Favor Tumor Growth Research Articles

Abstract I22: New players and unique features of cancer lysosomes

Abstract Pancreatic ductal adenocarcinoma (PDA) cells and tumors constitutively activate nutrient scavenging pathways, namely, autophagy (cellular self-catabolism) and macropinocytosis (bulk uptake of extracellular material) in order to maintain metabolic homeostasis. Importantly, both of these pathways converge on the lysosome, an acidic organelle that harbors within its lumen a complex series of enzymatic reactions that enable degradation of incoming cargo material. Our prior work has shown that the lysosome is greatly expanded and qualitatively different in PDA cells compared to normal tissue. PDA tumors are effectively “addicted” to enhanced catabolic activity as blocking lysosome function specifically inhibits in vitro and in vivo growth of PDA cell lines and tumor xenografts with minimal effects on normal cells. How key changes in lysosome composition and content contribute to the aggressive nature of PDA remains an open question. We have optimized methodologies to isolate and purify lysosomes from normal human pancreatic epithelial cells and several established patient-derived PDA cancer cell lines, using affinity-based capture techniques that isolate 50-80% intact lysosomes free of cytoplasm and other contaminating organelles. By systematically implementing lysosome purification from normal, primary, and metastatic PDA tumors combined with mass spectrometry-based proteomics, we have identified both resident and substrate proteins that are uniquely associated with PDA lysosomes. Several cancer-specific lysosomal membrane proteins emerged from this analysis that may endow cancer lysosomes with enhanced functionality, including rapid membrane repair in the face of sustained mechanical and chemical insults. Similarly, we find that PDA lysosomes degrade select cellular proteins to enable dynamic remodeling of the cellular proteome in a manner that favors tumor growth and progression. Collectively, our data provide strong evidence in support of the lysosome as a central mediator of cellular adaptation to stress and promoter of tumorigenesis. Citation Format: Rushika M Perera. New players and unique features of cancer lysosomes [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr I22.

Vascular PPARβ/δ Promotes Tumor Angiogenesis and Progression.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors, which function as transcription factors. Among them, PPARβ/δ is highly expressed in endothelial cells. Pharmacological activation with PPARβ/δ agonists had been shown to increase their angiogenic properties. PPARβ/δ has been suggested to be involved in the regulation of the angiogenic switch in tumor progression. However, until now, it is not clear to what extent the expression of PPARβ/δ in tumor endothelium influences tumor progression and metastasis formation. We addressed this question using transgenic mice with an inducible conditional vascular-specific overexpression of PPARβ/δ. Following specific over-expression of PPARβ/δ in endothelial cells, we induced syngenic tumors. We observed an enhanced tumor growth, a higher vessel density, and enhanced metastasis formation in the tumors of animals with vessel-specific overexpression of PPARβ/δ. In order to identify molecular downstream targets of PPARβ/δ in the tumor endothelium, we sorted endothelial cells from the tumors and performed RNA sequencing. We identified platelet-derived growth factor receptor beta (Pdgfrb), platelet-derived growth factor subunit B (Pdgfb), and the tyrosinkinase KIT (c-Kit) as new PPARβ/δ -dependent molecules. We show here that PPARβ/δ activation, regardless of its action on different cancer cell types, leads to a higher tumor vascularization which favors tumor growth and metastasis formation.

Structural Insights into the Intracellular Region of the Human Magnesium Transport Mediator CNNM4.

The four member family of “Cyclin and Cystathionine β-synthase (CBS) domain divalent metal cation transport mediators”, CNNMs, are the least-studied mammalian magnesium transport mediators. CNNM4 is abundant in the brain and the intestinal tract, and its abnormal activity causes Jalili Syndrome. Recent findings show that suppression of CNNM4 in mice promotes malignant progression of intestinal polyps and is linked to infertility. The association of CNNM4 with phosphatases of the regenerating liver, PRLs, abrogates its Mg2+-efflux capacity, thus resulting in an increased intracellular Mg2+ concentration that favors tumor growth. Here we present the crystal structures of the two independent intracellular domains of human CNNM4, i.e., the Bateman module and the cyclic nucleotide binding-like domain (cNMP). We also derive a model structure for the full intracellular region in the absence and presence of MgATP and the oncogenic interacting partner, PRL-1. We find that only the Bateman module interacts with ATP and Mg2+, at non-overlapping sites facilitating their positive cooperativity. Furthermore, both domains dimerize autonomously, where the cNMP domain dimer forms a rigid cleft to restrict the Mg2+ induced sliding of the inserting CBS1 motives of the Bateman module, from a twisted to a flat disk shaped dimer.

