Colorectal Carcinoma Tissue Samples Research Articles

BRAF mutations and the association of V600E with CD133 and CDX2 expression in a Pakistani colorectal carcinoma cohort

BackgroundDespite a high incidence of colorectal carcinoma, data regarding genetic aberrations in colorectal carcinoma (CRC) patients in Pakistan is scarce. This study aimed to determine the frequency of BRAFV600E mutations in colorectal carcinoma tissue in the Pakistani population and to associate BRAFV600E expression with CD133, a marker of colorectal stem cells, and CDX2 marker of differentiation.MethodsSanger Sequencing of exon 15 (426 bp) including the hotspot V600E was performed on formalin-fixed-paraffin-embedded (FFPE) CRC tissue samples of 115 patients. The samples were subjected to immunohistochemistry (IHC) to assess the expression of BRAFV600E, CDX2, and CD133. Additionally, homology modelling and docking were performed to investigate novel deletions revealed in sequencing.ResultsTwenty-four (20.8%) BRAF variants were identified in the coding region, with V600E mutations detected in 14 (12.2% )cases (GenBank: PP003258.1; Pop Set: 2678087296). Moreover, a wide spectrum of novel non-V600E mutations (8.6%) were identified, including deletions and missense variations. In-silico analysis revealed that due to large deletions in the coding region of three samples, the affinity of the anti-BRAF drugs (Encorafenib and Vemurafenib) for the active site decreased in comparison to the wild type. The IHC analysis showed that BRAFV600E expression was significantly associated with CD133 expression (χ2(1, n=115) = 26.351; p = < 0.001) and with CDX2 expression (χ2(1, n=115) = 14.88; p = 0.001). Multivariate analysis using binary logistic regression revealed association of BRAFV600E mutations with age (OR = 1.123; CI = 1.024–1.232; p = 0.014), gender (OR = 0.071; CI = 0.006–0.831; p = 0.035), grade (0.007; CI = 0-0.644) and CD133 expression (OR = 65.649; CI = 2.153-2001.556; p = 0.016).ConclusionThe present study demonstrates a notably high V600E frequency (12.2%) in comparison to global reported data, which ranges from 0.4 to 18%. This finding reflects the importance of upfront BRAF testing of the genetically distinct population of Pakistan. Previously unreported mutations identified in the sample may be of clinical significance and warrant further investigation. The concomitant high expression and significant association between CD133 and BRAFV600E represent vital actionable genes that may be targeted together to improve CRC patient management.

GLI1 and PTTG1 expression in colorectal carcinoma patients undergoing radical surgery and their correlation with lymph node metastasis.

Few studies have investigated the expression of GLI1 and PTTG1 in patients undergoing radical surgery for colorectal carcinoma (CRC) and their association with lymph node metastasis (LNM). Therefore, more relevant studies and analyses need to be conducted. To explore GLI1 and PTTG1 expression in patients undergoing radical surgery for CRC and their correlation with LNM. This study selected 103 patients with CRC admitted to our hospital between April 2020 and April 2023. Sample specimens of CRC and adjacent tissues were collected to determine the positive rates and expression levels of GLI1 and PTTG1. The correlation of the two genes with patients' clinicopathological data (e.g., LNM) was explored, and differences in GLI1 and PTTG1 expression between patients with LNM and those without were analyzed. Receiver operating characteristic (ROC) curves were plotted to evaluate the predictive potential of the two genes for LNM in patients with CRC. Significantly higher positive rates and expression levels of GLI1 and PTTG1 were observed in CRC tissue samples compared with adjacent tissues. GLI1 and PTTG1 were strongly linked to LNM in patients undergoing radical surgery for CRC, with higher GLI1 and PTTG1 levels found in patients with LNM than in those without. The areas under the ROC curve of GLI1 and PTTG1 in assessing LNM in patients with CRC were 0.824 and 0.811, respectively. GLI1 and PTTG1 expression was upregulated in patients undergoing radical surgery for CRC and are significantly related to LNM in these patients. Moreover, high GLI1 and PTTG1 expression can indicate LNM in patients with CRC undergoing radical surgery. The expression of both genes has certain diagnostic and therapeutic significance.

