Tu1670 Inhibition of Corticotropin-Releasing Hormone Receptor 2 (CRHR2) Expression in Colorectal...
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AGA Abstracts a*sqrt(X(i,j) > μ(i) and "low" if, X(i,j) + a*sqrt(X(i,j) < μ(i), where a=6. Genes were grouped into pathways using Ingenuity pathway analysis. RESULTS: Based on their relative expression levels at any one of 4 time points studied, all the genes from the raw RNAseq reads were grouped into 24 gene sets (table1). Four gene-sets figure (1 a-d). appear most interesting and coincide with change of morphology in the BEC-40W and loss of adherence to substrate (colony formation) observed in BEC-60W cells. FPKM data analysis supported upregulation of stem cell, immune response and epigenetic signatures, oxidative phosphorylation and stress response pathways. Pathway analysis revealed these genes are members of VEGF, RB, PTEN, ATF2, TP53 and other known oncogenic pathways. CONCLUSIONS: A statistical model identified four interesting gene sets from RNA sequencing data of the BEC model. These gene sets comprise oncogenes, tumor suppressors as well as regulators of signal transduction that correlated with and may be responsible for the transformed phenotype observed in BEC-40W and BEC-60W cells. Our observation from the BEC model corroborate that chronic exposure to B4 leads to genetic changes that can promote carcinogenesis in BE. Characterization of these potential candidate genes and pathways may lead to innovative biomarkers and therapeutic targets for potentially progressive BE. Table 1: The number of genes in each of the four interesting states (out of the 14 possible states). The expression state is denoted by binary digits 1=upregulation, 0= down regulation. Tu1668 Highly Metastatic Murine Colorectal Cancer Cells Share Transcriptome Identity Indicative of Cancer Cell Fusion With Macrophages Charles E. Gast, Alain Silk, Mark Schmidt, Lara Riegler, Chris Harrington, Tomi Mori, Melissa H. Wong Introduction: The most deadly phase in cancer progression is attributed to the inappropriate acquisition of molecular machinery leading to aggressive behavior and spread of disease to distant organs. The mechanisms that underlie how cancer cells gain these behaviors that ultimately lead to therapeutic resistance, clonal evolution and recurrence is poorly under- stood. We have intriguing evidence that heterotypic fusion between macrophages (Macs) and cancer cells results in hybrid cells that have gained aggressive behavior, providing support that cell fusion is one mechanism for acquisition of properties that enhance tumori- genesis. Further, we have established an in vitro cell fusion system where Macs spontaneously fuse with murine colorectal cancer cells (MC38) to form unique fusion hybrid cells that retain features of both Macs and cancer cells. Interestingly, a metastatic murine colorectal cancer cell line, MC38-met, was derived from a parental cell line, MC38 that did not possess aggressive behavior. In this study, we hypothesized that the MC38-met cell line harbors transcriptome similarities with both Macs and our Mac-MC38 fusion hybrids. Methods: Isolated murine Macs harboring a Cre recombinase yellow fluorescent protein (YFP) reporter were co-cultured with MC38 murine colorectal cancer cells engineered to express Histone2B- red fluorescent protein and Cre recombinase in a 2:1 ratio to permit spontaneous fusion. Mac-MC38 fusion hybrids were FACS-isolated based upon YFP expression. Gene tran- scriptome analysis was performed on MC38s, Macs and Mac-MC38 fusions and gene sets were identified that were up or down-regulated in Macs and Mac-MC38 fusions relative to the parental cancer cells. Analyses of these genes were compared to the MC38-met relative to their parental transcriptome. Results and Conclusion: The aggressive colorectal cancer MC38-met cells harbor a subset of genes that are expressed similarly to Macs and Mac- MC38 fusion hybrid cells, supporting the concept that aggressive tumor behavior may, in part, be a