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Genome-Wide Analysis Revealed a Robust Gene Expression Signature to Identify Lymph Node Metastasis in Submucosal Colorectal Cancer
AGA Abstracts
Tu1963
Tu1965
GENOME-WIDE ANALYSIS REVEALED A ROBUST GENE EXPRESSION SIGNATURE TO IDENTIFY LYMPH NODE METASTASIS IN SUBMUCOSAL COLORECTAL CANCER Raju Kandimalla, Tsuyoshi Ozawa, Feng Gao, Keisuke Hata, Hiroaki Nozawa, Hiroshi Nagata, Daisuke Izumi, Hideo Baba, James W. Fleshman, Xin Wang, Toshiaki Watanabe, Ajay Goel
EPIGENETIC SILENCING OF MIR-193A, A STAT3 TARGETED MICRORNA BY CONSTITUTIVE ACTIVATION OF JAK/STAT SIGNALING LEAD TO TUMOR PROGRESSION THROUGH OVEREXPRESSION OF YWHAZ IN GASTRIC CANCER Kuo-Liang Wei, Cheng-Shyong Wu, Jian-Liang Chou, Pearlly S. Yan, Chin Li, Michael W.Y. Chan
Background: Due to recent advances in colonoscopic techniques, submucosal colorectal cancers (T1 CRCs) can now be removed endoscopically. Among these, 70% of T1 CRCs are considered as "high risk" because they demonstrate presence of lymphovascular invasion, poor differentiation, and the depth of tumor is >1000um. However, post-surgical pathology results suggest that only ~10-15% of all T1 CRCs are truly lymph node positive, while majority of high risk patients undergo unnecessary surgical treatments with current criteria. Since current pathological criteria have limitations, availability of molecular biomarkers that can identify ‘genuine high risk patients with lymph node (LN) metastasis' will reduce the burden of surgical overtreatment. Since gene expression-based classification of CRC could identify patients with poor prognosis, we sought to identify a gene expression signature which can detect T1 CRCs with LN metastasis. Methods: Two independent publicly available genome-wide mRNA expression datasets were used for mRNA biomarker discovery (n= 125) and in-silico validation (n=56). Genome-wide unbiased gene expression signature was developed from The Cancer Genome Atlas (TCGA) RNA-Seq data by comparing the expression profiles between 16 LN-positive and 109 LN-negative T1/2 CRC patients. In addition to the selection of most differentially expressed genes between the two groups, we used (ROC) based back-step elimination methodology to identify a robust mRNA panel. The gene panel was validated in an independent publicly available dataset (n=56), followed by analytical validation in two independent T1 CRC patient cohorts (n=134 and n=67) using RT-PCR assays. Results: The in silico genome-wide comprehensive discovery led to the identification of an eight gene mRNA classifier that significantly predicted LN-metastasis with an AUC of 1.0, and the subsequent validation in an independent public data set resulted in an AUC of 0.93. The RT-PCR based training and validation of this eight gene classifier in two independent clinical cohorts robustly identified LN metastasis-positive T1 CRC patients with an AUC of 0.86 (95% CI 0.74-0.97, P=0.001) and 0.79 (95% CI 0.69-0.92, P=0.001) respectively. Conclusions: In conclusion, we have identified a novel mRNA-based classifier that can detect high risk T1 CRCs with Lymph node metastasis. Further validation of these biomarkers in endoscopically collected biopsies will aid in clinical decision making and improving the clinical management of such patients.
