakalyan Kotapalli, Raju SR Adduri, Vasantha Kumar Bhaskara, Swarnalata Gowrishankar, Leena Bashyam, Ajay Chaudhary, Mohana Vamsy Chigurupati, Sujith Patnaik, Mukta Srinivasulu, Regulagadda Sastry, Subramanyeshwar Rao, Anjayneyulu Vasala, NarasimhaRaju Kalidindi, Jonathan Pollack, Sudha Murthy, Murali D. Bashyam. Presence of non-canonical tumorigenesis pathways in early-onset sporadic rectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1198. doi:10.1158/1538-7445.AM2013-1198

Abstract 4774: JC virus T-antigen-dependent activation of Wnt target genes and cell cycle progression in colon cancer.

Abstract Background: The Wnt signaling pathway is aberrantly activated in many different types of cancer including colon cancer. Transcription of target genes in the Wnt pathway promotes strong proliferation and differentiation signals in the cell. This transcription is mediated by β-catenin, the central signaling protein in the Wnt pathway. β-catenin targets include potent transcription factors and cell cycle regulatory proteins such as c-myc and cyclin D1. We have previously shown that a strong regulator of β-catenin-binding is the polyomavirus JC virus protein large T-antigen (TAg). According to sero-epidemiological studies, JCV establishes a latent infection in 70-90% of the general population by adulthood and TAg has been associated with numerous types of cancer in the CNS and GI tract. We and others have previously shown that TAg is expressed in colon cancer samples and has a significant association with β-catenin in the nuclei of colon cancer cells. Our hypothesis is that the viral protein T-antigen (TAg) interacts with β-catenin in colonic epithelial cells to promote transcription of β-catenin target genes particularly c-myc and cyclin D1, which affect cell cycle progression and lead to unchecked cellular growth. Materials and Methods: To test this hypothesis we transfected colon cancer cell lines with a plasmid containing the TAg gene and measured β-catenin target gene transcription. We used the TOPflash reporter construct to determine general β-catenin / TCF4-mediated promoter activity and reporter constructs for the β-catenin target genes c-myc and cyclin D1. We performed RT-qPCR and Western blot analysis to determine mRNA and protein levels of c-myc and cyclin D1 in TAg- and empty vector-transfected cells. We also utilized β-catenin siRNA or the dominant negative TCF4 plasmid to inhibit Wnt signaling in these groups and measured the same endpoints. Results: TCF-dependent promoter activity with a TOPflash reporter plasmid was increased in cells transfected with the TAg gene. This increased activity resulted in elevated levels of β-catenin target genes c-myc and cyclin D1. Moreover, inhibition of β-catenin, either by β-catenin siRNA or co-transfection with DN-TCF4 ablated this response. Finally, we found that transfection with TAg altered cell cycle distribution in these colon cancer cells with an increased proportion of cells in the G2/M phase. Conclusions: We found that the TAg protein activates the Wnt signaling pathway in our cellular model of colon cancer. This activation was determined to be β-catenin-dependent and mediated by TCF4 binding to promoter regions. Moreover, because Wnt pathway activation in these cells leads to cell cycle progression we suggest an oncogenic role for TAg in colon cancer. Considering the large proportion of the population seropositive for JC virus, it is imperative to elucidate its role in the oncogenesis of colon cancer. Citation Format: Michael J. Ripple, Amanda Struckhoff, Robin McGoey, Luis Del Valle. JC virus T-antigen-dependent activation of Wnt target genes and cell cycle progression in colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4774. doi:10.1158/1538-7445.AM2013-4774

Abstract 4158: MicroRNA-mediated regulation of Bmi1 expression in gastric and colon cancer cells.

