Phase I Trial of Viral Vector-Based Personalized Vaccination Elicits Robust Neoantigen-Specific Antitumor T-Cell Responses.

PURPOSE: Personalized vaccines targeting multiple neoantigens (nAgs) are a promising strategy for eliciting a diversified antitumor T-cell response to overcome tumor heterogeneity. NOUS-PEV is a vector-based personalized vaccine, expressing 60 nAgs and consists of priming with a nonhuman Great Ape Adenoviral vector (GAd20) followed by boosts with Modified Vaccinia Ankara. Here, we report data of a phase Ib trial of NOUS-PEV in combination with pembrolizumab in treatment-naïve patients with metastatic melanoma (NCT04990479). PATIENTS AND METHODS: The feasibility of this approach was demonstrated by producing, releasing, and administering to 6 patients 11 of 12 vaccines within 8 weeks from biopsy collection to GAd20 administration. RESULTS: The regimen was safe, with no treatment-related serious adverse events observed and mild vaccine-related reactions. Vaccine immunogenicity was demonstrated in all evaluable patients receiving the prime/boost regimen, with detection of robust neoantigen-specific immune responses to multiple neoantigens comprising both CD4 and CD8 T cells. Expansion and diversification of vaccine-induced T-cell receptor (TCR) clonotypes was observed in the posttreatment biopsies of patients with clinical response, providing evidence of tumor infiltration by vaccine-induced neoantigen-specific T cells. CONCLUSIONS: These findings indicate the ability of NOUS-PEV to amplify and broaden the repertoire of tumor-reactive T cells to empower a diverse, potent, and durable antitumor immune response. Finally, a gene signature indicative of the reduced presence of activated T cells together with very poor expression of the antigen-processing machinery genes has been identified in pretreatment biopsies as a potential biomarker of resistance to the treatment.

Tumor Burden Dictates the Neoantigen Features Required to Generate an Effective Cancer Vaccine.

Tumor neoantigens (nAg) represent a promising target for cancer immunotherapy. The identification of nAgs that can generate T-cell responses and have therapeutic activity has been challenging. Here, we sought to unravel the features of nAgs required to induce tumor rejection. We selected clinically validated Great Ape-derived adenoviral vectors (GAd) as a nAg delivery system for differing numbers and combinations of nAgs. We assessed their immunogenicity and efficacy in murine models of low to high disease burden, comparing multi-epitope versus mono-epitope vaccines. We demonstrated that the breadth of immune response is critical for vaccine efficacy and having multiple immunogenic nAgs encoded in a single vaccine improves efficacy. The contribution of each single neoantigen was examined, leading to the identification of 2 nAgs able to induce CD8+ T cell-mediated tumor rejection. They were both active as individual nAgs in a setting of prophylactic vaccination, although to different extents. However, the efficacy of these single nAgs was lost in a setting of therapeutic vaccination in tumor-bearing mice. The presence of CD4+ T-cell help restored the efficacy for only the most expressed of the two nAgs, demonstrating a key role for CD4+ T cells in sustaining CD8+ T-cell responses and the necessity of an efficient recognition of the targeted epitopes on cancer cells by CD8+ T cells for an effective antitumor response. This study provides insight into understanding the determinants of nAgs relevant for effective treatment and highlights features that could contribute to more effective antitumor vaccines. See related Spotlight by Slingluff Jr, p. 382.

The Past and Future of Inflammation as a Target to Cancer Prevention.

Inflammation is an essential defense mechanism in which innate immune cells are coordinately activated on encounter of harmful stimuli, including pathogens, tissue injury, and toxic compounds and metabolites to neutralize and eliminate the instigator and initiate healing and regeneration. Properly terminated inflammation is vital to health, but uncontrolled runaway inflammation that becomes chronic begets a variety of inflammatory and metabolic diseases and increases cancer risk. Making damaged tissues behave as "wounds that do not heal" and sustaining the production of growth factors whose physiologic function is tissue healing, chronic inflammation accelerates cancer emergence from premalignant lesions. In 1863, Rudolf Virchow, a leading German pathologist, suggested a possible association between inflammation and tumor formation, but it took another 140 years to fully elucidate and appreciate the tumorigenic role of inflammation. Key findings outlined molecular events in the inflammatory cascade that promote cancer onset and progression and enabled a better appreciation of when and where inflammation should be inhibited. These efforts triggered ongoing research work to discover and develop inflammation-reducing chemopreventive strategies for decreasing cancer risk and incidence.

