The Vasopressin Receptor Antagonist Tolvaptan Counteracts Tumor Growth in a Murine Xenograft Model of Small Cell Lung Cancer.

We have previously demonstrated that the vasopressin type 2 receptor (AVPR2) antagonist tolvaptan reduces cell proliferation and invasion and triggers apoptosis in different human cancer cell lines. To study this effect in vivo, a xenograft model of small cell lung cancer was developed in Fox1nu/nu nude mice through the subcutaneous inoculation of H69 cells, which express AVPR2. One group of mice (n = 5) was treated with tolvaptan for 60 days, whereas one group (n = 5) served as the control. A reduced growth was observed in the tolvaptan group in which the mean tumor volume was significantly smaller on day 60 compared to the control group. In the latter group, a significantly lower survival was observed. The analysis of excised tumors revealed that tolvaptan effectively inhibited the cAMP/PKA and PI3K/AKT signaling pathways. The expression of the proliferative marker proliferating cell nuclear antigen (PCNA) was significantly lower in tumors excised from tolvaptan-treated mice, whereas the expression levels of the apoptotic marker caspase-3 were higher than those in control animals. Furthermore, tumor vascularization was significantly lower in the tolvaptan group. Overall, these findings suggest that tolvaptan counteracts tumor progression in vivo and, if confirmed, might indicate a possible role of this molecule as an adjuvant in anticancer strategies.

Hyponatremia Promotes Cancer Growth in a Murine Xenograft Model of Neuroblastoma.

In cancer patients, hyponatremia is detected in about 40% of cases at hospital admission and has been associated to a worse outcome. We have previously observed that cancer cells from different tissues show a significantly increased proliferation rate and invasion potential, when cultured in low extracellular [Na+]. We have recently developed an animal model of hyponatremia using Foxn1nu/nu mice. The aim of the present study was to compare tumor growth and invasivity of the neuroblastoma cell line SK-N-AS in hyponatremic vs. normonatremic mice. Animals were subcutaneously implanted with luciferase-expressing SK-N-AS cells. When masses reached about 100 mm3, hyponatremia was induced in a subgroup of animals via desmopressin infusion. Tumor masses were significantly greater in hyponatremic mice, starting from day 14 and until the day of sacrifice (day 28). Immunohistochemical analysis showed a more intense vascularization and higher levels of expression of the proliferating cell nuclear antigen, chromogranin A and heme oxigenase-1 gene in hyponatremic mice. Finally, metalloproteases were also more abundantly expressed in hyponatremic animals compared to control ones. To our knowledge, this is the first demonstration in an experimental animal model that hyponatremia is associated to increased cancer growth by activating molecular mechanisms that promote proliferation, angiogenesis and invasivity.

Growth arrest-specific 5 lncRNA as a valuable biomarker of chemoresistance in osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumour in children and teenagers, and it is characterised by drug resistance and high metastatic potential. Increasing studies have highlighted the critical roles of long noncoding RNAs (lncRNAs) as oncogenes or tumour suppressors as well as new biomarkers and therapeutic targets in osteosarcoma. The growth arrest-specific 5 (GAS5) lncRNA can function as a tumour suppressor in several cancers. The present study aimed to validate GAS5 and other chemoresistance-associated lncRNAs as biomarkers in a cohort of primary osteosarcoma samples, to obtain predictive information on resistance or sensitivity to treatment. The GAS5 and a panel of lncRNAs related to chemoresistance [SNGH1, FOXD2-AS1, deleted in lymphocytic leukemia (DLEU2) and LINC00963] were evaluated in a cohort of osteosarcoma patients enrolled at the Careggi University Hospital. Total RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue sections and the expression levels of the lncRNAs were quantified by qPCR. A bioinformatic analysis on deposited RNA-seq data was performed to validate the qPCR results. Clustering analysis shows that GAS5 could be linked to the expression of isoforms 02 and 04 of the lncRNA DLEU2, whereas the DLEU2 isoform 08 is linked to the lncRNA LINC00963. We found that GAS5 is significantly increased in patients with a good prognosis and is expressed differently between chemosensitive and chemoresistant osteosarcoma patients. However, the results obtained are not concordant with the in-silico analysis performed on the TARGET osteosarcoma dataset. In the future, we would enlarge the case series, including different disease settings.

