Conventional Colorectal Carcinoma Research Articles

Identification of the epigenetic mechanism for colorectal cancer stem cell regulation through the comprehensive microRNA expression profiling of human colorectal cancer tissues.

42 Background: MicroRNAs (miRNAs) are key regulators of cancer stem cell (CSC) functions, including self-renewal and differentiation. However, little is known about miRNAs that regulate such properties in human colorectal cancer. Methods: We compared the expression levels of 754 miRNAs between paired samples of EpCAM+/CD44+ cancer cells (enriched in CSCs) and EpCAM+/CD44neg cancer cells (non-tumorigenic cancer cells (NTCs)) sorted in parallel from human primary colorectal cancers by multiplex semi-quantitative PCR. Results: Comparison of the expression levels of miRNAs in the CSCs and NTCs resulted in the identification of 10 miRNAs upregulated (miR-221, miR-218, miR-500 and miR-195) or downregulated (miR-185, miR-19a, miR-337, miR-10a, miR-21 and miR-24) in CSCs compared to NTCs. Among them, miR-221 was most highly expressed in CSCs; and its expression level was very low in NTCs and normal colon epithelial cells (n=6, p<0.05). Kaplan-Myer estimates and univariate and multivariate analyses showed that higher miR-221 expression was an independent prognosis factor for poorer outcome. Constitutive overexpression of miR-221 enhanced organoid-forming capacity of both conventional colorectal carcinoma cell lines and patient-derived xenografts (PDX) in vitro. Importantly, constitutive downregulation of miR-221 suppressed organoid-forming capacity in vitro and substantially reduced the tumorigenic capacity of CSC populations from PDX lines in vivo. miR-221 directly targeted Quaking (QKI) 5, the most abundant splicing isoform of the human QKI gene, and suppressed its protein level. Knockdown of miR-221 suppressed the clonogenicity of colorectal CSCs in vitro in a QKI-expression dependent manner and ability to form tumors in vivo. Conclusions: Our study reveals a mechanistic link between miR-221 and QKI and highlights their key role in regulating CSC properties in human colorectal cancer and proposes that miR-221 will be a promising marker and a specific therapeutic target for human colorectal CSCs.

Colorectal adenosquamous carcinoma: genomic profiling of a rare histotype of colorectal cancer

Colorectal adenosquamous carcinoma (ASC) is exceedingly rare, comprising less than 0.1% of all colorectal malignancies, and is characterized by an aggressive disease course, with a higher metastatic rate and worse outcome than conventional colorectal adenocarcinoma. A comprehensive molecular profile of this group of neoplasms is still lacking. A total of 22 cases of colorectal ASCs (with 22 primary lesions and 7 metastases matched with 4 primaries) were subject to NGS targeting 67 cancer-related genes (VariantPlex solid tumor; Archer). Mismatch repair (MMR), p53, and V600EBRAF status were also investigated by immunohistochemistry. In 28 of 29 (96.6%) ASC samples, at least one single-nucleotide variant (SNV) or copy number variation (CNV) was detected. Among the 22 primary tumors, the most frequently mutated genes were TP53 (59.1%), APC (40.9%), KRAS (27.3%), BRAF (13.6%), and GNAS (9.1%). Only 1/22 (4.5%) primary ASC was MMR-deficient (MMRd) and harbored a BRAF mutation. Limited differences in SNVs were observed between primary and metastatic diseases. This study sheds light on the molecular landscape of colorectal ASCs. According to our data, the genomic profile of colorectal ASC is similar to that of conventional colorectal carcinoma, with significant druggable genetic alterations. Further studies are required to understand the more aggressive clinical behavior of this neoplasm.

