Metabolic reprogramming in Nrf2-driven proliferation of normal rat hepatocytes.

BACKGROUND AND AIMS: Cancer cells reprogram their metabolic pathways to support bioenergetic and biosynthetic needs and to maintain their redox balance. In several human tumors, the Keap1-Nrf2 system controls proliferation and metabolic reprogramming by regulating the pentose phosphate pathway (PPP). However, whether this metabolic reprogramming also occurs in normal proliferating cells is unclear. APPROACH AND RESULTS: To define the metabolic phenotype in normal proliferating hepatocytes, we induced cell proliferation in the liver by 3 distinct stimuli: liver regeneration by partial hepatectomy and hepatic hyperplasia induced by 2 direct mitogens: lead nitrate (LN) or triiodothyronine. Following LN treatment, well-established features of cancer metabolic reprogramming, including enhanced glycolysis, oxidative PPP, nucleic acid synthesis, NAD + /NADH synthesis, and altered amino acid content, as well as downregulated oxidative phosphorylation, occurred in normal proliferating hepatocytes displaying Nrf2 activation. Genetic deletion of Nrf2 blunted LN-induced PPP activation and suppressed hepatocyte proliferation. Moreover, Nrf2 activation and following metabolic reprogramming did not occur when hepatocyte proliferation was induced by partial hepatectomy or triiodothyronine. CONCLUSIONS: Many metabolic changes in cancer cells are shared by proliferating normal hepatocytes in response to a hostile environment. Nrf2 activation is essential for bridging metabolic changes with crucial components of cancer metabolic reprogramming, including the activation of oxidative PPP. Our study demonstrates that matured hepatocytes exposed to LN undergo cancer-like metabolic reprogramming and offers a rapid and useful in vivo model to study the molecular alterations underpinning the differences/similarities of metabolic changes in normal and neoplastic hepatocytes.

Biological and targeting differences between the rare KRAS A146T and canonical KRAS mutants in gastric cancer models.

BACKGROUND: Gastric cancer (GC) is the third leading cause of cancer-related death worldwide, with a poor prognosis for patients with advanced disease. Since the oncogenic role of KRAS mutants has been poorly investigated in GC, this study aims to biochemically and biologically characterize different KRAS-mutated models and unravel differences among KRAS mutants in response to therapy. METHODS: Taking advantage of a proprietary, molecularly annotated platform of more than 200 GC PDXs (patient-derived xenografts), we identified KRAS-mutated PDXs, from which primary cell lines were established. The different mutants were challenged with KRAS downstream inhibitors in in vitro and in vivo experiments. RESULTS: Cells expressing the rare KRAS A146T mutant showed lower RAS-GTP levels compared to those bearing the canonical G12/13D mutations. Nevertheless, all the KRAS-mutated cells displayed KRAS addiction. Surprisingly, even if the GEF SOS1 is considered critical for the activation of KRAS A146T mutants, its abrogation did not significantly affect cell viability. From the pharmacologic point of view, Trametinib monotherapy was more effective in A146T than in G12D-mutated models, suggesting a vulnerability to MEK inhibition. However, in the presence of mutations in the PI3K pathway, more frequently co-occurrent in A146T models, the association of Trametinib and the AKT inhibitor MK-2206 was required to optimize the response. CONCLUSION: A deeper genomic and biological characterization of KRAS mutants might sustain the development of more efficient and long-lasting therapeutic options for patients harbouring KRAS-driven GC.

The iBuddy experience: A digital simulation-based approach to enhance secondary school students' privacy awareness.

