Colon Cancer Tissue Microarrays Research Articles

SIRT2 deacetylates and decreases the expression of FOXM1 in colon cancer.

New FOXM1-specific inhibitors with the potential to be used for therapeutic purposes are under extensive research. We hypothesized that deacetylation of FOXM1 would decrease protein expression, thus providing novel therapeutic management of colon cancers. Immunostaining was used to determine FOXM1 and SIRT2 expressions in human colon cancer tissue microarrays (n = 90) from Stage I to Stage IV. SIRT2-FOXM1 interaction was evaluated in colon cancer cells using immunoprecipitation. Deacetylation of FOXM1 via SIRT2 was determined using in vitro deacetylation assays. FOXM1 could be hyper-acetylated when p300 and pCAF histone acetyltransferases were administered alongside deacetylase inhibitors. We detected that SIRT2 and FOXM1 physically interacted, and SIRT2 deacetylated FOXM1 in vitro. SIRT2 overexpression led to a significant decrease while knockdown of SIRT2 increased the FOXM1 expression in HCT116 human colon carcinoma cells. In the analysis of 90 human colorectal cancer samples, high SIRT2 expression was observed in about 49% of colorectal cancer, intermediate in 29%, and low or no staining in 22%. Strong SIRT2 expression was found to be negatively associated with the FOXM1 staining in our clinical cohort. This study reveals a molecular interaction and association between SIRT2 and FOXM1 expression in colon cancer cell lines and human colon cancer samples, and suggests that targeting SIRT2 activity using small molecule modulators may be a promising therapeutic approach for colorectal cancer.

Abstract C051: Deciphering the role of stress-survival pathway genes and mutations in Hispanic colorectal cancer disparities

Abstract This study aims to determine the role of stress-survival pathway (SSP) genes and the associated mutations in Hispanic colorectal cancer (CRC) disparities, particularly in young and late-stage Hispanic CRC. CRC is the second most-deadliest among all cancers and the third leading cause of cancer in the United States (US). An overall reduction in CRC cases among individuals aged 50 and older has been reported, albeit there is an alarming rise in the incidence of early-onset (patients under the age of 50) CRC. The increase in the early onset of CRC is concerning as these cases tend to appear with higher pathologic grades and have a higher risk of recurrence and metastasis. The incidence of early-onset CRC is almost two times in Hispanics (16.5%) when compared to that in non-Hispanic White (NHWs; 8.7%). Access to health care for Hispanics is limited, as a result, they also tend to present with later-stage CRC when compared to NHWs (32% versus 19% of Stage IV), which likely accounts for their inferior age-adjusted survival after CRC diagnosis. Since Hispanics have the highest percentage of young people and comprise ~18% of the US population, the rising incidence of early-onset and late-stage detection of CRC among Hispanics coupled with the rapid growth in this segment of the population makes it imperative to correctly assess the causes behind the observed disparities in Hispanics with early-onset and late-stage CRC. Our previous study, using in-silico analyses of CRC datasets, helped us identify twenty-eight oxidative stress-response and apoptosis-associated genes that are expressed differently in CRC when compared to normal colon tissues. Expressions of these SSP genes (mRNA and protein levels) were validated in CRC cell lines, cDNA arrays, and human colon cancer tissue microarrays (TMAs). Finally, we have confirmed the differential expression of all twenty-eight transcripts in Hispanic and NHW CRC, and corresponding normal adjacent to the tumor (NAT) tissues. Interestingly, we have identified distinct transcripts that are differentially expressed in Hispanics with early-onset (PDCD2L, CDK1, and PRDX4) and late-stage (CCNB1, CDK4, CHEK1, and MCM10) CRC. We have also identified the mutations associated with the SSP genes by analyzing the ClinVar and NHGRI-EBI GWAS databases. These findings have laid the foundation to characterize further, the roles played by these genes in the molecular basis for Hispanic CRC disparities at early-onset and late stages. Differential Expression of mRNAs in CRC tissues between the two ethnic groups (Hispanics and NHW) and corresponding NATs has been determined by RNA-seq. Ingenuity Pathway Analysis (IPA) has been performed to correlate SSP genes with the canonical CRC pathways to better understand the roles played by these genes, their effects on the downstream cancer-related pathways, and on the early-onset and late-stage Hispanic CRC. These putative ethnicity-specific biomarkers or targets could be used for developing novel prognostic tools or therapeutic interventions for Hispanic CRC patients in the future. Citation Format: Sourav Roy, Brian I. Grajeda, Md Zahirul Islam Khan, Urbashi Basnet, Soumya Nair. Deciphering the role of stress-survival pathway genes and mutations in Hispanic colorectal cancer disparities [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C051.