Gastric cancer is the second leading cause of cancer worldwide. Epigenetic silencing of tumorsuppressors has emerged as an important underlying mechanism in gastric carcinogenesis. Previous studies showed that infection of H. pylori activates JAK/STAT3 signaling pathway in gastric cancer. However, the role of this aberrant signaling in epigenetic silencing of tumor suppressor genes remains unclear. In this study, we performed combined expression microarray and MBDcap-Seq to analyze the expression and methylation profile in gastric cancer cell lines depleted with STAT3. As compared with parental cells, knock down of STAT3 resulted in demethylation and re-expression of a putative STAT3 targeted microRNA, miR-193a in AGS gastric cancer cells. Consistently, reciprocal experiments revealed that promoter region of miR-193a was hypermethylated in MKN28 cells overexpressed with constitutive activated STAT3 mutant. Expression analysis in a panel of gastric cancer cell lines found that miR-193a was down-regulated in AGS, MKN28 and KATOIII. These reduced expression was concomitant to promoter hypermethylation of miR-193a as revealed by bisulphite pyrosequencing. Combination treatment of DNMT inhibitor (5-azaDC) and HDAC inhibitor (TSA) resulted in a robust re-expression of miR-193a in AGS gastric cancer cells. Ectopic expression of miR-193a in AGS cells reduced gastric cancer cell growth and migration in colony formation and wound healing assay. Further microarray and bioinformatics analysis found that YWHAZ, a predicted miR-193a target, was suppressed in AGS cells overexpressed with miR-193a. Depletion of YWHAZ reduced migration in in AGS cells, while its overexpression increased invasion in MKN45 cells. Clinically, bisulphite pyrosequencing revealed that promoter methylation of miR-193a was significantly higher in human gastric cancer tissues (n=11) as compared to gastritis (n=8, p<0.05). Interestingly, tumor adjacent normal tissues infected with H. pylori showed a significantly higher promoter methylation of miR-193a that tissues without H. pylori infection (p < 0.05). Immunohistochemisty also showed a positive correlation between the expression of STAT3 and YWHAZ in tissue microarray of gastric cancer patients (n=64, p<0.005). As compared to low-stage tumor samples, higher expression of YWHAZ was also observed in high-stage patient samples (p<0.05). Taken together, constitutive activation of JAK/STAT signaling may confer epigenetic silencing of the putative STAT3 target and tumor suppressor microRNA, miR-193a in gastric cancer. Transcriptional suppression of miR-193a may led to overexpression of YWHAZ resulting in tumor progression. Targeted demethylation of miR-193a may be a novel therapeutic strategy against gastric cancer.
Tu1964 SERUM MICRORNAS CAN DETECT COLORECTAL CANCER; TWO VALIDATION STUDY AFTER IN-SILICO ANALYSIS Hiroyuki Takamaru, Yutaka Saito, Taku Sakamoto, Seiichiro Abe, Masayoshi Yamada, Takeshi Nakajima, Takahisa Matsuda, Ochiai Hiroki, Yukihide Kanemitsu, Kazuki Sudo, Ken Kato, Junpei Kawauchi, Satoko Takizawa, Hiromi Sakamoto, Motohiro Kojima, Atsushi Ochiai, Shumpei Niida, Hideshi Ishii, Juntaro Matsuzaki, Takahiro Ochiya
Tu1966 NFKB IS ESSENTIAL FOR ACTIVIN-INDUCED COLORECTAL CANCER MIGRATION VIA PI3K-MDM2 MEDIATED P21 DEGRADATION Arundhati Jana, Nancy Krett, Grace Guzman, Ahmer Khalid, Ozkan Ozden, Jonas J. Staudacher, Jessica I. Bauer, Seung Baik, Cemal Yazici, Barbara Jung
Introduction: Recent studies have reported that serum microRNAs (miRNAs) are potentially useful biomarkers for cancer. However, a confirmed the detection system using serum miRNAs is not yet established in colorectal cancer (CRC). The aims of this study are 1) To identify specific serum miRNAs, and establish a discriminant model for CRC detection and 2) To validate the miRNAs and the discriminant model using two large cohorts. Methods: We identified serum miRNAs associated with the presence of CRC by in-silico analysis. We trawled publicly-released serum miRNA database including 50 patients with CRC, 150 healthy individuals and 538 other cancers or benign disease (GSE59856 and GSE73002). Serum miRNA levels were compared between CRC and non-CRC patients. Fisher's linear discriminant analysis was performed to construct the discriminant model for CRC detection. The first validation cohort consisted of 742 patients of CRC managed at the National Cancer Center Hospital (NCCH) from 2007 to 2011 and Osaka University Hospital, and 1061 patients without cancer from Yokohama Minoru Clinic. The second validation cohort consisted of 311 patients CRC