Yolk Sac Tumor in a Recurrence of Colonic Adenocarcinoma With Shared Mutations in APC and TP53 Genes: A Case Report

Only four cases of colorectal adenocarcinoma with a yolk sac tumor (YST) component have been reported in the English literature. No genetic investigation has been performed in these cases. We report a case of colorectal adenocarcinoma in which the recurrent tumor had a YST component. A 49-year-old woman presented with a pelvic tumor three years after endoscopic mucosal resection of sigmoid colon adenocarcinoma. The pelvic tumor consisted of an undifferentiated carcinoma component and a YST component. The serum alpha-fetoprotein level was elevated to 42 ng/mL. Treatment as conventional colorectal carcinoma produced some anticancer effects, but the patient died 14 months after the recurrence and 49 months after the EMR. With the help of the next-generation sequencing results of the recurrent tumor, APC c.835 - 8A > G and TP53 c.524G > A (p.R175H) mutations were identified by direct sequencing in both the primary and the recurrent tumors, confirming the relationship between the two metachronous tumors.

Molecular and clinicopathological features of colorectal adenocarcinoma with enteroblastic differentiation.

Colorectal adenocarcinoma with enteroblastic differentiation (CAED) is a rare subtype of colorectal malignancy with expression of enteroblastic markers (glypican 3, SALL4, AFP); however, the clinicopathological and epidemiological features are not fully elucidated. The aims of this study were to elucidate and establish the molecular and clinicopathological characteristics of CAED. In addition to three cases recently diagnosed as CAED, colorectal carcinoma (CRC) with expression of enteroblastic markers were selected by using immunohistochemistry (IHC) on tissue microarrays of 988 advanced CRC. We employed next-generation sequencing (NGS) and Sanger sequencing for the detection of genetic alterations. IHC for p53 and HER2, HER2-FISH and MSI status was also investigated. Survival analyses for clinicopathological parameters were performed using Kaplan-Meier methods. Thirty-nine cases (4.0%) were positive for at least one enteroblastic marker. Histological evaluation of the total of 42 cases revealed that 10 contained tumour cells with clear cytoplasm. Enteroblastic marker-positive cases had aggressive behaviour and poor prognosis. NGS revealed TP53 as the most frequently mutated gene. The rate of HER2-positive cases and MSI-H cases was 9.5% (four of 42) and 12.2% (five of 41), respectively. Among these 42 cases, there were no molecular and clinicopathological differences according to the presence of tumour cells with clear cytoplasm. Enteroblastic marker-positive CRC could be grouped together as CAED regardless of clear cell cytoplasm. Using this definition, the frequency of CAED is 4.0% and has a poorer prognosis than that for conventional CRCs. HER2 targeted therapy would be a meaningful treatment for CAED, and CAEDs contain both MSI-H and MSI-stable CRCs, although the MSS phenotype is dominant.

Patient-Derived In Vitro Models for Drug Discovery in Colorectal Carcinoma.

Lack of relevant preclinical models that reliably recapitulate the complexity and heterogeneity of human cancer has slowed down the development and approval of new anti-cancer therapies. Even though two-dimensional in vitro culture models remain widely used, they allow only partial cell-to-cell and cell-to-matrix interactions and therefore do not represent the complex nature of the tumor microenvironment. Therefore, better models reflecting intra-tumor heterogeneity need to be incorporated in the drug screening process to more reliably predict the efficacy of drug candidates. Classic methods of modelling colorectal carcinoma (CRC), while useful for many applications, carry numerous limitations. In this review, we address the recent advances in in vitro CRC model systems, ranging from conventional CRC patient-derived models, such as conditional reprogramming-based cell cultures, to more experimental and state-of-the-art models, such as cancer-on-chip platforms or liquid biopsy.

New role of the antidepressant imipramine as a Fascin1 inhibitor in colorectal cancer cells

Serrated adenocarcinoma (SAC) is more invasive, has worse outcomes than conventional colorectal carcinoma (CRC), and is characterized by frequent resistance to anti-epidermal growth factor receptor (EGFR) and overexpression of fascin1, a key protein in actin bundling that plays a causative role in tumor invasion and is overexpressed in different cancer types with poor prognosis. In silico screening of 9591 compounds, including 2037 approved by the Food and Drug Administration (FDA), was performed, and selected compounds were analyzed for their fascin1 binding affinity by differential scanning fluorescence. The results were compared with migrastatin as a typical fascin1 inhibitor. In silico screening and differential scanning fluorescence yielded the FDA-approved antidepressant imipramine as the most evident potential fascin1 blocker. Biophysical and different in vitro actin-bundling assays confirm this activity. Subsequent assays investigating lamellipodia formation and migration and invasion of colorectal cancer cells in vitro using 3D human tissue demonstrated anti-fascin1 and anti-invasive activities of imipramine. Furthermore, expression profiling suggests the activity of imipramine on the actin cytoskeleton. Moreover, in vivo studies using a zebrafish invasion model showed that imipramine is tolerated, its anti-invasive and antimetastatic activities are dose-dependent, and it is associated with both constitutive and induced fascin1 expression. This is the first study that demonstrates an antitumoral role of imipramine as a fascin1 inhibitor and constitutes a foundation for a molecular targeted therapy for SAC and other fascin1-overexpressing tumors.