Targeted knock-in mice expressing the oxidase-fixed form of xanthine oxidoreductase favor tumor growth

Xanthine oxidoreductase has been implicated in cancer. Nonetheless, the role played by its two convertible forms, xanthine dehydrogenase (XDH) and oxidase (XO) during tumorigenesis is not understood. Here we produce XDH-stable and XO-locked knock-in (ki) mice to address this question. After tumor transfer, XO ki mice show strongly increased tumor growth compared to wild type (WT) and XDH ki mice. Hematopoietic XO expression is responsible for this effect. After macrophage depletion, tumor growth is reduced. Adoptive transfer of XO-ki macrophages in WT mice increases tumor growth. In vitro, XO ki macrophages produce higher levels of reactive oxygen species (ROS) responsible for the increased Tregs observed in the tumors. Blocking ROS in vivo slows down tumor growth. Collectively, these results indicate that the balance of XO/XDH plays an important role in immune surveillance of tumor development. Strategies that inhibit the XO form specifically may be valuable in controlling cancer growth.

Altered Lipid Tumor Environment and Its Potential Effects on NKT Cell Function in Tumor Immunity.

Natural killer T (NKT) cells are CD1d restricted T cells that mostly recognize lipid antigens. These cells share characteristics with both adaptive and innate immune cells and have multiple immunoregulatory roles. In a manner similar to innate immune cells, they respond quickly to stimuli and secrete large amounts of cytokines, amplifying and modulating the immune response. As T cells, they express T cell receptors (TCRs) and respond in an antigen-specific manner like conventional T cells. There are at least two subtypes of NKT cells, type I and type II, that differ in the nature of their TCR, either semi-invariant (type I) or diverse (type II). The two sub-types generally have opposing functions in tumor immunity, with type I promoting and type II suppressing tumor immunity, and they cross-regulate each other, forming an immunoregulatory axis. The tumor has multiple mechanisms by which it can evade immune-surveillance. One such mechanism involves alteration in tumor lipid repertoire and accumulation of lipids and fatty acids that favor tumor growth and evade anti-tumor immunity. Since NKT cells mostly recognize lipid antigens, an altered tumor lipid metabolic profile will also alter the repertoire of lipid antigens that can potentially affect their immune-modulatory function. In this review, we will explore the effects of alterations in the lipid metabolites on tumor growth, antigen cross-presentation, and overall effect on anti-tumor immunity, especially in the context of NKT cells.

P11.61 Development of a novel preclinical GBM model and therapeutic impact of IRE1 inhibition

Abstract Glioblastoma Multiforme (GBM) is the most severe primary brain tumor and represents more than 15% of all brain tumors. Despite an aggressive treatment comprising surgical resection and radio/chemotherapy, patient’s survival post diagnosis remains short with a median overall survival of 15 months. The lack of efficacy of the current treatments is mostly due to the tumor heterogeneity with different tumor cell types that exhibit various sensitivity to anti-cancer agents and to the diffuse feature of GBM that complicates the efficacy of complete resection. Another limitation for finding new valuable therapy approaches is the lack of relevant animal models that extensively recapitulate the current GBM patients’ standard of care. In the past couple of years, it has been demonstrated that the Unfolded Protein Response (UPR) plays an instrumental role in GBM development. It has been shown that IRE1, the most conserved UPR sensor, signals in tumor cells to reshape the tumor microenvironment to favor tumor growth and most likely to alter the response to treatment. The IRE1/XBP1s signaling axis exhibits pro-oncogenic properties and has a direct impact on patients’ survival. These observations point toward the IRE1/XBP1s axis as a potentially relevant therapeutic target. To further test the potential impact of the pharmacological targeting IRE1/XBP1 signaling, we proposed to use MKC8866, an inhibitors of IRE1, in preclinical models of GBM. As such we developed a novel GBM animal model that recapitulates the different steps engaged in GBM patient clinical handling (including surgical resection, irradiation and chemotherapy), and use this to demonstrate the relevance of IRE1 inhibition in GBM. Considering that IRE1 inhibitors do not cross the blood-brain barrier, we proposed an intraoperative delivery of the drugs though fibrin glue plugs applicated within the resection cavity, and then studied the impact on survival and tumor microenvironment. We showed that local delivery of IRE1 inhibitors combined with radio/chemotherapy had a positive effect on survival and induced tumoral and microenvironment remodeling, pointing at its high therapeutic potential and the need for an early clinical trial.