Abstract Background: MicroRNAs (miRs) target the 3’-untranslated region (3’-UTR) of mRNA and induce translational suppression or mRNA degradation. Bmi1 is a member of the polycomb-repressive complex 1 (PRC1) that has an essential role in maintaining chromatin silencing, and is involved in the self-renewal of neuronal, haematopoietic and intestinal cells through repression of the INK4A-ARF locus. The regulatory mechanism of Bmi1 in cancer cells is still unknown. The aim of this study is to clarify the miR-mediated regulatory mechanism of Bmi1 expression in gastric and colon cancer cells. Methods and Results: We searched for miRNAs that might regulate Bmi1 using miRBase and selected 64 miRNAs as a candidate. In these miRNAs, we focused miR-30e-3p that was known to be down-regulated in colon, breast and esophageal cancer cells. First, we examined the expression of miR-30e-3p in matched cancer tissue and non-tumor tissue using formalin-fixed paraffin-embedded(FFPE) samples of gastric and colon cancer. We confirmed the expression of miR-30e-3p was significantly lower in cancer tissue than in non-tumor tissue. Next, we measured the expression of miR-30e-3p and Bmi1 by real time PCR and western blotting using gastric and colon cancer cell lines. The endogenous expression of Bmi1 and miR-30e-3p showed inverse correlation in each cell lines. Then we examined the effect of overexpression or down regulation of miR-30e-3p on the expression of Bmi1. The suppression of miR-30e-3p increased Bmi1 expression, and the overexpression of miR-30e-3p decreased Bmi1 expression. Conclusion: Our current study revealed that the expression of Bmi1 was affected by miR-30e-3p in gastric and colon cancer. Mir-30e-3p may be a therapeutic target for the treatment of gastric and colon cancer. Citation Format: Hidetaka Sugihara, Takatsugu Ishimoto, Masayuki Watanabe, Hideo Baba. MicroRNA-mediated regulation of Bmi1 expression in gastric and colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4158. doi:10.1158/1538-7445.AM2013-4158

Abstract 5586: Interpatient differences in colon tumors reflected in kinase activity and inhibition profiles related to KRAS and BRAF mutation status.

Abstract Background. Due to the biological heterogeneity, response to systemic therapy is variable in colon cancer. Chemotherapy improves median survival from 6-12 months to 18-24 months. Epidermal growth factor receptor (EGFR) antibodies have shown to improve survival, although modest, even further. KRAS mutations were identified as negative predictive markers for EGFR directed therapy. The response rate to EGFR targeted therapy remains low. Therefore there is an urgent need to find additional therapeutic options and accompanying biomarkers. The analysis of kinase activity in colon tumors may yield tumor-specific information on aberrant cell signaling pathways and may be usefull in a better patient stratification. Aim of this study is to generate overall kinase activity profiles from colon cancer tissues to explore if subtypes of this cancer show distinct kinase activity profiles. This can help to find new therapeutic options or predictive markers for response to therapy. Methods. Normal and tumorous colon tissue from patients who had given informed consent was fresh frozen after surgical resection. All tumor specimens were analyzed for BRAF and KRAS mutation status. Tissue cryosections were lysed in buffer with phosphatase and protease inhibitors. Tyrosine kinase (PTK) and serine/threonine kinase (STK) activity profiles of normal and tumor tissue were generated on PamChip® peptide microarrays comprising peptide sequences derived from known human phosphorylation sites. The ex vivo effect of kinase inhibitors on these kinase activity profiles was also determined. Peptide phosphorylation was monitored using fluorescently labeled antibodies. Data were analysed with Bionavigator software. Results. Tumor tissue has a higher PTK activity than normal tissue, whereas STK activity was equal to or lower than in normal tissue. STK activity profiles of 23 colon tumors could not be grouped according to mutation status, percentage of tumor tissue, differentiation grade or TNM classification. The STK activity profiles showed that within the group of wild type KRAS tumors, clearly different clusters were found. In the group with KRAS mutations (G12V, G12D, G12C, G12R) the STK activity profiles differed between the tumors. A subset of tumors, (two KRAS mutants, a BRAF mutant and a WT tumor) were tested ex vivo with a selection of 11 kinase inhibitors and showed different sensitivity to these inhibitors. Conclusions. Kinase activity and the response to inhibitors can be measured ex vivo in colon tumor tissue to capture the diversity in kinase activity and response to inhibitors between patients. The differences in kinase activity profiles of colon cancer samples could not be explained by subtypes according to mutation (KRAS, BRAF) status. Interesting subclusters could be identified using the kinase activity profiling providing a higher granularity than KRAS mutation scoring allows. Citation Format: Rosanne van den Oord, Hans Pruijt, Adriaan van den Brule, Peet Nooijen, Rik de Wijn, Monique Mommersteeg, Riet Hilhorst, Rob Ruijtenbeek. Interpatient differences in colon tumors reflected in kinase activity and inhibition profiles related to KRAS and BRAF mutation status. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5586. doi:10.1158/1538-7445.AM2013-5586

Abstract 5533: Novel targets and monoclonal antibodies for cancer therapy.

