- Email: [email protected]
Cellular Interactions of TGF-β Pathway Members and Epigenetic Regulators of Liver and Gastrointestinal Cancers
Mo1372
the mRNA expression of FXR target genes, IBABP, OSTα, OSTβ, SHP, and FGF-19, by 104, 4.78-, 8.67-, 2.03-, and 11.5-fold, relative to controls. In contrast, glyco-CDCA modestly increased IBABP and FGF-19 expression (2.65-fold and 2.23-fold), decreased SHP expression (3.77-fold), and had no inductive effects (<2 fold) on OSTα or OSTβ expression. Unconjugated OCA and CDCA demonstrated high permeability in both the A→B and B→A directions (Papp ≥1.0x 10-6 cm/s) with no significant efflux. Papp (A→B) was greater than Papp (B→A) for glycine or taurine conjugates of OCA and CDCA. GSK2330672 significantly reduced Papp (A→B) of glyco-OCA and glyco-CDCA. Conclusion: The Caco-2 cell line appears to be an intestinal model sensitive to FXR activation, and could be used to investigate mechanisms of action of FXR agonists and to differentiate their potencies of FXR activation. Furthermore, in Caco-2 cells, unconjugated OCA and CDCA are absorbed by passive diffusion across membranes, while the absorption of their glycine and taurine conjugates are transporterdependent (likely via ASBT) which is consistent with the current understanding of bile acid kinetics.
THE MOLECULAR CROSSTALK BETWEEN HEPATIC TISSUE AND VISCERAL ADIPOSE TISSUE IN PATIENTS WITH OBESITY-RELATED NONALCOHOLIC FATTY LIVER DISEASE (NAFLD): THE INTERACTIONS BETWEEN INFLAMMATION AND IMMUNE ACTIVATION Aybike Birerdinc, Azza Karrar, Elzafir Elsheikh, Rohini Mehta, James M. Estep, Mariaelena Pierobon, Maria Stepanova, Leo Druker, Kianoush Jeiran, Alex Hodge, Fanny Monge, Lakshmi P. Alaparthi, Dinan Abdelatif, Zachary Goodman, Emanuel Petricoin, Zobair M. Younossi Background: NALFD is the most common cause of liver disease and is associated with visceral obesity. Although visceral adipose tissue (VAT) contributes to the pathogenesis of NAFLD, its exact contribution is yet to be elucidated. Aim: Our aim was to examine the association of Phosphoproteomic analysis of hepatic tissue with the gene expression (GE) of VAT and serum protein levels in NAFLD patients with liver morphometry. Methods: Snap frozen hepatic tissue (HP) and VAT were used from biopsy-proven NAFLD patients. Biopsies (H/E/Trichrome Stains) were read by a single hepatopathologist. Computer assisted morphometry (CAM) was used to quantify collagen deposition (% collagen). Activation/ phosphorylation of ~150 key proteins, was performed by Reverse Phase Protein Microarray (RPPA) on HP. Phosphoproteins associated with histologic NASH, Fibrosis, Portal Fibrosis, Pericellular Fibrosis, and % Collagen were identified using multiple logistic regression (bidirectional stepwise selection, p<0.05). These proteins were used for correlation analysis with GE from VAT and serum cytokines. RNA was extracted from VAT samples (Bio-Rad Aurum Total RNA Fatty & Fibrous Tissue Kit), converted to cDNA (SABiosciences RT2 First Strand Kit) and used for qPCR with primers for genes for cell cycle regulation/inflammation. Bio-Plex Pro™ Human Cytokine 17-plex Assay was used to quantify cytokines. Results: 19 biopsy-proven NAFLD patients (AGE was 42.789+/-9.337, BMI was 48.599+/-12.486) were included. VAT gene expression showed that FOXP3, a transcriptional regulator crucial for the function of regulatory T-cells, negatively correlated with FADD S194 (r=-0.524(1.78E02)). KCTD12 [family of proteins involved in the Activation of cAMP-Dependent PKA pathways] negatively correlated with IL-10 [r=-0.627(2.90E-02)], KCTD9 negatively correlated with p38 MAPK T180/Y182 [r=-0.588 (4.41E-02)] and KCTD15 positively correlated with ErbB2/HER2 [r=0.584(4.60E-02)]. All the KCTD genes exclusively correlated with % hepatic collagen by CAM. GATA3, a transcriptional activator for T-helper 2 differentiation, correlated negatively with FADD S194 [r=-0.492(2.74E-02)] and PKC alpha S657 [r=0.497(2.58E-02)] but correlated positively with Stat5 Y694 [r=0.590(6.12E-03)]. CD3e, which plays a role in coupling antigen recognition to intracellular signal-transduction pathways, negatively correlated with PKC alpha S657 [r=-0.449 (4.69E-02)]. Circulating levels of IL-4, IL-18, IL-5, IL-17 and IL-7 correlated most frequently with phosphorylation of hepatic proteins (See table 1). Conclusions: This pilot study elucidates some pivotal points of cross talk between VAT gene expression, serum cytokine levels and protein phosphorylation from liver tissue, highlighting focal points of interaction between inflammation and immune activation. These molecules are possible targets for future studies in NAFLD.