Intrahepatic immune changes after hepatitis c virus eradication by direct-acting antiviral therapy.

The recent approval of direct acting anti-virals (DAA) has dramatically changed the landscape of hepatitis C virus (HCV) therapy. Whether viral clearance could promote liver carcinogenesis is debated. It has been hypothesized that changes in intrahepatic immune surveillance following viral cure could favour tumour growth. This study aimed at characterizing the intrahepatic immune changes induced by HCV cure following DAA therapy. Patients with compensated cirrhosis who underwent surgical resection for hepatocellular carcinoma (HCC) after sustained virological response (SVR) to DAA therapy were included. A control group of untreated HCV-infected patients with compensated cirrhosis was selected. RNA was extracted from tumoral and non-tumoral tissues and analysed using the Nanostring Immuno-Oncology-360 panel. Immune cells were quantified by immunohistochemistry. Twenty patients were included: 10 patients with a DAA-induced SVR and 10 untreated controls. All of them had a de novo BCLC 0/A HCC. Non-tumoral tissue profiling showed down-regulation of interferon-related genes (including MX1, ISG15 and IFIT1) after DAA therapy. No other differences in immune profiles/immune cell densities were identified between the two groups. The intra-tumoral immune profiles of HCCs that occurred after DAA therapy were not qualitatively or quantitatively different from those of tumours occurring in untreated patients. In conclusion, removal of HCV infection after DAA-based therapy results only in a down-regulation of interferon-stimulated genes in non-tumoral tissues from patients with cirrhosis who develop HCC. These minor changes in the liver immune microenvironment are unlikely to favour HCC occurrence or recurrence after DAA-induced SVR.

Proteomic Profiling of Rhabdomyosarcoma-Derived Exosomes Yield Insights into Their Functional Role in Paracrine Signaling.

Exosomes are important intercellular communication vehicles, secreted into body fluids by multiple cell types, including tumor cells. They have been demonstrated to contribute to the metastatic progression of tumor cells through paracrine signaling. Tumor exosomes contain intact and functional proteins, mRNA and miRNA that may alter the cellular environment to favor tumor growth. We evaluated the protein cargo of exosomes derived from the childhood tumor rhabdomyosarcoma (RMS) and the molecular pathways they are implicated in to decipher their role in the progression of this aggressive disease. We conducted a mass spectrometry analysis of exosome content isolated from five RMS cell lines: three of embryonal RMS (ERMS) and two of alveolar RMS (ARMS) histology and verified results by multiple reaction monitoring and western blot analyses. Results revealed 161 common proteins in ERMS-derived exosomes and 122 common proteins in ARMS-derived exosomes, of which 81 proteins were common to both subtypes. Using both PANTHER gene classification and Pathway Studio software, we assessed the perturbed biological processes and altered pathways in which the exosomal proteins are involved. The 81 commonly expressed proteins included those involved in "cell-signaling," "cell-movement," and "cancer." Pathways engaging the identified proteins revealed 37 common pathways including "integrin signaling pathway," "inflammation mediated by chemokine and cytokine signaling pathway," and "angiogenesis." Finally, a comparison of exosomal proteins of RMS cells with publicly available datasets from other cancer cells revealed that 36 proteins are specific and endogenous to the RMS-exosomes. Taken together, our results reveal that RMS-derived exosomes carry a protein cargo that contributes to conserved cellular signaling networks across multiple cell lines, and we also identify RMS exosome-specific proteins that should be further evaluated as possible novel biomarkers for this tumor.

Local Administration of Caloric Restriction Mimetics to Promote the Immune Control of Lung Metastases.