product of cell fusion between cancer cells and Macs. This finding has implications on defining novel mechanisms for acquisition of aggressive disease and has potential to reveal new therapeutic targets. S-814 AGA Abstracts Tu1669 Role of MicroRNA-31 Expression and CpG Island Methylator Phenotype in the Progression of Serrated Lesion Katsuhiko Nosho, Hisayoshi Igarashi, Shinji Yoshii, Miki Ito, Takafumi Naito, Kei Mitsuhashi, Eiichiro Yamamoto, Taiga Takahashi, Yasutaka Sukawa, Masanori Nojima, Reo Maruyama, Hiromu Suzuki, Kohzoh Imai, Hiroyuki Yamamoto, Yasuhisa Shinomura Background & Aims: The serrated pathway has attracted considerable attention as an alterna- tive pathway of colorectal cancer (CRC). In contrast, microRNAs have been increasingly recognized as useful biomarkers for CRC. We recently found that microRNA-31 (miR-31) was associated with BRAF mutation and poor prognosis in CRC. However, no studies have described its role in the progression of serrated lesions. We therefore examined miR-31 expression level as well as other molecular alterations including CpG island methylator phenotype (CIMP) status in serrated lesions. Methods: Hyperplastic polyps (HPs) (N = 142), sessile serrated adenomas (SSAs) (N = 122), SSAs with cytological dysplasia (N = 10), traditional serrated adenomas (TSAs) (N = 102), and TSAs with high-grade dysplasia (HGD) (N = 16) were collected. We examined miR-31 expression in serrated lesions by quantitative RT-PCR. Tumor specimens were analyzed for KRAS and BRAF mutations and microsatellite instability (MSI). We quantified promoter methylation in five CIMP-specific genes (CAC- NA1G, CDKN2A, IGF2, MLH1, and RUNX3) by MethyLight. CIMP-high was defined as three to five methylated promoters. Results: The distributions of miR-31 expression in the 392 serrated lesion specimens were as follows: median, 12.4; range, 0.06-215; interquartile range, 2.6-35.5. Cases with miR-31 expression were then divided into quartiles for further analysis: Q1 (<2.6), Q2 (2.6-12.3), Q3 (12.4-35.4), and Q4 (≥35.5). miR-31 expression was associated with BRAF mutation and CIMP-high status in serrated lesions (P < 0.0001). After serrated lesions were stratified by BRAF and CIMP status, the relationship between miR-31 expression and BRAF mutation persisted (P ≤ 0.03), whereas the relationship between miR-31 expression and CIMP-high did not. No significant difference was observed between miR-31 expression and MSI status. With regard to the histopathology, high miR-31 expression (Q4) and CIMP-high was observed in 12% and 7.9% of HPs, respectively. In contrast, high miR-31 expression and CIMP-high were highly detected in SSAs with cytological dysplasia (70% and 100%, respectively) compared with SSAs (27% and 39%, respectively) (P < 0.0001). Likewise, CIMP-high was frequently observed in TSAs with HGD (79%) compared with TSAs (7.7%) (P < 0.0001); whereas no significant difference in miR-31 expression was found between TSAs with HGD (38%) and TSAs (36%). Conclusion: miR-31 expression was associated with BRAF mutation regardless of CIMP status. CIMP-high was highly detected in SSA with cytological dysplasia and TSA with HGD, suggesting its role in the progression of serrated lesion. Moreover, our data indicate that in SSA miR-31 may be a key molecule in the evolution to SSA with cytological dysplasia. Tu1670 Inhibition of Corticotropin-Releasing Hormone Receptor 2 (CRHR2) Expression in Colorectal Cancer Correlates With Tumor Growth and EMT In Vitro and In Vivo, Poor Patient Survival and Increased Risk for Distant Metastases Jorge Rodriguez, Dimitrios Iliopoulos, Sara huerta Ypez, Jill M. Hoffman, Guillermina J. Baay-Gusman, Ana B. Tirado-Rodriguez, Ivy Ka Man Law, Daniel W. Hommes, Hein W. Verspaget, Lin Chang, Charalabos Pothoulakis, Stavroula Baritaki BACKGROUND & AIMS: Chronic inflammation is a driving force for development and progression of colorectal carcinoma (CRC). We explored the contribution of the corticotropin- releasing-hormone (CRH) family of peptides and receptors in