Targeting inhibition of prognosis-related lipid metabolism genes including CYP19A1 enhances immunotherapeutic response in colon cancer

BackgroundLipid metabolic reprogramming in colon cancer shows a potential impact on tumor immune microenvironment and is associated with response to immunotherapy. Therefore, this study aimed to develop a lipid metabolism-related prognostic risk score (LMrisk) to provide new biomarkers and combination therapy strategies for colon cancer immunotherapy.MethodsDifferentially expressed lipid metabolism-related genes (LMGs) including cytochrome P450 (CYP) 19A1 were screened to construct LMrisk in TCGA colon cancer cohort. The LMrisk was then validated in three GEO datasets. The differences of immune cell infiltration and immunotherapy response between LMrisk subgroups were investigated via bioinformatic analysis. These results were comfirmed by in vitro coculture of colon cancer cells with peripheral blood mononuclear cells, human colon cancer tissue microarray analysis, multiplex immunofluorescence staining and mouse xenograft models of colon cancer.ResultsSix LMGs including CYP19A1, ALOXE3, FABP4, LRP2, SLCO1A2 and PPARGC1A were selected to establish the LMrisk. The LMrisk was positively correlated with the abundance of macrophages, carcinoma-associated fibroblasts (CAFs), endothelial cells and the levels of biomarkers for immunotherapeutic response including programmed cell death ligand 1 (PD-L1) expression, tumor mutation burden and microsatellite instability, but negatively correlated with CD8+ T cell infiltration levels. CYP19A1 protein expression was an independent prognostic factor, and positively correlated with PD-L1 expression in human colon cancer tissues. Multiplex immunofluorescence analyses revealed that CYP19A1 protein expression was negatively correlated with CD8+ T cell infiltration, but positively correlated with the levels of tumor-associated macrophages, CAFs and endothelial cells. Importantly, CYP19A1 inhibition downregulated PD-L1, IL-6 and TGF-β levels through GPR30-AKT signaling, thereby enhancing CD8+ T cell-mediated antitumor immune response in vitro co-culture studies. CYP19A1 inhibition by letrozole or siRNA strengthened the anti-tumor immune response of CD8+ T cells, induced normalization of tumor blood vessels, and enhanced the efficacy of anti-PD-1 therapy in orthotopic and subcutaneous mouse colon cancer models.ConclusionA risk model based on lipid metabolism-related genes may predict prognosis and immunotherapeutic response in colon cancer. CYP19A1-catalyzed estrogen biosynthesis promotes vascular abnormality and inhibits CD8+ T cell function through the upregulation of PD-L1, IL-6 and TGF-β via GPR30-AKT signaling. CYP19A1 inhibition combined with PD-1 blockade represents a promising therapeutic strategy for colon cancer immunotherapy.

N-acetyltransferase 10 promotes colon cancer progression by inhibiting ferroptosis through N4-acetylation and stabilization of ferroptosis suppressor protein 1 (FSP1) mRNA.

N-acetyltransferase 10 (NAT10) is the only enzyme known to mediate the N4-acetylcytidine (ac4C) modification of mRNA and is crucial for mRNA stability and translation efficiency. However, its role in cancer development and prognosis has not yet been explored. This study aimed to examine the possible role of NAT10 in colon cancer. The expression levels of NAT10 were evaluated by immunohistochemical analyses with a colon cancer tissue microarray, and its prognostic value in patients was further analyzed. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were performed to analyze NAT10 expression in harvested colon cancer tissues and cell lines. Stable NAT10-knockdown and NAT10-overexpressing colon cancer cell lines were constructed using lentivirus. The biological functions of NAT10 in colon cancer cell lines were analyzed in vitro by Cell Counting Kit-8 (CCK-8), wound healing, Transwell, cell cycle, and ferroptosis assays. Xenograft models were used to analyze the effect of NAT10 on the tumorigenesis and metastasis of colon cancer cells in vivo. Dot blotting, acetylated RNA immunoprecipitation-qPCR, and RNA stability analyses were performed to explore the mechanism by which NAT10 functions in colon cancer progression. NAT10 was upregulated in colon cancer tissues and various colon cancer cell lines. This increased NAT10 expression was associated with shorter patient survival. Knockdown of NAT10 in two colon cancer cell lines (HT-29 and LoVo) impaired the proliferation, migration, invasion, tumor formation and metastasis of these cells, whereas overexpression of NAT10 promoted these abilities. Further analysis revealed that NAT10 exerted a strong effect on the mRNA stability and expression of ferroptosis suppressor protein 1 (FSP1) in HT-29 and LoVo cells. In these cells, FSP1 mRNA was found to be modified by ac4C acetylation, and this epigenetic modification was associated with the inhibition of ferroptosis. Our study revealed that NAT10 plays a critical role in colon cancer development by affecting FSP1 mRNA stability and ferroptosis, suggesting that NAT10 could be a novel prognostic and therapeutic target in colon cancer.