α-Tocopherol Protects Lipopolysaccharide-Activated BV2 Microglia.

Microglia, the resident macrophage-like population in the central nervous system, play a crucial role in the pathogenesis of many neurodegenerative disorders by triggering an inflammatory response that leads to neuronal death. Neuroprotective compounds to treat or prevent neurodegenerative diseases are a new field of study in modern medicine. Microglia are activated in response to inflammatory stimuli. The pathogenesis of various neurodegenerative diseases is closely related to the constant activation of microglia due to their fundamental role as a mediator of inflammation in the brain environment. α-Tocopherol, also known as vitamin E, is reported to possess potent neuroprotective effects. The goal of this study was to investigate the biological effects of vitamin E on BV2 microglial cells, as a possible neuroprotective and anti-inflammatory agent, following stimulation with lipopolysaccharide (LPS). The results showed that the pre-incubation of microglia with α-tocopherol can guarantee neuroprotective effects during microglial activation induced by LPS. α-Tocopherol preserved the branched morphology typical of microglia in a physiological state. It also reduced the migratory capacity; the production of pro-inflammatory and anti-inflammatory cytokines such as TNF-α and IL-10; and the activation of receptors such as TRL4 and CD40, which modulate the PI3K-Akt signaling pathway. The results of this study require further insights and research, but they present new scenarios for the application of vitamin E as an antioxidant for the purpose of greater neuroprotection in vivo for the prevention of possible neurodegenerative diseases.

CAR-T Therapy for Pediatric High-Grade Gliomas: Peculiarities, Current Investigations and Future Strategies.

High-Grade Gliomas (HGG) are among the deadliest malignant tumors of central nervous system (CNS) in pediatrics. Despite aggressive multimodal treatment - including surgical resection, radiotherapy and chemotherapy - long-term prognosis of patients remains dismal with a 5-year survival rate less than 20%. Increased understanding of genetic and epigenetic features of pediatric HGGs (pHGGs) revealed important differences with adult gliomas, which need to be considered in order to identify innovative and more effective therapeutic approaches. Immunotherapy is based on different techniques aimed to redirect the patient own immune system to fight specifically cancer cells. In particular, T-lymphocytes can be genetically modified to express chimeric proteins, known as chimeric antigen receptors (CARs), targeting selected tumor-associated antigens (TAA). Disialoganglioside GD2 (GD-2) and B7-H3 are highly expressed on pHGGs and have been evaluated as possible targets in pediatric clinical trials, in addition to the antigens common to adult glioblastoma - such as interleukin-13 receptor alpha 2 (IL-13α2), human epidermal growth factor receptor 2 (HER-2) and erythropoietin-producing human hepatocellular carcinoma A2 receptor (EphA2). CAR-T therapy has shown promise in preclinical model of pHGGs but failed to achieve the same success obtained for hematological malignancies. Several limitations, including the immunosuppressive tumor microenvironment (TME), the heterogeneity in target antigen expression and the difficulty of accessing the tumor site, impair the efficacy of T-cells. pHGGs display an immunologically cold TME with poor T-cell infiltration and scarce immune surveillance. The secretion of immunosuppressive cytokines (TGF-ß, IL-10) and the presence of immune-suppressive cells - like tumor-associated macrophages/microglia (TAMs) and myeloid-derived suppressor cells (MDSCs) - limit the effectiveness of immune system to eradicate tumor cells. Innovative immunotherapeutic strategies are necessary to overcome these hurdles and improve ability of T-cells to eradicate tumor. In this review we describe the distinguishing features of HGGs of the pediatric population and of their TME, with a focus on the most promising CAR-T therapies overcoming these hurdles.

Periprosthetic femoral fractures in Total Hip Arthroplasty (THA): a comparison between osteosynthesis and revision in a retrospective cohort study.