CRC growth and metastatic potential through regulation of inflammatory responses. METHODS: We analyzed the expres- sion of all CRH family members in human CRC (N=56) and control tissues (N=46) as well as in 7 CRC cell lines by qRT-PCR. Expression of pro-inflammatory cytokines, cell proliferation, migration, invasion and colony formation were compared between parental and CRHR2- overexpressing (CRHR2high) CRC cell lines. Targets of CRHR2/Ucn2 signaling were identi- fied by gene superarray and immunoblot analyses. CRHR2/Ucn2-targeted effects on tumor proliferation and epithelial-to-mesenchymal transition (EMT) were validated in nude mice (n=6/group) carrying parental- or CRHR2high-SW620 xenografts and treated with Ucn2. RESULTS: CRC tissues and cells had diminished CRHR2 (>4-fold) and elevated Ucn2 (>5- fold) mRNA expression compared to control specimens and the immortalized epithelial colonic NCM460 cells, respectively. Ucn2 stimulation of CRHR2high cells reduced IL1b (p<0.03), IL6 (p<0.001) and IL6R (p<0.03) mRNAs, IL6-mediated cell proliferation (p<0.03), migration (p<0.01), invasion (p<0.03) and colony formation (p<0.02). At the molecular level, CRHR2/Ucn2 signaling in CRHR2high cells inhibited (>2 fold) the expression of cell cycle and EMT inducing genes in the presence of IL6 stimulation, while it augmented (>2 fold) the expression of cell cycle- and EMT-suppressors under the same conditions. In the SW620 xenograft model, CRHR2high tumors had significantly decreased growth and proliferation rates, increased apoptosis and necrosis, as well as reduced expression of EMT- inducers and elevated levels of EMT-suppressors (>2 fold). All the above changes were further augmented following the same trend under Ucn2 treatment conditions (all p values<0.02). In CRC clinical samples, CRHR2 mRNA expression was inversely correlated with IL6R and vimentin levels, as well as metastasis occurrence, while positively correlated with E-cadherin expression and 5-year survival post surgery (all p values<0.03). CONCLUSIONS: CRHR2 reduction in CRC has a protective role in tumor progression, expansion and metastatic potential through maintenance or promotion of the existing colonic inflammation. In addi- tion, CRHR2low CRC phenotypes are linked with distant metastases and poor clinical out- comes.
Transcript of Tu1670 Inhibition of Corticotropin-Releasing Hormone Receptor 2 (CRHR2) Expression in Colorectal...
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sa*sqrt(X(i,j) > μ(i) and "low" if, X(i,j) + a*sqrt(X(i,j) < μ(i), where a=6. Genes were groupedinto pathways using Ingenuity pathway analysis. RESULTS: Based on their relative expressionlevels at any one of 4 time points studied, all the genes from the raw RNAseq reads weregrouped into 24 gene sets (table1). Four gene-sets figure (1 a-d). appear most interestingand coincide with change of morphology in the BEC-40W and loss of adherence to substrate(colony formation) observed in BEC-60W cells. FPKM data analysis supported upregulationof stem cell, immune response and epigenetic signatures, oxidative phosphorylation andstress response pathways. Pathway analysis revealed these genes are members of VEGF, RB,PTEN, ATF2, TP53 and other known oncogenic pathways. CONCLUSIONS: A statisticalmodel identified four interesting gene sets from RNA sequencing data of the BEC model.These gene sets comprise oncogenes, tumor suppressors as well as regulators of signaltransduction that correlated with and may be responsible for the transformed phenotypeobserved in BEC-40W and BEC-60W cells. Our observation from the BEC model corroboratethat chronic exposure to B4 leads to genetic changes that can promote carcinogenesis inBE. Characterization of these potential candidate genes and pathways may lead to innovativebiomarkers and therapeutic targets for potentially progressive BE.Table 1: The number of genes in each of the four interesting states (out of the 14 possiblestates). The expression state is denoted by binary digits 1=upregulation, 0= down regulation.