Loss of CHGA Protein as a Potential Biomarker for Colon Cancer Diagnosis: A Study on Biomarker Discovery by Machine Learning and Confirmation by Immunohistochemistry in Colorectal Cancer Tissue Microarrays.

Simple SummaryThe identification of effective novel biomarkers is emergently needed in colon cancer patients. In the present study, firstly we predicted that CHGA could be a biomarker for colon cancer based on the protein–protein interaction network of all the reported biomarkers that were collected from our colorectal cancer biomarker database (CBD). Then we verified our results using a diagnostic test in gene expression data and an immunohistochemistry test. The results of this study suggest that a loss of CHGA expression from the normal colon and adjacent mucosa to colon cancer may be used as a valuable biomarker for early diagnosis of colon cancer patients.Background. The incidence of colorectal cancers has been constantly increasing. Although the mortality has slightly decreased, it is far from satisfaction. Precise early diagnosis for colorectal cancer has been a great challenge in order to improve patient survival. Patients and Methods. We started with searching for protein biomarkers based on our colorectal cancer biomarker database (CBD), finding differential expressed genes (GEGs) and non-DEGs from RNA sequencing (RNA-seq) data, and further predicted new biomarkers of protein–protein interaction (PPI) networks by machine learning (ML) methods. The best-selected biomarker was further verified by a receiver operating characteristic (ROC) test from microarray and RNA-seq data, biological network, and functional analysis, and immunohistochemistry in the tissue arrays from 198 specimens. Results. There were twelve proteins (MYO5A, CHGA, MAPK13, VDAC1, CCNA2, YWHAZ, CDK5, GNB3, CAMK2G, MAPK10, SDC2, and ADCY5) which were predicted by ML as colon cancer candidate diagnosis biomarkers. These predicted biomarkers showed close relationships with reported biomarkers of the PPI network and shared some pathways. An ROC test showed the CHGA protein with the best diagnostic accuracy (AUC = 0.9 in microarray data and 0.995 in RNA-seq data) among these candidate protein biomarkers. Furthermore, immunohistochemistry examination on our colon cancer tissue microarray samples further confirmed our bioinformatical prediction, indicating that CHGA may be used as a potential biomarker for early diagnosis of colon cancer patients. Conclusions. CHGA could be a potential candidate biomarker for diagnosing earlier colon cancer in the patients.

Evolutionary genetic algorithm identifies IL2RB as a potential predictive biomarker for immune-checkpoint therapy in colorectal cancer.

Identifying robust predictive biomarkers to stratify colorectal cancer (CRC) patients based on their response to immune-checkpoint therapy is an area of unmet clinical need. Our evolutionary algorithm Atlas Correlation Explorer (ACE) represents a novel approach for mining The Cancer Genome Atlas (TCGA) data for clinically relevant associations. We deployed ACE to identify candidate predictive biomarkers of response to immune-checkpoint therapy in CRC. We interrogated the colon adenocarcinoma (COAD) gene expression data across nine immune-checkpoints (PDL1, PDCD1, CTLA4, LAG3, TIM3, TIGIT, ICOS, IDO1 and BTLA). IL2RB was identified as the most common gene associated with immune-checkpoint genes in CRC. Using human/murine single-cell RNA-seq data, we demonstrated that IL2RB was expressed predominantly in a subset of T-cells associated with increased immune-checkpoint expression (P < 0.0001). Confirmatory IL2RB immunohistochemistry (IHC) analysis in a large MSI-H colon cancer tissue microarray (TMA; n = 115) revealed sensitive, specific staining of a subset of lymphocytes and a strong association with FOXP3+ lymphocytes (P < 0.0001). IL2RB mRNA positively correlated with three previously-published gene signatures of response to immune-checkpoint therapy (P < 0.0001). Our evolutionary algorithm has identified IL2RB to be extensively linked to immune-checkpoints in CRC; its expression should be investigated for clinical utility as a potential predictive biomarker for CRC patients receiving immune-checkpoint blockade.

Cyclophilin B overexpression predicts a poor prognosis and activates metastatic pathways in colon cancer.