MiR-221 Targets QKI to Enhance the Tumorigenic Capacity of Human Colorectal Cancer Stem Cells.

miRNAs are key players in the integrated regulation of cellular processes and shape many of the functional properties that define the "cancer stem cell" (CSC) phenotype. Little is known, however, about miRNAs that regulate such properties in human colorectal carcinoma. In this study, we compared the expression levels of 754 miRNAs between paired samples of EpCAM+/CD44+ cancer cells (enriched in CSCs) and EpCAM+/CD44neg cancer cells (with CSC depletion) sorted in parallel from human primary colorectal carcinomas and identified miR-221 as the miRNA that displayed the highest level of preferential expression in EpCAM+/CD44+ cancer cells. High levels of miR-221 expression were associated with Lgr5+ cells in mouse colon crypts and reduced survival in patients with colorectal carcinoma. Constitutive overexpression of miR-221 enhanced organoid-forming capacity of both conventional colorectal carcinoma cell lines and patient-derived xenografts (PDX) in vitro. Importantly, constitutive downregulation of miR-221 suppressed organoid-forming capacity in vitro and substantially reduced the tumorigenic capacity of CSC populations from PDX lines in vivo. Finally, the most abundant splicing isoform of the human Quaking (QKI) gene, QKI-5, was identified as a functional target of miR-221; overexpression of miR-221-reduced QKI-5 protein levels in human colorectal carcinoma cells. As expected, overexpression of QKI-5 suppressed organoid-forming capacity in vitro and tumorigenic capacity of colorectal carcinoma PDX cells in vivo. Our study reveals a mechanistic link between miR-221 and QKI and highlights their key role in regulating CSC properties in human colorectal cancer. SIGNIFICANCE: These findings uncover molecular mechanisms underlying the maintenance of cancer stem cell properties in colon cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5151/F1.large.jpg.

A Comprehensive Evaluation of Special AT-rich Sequence-binding Protein 2 (SATB2) Immunohistochemical Staining in Mucinous Tumors From Gastrointestinal and Nongastrointestinal Sites

Special AT-rich sequence-binding protein 2 (SATB2) is an accurate marker for conventional colorectal carcinoma (CRC), although its sensitivity and specificity in mucinous tumors from the colon and other sites remains unknown. The objective of this study is to evaluate the accuracy of SATB2 expression detected by immunohistochemical assay, as a marker of primary CRC in mucinous adenocarcinomas. SATB2 immunohistochemical stains were performed on whole sections from 63 conventional CRCs (controls), 47 mucinous CRCs (mCRC), and 182 noncolorectal mucinous tumors. SATB2 intensity was scored as 1 to 3 based on the estrogen receptor/progesterone receptor grading system, and the percent positive cells was scored in broad categories as follows: 0 (negative)≤5%, 1=5% to 49%, 2≥50%. An optimal sensitivity/specificity pairing (83% and 95%, respectively) was achieved in the mCRCs when the additive intensity and percent score was ≥3 (ie, intensity score+percent score=total score). Defining this total score (histologic score/"H score") as a "positive" result, the sensitivity of SATB2 for conventional CRC was 98% (62/63) versus 83% (39/47) for mCRCs (P=0.02); whereas 5% (9/182) of all noncolorectal mucinous tumors were considered positive. SATB2 especially demonstrated reduced specificity when applied to mucinous gastroesophageal and breast carcinomas, which showed significant expression in 27% and 9% of cases, respectively. In summary, SATB2 is a less sensitive marker of colorectal origin in mCRC compared with conventional CRC and shows significantly reduced specificity in mucinous gastroesophageal and breast primaries.