Liver haploinsufficiency of RuvBL1 causes hepatic insulin resistance and enhances hepatocellular carcinoma progression.

RuvBL1 is an AAA+ ATPase whose expression in hepatocellular carcinoma (HCC) correlates with a poor prognosis. In vitro models suggest that targeting RuvBL1 could be an effective strategy against HCC. However, the role of RuvBL1 in the onset and progression of HCC remains unknown. To address this question, we developed a RuvBL1hep+/- mouse model and evaluated the outcome of DEN-induced liver carcinogenesis up to 12 months of progression. We found that RuvBL1 haploinsufficiency initially delayed the onset of liver cancer, due to a reduced hepatocyte turnover in RuvBL1hep+/- mice. However, RuvBL1hep+/- mice eventually developed HCC nodules that, with aging, grew larger than in the control mice. Moreover, RuvBL1hep+/- mice developed hepatic insulin resistance and impaired glucose homeostasis. We could determine that RuvBL1 regulates insulin signaling through the Akt/mTOR pathway in liver physiology in vivo as well as in normal hepatocytic and HCC cells in vitro. Whole transcriptome analysis of mice livers confirmed the major role of RuvBL1 in the regulation of hepatic glucose metabolism. Finally, RuvBL1 expression was found significantly correlated to glucose metabolism and mTOR signaling by bioinformatic analysis of human HCC sample from the publicly available TGCA database. These data uncover a role of RuvBL1 at the intersection of liver metabolism, hepatocyte proliferation and HCC development, providing a molecular rationale for its overexpression in liver cancer.

Single-centre comparison of robotic and open pancreatoduodenectomy: a propensity score-matched study.

BACKGROUND: Pancreatoduodenectomy for pancreatic head and periampullary cancers is still associated with high perioperative morbidity and mortality. The aim of this study was to compare the short-term outcomes of robot-assisted pancreatoduodenectomy (RAPD) and open pancreatoduodenectomy (OPD) performed in a high-volume centre. METHODS: A single-centre, prospective database was used to retrospectively compare the early outcomes of RAPD procedures to standard OPD procedures completed between January 2014 and December 2018. Of the 121 included patients, 78 underwent RAPD and 43 underwent OPD. After propensity score matching (PSM), 35 RAPD patients were matched with 35 OPD patients with similar preoperative characteristics. RESULTS: There were no statistically significant differences in most of the baseline demographics and perioperative outcomes in the two groups after PSM optimization with the exception of the operative time (530 min (RAPD) versus 335 min (OPD) post-match, p < 0.000). No differences were found between the two groups in terms of complications (including pancreatic leaks, 11.4% in both OPD and RAPD), perioperative mortality, reoperations or readmissions. Earlier refeeding was obtained in the RAPD group vs. the OPD group (3 vs. 4 days, p = 0.002). Although the differences in the length of the hospital stay and blood transfusions were not statistically significant, both parameters showed a positive trend in favour of RAPD. The number of harvested lymph nodes was similar and oncologically adequate. CONCLUSIONS: RAPD is a safe and oncologically adequate technique to treat malignancies arising from the pancreatic head and periampullary region. Several perioperative parameters resulted in trends favouring RAPD over OPD, at the price of longer operating time. Data should be reinforced with a larger sample to guarantee statistical significance.

Correction to: Single-centre comparison of robotic and open pancreatoduodenectomy: a propensity score-matched study.