To Correlate the Expression of KRAS and BRAF V600e with Histological Grades and Variants in Tissue Samples of Colorectal Carcinoma

OBJECTIVES This study aimed to correlate the expression of KRAS and BRAF V600E with histological grades and variants in tissue samples of colorectal carcinoma.METHODOLOGY In this cross-sectional study total of 51 cases of Colorectal cancer (CRC) were analyzed for immunohistochemical staining using KRAS and BRAF antibodies on representative tissue blocks. Clinical and pathological records were retrieved for the collection of data. The results of the immunohistochemical analysis were correlated with the recorded clinicopathological parameters.RESULTSFifty-one cases of CRC were analyzed for immunoexpression of KRAS and BRAF V600E. The age of the patients ranged from 14 to 85 years, with a mean age of 60.96 years. Among the 51 cases, 37(72.5%) cases were males and 14(27.4%) were females. 37(72.5%) were localized to left side colon and 14(27.4%) were found in the right colon. For KRAS immunostaining, 41(80.3%) out of 51 cases showed overexpression, while the remaining 10(19.6%) cases revealed negative expression. In the case of BRAF V600E, positive expression was seen in 20(39.2%) cases, whereas 31(60.7%) cases showed negative expression of BRAFV600E. A significant association was seen between KRAS overexpression and histological variants, i.e. glandular carcinomas. CONCLUSIONOver-expression of KRAS was observed in advanced tumors. The presence of BRAF V600E mutation in the present study signifies the importance of BRAF V600E inhibitors as a potential alternate therapeutic tool in EGFR inhibitors and chemotherapy-resistant tumors.

Expression of KRAS in tissue samples of colorectal carcinoma and its correlation with various histo-pathological parameters.

Objective: To assess the expression of KRAS in tissue samples of colorectal carcinoma and to correlate it with histopathological parameters. Study Design: Cross Sectional study. Setting: Department of Pathology, PNS Shifa Hospital Karachi. Period: March 2016 to February 2019. Material &amp; Methods: A total of 51 cases of CRC were analyzed for immunohistochemical staining using KRAS antibody on representative tissue blocks. Clinical and pathological records were retrieved for collection of data. The results of immunohistochemical analysis were correlated with the recorded clinico-pathological parameters. Results: In this study 51 cases of CRC were analyzed for immunoexpression of KRAS. The age of the patients ranged from 14 to 85 years with the mean age of 60.96 years. Among the 51 cases, 37(72.5%) cases were males and 14(27.4%) were females. 37(72.5%) were localized to left side colon and 14(27.4%) were found in the right colon. For KRAS immunostaining, 41(80.3%) out of 51 cases showed overexpression. Significant association was seen between KRAS overexpression and histological variants i.e. glandular carcinomas. Conclusion: In the present study over expression of KRAS was observed in advanced tumors. Majority of these cases were adenocarcinomas with few showed mucinous histology. The present study signifies that established KRAS expression is usually seen in rapidly dividing cells with association of advanced tumors.

Soluble Epoxide Hydrolase as an Important Player in Intestinal Cell Differentiation

There is growing evidence that soluble epoxide hydrolase (sEH) may play a role in cell differentiation. sEH metabolizes biologically highly active and generally cytoprotective epoxyeicosatrienoic acids (EETs), generated from arachidonic acid metabolism by CYP epoxygenases (CYP2C and CYP2J subfamilies), to less active corresponding diols. We investigated the effect of sEH inhibitor (TPPU) on the expression of villin, CYP2C8, CYP2C9, CYP2J2, and sEH in undifferentiated and in vitro differentiated HT-29 and Caco2 cell lines. The administration of 10 μM TPPU on differentiated HT-29 and Caco2 cells resulted in a significant decrease in expression of villin, a marker for intestinal cell differentiation. It was accompanied by a disruption of the brush border when microvilli appeared sparse and short in atomic force microscope scans of HT-29 cells. Although inhibition of sEH in differentiated HT-29 and Caco2 cells led to an increase in sEH expression in both cell lines, this treatment had an opposite effect on CYP2J2 expression in HT-29 and Caco2 cells. In addition, tissue samples of colorectal carcinoma and adjacent normal tissues from 45 patients were immunostained for sEH and villin. We detected a significant decrease in the expression of both proteins in colorectal carcinoma in comparison to adjacent normal tissue, and the decrease in both sEH and villin expression revealed a moderate positive association. Taken together, our results showed that sEH is an important player in intestinal cell differentiation.