BackgroundCyclophilin B (CypB) has been found overexpressed in various malignant tumors. To date, there are few studies on CypB in colon cancer. In this study, we aimed to analyze the CypB expression pattern and to further evaluate its clinical significance, especially its prognostic value for colon cancer.MethodsCypB expression was investigated in colon cancer tissue microarrays (TMA) by RNAscope in situ hybridization and immunohistochemical (IHC) staining. The correlation between CypB and clinicopathological characteristics was analyzed. The Cancer Genome Atlas (TCGA) RNA-seq dataset of colon adenocarcinoma (COAD) was further analyzed to validate our main findings. Gene Set Enrichment Analysis (GSEA) and Search Tool for the Retrieval of Interacting Genes (STRING) analysis were performed to enrich CypB related biological pathways. In vitro experiments by knockdown of CypB in colon cancer cell HCT116 were performed to verify the bioinformatics results and analyze its role in the metastatic pathways in colon cancer.ResultsWe found that CypB expression was highly upregulated in colon cancer tissues (P<0.05). Importantly, the overall survival (OS) time of patients with high CypB expression was significantly shorter than those with low CypB expression, and overexpressed CypB was identified as an independent prognostic indicator for poor survival (P=0.015). Subgroup analysis indicated that a high level of CypB was associated with a shorter OS time, especially for advanced cancer patients, such as later T stage, lymph node metastasis, larger tumor size (P<0.05). Analysis of TCGA RNA-seq dataset of COAD provided us with a larger clinical sample verification. Bioinformatics analysis and the following in vitro study revealed that CypB was involved in tumor metastatic associated signaling pathways.ConclusionsCypB overexpression predicts a poor prognosis and may activate metastatic pathways in colon cancer.

RNF8 induces β-catenin-mediated c-Myc expression and promotes colon cancer proliferation

DNA damage signals transducer RING finger protein 8 (RNF8) is involved in maintaining genomic stability by facilitating the repair of DNA double-strand breaks (DSB) via ubiquitin signaling. By analyzing the TCGA database and colon cancer tissue microarrays, we found that the expression level of RNF8 was positively correlated with that of c-Myc in colon cancer, which were closely associated with poor survival of colon cancer patients. Furthermore, overexpressing and knocking down RNF8 increased and decreased the expression of c-Myc in colon cancer cells, respectively. In addition, RNF8 interacted with β-catenin and facilitated its nuclear translocation by conjugating K63 polyubiquitination on it. These observations suggested a de novo role of RNF8 in promoting the progression of colon cancer by inducing β-catenin-mediated c-Myc expression.

Abstract 513: Investigating the expression of indolamine deoxygenase I (IDO1) and other immune markers in tissue microarrays

Abstract Purpose of the study: To gain insight to the proportion of solid tumors that express IDO1, CD73 and PD-L1 in lung cancer, colon adenocarcinoma, renal cell carcinoma (RCC) and melanoma, and the correlation between expression of these immune checkpoints and immune cell infiltration within the tumor. Experimental procedures: Using a battery of properly validated antibodies, we have performed an in situ analysis of IDO1, CD73 and PD-L1 expression by immunohistochemistry in a panel of colon cancer (n=68), lung cancer (n=36), RCC (n=67) and melanoma (n=48) tissue microarray samples. Tumor infiltrating T cells, CD3+ and CD8+, were also detected by IHC and counted in serial sections for each sample. We search for a possible a correlation between the expression of these immunosuppressor proteins and the number of infiltrating CD3+ and CD8+ T cells as well as their expression in the different tumor components: tumor cells, stroma and endothelium. Results: Our IHC analysis allowed us to classify the analyzed samples into two main groups: those with expression of IDO1, CD73 and PD-L1, which were also the ones with a high presence of immune cells within the tumors, “hot tumors”, and those that showed to be negative for all three proteins, and that correlated with lower infiltration of T cells, “cold tumors”. We also analyzed the location of the proteins within the tumors, and found an effect between endothelial expression of Ido1 and reduced infiltration in lung and RCC tumors. Conclusions: The response to combined immune checkpoint and IDO1 therapy could depend on the proper stratification of patients; therefore, it is of significant interest to determine the proportion of solid tumors that express IDO1, CD73 and PD-L1, and the correlation of the expression of these three immune check points to immune cell infiltration within the tumor. Thus, proper patient stratification based on the expression of these immune checkpoints may improve the efficacy of IDO1 inhibitor based immune therapies. Citation Format: Ana Cerezo, Gloria Martinez-del Hoyo, Sandra Peregrina, Tamara Mondejar, Eduardo Caleiras, Patricia Gonzalez, Eva Lospitao, Sonia Hernandez-Tiedra, Laura Diezma, Estela Casas, Frank Dorsey, Karim Benhadji, Raymond Gilmour, Sandaruwan Geeganage, Susana Velasco. Investigating the expression of indolamine deoxygenase I (IDO1) and other immune markers in tissue microarrays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 513.

