Molecular Pathways Leading to Induction of Cell Death and Anti-Proliferative Properties by Tacrolimus and mTOR Inhibitors in Liver Cancer Cells.

BACKGROUND/AIMS: Orthotopic liver transplantation (OLT) is the recommended treatment for patients at early stages of hepatocarcinoma (HCC) with portal hypertension and/or increased bilirubinemia, but without vascular-associated diseases. Tumor recurrence, which is the main drawback for the survival of patients submitted to OLT for HCC, has been related to tumor-related variables and the immunosuppressive therapies. We have previously shown that Tacrolimus (FK506) exerts a more potent pro-apoptotic and anti-proliferative effects than the mammalian target of rapamycin (mTOR) inhibitors (Sirolimus and Everolimus) in liver cancer cells. This study identified the role of the immunosuppressant partners such as FK506-binding proteins (FKBPs) in the induction of cell death and arrest of cell proliferation by immunosuppressants in two representative liver cancer cells. METHODS: The regulation of endoplasmic reticulum (ER) stress, apoptosis/autophagy, cell proliferation, and FKBPs expression was determined in Tacrolimus-, Sirolimus- and Everolimus-treated primary human hepatocytes, and hepatoma HepG2 and Huh7 cell lines. The functional repercussion of FKBPs on cell death and proliferation was also addressed using the siRNA technology. The assessed antitumoral properties of the immunosuppressants were associated to microRNAs (miRNAs) pattern. RESULTS: The enhanced pro-apoptotic and anti-proliferative properties of Tacrolimus versus mTOR inhibitors were associated with increased protein kinase RNA-like endoplasmic reticulum kinase (PERK)-related ER stress, Ser15P-p53/p53 ratio and p21 protein expression that may counterbalance the risk of proliferative upregulation caused by enhanced Thr172P-Cdk4/Cdk4 activation in liver cancer cells. The inhibition of the mTOR pathway by Sirolimus and Everolimus was related to an induction of autophagy; and at a high dose, these drugs impaired translation likely at a very early step of the elongation phase. Tacrolimus and mTOR inhibitors increased the protein expression of FKBP12 and FKBP51 that appeared to play pro-survival role. Interestingly, the administration of immunosuppressants yields a specific pattern of miRNAs. Tacrolimus and mTOR inhibitors decreased miR-92a-1-5p, miR-197-3p, miR-483-3p and miR-720, and increased miR-22-3p, miR-376a-3p, miR-663b, miR-886-5p, miR-1300 and miR-1303 expressions in HepG2 cells. CONCLUSION: The more potent pro-apoptotic and anti-proliferative properties of Tacrolimus versus mTOR inhibitors were associated with an increased activation of PERK and p53 signaling, and p21 protein expression. FKBP12 and FKBP51 appeared to be the most relevant partners of Tacrolimus and mTOR inhibitors exerting a pro-survival effect in HepG2 cells. The observed effects of immunosuppressants were related to a specific miRNA signature in liver cancer cells.

Molecular characterization of autophagic and apoptotic signaling induced by sorafenib in liver cancer cells.

Sorafenib is the unique accepted molecular targeted drug for the treatment of patients in advanced stage of hepatocellular carcinoma. The current study evaluated cell signaling regulation of endoplasmic reticulum (ER) stress, c-Jun-N-terminal kinase (JNK), Akt, and 5'AMP-activated protein kinase (AMPK) leading to autophagy and apoptosis induced by sorafenib. Sorafenib induced early (3-12 hr) ER stress characterized by an increase of Ser51 P-eIF2α/eIF2α, C/EBP homologous protein (CHOP), IRE1α, and sXBP1, but a decrease of activating transcription factor 6 expression, overall temporally associated with the increase of Thr183,Tyr185 P-JNK1/2/JNK1/2, Thr172 P-AMPKα, Ser413 P-Foxo3a, Thr308 P-AKt/AKt and Thr32 P-Foxo3a/Foxo3a ratios, and reduction of Ser2481 P-mammalian target of rapamycin (mTOR)/mTOR and protein translation. This pattern was related to a transient increase of tBid, Bim EL , Beclin-1, Bcl-xL, Bcl-2, autophagy markers, and reduction of myeloid cell leukemia-1 (Mcl-1) expression. The progressive increase of CHOP expression, and reduction of Thr308 P-AKt/AKt and Ser473 P-AKt/AKt ratios were associated with the reduction of autophagic flux and an additional upregulation of Bim EL expression and caspase-3 activity (24 hr). Small interfering-RNA (si-RNA) assays showed that Bim, but not Bak and Bax, was involved in the induction of caspase-3 in sorafenib-treated HepG2 cells. Sorafenib increased autophagic and apoptotic markers in tumor-derived xenograft model. In conclusion, the early sorafenib-induced ER stress and regulation of JNK and AMPK-dependent signaling were related to the induction of survival autophagic process. The sustained drug treatment induced a progressive increase of ER stress and PERK-CHOP-dependent rise of Bim EL , which was associated with the shift from autophagy to apoptosis. The kinetic of Bim EL expression profile might also be related to the tight balance between AKt- and AMPK-related signaling leading to Foxo3a-dependent BIM EL upregulation.