Abstract Cancer therapeutic targets is an extremely active research field in both academia and pharmaceutical companies. In our recent research activities we identified by a high-throughput immunohistochemistry screening of Tissue microarray (TMA), a panel of 89 novel candidate tumor markers for prevalent cancers representing breast, lung colon ovary and prostate carcinomas. The novel marker candidates were found over-expressed in one or more of the five tumors under analysis, with significant frequency. Three of them seems to be promising therapeutic targets for monoclonal antibody therapy, being exposed on the surface of cancer cells. They include: 1) a lectin binding protein, over-expressed in breast, lung and ovary cancer; 2) a protein involved in iron homoeostasis and over-expressed in breast, colon, lung and ovary cancers, 3) a cadherin homologous protein detected in colon, lung and ovary cancers. Gene silencing using siRNA technology and/or over-expression experiments using marker-encoding plasmids significantly alter cell proliferation, migration, invasiveness and clonal growth in vitro, indicating that expression of the three proteins confers cell phenotypes relevant for tumor progression. Highly specific murine monoclonal antibodies (mAbs) were generated against the three proteins and proved to bind the surface of cancer cells lines. Of particular interest is a murine mAb targeting the cadherin-like protein that, upon binding to the cell surface, is efficiently internalized by cancer cells, suggesting that it is amenable to the development of antibody-drug conjugates. This mAb did not show any relevant IHC cross-reactivity in the 35 human tissues requested by FDA to demonstrate antibody specificity. Furthermore this mAb significantly inhibited tumor growth in athymic nude mice bearing HCT15 and HT29 colon cancer xenograft models. Finally, IHC analysis of approximately 300 colon cancer clinical samples indicates that the antibody stains a large fraction of colon cancer samples, and gives intense membranous staining in specific patients’ group. Overall, data indicate that this antibody could be developed as a novel tool for a targeted therapy of colo-rectal cancer, alone or in combination with other treatments Citation Format: Alberto Grandi, Matteo Parri, Susanna Campagnoli, Renzo Nogarotto, Elisa De Camilli, Ilaria Naldi, Caterina Cinti, Paolo Sarmientos, Guido Grandi, Luigi Terracciano, Giuseppe Viale, Piero Pileri, Renata Maria Grifantini. Novel targets and monoclonal antibodies for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5533. doi:10.1158/1538-7445.AM2013-5533

Abstract 59: Identifying tumor driving signaling pathways for companion diagnostics using computational pathway models.

Abstract Introduction Targeted drug treatment requires reliable companion diagnostics for therapy selection. Genomic and transcriptomic data can provide input for this, provided tools exist to convert this complex data into meaningful clinical information. We develop computational models of oncogenic pathways, to assess which one drives tumor growth in an individual patient and what is the causing (epi)genetic defect. Computational pathway models Based on a selection of experimentally validated direct target genes, we built initial models of the Wnt, ER, AR and Hedgehog pathways, covering their transcriptional program. We have modeled each pathway by a Bayesian network, which interprets the target genes’ mRNA levels (Affymetrix U133Plus2.0), and infers a probability that the respective pathway is active in a certain sample. Model parameters are based on literature insights and experimental data. Results A first Wnt model, calibrated on cell line data, validated perfectly on 32 normal colon samples and 32 colon adenomas from patients (GSE8671). A second Wnt model, calibrated on these 64 patient samples, correctly predicted no Wnt activity in all 44 normal colon samples, and Wnt activity in 97 of 101 colon cancer samples from GSE20916. Next, we tested the second Wnt model on other cancer types. On 25 breast cancer cell lines from GSE12777 with known Wnt status, the model correctly identified the two samples with an active pathway. On two patient data sets (GSE12276, n=204; GSE21653, n=266) Wnt activity was predicted in a higher number of basal samples compared to other subtypes (p=0.021 and p=2.7e-5, respectively), in line with increasing evidence for Wnt activity in this subtype. Finally, tests on liver (GSE9843, GSE6764) and medulloblastoma sets (GSE10327) confirm the power of these models to predict Wnt pathway activity. A first ER model was calibrated on estrogen-deprived and -stimulated MCF7 cell lines (GSE8697). Applied on breast cancer cell line data from GSE21618, increased incidence of ER pathway activity was found in tamoxifen-sensitive cell lines compared to resistant ones. On breast cancer patient data (GSE12276, GSE9195, GSE6532) the model showed no pathway activity in ER- samples, and an active ER pathway in 26-38% of the ER+ samples. Within the latter group, model-predicted ER activity correlated with improved survival. Clinical utility studies to correlate ER activity to hormone therapy response are in progress. Finally, the AR model showed promising results on prostate cancer cell lines (GSE34211, GSE36133), as did the Hedgehog model on medulloblastoma samples (GSE10327). Conclusion Our computational pathway models predict functional activity of oncogenic pathways for an individual patient based on mRNA data, complementary to existing molecular and histopathology staining tests. Clinical utility for therapy response prediction is currently being validated with clinical partners. Citation Format: Wim Verhaegh, Henk van Ooijen, Marcia Alves de Inda, Kalyan Dulla, Ralf Hoffmann, Dianne van Strijp, Pantelis Hatzis, Hans Clevers, Anja van de Stolpe. Identifying tumor driving signaling pathways for companion diagnostics using computational pathway models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 59. doi:10.1158/1538-7445.AM2013-59