Increased expression of GEF-H1 promotes colon cancer progression by RhoA signaling

Colorectal cancer (CRC) is the third most common malignancy and a leading cause of cancer-related death worldwide. GEF-H1 is considered a RhoA-specific guanine nucleotide exchange factor. GEF-H1 upregulation may contribute to cancer cell migration and invasion and tumor progression. However, the expression and role of GEF-H1 in CRC have not yet been elucidated. This study attempted to elucidate how GEF-H1 drives tumor formation, motility, invasion and metastasis in colon cancer (CC). The expression of GEF-H1 in CC tissue microarrays (TMAs) was analyzed by immunohistochemistry (IHC). GEF-H1 was upregulated in CC tissues compared with adjacent non-tumoral tissues. In addition, we found that high GEF-H1 expression correlated with shorter overall survival and distant metastasis. Migration and invasion assays showed that GEF-H1 upregulation increased CC cell motility, invasion and metastasis. In contrast, functional knockdown of GEF-H1 by RNAi rescued the effects caused by GEF-H1 overexpression in CC cells. Overexpression of GEF-H1 re-organized the actin cytoskeleton, with increased punctate paxillin staining and F-actin stress fibers. Furthermore, western blotting showed that RhoA activation triggered by GEF-H1 overexpression caused phosphorylation of its downstream target, MLC2, in CC cells. In summary, the present study revealed that GEF-H1 is upregulated in CC tissues and plays a key role in CC metastasis through the GEF-H1-RhoA-MLC2 signaling pathway.

Comparative Proteomics Analysis Identifies Cdc42-Cdc42BPA Signaling as Prognostic Biomarker and Therapeutic Target for Colon Cancer Invasion.

Metastasis is one of the major causes of treatment failure in the patients with colon cancer. The aim of our study is to find key proteins and pathways that drive invasion and metastasis in colon cancer. Eight rounds of selection of cancer cells invading through matrigel-coated chamber were performed to obtain highly invasive colon cancer sublines HCT116-I8 and RKO-I8. Stable Isotope Labeling by Amino Acids in Cell Culture technology was used to identify the differently expressed proteins, and the proteomics data were analyzed by ingenuity pathway analysis. PAK1-PBD immunoprecipitation combined with Western blot were carried out to determine Cdc42 activity, and qRT-PCR and Western blot were used to determine gene expression. The functional role of Cdc42BPA and Cdc42 pathway in colon cancer invasion was studied by loss-of-function experiments including pharmacological blockade, siRNA knockdown, chamber invasion, and WST-1 assays. Human colon cancer tissue microarray was analyzed by immunohistochemistry for overexpression of Cdc42BPA and its correlation with clinicopathological parameters and patient survival outcomes. HCT116-I8 and RKO-I8 cells showed significantly stronger invasive potential as well as decreased E-cadherin and increased vimentin expressions compared with parental cells. The differently expressed proteins in I8 cells compared with parental cells were identified. Bioinformatics analysis of proteomics data suggested that Cdc42BPA protein and Cdc42 signaling pathway are important for colon cancer invasion, which was confirmed by experimental data showing upregulation of Cdc42BPA and higher expression of active GTP-bound form of Cdc42 in HCT116-I8 and RKO-I8 cells. Functionally, pharmacological and genetic blockade of Cdc42BPA and Cdc42 signaling markedly suppressed colon cancer cell invasion and reversed epithelial mesenchymal transition process. Furthermore, compared with adjacent normal tissues, Cdc42BPA expression was significantly higher in colon cancer tissues and further upregulated in metastatic tumors in lymph nodes. More importantly, Cdc42BPA expression was correlated with metastasis and poor survival of the patients with colon cancer. This study provides the first evidence that Cdc42BPA and Cdc42 signaling are important for colon cancer invasion, and Cdc42BPA has potential implications for colon cancer prognosis and treatment.