Caloric restriction mimetics (CRMs), compounds that mimic the biochemical effects of nutrient deprivation, administered via systemic route promote antitumor effects through the induction of autophagy and the modulation of the immune microenvironment; however, collateral effects due to metabolic changes and the possible weight loss might potentially limit their administration at long term. Here, we investigated in mice local administration of CRMs via aerosol to reduce metastasis implantation in the lung, whose physiologic immunosuppressive status favors tumor growth. Hydroxycitrate, spermidine, and alpha-lipoic acid, CRMs that target different metabolic enzymes, administered by aerosol, strongly reduced implantation of intravenously injected B16 melanoma cells without overt signs of toxicity, such as weight loss and changes in lung structure. Cytofluorimetric analysis of lung immune infiltrates revealed a significant increase of alveolar macrophages and CD103+ dendritic cells in mice treated with CRMs that paralleled an increased recruitment and activation of both CD3 T lymphocytes and NK cells. These effects were associated with the upregulation of genes related to M1 phenotype, as IL-12 and STAT-1, and to the decrease of M2 genes, as IL-10 and STAT-6, in adherent fraction of lung immune infiltrate, as revealed by real-time PCR analysis. Thus, in this proof-of-principle study, we highlight the antitumor effect of CRM aerosol delivery as a new and noninvasive therapeutic approach to locally modulate immunosurveillance at the tumor site in the lung.

The role of immunotherapy in mesothelioma

Malignant pleural mesothelioma (MPM) is a rare tumour occurring after asbestos exposure. Most cases are unresectable at the time of diagnosis and the prognosis is very poor. Platinum based chemotherapy, with or without added antiangiogenics is standard first-line treatment. However, its efficacy is limited, patients with unresectable disease have a median 12 months overall survival and the benefit of treatment is only 3 months. The immune micro-environment plays a crucial role in tumour development. Local immunosuppression involving, but not limited to, the expression of T cell inhibitory receptors, favours tumour growth. Moreover, chronic stromal inflammatory response and the presence of lymphocytic infiltration and macrophages are all prognostic factors. Immune checkpoint inhibitors (ICIs) targeting programmed death protein 1 (PD-1), its ligand (PD-L1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) are able to block inhibitory signals on T cells, thus enhancing the immune anti-tumour response. PD-L1 is expressed in 60% of MPM and associated with poor prognosis. Several studies have shown encouraging results in single agent or combination checkpoint inhibition in MPM. Furthermore, T cell-mediated treatments are being developed with promising results, as are chimeric antigen receptor (CAR) T cells targeting mesothelin, a highly expressed protein in MPM. In this review, we examine currently available data, as well as ongoing trials in the field of immuno-oncology for MPM.

Chinese‐German Cooperation Group Tumor Immunology: Another inspiring Meeting in Deidesheim

Chinese‐German Cooperation Group Tumor Immunology: Another inspiring Meeting in Deidesheim

The supernatant of cervical carcinoma cells lines induces a decrease in phosphorylation of STAT-1 and NF-κB transcription factors associated with changes in profiles of cytokines and growth factors in macrophages derived from U937 cells.

Macrophages are presents in the tumor microenvironment and acquire different phenotypic and functional characteristics in response to microenvironmental signals. Macrophages can be differentiated into two phenotypes: M1 or pro-inflammatory (classically activated), and M2 or anti-inflammatory macrophage (alternatively activated). In response to the microenvironment, macrophages activate transcription factors as STAT1 and NF-κB-p65 for M1 macrophages or STAT3 and STAT6 for M2 macrophages; activation impacts on the profile of cytokine, chemokines and growth factors secreted by macrophages. We evaluated the effect of the supernatant of cervical-derived carcinoma cell lines HeLa, SiHa, and C-33A on the phosphorylation of transcriptional factors STAT1, NF-κB-p65, and STAT6, and their impact in the profile of secretion of cytokines and growth factors by macrophages derived from the U937 cell line. The results show that in macrophages, these supernatants induce a decrease in the phosphorylation of NF-κB-p65 and STAT1 in U937-macrophages accompanied by an increase in the secretion of IL-10, IL-6, MCP-1, and IL-8, as well as GM-CSF, G-CSF, PDGF-AA, PDGF-BB, and VEGF. Our results suggest that HeLa, SiHa, and C-33A cell lines down-regulate the activation of transcription factors characteristic of M1 macrophages (STAT1, NF-κB-p65) and induce the secretion of factors that favor tumor growth.

Regulation of death receptor signaling by the autophagy protein TP53INP2.

