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Mo1642 Talin-1 Regulates TGFβ1-Mediated Focal Adhesion Assembly and Hepatocellular Carcinoma Cell Migration In Vitro
HepWT mice. Furthermore, hepatic proliferation in HepFAK KO mice was dramatically decreased compared with HepWT mice. Conclusions: Deletion of FAK decreases tumor proliferation and HCC development in MET/CAT-induced HCC model, suggesting that inhibition of FAK may be a promising strategy to treat HCC.
development and poor prognosis of various cancers including hepatocellular carcinoma (HCC). Pro-inflammatory TAMs (M1-type) are considered classically activated macrophages and enhance cancer immunity while M2-type macrophages, are immunosuppressive and have the potential to accelerate cancer progression. TAMs demonstrate a high degree of plasticity and can revert from one phenotype to another under appropriate microenvironmental cues. TAMs are mainly derived from the peripheral blood under the influence of various tumor-mediated factors including colony stimulating factor-1 (CSF-1), however there is emerging evidence for local proliferation of TAMs in tumors and surrounding tissue. While locally proliferating TAMs have been linked with chronic inflammatory disease and various cancers, they have not been well studied in HCC. We hypothesized that TAMs can proliferate in response to HCC cell derived CSF-1. Our in vitro experiments showed proliferation of macrophages derived from mouse bone marrow when cultured in serum-free conditioned media derived from the murine HCC cell line hepa 1-6. This activity was found to be mainly influenced by hepa1-6 derived CSF-1, since inhibition with a small molecule inhibitor of the CSF-1 receptor led to a significant decrease in the number of viable macrophages determined by trypan blue viability assay as well as decreased levels of proliferating cell nuclear antigen (PCNA) determined by western blot. Next, we performed an in-vivo analysis of TAM proliferation in the diethylnitrosamine- induced murine model of hepatocellular carcinoma. Immuno-fluorescent staining using F4/80 as pan-macrophage marker and PCNA as proliferation marker was performed on the tumor tissues and compared with the wild type control liver tissue. We identified a population of F4/80-PCNA double stained cells in the tumor tissues compared with the controls which suggests local proliferation of TAMs occurring in tumor-bearing livers. FACS analysis using the proliferation marker Ki-67 and macrophage marker f4/80 also revealed a sub-population of proliferating macrophages in orthotopically implanted hepa1-6 liver tumors and peri-tumoral liver tissue. These data provide evidence for local proliferation of TAMs in the HCC tumor microenvironment. Further studies to define the role of local TAM proliferation could facilitate the development of prognostic and therapeutic modalities targeting pro-tumorigenic TAMs in HCC.
Mo1642
AASLD Abstracts
Talin-1 Regulates TGFβ1-Mediated Focal Adhesion Assembly and Hepatocellular Carcinoma Cell Migration In Vitro Michael Duncan, James McPhail, Priyanka Thakur Cirrhotic liver disease, typified by the extensive deposition of extracellular matrix (ECM) and tissue remodeling, often precedes hepatocellular carcinoma (HCC). Cancer cells are in constant contact with the surrounding stromal ECM and respond to these changes via the integrin family of cell surface receptors. The activation of integrin receptors via ECM binding initiates assembly of focal adhesions, which promotes a variety of signaling pathways and cellular responses. A key molecular constituent of focal adhesion complex is the scaffolding protein talin. At the molecular level talin is involved in focal adhesion assembly, cell-cell adhesions, and links the actin cytoskeleton with integrins that bind to extracellular matrix proteins. A recent proteomics based study revealed that increased HCC cell talin-1 levels correlate strongly with aggressive HCC tumor phenotype and poor clinical outcomes. The report suggests that increased talin-1 expression is associated with poorly differentiated tumors, portal vein invasion and decreased time of recurrence after surgical resection. These correlative findings suggest a pro-tumorigenic role for talin in HCC, however the molecular mechanisms remain unresolved. Transforming growth factor beta (TGF β) is a pleiotropic cytokine and plays important roles HCC cellular phenotypes, including cell adhesion and migration, that impact liver cancer progression. We wanted to determine if TGF β signaling impacts talin expression and activity in HCC cancer cells. Using the hepa1-6 murine HCC cell line, we observed by Western blot and immunocytochemistry that while TGF β1 does not impact talin protein levels, a significant redistribution of talin to focal and nascent adhesions does occur. This suggests that TGFβ1 stimulates talin localization at focal adhesions of Hepa1-6 HCC cells. Since the formation of nascent focal adhesions plays a key role in cell migration, we utilized an RNA inhibition strategy to deplete hepa1-6 cells of talin-1 in order to determine its impact on TGFβ1 mediated cell migration. Using a lentiviral transduction strategy, we generated stable hepa1-6 cell lines that express short hairpin RNA targeting talin-1 (hepa1-6shTln1) and a control scramble short hairpin RNA sequence (hepa1-6shScr). We observed reduced talin levels in the hepa1-6shTln1 cells compared to hepa1-6shScr controls. Hepa1-6shTln1 displayed a rounded morphology and F-actin levels did not increase in response to TGFβ1. Futhermore, talin-1 depletion resulted in decreased TGF β1 dependent migration of hepa1-6 cells as determined by an in vitro scratch assay. Our collective results indicate that talin-1 is an important mediator of HCC focal adhesion assembly, cell morphology, and migration. These results are consistent with advanced HCC progression that has been observed clinically when HCC cells express high levels of talin-1.