HOME / ARCHIVES / VOL. 10 NO. 4 (2020): VOLUME 10 ISSUE 4 / Original Articles Expression of BRAF V600E in Tissue Samples of Colorectal Carcinoma and Its Correlation with Various Clinico-Pathological Parameters

Objective: To determine the expression of BRAF V600E in tissue samples of colorectal carcinoma and to correlate it with various clinico-pathological parameters. Study design and setting: Cross-sectional study was conducted at department of Pathology, Pakistan Navy Station Shifa hospital Karachi from 1st March 2016 to 28th February 2019 Methodology: Total of 51 cases of colorectal cancer were analyzed for immunohistochemical staining using BRAF antibodies on representative tissue blocks. Clinical and pathological records were retrieved for data collection. The results of immunohistochemical analysis were correlated with the recorded clinico-pathological parameters. Results: In this study 51 cases of colorectal cancer were analyzed for immune expression of BRAF V600E. The age of the patients ranged from 14 to 85 years with the mean age of 60.96 years. Among the 51 cases, 37(72.5%) cases were males and 14(27.4%) were females. 37(72.5%) were localized to left side colon and 14(27.4%) were found in the right colon. For BRAF V600E, positive expression was seen in 20(39.2%) cases, whereas 31(60.7%) cases showed negative expression of BRAFV600E. No significant association was seen between BRAF V600E expression and histological variants like age, gender, tumor location and glandular carcinomas. Conclusion: BRAF V600E immunosuppression was seen in 39.2% of colorectal carcinoma in this study. No significant association was seen in BRAF V600E expression and histological variants

Integrin Subunit Alpha 5 (ITGA5) Gene Circular RNA Sponges microRNA-107 in Colorectal Carcinoma Cells and Tissues and Regulates the Expression of the Forkhead Box J3 (FOXJ3) Gene.

BackgroundCircular RNAs (circRNAs) can function as sponges for microRNA (miRNA) in carcinogenesis. This study aimed to investigate the role of the circRNA of the integrin subunit alpha 5 (ITGA5) gene and microRNA-107 (miR-107) in human colorectal carcinoma (CRC) cells in vitro and tissue samples from patients with CRC and the expression of forkhead box J3 (FOXJ3) protein.Material/MethodsThirty paired CRC tissue samples and adjacent normal colon tissue samples were studied. Human CRC cell lines, including HT29, SW480, LoVo, and HIEC cells, were studied for cell proliferation using the cell counting kit-8 (CCK-8) assay. Cell migration was studied using a transwell assay, and cell apoptosis was determined using flow cytometry. The luciferase reporter assay was used to study the interactions between ITGA5 circRNA, FOXJ3, and miR-107 in human CRC cells. The expression of ITGA5 circRNA and miR-107 was determined using quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of FOXJ3 were measured by Western blot.ResultsThe expression of ITGA5 circRNA was significantly reduced in CRC tissues and CRC cell lines. High ITGA5 circRNA expression inhibited the proliferation and cell migration of CRC cells and promoted the apoptosis of CRC cells. The luciferase reporter assay confirmed that ITGA5 circRNA bound to miR-107, which directly targeted FOXJ3.ConclusionsITGA5 circRNA may act as a sponge for miR-107 to upregulate FOXJ3 expression and act as a tumor suppressor in CRC cells.

