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864 KLF4 Controls Esophageal Epithelial Inflammation and Carcinogenesis via Rho GTPase Activation of NfκB Signaling
AGA Abstracts
Table 1 Characteristics of 192 patients with longstanding inflammatory bowel disease
865 ACVR1B-Dependent Signaling Inhibits Esophageal Squamous Cancer Cell Invasion Claudia D. Andl, Holli A. Loomans, Laura L. Quast Background: Similar to TGFb receptor type II, Activin receptors have been reported to be prone to inactivation by mutation in colorectal and pancreatic cancer. We hypothesized that loss of Activin receptor mediated signaling plays a role in the progression of esophageal tumorigenesis. Materials and Methods: We used the esophageal squamous cell carcinoma (ESCC) cell line series TE and KYSE to broadly assess the expression of Activin receptors. To investigate the functional consequences of disruption of Activin signaling, we selected the TE2 cell line, which is ACVR1B-positive and the ACVR1B-negative cell line TE11. These cell lines were grown in a physiological context with Activin A (Act A)-overexpressing or control human esophageal fibroblasts in organotypic cultures. Western Blot analysis, qRTPCR and Immunofluorescence staining were performed separately for tumor and fibroblast layers, and 208 ESCC tissue cores on a tissue microarray. Results: The majority of the ESCC cell lines showed low ACVR1B expression by Western Blot analysis. When comparing the ACVR1B-positive cell line TE2 with ACVR1B-negative TE11, TE2 cells responded to Act A signaling induction with changes in downstream signaling and rearrangements of the surrounding extracellular matrix resulting in a reduction of cell invasion. By contrast, TE11 cells lacked an inhibitory response to Act A, but showed functional downstream activation with TGFb1 control stimulation, demonstrating the ACVR1B-dependency of cell invasion inhibition. RNA profiling of TE2 and TE11 organotypic cultures identified differential regulation of extracellular matrix proteins involved in cell invasion such as matrix metalloproteinases and integrins. Of particular interest, we identified ACVR1B-dependent dowregulation of MMP1, MMP9 and MMP12 in TE2 cultures with Act A-overexpressing fibroblast compared to empty vector fibroblast control. TE11/Act A cultures compared to control showed increased expression of MMP1 3.95-fold, MMP9 15.89-fold, and MMP12 3.95-fold, indicating the upregulation is ACVR1B-independent. ITGA6 was downregulated in TE11/Activin A cultures but increased in TE2/Activin A. Analysis of the ESCC tissues identified an increase in Act A expression with stage, associated with a concomitant loss of ACVR1B. Interestingly, Act A and ACVR1B expression was generally lower in the stroma, compared to the tumor. Conclusions: This is the first time Activin receptors have been implicated in esophageal squamous tumorigenesis. We show that inhibition of ESCC invasion is ACVR1B-dependent and are currently restoring ACVR1B-mediated signaling in TE11 cells. At the same time we are inducing AVCR1B loss in TE2 cell via CRISPR/Cas9 knock-out to delineate the mechanism in confirmation of the RNA profile analysis, which identified matrix metalloproteinase and integrins as potential mediators of ESCC invasion.
*Extent of inflammation **2/37 Missing data on extent of inflammation
864 KLF4 Controls Esophageal Epithelial Inflammation and Carcinogenesis via Rho GTPase Activation of NfkB Signaling Khvaramze Shaverdashvili, Daniel Weinblatt, Marie-Pier Tetreault, Jonathan P. Katz Background: Understanding the regulation of esophageal epithelial homeostasis is essential to gain insight into the pathogenesis of esophageal diseases such as esophageal squamous cell cancer (ESCC). KLF4 (Krüppel-like factor 4, previously known as GKLF) is a DNA-binding transcriptional regulator expressed at high levels in gastrointestinal epithelia, specifically in regions of cellular differentiation. Previously, we demonstrated that overexpression of Klf4 in esophageal epithelia leads to the development of ESCC arising in the context of epithelial hyperplasia and chronic inflammation; these changes appear to be mediated by NF kB activation downstream of KLF4. However, the mechanisms of NF kB activation by KLF4 and the specific contribution of NF kB signaling to ESCC are not known. Small Rho GTPases can activate NFkB, and we identified several Rho family members and guanine nucleotide exchange factors (GEFs) that activate Rho proteins as putative KLF4 targets by transcriptional profiling. Thus, we hypothesized that KLF4 controls esophageal epithelial inflammation and carcinogenesis by activation of NFkB via the small Rho GTPases. Methods: We utilized murine and human primary esophageal keratinocytes for in vitro studies and employed mice with esophageal epithelial-specific Klf4 overexpression (ED-L2/Klf4 mice) or deletion (ED-L2/ Cre;Klf4loxP/loxP mice) for in vivo analyses. ED-L2/Klf4 mice were also crossed with esophagealspecific IKK2 null mice in order to define the contribution of NF kB signaling to esophageal inflammation, hyperplasia, and cancer. Results: Overexpression of Klf4 in esophageal epithelia of ED-L2/KLF4 mice increased IKK1, IKK2, and NEMO, key regulators of the NFkB pathway, while ED-L2/Cre;Klf4loxP/loxP mice had reduced NFkB signaling and decreased levels of the small Rho GTPase RhoF ( Figure 1). In primary esophageal keratinocytes, KLF4 knockdown by shRNA decreased expression of RhoA and RhoF, as well the GEF Arhgef17. In addition, shRNA knockdown of RhoF or Arhgef17 decreased IKK1, IKK2, and NEMO expression. Crosses of ED-L2/Klf4 mice and esophageal-specific IKK2 null mice are currently being analyzed. Conclusion: KLF4 transcriptionally activates small Rho GTPases, which then activate NFkB signaling, providing a potentially important mechanism for the regulation of esophageal epithelial inflammation and carcinogenesis.