managed at NCCH from 2012 to 2014 and 1193 patients of without cancer from the National Center for Geriatrics and Gerontology. Total RNA was extracted from serum and comprehensive miRNA expression analysis was performed using a "3D-Gene" microarray. Measured values of each miRNAs were extrapolated into the discriminant formulas. We performed ROC analysis to evaluate the diagnostic ability of these formulas in each validation cohort. The sensitivity of each colon segment was also reported. This research is partially supported by the "Development of Diagnostic Technology for Detection of miRNA in Body Fluids" grant from the Japan Agency for Medical Research and Development (AMED). Results: First we picked up 28 miRNAs for CRC diagnosis by in-silico analysis. Fisher's analysis revealed about 320,000 candidates of numerical formulas, that showed > 80% of sensitivity and specificity. We could narrow to 14 candidate formulas with first validation analysis. Five or six miRNAs were incorporated in these formulas. One of the best AUC, sensitivity and specificity of the discriminant formula in first validation was 0.88, 0.79 and 0.87, respectively by ROC analysis. The sensitivity of CRC at right and left colon segment was 91.8% and 88.9%, respectively. Second validation showed the AUC, sensitivity and specificity of the same discriminant formula with the same cut-off point was 0.97, 0.81 and 0.97, respectively. The sensitivity of CRC at right and left colon segment was 84.8% and 92.5%, respectively. Conclusions: We identified novel serum miRNAs for CRC detection. Our discriminant using these miRNAs can diagnose CRC. Furthermore, the sensitivity was high irrespective of the tumor location.
AGA Abstracts
Background and Aims: Colorectal cancer (CRC) remains a common and deadly cancer due to metastatic disease. Activin and TGFB signaling are growth suppressive pathways however they exert non-canonical pro-metastatic effects late in CRC carcinogenesis. Both activin and NFkB signaling are important in inflammation and we have previously shown that in CRC, activin downregulates p21 via PI3K. Since NFkB regulates expression of the E3 ubiquitin ligase MDM2 (Mouse double minute 2 homolog) and activin-induced p21 degradation is through ubiquitination we hypothesized that activin and NFkB signaling may be linked in CRC. Methods: Correlation of activated NFkB signaling and activin ligand expression was determined by immunohistochemistry on a tissue microarray (TMAs) generated from 131 colorectal cancer patients. Colon cancer cell lines including activin receptor type 2 (ACVR2) mutated HCT116, ACVR2-restored HCT116+chr2, ACVR2 and SMAD4 positive FET and ACVR2 positive but SMAD4-null SW480 colon cancer cells were utilized to dissect linkage of activin and NFkB signaling. DNA-Binding activity of NFkB was assessed by electrophoretic mobility shift assay (EMSA). Recruitment of p65 to the promoter of MDM2 was measured by chromatin immunoprecipitation assay (ChIP). The requirement for NFkB and PI3K activation was confirmed through use of pharmacological inhibitors of NFkB and PI3K. Migration was assessed by trans well experiment and relative cell viability by MTT. Results: Activated NFkB as evidenced by phospho-p65 staining and activin ligand expression correlate in primary CRC (correlation coefficient =0.542, p<0.001) with high dual staining correlating to metastasis (p=0.0001). Activin induced activation of NFkB is shown by EMSA and a reduction in IKB alpha protein. Furthermore, this regulation is dependent on activation of PI3K. The overexpression of wild type p65 in colon cancer cells increased MDM2 paralleled by a decrease in p21 expression while NFkB inhibition blocked the activin-induced increase in MDM2. Moreover, activin induced MDM2 expression is independent of SMAD4 and activin induced recruitment of p65 to two NFkB binding sites on the MDM2 promoter. Functionally, NFkB inhibition decreased activin-induced cell migration and also activininduced decrease in cell viability in various colon cancer cells in an ACVR2-dependent, but SMAD4- independent manner. Conclusions: Here, we provide evidence that in colon cancer, activin activates NFkB via PI3K enhancing migration via p21 down regulation though the MDM2 ubiquitin ligase independent of SMAD4. These results support a new mechanistic understanding linking inflammation and advanced colon cancer through activin-induced NFkB signaling. In addition, our current data support the hypothesis that CRC patients with high tumor expression of activin ligand may benefit from clinical inhibition of NFkB.