Privacy is a central issue in the digitalization of society and directly concerns all Internet users. Privacy education is part of the picture of a more just digital society: it aims at making users more aware of the importance of their data and of the technical and financial tools and processes that involve their personal data. Nonetheless, privacy education is confronted with a paradox: while people perceive the importance of privacy, they seldom take action to actually protect their personal data. iBuddy is a narrative simulation-based session inspired by research evidence about the privacy paradox and aims to (a) enhancing awareness and (b) promoting the uptake of privacy-safe behaviors for secondary and higher students (age range 11-20). The paper presents the design and development of the simulation and of the following modular debriefing, as a case study in evidence-based collaborative instructional design and in the instructional used of digital technology. The evaluation of iBuddy, which combined a post-session satisfaction and perceived learning survey (N = 978) and a follow-up survey (N = 124), provides insights in the novel domain of privacy education. Results suggests that iBuddy sessions are engaging, effective and conducive to medium-term behavioral change, thus indirectly confirming the design assumptions about how to tackle the privacy paradox through a simulation-based approach.

Reflux conditions induce E-cadherin cleavage and EMT via APE1 redox function in oesophageal adenocarcinoma.

OBJECTIVE: Chronic gastro-oesophageal reflux disease, where acidic bile salts (ABS) reflux into the oesophagus, is the leading risk factor for oesophageal adenocarcinoma (EAC). We investigated the role of ABS in promoting epithelial-mesenchymal transition (EMT) in EAC. DESIGN: RNA sequencing data and public databases were analysed for the EMT pathway enrichment and patients' relapse-free survival. Cell models, pL2-IL1ß transgenic mice, deidentified EAC patients' derived xenografts (PDXs) and tissues were used to investigate EMT in EAC. RESULTS: Analysis of public databases and RNA-sequencing data demonstrated significant enrichment and activation of EMT signalling in EAC. ABS induced multiple characteristics of the EMT process, such as downregulation of E-cadherin, upregulation of vimentin and activation of ß-catenin signalling and EMT-transcription factors. These were associated with morphological changes and enhancement of cell migration and invasion capabilities. Mechanistically, ABS induced E-cadherin cleavage via an MMP14-dependent proteolytic cascade. Apurinic/apyrimidinic endonuclease (APE1), also known as redox factor 1, is an essential multifunctional protein. APE1 silencing, or its redox-specific inhibitor (E3330), downregulated MMP14 and abrogated the ABS-induced EMT. APE1 and MMP14 coexpression levels were inversely correlated with E-cadherin expression in human EAC tissues and the squamocolumnar junctions of the L2-IL1ß transgenic mouse model of EAC. EAC patients with APE1high and EMThigh signatures had worse relapse-free survival than those with low levels. In addition, treatment of PDXs with E3330 restrained EMT characteristics and suppressed tumour invasion. CONCLUSION: Reflux conditions promote EMT via APE1 redox-dependent E-cadherin cleavage. APE1-redox function inhibitors can have a therapeutic role in EAC.

FGFR2 fusion proteins drive oncogenic transformation of mouse liver organoids towards cholangiocarcinoma.