GATA4 loss in the septum transversum mesenchyme promotes liver fibrosis in mice.

UNLABELLED: The zinc finger transcription factor GATA4 controls specification and differentiation of multiple cell types during embryonic development. In mouse embryonic liver, Gata4 is expressed in the endodermal hepatic bud and in the adjacent mesenchyme of the septum transversum. Previous studies have shown that Gata4 inactivation impairs liver formation. However, whether these defects are caused by loss of Gata4 in the hepatic endoderm or in the septum transversum mesenchyme remains to be determined. In this study, we have investigated the role of mesenchymal GATA4 activity in liver formation. We have conditionally inactivated Gata4 in the septum transversum mesenchyme and its derivatives by using Cre/loxP technology. We have generated a mouse transgenic Cre line, in which expression of Cre recombinase is controlled by a previously identified distal Gata4 enhancer. Conditional inactivation of Gata4 in hepatic mesenchymal cells led to embryonic lethality around mouse embryonic stage 13.5, likely as a consequence of fetal anemia. Gata4 knockout fetal livers exhibited reduced size, advanced fibrosis, accumulation of extracellular matrix components and hepatic stellate cell (HSC) activation. Haploinsufficiency of Gata4 accelerated CCl4 -induced liver fibrosis in adult mice. Moreover, Gata4 expression was dramatically reduced in advanced hepatic fibrosis and cirrhosis in humans. CONCLUSIONS: Our data demonstrate that mesenchymal GATA4 activity regulates HSC activation and inhibits the liver fibrogenic process.

Development of a liver graft assessment expert machine-learning system: when the artificial intelligence helps liver transplant surgeons.

Background: The complex process of liver graft assessment is one point for improvement in liver transplantation. The main objective of this study is to develop a tool that supports the surgeon who is responsible for liver donation in the decision-making process whether to accept a graft or not using the initial variables available to it. Material and method: Liver graft samples candidate for liver transplantation after donor brain death were studied. All of them were evaluated "in situ" for transplantation, and those discarded after the "in situ" evaluation were considered as no transplantable liver grafts, while those grafts transplanted after "in situ" evaluation were considered as transplantable liver grafts. First, a single-center, retrospective and cohort study identifying the risk factors associated with the no transplantable group was performed. Then, a prediction model decision support system based on machine learning, and using a tree ensemble boosting classifier that is capable of helping to decide whether to accept or decline a donor liver graft, was developed. Results: A total of 350 liver grafts that were evaluated for liver transplantation were studied. Steatosis was the most frequent reason for classifying grafts as no transplantable, and the main risk factors identified in the univariant study were age, dyslipidemia, personal medical history, personal surgical history, bilirubinemia, and the result of previous liver ultrasound (p < 0.05). When studying the developed model, we observe that the best performance reordering in terms of accuracy corresponds to 76.29% with an area under the curve of 0.79. Furthermore, the model provides a classification together with a confidence index of reliability, for most cases in our data, with the probability of success in the prediction being above 0.85. Conclusion: The tool presented in this study obtains a high accuracy in predicting whether a liver graft will be transplanted or deemed non-transplantable based on the initial variables assigned to it. The inherent capacity for improvement in the system causes the rate of correct predictions to increase as new data are entered. Therefore, we believe it is a tool that can help optimize the graft pool for liver transplantation.

Impact of Early Initiated Everolimus on the Recurrence of Hepatocellular Carcinoma After Liver Transplantation.