S-1018
Tu1963
Tu1965
GENOME-WIDE ANALYSIS REVEALED A ROBUST GENE EXPRESSION SIGNATURE TO IDENTIFY LYMPH NODE METASTASIS IN SUBMUCOSAL COLORECTAL CANCER Raju Kandimalla, Tsuyoshi Ozawa, Feng Gao, Keisuke Hata, Hiroaki Nozawa, Hiroshi Nagata, Daisuke Izumi, Hideo Baba, James W. Fleshman, Xin Wang, Toshiaki Watanabe, Ajay Goel
EPIGENETIC SILENCING OF MIR-193A, A STAT3 TARGETED MICRORNA BY CONSTITUTIVE ACTIVATION OF JAK/STAT SIGNALING LEAD TO TUMOR PROGRESSION THROUGH OVEREXPRESSION OF YWHAZ IN GASTRIC CANCER Kuo-Liang Wei, Cheng-Shyong Wu, Jian-Liang Chou, Pearlly S. Yan, Chin Li, Michael W.Y. Chan
Background: Due to recent advances in colonoscopic techniques, submucosal colorectal cancers (T1 CRCs) can now be removed endoscopically. Among these, 70% of T1 CRCs are considered as "high risk" because they demonstrate presence of lymphovascular invasion, poor differentiation, and the depth of tumor is >1000um. However, post-surgical pathology results suggest that only ~10-15% of all T1 CRCs are truly lymph node positive, while majority of high risk patients undergo unnecessary surgical treatments with current criteria. Since current pathological criteria have limitations, availability of molecular biomarkers that can identify ‘genuine high risk patients with lymph node (LN) metastasis' will reduce the burden of surgical overtreatment. Since gene expression-based classification of CRC could identify patients with poor prognosis, we sought to identify a gene expression signature which can detect T1 CRCs with LN metastasis. Methods: Two independent publicly available genome-wide mRNA expression datasets were used for mRNA biomarker discovery (n= 125) and in-silico validation (n=56). Genome-wide unbiased gene expression signature was developed from The Cancer Genome Atlas (TCGA) RNA-Seq data by comparing the expression profiles between 16 LN-positive and 109 LN-negative T1/2 CRC patients. In addition to the selection of most differentially expressed genes between the two groups, we used (ROC) based back-step elimination methodology to identify a robust mRNA panel. The gene panel was validated in an independent publicly available dataset (n=56), followed by analytical validation in two independent T1 CRC patient cohorts (n=134 and n=67) using RT-PCR assays. Results: The in silico genome-wide comprehensive discovery led to the identification of an eight gene mRNA classifier that significantly predicted LN-metastasis with an AUC of 1.0, and the subsequent validation in an independent public data set resulted in an AUC of 0.93. The RT-PCR based training and validation of this eight gene classifier in two independent clinical cohorts robustly identified LN metastasis-positive T1 CRC patients with an AUC of 0.86 (95% CI 0.74-0.97, P=0.001) and 0.79 (95% CI 0.69-0.92, P=0.001) respectively. Conclusions: In conclusion, we have identified a novel mRNA-based classifier that can detect high risk T1 CRCs with Lymph node metastasis. Further validation of these biomarkers in endoscopically collected biopsies will aid in clinical decision making and improving the clinical management of such patients.