Efficient recovery of proteins from multiple source samples after trizol® or trizol®LS RNA extraction and long-term storage

BackgroundSimultaneous isolation of nucleic acids and proteins from a single biological sample facilitates meaningful data interpretation and reduces time, cost and sampling errors. This is particularly relevant for rare human and animal specimens, often scarce, and/or irreplaceable. TRIzol® and TRIzol®LS are suitable for simultaneous isolation of RNA, DNA and proteins from the same biological sample. These reagents are widely used for RNA and/or DNA isolation, while reports on their use for protein extraction are limited, attributable to technical difficulties in protein solubilisation.ResultsTRIzol®LS was used for RNA isolation from 284 human colon cancer samples, including normal colon mucosa, tubulovillous adenomas, and colon carcinomas with proficient and deficient mismatch repair system. TRIzol® was used for RNA isolation from human colon cancer cells, from brains of transgenic Alzheimer’s disease mice model, and from cultured mouse cortical neurons. Following RNA extraction, the TRIzol®-chloroform fractions from human colon cancer samples and from mouse hippocampus and frontal cortex were stored for 2 years and 3 months, respectively, at −80°C until used for protein isolation.Simple modifications to the TRIzol® manufacturer’s protocol, including Urea:SDS solubilization and sonication, allowed improved protein recovery yield compared to the TRIzol® manufacturer’s protocol. Following SDS-PAGE and Ponceau and Coomassie staining, recovered proteins displayed wide molecular weight range and staining pattern comparable to those obtainable with commonly used protein extraction protocols. We also show that nuclear and cytosolic proteins can be easily extracted and detected by immunoblotting, and that posttranslational modifications, such as protein phosphorylation, are detectable in proteins recovered from TRIzol®-chloroform fractions stored for up to 2 years at −80°C.ConclusionsWe provide a novel approach to improve protein recovery from samples processed for nucleic acid extraction with TRIzol® and TRIzol®LS compared to the manufacturer`s protocol, allowing downstream immunoblotting and evaluation of steady-state relative protein expression levels. The method was validated in large sets of samples from multiple sources, including human colon cancer and brains of transgenic Alzheimer’s disease mice model, stored in TRIzol®-chloroform for up to two years. Collectively, we provide a faster and cheaper alternative to the TRIzol® manufacturer`s protein extraction protocol, illustrating the high relevance, and wide applicability, of the present protein isolation method for the immunoblot evaluation of steady-state relative protein expression levels in samples from multiple sources, and following prolonged storage.

Nuclear expression of Glycogen synthase kinase‐3β and lack of membranous β‐catenin is correlated with poor survival in colon cancer

Dysregulation of Wnt/β-catenin signaling is a hallmark of colon cancer. Glycogen synthase kinase-3β (GSK-3β) can be a positive regulator of survival and proliferation of cultured colon cancer cell but its role in clinical colon cancer is unknown. Our objectives were to evaluate the role of GSK-3β in colon cancer. A tumor tissue microarray of primary colon cancers and metastases was used to evaluate expression and subcellular localization of GSK-3β and β-catenin. In total, 85 primary colon cancer samples were evaluated by immunohistochemistry. Immunoreactivity was correlated to known markers of adverse prognosis. Overall survival was the primary end-point. We found nuclear accumulation of GSK-3β in 39% (33/85) of evaluated tumors. Nuclear GSK-3β was significantly associated with shorter overall survival (p = 0.008), larger tumor size (p = 0.015), distant metastasis (p = 0.029) and loss of membranous β-catenin (p = 0.007). Loss of membranous β-catenin occurred in 37% (30/82) of the tumors and was associated with poor survival (p = 0.016). The combination of nuclear GSK-3β and lack of membrane β-catenin occurred in a total of 26% of the studied tumors (21/61) and was significantly and independently associated with poor prognosis. Our results suggest that nuclear expression of GSK-3β and loss of membrane β-catenin identify a subset of colon carcinomas with worse prognosis.