BACKGROUND: Periprosthetic femoral fractures are challenging complications of hip arthroplasty. They are supposed to be a rare complication, but their incidence is rapidly increasing. Surgical treatment aims to achieve early mobilization and avoid the complications of prolonged bed rest. Aim of this study is to evaluate the clinical outcomes of surgical treatment comparing two surgical approaches: revision arthroplasty (RA) versus open reduction and internal fixation (ORIF). METHODS: Authors retrospectively reviewed a series of 117 patients with total hip arthroplasty treated for periprosthetic femur fractures in the period between January 2013 and March 2018 at a single tertiary referral center. Of these, 70 patients satisfied strict inclusion criteria. Patients were classified according to the Unified Classification System (UCS) and distributed in two groups according to surgical treatment. Clinical outcomes were assessed using the Oxford Hip recorded preoperatively and post operatively, Barthel Score, CIRS score (Cumulative illness rating scale), type of fracture and post-operative complications with a minimum follow up of 1 year. RESULTS: Nominal univariate statistical analysis revealed significant differences between the post and pre-operative Oxford Hip Score (Δ Oxford) and the surgical treatment (p = 0.008) and CIRS score (p = 0.048). Moreover, we observed a significant relationship between type of treatment and type of fracture (p = 0.0001). Multivariate analyses revealed that CIRS score was independently associated with Oxford Score improvement after surgery (p = 0.024). CONCLUSIONS: Data from this case series confirmed that surgical treatment was correlated to type of fracture, according to UCS classification. Patients treated by RA had a better functional outcome than patients treated with ORIF, but these results are strongly influenced from the patients' age, Barthel index and CIRS score. Also, authors found a correlation between functional outcome and comorbidities evaluated by CIRS score. Based on these data we suggest a multimodal approach to these patients, like those used for proximal femoral fractures.

Polymorphonuclear myeloid-derived suppressor cells impair the anti-tumor efficacy of GD2.CAR T-cells in patients with neuroblastoma.

The outcome of patients affected by high-risk or metastatic neuroblastoma (NB) remains grim, with ≥ 50% of the children experiencing relapse or progression of the disease despite multimodal, intensive treatment. In order to identify new strategies to improve the overall survival and the quality of life of these children, we recently developed and optimized a third-generation GD2-specific chimeric antigen receptor (CAR) construct, which is currently under evaluation in our Institution in a phase I/II clinical trial (NCT03373097) enrolling patients with relapsed/refractory NB. We observed that our CAR T-cells are able to induce marked tumor reduction and even achieve complete remission with a higher efficiency than that of other CAR T-cells reported in previous studies. However, often responses are not sustained and relapses occur. Here, we demonstrate for the first time a mechanism of resistance to GD2.CAR T-cell treatment, showing how polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) increase in the peripheral blood (PB) of NB patients after GD2.CAR T-cell treatment in case of relapse and loss of response. In vitro, isolated PMN-MDSC demonstrate to inhibit the anti-tumor cytotoxicity of different generations of GD2.CAR T-cells. Gene-expression profiling of GD2.CAR T-cells "conditioned" with PMN-MDSC shows downregulation of genes involved in cell activation, signal transduction, inflammation and cytokine/chemokine secretion. Analysis of NB gene-expression dataset confirms a correlation between expression of these genes and patient outcome. Moreover, in patients treated with GD2.CAR T-cells, the frequency of circulating PMN-MDSC inversely correlates with the levels of GD2.CAR T-cells, resulting more elevated in patients who did not respond or lost response to the treatment. The presence and the frequency of PMN-MDSC in PB of high-risk and metastatic NB represents a useful prognostic marker to predict the response to GD2.CAR T-cells and other adoptive immunotherapy. This study underlines the importance of further optimization of both CAR T-cells and clinical trial in order to target elements of the tumor microenvironment.

Oncolytic adenovirus and gene therapy with EphA2-BiTE for the treatment of pediatric high-grade gliomas.

BACKGROUND: Pediatric high-grade gliomas (pHGGs) are among the most common and incurable malignant neoplasms of childhood. Despite aggressive, multimodal treatment, the outcome of children with high-grade gliomas has not significantly improved over the past decades, prompting the development of innovative approaches. METHODS: To develop an effective treatment, we aimed at improving the suboptimal antitumor efficacy of oncolytic adenoviruses (OAs) by testing the combination with a gene-therapy approach using a bispecific T-cell engager (BiTE) directed towards the erythropoietin-producing human hepatocellular carcinoma A2 receptor (EphA2), conveyed by a replication-incompetent adenoviral vector (EphA2 adenovirus (EAd)). The combinatorial approach was tested in vitro, in vivo and thoroughly characterized at a molecular level. RESULTS: After confirming the relevance of EphA2 as target in pHGGs, documenting a significant correlation with worse clinical outcome of the patients, we showed that the proposed strategy provides significant EphA2-BiTE amplification and enhanced tumor cell apoptosis, on coculture with T cells. Moreover, T-cell activation through an agonistic anti-CD28 antibody further increased the activation/proliferation profiles and functional response against infected tumor cells, inducing eradication of highly resistant, primary pHGG cells. The gene-expression analysis of tumor cells and T cells, after coculture, revealed the importance of both EphA2-BiTE and costimulation in the proposed system. These in vitro observations translated into significant tumor control in vivo, in both subcutaneous and a more challenging orthotopic model. CONCLUSIONS: The combination of OA and EphA2-BiTE gene therapy strongly enhances the antitumor activity of OA, inducing the eradication of highly resistant tumor cells, thus supporting the clinical translation of the approach.