Mo1807 Kupffer Cells (KC) Contribute to Early Development of Acetaminophen (APAP) Hepatotoxicity, but Their Role in the Late Phase of Liver Injury and Regeneration Is Replaced by Newly Recruited Inflammatory Monocytes/ Macrophages Luoluo Yang, Jo Suda, Xiangwei Meng, Neil Kaplowitz, Zhang-Xu Liu Background&aim: Dying hepatocytes release DAMPs, which can activate Kupffer cells(KC). The pro-inflammatory role of KC has been implicated in numerous models of liver disease. However, the role of KC in APAP hepatotoxicity has been controversial. This report provides detailed investigation on the function of KC in the development and resolution of APAP hepatotoxicity. Methods: C57BL/6J mice without food restriction were i.p. injected 500mg/ kg of APAP dissolved in PBS to induce liver injury. KC were depleted by i.v. injection of 150ul of liposome/clodronate(CLO) 24h prior to APAP challenge; liposome/PBS was used as vehicle control. Hepatic non-parenchymal cells (NPC) and leukocytes were isolated and assessed for cell subtypes by flowcytometry. Immunohistochemistry(IHC) of liver sections for F4/80 and PCNA were performed to identify KC and proliferating hepatocytes, respectively. Results: KC were effectively depleted at 24h after CLO treatment, which was confirmed by hepatic F4/80 IHC staining and CD11b+F4/80hi subtype in hepatic NPC. CLO treatment had no effect on the numbers of hepatic NK, NKT, T and B cells. Compared with APAPtreated control mice, KC depletion by CLO pretreatment conferred protection against APAPinduced early liver injury at 6h evidenced by significantly reduced levels of serum ALT(1598±681 U/L in Controls vs. 808±493 U/L in CLO, p<0.05) and centrilobular necrosis area(35.5±7.0% vs. 14.5±7.7%, p<0.05). The protection was associated with decreased hepatic infiltration of inflammatory monocytes (CD11b+Ly6Chi) and neutrophils (CD11b+Ly6Ghi), and reduced hepatic mRNA expressions for chemokines(CCL2, CXCL1) and cytokines(IL-6, TNFa, IFNg) in CLO-treated mice. However, at 24h after APAP, there were no significant differences of liver injury in terms of serum ALT (1062±462 vs. 1054±683, p>0.05) and liver histology between these two groups. Interestingly, the numbers of hepatic inflammatory monocytes and neutrophils in CLO-treated mice reached to the similar levels of APAP-treated control mice at 24h after APAP. APAP-induced liver injury was resolved by 72h in both groups and no differences of serum ALT and liver histology were observed at 48 and 72h (p>0.05) despite the continued absence of KC in the liver of CLO-treated mice. Furthermore, the numbers of PCNA positive hepatocytes in the liver displayed no differences at 24, 48 and 72h (p>0.05). Hepatic CYP2E1 proteins, APAP adducts and GSH depletions at 2 and 4h after APAP exhibited no differences between control and CLO-treated mice. Conclusion: The findings suggest that KC contribute to the early development of APAP-induced liver injury by producing inflammatory chemokines/cytokines to recruit inflammatory monocytes and neutrophils, but their role in the late phase of liver injury and regeneration could be replaced by newly recruited inflammatory monocytes/macrophages.