BACKGROUND & AIMS: About 15% of intrahepatic cholangiocarcinomas (iCCAs) express fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs), usually alongside mutational inactivation of TP53, CDKN2A or BAP1. In FFs, FGFR2 residues 1-768 fuse to sequences encoded by a diverse array of partner genes (>60) causing oncogenic FF activation. While FGFR-specific tyrosine kinase inhibitors (F-TKI) provide clinical benefit in FF+ iCCA, responses are partial and/or limited by resistance mechanisms, such as the V565F substitution in the FGFR2 gatekeeper residue. Improving on FF targeting in iCCA therefore remains a critical unmet need. Herein, we aimed to generate a murine model of FF-driven iCCA and use this to uncover actionable FF-associated dependencies. METHODS: Four iCCA FFs carrying different fusion sequences were expressed in Tp53-/- mouse liver organoids. Tumorigenic properties of genetically modified liver organoids were assessed by transplantation into immuno-deficient mice. Cellular models derived from neoplastic lesions were exploited for pre-clinical studies. RESULTS: Transplantation of FF-expressing liver organoids yielded tumors diagnosed as CCA based on histological, phenotypic and transcriptomic analyses. The penetrance of this tumorigenic phenotype was influenced by FF identity. Tumor organoids and 2D cell lines derived from CCA lesions were addicted to FF signaling via Ras-Erk, regardless of FF identity or V565F mutation. Dual blockade of FF and the Ras-Erk pathway by concomitant pharmacological inhibition of FFs and Mek1/2 provided greater therapeutic efficacy than single agent F-TKI in vitro and in vivo. CONCLUSIONS: FF-driven iCCA pathogenesis was successfully modeled on a Tp53-/- murine background, revealing biological heterogeneity among structurally different FFs. Double blockade of FF-ERK signaling deserves consideration for precision-based approaches against human FF+ iCCA. LAY SUMMARY: Intrahepatic cholangiocarcinoma (iCCA) is a rare cancer that is difficult to treat. A subtype of iCCA is caused by genomic alterations that generate oncogenic drivers known as FGFR2 fusions. Patients with FGFR2 fusions respond to FGFR inhibitors, but clinical responses are often of modest duration. We used animal and cellular models to show that FGFR2 fusions require the activity of a downstream effector named Mek1/2. We found that dual blockade of FGFR2 fusions and Mek1/2 was more effective than isolated inhibition of FGFR2 fusions, pointing to the potential clinical utility of dual FGFR2-MEK1/2 blockade in patients with iCCA.

Personalized therapeutic strategies in HER2-driven gastric cancer.

BACKGROUND: Trastuzumab is the only approved targeted therapy in patients with HER2-amplified metastatic gastric cancer (GC). Regrettably, in clinical practice, only a fraction of them achieves long-term benefit from trastuzumab-based upfront strategy. To advance precision oncology, we investigated the therapeutic efficacy of different HER2-targeted strategies, in HER2 "hyper"-amplified (≥ 8 copies) tumors. METHODS: We undertook a prospective evaluation of HER2 targeting with monoclonal antibodies, tyrosine kinase inhibitors and antibody-drug conjugates, in a selected subgroup of HER2 "hyper"-amplified gastric patient-derived xenografts (PDXs), through the design of ad hoc preclinical trials. RESULTS: Despite the high level of HER2 amplification, trastuzumab elicited a partial response only in 2 out of 8 PDX models. The dual-HER2 blockade with trastuzumab plus either pertuzumab or lapatinib led to complete and durable responses in 5 (62.5%) out of 8 models, including one tumor bearing a concomitant HER2 mutation. In a resistant PDX harboring KRAS amplification, the novel antibody-drug conjugate trastuzumab deruxtecan (but not trastuzumab emtansine) overcame KRAS-mediated resistance. We also identified a HGF-mediated non-cell-autonomous mechanism of secondary resistance to anti-HER2 drugs, responsive to MET co-targeting. CONCLUSION: These preclinical randomized trials clearly indicate that in HER2-driven gastric tumors, a boosted HER2 therapeutic blockade is required for optimal efficacy, leading to complete and durable responses in most of the cases. Our results suggest that a selected subpopulation of HER2-"hyper"-amplified GC patients could strongly benefit from this strategy. Despite the negative results of clinical trials, the dual blockade should be reconsidered for patients with clearly HER2-addicted cancers.

Thyroid hormone inhibits hepatocellular carcinoma progression via induction of differentiation and metabolic reprogramming.