Tu1668
Highly Metastatic Murine Colorectal Cancer Cells Share TranscriptomeIdentity Indicative of Cancer Cell Fusion With MacrophagesCharles E. Gast, Alain Silk, Mark Schmidt, Lara Riegler, Chris Harrington, Tomi Mori,Melissa H. Wong
Introduction: The most deadly phase in cancer progression is attributed to the inappropriateacquisition of molecular machinery leading to aggressive behavior and spread of disease todistant organs. The mechanisms that underlie how cancer cells gain these behaviors thatultimately lead to therapeutic resistance, clonal evolution and recurrence is poorly under-stood. We have intriguing evidence that heterotypic fusion between macrophages (Macs)and cancer cells results in hybrid cells that have gained aggressive behavior, providingsupport that cell fusion is one mechanism for acquisition of properties that enhance tumori-genesis. Further, we have established an in vitro cell fusion system where Macs spontaneouslyfuse with murine colorectal cancer cells (MC38) to form unique fusion hybrid cells thatretain features of both Macs and cancer cells. Interestingly, a metastatic murine colorectalcancer cell line, MC38-met, was derived from a parental cell line, MC38 that did not possessaggressive behavior. In this study, we hypothesized that the MC38-met cell line harborstranscriptome similarities with both Macs and our Mac-MC38 fusion hybrids. Methods:Isolated murine Macs harboring a Cre recombinase yellow fluorescent protein (YFP) reporterwere co-cultured with MC38 murine colorectal cancer cells engineered to express Histone2B-red fluorescent protein and Cre recombinase in a 2:1 ratio to permit spontaneous fusion.Mac-MC38 fusion hybrids were FACS-isolated based upon YFP expression. Gene tran-scriptome analysis was performed on MC38s, Macs and Mac-MC38 fusions and gene setswere identified that were up or down-regulated in Macs and Mac-MC38 fusions relative tothe parental cancer cells. Analyses of these genes were compared to the MC38-met relativeto their parental transcriptome. Results and Conclusion: The aggressive colorectal cancerMC38-met cells harbor a subset of genes that are expressed similarly to Macs and Mac-MC38 fusion hybrid cells, supporting the concept that aggressive tumor behavior may, inpart, be a product of cell fusion between cancer cells and Macs. This finding has implicationson defining novel mechanisms for acquisition of aggressive disease and has potential toreveal new therapeutic targets.
S-814AGA Abstracts
Tu1669
Role of MicroRNA-31 Expression and CpG Island Methylator Phenotype in theProgression of Serrated LesionKatsuhiko Nosho, Hisayoshi Igarashi, Shinji Yoshii, Miki Ito, Takafumi Naito, KeiMitsuhashi, Eiichiro Yamamoto, Taiga Takahashi, Yasutaka Sukawa, Masanori Nojima, ReoMaruyama, Hiromu Suzuki, Kohzoh Imai, Hiroyuki Yamamoto, Yasuhisa Shinomura
Background & Aims: The serrated pathway has attracted considerable attention as an alterna-tive pathway of colorectal cancer (CRC). In contrast, microRNAs have been increasinglyrecognized as useful biomarkers for CRC. We recently found that microRNA-31 (miR-31)was associated with BRAF mutation and poor prognosis in CRC. However, no studies havedescribed its role in the progression of serrated lesions. We therefore examined miR-31expression level as well as other molecular alterations including CpG island methylatorphenotype (CIMP) status in serrated lesions. Methods: Hyperplastic polyps (HPs) (N = 142),sessile serrated adenomas (SSAs) (N = 122), SSAs with cytological dysplasia (N = 10),traditional serrated adenomas (TSAs) (N = 102), and TSAs with high-grade dysplasia (HGD)(N = 16) were collected. We examined miR-31 expression in serrated lesions by quantitativeRT-PCR. Tumor specimens were analyzed for KRAS and BRAF mutations and microsatelliteinstability (MSI). We quantified promoter methylation in five CIMP-specific genes (CAC-NA1G, CDKN2A, IGF2, MLH1, and RUNX3) by MethyLight. CIMP-high was defined asthree to five methylated promoters. Results: The distributions of miR-31 expression in the392 serrated lesion specimens were as follows: median, 12.4; range, 0.06-215; interquartilerange, 2.6-35.5. Cases with miR-31 expression were then divided into quartiles for furtheranalysis: Q1 (<2.6), Q2 (2.6-12.3), Q3 (12.4-35.4), and Q4 (≥35.5). miR-31 expressionwas associated with BRAF mutation and CIMP-high status in serrated lesions (P < 0.0001).After serrated lesions were stratified by BRAF and CIMP status, the relationship betweenmiR-31 expression and BRAF mutation persisted (P ≤ 0.03), whereas the relationship betweenmiR-31 expression and CIMP-high did not. No significant difference was observed betweenmiR-31 expression and MSI status. With regard to the histopathology, high miR-31 expression(Q4) and CIMP-high was observed in 12% and 7.9% of HPs, respectively. In contrast, highmiR-31 expression and CIMP-high were highly detected in SSAs with cytological dysplasia(70% and 100%, respectively) compared with SSAs (27% and 39%, respectively) (P <0.0001). Likewise, CIMP-high was frequently observed in TSAs with HGD (79%) comparedwith TSAs (7.7%) (P < 0.0001); whereas no significant difference in miR-31 expression wasfound between TSAs with HGD (38%) and TSAs (36%). Conclusion: miR-31 expressionwas associated with BRAF mutation regardless of CIMP status. CIMP-high was highly detectedin SSA with cytological dysplasia and TSA with HGD, suggesting its role in the progressionof serrated lesion. Moreover, our data indicate that in SSA miR-31 may be a key moleculein the evolution to SSA with cytological dysplasia.