Cancer cells escape autophagy inhibition via NRF2-induced macropinocytosis.

Many cancers, including pancreatic ductal adenocarcinoma (PDAC), depend on autophagy-mediated scavenging and recycling of intracellular macromolecules, suggesting that autophagy blockade should cause tumor starvation and regression. However, until now autophagy-inhibiting monotherapies have not demonstrated potent anti-cancer activity. We now show that autophagy blockade prompts established PDAC to upregulate and utilize an alternative nutrient procurement pathway: macropinocytosis (MP) that allows tumor cells to extract nutrients from extracellular sources and use them for energy generation. The autophagy to MP switch, which may be evolutionarily conserved and not cancer cell restricted, depends on activation of transcription factor NRF2 by the autophagy adaptor p62/SQSTM1. NRF2 activation by oncogenic mutations, hypoxia, and oxidative stress also results in MP upregulation. Inhibition of MP in autophagy-compromised PDAC elicits dramatic metabolic decline and regression of transplanted and autochthonous tumors, suggesting the therapeutic promise of combining autophagy and MP inhibitors in the clinic.

Activation of NF-κB and p300/CBP potentiates cancer chemoimmunotherapy through induction of MHC-I antigen presentation.

Many cancers evade immune rejection by suppressing major histocompatibility class I (MHC-I) antigen processing and presentation (AgPP). Such cancers do not respond to immune checkpoint inhibitor therapies (ICIT) such as PD-1/PD-L1 [PD-(L)1] blockade. Certain chemotherapeutic drugs augment tumor control by PD-(L)1 inhibitors through potentiation of T-cell priming but whether and how chemotherapy enhances MHC-I-dependent cancer cell recognition by cytotoxic T cells (CTLs) is not entirely clear. We now show that the lysine acetyl transferases p300/CREB binding protein (CBP) control MHC-I AgPPM expression and neoantigen amounts in human cancers. Moreover, we found that two distinct DNA damaging drugs, the platinoid oxaliplatin and the topoisomerase inhibitor mitoxantrone, strongly up-regulate MHC-I AgPP in a manner dependent on activation of nuclear factor kappa B (NF-κB), p300/CBP, and other transcription factors, but independently of autocrine IFNγ signaling. Accordingly, NF-κB and p300 ablations prevent chemotherapy-induced MHC-I AgPP and abrogate rejection of low MHC-I-expressing tumors by reinvigorated CD8+ CTLs. Drugs like oxaliplatin and mitoxantrone may be used to overcome resistance to PD-(L)1 inhibitors in tumors that had "epigenetically down-regulated," but had not permanently lost MHC-I AgPP activity.

NRF2 as a regulator of cell metabolism and inflammation in cancer.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a master transcriptional regulator of genes whose products defend our cells for toxic and oxidative insults. Although NRF2 activation may reduce cancer risk by suppressing oxidative stress and tumor-promoting inflammation, many cancers exhibit elevated NRF2 activity either due to mutations that disrupt the negative control of NRF2 activity or other factors. Importantly, NRF2 activation is associated with poor prognosis and NRF2 has turned out to be a key activator of cancer-supportive anabolic metabolism. In this review, we summarize the diverse roles played by NRF2 in cancer focusing on metabolic reprogramming and tumor-promoting inflammation.

NRF2 activates growth factor genes and downstream AKT signaling to induce mouse and human hepatomegaly.