Abstract 1701: CCR6 associates with colon cancer metastasis

Abstract Despite established benefits of screening, colon cancer remains a leading cause of cancer death in the U.S. Majority of colon cancer deaths result from metastasis. Effective treatments are not available for advanced disease because molecular mechanisms of initiation and progression of this disease are yet to be defined. Chemokine-chemokine receptor interaction plays an important role in cancer progression. In this study, using colon cancer tissue microarray, we have shown that expression of CCR6 was significantly higher in advanced colon cancer (p&amp;lt;0.0001) with distant and regional lymph node metastasis as compared to non-metastatic and adjacent normal tissues. Expression of CCR6 was further confirmed in cell lines derived from Dukes's type C and type D colon cancer patients using flow cytometric analysis. Like tissues, CCR6 expression was significantly (p&amp;lt;0.0001) higher in colon cancer cell lines compared to normal colon epithelial cells. Furthermore, colon cancer cells showed higher migratory potential toward chemotactic gradient of its only known natural ligand, CCL20. Cell proliferation was also inhibited in presence of CCL20. Significant decrease in E-cadherin, increased expression of vimentin, β-catenin, N-cadherin, α-SMA, SNAIL and ZEB1 was observed following CCL20 treatment. These results suggest the importance of CCR6-CCL20 axis in the etiopathogenesis of colon cancer and highlight its potential as therapeutic target. Citation Format: Neeraj Kapur, Hina Mir, Clarence E. Clark, Uma Krishnamurti, Derrick J. Beech, James W. Lillard, Shailesh Singh. CCR6 associates with colon cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1701.

Ubiquitin-specific peptidase 22 inhibits colon cancer cell invasion by suppressing the signal transducer and activator of transcription 3/matrix metalloproteinase 9 pathway.

Colon cancer is associated with increased cell migration and invasion. In the present study, the role of ubiquitin-specific peptidase 22 (USP22) in signal transducer and activator of transcription 3 (STAT3)-mediated colon cancer cell invasion was investigated. The messenger RNA levels of STAT3 target genes were measured by reverse transcription-quantitative polymerase chain reaction, following USP22 knockdown by RNA interference in SW480 colon cancer cells. The matrix metalloproteinase 9 (MMP9) proteolytic activity and invasion potential of SW480 cells were measured by zymography and Transwell assay, respectively, following combined USP22 and STAT3 short interfering (si)RNA treatment or STAT3 siRNA treatment alone. Similarly, a cell counting kit-8 assay was used to detect the proliferation potential of SW480 cells. The protein expression levels of USP22, STAT3 and MMP9 were detected by immunohistochemistry in colon cancer tissue microarrays (TMAs) and the correlation between USP22, STAT3 and MMP9 was analyzed. USP22/STAT3 co-depletion partly rescued the MMP9 proteolytic activity and invasion of SW480 cells, compared with that of STAT3 depletion alone. However, the proliferation of USP22/STAT3si-SW480 cells was decreased compared with that of STAT3si-SW480 cells. USP22 expression was positively correlated with STAT3 and MMP9 expression in colon cancer TMAs. In conclusion, USP22 attenuated the invasion capacity of colon cancer cells by inhibiting the STAT3/MMP9 signaling pathway.

LIN28B promotes colon cancer migration and recurrence.

LIN28B is involved in “stemness” and tumourigenesis by negatively regulating the maturation of let-7 microRNA family members. In this study, we showed that LIN28B expression promotes migration and recurrence of colon cancer. Immunohistochemistry and reverse-transcription polymerase chain reactions were performed to detect LIN28B expression in colon cancer tissue microarrays, paraffin-embedded surgical resected tissues and cancer cells. Loss-of-function, migration and proliferation analyses were performed to delineate the potential roles of LIN28B in colon cancer. LIN28B was upregulated in colon cancer tissue compared to normal mucosa, and its overexpression correlated with reduced patient survival and increased tumour recurrence. LIN28B suppression inhibited the migration of SW480 colon cancer cells and facilitated the cytotoxicity induced by oxaliplatin in SW480 and HCT116 colon cancer cells. In conclusion, LIN28B overexpression contributes to colon tumourigenesis, and LIN28B may serve as a diagnostic tool and therapeutic target for colon cancer.