BACKGROUND & AIMS: Only limited therapeutic options are currently available for hepatocellular carcinoma (HCC), making the development of effective alternatives essential. Based on the recent finding that systemic or local hypothyroidism is associated with HCC development in humans and rodents, we investigated whether the thyroid hormone triiodothyronine (T3) could inhibit the progression of HCCs. METHODS: Different rat and mouse models of hepatocarcinogenesis were investigated. The effect of T3 on tumorigenesis and metabolism/differentiation was evaluated by transcriptomic analysis, quantitative reverse transcription PCR, immunohistochemistry, and enzymatic assay. RESULTS: A short treatment with T3 caused a shift in the global expression profile of the most aggressive preneoplastic nodules towards that of normal liver. This genomic reprogramming preceded the disappearance of nodules and involved reprogramming of metabolic genes, as well as pro-differentiating transcription factors, including Kruppel-like factor 9, a target of the thyroid hormone receptor ß (TRß). Treatment of HCC-bearing rats with T3 strongly reduced the number and burden of HCCs. Reactivation of a local T3/TRß axis, a switch from Warburg to oxidative metabolism and loss of markers of poorly differentiated hepatocytes accompanied the reduced burden of HCC. This effect persisted 1 month after T3 withdrawal, suggesting a long-lasting effect of the hormone. The antitumorigenic effect of T3 was further supported by its inhibitory activity on cell growth and the tumorigenic ability of human HCC cell lines. CONCLUSIONS: Collectively, these findings suggest that reactivation of the T3/TRß axis induces differentiation of neoplastic cells towards a more benign phenotype and that T3 or its analogs, particularly agonists of TRß, could be useful tools in HCC therapy. LAY SUMMARY: Hepatocellular carcinoma (HCC) represents an important challenge for global health. Recent findings showed that systemic or local hypothyroidism is associated with HCC development. In rat models, we showed that administration of the thyroid hormone T3 impaired HCC progression, even when given at late stages. This is relevant from a translational point of view as HCC is often diagnosed at an advanced stage when it is no longer amenable to curative treatments. Thyroid hormones and/or thyromimetics could be useful for the treatment of patients with HCC.

Rapid automated diagnosis of primary hepatic tumour by mass spectrometry and artificial intelligence.

BACKGROUND AND AIMS: Complete surgical resection with negative margin is one of the pillars in treatment of liver tumours. However, current techniques for intra-operative assessment of tumour resection margins are time-consuming and empirical. Mass spectrometry (MS) combined with artificial intelligence (AI) is useful for classifying tissues and provides valuable prognostic information. The aim of this study was to develop a MS-based system for rapid and objective liver cancer identification and classification. METHODS: A large dataset derived from 222 patients with hepatocellular carcinoma (HCC, 117 tumours and 105 non-tumours) and 96 patients with mass-forming cholangiocarcinoma (MFCCC, 50 tumours and 46 non-tumours) were analysed by Probe Electrospray Ionization (PESI) MS. AI by means of support vector machine (SVM) and random forest (RF) algorithms was employed. For each classifier, sensitivity, specificity and accuracy were calculated. RESULTS: The overall diagnostic accuracy exceeded 94% in both the AI algorithms. For identification of HCC vs non-tumour tissue, RF was the best, with 98.2% accuracy, 97.4% sensitivity and 99% specificity. For MFCCC vs non-tumour tissue, both algorithms gave 99.0% accuracy, 98% sensitivity and 100% specificity. CONCLUSIONS: The herein reported MS-based system, combined with AI, permits liver cancer identification with high accuracy. Its bench-top size, minimal sample preparation and short working time are the main advantages. From diagnostics to therapeutics, it has the potential to influence the decision-making process in real-time with the ultimate aim of improving cancer patient cure.

Colorectal cancer early methylation alterations affect the crosstalk between cell and surrounding environment, tracing a biomarker signature specific for this tumor.

Colorectal cancer (CRC) develops through the accumulation of both genetic and epigenetic alterations. However, while the former are already used as prognostic and predictive biomarkers, the latter are less well characterized. Here, performing global methylation analysis on both CRCs and adenomas by Illumina Infinium HumanMethylation450 Bead Chips, we identified a panel of 74 altered CpG islands, demonstrating that the earliest methylation alterations affect genes coding for proteins involved in the crosstalk between cell and surrounding environment. The panel discriminates CRCs and adenomas from peritumoral and normal mucosa with very high specificity (100%) and sensitivity (99.9%). Interestingly, over 70% of the hypermethylated islands resulted in downregulation of gene expression. To establish the possible usefulness of these non-invasive markers for detection of colon cancer, we selected three biomarkers and identified the presence of altered methylation in stool DNA and plasma cell-free circulating DNA from CRC patients.