Regulation of cancer stem cell properties, angiogenesis, and vasculogenic mimicry by miR-450a-5p/SOX2 axis in colorectal cancer

Growing evidence indicates that a small number of cancer cells express stem cell markers and possess stem cell-like properties that promote malignant progression. Sex-determining region Y-box2 (SOX2) is a stem cell transcription factor essential for maintaining the properties of cancer stem cell (CSC). As CSC properties have been associated with angiogenesis and vasculogenic mimicry (VM), we aimed to comprehensively investigate whether SOX2 regulates CSC properties, angiogenesis, and VM in colorectal carcinoma (CRC) and its potential mechanism in this study. For this study, sphere formation assay, flow cytometry, cell survival analysis, tube formation, 3D culture, immunoblot, mouse model, and luciferase reporter assay were performed in vivo and in vitro. Expressions of SOX2 and miR-450a-5p in CRC tissue samples were examined through immunohistochemistry. First, the expression of SOX2 was not only associated with poor differentiation and prognosis but also promoted angiogenesis and VM. Knockdown of SOX2 ceased stemness properties, angiogenesis, and VM, along with decreased expression of CD133, CD31, and VE-cadherin as observed in functional experiments. Downregulation of SOX2 was found to inhibit tumorigenesis in vivo. Second, miR-450a-5p suppressed the expression of SOX2 by targeting its 3’UTR region directly and hence restrained SOX2-induced CSC properties, angiogenesis, and VM. Moreover, SOX2 overexpression preserved the miR-450a-5p-induced inhibition of CRC properties, angiogenesis, and VM. Finally, clinical samples exhibited a negative correlation between miR-450a-5p and SOX2. Patients with higher SOX2 and lower miR-450a-5p expressions had a poorer prognosis than patients with inverse expressions. Conclusively, we elucidated a unique mechanism of miR-450a-5p-SOX2 axis in the regulation of stemness, angiogenesis, and VM, which may act as a potential therapeutic practice in CRC.

MicroRNA-760 inhibits the biological progression of colorectal carcinoma by directly targeting FOXA1 and regulating epithelial-to-mesenchymal transition and PI3K/AKT signaling pathway.

Colorectal carcinoma (CRC) is one of the most common factors for tumor-associated mortalities globally. In recent years, microRNAs (miRNAs) have been identified as novel therapeutic biomarkers for cancer treatment. The purpose of the current study was to unravel the clinical significance and underlying molecular mechanisms of miR-760 in CRC progression. Fifty-four pairs of CRC tissue samples and adjacent para-carcinoma tissue samples were collected from CRC patients who underwent surgical resection. We measured miR-760 expressions in CRC using quantitative Real-time polymerase chain reaction (qRT-PCR) analysis. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assays and transwell assays were performed to determine the functions of miR-760 in CRC cell proliferation, invasion and migration. Dual-luciferase reporter assays and Western blots were used to investigate the underlying molecular mechanisms. Moreover, the association between miR-760 expressions and clinicopathological features was analyzed. In this study, the results showed that the down-regulated miR-760 expressions were related to the poor prognosis and malignant clinicopathologic features of CRC patients. Furthermore, functional assays revealed that miR-760 restoration obviously suppressed CRC cell proliferation, migration and invasion through modulating phosphatidylinositol 3-kinase/ protein kinase B (PI3K/AKT) pathway and epithelial-mesenchymal transition (EMT). FOXA1 was also considered as a functional target of miR-760 in CRC cells. Furthermore, miR-760 up-regulation also significantly repressed tumorigenesis in vivo. These results suggested that miR-760 exerted cancer-suppressive functions in CRC, providing a therapeutic strategy for CRC treatment.

FBX8 degrades GSTP1 through ubiquitination to suppress colorectal cancer progression

F-box only protein 8 (FBX8), as a critical component of the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases, has been associated with several malignancies through interacting with a member of proteins. However, the substrates of FBX8 for destruction in the progression of colorectal carcinoma (CRC) need to be explored. Here, we show that loss of FBX8 accelerates chemical-induced colon tumorigenesis. FBX8 directly targets GSTP1 for ubiquitin-mediated proteasome degradation in CRC. GSTP1 promotes the proliferation, invasion, and metastasis of CRC cells. Furthermore, GSTP1 is upregulated in CRC tissue samples and predicts poor prognosis of CRC patients. The inactivation of FBX8 negatively correlated with increased levels and stability of GSTP1 in clinical CRC tissues and FBX8 knockout transgenic mice. These findings identify a novel ubiquitination pathway as FBX8-GSTP1 axis that regulates the progression of CRC, which might be a potential prognostic biomarker for CRC patients.