Gastric cancer is the second leading cause of cancer worldwide. Epigenetic silencing of tumorsuppressors has emerged as an important underlying mechanism in gastric carcinogenesis. Previous studies showed that infection of H. pylori activates JAK/STAT3 signaling pathway in gastric cancer. However, the role of this aberrant signaling in epigenetic silencing of tumor suppressor genes remains unclear. In this study, we performed combined expression microarray and MBDcap-Seq to analyze the expression and methylation profile in gastric cancer cell lines depleted with STAT3. As compared with parental cells, knock down of STAT3 resulted in demethylation and re-expression of a putative STAT3 targeted microRNA, miR-193a in AGS gastric cancer cells. Consistently, reciprocal experiments revealed that promoter region of miR-193a was hypermethylated in MKN28 cells overexpressed with constitutive activated STAT3 mutant. Expression analysis in a panel of gastric cancer cell lines found that miR-193a was down-regulated in AGS, MKN28 and KATOIII. These reduced expression was concomitant to promoter hypermethylation of miR-193a as revealed by bisulphite pyrosequencing. Combination treatment of DNMT inhibitor (5-azaDC) and HDAC inhibitor (TSA) resulted in a robust re-expression of miR-193a in AGS gastric cancer cells. Ectopic expression of miR-193a in AGS cells reduced gastric cancer cell growth and migration in colony formation and wound healing assay. Further microarray and bioinformatics analysis found that YWHAZ, a predicted miR-193a target, was suppressed in AGS cells overexpressed with miR-193a. Depletion of YWHAZ reduced migration in in AGS cells, while its overexpression increased invasion in MKN45 cells. Clinically, bisulphite pyrosequencing revealed that promoter methylation of miR-193a was significantly higher in human gastric cancer tissues (n=11) as compared to gastritis (n=8, p<0.05). Interestingly, tumor adjacent normal tissues infected with H. pylori showed a significantly higher promoter methylation of miR-193a that tissues without H. pylori infection (p < 0.05). Immunohistochemisty also showed a positive correlation between the expression of STAT3 and YWHAZ in tissue microarray of gastric cancer patients (n=64, p<0.005). As compared to low-stage tumor samples, higher expression of YWHAZ was also observed in high-stage patient samples (p<0.05). Taken together, constitutive activation of JAK/STAT signaling may confer epigenetic silencing of the putative STAT3 target and tumor suppressor microRNA, miR-193a in gastric cancer. Transcriptional suppression of miR-193a may led to overexpression of YWHAZ resulting in tumor progression. Targeted demethylation of miR-193a may be a novel therapeutic strategy against gastric cancer.
Tu1964 SERUM MICRORNAS CAN DETECT COLORECTAL CANCER; TWO VALIDATION STUDY AFTER IN-SILICO ANALYSIS Hiroyuki Takamaru, Yutaka Saito, Taku Sakamoto, Seiichiro Abe, Masayoshi Yamada, Takeshi Nakajima, Takahisa Matsuda, Ochiai Hiroki, Yukihide Kanemitsu, Kazuki Sudo, Ken Kato, Junpei Kawauchi, Satoko Takizawa, Hiromi Sakamoto, Motohiro Kojima, Atsushi Ochiai, Shumpei Niida, Hideshi Ishii, Juntaro Matsuzaki, Takahiro Ochiya
Tu1966 NFKB IS ESSENTIAL FOR ACTIVIN-INDUCED COLORECTAL CANCER MIGRATION VIA PI3K-MDM2 MEDIATED P21 DEGRADATION Arundhati Jana, Nancy Krett, Grace Guzman, Ahmer Khalid, Ozkan Ozden, Jonas J. Staudacher, Jessica I. Bauer, Seung Baik, Cemal Yazici, Barbara Jung
Introduction: Recent studies have reported that serum microRNAs (miRNAs) are potentially useful biomarkers for cancer. However, a confirmed the detection system using serum miRNAs is not yet established in colorectal cancer (CRC). The aims of this study are 1) To identify specific serum miRNAs, and establish a discriminant model for CRC detection and 2) To validate the miRNAs and the discriminant model using two large cohorts. Methods: We identified serum miRNAs associated with the presence of CRC by in-silico analysis. We trawled publicly-released serum miRNA database including 50 patients with CRC, 150 healthy individuals and 538 other cancers or benign disease (GSE59856 and GSE73002). Serum miRNA levels were compared between CRC and non-CRC patients. Fisher's linear discriminant analysis was performed to construct the discriminant model for CRC detection. The first validation cohort consisted of 742 patients of CRC managed at the National Cancer Center Hospital (NCCH) from 2007 to 2011 and Osaka University Hospital, and 1061 patients without cancer from Yokohama Minoru Clinic. The second validation cohort consisted of 311 patients CRC