PO-415 New anti-migratory and anti-invasive effects of a fascin inhibitor on colorrectal cancer cells

IntroductionSerrated adenocarcinoma (SAC) is a histological subtype of colorrectal carcinoma characterised by its poor prognosis, prominent invasive front and over-expression of fascin, the key protein involved in actine bundling needed for cell migration and invasion. Given that the frequency of KRAS or BRAF mutations in SAC are higher than in conventional colorectal carcinoma, this tumour type is usually resistant to anti-EGFR therapy. For these reason, anti-fascin treatment could be an interesting aproach to treat SAC and other tumours over-expressing fascin. In this work we have charaterized the anti-migratory and anti-invasive properties of compound G2 (N-(1-(4-(trifluoromethyl)benzyl)−1H-indazol-3-yl)furan-2-carboxamide), a patented anti-fascin molecule and compared its effect with migrastatine, a typical fascin inhibitor.Material and methodsWe have used molecular modelling to predict the fascin aminoacids involved in G2 binding. A qPCR assay was carried to find out which from eigth different colorectal cancer cell lines expressed the highest amount of fascin. After cell viability assay, scratch and IBIDI assays were performed to evaluate the anti-migratory effect of the compounds. Immunofluoresce for fascin was used for assessing lamelipodia formation. Anti-invasive effect was evaluated using cell invasion assay Transwell with matrigel and a myoma organotypic invasion model.Results and discussionsMolecular modelling using blind docking calculations identified a region in fascin possibly involved in G2 binding. HCT116 cells expressed the highest fascin levels and its migration capacity was clearly reduced by migrastatin and G2, the latter even at lower concentrations. Still, G2 inhibited the migration of all the cell lines demonstrating that G2 affected fascin functional capacity.Invasion and confocal studies were performed with HCT116 cells and both inhibitors strongly abolished the protrusion of lamellipodium (p=0.003). The Transwell Matrigel invasion assay also evidenced the anti-invasive effects of migrastatin and G2. Myoma discs showed that both compounds were similarly able to significantly decrease both the invasion depth and invasion area of HCT116 cells at 50% (p<0.001).ConclusionThis study demonstrates an interesting anti-migratory and anit-invasive effect of G2 in a similar extent to migrastatin and provides significant evidence that G2 is an interesting candidate for further investigation/chemical modifications to develop new fascin-specific therapies for colorectal cancer.

Micropapillary colorectal carcinoma: clinical, pathological and molecular properties, including evidence of epithelial-mesenchymal transition.

Colorectal carcinoma (CRC) with micropapillary (MP) features has only been described recently and is still being characterized. We reviewed the clinicopathological and molecular features of 42 CRC with MP features. Twenty-nine cases were also evaluated for immunohistochemical evidence of epithelial-mesenchymal transition (EMT). The extent of MP features within our cohort ranged from 5% (13 cases) to 100% (one case). Twenty-seven cases featured prominent cribriforming with dirty necrosis in the non-MP component; nine displayed mucinous features. Twenty-four of 29 cases (83%) demonstrated evidence of EMT. Thirty-six cases (86%) showed advanced T-category (pT3 or pT4), 31 (74%) had lymph node metastases and 23 (55%) had distant metastases. Median overall follow-up was 36 months. Seventeen patients (40%) died of disease, with median survival of 23 months. Mutations were seen in 17 of 31 tested cases (55%), including 11 KRAS mutations and four BRAF V600E mutations. Microsatellite instability testing was performed on 21 cases; all were microsatellite-stable. Compared to a cohort of 972 conventional CRC, MP CRC was more likely to present as stage IV disease (P < 0.001), but patients with MP CRC showed no significant differences in overall survival after adjusting for stage. Micropapillary features in CRC portend a high likelihood of advanced local disease and distant metastases. MP CRC is often associated with a cribriform pattern elsewhere in the tumour and cystic nodal metastases with prominent necrosis. They also show frequent mutations in KRAS and BRAF. Immunohistochemical evidence of EMT is common in MP CRC.