In the Abstract, in the Methods section the sentence "Of the 121 included patients, 78 underwent RAPD and 43 underwent OPD." Should read: Of the 121 included patients, 77 underwent OPD and 44 underwent RAPD."

Telomerase reactivation is associated with hepatobiliary and pancreatic cancers.

BACKGROUND: Human telomerase reverse transcriptase (hTERT) and its components play a significant role in cancer progression, but recent data demonstrated that telomeres and telomerase alterations could be found in other diseases; increasing evidence suggests a key role of this enzyme in the fields of hepatobiliary and pancreatic diseases. DATA SOURCES: We performed a PubMed search with the following keywords: telomerase, hepatocellular carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma by December 2019. We reviewed the relevant publications that analyzed the correlation between telomerase activity and hepatobiliary and pancreatic diseases. RESULTS: Telomerase reactivation plays a significant role in the development and progression of hepatobiliary and pancreatic tumors and could be used as a diagnostic biomarker for hepatobiliary and pancreatic cancers, as a predictor for prognosis and a promising therapeutic target. CONCLUSIONS: Our review summarized the evidence about the critical role of hTERT in cancerous and precancerous lesions of the alteration and its activity in hepatobiliary and pancreatic diseases.

The orphan nuclear receptor COUP-TFII coordinates hypoxia-independent proangiogenic responses in hepatic stellate cells.

BACKGROUND & AIMS: Hepatic stellate cell (HSC) transdifferentiation into collagen-producing myofibroblasts is a key event in hepatic fibrogenesis, but the transcriptional network that controls the acquisition of the activated phenotype is still poorly understood. In this study, we explored whether the nuclear receptor chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is involved in HSC activation and in the multifunctional role of these cells during the response to liver injury. METHODS: COUP-TFII expression was evaluated in normal and cirrhotic livers by immunohistochemistry and Western blot. The role of COUP-TFII in HSC was assessed by gain and loss of function transfection experiments and by generation of mice with COUP-TFII deletion in HSC. Molecular changes were determined by gene expression microarray and RT-qPCR. RESULTS: We showed that COUP-TFII is highly expressed in human fibrotic liver and in mouse models of hepatic injury. COUP-TFII expression rapidly increased upon HSC activation and it was associated with the regulation of genes involved in cell motility, proliferation and angiogenesis. Inactivation of COUP-TFII impairs proliferation and invasiveness in activated HSC and COUP-TFII deletion in mice abrogate HSC activation and angiogenesis. Finally, co-culture experiments with HSC and liver sinusoidal endothelial cells (SEC) showed that COUP-TFII expression in HSC influenced SEC migration and tubulogenesis via a hypoxia-independent and nuclear factor kappaB-dependent mechanism. CONCLUSION: This study elucidates a novel transcriptional pathway in HSC that is involved in the acquisition of the proangiogenic phenotype and regulates the paracrine signals between HSC and SEC during hepatic wound healing. LAY SUMMARY: In this study, we identified an important regulator of HSC pathobiology. We showed that the orphan receptor COUP-TFII is an important player in hepatic neoangiogenesis. COUP-TFII expression in HSC controls the crosstalk between HSC and endothelial cells coordinating vascular remodelling during liver injury. TRANSCRIPT PROFILING: ArrayExpress accession E-MTAB-1795.

Peroxisome proliferator-activated receptor-γ agonist pioglitazone reduces the development of necrotizing enterocolitis in a neonatal preterm rat model.