Nuclear lncRNA HOXD-AS1 suppresses colorectal carcinoma growth and metastasis via inhibiting HOXD3-induced integrin \u03b23 transcriptional activating and MAPK/AKT signalling

BackgroundLong noncoding RNAs (lncRNAs) have been indicated to play critical roles in cancer development and progression. LncRNA HOXD cluster antisense RNA1 (HOXD-AS1) has recently been found to be dysregulated in several cancers. However, the expression levels, cellular localization, precise function and mechanism of HOXD-AS1 in colorectal carcinoma (CRC) are largely unknown.MethodsReal-time PCR and in situ hybridization were used to detect the expression of HOXD-AS1 in CRC tissue samples and cell lines. Gain- and loss-of-function experiments were performed to investigate the biological roles of HOXD-AS1 in CRC cell line. RNA pull down, RNA immunoprecipitation and chromatin immunoprecipitation assays were conducted to investigate the mechanisms underlying the functions of HOXD-AS1 in CRC.ResultsWe observed that HOXD-AS1 was located in the nucleus of CRC cells and that nuclear HOXD-AS1 was downregulated in most CRC specimens and cell lines. Lower levels of nuclear HOXD-AS1 expression were associated with poor outcomes of CRC patients. HOXD-AS1 downregulation enhanced proliferation and migration of CRC cells in vitro and facilitated CRC tumourigenesis and metastasis in vivo. Mechanistic investigations revealed that HOXD-AS1 could suppress HOXD3 transcription by recruiting PRC2 to induce the accumulation of the repressive marker H3K27me3 at the HOXD3 promoter. Subsequently, HOXD3, as a transcriptional activator, promoted Integrin β3 transcription, thereby activating the MAPK/AKT signalling pathways.ConclusionOur results reveal a previously unrecognized HOXD-AS1-HOXD3-Integrin β3 regulatory axis involving in epigenetic and transcriptional regulation constitutes to CRC carcinogenesis and progression.

Expression of Zinc Finger and BTB Domain-containing 7A in Colorectal Carcinoma.

Previous studies have revealed that zinc finger and BTB domain-containing 7A (ZBTB7A), an important proto-oncogene, plays multiple roles in carcinogenesis and is up-regulated in several human malignancies. However, the expression of ZBTB7A in colorectal carcinoma (CRC) has seldom been documented. In this study, we investigated the differential expression of ZBTB7A in CRC cell lines and tissues. Expression levels of ZBTB7A mRNA and protein were examined in CRC cell lines. ZBTB7A protein expression was also evaluated in tissue samples of normal colonic mucosa, high-grade dysplasia, and CRC using immunohistochemical staining. All CRC cell lines exhibited significantly higher ZBTB7A mRNA expression levels than did normal colonic epithelial cells. The ZBTB7A protein expression levels were clearly higher in the CRC cell lines than in the normal colonic epithelial cells. Consistent with the cell line data, immunostaining revealed that there were significant differences in ZBTB7A protein expression between tissue samples of CRC and normal colonic mucosa (p=0.048) and high-grade dysplasia (p=0.015). In addition, metastatic CRC exhibited significantly higher ZBTB7A protein expression levels than primary CRC (p=0.027). We demonstrated that ZBTB7A expression is up-regulated in CRC cell lines and tissues. Our data suggest that ZBTB7A is involved in the development and progression of CRC.

Down-regulation of B-Cell Translocation Gene 1 by Promoter Methylation in Colorectal Carcinoma.