Drug-Homogeneity Index in Mass-Spectrometry Imaging.

Enhancing drug penetration in solid tumors is an interesting clinical issue of considerable importance. In preclinical research, mass-spectrometry imaging is a promising technique for visualizing drug distribution in tumors under different treatment conditions and its application in this field is rapidly increasing. However, in view of the huge variability among MSI data sets, drug homogeneity is usually manually assessed by an expert, and this approach is biased by interobserver variability and lacks reproducibility. We propose a new texture-based feature, the drug-homogeneity index (DHI), which provides an objective, automated measure of drug homogeneity in MSI data. A simulation study on synthetic data sets showed that previously known texture features do not give an accurate picture of intratumor drug-distribution patterns and are easily influenced by the tumor-tissue morphology. The DHI has been used to study the distribution profile of the anticancer drug paclitaxel in various xenograft models, which were either pretreated or not pretreated with antiangiogenesis compounds. The conclusion is that drug homogeneity is better in the pretreated condition, which is in agreement with previous experimental findings published by our group. This study shows that DHI could be useful in preclinical studies as a new parameter for the evaluation of protocols for better drug penetration.

The metabolic gene HAO2 is downregulated in hepatocellular carcinoma and predicts metastasis and poor survival.

BACKGROUND & AIMS: l-2-Hydroxy acid oxidases are flavin mononucleotide-dependent peroxisomal enzymes, responsible for the oxidation of l-2-hydroxy acids to ketoacids, resulting in the formation of hydrogen peroxide. We investigated the role of HAO2, a member of this family, in rat, mouse and human hepatocarcinogenesis. METHODS: We evaluated Hao2 expression by qRT-PCR in the following rodent models of hepatocarcinogenesis: the Resistant-Hepatocyte, the CMD and the chronic DENA rat models, and the TCPOBOP/DENA and TCPOBOP only mouse models. Microarray and qRT-PCR analyses were performed on two cohorts of human hepatocellular carcinoma (HCC) patients. Rat HCC cells were transduced by a Hao2 encoding lentiviral vector and grafted in mice. RESULTS: Downregulation of Hao2 was observed in all investigated rodent models of hepatocarcinogenesis. Interestingly, Hao2 mRNA levels were also profoundly downregulated in early preneoplastic lesions. Moreover, HAO2 mRNA levels were strongly downregulated in two distinct series of human HCCs, when compared to both normal and cirrhotic peri-tumoral liver. HAO2 levels were inversely correlated with grading, overall survival and metastatic ability. Finally, exogenous expression of Hao2 in rat cells impaired their tumorigenic ability. CONCLUSION: Our work identifies for the first time the oncosuppressive role of the metabolic gene Hao2. Indeed, its expression is severely decreased in HCC of different species and etiology, and its reintroduction in HCC cells profoundly impairs tumorigenesis. We also demonstrate that dysregulation of HAO2 is a very early event in the development of HCC and it may represent a useful diagnostic and prognostic marker for human HCC.

Local hypothyroidism favors the progression of preneoplastic lesions to hepatocellular carcinoma in rats.