BACKGROUND: Factors affecting innate immunity and acting as inflammatory regulators, such as the nuclear peroxisome proliferator-activated receptors (PPAR) could be crucial in the pathogenesis of necrotizing enterocolitis (NEC). We hypothesized that the PPARγ agonist pioglitazone (PIO) might be effective in preventing the development of NEC and/or reducing its severity. METHODS: We studied preterm rats in which NEC was induced using the hypoxia-hypothermia model. The treatment group (TG; n = 30) received enteral PIO (10 mg/kg/d) for 72 h and the control group (CG; n = 30) did not. Animals were sacrificed 96 h after birth. NEC was diagnosed evaluating histological ileum changes, and mRNA levels of IL-4, IL-12, IL-6, IL-10, INF-γ, and TNF-α cytokines were measured. RESULTS: NEC occurrence was higher in the CG (18/30; 60%) than in the TG (5/30; 16.7%) and was more severe. Proinflammatory IL-12 and INF-γ mRNA levels were significantly lower in the TG than in the CG; conversely, the anti-inflammatory IL-4 mRNA level was significantly higher in the TG than in the CG. CONCLUSION: Our results demonstrate for the first time that PIO is effective in reducing the incidence and severity of NEC and in decreasing renal injuries in a preterm rat model.

Magnetic Hyperthermia and Oxidative Damage to DNA of Human Hepatocarcinoma Cells.

Nanotechnology is addressing major urgent needs for cancer treatment. We conducted a study to compare the frequency of 3-(2-deoxy-ß-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) adducts, biomarkers of oxidative stress and/or lipid peroxidation, on human hepatocarcinoma HepG2 cells exposed to increasing levels of Fe3O4-nanoparticles (NPs) versus untreated cells at different lengths of incubations, and in the presence of increasing exposures to an alternating magnetic field (AMF) of 186 kHz using 32P-postlabeling. The levels of oxidative damage tended to increase significantly after ≥24 h of incubations compared to controls. The oxidative DNA damage tended to reach a steady-state after treatment with 60 µg/mL of Fe3O4-NPs. Significant dose-response relationships were observed. A greater adduct production was observed after magnetic hyperthermia, with the highest amounts of oxidative lesions after 40 min exposure to AMF. The effects of magnetic hyperthermia were significantly increased with exposure and incubation times. Most important, the levels of oxidative lesions in AMF exposed NP treated cells were up to 20-fold greater relative to those observed in nonexposed NP treated cells. Generation of oxidative lesions may be a mechanism by which magnetic hyperthermia induces cancer cell death.

COUP-TFII in pancreatic adenocarcinoma: clinical implication for patient survival and tumor progression.

Despite the accumulating knowledge of alterations in pancreatic cancer molecular pathways, no substantial improvements in the clinical prognosis have been made and this malignancy continues to be a leading cause of cancer death in the Western World. The orphan nuclear receptor COUP-TFII is a regulator of a wide range of biological processes and it may exert a pro-oncogenic role in cancer cells; interestingly, indirect evidences suggest that the receptor could be involved in pancreatic cancer. The aim of this study was to evaluate the expression of COUP-TFII in human pancreatic tumors and to unveil its role in the regulation of pancreatic tumor growth. We evaluated COUP-TFII expression by immunohistochemistry on primary samples. We analyzed the effect of the nuclear receptor silencing in human pancreatic cancer cells by means of shRNA expressing cell lines. We finally confirmed the in vitro results by in vivo experiments on nude mice. COUP-TFII is expressed in 69% of tested primary samples and correlates with the N1 and M1 status and clinical stage; Kaplan-Meier and Cox regression analysis show that it may be an independent prognostic factor of worst outcome. In vitro silencing of COUP-TFII reduces the cell growth and invasiveness and it strongly inhibits angiogenesis, an effect mediated by the regulation of VEGF-C. In nude mice, COUP-TFII silencing reduces tumor growth by 40%. Our results suggest that COUP-TFII might be an important regulator of the behavior of pancreatic adenocarcinoma, thus representing a possible new target for pancreatic cancer therapy.