BACKGROUND & AIMS: Hepatomegaly can be triggered by insulin and insulin-unrelated etiologies. Insulin acts via AKT, but how other challenges cause hepatomegaly is unknown. METHODS: Since many hepatomegaly-inducing toxicants and stressors activate NRF2, we examined the effect of NRF2 activation on liver size and metabolism using a conditional allele encoding a constitutively active NRF2 variant to generate Nrf2Act-hep mice in which NRF2 is selectively activated in hepatocytes. We also used adenoviruses encoding variants of the autophagy adaptor p62/SQSTM1, which activates liver NRF2, as well as liver-specific ATG7-deficient mice (Atg7Δhep) and liver specimens from patients with hepatic sinusoidal obstruction syndrome (HSOS) and autoimmune hepatitis (AIH). RNA sequencing and cell signaling analyses were used to determine cellular consequences of NRF2 activation and diverse histological analyses were used to study effects of the different manipulations on liver and systemic pathophysiology. RESULTS: Hepatocyte-specific NRF2 activation, due to p62 accumulation or inhibition of KEAP1 binding, led to hepatomegaly associated with enhanced glycogenosis, steatosis and G2/M cell cycle arrest, fostering hyperplasia without cell division. Surprisingly, all manipulations that led to NRF2 activation also activated AKT, whose inhibition blocked NRF2-induced hepatomegaly and glycogenosis, but not NRF2-dependent antioxidant gene induction. AKT activation was linked to NRF2-mediated transcriptional induction of PDGF and EGF receptor ligands that signaled through their cognate receptors in an autocrine manner. Insulin and insulin-like growth factors were not involved. The NRF2-AKT signaling axis was also activated in human HSOS- and AIH-related hepatomegaly. CONCLUSIONS: NRF2, a transcription factor readily activated by xenobiotics, oxidative stress and autophagy disruptors, may be a common mediator of hepatomegaly; its effects on hepatic metabolism can be reversed by AKT/tyrosine kinase inhibitors. LAY SUMMARY: Hepatomegaly can be triggered by numerous etiological factors, including infections, liver cancer, metabolic disturbances, toxicant exposure, as well as alcohol abuse or drug-induced hepatitis. This study identified the oxidative stress response transcription factor NRF2 as a common mediator of hepatomegaly. NRF2 activation results in elevated expression of several growth factors. These growth factors are made by hepatocytes and activate their receptors in an autocrine fashion to stimulate the accumulation of glycogen and lipids that lead to hepatocyte and liver enlargement. The protein kinase AKT plays a key role in this process and its inhibition leads to reversal of hepatomegaly.

NF-κB-p62-NRF2 survival signaling is associated with high ROR1 expression in chronic lymphocytic leukemia.

Progression of chronic lymphocytic leukemia (CLL) and resistance to therapy are affected by tumor microenvironmental factors. One such factor is B-cell activating factor (BAFF), a cytokine that is produced mainly by nurse-like cells (NLC) and enhances CLL cells survival and modulates response to therapy. In CLL cells, BAFF activates NF-κB signaling, but how NF-κB supports CLL survival is not entirely clear. In this study we show that BAFF induces accumulation of the signaling and autophagy adaptor p62/SQSTM1 in a manner dependent on NF-κB activation. p62 potentiates mTORC1 signaling and activates NRF2, the master regulator of the anti-oxidant response. We found that expression of NRF2 target genes, such as NAD(P)H quinone oxidoreductase 1 (NQO1), is particularly enriched in CLL cells with high ROR1 surface expression (ROR1Hi). ROR1Hi CLL cells with elevated NQO1 expression exhibit resistance to drugs that induce ROS accumulation, such venetoclax. However, such cells are more sensitive to compound 29h, a pro-drug that only becomes active after being metabolized by NQO1. Accordingly, 29h sensitizes high NQO1 CLL cells to venetoclax. Collectively, our study unravels a previously unknown signaling network through which the NF-κB-p62-NRF2 axis protects ROR1-high CLL cells from ROS-inducing therapeutics.

Universal Ready-to-Use Immunotherapeutic Approach for the Treatment of Cancer: Expanded and Activated Polyclonal γδ Memory T Cells.