UNLABELLED: Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that mediate most of the effects elicited by the thyroid hormone, 3,5,3'-L-triiodothyronine (T3). TRs have been implicated in tumorigenesis, although it is unclear whether they act as oncogenes or tumor suppressors, and at which stage of tumorigenesis their dysregulation occurs. Using the resistant-hepatocyte rat model (R-H model), we found down-regulation of TRß1 and TRα1 and their target genes in early preneoplastic lesions and hepatocellular carcinoma (HCCs), suggesting that a hypothyroid status favors the onset and progression of preneoplastic lesions to HCC. Notably, TRß1 and, to a lesser extent, TRα1 down-regulation was observed only in preneoplastic lesions positive for the progenitor cell marker, cytokeratin-19 (Krt-19) and characterized by a higher proliferative activity, compared to the Krt-19 negative ones. TRß1 down-regulation was observed also in the vast majority of the analyzed human HCCs, compared to the matched peritumorous liver or to normal liver. Hyperthyroidism induced by T3 treatment caused up-regulation of TRß1 and of its target genes in Krt-19(+) preneoplastic rat lesions and was associated with nodule regression. In HCC, TRß1 down-regulation was not the result of hypermethylation of its promoter, but was associated with an increased expression of TRß1-targeting microRNAs ([miR]-27a, -181a, and -204). An inverse correlation between TRß1 and miR-181a was also found in human cirrhotic peritumoral tissue, compared to normal liver. CONCLUSION: Down-regulation of TRs, especially TRß1, is an early and relevant event in liver cancer development and is species and etiology independent. The results also suggest that a hypothyroid status of preneoplastic lesions may contribute to their progression to HCC and that the reversion of this condition may represent a possible therapeutic goal to interfere with the development of this tumor.

Nrf2, but not ß-catenin, mutation represents an early event in rat hepatocarcinogenesis.

UNLABELLED: Hepatocellular carcinoma (HCC) develops through a multistage process, but the nature of the molecular changes associated with the different steps, the very early ones in particular, is largely unknown. Recently, dysregulation of the NRF2/KEAP1 pathway and mutations of these genes have been observed in experimental and human tumors, suggesting their possible role in cancer development. To assess whether Nrf2/Keap1 mutations are early or late events in HCC development, we investigated their frequency in the rat Resistant Hepatocyte model, consisting of the administration of diethylnitrosamine followed by a brief exposure to 2-acetylaminofluorene. This model enables the dissection of all stages of hepatocarcinogenesis. We found that Nrf2/Keap1 mutations were present in 71% of early preneoplastic lesions and in 78.6% and 59.3% of early and advanced HCCs, respectively. Mutations of Nrf2 were more frequent, missense, and located in the Nrf2-Keap1 binding region. Mutations of Keap1 occurred at a much lower frequency in both preneoplastic lesions and HCCs and were mutually exclusive with those of Nrf2. Functional in vitro and in vivo studies showed that Nrf2 silencing inhibited the ability of tumorigenic rat cells to grow in soft agar and to form tumors. Unlike Nrf2 mutations, those of Ctnnb1, which are frequent in human HCC, were a later event as they appeared only in fully advanced HCCs (18.5%). CONCLUSION: In the Resistant Hepatocyte model of hepatocarcinogenesis the onset of Nrf2 mutations is a very early event, likely essential for the clonal expansion of preneoplastic hepatocytes to HCC, while Ctnnb1 mutations occur only at very late stages. Moreover, functional experiments demonstrate that Nrf2 is an oncogene critical for HCC progression and development.

MicroRNA/gene profiling unveils early molecular changes and nuclear factor erythroid related factor 2 (NRF2) activation in a rat model recapitulating human hepatocellular carcinoma (HCC).