B-cell translocation gene 1 (BTG1) acts as a tumour suppressor in human malignancies. However, the precise mechanism of BTG1 down-regulation in colorectal carcinoma (CRC) remains unclear. We analyzed BTG1 expression in CRC cell lines and tissues and investigated the mechanism underlying the observed alterations. Real-time polymerase chain reaction (PCR) and western blot analyses were performed to analyze BTG1 expression in CRC cell lines. The methylation status of the BTG1 promoter region in cell lines was determined by methylation-specific PCR, and the effect of demethylation on BTG1 expression was explored with 5-aza-deoxycytidine treatment. BTG1 protein expression in CRC tissue samples was evaluated using immunostaining. CRC cell lines and tissue samples expressed lower levels of BTG1 compared to controls, and BTG1 levels were significantly lower in metastatic than primary CRC. In BTG1-down-regulated CRC cell lines, the BTG1 promoter was highly methylated, and 5-aza-deoxycytidine significantly restored BTG1 expression. BTG1 down-regulation in CRC occurs through epigenetic repression, which is involved in the development and progression of CRC.

Down-regulation of Inositol Polyphosphate 4-Phosphatase Type II Expression in Colorectal Carcinoma.

Aberrant expression of survival signaling pathways causes deregulation of cellular proliferation and resistance to apoptosis, and plays a crucial role in the development, progression and metastasis of cancer. Inositol polyphosphate 4-phosphatase type II (INPP4B) negatively regulates phosphatidylinositol 3-kinase signaling and has a tumor-suppressive role in several human malignancies. We analyzed the expression levels of INPP4B mRNA and protein in colorectal carcinoma (CRC) cell lines and tissue samples using western blot, quantitative real-time reverse-transcriptase polymerase chain reaction, and immunohistochemical staining. Western blot analysis revealed that the CRC cell lines HCT 116, SW620, DLD-1, and WiDr expressed significantly lower levels of INPP4B protein than the normal colonic epithelial cell lines CCD 841 CoTr and FHC. Consistent with these results, INPP4B mRNA expression in the CRC cell lines was significantly lower than in the normal colonic epithelial cells. Immunohistochemical staining revealed that normal colonic mucosa displayed uniform and strong-to-moderate INPP4B immunoreactivity, whereas 60.7% (71/117; p<0.001) and 76.5% (62/81; p<0.001) of the primary and metastatic CRC tissue samples exhibited reduced INPP4B expression, respectively. Our results indicate that INPP4B is down-regulated in CRC and that INPP4B is involved in the development and progression of CRC.

Evaluation of the correlation of vasculogenic mimicry, ALDH1, KAI1 and microvessel density in the prediction of metastasis and prognosis in colorectal carcinoma

BackgroundMetastasis and recurrence are the most common reasons for treatment failure of colorectal carcinoma (CRC). Vasculogenic mimicry (VM, blood supply formation often seen in highly aggressive tumors), Aldehyde dehydrogenase 1 (ALDH1, a biomarker of cancer stem cells), KAI1 (a suppressor gene of tumor metastasis) are all valuable factors for metastasis and prognosis in diverse human cancers. However, the correlation of VM, ALDH1, KAI1 and microvessel density (MVD) in CRC is unclear. In this study, we analyzed the correlations among VM, ALDH1, KAI1 and MVD, as well as their respective correlations with clinicopathological parameters and survival in CRC.MethodsThe level of VM, ALDH1, KAI1 and MVD in 204 whole tissue samples of CRC were examined by immunhistochemistry. Clinical data was also collected.ResultsLevels of VM, ALDH1 and MVD were significantly higher, and levels of KAI1 significantly lower, in CRC tissues than in normal colorectal tissues. Levels of VM, ALDH1 and MVD were positively associated with invasion of depth, lymph node metastasis (LNM), distant metastasis and tumor-node-metastasis (TNM) stages, and negatively with patients’ overall survival (OS). Levels of KAI1 was negatively correlated with invasion of depth, LNM, distant metastasis and TNM stages, and the KAI1 positive expression subgroup had significantly longer OS than did the KAI1- subgroup. In multivariate analysis, high levels of VM, ALDH1 and KAI1, as well as TNM stages were independently correlated with lower OS in patients with CRC.ConclusionsVM, MVD and the expression of ALDH1 and KAI1 may represent promising metastatic and prognostic biomarkers, as well as potential therapeutic targets for CRC.