Mo1643 Diets With Subnormal Supplementation of Vitamin D Regimen Promote Hepatocellular Carcinoma Tumor Growth in TGF-β Impaired Mice With SMAD3 Heterozygous Deletion Nina M. Muñoz, Lior H. Katz, Keigo Machida, Hidekazu Tsukamoto, Kirti Shetty, Aiwu R. He, Lynt B. Johnson, Asif Rashid, Sang-Bae Kim, Ju-Seog Lee, Lopa Mishra Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. Vitamin D (VD) has been implicated in the prevention of multiple cancers, some of which are characterized by inactivation of TGF- β signaling. Recently, inactivation of TGFβ signaling has been associated with development of HCC. We examined the potential role of VD in HCC chemoprevention in the context of TGF- β inactivation in a mouse model. Methods: Wild type, Sptbn1+/- and Smad3+/- male mice were treated with 50 mg/kg of the chemical carcinogen diethylnitrosamine (DEN) via i.p. injection at 14 days of age. At 8 months, the animals were changed from regular chow (2,200 IU/kg VD) to one of two diets: lower than normal VD (200 IU/kg) or high VD (10,000 IU/kg). At one year of age, the livers were harvested for tissue analysis. Several metrics were assessed including expression levels of 164 proteins from liver tumors by Reverse Phase Protein Array (RPPA). Results: Twenty three mice were treated with lower than normal dose VD while 26 received high dose VD. We did not find significant differences in blood calcium levels in animals on either diet. As expected, high VD intervention after 8 months of DEN injection did not affect the total number of tumors the mice developed; however, the lower than normal VD regimen promoted tumor growth in the context of Smad3 disruption (liver-to-body weight ratio 18.37% in Smad3+/- mice treated with low dose VD vs. 6.25% in Smad3+/- animals treated with high dose, p=0.028). Furthermore, RPPA data revealed that the lower than normal VD intake in DEN-treated Smad3 heterozygous null mice results in repression of tumor suppressing genes such as PDCD4 and concomitant up-regulation of tumor promoting genes like Stat5A, Bcl2-XL, PDGFRB and PEA15. Conclusions: Remarkably, lower than normal VD in the context of Smad3 disruption promotes tumor growth possibly through repression of tumor suppressing genes such as PDCD4 and up-regulation of oncogenes like Stat5A, Bcl2XL, PDGFRB and PEA15. These results suggest that VD treatment strategies could potentially be significant for chemoprevention and treatment of HCC in the context of inactivation of TGF-β signaling in patient populations with underlying deficiency of VD.
Mo1808 Association of Gender and Menopause With Injury Types and Histological Features of Drug-Induced Liver Injury Ayako Suzuki, Jiezhun Gu, Hans L. Tillmann, Herbert L. Bonkovsky, Robert J. Fontana, David E. Kleiner Background and Aims: Sex and sex hormones are known to impact drug metabolism, drug transporters, and immune response. Recent studies in brain and kidney injury animal models revealed that cellular response to stress and cell death program differ between males and females. We hypothesized that clinical presentations and histologic features of drug-induced liver injury (DILI) may be influenced by gender and menopausal state. We therefore looked for associations of gender and menopausal state with histologic features and biochemical injury of DILI in the Drug-Induced Liver Injury Network (DILIN). Methods: Of 1234 patients enrolled in DILIN (Sep 2004 - May 2013), we analyzed 200 adults who 1) had a DILIN causality score of at least ‘probable' and 2) had liver biopsies within 30 days of DILI onset which were scored in a standardized manner. The patients were classified into hepatocellular injury (HC) and cholestatic/mixed injury (CS/MIX) based on the laboratory
Mo1644 Colony Stimulating Growth Factor (CSF-1) Promotes Proliferation of TumorAssociated Macrophages in Hepatocellular Carcinoma Abdul Rehman, Priyanka Thakur, Grace Dougherty, Michael Duncan Abstract: Macrophages are an essential component of the innate immune response in cancer, however they are no longer considered to be entirely the agents of host defense against malignancy. Several studies have implicated tumor-associated macrophages (TAMs) in the
AASLD Abstracts
S-1000