Tu1670
Inhibition of Corticotropin-Releasing Hormone Receptor 2 (CRHR2)Expression in Colorectal Cancer Correlates With Tumor Growth and EMT InVitro and In Vivo, Poor Patient Survival and Increased Risk for DistantMetastasesJorge Rodriguez, Dimitrios Iliopoulos, Sara huerta Ypez, Jill M. Hoffman, Guillermina J.Baay-Gusman, Ana B. Tirado-Rodriguez, Ivy Ka Man Law, Daniel W. Hommes, Hein W.Verspaget, Lin Chang, Charalabos Pothoulakis, Stavroula Baritaki
BACKGROUND & AIMS: Chronic inflammation is a driving force for development andprogression of colorectal carcinoma (CRC). We explored the contribution of the corticotropin-releasing-hormone (CRH) family of peptides and receptors in CRC growth and metastaticpotential through regulation of inflammatory responses. METHODS: We analyzed the expres-sion of all CRH family members in human CRC (N=56) and control tissues (N=46) as well asin 7 CRC cell lines by qRT-PCR. Expression of pro-inflammatory cytokines, cell proliferation,migration, invasion and colony formation were compared between parental and CRHR2-overexpressing (CRHR2high) CRC cell lines. Targets of CRHR2/Ucn2 signaling were identi-fied by gene superarray and immunoblot analyses. CRHR2/Ucn2-targeted effects on tumorproliferation and epithelial-to-mesenchymal transition (EMT) were validated in nude mice(n=6/group) carrying parental- or CRHR2high-SW620 xenografts and treated with Ucn2.RESULTS: CRC tissues and cells had diminished CRHR2 (>4-fold) and elevated Ucn2 (>5-fold) mRNA expression compared to control specimens and the immortalized epithelialcolonic NCM460 cells, respectively. Ucn2 stimulation of CRHR2high cells reduced IL1b(p<0.03), IL6 (p<0.001) and IL6R (p<0.03) mRNAs, IL6-mediated cell proliferation (p<0.03),migration (p<0.01), invasion (p<0.03) and colony formation (p<0.02). At the molecularlevel, CRHR2/Ucn2 signaling in CRHR2high cells inhibited (>2 fold) the expression of cellcycle and EMT inducing genes in the presence of IL6 stimulation, while it augmented (>2fold) the expression of cell cycle- and EMT-suppressors under the same conditions. Inthe SW620 xenograft model, CRHR2high tumors had significantly decreased growth andproliferation rates, increased apoptosis and necrosis, as well as reduced expression of EMT-inducers and elevated levels of EMT-suppressors (>2 fold). All the above changes were furtheraugmented following the same trend under Ucn2 treatment conditions (all p values<0.02). InCRC clinical samples, CRHR2 mRNA expression was inversely correlated with IL6R andvimentin levels, as well as metastasis occurrence, while positively correlated with E-cadherinexpression and 5-year survival post surgery (all p values<0.03). CONCLUSIONS: CRHR2reduction in CRC has a protective role in tumor progression, expansion and metastaticpotential through maintenance or promotion of the existing colonic inflammation. In addi-tion, CRHR2low CRC phenotypes are linked with distant metastases and poor clinical out-comes.
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