In the last years, important progresses have been registered in the treatment of patients suffering from oncological/haematological malignancies, but more still needs to be done to reduce toxicity and side effects, improve outcome and offer new strategies for relapsed or refractory disease. A remarkable part of these clinical benefits is due to advances in immunotherapy. Here, we investigate the generation of a novel, universal and ready-to-use immunotherapeutic product based on γδ-T lymphocytes. These cells are part of the innate immune system, exerting potent natural cytotoxicity against bacteria, viruses and tumours. This ability, coupled with their negligible alloreactivity, makes them attractive for adoptive immunotherapy approaches. To achieve a cell product suitable for clinical use, we developed a strategy capable to generate polyclonal γδ-T cells with predominant memory-Vδ1 phenotype in good manufacturing practice (GMP) procedures with the additional possibility of gene-modification to improve their anti-tumour activity. Irradiated, engineered artificial antigen-presenting cells (aAPCs) expressing CD86/41BBL/CD40L and the cytomegalovirus (CMV)-antigen-pp65 were used. The presence of CMV-pp65 and CD40L proved to be crucial for expansion of the memory-Vδ1 subpopulation. To allow clinical translation and guarantee patient safety, aAPCs were stably transduced with an inducible suicide gene. Expanded γδ-T cells showed high expression of activation and memory markers, without signs of exhaustion; they maintained polyclonality and potent anti-tumour activity both in vitro (against immortalised and primary blasts) and in in vivo studies without displaying alloreactivity signals. The molecular characterisation (phophoproteomic and gene-expression) of these cell products underlines their unique properties. These cells can further be armed with chimeric antigen receptors (CAR) to improve anti-tumour capacity and persistence. We demonstrate the feasibility of establishing an allogeneic third-party, off-the-shelf and ready-to-use, γδ-T-cell bank. These γδ-T cells may represent an attractive therapeutic option endowed with broad clinical applications, including treatment of viral infections in highly immunocompromised patients, treatment of aggressive malignancies refractory to conventional approaches, bridging therapy to more targeted immunotherapeutic approaches and, ultimately, an innovative platform for the development of off-the-shelf CAR-T-cell products.

Choline Uptake and Metabolism Modulate Macrophage IL-1ß and IL-18 Production.

Choline is a vitamin-like nutrient that is taken up via specific transporters and metabolized by choline kinase, which converts it to phosphocholine needed for de novo synthesis of phosphatidylcholine (PC), the main phospholipid of cellular membranes. We found that Toll-like receptor (TLR) activation enhances choline uptake by macrophages and microglia through induction of the choline transporter CTL1. Inhibition of CTL1 expression or choline phosphorylation attenuated NLRP3 inflammasome activation and IL-1ß and IL-18 production in stimulated macrophages. Mechanistically, reduced choline uptake altered mitochondrial lipid profile, attenuated mitochondrial ATP synthesis, and activated the energy sensor AMP-activated protein kinase (AMPK). By potentiating mitochondrial recruitment of DRP1, AMPK stimulates mitophagy, which contributes to termination of NLRP3 inflammasome activation. Correspondingly, choline kinase inhibitors ameliorated acute and chronic models of IL-1ß-dependent inflammation.

Mitogen-activated kinase kinase kinase 1 inhibits hedgehog signaling and medulloblastoma growth through GLI1 phosphorylation.

The aberrant activation of hedgehog (HH) signaling is a leading cause of the development of medulloblastoma, a pediatric tumor of the cerebellum. The FDA­approved HH inhibitor, Vismodegib, which targets the transmembrane transducer SMO, has shown limited efficacy in patients with medulloblastoma, due to compensatory mechanisms that maintain an active HH­GLI signaling status. Thus, the identification of novel actionable mechanisms, directly affecting the activity of the HH­regulated GLI transcription factors is an important goal for these malignancies. In this study, using gene expression and reporter assays, combined with biochemical and cellular analyses, we demonstrate that mitogen­activated kinase kinase kinase 1 (MEKK1), the most upstream kinase of the mitogen­activated protein kinase (MAPK) phosphorylation modules, suppresses HH signaling by associating and phosphorylating GLI1, the most potent HH­regulated transcription factor. Phosphorylation occurred at multiple residues in the C­terminal region of GLI1 and was followed by an increased association with the cytoplasmic proteins 14­3­3. Of note, the enforced expression of MEKK1 or the exposure of medulloblastoma cells to the MEKK1 activator, Nocodazole, resulted in a marked inhibitory effect on GLI1 activity and tumor cell proliferation and viability. Taken together, the results of this study shed light on a novel regulatory mechanism of HH signaling, with potentially relevant implications in cancer therapy.

Liver Cancer Initiation Requires p53 Inhibition by CD44-Enhanced Growth Factor Signaling.

