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P0305 : HOXA13 expression is associated to worst prognosis in HCC and modulates HCC-derived cells response to sorafenib in vitro
POSTERS reaction (PCR), immunohistochemistry and western blot. Albumin secretion by cells grown within scaffolds, monolayer culture and on Matrigel was chekced by ELISA. We also evaluated the function of HepG2 cells grown on scaffolds in the presence of a wellknown anti-cancer drug; methotrexate, to investigate the potential application of our system for drug screening. Results: Histological and scanning electron microscopy examinations of decellularized scaffold revealed total removal of the cytoplasmic and nuclear materials. Perfusion of decellularized liver by fluroscent dextran showed the preservation of the vascular tree in scaffolds. Biochemical analysis showed the presence of a negligible amount of DNA and preserving of the important extracellular matrix components such as collagen, elastin and glycosaminoglycans. HepG2 cells grew well on the scaffolds. PCR, immunohistochemical examination and western blotting showed the ability of HepG2 cell grown within scaffolds to maintain their function and tumorgenicity at significantly higher levels than cells grown on two-dimensional (2-D) dishes or spheroids on Matrigel. Unlike the 2-D cultures, albumin secretion and alpha-fetoprotein expression in three-dimensional cultures were less susceptible to lower concentrations of the drug. Cells grown in scaffolds seemed to respond to the drug in an analogous manner to its known activity in vivo. Conclusions: In summary, using HepG2-reseeded scaffolds as in vitro 3-D cancer models provided a superior 3-D cell culture environment for tumor studies and will enable researchers to evaluate and predict the efficacy of new anti-cancer drugs. P0305 HOXA13 EXPRESSION IS ASSOCIATED TO WORST PROGNOSIS IN HCC AND MODULATES HCC-DERIVED CELLS RESPONSE TO SORAFENIB IN VITRO L. Quagliata1 , C. Quintavalle1 , V. Perrina1 , M. Matter1 , C. Cillo1 , L. Terracciano1 . 1 Institute of Pathology, University Hospital Basel, Basel, Switzerland E-mail: [email protected] Background and Aims: Despite significant advances in HCC diagnosis and management, for advanced stages no therapeutic options exist beside Sorafenib. Recently, using human liver biopsies, we showed that HOXA13 expression in HCC correlates with poor survival and metastasis presence. In addition, we observed that HOXA13 expression increases HCC cells proliferation in vitro. Here we seek to confirm our data on a larger cohort of samples and to investigate whether HOXA13 could modulate cells’ Sorafenib response. Methods: A liver TMA (tissue microarray) comprises a total of n = 305 specimens, n = 82 normal liver tissues, n = 108 cirrhotic patients and n = 115 HCCs, has been stained for HOXA13, CK-7, CK19, E-Cad. Protein levels have been correlated with patients’clinical data. In vitro experiments to stably modulate HOXA13 expression (gain and loss of function) have been performed using the HCC derived cell lines: Hep-G2, SNU449 and PLC5. Subsequently, cells have been treated with Sorafenib and cell cycle analysis, proliferation, migration and drug responce have been tested. Results: HOXA13 is altered in 41% of HCC tested samples, thus confirming our previous results obtained using iver biopsies. In addition, high HOXA13 levels are associated with poorer outcome and higher grading (Edmondson and BCLC). Increased HOXA13 expression is linked with stem progenitor markers, CK-7 and CK19. Furthermore, high HOXA13 expression is coupled with diminished levels of E-Cad, providing a molecular basis for its association with metastasis in HCC. Finally, in vitro experiments demonstrate that HOXA13 overexpression results in higher resistance to Sorafenib exposure. Conversely, HOXA13 downregulation sensitize HCC cells to Sorafenib.