TP53INP2 positively regulates autophagy by binding to Atg8 proteins. Here, we uncover a novel role of TP53INP2 in death-receptor signaling. TP53INP2 sensitizes cells to apoptosis induced by death receptor ligands. In keeping with this, TP53INP2 deficiency in cultured cells or mouse livers protects against death receptor-induced apoptosis. TP53INP2 binds caspase-8 and the ubiquitin ligase TRAF6, thereby promoting the ubiquitination and activation of caspase-8 by TRAF6. We have defined a TRAF6-interacting motif (TIM) and a ubiquitin-interacting motif in TP53INP2, enabling it to function as a scaffold bridging already ubiquitinated caspase-8 to TRAF6 for further polyubiquitination of caspase-8. Mutations of key TIM residues in TP53INP2 abrogate its interaction with TRAF6 and caspase-8, and subsequently reduce levels of death receptor-induced apoptosis. A screen of cancer cell lines showed that those with higher protein levels of TP53INP2 are more prone to TRAIL-induced apoptosis, making TP53INP2 a potential predictive marker of cancer cell responsiveness to TRAIL treatment. These findings uncover a novel mechanism for the regulation of caspase-8 ubiquitination and reveal TP53INP2 as an important regulator of the death receptor pathway.

Deciphering the Roles of Innate Lymphoid Cells in Cancer.

Cancer is a complex disease and the role played by innate lymphoid cells (ILCs) in cancer development has begun to be uncovered over recent years. We aim to provide an exhaustive summary of the knowledge acquired on the role of ILCs in cancer. ILCs are classified into 3 different categories, ILC1s, ILC2s, and ILC3s, each encompassing specific and unique functions. ILC1s exhibit NK cells characteristics and can exert anti-tumor functions, but surprisingly their IFNγ production is not associated with a better immune response. In response to TGF-β or IL-12, ILC1s were shown to exert pro-tumor functions and to favor tumor growth. ILC2s role in cancer immune response is dependent on cytokine context. The production of IL-13 by ILC2s is associated with a negative outcome in cancer. ILC2s can also produce IL-5, leading to eosinophil activation and an increased anti-tumor immune response in lung cancer. ILC3s produce IL-22, which could promote tumor growth. In contrast, ILC3s recognize tumor cells and facilitate leukocyte tumor entry, increasing anti-tumor immunity. In some contexts, ILC3s were found at the edge of tertiary lymphoid structures, associated with a good prognostic. We are at the dawn of our understanding of ILCs role in cancer. This review aims to thoroughly analyze existing data and to provide a comprehensive overview of our present knowledge on the impact of ILCs in cancer.

Lysosomal Pathways and Autophagy Distinctively Control Endothelial Cell Behavior to Affect Tumor Vasculature

Cancer cell-stromal cell crosstalk is orchestrated by a plethora of ligand-receptor interactions generating a tumor microenvironment (TME) which favors tumor growth. The high pro-angiogenic nature of the TME perpetuates the chaotic network of structurally immature, low pericyte-covered vessels characteristic of the tumor vasculature. We previously demonstrated that chloroquine (CQ) -a lysosomotropic agent used as first-generation autophagy blocker in clinical trials- induced tumor vessel normalization and reduced tumor hypoxia. CQ improved both vessel structure and maturation, whereas the conditional knockout of the crucial autophagy gene Atg5 in endothelial cells (ECs) did not, thus highlighting a potential differential role for EC-associated autophagy and the lysosomes in pathological tumor angiogenesis. However, how CQ or ATG5-deficiency in ECs affect angiogenic signals regulating EC-pericyte interface and therefore vessel maturation, remains unknown. Here, we show that in ECs CQ constrained VEGF-A-mediated VEGF receptor (VEGFR)2 phosphorylation, a driver of angiogenic signaling. In the presence of CQ we observed increased expression of the decoy receptor VEGFR1 and of a lower molecular weight form of VEGFR2, suggesting receptor cleavage. Consequently, VEGF-A-driven EC spheroid sprouting was reduced by CQ treatment. Furthermore, CQ significantly affected the transcription and secretion of platelet-derived growth factor (PDGF)-AB/BB (upregulated) and Endothelin-1 (EDN1, downregulated), both modulators of perivascular cell (PC) behavior. In contrast, silencing of ATG5 in ECs had no effect on VEGFR2 to VEGFR1 ratio nor on PDGFB and EDN1 expression. Accordingly, mice harboring B16F10 melanoma tumors treated with CQ, displayed both an increased number of αSMA+ PCs covering tumor vessels and co-expressed PDGF receptor-β, enabling PDGF ligand dependent recruitment. Moreover, upon CQ treatment the tumoral expression of angiopoietin-1 (Angpt1), which retains mural cells, and induces vessel stabilization by binding to the EC-localized cognate receptor (TIE2), was increased thus supporting the vessel normalization function of CQ. These features associated with improved tumor vasculature were not phenocopied by the specific deletion of Atg5 in ECs. In conclusion, this study further unravels endothelial cell autonomous and non-autonomous mechanisms by which CQ “normalizes” the intercellular communication in the tumor vasculature independent of autophagy.