866 N-MYC Downregulated Gene 4 (NDRG4) Is a Potential Tumor Suppressor Gene in Esophageal Adenocarcinoma DunFa Peng, TianLing Hu, Abbes Belkhiri, Alexander Zaika, Wael El-Rifai Background: The incidence of esophageal adenocarcinoma (EAC) has been rising dramatically during past decades in the US and the Western world. The only known precancerous lesion of EAC is Barrett's esophagus (BE). However, only less than 2% of BE progress to adenocarcinoma through low-grade and high-grade dysplasia. The mechanisms that underlie this process remain largely unclear. The N-myc downregulated gene 4 (NDRG4) is a recently identified human NDRG member. Its functions have been controversial because both oncogenic and tumor suppressor functions have been claimed with a tissue specific pattern. In this study, we aimed to identify the expression levels, regulation, and functions of NDRG4 in EAC. Methods and Results: In our pilot screening experiment to identify gene expression profile of esophageal adenocarcinoma, we examined EAC cell lines and 10 paired normal tissues and primary tumors using cDNA microarrays analysis. We found that NDRG4 expression was significantly downregulated in EAC samples as compared to normal samples (P<.01). To confirm and validate this finding, we examined the NDRG4 mRNA expression in 55 normal esophageal and adenocarcinoma samples, including 23 matched normal and
AGA Abstracts
S-186
Table 1 Characteristics of 192 patients with longstanding inflammatory bowel disease
865 ACVR1B-Dependent Signaling Inhibits Esophageal Squamous Cancer Cell Invasion Claudia D. Andl, Holli A. Loomans, Laura L. Quast Background: Similar to TGFb receptor type II, Activin receptors have been reported to be prone to inactivation by mutation in colorectal and pancreatic cancer. We hypothesized that loss of Activin receptor mediated signaling plays a role in the progression of esophageal tumorigenesis. Materials and Methods: We used the esophageal squamous cell carcinoma (ESCC) cell line series TE and KYSE to broadly assess the expression of Activin receptors. To investigate the functional consequences of disruption of Activin signaling, we selected the TE2 cell line, which is ACVR1B-positive and the ACVR1B-negative cell line TE11. These cell lines were grown in a physiological context with Activin A (Act A)-overexpressing or control human esophageal fibroblasts in organotypic cultures. Western Blot analysis, qRTPCR and Immunofluorescence staining were performed separately for tumor and fibroblast layers, and 208 ESCC tissue cores on a tissue microarray. Results: The majority of the ESCC cell lines showed low ACVR1B expression by Western Blot analysis. When comparing the ACVR1B-positive cell line TE2 with ACVR1B-negative TE11, TE2 cells responded to Act A signaling induction with changes in downstream signaling and rearrangements of the surrounding extracellular matrix resulting in a reduction of cell invasion. By contrast, TE11 cells lacked an inhibitory response to Act A, but showed functional downstream activation with TGFb1 control stimulation, demonstrating the ACVR1B-dependency of cell invasion inhibition. RNA profiling of TE2 and TE11 organotypic cultures identified differential regulation of extracellular matrix proteins involved in cell invasion such as matrix metalloproteinases and integrins. Of particular interest, we identified ACVR1B-dependent dowregulation of MMP1, MMP9 and MMP12 in TE2 cultures with Act A-overexpressing fibroblast compared to empty vector fibroblast control. TE11/Act A cultures compared to control showed increased expression of MMP1 3.95-fold, MMP9 15.89-fold, and MMP12 3.95-fold, indicating the upregulation is ACVR1B-independent. ITGA6 was downregulated in TE11/Activin A cultures but increased in TE2/Activin A. Analysis of the ESCC tissues identified an increase in Act A expression with stage, associated with a concomitant loss of ACVR1B. Interestingly, Act A and ACVR1B expression was generally lower in the stroma, compared to the tumor. Conclusions: This is the first time Activin receptors have been implicated in esophageal squamous tumorigenesis. We show that inhibition of ESCC invasion is ACVR1B-dependent and are currently restoring ACVR1B-mediated signaling in TE11 cells. At the same time we are inducing AVCR1B loss in TE2 cell via CRISPR/Cas9 knock-out to delineate the mechanism in confirmation of the RNA profile analysis, which identified matrix metalloproteinase and integrins as potential mediators of ESCC invasion.