Detection of miR-34a Promoter Methylation in Combination with Elevated Expression of c-Met and β-Catenin Predicts Distant Metastasis of Colon Cancer

Here, we determined whether epigenetic inactivation of miR-34a and miR-34b/c genes may serve as a prognostic marker for distant metastases in colon cancer. Using a case-control study design of 94 primary colon cancer samples with and without liver metastases, we determined CpG methylation frequencies of miR-34a and miR-34b/c promoters, expression of miR-34a, and its targets c-Met, Snail, and β-catenin and their prognostic value. miR-34a methylation was detected in 45.1% (n = 42 of 93) of the samples and strongly associated with metastases to the liver (P = 0.003) and lymph nodes (P = 0.006). miR-34b/c methylation was detected in 91.9% of the samples (n = 79/86). A significant inverse correlation between miR-34a methylation and expression of mature miR-34a (P = 0.018) was detected. Decreased miR-34a expression was associated with upregulation of c-Met, Snail, and β-catenin protein levels (P = 0.031, 0.132, and 0.004), which were associated with distant metastases (P = 0.001, 0.017, and 0.005). In a confounder-adjusted multivariate regression model miR-34a methylation, high c-Met and β-catenin levels provided the most significant prognostic information about metastases to the liver (P = 0.014, 0.031, and 0.058) and matched pairs showed a higher prevalence of these risk factors in the samples with distant spread (P = 0.029). Finally, we obtained statistical evidence indicating that the simultaneous detection of these three markers has the highest prognostic value. Silencing of miR-34a and upregulation of c-Met, Snail, and β-catenin expression is associated with liver metastases of colon cancer. Detection of miR-34a silencing in resected primary colon cancer may be of prognostic value, especially in combination with detection of c-Met and β-catenin expression.

Quantitative analysis of FJ 194940.1 gene expression in colon cancer and its association with clinicopathological parameters

Aim of the studyThe FJ 194940.1 gene is located on chromosome 1 and consists of 6 exons and 5 introns. The gene undergoes alternative splicing and its isoforms appear during cancer development. Evidence suggests that expression of FJ 194940.1 splice variants relate to colorectal cancer progression. This paper discusses the quantitative analysis of the exon V expression level of FJ 194940.1 in colon cancer. The aim of the study is to carry out quantitative analysis by real-time PCR in a series of 102 colon cancer samples that had previously shown presence of exon V expression. To compare the exon V expression level with certain histological parameters and clinical staging of the neoplasm in order to assess its potential role as a prognostic factor in colon cancer.Material and methodsTissue specimens of colorectal cancer were obtained from the Oncological Centre of Lodz, Poland.Total RNA isolation was performed in accordance with the protocol enclosed in the Total RNA Prep Plus Minicolumn Kit (A&A Biotechnology, Poland). Reverse Transcriptase-PCR reaction was carried out using Enhanced Avian HSRT-PCR Kit, Sigma, according to the manufacturer's protocol.. Presence of cDNA in each sample was checked by PCR amplification of β-actin. Only samples showing the PCR product of this housekeeping gene were included in further tests. The amount of FJ 194940.1 transcript containing exon V was analysed by means of real-time PCR.ResultsExon V expression level is not significantly related to any clinicopathological features in colon cancer. However, there was a tendency towards a lower exon V expression level in a group of cases where vessel invasion was present (p = 0.0697). Additionally, the risk of death in patients with a low exon V expression level was more than two times higher when compared to patients with a high exon V expression level.ConclusionsFJ 194940.1 gene expression correlates with cancer progression independently of analysed clinicopathological parameters.

Hath1 Inhibits Proliferation of Colon Cancer Cells Probably Through Up-regulating Expression of Muc2 and p27 and Down-regulating Expression of Cyclin D1