Immunohistochemical Expression of Melatonin Receptor MT1 and Glucose Transporter GLUT1 in Human Breast Cancer.

Breast cancer is a heterogeneous disease and is the leading cause of cancer-related deaths among women. Even after diagnosis, the prognosis cannot be concluded since patients can develop resistance to therapy, which favors tumor growth, invasion and metastasis. In recent years, research has focused on identifying significant markers that can be used to determine the prognosis. Melatonin can act through G protein- coupled MT1 receptor, which controls selected protein kinases, influences the levels of transcription factor phosphorylation, specific genes expression, proliferation, angiogenesis, cell differentiation, migration, and indirectly controls the transport of glucose in cancer cells. It is known that glucose enters the cells by glucose transporters, such as GLUT1 which shows wide tissue distribution and appears to be altered in human breast carcinoma. High GLUT1 expression is associated with increased malignant potential, invasiveness and poor prognosis in some cancers including breast cancer. The aim of this study was to evaluate the expression of MT1 receptor and GLUT1 in breast tumors and correlate with molecular subtypes and prognostic characteristics. Protein expression was performed by an immunohistochemical procedure with specific antibodies and positive and negative controls. We found that MT1 high expression was associated with good prognosis subtype (Luminal A), while GLUT1 high expression was related to poor prognosis subtype (triple-negative). In addition, we found high expression of MT1 in ER+ and the inverse in GLUT1 expression. GLUT1 is also highly expressed in tumor ≥20mm. These results indicate MT1 and GLUT1 as potential targets for breast cancer subtypes and prognosis.

Regulation of senescence escape by TSP1 and CD47 following chemotherapy treatment

Senescence is a tumor-suppressive mechanism induced by telomere shortening, oncogenes, or chemotherapy treatment. Although it is clear that this suppressive pathway leads to a permanent arrest in primary cells, this might not be the case in cancer cells that have inactivated their suppressive pathways. We have recently shown that subpopulations of cells can escape chemotherapy-mediated senescence and emerge as more transformed cells that induce tumor formation, resist anoikis, and are more invasive. In this study, we characterized this emergence and showed that senescent cells favor tumor growth and metastasis, in vitro and in vivo. Senescence escape was regulated by secreted proteins produced during emergence. Among these, we identified thrombospondin-1 (TSP1), a protein produced by senescent cells that prevented senescence escape. Using SWATH quantitative proteomic analysis, we found that TSP1 can be detected in the serum of patients suffering from triple-negative breast cancer and that its low expression was associated with treatment failure. The results also indicate that senescence escape is explained by the emergence of CD47low cells that express a reduced level of CD47, the TSP1 receptor. The results show that CD47 expression is regulated by p21waf1. The cell cycle inhibitor was sufficient to maintain senescence since its downregulation in senescent cells increased cell emergence. This leads to the upregulation of Myc, which then binds to the CD47 promoter to repress its expression, allowing the generation of CD47low cells that escape the suppressive arrest. Altogether, these results uncovered a new function for TSP1 and CD47 in the control of chemotherapy-mediated senescence.

Immunonutritional consequences of different serine-type protease inhibitors in a C57BL/6 hepatocarcinoma model.