Mo1374
The Transforming Growth Factor-Beta (TGF-β) plays a pleiotropic role in regulating stem cell differentiation, proliferation, apoptosis, migration, and inflammation. The pathway orchestrates complex and sometimes paradoxical role in cancers, from tumor suppression to inflammation to tumor development and in later stages invasion and metastases. To date, the dichotomy of the multiple roles of TGF-β members at progressive stages of liver and gastrointestinal (GI) tumors remains poorly delineated. We performed an integrated analysis of the pathway in liver and GI cancers from mouse genetics to TCGA based analyses coupled with mechanistic insight. We first analyzed the transcriptome of 488 hepatocellular cancers (HCCs) and screened for mutations in The Cancer Genome Atlas (TCGA), and observed two different signatures (activated and inactivated) for 18 TGF-β pathway genes. While increased levels of TGF-β-related transcripts were associated with activation of hepatic fibrosis/immune/tumor microenvironment pathways, decreased levels of TGF-β members were associated with loss of TGF-β tumor suppressor function. HCCs characterized by the "inactivated" TGF-β signature were associated with a significantly poorer survival, compared to HCCs with the "activated" TGF-β signature (p=0.0027). We expanded our analysis of TGF-β pathway in a PanCancer setting across 33 TCGA tumor types to address this question. Liver and GI cancers are those tumor types with the largest percentage of samples having aberrations in at least one of the 39 core TGF-β pathway genes including β2-spectrin (β2SP, gene SPTBN1), a SMAD adaptor for TGF-β signaling and SMAD4, are some of the most frequently altered genes. Further, we found that epigenetic silencing of β2SP is causally associated with a human stem cell disorder Beckwith-Wiedemann syndrome (BWS). Patients with BWS have an 800-fold increased risk for cancers that develop from deregulation of a chromatin insulator CCCTC-binding factor (CTCF), and overexpression of oncogenic actors. Consistently, our double heterozygous β2SP+/−/Smad3+/− mice with defective TGF-β signaling phenocopy BWS, together with loss of expression and function of the chromatin insulator CCCTC-binding factor (CTCF). Our analysis also revealed that CTCF is regulated by TGFβ in a post-transcriptional manner and facilitates TGF-β-mediated repression of multiple growth promoting molecules, including IGF2 and hTERT via β2SP/SMAD3/CTCF interactions. We propose that the tumorigenesis occurs through a lifting of chromatin modulation and epigenetic alterations by defective TGF-β/CTCF-dependent regulation of tumor promoter genes. Our studies provide a framework for new animal models of liver and gastrointestinal cancers, and future new therapeutics.
Mo1375 ZBP-89 NEGATIVELY REGULATES CANCER STEMNESS IN HEPATOCELLULAR CARCINOMA VIA SUPPRESSION OF NOTCH1 SIGNALING PATHWAY Nuozhou Wang, Yi Liu, Rocky Ho, Paul B. Lai, George G. Chen
Table 1. Phosphoproteins independently associated with histologic NASH, Fibrosis, Portal Fibrosis, Pericellular Fibrosis, and % Collagen were assessed using multiple logistic regression (bidirectional stepwise selection, p<0.05 only). These were correlated to circulating cytokines in serum.