UNLABELLED: Studies on gene and/or microRNA (miRNA) dysregulation in the early stages of hepatocarcinogenesis are hampered by the difficulty of diagnosing early lesions in humans. Experimental models recapitulating human hepatocellular carcinoma (HCC) are then used to perform this analysis. We performed miRNA and gene expression profiling to characterize the molecular events involved in the multistep process of hepatocarcinogenesis in the resistant-hepatocyte rat model. A high percentage of dysregulated miRNAs/genes in HCC were similarly altered in early preneoplastic lesions positive for the stem/progenitor cell marker cytokeratin-19, indicating that several HCC-associated alterations occur from the very beginning of the carcinogenic process. Our analysis also identified miRNA/gene-target networks aberrantly activated at the initial stage of hepatocarcinogenesis. Activation of the nuclear factor erythroid related factor 2 (NRF2) pathway and up-regulation of the miR-200 family were among the most prominent changes. The relevance of these alterations in the development of HCC was confirmed by the observation that NRF2 silencing impaired while miR-200a overexpression promoted HCC cell proliferation in vitro. Moreover, T3-induced in vivo inhibition of the NRF2 pathway accompanied the regression of cytokeratin-19-positive nodules, suggesting that activation of this transcription factor contributes to the onset and progression of preneoplastic lesions towards malignancy. The finding that 78% of genes and 57% of dysregulated miRNAs in rat HCC have been previously associated with human HCC as well underlines the translational value of our results. CONCLUSION: This study indicates that most of the molecular changes found in HCC occur in the very early stages of hepatocarcinogenesis. Among these, the NRF2 pathway plays a relevant role and may represent a new therapeutic target.

Presarcopenia and sarcopenia in hip-fracture women: prevalence and association with ability to function in activities of daily living.

BACKGROUND AND AIMS: Sarcopenia staging systems have been proposed, but little is known on their application in hip-fracture patients. Our aim was to assess the prevalence of presarcopenia and sarcopenia and their association with ability to function in activities of daily living in hip-fracture women. METHODS: We investigated white women (N = 138 of 149) who were consecutively admitted to a rehabilitation hospital because of their first hip fracture. In each woman, we measured appendicular lean mass (aLM) by dual-energy X-ray absorptiometry, at a median of 18 days after hip fracture occurrence. On the same day, we assessed grip strength with a handheld dynamometer. Functional autonomy was assessed by the Barthel Index. We used the European Working Group on Sarcopenia in Older People (EWGSOP) definition to calculate the prevalence of presarcopenia and sarcopenia, taking into account both aLM/height(2) and handgrip strength. Gait speed was not considered, because of the recent hip fracture. RESULTS: Twenty-three (17 %) of the 138 women fulfilled the diagnostic criteria for presarcopenia whereas 80 (58 %) were sarcopenic. The women with presarcopenia were younger, healthier and with higher Barthel Index scores (median 65 vs. 55; interquartile range 60-75 and 50-60, respectively; p < 0.001) than those with sarcopenia. Significant differences in Barthel Index scores at the time of assessment (but not at the end of the rehabilitation course) persisted after multiple adjustments (p < 0.001). CONCLUSIONS: The prevalence of presarcopenia and sarcopenia was high in hip-fracture women. Presarcopenic women had higher ability to function in activities of daily living than sarcopenic women.

Shedding-generated Met receptor fragments can be routed to either the proteasomal or the lysosomal degradation pathway.

The receptor tyrosine kinase Met and its ligand, the hepatocyte growth factor/scatter factor, are essential for embryonic development, whereas deregulation of Met signaling pathways is associated with tumorigenesis and metastasis. The presenilin-regulated intramembrane proteolysis (PS-RIP) is involved in ligand-independent downregulation of Met. This proteolytic process involves shedding of the Met extracellular domain followed by γ-secretase cleavage, generating labile intracellular fragments degraded by the proteasome. We demonstrate here that upon shedding both generated Met N- and C-terminal fragments are degraded directly in the lysosome, with C-terminal fragments escaping γ-secretase cleavage. PS-RIP and lysosomal degradation are complementary, because their simultaneous inhibition induces synergistic accumulation of fragments. Met N-terminal fragments associate with the high-affinity domain of HGF/SF, confirming its decoy activity which could be reduced through their routing to the lysosome at the expense of extracellular release. Finally, the DN30 monoclonal antibody inducing Met shedding promotes receptor degradation through induction of both PS-RIP and the lysosomal pathway. Thus, we demonstrate that Met shedding initiates a novel lysosomal degradation which participates to ligand-independent downregulation of the receptor.