Methylome profiling reveals functions and genes which are differentially methylated in serrated compared to conventional colorectal carcinoma.

BackgroundSerrated adenocarcinoma (SAC) is a recently recognized colorectal cancer (CRC) subtype accounting for 7.5–8.7 % of CRCs. It has been shown that SAC has a worse prognosis and different histological and molecular features compared to conventional carcinoma (CC) but, to date, there is no study analysing its methylome profile.ResultsThe methylation status of 450,000 CpG sites using the Infinium Human Methylation 450 BeadChip array was investigated in 103 colorectal specimens, including 39 SACs and 34 matched CCs, from Spanish and Finnish patients. Microarray data showed a higher representation of morphogenesis-, neurogenesis-, cytoskeleton- and vesicle transport-related functions and also significant differential methylation of 15 genes, including the iodothyronine deiodinase DIO3 and the forkhead family transcription factor FOXD2 genes which were validated at the CpG, mRNA and protein level using pyrosequencing, methylation-specific PCR, quantitative polymerase chain reaction (qPCR) and immunohistochemistry. A quantification study of the methylation status of CpG sequences in FOXD2 demonstrated a novel region controlling gene expression. Moreover, differences in these markers were also evident when comparing SAC with CRC showing molecular and histological features of high-level microsatellite instability.ConclusionsThis methylome study demonstrates distinct epigenetic regulation patterns in SAC which are consistent to previous expression profile studies and that DIO3 and FOXD2 might be molecular targets for a specific histology-oriented treatment of CRC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0128-7) contains supplementary material, which is available to authorized users.

Serrated adenocarcinoma: A new look into colorectal carcinoma in Indian population

Context: Serrated adenocarcinoma (SAC) is a recently described, distinct variant of colorectal carcinoma (CRC), accounting for about 7.5% of all CRCs and up to 17.5% of most proximal CRCs. It arises by serrated pathway characterized by early involvement of oncogenic BRAF mutations, excess CpG island methylation and subsequent low- or high-level DNA microsatellite instability and has a poorer prognosis than conventional colorectal carcinoma. Aims: The present study is an attempt to review and reclassify colorectal carcinomas into serrated and nonserrated type, and finds its incidence and associated features. Materials and Methods: Totally, 81 histopathologically diagnosed colorectal carcinomas during the period of 2001-2008 were reviewed. Histopathological criteria by Tuppurainen et al . was used to identify SAC and its features were studied. Results: Out of the total 81 cases of colorectal carcinoma comprising of mainly adenocarcinoma (81.48%), 5 cases of SAC were identified giving an incidence of 6.17%. Female preponderance (60%) was noted with mean age of 61.8 years. Most cases were located in cecum (60%). Serrated growth pattern was most frequent, noted in 60% cases, followed by mucinous in 20% and trabecular in 20%. Serrated mucinous carcinoma case also showed the presence of papillary rods, cell balls, focal clear cell change, and intraluminal dirty necrosis. Conclusion: This study attempts to define serrated colorectal adenocarcinoma in Indian population, where it has rarely been studied, using Tuppurainen et al .'s criteria. Since it is associated with poor prognosis its recognition, it is important for appropriate follow-up and therapeutic intervention.

Protein-protein interaction analysis of distinct molecular pathways in two subtypes of colorectal carcinoma.