development and poor prognosis of various cancers including hepatocellular carcinoma (HCC). Pro-inflammatory TAMs (M1-type) are considered classically activated macrophages and enhance cancer immunity while M2-type macrophages, are immunosuppressive and have the potential to accelerate cancer progression. TAMs demonstrate a high degree of plasticity and can revert from one phenotype to another under appropriate microenvironmental cues. TAMs are mainly derived from the peripheral blood under the influence of various tumor-mediated factors including colony stimulating factor-1 (CSF-1), however there is emerging evidence for local proliferation of TAMs in tumors and surrounding tissue. While locally proliferating TAMs have been linked with chronic inflammatory disease and various cancers, they have not been well studied in HCC. We hypothesized that TAMs can proliferate in response to HCC cell derived CSF-1. Our in vitro experiments showed proliferation of macrophages derived from mouse bone marrow when cultured in serum-free conditioned media derived from the murine HCC cell line hepa 1-6. This activity was found to be mainly influenced by hepa1-6 derived CSF-1, since inhibition with a small molecule inhibitor of the CSF-1 receptor led to a significant decrease in the number of viable macrophages determined by trypan blue viability assay as well as decreased levels of proliferating cell nuclear antigen (PCNA) determined by western blot. Next, we performed an in-vivo analysis of TAM proliferation in the diethylnitrosamine- induced murine model of hepatocellular carcinoma. Immuno-fluorescent staining using F4/80 as pan-macrophage marker and PCNA as proliferation marker was performed on the tumor tissues and compared with the wild type control liver tissue. We identified a population of F4/80-PCNA double stained cells in the tumor tissues compared with the controls which suggests local proliferation of TAMs occurring in tumor-bearing livers. FACS analysis using the proliferation marker Ki-67 and macrophage marker f4/80 also revealed a sub-population of proliferating macrophages in orthotopically implanted hepa1-6 liver tumors and peri-tumoral liver tissue. These data provide evidence for local proliferation of TAMs in the HCC tumor microenvironment. Further studies to define the role of local TAM proliferation could facilitate the development of prognostic and therapeutic modalities targeting pro-tumorigenic TAMs in HCC.
Mo1642
AASLD Abstracts
Talin-1 Regulates TGFβ1-Mediated Focal Adhesion Assembly and Hepatocellular Carcinoma Cell Migration In Vitro Michael Duncan, James McPhail, Priyanka Thakur Cirrhotic liver disease, typified by the extensive deposition of extracellular matrix (ECM) and tissue remodeling, often precedes hepatocellular carcinoma (HCC). Cancer cells are in constant contact with the surrounding stromal ECM and respond to these changes via the integrin family of cell surface receptors. The activation of integrin receptors via ECM binding initiates assembly of focal adhesions, which promotes a variety of signaling pathways and cellular responses. A key molecular constituent of focal adhesion complex is the scaffolding protein talin. At the molecular level talin is involved in focal adhesion assembly, cell-cell adhesions, and links the actin cytoskeleton with integrins that bind to extracellular matrix proteins. A recent proteomics based study revealed that increased HCC cell talin-1 levels correlate strongly with aggressive HCC tumor phenotype and poor clinical outcomes. The report suggests that increased talin-1 expression is associated with poorly differentiated tumors, portal vein invasion and decreased time of recurrence after surgical resection. These correlative findings suggest a pro-tumorigenic role for talin in HCC, however the molecular mechanisms remain unresolved. Transforming growth factor beta (TGF β) is a pleiotropic cytokine and plays important roles HCC cellular phenotypes, including cell adhesion and migration, that impact liver cancer progression. We wanted to determine if TGF β signaling impacts talin expression and activity in HCC cancer cells. Using the hepa1-6 murine HCC cell line, we observed by Western blot and immunocytochemistry that while TGF β1 does not impact talin protein levels, a significant redistribution of talin to focal and nascent adhesions does occur. This suggests that TGFβ1 stimulates talin localization at focal adhesions of Hepa1-6 HCC cells. Since the formation of nascent focal adhesions plays a key role in cell migration, we utilized an RNA inhibition strategy to deplete hepa1-6 cells of talin-1 in order to determine its impact on TGFβ1 mediated cell migration. Using a lentiviral transduction strategy, we generated stable hepa1-6 cell lines that express short hairpin RNA targeting talin-1 (hepa1-6shTln1) and a control scramble short hairpin RNA sequence (hepa1-6shScr). We observed reduced talin levels in the hepa1-6shTln1 cells compared to hepa1-6shScr controls. Hepa1-6shTln1 displayed a rounded morphology and F-actin levels did not increase in response to TGFβ1. Futhermore, talin-1 depletion resulted in decreased TGF β1 dependent migration of hepa1-6 cells as determined by an in vitro scratch assay. Our collective results indicate that talin-1 is an important mediator of HCC focal adhesion assembly, cell morphology, and migration. These results are consistent with advanced HCC progression that has been observed clinically when HCC cells express high levels of talin-1.
