The miR-29 family facilitates the activation of NK-cell immune responses by targeting the B7-H3 immune checkpoint in neuroblastoma.

Neuroblastoma (NB) is a highly aggressive pediatric cancer that originates from immature nerve cells, presenting significant treatment challenges due to therapy resistance. Despite intensive treatment, approximately 50% of high-risk NB cases exhibit therapy resistance or experience relapse, resulting in poor outcomes often associated with tumor immune evasion. B7-H3 is an immune checkpoint protein known to inhibit immune responses. MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation. Our study aims to explore the impact of miRNAs on B7-H3 regulation, the anti-tumor immune response, and tumorigenicity in NB. Analysis of NB patients and patient-derived xenograft tumors revealed a correlation between higher B7-H3 expression and poorer patient survival. Notably, deceased patients exhibited a depletion of miR-29 family members (miR-29a, miR-29b, and miR-29c), which displayed an inverse association with B7-H3 expression in NB patients. Overexpression and knockdown experiments demonstrated that these miRNAs degrade B7-H3 mRNA, resulting in enhanced NK cell activation and cytotoxicity. In vivo, experiments provided further evidence that miR-29 family members reduce tumorigenicity, macrophage infiltration, and microvessel density, promote infiltration and activation of NK cells, and induce tumor cell apoptosis. These findings offer a rationale for developing more effective combination treatments that leverage miRNAs to target B7-H3 in NB patients.

Mechanisms of Sorafenib Resistance in HCC Culture Relate to the Impaired Membrane Expression of Organic Cation Transporter 1 (OCT1).

Introduction: Sorafenib, an FDA-approved drug for advanced hepatocellular carcinoma (HCC) treatment, encounters resistance in many patients. Deciphering the mechanisms underlying sorafenib resistance is crucial for devising alternative strategies to overcome it. Aim: This study aimed to investigate sorafenib resistance mechanisms using a diverse panel of HCC cell lines. Methods: HCC cell lines were subjected to continuous sorafenib treatment, and stable cell lines (Huh 7.5 and Huh 7PX) exhibiting sustained growth in its presence were isolated. The investigation of drug resistance mechanisms involved a comparative analysis of drug-targeted signal transduction pathways (EGFR/RAF/MEK/ERK/Cyclin D), sorafenib uptake, and membrane expression of the drug uptake transporter. Results: HCC cell lines (Huh 7.5 and Huh 7PX) with a higher IC50 (10µM) displayed a more frequent development of sorafenib resistance compared to those with a lower IC50 (2-4.8µM), indicating a potential impact of IC50 variation on initial treatment response. Our findings reveal that activated overexpression of Raf1 kinases and impaired sorafenib uptake, mediated by reduced membrane expression of organic cation transporter-1 (OCT1), contribute to sorafenib resistance in HCC cultures. Stable expression of the drug transporter OCT1 through cDNA transfection or adenoviral delivery of OCT1 mRNA increased sorafenib uptake and successfully overcame sorafenib resistance. Additionally, consistent with sorafenib resistance in HCC cultures, cirrhotic liver-associated human HCC tumors often exhibited impaired membrane expression of OCT1 and OCT3. Conclusion: Intrinsic differences among HCC cell clones, affecting sorafenib sensitivity at the expression level of Raf kinases, drug uptake, and OCT1 transporters, were identified. This study underscores the potential of HCC tumor targeted OCT1 expression to enhance sorafenib treatment response.

Physiological and regenerative functions of sterile-α motif protein-14 in hematopoiesis.

Sterile α-motif domain-14 (Samd14) protein expression increases the regenerative capacity of the erythroid system. Samd14 is transcriptionally upregulated and promotes cell signaling via the receptor tyrosine kinase Kit in a critical window of acute erythroid regeneration. We generated a hematopoietic-specific conditional Samd14 knockout mouse model (Samd14-CKO) to study the role of Samd14 in hematopoiesis. The Samd14-CKO mouse was viable and exhibited no steady-state hematopoietic phenotype. Samd14-CKO mice were hypersensitive to 5-fluorouracil, resulting in more severe anemia during recovery and impaired erythroid progenitor colony formation. Ex vivo, Samd14-CKO hematopoietic progenitors were defective in their ability to form mast cells. Samd14-CKO mast cells exhibited altered Kit/stem cell factor (SCF), IL-3/IL-3R signaling, and less granularity than Samd14-FL/FL cells. Our findings indicate that Samd14 promotes both erythroid and mast cell functions. The Samd14-CKO mouse phenotype exhibits striking similarities to the KitW/W-v mice, which carry Kit mutations resulting in reduced tyrosine kinase-dependent signaling, causing mast cell and erythroid abnormalities. The Samd14-CKO mouse model is a new tool for studying hematologic pathologies involving Kit signaling.

Acute ethanol-induced liver injury is prevented by betaine administration.