Expression of Wntless in colorectal carcinomas is associated with invasion, metastasis, and poor survival.

Wntless also known as WLS, GPR177, or Evi, is a key modulator of Wnt protein secretion. Its overexpression is found in certain types of human cancers such as malignant astrocytoma and breast cancers. We hypothesized that this protein may be aberrantly expressed in colorectal carcinoma which also possesses aberrant Wnt signaling. To investigate the association between the expression of Wnt and clinicopathological parameters in colorectal carcinomas, a set of colorectal carcinoma tissue samples was analyzed for the expression of WLS using an anti-GPR177 monoclonal antibody specific for the WLS protein. High expression of WLS protein was observed in most colorectal carcinoma samples compared with nontumor mucosa in the same patients (117/201, 58.2%). High expression of WLS was associated with sex (p = 0.005), age (p = 0.009), depth of invasion (p < 0.001), lymph node metastasis (p = 0.026), and tumor-node-metastasis (TNM) stage (p = 0.003). No significant relationship between the expression of WLS and tumor location, size, and differentiation was found. The survival analyses showed WLS was an independent prognostic marker and that patients whose carcinoma exhibited high expression of WLS had a poorer outcome (p = 0.033). Our results indicate that WLS may play a role in invasion and metastasis of colorectal carcinoma. The WLS protein expression level may be used as a potential prognostic marker in colorectal carcinoma. Furthermore, the WLS gene may provide a novel target for therapy of colorectal carcinoma.

Down-regulation of osteoprotegerin expression as a novel biomarker for colorectal carcinoma.

A better understanding of tumor biology is important in the identification of molecules that are down-regulated in malignancy and in determining their role in tumor suppression. The aim of this study was to analyze osteoprotegerin (OPG) expression in colorectal carcinoma (CRC) and to investigate the underlying mechanism for changes in the expression of OPG. OPG expression was assessed in CRC tissue samples and cell lines. The methylation status of the OPG promoter region was determined, and the effects of demethylation on OPG expression were analyzed. The effects of recombinant OPG (rOPG) administration on cellular functions were also investigated. Clinical and prognostic implications of OPG protein expression in CRC patients were analyzed. The CRC tissues and cells showed significantly lower OPG expression. Pyrosequencing of OPG-silenced CRC cells revealed that the OPG gene promoter was highly methylated. Treatment with demethylating agent significantly elevated OPG mRNA and protein expression. rOPG significantly decreased cell viability and MMP-2 and VEGF-A production in CRC cells. Reduced OPG immunoreactivity was associated with aggressive oncogenic behavior in CRC. Also, OPG expression was found to be an independent predictor of recurrent hepatic metastasis and independent prognostic factor for worse survival rates. We demonstrated that OPG silencing in CRC occurs through epigenetic repression, and is involved in the development and progression of CRC. Our data suggest that OPG is a novel prognostic biomarker and a new therapeutic target for the treatment of patients with CRC.

MicroRNA-155 enhances the activation of Wnt/β-catenin signaling in colorectal carcinoma by suppressing HMG-box transcription factor 1

Colorectal carcinoma (CRC) is a malignant solid tumor arising from the large intestine and is associated with an increasing incidence and poor prognosis. Further understanding of the molecular mechanisms underlying CRC may contribute to the development of novel effective therapeutic strategies. MicroRNAs (miRs), including miR‑155, have been reported to be associated with the etiology and biology of CRC; however, the molecular mechanisms by which miR‑155 affect CRC remain to be fully elucidated. The present study used a multidisciplinary approach, involving reverse transcription‑quantitative polymerase chain reaction, northern blotting, MTT assay, cell cycle progression analysis, immunoblotting, and animal experiments, to determine the possible targets of miR‑155 in CRC cells. miR‑155 was found to be overexpressed in CRC tissue samples, compared with paired normal colon tissue samples. In addition, the inhibition of miR‑155 induced a deceleration in CRC cell proliferation and inactivation of the Wnt/β‑catenin signaling pathway. miR‑155 suppression also reduced the growth of CRC xenografts in an animal model. HMG‑box transcription factor 1 (HBP1) was identified as a novel target of miR‑155, which mediated its effect on CRC via the Wnt/β‑catenin pathway. Furthermore, patients with CRC exhibiting higher serum levels of miR‑155 exhibited reduced survival rates. In conclusion, the present study demonstrated that miR‑155 may contribute to the progression and growth of CRC by enhancing the Wnt/β‑catenin pathway in an HBP1‑associated mechanism. Therefore, miR‑155 may be considered a promising therapeutic target for the treatment of CRC.