Introduction: Cancer stem cells (CSCs) play a critical role in tumor initiation, metastasis, invasion and chemoresistance. The transcription factor ZBP-89 was reported to be involved in cell growth and apoptosis in tumor and acts as a potential tumor suppressor in hepatocellular carcinoma (HCC). Prior studies have shown a statistical significance between high levels of ZBP-89 expression and better survival rates among HCC patients, although the regulatory functions of ZBP-89 during the formation of such set of CSCs is still unclear. In this study, we investigated the role of ZBP-89 in regulating cancer stemness and drug resistance in HCC. Methods: We analyzed a set of human hepatic cells from induced-pluripotent stem cells using database mining tools. The levels of ZBP-89 and EpCAM were measured using q-PCR in human HCC tissue samples. Lentiviral-based overexpression and silencing of ZBP89 were developed to modulate ZBP-89 levels in HCC cell lines. We investigated the regulatory effects of ZBP-89 on CSC phenotype through q-RCP, immunoblotting, immunofluorescence and tumor sphere formation assay. Genes expressions and protein interaction in stemness signaling pathways were analyzed. In addition, sensitizing HCC cell lines to the treatment of sorafenib in the existence of ZBP-89 was also studied using MTT assay and drug-resistant colony formation assay. Results: Using the data from Gene Expression Omnibus (GEO) database, we found that the expression levels of ZBP-89 varied during hepaticdifferentiation from ES cells. In our study, we found that ZBP-89 expression was highly correlated with CSC marker EpCAM in human HCC tissues. In vitro study indicated that tumor sphere formation capacity was impaired upon stable overexpression of ZBP-89, suggesting that ZBP-89 was involved in suppression of CSC phenotype. Further investigation against control cells showed that overexpression of ZBP-89 resulted in reduced expression of CSC markers EpCAM, CD133, Sox2 and c-myc at both mRNA and protein levels; whereas ZBP-89 shRNA depletion enhanced the expressions of CSC markers associated with more number of and larger tumor spheres. Mechanistic studies revealed that ZBP-89 was able to
Mo1373 EVIDENCE OF FXR ACTIVATION WITH OBETICHOLIC ACID IN AN IN VITRO INTESTINAL MODEL Yuanyuan Zhang, Carl LaCerte, Kenneth R. Brouwer, Jonathan P. Jackson, Sanjay Kansra, Jeffrey E. Edwards Background: The Caco-2 cell line is commonly used as a model for intestinal absorption and metabolism of therapeutic agents. Obeticholic acid (OCA) is a potent and selective farnesoid X receptor (FXR) agonist currently in development for several chronic, non-viral liver diseases. The effects of FXR activation in Caco-2 cells have not yet been completely characterized. The objective of this study was to more fully understand the effects of FXR activation in Caco-2 cells with OCA. Methods: Caco-2 human intestinal epithelial cells were cultured to confluence in monolayers and treated with 10 µM of glycine conjugates (glyco) of OCA or chenodeoxycholic acid (CDCA); vehicle-treated cells were used as controls. Cells were harvested and assessed for changes in mRNA expression of key FXR regulated genes (ASBT, OSTα, OSTβ, SHP, IBABP, and FGF-19). The apparent permeability (Papp) of OCA and CDCA as well as their glycine and taurine conjugates (tauro-) were also assessed in both apical to basolateral (A→B) or basolateral to apical (B→A) directions. The Papp (A→B) of glyco-OCA and glyco-CDCA was assessed in the absence or in the presence of an apical sodium dependent bile acid transporter (ASBT) inhibitor (GSK2330672, 3 µM). Results: Expression of FXR in Caco-2 cells was confirmed by mRNA analysis. Glyco-OCA increased
S-1159
AASLD Abstracts
AASLD Abstracts
CELLULAR INTERACTIONS OF TGF-β PATHWAY MEMBERS AND EPIGENETIC REGULATORS OF LIVER AND GASTROINTESTINAL CANCERS Shuyun Rao, Jian Chen, Rehan Akbani, Jon White, Heather Levin, Wilma Jogunoori, Shoujun Gu, Kazufuni Ohshiro, Sobia Zaidi, Bibhuti Mishra, Asif Rashid, Shulin Li, Lopa Mishra