Telomerase activated thymidine analogue pro-drug is a new molecule targeting hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Although hepatectomy and transplantation have significantly improved survival, there is no effective chemotherapeutic treatment for HCC and its prognosis remains poor. Sustained activation of telomerase is essential for the growth and progression of HCC, suggesting that telomerase is a rational target for HCC therapy. Therefore, we developed a thymidine analogue pro-drug, acycloguanosyl-5'-thymidyltriphosphate (ACV-TP-T), which is specifically activated by telomerase in HCC cells and investigated its anti-tumour efficacy. METHODS: First, we verified in vitro whether ACV-TP-T was a telomerase substrate. Second, we evaluated proliferation and apoptosis in murine (Hepa1-6) and human (Hep3B, HuH7, HepG2) hepatic cancer cells treated with ACV-TP-T. Next, we tested the in vivo treatment efficacy in HBV transgenic mice that spontaneously develop hepatic tumours, and in a syngeneic orthotopic murine model where HCC cells were implanted directly in the liver. RESULTS: In vitro characterization provided direct evidence that the pro-drug was actively metabolized in liver cancer cells by telomerase to release the active form of acyclovir. Alterations in cell cycle and apoptosis were observed following in vitro treatment with ACV-TP-T. In the transgenic and orthotopic mouse models, treatment with ACV-TP-T reduced tumour growth, increased apoptosis, and reduced the proliferation of tumour cells. CONCLUSIONS: ACV-TP-T is activated by telomerase in HCC cells and releases active acyclovir that reduces proliferation and induces apoptosis in human and murine liver cancer cells. This pro-drug holds a great promise for the treatment of HCC.

The Impact of PAD4-dependent Neutrophil Extracellular Trap Formation on the Early Development of Intestinal Fibrosis in Crohn's Disease.

BACKGROUND AND AIMS: During early phases of inflammation, activated neutrophils extrude neutrophil extracellular traps (NETs) in a PAD4-dependent manner, aggravating tissue injury and remodelling. In this study, we investigated the potential pro-fibrotic properties and signalling of NETs in Crohn's disease (CD). METHODS: NETs and activated fibroblasts were labelled on resected ileum from CD patients by multiplex immunofluorescence staining. NETs-treated human primary intestinal fibroblasts were analysed by bulk RNA-sequencing to uncover cell signalling pathways, and by high-throughput imaging to assess collagen production and migratory activity. Consequentially, TLR2/NF-kB pathway was evaluated by transfection of CCD-18Co fibroblasts with NF-kB-luciferase reporter plasmid, incorporating C29 to block TLR2 signalling. A chronic DSS mouse model was used to define the specific role of PAD4 deletion in neutrophils (MRP8-Cre, Pad4fl/fl). RESULTS: Immunofluorescence showed spatial co-localisation of NETs and activated fibroblasts in ileal ulcerations of CD patients. Transcriptomic analysis revealed upregulation of pro-fibrotic genes and activation of TLR-signalling pathways in NETs-treated fibroblasts. NETs treatment induced fibroblast proliferation, diminished migratory capability, and increased collagen release. Transfection experiments indicated a substantial increase in NF-kB expression with NETs, whereas C29 led to decreased expression and release of collagen. In line, a significantly reduction in collagen content was observed in the colon of MRP8-Cre, Pad4fl/fl mice subjected to chronic DSS colitis. CONCLUSIONS: NETs potentially serve as an initial stimulus for pathological activation of fibroblasts within the intestine via the TLR2/NF-kB pathway. Given their early involvement in inflammation, inhibition of PAD4 might offer a strategy to modulate both inflammation and fibrogenesis in CD.

KRAS-related miR-143 expression is associated with lymph node involvement and correlates with outcome in pancreatic adenocarcinoma patients.