The aim of this study was to identify the molecular events that distinguish serrated colorectal carcinoma (SCRC) from conventional colorectal carcinoma (CCRC) through differential gene expression, pathway and protein-protein interaction (PPI) network analysis. The GSE4045 and GSE8671 microarray datasets were downloaded from the Gene Expression Omnibus database. We identified the genes that are differentially expressed between SCRC and normal colon tissues, CCRC and healthy tissues, and between SCRC and CCRC using Student’s t-tests and Benjamini-Hochberg (BH) multiple testing corrections. The differentially expressed genes (DEGs) were then mapped to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and their enrichment for specific pathways was investigated using the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool with a significance threshold of 0.1. Analysis of the potential interactions between the protein products of 220 DEGs (between CCRC and SCRC) was performed by constructing a PPI network using data from the high performance RDF database (P<0.1). The interaction between pathways was also analyzed in CCRC based on the PPI network. Our study identified thousands of genes differentially expressed in SCRC and CCRC compared to healthy tissues. The DEGs in SCRC and CCRC were enriched in cell cycle, DNA replication, and base excision repair pathways. The proteasome pathway was significantly enriched in SCRC but not in CCRC after BH adjustment. The PPI network showed that tumour necrosis factor receptor-associated factor 6 (TRAF6) and atrophin 1 (ATN1) were the most central genes in the network, with respective degrees of node predicted at 90 and 88. In conclusion, the preoteasome pathway was shown to be specifically enriched in SCRC. Furthermore, TRAF6 and ATN1 may be promising biomarkers for the distinction between serrated and conventional CRC.

Clinicopathological Features of Serrated Adenocarcinoma Defined by Mäkinen in Dukes’ B Colorectal Carcinoma

Objective: Serrated adenocarcinoma (SAC), proposed as a new pathologic type, arises predominantly in the right side of the colon and has a poorer prognosis than conventional colorectal carcinoma. The prognosis of colorectal carcinoma is variable in Dukes’ B, so the aim of this study was to determine whether or not SAC has a poor prognosis in Dukes’ B. Methods: The study group comprised 64 patients who underwent surgery for colorectal carcinoma. We undertook a statistical analysis of the association of SAC and non-SAC with sex, age, histologic type, depth of tumor, location of tumor, venous invasion and lymphatic invasion. Results: SACs were encountered in 17.5% of cases (n = 11). SAC had a less favorable 5-year survival than non-SAC (p = 0.0396 log-rank, Kaplan-Meier). The factors that achieved statistical significance in the univariate analysis were subsequently included in a multivariate analysis and we found that SAC was an independent factor (p = 0.027). Conclusions: SAC has a poor prognosis and is not affected by other factors confirming that SAC is an independently less favorable prognostic factor.

Clear cell adenocarcinoma of the colon is a unique morphological variant of intestinal carcinoma: Case report with molecular analysis

Here we report a new case of clear cell adenocarcinoma (CCA) of the colon in a 54-year-old Caucasian man. Despite of the previous reported cases, the lesion was located in the right colon and was not associated with the conventional adenoma. We performed immunohistochemical and molecular analyses in order to explore whether the CCA had the molecular features generally associated with conventional colorectal carcinoma. The immunohistochemical and molecular analyses showed that the different morphology of CCA does not reflect a distinct biological entity but only an unusual morphological variant of intestinal carcinoma.

Nuclear ??-Catenin Expression Is Rare and its Potential Association With Short Survival in Colorectal Signet-Ring Cell Carcinoma

Colorectal signet-ring cell carcinoma (SRCC) is a rare cancer and the prognosis is usually very poor. The biologic pathways involved in its oncogenesis are unknown. beta-catenin, a key target in the Wnt-signaling pathway, is recognized to play an important role in the carcinogenesis in conventional colorectal carcinoma. This study explores the involvement of Wnt-signaling molecules beta-catenin and cyclin D1, cell cycle regulators cyclin D3, proliferative index Ki-67, apoptotic index, and angiogenic indicator CD31 in 20 colorectal SRCC paraffin-embedded specimens. Results showed that there were 2 specimens with nuclear beta-catenin and higher expression of cyclin D1 than the remaining 18 specimens. Surprisingly, those 2 patients had a much shorter survival of 6 months than the remaining 15 patients, who had around 24 months. Moreover, all colorectal SRCC specimens had an overexpression of cyclin D1, cyclin D3, and Ki-67, as well as much more angiogenesis and apoptosis than adjacent normal epithelial tissues. The authors make the preliminary comment that nuclear beta-catenin is a rare phenomenon in colorectal SRCC, but the involvement of it may indicate a worse prognosis with shorter survival than colorectal SRCC without nuclear beta-catenin expression. Besides, overexpression of cyclin D1, cyclin D3, Ki-67, and increased angiogenesis and apoptosis may play a vital role in promoting colorectal SRCC development.