the mRNA expression of FXR target genes, IBABP, OSTα, OSTβ, SHP, and FGF-19, by 104, 4.78-, 8.67-, 2.03-, and 11.5-fold, relative to controls. In contrast, glyco-CDCA modestly increased IBABP and FGF-19 expression (2.65-fold and 2.23-fold), decreased SHP expression (3.77-fold), and had no inductive effects (<2 fold) on OSTα or OSTβ expression. Unconjugated OCA and CDCA demonstrated high permeability in both the A→B and B→A directions (Papp ≥1.0x 10-6 cm/s) with no significant efflux. Papp (A→B) was greater than Papp (B→A) for glycine or taurine conjugates of OCA and CDCA. GSK2330672 significantly reduced Papp (A→B) of glyco-OCA and glyco-CDCA. Conclusion: The Caco-2 cell line appears to be an intestinal model sensitive to FXR activation, and could be used to investigate mechanisms of action of FXR agonists and to differentiate their potencies of FXR activation. Furthermore, in Caco-2 cells, unconjugated OCA and CDCA are absorbed by passive diffusion across membranes, while the absorption of their glycine and taurine conjugates are transporterdependent (likely via ASBT) which is consistent with the current understanding of bile acid kinetics.
THE MOLECULAR CROSSTALK BETWEEN HEPATIC TISSUE AND VISCERAL ADIPOSE TISSUE IN PATIENTS WITH OBESITY-RELATED NONALCOHOLIC FATTY LIVER DISEASE (NAFLD): THE INTERACTIONS BETWEEN INFLAMMATION AND IMMUNE ACTIVATION Aybike Birerdinc, Azza Karrar, Elzafir Elsheikh, Rohini Mehta, James M. Estep, Mariaelena Pierobon, Maria Stepanova, Leo Druker, Kianoush Jeiran, Alex Hodge, Fanny Monge, Lakshmi P. Alaparthi, Dinan Abdelatif, Zachary Goodman, Emanuel Petricoin, Zobair M. Younossi Background: NALFD is the most common cause of liver disease and is associated with visceral obesity. Although visceral adipose tissue (VAT) contributes to the pathogenesis of NAFLD, its exact contribution is yet to be elucidated. Aim: Our aim was to examine the association of Phosphoproteomic analysis of hepatic tissue with the gene expression (GE) of VAT and serum protein levels in NAFLD patients with liver morphometry. Methods: Snap frozen hepatic tissue (HP) and VAT were used from biopsy-proven NAFLD patients. Biopsies (H/E/Trichrome Stains) were read by a single hepatopathologist. Computer assisted morphometry (CAM) was used to quantify collagen deposition (% collagen). Activation/ phosphorylation of ~150 key proteins, was performed by Reverse Phase Protein Microarray (RPPA) on HP. Phosphoproteins associated with histologic NASH, Fibrosis, Portal Fibrosis, Pericellular Fibrosis, and % Collagen were identified using multiple logistic regression (bidirectional stepwise selection, p<0.05). These proteins were used for correlation analysis with GE from VAT and serum cytokines. RNA was extracted from VAT samples (Bio-Rad Aurum Total RNA Fatty & Fibrous Tissue Kit), converted to cDNA (SABiosciences RT2 First Strand Kit) and used for qPCR with primers for genes for cell cycle regulation/inflammation. Bio-Plex Pro™ Human Cytokine 17-plex Assay was used to quantify cytokines. Results: 19 biopsy-proven NAFLD patients (AGE was 42.789+/-9.337, BMI was 48.599+/-12.486) were included. VAT gene expression showed that FOXP3, a transcriptional regulator crucial for the function of regulatory T-cells, negatively correlated with FADD S194 (r=-0.524(1.78E02)). KCTD12 [family of proteins involved in the Activation of cAMP-Dependent PKA pathways] negatively correlated with IL-10 [r=-0.627(2.90E-02)], KCTD9 negatively correlated with p38 MAPK T180/Y182 [r=-0.588 (4.41E-02)] and KCTD15 positively correlated with ErbB2/HER2 [r=0.584(4.60E-02)]. All the KCTD genes exclusively correlated with % hepatic collagen by CAM. GATA3, a transcriptional activator for T-helper 2 differentiation, correlated negatively with FADD S194 [r=-0.492(2.74E-02)] and PKC alpha S657 [r=0.497(2.58E-02)] but correlated positively with Stat5 Y694 [r=0.590(6.12E-03)]. CD3e, which plays a role in coupling antigen recognition to intracellular signal-transduction pathways, negatively correlated with PKC alpha S657 [r=-0.449 (4.69E-02)]. Circulating levels of IL-4, IL-18, IL-5, IL-17 and IL-7 correlated most frequently with phosphorylation of hepatic proteins (See table 1). Conclusions: This pilot study elucidates some pivotal points of cross talk between VAT gene expression, serum cytokine levels and protein phosphorylation from liver tissue, highlighting focal points of interaction between inflammation and immune activation. These molecules are possible targets for future studies in NAFLD.