Previous studies showed that Math1 homologous to human Hath1 can cause mouse goblet cells to differentiate. In this context it is important that the majority of colon cancers have few goblet cells. In the present study, the potential role of Hath1 in colon carcinogenesis was investigated. Sections of paraffin-embedded tissues were used to investigate the goblet cell population of normal colon mucosa, mucosa adjacent colon cancer and colon cancer samples from 48 patients. Hath1 and Muc2 expression in these samples were tested by immunohistochemistry, quantitative real-time reverse transcription -PCR and Western blotting. After the recombinant plasmid, pcDNA3.1(+)-Hath1 had been transfected into HT29 colon cancer cells, three clones were selected randomly to test the levels of Hath1 mRNA, Muc2 mRNA, Hath1, Muc2, cyclin D1 and p27 by quantitative real-time reverse transcription-PCR and Western blotting. Moreover, the proliferative ability of HT29 cells introduced with Hath1 was assessed by means of colony formation assay and xenografting. Expression of Hath1, Muc2, cyclin D1 and p27 in the xenograft tumors was also detected by Western blotting. No goblet cells were to be found in colon cancer and levels of Hath1 mRNA and Hath1, Muc2 mRNA and Muc2 were significantly down-regulated. Hath1 could decrease cyclin D1, increase p27 and Muc2 in HT29 cells and inhibit their proliferation. Hath1 may be an anti-oncogene in colon carcinogenesis.

Global mutational profiling of formalin-fixed human colon cancers from a pathology archive

The advent of Next-Generation sequencing technologies, which significantly increases the throughput and reduces the cost of large-scale sequencing efforts, provides an unprecedented opportunity for discovery of novel gene mutations in human cancers. However, it remains a challenge to apply Next-Generation technologies to DNA extracted from formalin-fixed paraffin-embedded cancer specimens. We describe here the successful development of a custom DNA capture method using Next-Generation for detection of 140 driver genes in five formalin-fixed paraffin-embedded human colon cancer samples using an improved extraction process to produce high-quality DNA. Isolated DNA was enriched for targeted exons and sequenced using the Illumina Next-Generation platform. An analytical pipeline using 3 software platforms to define single-nucleotide variants was used to evaluate the data output. Approximately 250 × average coverage was obtained with >96% of target bases having at least 30 sequence reads. Results were then compared with previously performed high-throughput Sanger sequencing. Using an algorithm of needing a positive call from all three callers to give a positive result, 98% of the verified Sanger sequencing somatic driver gene mutations were identified by our method with a specificity of 90%. In all, 13 insertions and deletions identified by Next-Generation were confirmed by Sanger sequencing. We also applied this technology to two components of a biphasic colon cancer, which had strikingly differing histology. Remarkably, no new driver gene mutation accumulation was identified in the more undifferentiated component. Applying this method to profiling of formalin-fixed paraffin-embedded colon cancer tissue samples yields equivalent sensitivity and specificity for mutation detection as Sanger sequencing of matched cell lines derived from these cancers. This method directly enables high-throughput comprehensive mutational profiling of colon cancer samples, and is easily extendable to enable targeted sequencing from formalin-fixed paraffin-embedded material for other tumor types.

The downregulation of Mcl-1 via USP9X inhibition sensitizes solid tumors to Bcl-xl inhibition

BackgroundIt has been shown in many solid tumors that the overexpression of the pro-survival Bcl-2 family members Bcl-xL and Mcl-1 confers resistance to a variety of chemotherapeutic agents. Mcl-1 is a critical survival protein in a variety of cell lineages and is critically regulated via ubiquitination.MethodsThe Mcl-1, Bcl-xL and USP9X expression patterns in human lung and colon adenocarcinomas were evaluated via immunohistochemistry. Interaction between USP9X and Mcl-1 was demonstrated by immunoprecipitation-western blotting. The protein expression profiles of Mcl-1, Bcl-xL and USP9X in multiple cancer cell lines were determined by western blotting. Annexin-V staining and cleaved PARP western blotting were used to assay for apoptosis. The cellular toxicities after various treatments were measured via the XTT assay.ResultsIn our current analysis of colon and lung cancer samples, we demonstrate that Mcl-1 and Bcl-xL are overexpressed and also co-exist in many tumors and that the expression levels of both genes correlate with the clinical staging. The downregulation of Mcl-1 or Bcl-xL via RNAi was found to increase the sensitivity of the tumor cells to chemotherapy. Furthermore, our analyses revealed that USP9X expression correlates with that of Mcl-1 in human cancer tissue samples. We additionally found that the USP9X inhibitor WP1130 promotes Mcl-1 degradation and increases tumor cell sensitivity to chemotherapies. Moreover, the combination of WP1130 and ABT-737, a well-documented Bcl-xL inhibitor, demonstrated a chemotherapeutic synergy and promoted apoptosis in different tumor cells.ConclusionMcl-1, Bcl-xL and USP9X overexpression are tumor survival mechanisms protective against chemotherapy. USP9X inhibition increases tumor cell sensitivity to various chemotherapeutic agents including Bcl-2/Bcl-xL inhibitors.