BACKGROUND: Many centers implement everolimus-based immunosuppression in liver transplant patients with hepatocellular carcinoma. We aimed to explore the potential impact of early initiated everolimus on tumor recurrence after liver transplantation. METHODS: This study included 192 patients with hepatocellular carcinoma undergoing liver transplantation among who 64 individuals were prospectively enrolled (2012-2015) and received early initiated everolimus (ie, started between postoperative day 15 to 21), whereas the remaining 128 patients acted as historical controls without everolimus. Propensity score matching was performed to ensure comparability. Multivariate Cox regression and competing risks analysis were used to control for potential confounders. RESULTS: Patients with and without everolimus were comparable in terms of number of nodules (P = 0.37), total tumor diameter (P = 0.44), Milan criteria fulfillment (P = 0.56), and histological differentiation (P = 0.61), but there were increased microvascular invasion rates in the everolimus group (26.5% vs 13.3%; P = 0.026). Tumor recurrence rates were similar with and without everolimus (10.9% vs 9.9% at 36 months respectively; P = 0.18). After controlling for microvascular invasion among other potential confounders, everolimus had no significant impact on tumor recurrence, neither in the multivariate Cox regression (relative risk = 3.23; P = 0.09), nor in the competing risks analysis for tumor recurrence-death (relative risk = 1.02; P = 0.94). Patients receiving everolimus had reduced tacrolimus trough concentrations and lower serum creatinine within the first 18 months postliver transplantation. CONCLUSION: Everolimus may not be universally prescribed to prevent tumor recurrence in liver transplant patients with hepatocellular carcinoma. Future randomized trials should be focused on patients with histological features of increased tumor aggressiveness, in whom the potential benefit would be higher.

Differential Antitumoral Properties and Renal-Associated Tissue Damage Induced by Tacrolimus and Mammalian Target of Rapamycin Inhibitors in Hepatocarcinoma: In Vitro and In Vivo Studies.

Orthotopic liver transplantation (OLT) is the recommended treatment for patients at early stages of hepatocarcinoma (HCC) with potential portal hypertension and/or bilirubinemia, but without vascular-associated diseases. The patients are receiving immunosuppressive therapy to reduce graft rejection, but differential side effects have been related to calcineurin and mTOR inhibitor administration regarding tumor recurrence and nephrotoxicity. The in vitro studies showed that Tacrolimus exerted a more potent pro-apoptotic effect than Everolimus (Huh 7>Hep 3B>HepG2), being sirolimus only active in Hep3B cell line. Tacrolimus and Everolimus exerted potent antiproliferative properties in Huh 7 and Hep3B in which cells Sirolimus was inactive. Interestingly, Tacrolimus- and Everolimus-dependent G0/G1 cell accumulation occurred as a consequence of drastic reduction in S, as well as in S and G2+M phases, respectively. The in vivo studies support data on the more effective antitumoral properties of Everolimus, eventual risk of pro-angiogenic tumoral properties and nephrotoxicity of Tacrolimus, and pro-proliferative properties of Sirolimus in tumors developed in nude mice.

[Retroperitoneal sarcomas. Report of five new cases and review of the current situation]. / Sarcomas retroperitoneales. Aportación de cinco nuevos casos y revisión de la situación actual.

INTRODUCTION: Soft-tissue sarcomas represent a heterogeneous group of rare tumors arising from the mesenchymal cells of the connective tissue. Approximately 15% of these tumors arise in the retroperitoneum. These neoplasms are locally aggressive and the only curative treatment is surgical resection "en-bloc". The main cause of mortality is locoregional recurrence. Five-year survival is 3-58%, depending on the histologic subtype and grade. PATIENTS AND METHOD: Over a 5-year period (2001 to 2006), we performed surgery in five patients with retroperitoneal sarcomas. The mean age was 59 years (range, 46-76) with a male-to-female ratio of 3:2. The most frequent signs were abdominal pain and the appearance of a mass. In 2 patients, an incidental diagnosis was made during the surgical intervention. RESULTS: Surgical resection was performed in 5 patients but was incomplete in two patients. We removed 2 liposarcomas, 1 leiomyosarcoma, 1 chondrosarcoma and 1 fusocellular sarcoma arising in the kidney. Locoregional recurrence occurred in four patients, requiring between 1 and 2 new relaparotomies (using the retroperitoneal approach in 2 patients). There was one death. CONCLUSIONS: Retroperitoneal sarcomas are a heterogeneous group of tumors, in which the only common factor is the anatomical location. New randomized, prospective, multicenter trials are required to apply different therapeutic options according to the subtype of retroperitoneal sarcoma.