*Extent of inflammation **2/37 Missing data on extent of inflammation
864 KLF4 Controls Esophageal Epithelial Inflammation and Carcinogenesis via Rho GTPase Activation of NfkB Signaling Khvaramze Shaverdashvili, Daniel Weinblatt, Marie-Pier Tetreault, Jonathan P. Katz Background: Understanding the regulation of esophageal epithelial homeostasis is essential to gain insight into the pathogenesis of esophageal diseases such as esophageal squamous cell cancer (ESCC). KLF4 (Krüppel-like factor 4, previously known as GKLF) is a DNA-binding transcriptional regulator expressed at high levels in gastrointestinal epithelia, specifically in regions of cellular differentiation. Previously, we demonstrated that overexpression of Klf4 in esophageal epithelia leads to the development of ESCC arising in the context of epithelial hyperplasia and chronic inflammation; these changes appear to be mediated by NF kB activation downstream of KLF4. However, the mechanisms of NF kB activation by KLF4 and the specific contribution of NF kB signaling to ESCC are not known. Small Rho GTPases can activate NFkB, and we identified several Rho family members and guanine nucleotide exchange factors (GEFs) that activate Rho proteins as putative KLF4 targets by transcriptional profiling. Thus, we hypothesized that KLF4 controls esophageal epithelial inflammation and carcinogenesis by activation of NFkB via the small Rho GTPases. Methods: We utilized murine and human primary esophageal keratinocytes for in vitro studies and employed mice with esophageal epithelial-specific Klf4 overexpression (ED-L2/Klf4 mice) or deletion (ED-L2/ Cre;Klf4loxP/loxP mice) for in vivo analyses. ED-L2/Klf4 mice were also crossed with esophagealspecific IKK2 null mice in order to define the contribution of NF kB signaling to esophageal inflammation, hyperplasia, and cancer. Results: Overexpression of Klf4 in esophageal epithelia of ED-L2/KLF4 mice increased IKK1, IKK2, and NEMO, key regulators of the NFkB pathway, while ED-L2/Cre;Klf4loxP/loxP mice had reduced NFkB signaling and decreased levels of the small Rho GTPase RhoF ( Figure 1). In primary esophageal keratinocytes, KLF4 knockdown by shRNA decreased expression of RhoA and RhoF, as well the GEF Arhgef17. In addition, shRNA knockdown of RhoF or Arhgef17 decreased IKK1, IKK2, and NEMO expression. Crosses of ED-L2/Klf4 mice and esophageal-specific IKK2 null mice are currently being analyzed. Conclusion: KLF4 transcriptionally activates small Rho GTPases, which then activate NFkB signaling, providing a potentially important mechanism for the regulation of esophageal epithelial inflammation and carcinogenesis.
866 N-MYC Downregulated Gene 4 (NDRG4) Is a Potential Tumor Suppressor Gene in Esophageal Adenocarcinoma DunFa Peng, TianLing Hu, Abbes Belkhiri, Alexander Zaika, Wael El-Rifai Background: The incidence of esophageal adenocarcinoma (EAC) has been rising dramatically during past decades in the US and the Western world. The only known precancerous lesion of EAC is Barrett's esophagus (BE). However, only less than 2% of BE progress to adenocarcinoma through low-grade and high-grade dysplasia. The mechanisms that underlie this process remain largely unclear. The N-myc downregulated gene 4 (NDRG4) is a recently identified human NDRG member. Its functions have been controversial because both oncogenic and tumor suppressor functions have been claimed with a tissue specific pattern. In this study, we aimed to identify the expression levels, regulation, and functions of NDRG4 in EAC. Methods and Results: In our pilot screening experiment to identify gene expression profile of esophageal adenocarcinoma, we examined EAC cell lines and 10 paired normal tissues and primary tumors using cDNA microarrays analysis. We found that NDRG4 expression was significantly downregulated in EAC samples as compared to normal samples (P<.01). To confirm and validate this finding, we examined the NDRG4 mRNA expression in 55 normal esophageal and adenocarcinoma samples, including 23 matched normal and
AGA Abstracts
S-186