Mo1374
The Transforming Growth Factor-Beta (TGF-β) plays a pleiotropic role in regulating stem cell differentiation, proliferation, apoptosis, migration, and inflammation. The pathway orchestrates complex and sometimes paradoxical role in cancers, from tumor suppression to inflammation to tumor development and in later stages invasion and metastases. To date, the dichotomy of the multiple roles of TGF-β members at progressive stages of liver and gastrointestinal (GI) tumors remains poorly delineated. We performed an integrated analysis of the pathway in liver and GI cancers from mouse genetics to TCGA based analyses coupled with mechanistic insight. We first analyzed the transcriptome of 488 hepatocellular cancers (HCCs) and screened for mutations in The Cancer Genome Atlas (TCGA), and observed two different signatures (activated and inactivated) for 18 TGF-β pathway genes. While increased levels of TGF-β-related transcripts were associated with activation of hepatic fibrosis/immune/tumor microenvironment pathways, decreased levels of TGF-β members were associated with loss of TGF-β tumor suppressor function. HCCs characterized by the "inactivated" TGF-β signature were associated with a significantly poorer survival, compared to HCCs with the "activated" TGF-β signature (p=0.0027). We expanded our analysis of TGF-β pathway in a PanCancer setting across 33 TCGA tumor types to address this question. Liver and GI cancers are those tumor types with the largest percentage of samples having aberrations in at least one of the 39 core TGF-β pathway genes including β2-spectrin (β2SP, gene SPTBN1), a SMAD adaptor for TGF-β signaling and SMAD4, are some of the most frequently altered genes. Further, we found that epigenetic silencing of β2SP is causally associated with a human stem cell disorder Beckwith-Wiedemann syndrome (BWS). Patients with BWS have an 800-fold increased risk for cancers that develop from deregulation of a chromatin insulator CCCTC-binding factor (CTCF), and overexpression of oncogenic actors. Consistently, our double heterozygous β2SP+/−/Smad3+/− mice with defective TGF-β signaling phenocopy BWS, together with loss of expression and function of the chromatin insulator CCCTC-binding factor (CTCF). Our analysis also revealed that CTCF is regulated by TGFβ in a post-transcriptional manner and facilitates TGF-β-mediated repression of multiple growth promoting molecules, including IGF2 and hTERT via β2SP/SMAD3/CTCF interactions. We propose that the tumorigenesis occurs through a lifting of chromatin modulation and epigenetic alterations by defective TGF-β/CTCF-dependent regulation of tumor promoter genes. Our studies provide a framework for new animal models of liver and gastrointestinal cancers, and future new therapeutics.
Mo1375 ZBP-89 NEGATIVELY REGULATES CANCER STEMNESS IN HEPATOCELLULAR CARCINOMA VIA SUPPRESSION OF NOTCH1 SIGNALING PATHWAY Nuozhou Wang, Yi Liu, Rocky Ho, Paul B. Lai, George G. Chen
Table 1. Phosphoproteins independently associated with histologic NASH, Fibrosis, Portal Fibrosis, Pericellular Fibrosis, and % Collagen were assessed using multiple logistic regression (bidirectional stepwise selection, p<0.05 only). These were correlated to circulating cytokines in serum.