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

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

Abstract 1466: Regulation of colon cancer metastasis by sprouty-2

Abstract Introduction: We earlier established that sprouty-2 is overexpressed in human colon cancer and up-regulates c-met and thereby increases the growth phenotype and metastatic potential of colon cancer cells (Oncogene, 29: 5241-53, 2010). To our knowledge, this was the first study to demonstrate the tumor oncogene phenotype of sprouty-2 in colon cancer metastasis. On the contrary, cancer protective role of sprouty-2 was noted in breast, liver and prostate cancers. However, a recent report on non-small cell lung cancer (NSCLC) discovered that silencing of sprouty-2 reduced c-met expression and thus confirmed our findings in colorectal cancer (PNAS, 110: 8573-78, 2013). We further extended our studies to demonstrate that this pathway modulates gastrointestinal specific microRNAs (miRNAs) and regulates various oncogenes and tumor suppressor genes in colon cancer cells. Methods: RNA from control and shRNA sprouty-2 transfected cells were labeled with Hy5 and Hy3 respectively and hybridized to miRCURY LNA arrays. Unsupervised miRNA clustering was carried out and a heat map prepared with Java TreeView. MicroRNA targets were identified by Real-time PCR, western blotting and 3′UTR luciferase assays. Migratory phenotype of colon cancer cells was confirmed by cell migration and invasion assays in trans-well chambers. Cancer cells were transfected by miRNA mimics to assess the expression levels of target genes. Human colon cancer tissue microarrays were utilized to assess the expression of miRNA target genes. Human tumor sections were evaluated by immunofluorescence and immunohistochemical staining. Results: Sprouty-2 shRNA transfected stable cell lines demonstrated upregulation of tumor suppressive miRNAs and down-regulation of oncogenic miRNAs. Overexpression of gastrointestinal specific miRNA mimics inhibits colon cancer cells proliferation and metastasis. On the contrary, overexpression of miRNA inhibitor in colon cancer cells promotes proliferation and colonization. Studies further indicated that miRNA down-regulates expression of CDK7, RB1, AKT, FASL, IGF1R, EGFR and N-cadherin and up-regulates expression of P21 and E-cadherin. MiRNA regulated genes were responsible for cell cycle arrest, enhanced apoptosis and decreased metastasis. AKT was upregulated whereas E-cadherin was downregulated at the invasive fronts in human colon tumors. Conclusion: Sprouty-2/miRNA expression levels could be used to risk-stratify patients for CRC recurrence and sprouty-2 axis regulation may lead to the development of therapeutics in colon and lung cancer. Citation Format: Qiong Zhang, Vivek Vaish, Sharad Khare. Regulation of colon cancer metastasis by sprouty-2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1466. doi:10.1158/1538-7445.AM2014-1466

Tu1677 Upregulation of Fas Ligand Expression Promotes Colon Cancer Cell Migration and Metastasis In Vitro and In Vivo

Introduction: We earlier established that sprouty-2 is overexpressed in human colon cancer and up-regulates c-met and thereby increases the growth phenotype and metastatic potential of colon cancer cells (Oncogene, 29: 5241-53, 2010). To our knowledge, this was the first study to demonstrate the tumor oncogene phenotype of sprouty-2 in colon cancer metastasis. On the contrary, cancer protective role of sprouty-2 was noted in breast, liver and prostate cancers. However, a recent report on non-small cell lung cancer (NSCLC) discovered that silencing of sprouty-2 reduced c-met expression and thus confirmed our findings in colorectal cancer (PNAS, 110: 8573-78, 2013). We further extended our studies to demonstrate that this pathway modulates gastrointestinal specific microRNAs (miRNAs) and regulates various oncogenes and tumor suppressor genes in colon cancer cells. Methods: RNA from control and shRNA sprouty-2 transfected cells were labeled with Hy5 and Hy3 respectively and hybridized to miRCURY LNA arrays. Unsupervised miRNA clustering was carried out and a heat map prepared with Java TreeView. MicroRNA targets were identified by Real-time PCR, western blotting and 3'UTR luciferase assays. Migratory phenotype of colon cancer cells was confirmed by cell migration and invasion assays in trans-well chambers. Cancer cells were transfected by miRNA mimics to assess the expression levels of target genes. Human colon cancer tissue microarrays were utilized to assess the expression of miRNA target genes. Human tumor sections were evaluated by immunofluorescence and immunohistochemical staining. Results: Sprouty-2 shRNA transfected stable cell lines demonstrated upregulation of tumor suppressive miRNAs and down-regulation of oncogenic miRNAs. Transfection of miRNA mimics regulated AKT, p21, N-cadherin and E-cadherin expression in western blotting and RTPCR experiments. Both shRNA sprouty-2 and miRNA mimics blocked cell migration and invasion in cancer cells. AKT was upregulated whereas E-cadherin was downregulated at the invasive fronts in human colon tumors. Conclusion: Sprouty-2/ AKT/E-cadherin expression levels could be used to risk-stratify patients for CRC recurrence and sprouty-2 axis regulation may lead to the development of therapeutics in colon and lung cancer.