Conclusions: Here we show that HOXA13 IHC-based protein levels can reliably predict HCC outcome and correlates with a number of tumour features (e.g. grade). Thus the usage of HOXA13 as a marker for HCC aggressiveness deserves further investigations. In addition, our in vitro data concerning HOXA13 modulation of Sorafenib response prompt us to validate this finding in vivo using HCC samples. P0306 IMPAIRMENT OF AUTOPHAGY IN THE EARLY STAGES OF HEPATOCARCINOGENESIS M.A. Kowalik1 , A. Perra1 , L. Falasca2 , M. Piacentini3 , G.M. LeddaColumbano1 , A. Columbano1 . 1 Department of Biomedical Sciences, University of Cagliari, Cagliari, 2 National Institute for Infectious Diseases I.R.C.C.S. ‘L. Spallanzani’, 3 Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy E-mail: [email protected] Background and Aims: Hepatocellular carcinoma (HCC), the third leading cause of cancer-related mortality worldwide, represents a major health problem. Recent studies highlight the importance of autophagy, a catabolic process by which cells remove protein aggregates and damaged organelles, in hepatocarcinogenesis. Autophagy dysfunction is also associated with neurodegeneration, aging and autoimmune disorders, however, its role in carcinogenesis remains complex, dependent on cancer stage and still controversial. Therefore, the aim of this study was to investigate the role of autophagy in the early stages of HCC development. Methods: The Resistant-Hepatocyte (R-H) rat model [1], which offers the possibility to identify distinct lesions (preneoplastic nodules, early and fully advanced HCCs) at well-defined timings, was used. Autophagy was investigated by immunohistochemistry, electron microscopy and qRT-PCR. Results: First of all, the expression of different autophagy markers (Ambra1, p62, Becn1, VpS34, UVRAG) was investigated in preneoplastic nodules. The results indicate that while the mRNA levels of Ambra1, p62, Becn1, Ulk1, Atg5 and Atg12 were not increased in the preneoplastic lesions, immunohistochemistry analysis demonstrated a marked accumulation of all analyzed proteins in preneoplastic nodules when compared with the surrounding tissue, suggesting impairment of the autophagic process. The results of electron microscopy analysis confirmed the autophagy blockage, as demonstrated not only by the lack of autophagic vacuoles but also by the presence of strongly altered, swollen and unremoved mitochondria. Recent studies [2] reported that p62 interacts with the Nrf2-binding site on Keap1, resulting in stabilization of Nrf2 followed by transcriptional activation of its target genes. Accordingly, p62 accumulation in preneoplastic nodules was associated with a significant induction of NQO1, GCLC and GSTA4 (Nrf2 target genes). Finally, the expression levels of miRNA-224, a miR whose expression is inversely correlated with autophagy [3], was about 100-fold higher in preneoplastic nodules when compared to the surrounding tissue. Conclusions: These results strongly suggest that autophagy results impaired in the early stages of hepatocarcinogenesis induced by R-H model and its modulation may represent a promising therapeutic target in HCC. Reference(s) [1] Solt et al. Am J Pathol. 1977; 88(3): 595–618. [2] Inami et al. J Cell Biol. 2011; 193(2): 275–84. [3] Lan et al. Hepatology. 2014; 59(2): 505–17.
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Conclusions: Here we show that HOXA13 IHC-based protein levels can reliably predict HCC outcome and correlates with a number of tumour features (e.g. grade). Thus the usage of HOXA13 as a marker for HCC aggressiveness deserves further investigations. In addition, our in vitro data concerning HOXA13 modulation of Sorafenib response prompt us to validate this finding in vivo using HCC samples. P0306 IMPAIRMENT OF AUTOPHAGY IN THE EARLY STAGES OF HEPATOCARCINOGENESIS M.A. Kowalik1 , A. Perra1 , L. Falasca2 , M. Piacentini3 , G.M. LeddaColumbano1 , A. Columbano1 . 1 Department of Biomedical Sciences, University of Cagliari, Cagliari, 2 National Institute for Infectious Diseases I.R.C.C.S. ‘L. Spallanzani’, 3 Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy E-mail: [email protected] Background and Aims: Hepatocellular carcinoma (HCC), the third leading cause of cancer-related mortality worldwide, represents a major health problem. Recent studies highlight the importance of autophagy, a catabolic process by which cells remove protein aggregates and damaged organelles, in hepatocarcinogenesis. Autophagy dysfunction is also associated with neurodegeneration, aging and autoimmune disorders, however, its role in carcinogenesis remains complex, dependent on cancer stage and still controversial. Therefore, the aim of this study was to investigate the role of autophagy in the early stages of HCC development. Methods: The Resistant-Hepatocyte (R-H) rat model [1], which offers the possibility to identify distinct lesions (preneoplastic nodules, early and fully advanced HCCs) at well-defined timings, was used. Autophagy was investigated by immunohistochemistry, electron microscopy and qRT-PCR. Results: First of all, the expression of different autophagy markers (Ambra1, p62, Becn1, VpS34, UVRAG) was investigated in preneoplastic nodules. The results indicate that while the mRNA levels of Ambra1, p62, Becn1, Ulk1, Atg5 and Atg12 were not increased in the preneoplastic lesions, immunohistochemistry analysis demonstrated a marked accumulation of all analyzed proteins in preneoplastic nodules when compared with the surrounding tissue, suggesting impairment of the autophagic process. The results of electron microscopy analysis confirmed the autophagy blockage, as demonstrated not only by the lack of autophagic vacuoles but also by the presence of strongly altered, swollen and unremoved mitochondria. Recent studies [2] reported that p62 interacts with the Nrf2-binding site on Keap1, resulting in stabilization of Nrf2 followed by transcriptional activation of its target genes. Accordingly, p62 accumulation in preneoplastic nodules was associated with a significant induction of NQO1, GCLC and GSTA4 (Nrf2 target genes). Finally, the expression levels of miRNA-224, a miR whose expression is inversely correlated with autophagy [3], was about 100-fold higher in preneoplastic nodules when compared to the surrounding tissue. Conclusions: These results strongly suggest that autophagy results impaired in the early stages of hepatocarcinogenesis induced by R-H model and its modulation may represent a promising therapeutic target in HCC. Reference(s) [1] Solt et al. Am J Pathol. 1977; 88(3): 595–618. [2] Inami et al. J Cell Biol. 2011; 193(2): 275–84. [3] Lan et al. Hepatology. 2014; 59(2): 505–17.
Journal of Hepatology 2015 vol. 62 | S263–S864
S423