EZH2 is regulated by ERK/AKT and targets integrin alpha2 gene to control Epithelial–Mesenchymal Transition and anoikis in colon cancer cells

Epithelial–Mesenchymal Transition is a good example of cell plasticity. In tumorigenesis, this process has been associated with metastasis. Overexpression of EZH2 has been detected in most malignant human tumors, including colorectal carcinomas. Herein, we provide evidence supporting the idea that oncogenic Epithelial–Mesenchymal Transition in colon cancer cell models is partially controlled by epigenetic factors such as the transcription regulator EZH2. Evaluation of EZH2 mRNA and protein levels revealed overexpression in cell lines with metastatic traits. Analysis of EZH2 mRNA expression was expanded in clinical samples of colon cancer, and high level of EZH2 correlates with appearance of metastasis. Furthermore, inhibition of ERK and AKT pathways in metastatic colon cancer cell lines attenuates EZH2 overexpression. EZH2 promoter analysis illustrates presence of putative AP-1 binding sites and occupancy of transcription factors such as FRA-1 and C-JUN is demonstrated here on EZH2 promoter. Abrogation of EZH2 expression impairs the ability of colon cancer cells to move associated with anoikis in three-dimensional environment. Integrin alpha2 was identified to be a novel EZH2 target by chromatin immunoprecipitation and short hairpin RNA analysis. This study proposes that activation of ERK/AKT pathways and FRA1/C-JUN induce EZH2 overexpression, which results in Integrin alpha2 silencing. Our results show how deregulation of epigenetic factors and mechanisms can affect cancer cell aggressiveness and propose EZH2 as a potential metastasis marker and/or therapeutic target for colorectal cancer treatment.

Optimizing multi-dimensional terahertz imaging analysis for colon cancer diagnosis

Terahertz reflection imaging (at frequencies ∼0.1–10THz/1012Hz) is non-ionizing and has potential as a medical imaging technique; however, there is currently no consensus on the optimum imaging parameters to use and the procedure for data analysis. This may be holding back the progress of the technique. This article describes the use of various intelligent analysis methods to choose relevant imaging parameters and optimize the processing of terahertz data in the diagnosis of ex vivo colon cancer samples. Decision trees were used to find important parameters, and neural networks and support vector machines were used to classify the terahertz data as indicating normal or abnormal samples. This work reanalyzes the data described in Reid et al. (2011) (Physics in Medicine and Biology, 56, 4333–4353), and improves on their reported diagnostic accuracy, finding sensitivities of 90–100% and specificities of 86–90%. This optimization of the analysis of terahertz data allows certain recommendations to be suggested concerning terahertz reflection imaging of colon cancer samples.

CD66c is a novel marker for colorectal cancer stem cell isolation, and its silencing halts tumor growth in vivo

Despite the well recognized expression of the cell surface markers cluster of differentiation 44 (homing cell adhesion molecule) and CD133 (Prominin 1) on human colorectal cancer stem cells (CCSCs), these molecules do not appear to be effective targets for stem cell-directed therapies. Because the surface marker CD66c (also known as carcinoembryonic antigen-related cell adhesion molecule 6) has demonstrated promise as a therapeutic target in pancreatic malignancy, the authors evaluated its potential as a target for stem cell-directed treatment of colorectal cancer. First, the authors characterized CD66c expression by flow cytometry and immunohistochemistry in colon cancer samples and in normal colon tissues. Then, the coexpression of CD66c and CD133 was evaluated on putative CCSCs. CD66c expression also was measured in stem cell-enriched colon spheres. Finally, the effects of small-interfering RNA-mediated CD66c silencing on the in vitro and in vivo growth of Caco2 colon cancer cells were evaluated. CD66c expression was significantly higher in colon cancers than in contiguous normal colon tissues and paralleled cancer stage. CD66c was absent in CD133-positive cells that were isolated from normal colon, whereas its expression was brightest (CD66c(bright) ) in CD133-positive cells from colon cancer samples. In vitro experiments demonstrated that colon spheres were considerably enriched in a CD66c(bright) population in a fashion comparable to the enrichment observed in fresh liver metastases. In vitro proliferation and clonogenic potential were hampered when CD66c was silenced in Caco2 cells. Finally, in vivo xenograft experiments demonstrated that CD66c silencing almost completely abrogated the tumorigenic potential of Caco2 cells. CD66c(bright) expression was associated with colon cancer stem cells and CD66c silencing blocked tumor growth, thereby opening the way to a potential new treatment for colon cancer.