Tu1676 Role of Sprouty-2 and MicroRNA in Colon Cancer

Introduction: We earlier established that sprouty-2 is overexpressed in human colon cancer and up-regulates c-met and thereby increases the growth phenotype and metastatic potential of colon cancer cells (Oncogene, 29: 5241-53, 2010). To our knowledge, this was the first study to demonstrate the tumor oncogene phenotype of sprouty-2 in colon cancer metastasis. On the contrary, cancer protective role of sprouty-2 was noted in breast, liver and prostate cancers. However, a recent report on non-small cell lung cancer (NSCLC) discovered that silencing of sprouty-2 reduced c-met expression and thus confirmed our findings in colorectal cancer (PNAS, 110: 8573-78, 2013). We further extended our studies to demonstrate that this pathway modulates gastrointestinal specific microRNAs (miRNAs) and regulates various oncogenes and tumor suppressor genes in colon cancer cells. Methods: RNA from control and shRNA sprouty-2 transfected cells were labeled with Hy5 and Hy3 respectively and hybridized to miRCURY LNA arrays. Unsupervised miRNA clustering was carried out and a heat map prepared with Java TreeView. MicroRNA targets were identified by Real-time PCR, western blotting and 3'UTR luciferase assays. Migratory phenotype of colon cancer cells was confirmed by cell migration and invasion assays in trans-well chambers. Cancer cells were transfected by miRNA mimics to assess the expression levels of target genes. Human colon cancer tissue microarrays were utilized to assess the expression of miRNA target genes. Human tumor sections were evaluated by immunofluorescence and immunohistochemical staining. Results: Sprouty-2 shRNA transfected stable cell lines demonstrated upregulation of tumor suppressive miRNAs and down-regulation of oncogenic miRNAs. Transfection of miRNA mimics regulated AKT, p21, N-cadherin and E-cadherin expression in western blotting and RTPCR experiments. Both shRNA sprouty-2 and miRNA mimics blocked cell migration and invasion in cancer cells. AKT was upregulated whereas E-cadherin was downregulated at the invasive fronts in human colon tumors. Conclusion: Sprouty-2/ AKT/E-cadherin expression levels could be used to risk-stratify patients for CRC recurrence and sprouty-2 axis regulation may lead to the development of therapeutics in colon and lung cancer.

MiR-126 inhibits colon cancer proliferation and invasion through targeting IRS1, SLC7A5 and TOM1 gene

To explore the expression pattern and function of miR-126 in human colon cancer and the underlying mechanisms. The expression pattern of miR-126 in high-density human colon cancer tissue microarray was analyzed by in situ hybridization. Further more, the biological function of miR-126 in colon cancer in vitro was investigated by establishing a stable miR-126 over-expression cell lines. The expression of miR-126 was lower in the tumor tissue, especially in metastasis tissue. The down-regulation of miR-126 was more obvious in the patients who displayed bad prognosis (P=0.025). Over-expression of miR-126 in colon cancer cell was able to inhibit cell proliferation, promote cell apoptosis and reduce the invasive ability. MiR-126 significantly enhanced the sensitivity of the colon cancer cell to chemotherapeutic drug. It has been shown that IRS1, SLC75A and TOM1 were the potential target genes of miR-126 in colon cancer. MiR-126 was able to inhibit the development of colon cancer and its level was closely related with the prognosis of patients with colon cancer. The potential target genes for miR-126 might include IRS1, SLC7A5 and TOM1. Therefore, miR-126 might be a therapeutic target for colon cancer diagnosis and treatment.

Retinoic acid-induced protein 3: Identification and characterisation of a novel prognostic colon cancer biomarker

Aim of the studyValidated molecular biomarkers are urgently required in colon cancer (CC) to accurately define prognosis and, ideally, to predict response to therapeutic modalities such as adjuvant chemotherapy. We aimed to identify and characterise a novel membrane-associated protein in CC tissues which may offer diagnostic and, potentially, therapeutic targeting opportunities. MethodsLabel-free mass spectrometric (MS) quantitation was employed to profile matched colon tissues for malignancy-associated proteins. The putative diagnostic utility of a chosen marker was evaluated using immunohistochemistry (IHC) on 367 CC tissue samples contained within the NCI Progression Colon Cancer tissue microarray (TMA) set. ResultsRetinoic acid-induced protein 3 (RAI3) was initially identified as a plasma membrane protein overexpressed in CC. Cancer-associated RAI3 over-expression was confirmed by RAI3 IHC. Although RAI3 IHC expression patterns were variable within neoplastic epithelium, 76% (n=236) of interpretable CC cases (n=312) displayed diffuse cytoplasmic expression. Of note, a sub-set of CC tissues (n=23, 7.4%) displayed very strong cytoplasmic expression, a feature significantly associated with disease recurrence in Dukes’ A–C (stage I–III) patients (hazard ratio (HR)=3.076, [95%confidence interval (CI)=1.738–5.445]; p<0.001) when compared to low or negative expression of RAI3. This association retained univariate significance in Dukes’ B/stage II patients only (HR=3.494, [95%CI=1.197–10.20]; p<0.022). Significantly, the prognostic capacity of RAI3 was maintained in the stage I–III cohort following multivariate modelling (HR=2.11, [95%CI 1.109–4.017], p=0.023). ConclusionRAI3 is a putative prognostic marker that identifies a small subset of CC patients with high recurrence risk. This study demonstrates the potential value of modern proteomic technology in clinically relevant applications.