Mo1807 Kupffer Cells (KC) Contribute to Early Development of Acetaminophen (APAP) Hepatotoxicity, but Their Role in the Late Phase of Liver Injury and Regeneration Is Replaced by Newly Recruited Inflammatory Monocytes/ Macrophages Luoluo Yang, Jo Suda, Xiangwei Meng, Neil Kaplowitz, Zhang-Xu Liu Background&aim: Dying hepatocytes release DAMPs, which can activate Kupffer cells(KC). The pro-inflammatory role of KC has been implicated in numerous models of liver disease. However, the role of KC in APAP hepatotoxicity has been controversial. This report provides detailed investigation on the function of KC in the development and resolution of APAP hepatotoxicity. Methods: C57BL/6J mice without food restriction were i.p. injected 500mg/ kg of APAP dissolved in PBS to induce liver injury. KC were depleted by i.v. injection of 150ul of liposome/clodronate(CLO) 24h prior to APAP challenge; liposome/PBS was used as vehicle control. Hepatic non-parenchymal cells (NPC) and leukocytes were isolated and assessed for cell subtypes by flowcytometry. Immunohistochemistry(IHC) of liver sections for F4/80 and PCNA were performed to identify KC and proliferating hepatocytes, respectively. Results: KC were effectively depleted at 24h after CLO treatment, which was confirmed by hepatic F4/80 IHC staining and CD11b+F4/80hi subtype in hepatic NPC. CLO treatment had no effect on the numbers of hepatic NK, NKT, T and B cells. Compared with APAPtreated control mice, KC depletion by CLO pretreatment conferred protection against APAPinduced early liver injury at 6h evidenced by significantly reduced levels of serum ALT(1598±681 U/L in Controls vs. 808±493 U/L in CLO, p<0.05) and centrilobular necrosis area(35.5±7.0% vs. 14.5±7.7%, p<0.05). The protection was associated with decreased hepatic infiltration of inflammatory monocytes (CD11b+Ly6Chi) and neutrophils (CD11b+Ly6Ghi), and reduced hepatic mRNA expressions for chemokines(CCL2, CXCL1) and cytokines(IL-6, TNFa, IFNg) in CLO-treated mice. However, at 24h after APAP, there were no significant differences of liver injury in terms of serum ALT (1062±462 vs. 1054±683, p>0.05) and liver histology between these two groups. Interestingly, the numbers of hepatic inflammatory monocytes and neutrophils in CLO-treated mice reached to the similar levels of APAP-treated control mice at 24h after APAP. APAP-induced liver injury was resolved by 72h in both groups and no differences of serum ALT and liver histology were observed at 48 and 72h (p>0.05) despite the continued absence of KC in the liver of CLO-treated mice. Furthermore, the numbers of PCNA positive hepatocytes in the liver displayed no differences at 24, 48 and 72h (p>0.05). Hepatic CYP2E1 proteins, APAP adducts and GSH depletions at 2 and 4h after APAP exhibited no differences between control and CLO-treated mice. Conclusion: The findings suggest that KC contribute to the early development of APAP-induced liver injury by producing inflammatory chemokines/cytokines to recruit inflammatory monocytes and neutrophils, but their role in the late phase of liver injury and regeneration could be replaced by newly recruited inflammatory monocytes/macrophages.