Correlation of Vascular Endothelial Growth Factor Expression and Neovascularization with Colorectal Carcinoma: A Pilot Study

Background: In Egypt, there is an increasing incidence of colorectal cancer (CRC), especially among patients under 40 years of age, affecting a very important age group in our community. The basic pathogenic step in the process of tumor growth, invasion and metastasis is tumor induced angiogenesis. The aim of this study was to evaluate the angiogenesis in colorectal carcinoma by detect the expression of VEGF vascular endothelial growth factor and micro vascular density (MVD) determination with IHC (immunohistochemistry) method and to determine if and how angiogenesis correlates with clinicopathologic parameters. Methods and findings: Sixty five archival, paraffin embedded tissue samples of colorectal carcinoma from Pathology Lab of Suez Canal University Teaching Hospital (Egypt) with complete follow-up files in Oncology Unit from. VEGF and micro vessels were identified immunohistochemically, using monoclonal VEGF antibody and CD34 antibody, respectively. VEGF IHC expression was positive in 94.7% of cases and MVD ranged from 9 to 42 mean 24.55 ± 13.79. A significant positive relation was found between VEGF expression; and histological type, tumor grade, lymph node (LN) involvement, UICC TNM classification, perineural and lympho-vascular invasions (p<0.05). A significant positive relation was found between VEGF expression and MVD (p<0.001). There is a significant correlation has been found between MVD and size of the tumor, histological subtype, tumor grade, LN involvement, UICC TNM classification, vascular and perineural invasion and survival (p<0.05). Conclusions: This study has highlighted the prognostic significance of VEGF and MVD to predict survival in CRC patients, as well as some of traditional prognostic factors as tumor histologic type, tumor size, tumor grade, LN involvement, tumor stage and lympho-vascular and perineural invasion to identify patients at high risk for relapse who may benefit from adjuvant treatment including new therapeutic strategies in the future.

Identification and Validation of a Potential Marker of Tissue Quality Using Gene Expression Analysis of Human Colorectal Tissue.

Correlative studies have identified numerous biomarkers that are individualizing therapy across many medical specialties, including oncology. Accurate interpretation of these studies requires the collection of tissue samples of sufficient quality. Tissue quality can be measured by changes in levels of gene expression and can be influenced by many factors including pre-analytical conditions, ischemic effects and the surgical collection procedure itself. However, as yet there are no reliable biomarkers of tissue quality at researchers’ disposal. The aim of the current study was to identify genes with expression patterns that fluctuated reproducibly in response to typical post-surgical stress (ischemia) in order to identify a specific marker of tissue quality. All tissue samples were obtained from patients with primary colorectal carcinoma (CRC) (N = 40) either via colonoscopy prior to surgery, or by surgical resection. Surgically resected tissue samples were divided into three groups and subjected to cold ischemia for 10, 20 or 45 minutes. Normal colorectal tissue and CRC tissue was analyzed using microarray and quantitative real-time PCR (qPCR). Comparing changes in gene expression between pre- and post-surgical tissue using microarray analysis identified a list of potential tissue quality biomarkers and this list was validated using qPCR. Results revealed that post-operative ischemia significantly alters gene expression in normal and CRC tissue samples. Both microarray analysis and qPCR revealed regulator of G-protein signaling 1 (RGS1) as a potential marker of CRC tissue quality and eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) as a potential reference gene of post-operative tissue quality. Larger studies with additional time points and endpoints will be needed to confirm these results.