How fully differentiated cells that experience carcinogenic insults become proliferative cancer progenitors that acquire multiple initiating mutations is not clear. This question is of particular relevance to hepatocellular carcinoma (HCC), which arises from differentiated hepatocytes. Here we show that one solution to this problem is provided by CD44, a hyaluronic acid receptor whose expression is rapidly induced in carcinogen-exposed hepatocytes in a STAT3-dependent manner. Once expressed, CD44 potentiates AKT activation to induce the phosphorylation and nuclear translocation of Mdm2, which terminates the p53 genomic surveillance response. This allows DNA-damaged hepatocytes to escape p53-induced death and senescence and respond to proliferative signals that promote fixation of mutations and their transmission to daughter cells that go on to become HCC progenitors.

Copper/MYC/CTR1 interplay: a dangerous relationship in hepatocellular carcinoma.

Free serum copper correlates with tumor incidence and progression of human cancers, including hepatocellular carcinoma (HCC). Copper extracellular uptake is provided by the transporter CTR1, whose expression is regulated to avoid excessive intracellular copper entry. Inadequate copper serum concentration is involved in the pathogenesis of Non Alcoholic Fatty Liver Disease (NAFLD), which is becoming a major cause of liver damage progression and HCC incidence. Finally, MYC is over-expressed in most of HCCs and is a critical regulator of cellular growth, tumor invasion and metastasis. The purpose of our study was to understand if higher serum copper concentrations might be involved in the progression of NAFLD-cirrhosis toward-HCC. We investigated whether high exogenous copper levels sensitize liver cells to transformation and if it exists an interplay between copper-related proteins and MYC oncogene. NAFLD-cirrhotic patients were characterized by a statistical significant enhancement of serum copper levels, even more evident in HCC patients. We demonstrated that high extracellular copper concentrations increase cell growth, migration, and invasion of liver cancer cells by modulating MYC/CTR1 axis. We highlighted that MYC binds a specific region of the CTR1 promoter, regulating its transcription. Accordingly, CTR1 and MYC proteins expression were progressively up-regulated in liver tissues from NAFLD-cirrhotic to HCC patients. This work provides novel insights on the molecular mechanisms by which copper may favor the progression from cirrhosis to cancer. The Cu/MYC/CTR1 interplay opens a window to refine HCC diagnosis and design new combined therapies.

Stress-Activated NRF2-MDM2 Cascade Controls Neoplastic Progression in Pancreas.

Despite expression of oncogenic KRAS, premalignant pancreatic intraepithelial neoplasia 1 (PanIN1) lesions rarely become fully malignant pancreatic ductal adenocarcinoma (PDAC). The molecular mechanisms through which established risk factors, such as chronic pancreatitis, acinar cell damage, and/or defective autophagy increase the likelihood of PDAC development are poorly understood. We show that accumulation of the autophagy substrate p62/SQSTM1 in stressed KrasG12D acinar cells is associated with PDAC development and maintenance of malignancy in human cells and mice. p62 accumulation promotes neoplastic progression by controlling the NRF2-mediated induction of MDM2, which acts through p53-dependent and -independent mechanisms to abrogate checkpoints that prevent conversion of differentiated acinar cells to proliferative ductal progenitors. MDM2 targeting may be useful for preventing PDAC development in high-risk individuals.

Non-Alcoholic Fatty Liver Disease and Nutritional Implications: Special Focus on Copper.

Non-alcoholic fatty liver disease (NAFLD) is characterized by excess lipids in hepatocytes, due to excessive fatty acid influx from adipose tissue, de novo hepatic lipogenesis, in addition to excessive dietary fat and carbohydrate intake. Chronic hepatic lipid overload induces mitochondrial oxidative stress and cellular damage leading the development of NAFLD into a more severe liver disease condition, non-alcoholic steato-hepatitis (NASH). In turn, this can progress to cirrhosis and hepatocellular carcinoma (HCC). Among others, copper is one of the main bio-metals required for the preponderance of the enzymes involved in physiological redox reactions, which primarily occurs during mitochondrial respiration. Thus, copper homeostasis could be considered a target point for counteracting the progression of NAFLD. Accordingly, many diseases are correlated to unbalanced copper levels and, actually, some clinical trials are examining the use of copper chelating agents. Currently, no pharmacological interventions are approved for NAFLD, but nutritional and lifestyle modifications are always recommended. Fittingly, antioxidant food agents recognized to improve NAFLD and its complications have been described in the literature to bind copper. Therefore, this review describes the role of nutrition in the development and progression of NAFLD with a particular focus on copper and copper-binding antioxidant compounds against NAFLD.