Introduction: Pancreatic adenocarcinoma (PC) is one of the most lethal malignancies; even after resection the patients' 5-year disease-free survival (DFS) is lower than 26%. The genetic mutational landscape of PC is dominated by activating KRAS mutations, that have been reported in approximately 90% of cases; however, beyond KRAS - direct mutations, several KRAS-targeting miRNAs appear to be downregulated, strengthening the already activated RAS signaling. In addition, the interplay between miRNAs and RAS includes poorly investigated downstream miRNAs. The aim of this study was to determine the prognostic value of some of these candidate KRAS-related miRNAs. Patients and methods: Between 2015 and 2022, 44 patients with pathologically confirmed PC, who received surgery and were enrolled by the Clinical Oncology Unit, Careggi University Hospital, Florence (Italy). PC Total RNA was extracted from FFPE sections, retro-transcribed and the resulting cDNA was then used for qPCR analysis. A panel of KRAS-related miRNA (miR-155, miR-206 and miR-143) was analyzed. Results: In this observational study patients sex distribution was unequal with 34.1% being male and 65.9% female. The most frequent tumor localization was the head of the pancreas (65.9%) and the pathological stages were pT1-2 (45.5%), pT3 (54.5%), pN0 (22.7%), pN+ (77.3%). Adjuvant therapy was administered to 63.6% of patients; disease recurrence was observed in 69% of cases. Twenty-three patients, whose RNA was of adequate quality, were used in the mRNAs expression studies. When comparing the miRNA expression between PC and a pool of healthy tissues, miR-155 was overexpressed and miR-206 downregulated in PC, while miR-143 expression was unchanged. However, when categorized in low- and high- miR-143 expressing PC (according to the median value), high miR-143 was associated with nodal involvement (pN+) (p=0.029), who in turn was linked with shorter DFS (p=0.009) and overall survival (OS) (p=0.021) compared to pN0. A trend toward inferior DFS was observed for higher expression of miR-206 (p=0.095) and miR-143 (p=0.092). Finally, responders to a first-line treatment for advanced disease had miR-155 overexpressed (p=0.048). Conclusions: miRNAs are involved in PC tumorigenesis and metastatic spread. In light of miR-143 association with lymphatic spread and poor prognosis, a comprehensive analysis of miRNA interplay with KRAS deserves further investigation.

Acycloguanosyl 5'-thymidyltriphosphate, a thymidine analogue prodrug activated by telomerase, reduces pancreatic tumor growth in mice.

BACKGROUND & AIMS: Gemcitabine is the standard of care for metastatic and nonresectable pancreatic tumors. Phase II and III trials have not demonstrated efficacy of recently developed reagents, compared with gemcitabine alone; new chemotherapic agents are needed. Ninety percent of pancreatic tumors have telomerase activity, and expression correlates with tumor stage. We developed a thymidine analogue prodrug, acycloguanosyl 5'-thymidyltriphosphate (ACV-TP-T), that is metabolized by telomerase and releases the active form of acyclovir. We investigated the antitumor efficacy of ACV-TP-T in vitro and in vivo. METHODS: We evaluated proliferation and apoptosis of human pancreatic cancer cells (PANC-1, MiaPaca2, BxPc3, PL45, and Su.86.86) incubated with ACV-TP-T. The presence of ACV-TP-T and its metabolite inside the cells were analyzed by mass spectrometry. In vivo efficacy was evaluated in nude mice carrying PANC-1 or MiaPaca2 pancreatic xenograft tumors. RESULTS: The prodrug of ACV-TP-T was actively metabolized inside pancreatic cancer cells into the activated form of acyclovir; proliferation was reduced, apoptosis was increased, and the cell cycle was altered in pancreatic cancer incubated with ACV-TP-T, compared with controls. Administration of ACV-TP-T to mice reduced growth, increased apoptosis, and reduced proliferation and vascularization of pancreatic xenograft tumors. CONCLUSIONS: ACV-TP-T, a thymidine analogue that is metabolized by telomerase and releases the active form of acyclovir, reduces proliferation and induces apoptosis of human pancreatic cancer cell lines in vitro and pancreatic xenograft tumors in mice.

Isoforms of the orphan nuclear receptor COUP­TFII differentially modulate pancreatic cancer progression.