Met as a therapeutic target in HCC: facts and hopes.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide, and its burden is expected to increase further in the next years. In spite of the advances of classical therapies, such as surgery, transplantation, use of radiofrequency and transarterial embolization, the prognosis of this neoplasm has not considerably improved over the past few years. The advent of targeted therapies and the approval of the systemic treatment of advanced HCC with the kinase inhibitor sorafenib have provided some hope for the future. Even if the molecular mechanisms responsible for the onset and progression of HCC are still largely unknown, new therapeutic targets have recently come to the spotlight. One of these targets is the tyrosine kinase receptor for the Hepatocyte Growth Factor, encoded by the MET gene, known to promote tumor growth and metastasis in many human organs. In this review we will summarize the contrasting results obtained in vitro (in HCC cell lines) and in animal experimental models and we will also try to analyze the reasons for the opposite findings, suggesting that the HGF/MET axis can have either a promoting or a suppressive role in the development of HCC. We will also reconsider the evidence of activation of this pathway in human HCCs and discuss the results of the clinical trials performed with MET inhibitors. The final purpose is to better clarify which can be the role of MET as a therapeutic target in HCC.

YAP activation is an early event and a potential therapeutic target in liver cancer development.

BACKGROUND & AIMS: Although the growth suppressing Hippo pathway has been implicated in hepatocellular carcinoma (HCC) pathogenesis, it is unknown at which stage of hepatocarcinogenesis its dysregulation occurs. We investigated in rat and human preneoplastic lesions whether overexpression of the transcriptional co-activator Yes-associated protein (YAP) is an early event. METHODS: The experimental model used is the resistant-hepatocyte (R-H) rat model. Gene expression was determined by qRT-PCR or immunohistochemistry. Forward genetic experiments were performed in human HCC cells and in murine oval cells. RESULTS: All foci of preneoplastic hepatocytes, generated in rats 4weeks after diethylnitrosamine (DENA) treatment, displayed YAP accumulation. This was associated with down-regulation of the ß-TRCP ligase, known to mediate YAP degradation, and of microRNA-375, targeting YAP. YAP accumulation was paralleled by the up-regulation of its target genes. Increased YAP expression was also observed in human early dysplastic nodules and adenomas. Animal treatment with verteporfin (VP), which disrupts the formation of the YAP-TEAD complex, significantly reduced preneoplastic foci and oval cell proliferation. In vitro experiments confirmed that VP-mediated YAP inhibition impaired cell growth in HCC and oval cells; notably, oval cell transduction with wild type or active YAP conferred tumorigenic properties in vitro and in vivo. CONCLUSIONS: These results suggest that (i) YAP overexpression is an early event in rat and human liver tumourigenesis; (ii) it is critical for the clonal expansion of carcinogen-initiated hepatocytes and oval cells, and (iii) VP-induced disruption of the YAP-TEAD interaction may provide an important approach for the treatment of YAP-overexpressing cancers.

MicroRNAs: new tools for diagnosis, prognosis, and therapy in hepatocellular carcinoma?

MicroRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs involved in the regulation of gene expression and protein translation. Many studies have shown that they play a crucial role in driving organ and tissue differentiation during embryogenesis and in the fine-tuning of fundamental biological processes, such as proliferation and apoptosis. Growing evidence indicates that their deregulation plays an important role in cancer onset and progression as well, where they act as oncogenes or oncosuppressors. In this review, we highlight the most recent findings regarding the role of miRNAs in hepatocellular carcinoma (HCC) by analyzing the possible mechanisms by which they contribute to this neoplasm. Moreover, we discuss the possible role of circulating miRNAs as biomarkers, a field that needs urgent improvement in the clinical surveillance of HCC, and the fascinating possibility of using them as therapeutic targets or drugs themselves.