Mo1643 Diets With Subnormal Supplementation of Vitamin D Regimen Promote Hepatocellular Carcinoma Tumor Growth in TGF-β Impaired Mice With SMAD3 Heterozygous Deletion Nina M. Muñoz, Lior H. Katz, Keigo Machida, Hidekazu Tsukamoto, Kirti Shetty, Aiwu R. He, Lynt B. Johnson, Asif Rashid, Sang-Bae Kim, Ju-Seog Lee, Lopa Mishra Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. Vitamin D (VD) has been implicated in the prevention of multiple cancers, some of which are characterized by inactivation of TGF- β signaling. Recently, inactivation of TGFβ signaling has been associated with development of HCC. We examined the potential role of VD in HCC chemoprevention in the context of TGF- β inactivation in a mouse model. Methods: Wild type, Sptbn1+/- and Smad3+/- male mice were treated with 50 mg/kg of the chemical carcinogen diethylnitrosamine (DEN) via i.p. injection at 14 days of age. At 8 months, the animals were changed from regular chow (2,200 IU/kg VD) to one of two diets: lower than normal VD (200 IU/kg) or high VD (10,000 IU/kg). At one year of age, the livers were harvested for tissue analysis. Several metrics were assessed including expression levels of 164 proteins from liver tumors by Reverse Phase Protein Array (RPPA). Results: Twenty three mice were treated with lower than normal dose VD while 26 received high dose VD. We did not find significant differences in blood calcium levels in animals on either diet. As expected, high VD intervention after 8 months of DEN injection did not affect the total number of tumors the mice developed; however, the lower than normal VD regimen promoted tumor growth in the context of Smad3 disruption (liver-to-body weight ratio 18.37% in Smad3+/- mice treated with low dose VD vs. 6.25% in Smad3+/- animals treated with high dose, p=0.028). Furthermore, RPPA data revealed that the lower than normal VD intake in DEN-treated Smad3 heterozygous null mice results in repression of tumor suppressing genes such as PDCD4 and concomitant up-regulation of tumor promoting genes like Stat5A, Bcl2-XL, PDGFRB and PEA15. Conclusions: Remarkably, lower than normal VD in the context of Smad3 disruption promotes tumor growth possibly through repression of tumor suppressing genes such as PDCD4 and up-regulation of oncogenes like Stat5A, Bcl2XL, PDGFRB and PEA15. These results suggest that VD treatment strategies could potentially be significant for chemoprevention and treatment of HCC in the context of inactivation of TGF-β signaling in patient populations with underlying deficiency of VD.
Mo1808 Association of Gender and Menopause With Injury Types and Histological Features of Drug-Induced Liver Injury Ayako Suzuki, Jiezhun Gu, Hans L. Tillmann, Herbert L. Bonkovsky, Robert J. Fontana, David E. Kleiner Background and Aims: Sex and sex hormones are known to impact drug metabolism, drug transporters, and immune response. Recent studies in brain and kidney injury animal models revealed that cellular response to stress and cell death program differ between males and females. We hypothesized that clinical presentations and histologic features of drug-induced liver injury (DILI) may be influenced by gender and menopausal state. We therefore looked for associations of gender and menopausal state with histologic features and biochemical injury of DILI in the Drug-Induced Liver Injury Network (DILIN). Methods: Of 1234 patients enrolled in DILIN (Sep 2004 - May 2013), we analyzed 200 adults who 1) had a DILIN causality score of at least ‘probable' and 2) had liver biopsies within 30 days of DILI onset which were scored in a standardized manner. The patients were classified into hepatocellular injury (HC) and cholestatic/mixed injury (CS/MIX) based on the laboratory
Mo1644 Colony Stimulating Growth Factor (CSF-1) Promotes Proliferation of TumorAssociated Macrophages in Hepatocellular Carcinoma Abdul Rehman, Priyanka Thakur, Grace Dougherty, Michael Duncan Abstract: Macrophages are an essential component of the innate immune response in cancer, however they are no longer considered to be entirely the agents of host defense against malignancy. Several studies have implicated tumor-associated macrophages (TAMs) in the
AASLD Abstracts
S-1000