The expression of the nuclear receptor transcription factor (TF) COUP­TFII is broadly associated with cell differentiation and cancer development, including of pancreatic ductal adenocarcinoma (PDAC), a devastating disease with one of the poorest prognoses among cancers worldwide. Recent studies have started to investigate the pathological and physiological roles of a novel COUP­TFII isoform (COUP­TFII_V2) that lacks the DNA­binding domain. As the role of the canonical COUP­TFII in PDAC was previously demonstrated, the present study evaluated whether COUP­TFII_V2 may have a functional role in PDAC. It was demonstrated that COUP­TFII_V2 naturally occurs in PDAC cells and in primary samples, where its expression is consistent with shorter overall survival and peripheral invasion. Of note, COUP­TFII_V2, exhibiting nuclear and cytosolic expression, is linked to epithelial to mesenchymal transition (EMT) and cancer progression, as confirmed by nude mouse experiments. The present results demonstrated that COUP­TFII_V2 distinctively regulates the EMT of PDAC and, similarly to its sibling, it is associated with tumor aggressiveness. The two isoforms have both overlapping and exclusive functions that cooperate with cancer growth and dissemination. By studying how PDAC cells switch from one isoform to the other, novel insight into cancer biology was gained, indicating that this receptor may serve as a novel possible target for PDAC management.

Thiazolidinediones inhibit hepatocarcinogenesis in hepatitis B virus-transgenic mice by peroxisome proliferator-activated receptor gamma-independent regulation of nucleophosmin.

UNLABELLED: Antidiabetic thiazolidinediones (TZD) have in vitro antiproliferative effect in epithelial cancers, including hepatocellular carcinoma (HCC). The effective anticancer properties and the underlying molecular mechanisms of these drugs in vivo remain unclear. In addition, the primary biological target of TZD, the ligand-dependent transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma), is up-regulated in HCC and seems to provide tumor-promoting responses. The aim of our study was to evaluate whether chronic administration of TZD may affect hepatic carcinogenesis in vivo in relation to PPARgamma expression and activity. The effect of TZD oral administration for 26 weeks was tested on tumor formation in PPARgamma-expressing and PPARgamma-deficient mouse models of hepatic carcinogenesis. Proteomic analysis was performed in freshly isolated hepatocytes by differential in gel electrophoresis and mass spectrometry analysis. Identified TZD targets were confirmed in cultured PPARgamma-deficient hepatocytes. TZD administration in hepatitis B virus (HBV)-transgenic mice (TgN[Alb1HBV]44Bri) reduced tumor incidence in the liver, inhibiting hepatocyte proliferation and increasing apoptosis. PPARgamma deletion in hepatocytes of HBV-transgenic mice (Tg[HBV]CreKOgamma) did not modify hepatic carcinogenesis but increased the TZD antitumorigenic effect. Proteomic analysis identified nucleophosmin (NPM) as a TZD target in PPARgamma-deficient hepatocytes. TZD inhibited NPM expression at protein and messenger RNA levels and decreased NPM promoter activity. TZD inhibition of NPM was associated with the induction of p53 phosphorylation and p21 expression. CONCLUSION: These findings suggest that chronic administration of TZD has anticancer activity in the liver via inhibition of NPM expression and indicate that these drugs might be useful for HCC chemoprevention and treatment.

Low sodium and tolvaptan have opposite effects in human small cell lung cancer cells.