Epigenomics of Idiopathic Pulmonary Fibrosis

Epigenomics of Idiopathic Pulmonary Fibrosis

A Seven-Gene Signature Aggregates a Subgroup of Stage II Colon Cancers with Stage III

Colorectal cancer is one of the most common cancers in the world. Histological staging is efficient, but combination with molecular markers may improve tumor classification. Gene expression profiles have been defined as prognosis predictors among stage II and III tumors, but their implementation in medical practice remains controversial. Stage II tumors have been recognized as a heterogeneous group, and high-risk morphologic features have been used to justify adjuvant chemotherapy. We propose here the investigation of clinical features and expression profiles from stage II and stage III colon carcinomas without DNA mismatch repair defects. Two series of 130 and 66 colon cancer samples were obtained. Expression profiles were established on oligonucleotide microarrays and processed in the R/Bioconductor environment. Hierarchical, then supervised, analyses were successively performed by applying a data-sampling approach. A molecular signature of seven genes was found to cluster stage III tumors with adjusted p values lower than 10(-10). A subgroup of stage II tumors aggregated this cluster in both series. No correlation was found with disease severity, but the function of the discriminating genes suggests that tumors have been classified according to their putative response to adjuvant targeted or classic therapies. Further pharmacogenetic studies might verify this observation.

Increased Expression and Aberrant Localization of Mucin 13 in Metastatic Colon Cancer

MUC13 is a newly identified transmembrane mucin. Although MUC13 is known to be overexpressed in ovarian and gastric cancers, limited information is available regarding the expression of MUC13 in metastatic colon cancer. Herein, we investigated the expression profile of MUC13 in colon cancer using a novel anti-MUC13 monoclonal antibody (MAb, clone ppz0020) by immunohistochemical (IHC) analysis. A cohort of colon cancer samples and tissue microarrays containing adjacent normal, non-metastatic colon cancer, metastatic colon cancer, and liver metastasis tissues was used in this study to investigate the expression pattern of MUC13. IHC analysis revealed significantly higher (p<0.001) MUC13 expression in non-metastatic colon cancer samples compared with faint or very low expression in adjacent normal tissues. Interestingly, metastatic colon cancer and liver metastasis tissue samples demonstrated significantly (p<0.05) higher cytoplasmic and nuclear MUC13 expression compared with non-metastatic colon cancer and adjacent normal colon samples. Moreover, cytoplasmic and nuclear MUC13 expression correlated with larger and poorly differentiated tumors. Four of six tested colon cancer cell lines also expressed MUC13 at RNA and protein levels. These studies demonstrate a significant increase in MUC13 expression in metastatic colon cancer and suggest a correlation between aberrant MUC13 localization (cytoplasmic and nuclear expression) and metastatic colon cancer.

Expression Analysis of Aldehyde Dehydrogenase 1A1 (ALDH1A1) in Colon and Rectal Cancer in Association with Prognosis and Response to Chemotherapy

Aldehyde dehydrogenase 1A1 (ALDH1A1) has been described as a cancer stem cell marker and as a regulator of cellular chemoresistance. Therefore, ALDH1A1 has been suggested as potential biomarker to stratify patients into different risk categories for a "personalized" therapy approach. We have investigated the prognostic role of ALDH1A1 in primary colorectal cancer and its value in predicting response to chemotherapy in metastatic colorectal cancer. Immunostaining against ALDH1A1 was performed on a paraffin-embedded tissue microarray including 659 primary colon cancer samples and 338 rectal cancer samples. Likewise, tissue of 44 palliatively resected colorectal liver metastases on whole-mount tissue slides was immunostained against ALDH1A1. Cytoplasmic, nuclear, and stromal expression of ALDH1A1 was assessed and merged with histopathological and clinical data. Univariate and multivariate analysis revealed that cytoplasmic and stromal expression of ALDH1A1 is not significantly associated with prognosis either in colon or in rectal cancer. Furthermore, cytoplasmic expression of ALDH1A1 does not predict response to palliative chemotherapy in patients with metastatic diseases. Intriguingly, as a novel finding, nuclear expression of ALDH1A1 was observed in a small subgroup of patients with colon cancer and rectal cancer. In colon cancer, nuclear expression was significantly associated with shortened overall survival by univariate and multivariate analysis. Immunohistochemical expression analysis of ALDH1A1 in colon cancer is useful for the detection of nuclear expression in a small subpopulation of patients and is associated with shorter survival. Cytoplasmic expression fails to be of clinical relevance as prognostic or predictive marker in colorectal cancer.