Imbalances in innate immunity and the activity of innate immune cells are implicated in the development of hepatocellular carcinoma (HCC). Plant seeds are good sources of protease inhibitors, which can have a significant influence on human health disorders, especially in the field of cancer prevention. To elucidate the impact and preventive effects of immunonutritional serine-type protease inhibitors (STPIs) on HCC, it was used an established model of chemically induced liver injury. Injured livers induced Akt as well as hepatic infiltration of NKG2D+ and CD74+ cells. Feeding STPIs reduced size and number of intrahepatic nodes of mononuclear. These animals showed an inverse association of the severity of HCC with bioactive hepcidin levels, which was significantly correlated with the hepatic myeloperoxidase activity. According to their origin, administration of STPIs significantly induce increased numbers of F4/80+ cells in injured livers that can be responsible for the biological effects detected on the parenchyma and inflammatory markers under DEN/TAA treatment. These findings can have direct implications in HCC immunotherapy where enhanced response(s) in inflammation-driven cancer patients could help promoting inflammation-driven processes and favor tumor growth. Altogether, this study demonstrates that oral administration of STPIs modulate innate immunity response influencing HCC aggressiveness and progression. These results represent a path forward to develop durable, long-lasting response against hepatocarcinoma and open a future research path in the development of coadjutant intervention strategies to pharmacological therapies.

Preventive action of benztropine on platinum-induced peripheral neuropathies and tumor growth

The endogenous cholinergic system plays a key role in neuronal cells, by suppressing neurite outgrowth and myelination and, in some cancer cells, favoring tumor growth. Platinum compounds are widely used as part of first line conventional cancer chemotherapy; their efficacy is however limited by peripheral neuropathy as a major side-effect. In a multiple sclerosis mouse model, benztropine, that also acts as an anti-histamine and a dopamine re-uptake inhibitor, induced the differentiation of oligodendrocytes through M1 and M3 muscarinic receptors and enhanced re-myelination. We have evaluated whether benztropine can increase anti-tumoral efficacy of oxaliplatin, while preventing its neurotoxicity.We showed that benztropine improves acute and chronic clinical symptoms of oxaliplatin-induced peripheral neuropathies in mice. Sensory alterations detected by electrophysiology in oxaliplatin-treated mice were consistent with a decreased nerve conduction velocity and membrane hyperexcitability due to alterations in the density and/or functioning of both sodium and potassium channels, confirmed by action potential analysis from ex-vivo cultures of mouse dorsal root ganglion sensory neurons using whole-cell patch-clamp. These alterations were all prevented by benztropine. In oxaliplatin-treated mice, MBP expression, confocal and electronic microscopy of the sciatic nerves revealed a demyelination and confirmed the alteration of the myelinated axons morphology when compared to animals injected with oxaliplatin plus benztropine. Benztropine also prevented the decrease in neuronal density in the paws of mice injected with oxaliplatin. The neuroprotection conferred by benztropine against chemotherapeutic drugs was associated with a lower expression of inflammatory cytokines and extended to diabetic-induced peripheral neuropathy in mice.Mice receiving benztropine alone presented a lower tumor growth when compared to untreated animals and synergized the anti-tumoral effect of oxaliplatin, a phenomenon explained at least in part by benztropine-induced ROS imbalance in tumor cells.This report shows that blocking muscarinic receptors with benztropine prevents peripheral neuropathies and increases the therapeutic index of oxaliplatin. These results can be rapidly transposable to patients as benztropine is currently indicated in Parkinson’s disease in the United States.

MiR-517a promotes Warburg effect in HCC by directly targeting FBP1.

PurposeHepatocellular carcinoma (HCC) is one of the most aggressive malignancies worldwide. Our aim is to explore the expression and biological function of miR-517a in HCC.Materials and methodsWe performed qRT-PCR to detect the expression of miR-517a in clinical samples and cell lines. CKK-8 assay and colony formation assay were employed to detect the miR-517a regulated cell proliferation. Glucose uptake and lactate production were examined to determine the Warburg effect. We also performed ECAR assay using Seahorse system. Luciferase acitivy assay was used to examine the binding of FBP1 3′UTR by miR-517a.ResultsmiR-517a was upregulated in HCC samples in both genomic and mRNA levels. Moreover, overexpression of miR-517a promoted cell proliferation and Warburg effect. Mechanically, miR-517a could directly target the 3′-UTR of FBP1. In addition, restoring the expression of FBP1 inhibited cell growth.ConclusionWe demonstrated that miR-517a acts as an oncogene to promote Warburg effect in HCC, favoring tumor growth, and miR-517a/FBP1 could be a novel target for HCC treatment.