PURPOSE: Hyponatraemia is frequently observed in cancer patients and can be due to the syndrome of inappropriate anti-diuresis (SIAD), related to ectopic vasopressin secretion, particularly in small cell lung cancer (SCLC). Hyponatraemia is associated with a worse outcome in cancer patients. The vasopressin receptor antagonist tolvaptan effectively corrects hyponatraemia secondary to SIAD and there is in vitro evidence that it has also an antiproliferative effect in cancer cells. The purpose of this study was i) to analyse the effect of low serum sodium concentrations ([Na+]) in SCLC cells and ii) to determine whether tolvaptan counteracts tumor progression. METHODS: We evaluated cell proliferation, cell cycle, apoptosis, oxidative stress, invasivity in low [Na+] as well as after exposure to tolvaptan. We also analysed the intracellular signalling pathways involved. RESULTS: In reduced [Na+] cell proliferation was significantly increased compared to normal [Na+] and cells were mostly distributed in the G2/M phase. Apoptosis appeared reduced. In addition, the ability to cross matrigel-coated membranes markedly increased. As observed in other cancer cell models, the expression of the heme-oxigenase-1 gene was increased. Finally, we found that in cells cultured in low [Na+] the RhoA/ROCK1/2 pathway, which is involved in the regulation of actin cytoskeleton, was activated. On the other hand, we found that tolvaptan effectively inhibited cell proliferation, anchorage-independent growth, invasivity and promoted apoptosis. Accordingly, the RhoA/ROCK-1/2 pathway was inhibited. CONCLUSIONS: These findings demonstrate for the first time that low [Na+] favours tumor progression in SCLC cells, whereas tolvaptan effectively inhibits cell proliferation, survival and invasivity.

COUP-TFII in Health and Disease.

The nuclear receptors (NRs) belong to a vast family of evolutionary conserved proteins acting as ligand-activated transcription factors. Functionally, NRs are essential in embryogenesis and organogenesis and in adulthood they are involved in almost every physiological and pathological process. Our knowledge of NRs action has greatly improved in recent years, demonstrating that both their expression and activity are tightly regulated by a network of signaling pathways, miRNA and reciprocal interactions. The Chicken Ovalbumin Upstream Promoter Transcription Factor II (COUP-TFII, NR2F2) is a NR classified as an orphan due to the lack of a known natural ligand. Although its expression peaks during development, and then decreases considerably, in adult tissues, COUP-TFII is an important regulator of differentiation and it is variably implicated in tissues homeostasis. As such, alterations of its expression or its transcriptional activity have been studied and linked to a spectrum of diseases in organs and tissues of different origins. Indeed, an altered COUP-TFII expression and activity may cause infertility, abnormality in the vascular system and metabolic diseases like diabetes. Moreover, COUP-TFII is actively investigated in cancer research but its role in tumor progression is yet to be fully understood. In this review, we summarize the current understanding of COUP-TFII in healthy and pathological conditions, proposing an updated and critical view of the many functions of this NR.

Chemotherapy for hepatocellular carcinoma: The present and the future.

Hepatocellular carcinoma (HCC) is the most common primary tumor of the liver. Its relationship to chronic liver diseases, in particular cirrhosis, develops on a background of viral hepatitis, excessive alcohol intake or metabolic steatohepatitis, leads to a high incidence and prevalence of this neoplasia worldwide. Despite the spread of HCC, its treatment it's still a hard challenge, due to high rate of late diagnosis and to lack of therapeutic options for advanced disease. In fact radical surgery and liver transplantation, the most radical therapeutic approaches, are indicated only in case of early diagnosis. Even local therapies, such as transarterial chemoembolization, find limited indications, leading to an important problem regarding treatment of advanced disease. In this situation, until terminal HCC occurs, systemic therapy is the only possible approach, with sorafenib as the only standard treatment available. Anyway, the efficacy of this drug is limited and many efforts are necessary to understand who could benefit more with this treatment. Therefore, other molecules for a targeted therapy were evaluated, but only regorafenib showed promising results. Beside molecular target therapy, also cytotoxic drugs, in particular oxaliplatin- and gemcitabine-based regimens, and immune-checkpoint inhibitors were tested with interesting results. The future of the treatment of this neoplasia is linked to our ability to understand its mechanisms of resistance and to find novel therapeutic targets, with the objective to purpose individualized approaches to patients affected by advanced HCC.