managed at NCCH from 2012 to 2014 and 1193 patients of without cancer from the National Center for Geriatrics and Gerontology. Total RNA was extracted from serum and comprehensive miRNA expression analysis was performed using a "3D-Gene" microarray. Measured values of each miRNAs were extrapolated into the discriminant formulas. We performed ROC analysis to evaluate the diagnostic ability of these formulas in each validation cohort. The sensitivity of each colon segment was also reported. This research is partially supported by the "Development of Diagnostic Technology for Detection of miRNA in Body Fluids" grant from the Japan Agency for Medical Research and Development (AMED). Results: First we picked up 28 miRNAs for CRC diagnosis by in-silico analysis. Fisher's analysis revealed about 320,000 candidates of numerical formulas, that showed > 80% of sensitivity and specificity. We could narrow to 14 candidate formulas with first validation analysis. Five or six miRNAs were incorporated in these formulas. One of the best AUC, sensitivity and specificity of the discriminant formula in first validation was 0.88, 0.79 and 0.87, respectively by ROC analysis. The sensitivity of CRC at right and left colon segment was 91.8% and 88.9%, respectively. Second validation showed the AUC, sensitivity and specificity of the same discriminant formula with the same cut-off point was 0.97, 0.81 and 0.97, respectively. The sensitivity of CRC at right and left colon segment was 84.8% and 92.5%, respectively. Conclusions: We identified novel serum miRNAs for CRC detection. Our discriminant using these miRNAs can diagnose CRC. Furthermore, the sensitivity was high irrespective of the tumor location.
AGA Abstracts
Background and Aims: Colorectal cancer (CRC) remains a common and deadly cancer due to metastatic disease. Activin and TGFB signaling are growth suppressive pathways however they exert non-canonical pro-metastatic effects late in CRC carcinogenesis. Both activin and NFkB signaling are important in inflammation and we have previously shown that in CRC, activin downregulates p21 via PI3K. Since NFkB regulates expression of the E3 ubiquitin ligase MDM2 (Mouse double minute 2 homolog) and activin-induced p21 degradation is through ubiquitination we hypothesized that activin and NFkB signaling may be linked in CRC. Methods: Correlation of activated NFkB signaling and activin ligand expression was determined by immunohistochemistry on a tissue microarray (TMAs) generated from 131 colorectal cancer patients. Colon cancer cell lines including activin receptor type 2 (ACVR2) mutated HCT116, ACVR2-restored HCT116+chr2, ACVR2 and SMAD4 positive FET and ACVR2 positive but SMAD4-null SW480 colon cancer cells were utilized to dissect linkage of activin and NFkB signaling. DNA-Binding activity of NFkB was assessed by electrophoretic mobility shift assay (EMSA). Recruitment of p65 to the promoter of MDM2 was measured by chromatin immunoprecipitation assay (ChIP). The requirement for NFkB and PI3K activation was confirmed through use of pharmacological inhibitors of NFkB and PI3K. Migration was assessed by trans well experiment and relative cell viability by MTT. Results: Activated NFkB as evidenced by phospho-p65 staining and activin ligand expression correlate in primary CRC (correlation coefficient =0.542, p<0.001) with high dual staining correlating to metastasis (p=0.0001). Activin induced activation of NFkB is shown by EMSA and a reduction in IKB alpha protein. Furthermore, this regulation is dependent on activation of PI3K. The overexpression of wild type p65 in colon cancer cells increased MDM2 paralleled by a decrease in p21 expression while NFkB inhibition blocked the activin-induced increase in MDM2. Moreover, activin induced MDM2 expression is independent of SMAD4 and activin induced recruitment of p65 to two NFkB binding sites on the MDM2 promoter. Functionally, NFkB inhibition decreased activin-induced cell migration and also activininduced decrease in cell viability in various colon cancer cells in an ACVR2-dependent, but SMAD4- independent manner. Conclusions: Here, we provide evidence that in colon cancer, activin activates NFkB via PI3K enhancing migration via p21 down regulation though the MDM2 ubiquitin ligase independent of SMAD4. These results support a new mechanistic understanding linking inflammation and advanced colon cancer through activin-induced NFkB signaling. In addition, our current data support the hypothesis that CRC patients with high tumor expression of activin ligand may benefit from clinical inhibition of NFkB.
S-1018