Introduction: Cancer stem cells (CSCs) play a critical role in tumor initiation, metastasis, invasion and chemoresistance. The transcription factor ZBP-89 was reported to be involved in cell growth and apoptosis in tumor and acts as a potential tumor suppressor in hepatocellular carcinoma (HCC). Prior studies have shown a statistical significance between high levels of ZBP-89 expression and better survival rates among HCC patients, although the regulatory functions of ZBP-89 during the formation of such set of CSCs is still unclear. In this study, we investigated the role of ZBP-89 in regulating cancer stemness and drug resistance in HCC. Methods: We analyzed a set of human hepatic cells from induced-pluripotent stem cells using database mining tools. The levels of ZBP-89 and EpCAM were measured using q-PCR in human HCC tissue samples. Lentiviral-based overexpression and silencing of ZBP89 were developed to modulate ZBP-89 levels in HCC cell lines. We investigated the regulatory effects of ZBP-89 on CSC phenotype through q-RCP, immunoblotting, immunofluorescence and tumor sphere formation assay. Genes expressions and protein interaction in stemness signaling pathways were analyzed. In addition, sensitizing HCC cell lines to the treatment of sorafenib in the existence of ZBP-89 was also studied using MTT assay and drug-resistant colony formation assay. Results: Using the data from Gene Expression Omnibus (GEO) database, we found that the expression levels of ZBP-89 varied during hepaticdifferentiation from ES cells. In our study, we found that ZBP-89 expression was highly correlated with CSC marker EpCAM in human HCC tissues. In vitro study indicated that tumor sphere formation capacity was impaired upon stable overexpression of ZBP-89, suggesting that ZBP-89 was involved in suppression of CSC phenotype. Further investigation against control cells showed that overexpression of ZBP-89 resulted in reduced expression of CSC markers EpCAM, CD133, Sox2 and c-myc at both mRNA and protein levels; whereas ZBP-89 shRNA depletion enhanced the expressions of CSC markers associated with more number of and larger tumor spheres. Mechanistic studies revealed that ZBP-89 was able to
Mo1373 EVIDENCE OF FXR ACTIVATION WITH OBETICHOLIC ACID IN AN IN VITRO INTESTINAL MODEL Yuanyuan Zhang, Carl LaCerte, Kenneth R. Brouwer, Jonathan P. Jackson, Sanjay Kansra, Jeffrey E. Edwards Background: The Caco-2 cell line is commonly used as a model for intestinal absorption and metabolism of therapeutic agents. Obeticholic acid (OCA) is a potent and selective farnesoid X receptor (FXR) agonist currently in development for several chronic, non-viral liver diseases. The effects of FXR activation in Caco-2 cells have not yet been completely characterized. The objective of this study was to more fully understand the effects of FXR activation in Caco-2 cells with OCA. Methods: Caco-2 human intestinal epithelial cells were cultured to confluence in monolayers and treated with 10 µM of glycine conjugates (glyco) of OCA or chenodeoxycholic acid (CDCA); vehicle-treated cells were used as controls. Cells were harvested and assessed for changes in mRNA expression of key FXR regulated genes (ASBT, OSTα, OSTβ, SHP, IBABP, and FGF-19). The apparent permeability (Papp) of OCA and CDCA as well as their glycine and taurine conjugates (tauro-) were also assessed in both apical to basolateral (A→B) or basolateral to apical (B→A) directions. The Papp (A→B) of glyco-OCA and glyco-CDCA was assessed in the absence or in the presence of an apical sodium dependent bile acid transporter (ASBT) inhibitor (GSK2330672, 3 µM). Results: Expression of FXR in Caco-2 cells was confirmed by mRNA analysis. Glyco-OCA increased
S-1159
AASLD Abstracts
AASLD Abstracts
CELLULAR INTERACTIONS OF TGF-β PATHWAY MEMBERS AND EPIGENETIC REGULATORS OF LIVER AND GASTROINTESTINAL CANCERS Shuyun Rao, Jian Chen, Rehan Akbani, Jon White, Heather Levin, Wilma Jogunoori, Shoujun Gu, Kazufuni Ohshiro, Sobia Zaidi, Bibhuti Mishra, Asif Rashid, Shulin Li, Lopa Mishra