Binge drinking is the most common form of excessive alcohol use. Repeated episodes of binge drinking cause multiple organ injuries, including liver damage. We previously demonstrated that chronic ethanol administration causes a decline in the intrahepatic ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH). This decline causes impairments in essential methylation reactions that result in alcohol-induced fatty liver (steatosis) and other features of alcohol-associated liver disease (ALD). Co-treatment with betaine during chronic ethanol feeding, normalizes hepatocellular SAM:SAH ratio and alleviates many features of liver damage including steatosis. Here, we sought to examine whether betaine treatment similarly protects against liver injury in an alcohol binge-drinking model. We hypothesized that ethanol binge with prior or simultaneous betaine administration would prevent or attenuate acute alcohol-induced liver damage. Male C57Bl/6 mice were gavaged twice, 12 h apart, with either 6 g ethanol/kg BW or with an equal volume/kg BW of 0.9% NaCl. Two separate groups of mice (n = 5/group) were gavaged with 4 g betaine/kg BW, either 2 h before or simultaneously with the ethanol or saline gavages. All mice were sacrificed 8 h after the last gavage and serum and liver parameters were quantified. Ethanol binges caused a 50% decrease in hepatic SAM:SAH ratio and a >3-fold rise in liver triglycerides (p ≤ 0.05). These latter changes were accompanied by elevated serum AST and ALT activities and blood alcohol concentrations (BAC) that were ∼three-times higher than the legal limit of intoxication in humans. Mice that were treated with betaine 2 h before or simultaneously with the ethanol binges exhibited similar BAC as in mice given ethanol-alone. Both betaine treatments significantly elevated hepatic SAM levels thereby normalizing the SAM:SAH ratio and attenuating hepatic steatosis and other injury parameters, compared with mice given ethanol alone. Simultaneous betaine co-administration with ethanol was more effective in preventing or attenuating liver injury than betaine given before ethanol gavage. Our findings confirm the potential therapeutic value of betaine administration in preventing liver injury after binge drinking in an animal model.

miR-15a and miR-15b modulate natural killer and CD8+T-cell activation and anti-tumor immune response by targeting PD-L1 in neuroblastoma.

Neuroblastoma (NB) is an enigmatic and deadliest pediatric cancer to treat. The major obstacles to the effective immunotherapy treatments in NB are defective immune cells and the immune evasion tactics deployed by the tumor cells and the stromal microenvironment. Nervous system development during embryonic and pediatric stages is critically mediated by non-coding RNAs such as micro RNAs (miR). Hence, we explored the role of miRs in anti-tumor immune response via a range of data-driven workflows and in vitro & in vivo experiments. Using the TARGET, NB patient dataset (n=249), we applied the robust bioinformatic workflows incorporating differential expression, co-expression, survival, heatmaps, and box plots. We initially demonstrated the role of miR-15a-5p (miR-15a) and miR-15b-5p (miR-15b) as tumor suppressors, followed by their negative association with stromal cell percentages and a statistically significant negative regulation of T and natural killer (NK) cell signature genes, especially CD274 (PD-L1) in stromal-low patient subsets. The NB phase-specific expression of the miR-15a/miR-15b-PD-L1 axis was further corroborated using the PDX (n=24) dataset. We demonstrated miR-15a/miR-15b mediated degradation of PD-L1 mRNA through its interaction with the 3'-untranslated region and the RNA-induced silencing complex using sequence-specific luciferase activity and Ago2 RNA immunoprecipitation assays. In addition, we established miR-15a/miR-15b induced CD8+T and NK cell activation and cytotoxicity against NB in vitro. Moreover, injection of murine cells expressing miR-15a reduced tumor size, tumor vasculature and enhanced the activation and infiltration of CD8+T and NK cells into the tumors in vivo. We further established that blocking the surface PD-L1 using an anti-PD-L1 antibody rescued miR-15a/miR-15b induced CD8+T and NK cell-mediated anti-tumor responses. These findings demonstrate that miR-15a and miR-15b induce an anti-tumor immune response by targeting PD-L1 in NB.

Experimental Validation of Novel Glypican 3 Exosomes for the Detection of Hepatocellular Carcinoma in Liver Cirrhosis.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) developing in the context of preexisting cirrhosis is characterized by impaired autophagy that results in increased exosome release. This study was conducted to determine whether circulating exosomes expressing glypican 3 (GPC3) could be utilized as a biomarker for HCC detection and treatment response in patients with cirrhosis. METHODS: Immunohistochemistry was performed to assess p62 and GPC3 expression in the lesion and adjacent tissue from cirrhosis with HCC. GPC3-enriched exosomes were captured by an enzyme-linked immunosorbent assay (ELISA). The diagnostic specificity of serum exosome-derived GPC3 (eGPC3) was determined using samples obtained from malignancy-free controls, malignancy-free cirrhotics, cirrhotics with confirmed HCC, and patients with a non-HCC malignancy. The performance of eGPC3 was validated using serum samples of HCC patients received chemotherapy. RESULTS: We found that the expression of p62 and GPC3 was significantly increased in HCC tissues compared to adjacent cirrhotic liver. Impaired autophagy and exosome shedding were confirmed in HCC cell lines. Mass spectroscopic analysis revealed that GPC3 was enriched in exosomes isolated from HCC cell lines. An affinity ELISA assay was developed that specifically captures GPC3 positive exosomes in the serum. Total exosome concentration and eGPC3 were significantly elevated in cirrhotic patients with HCC as compared to the reference control groups. Furthermore, decreases in post-treatment exosome concentration and eGPC3 levels were more closely correlated with response to locoregional chemotherapy compared to change in serum AFP in HCC patients awaiting liver transplantation. CONCLUSION: We developed an affinity exosome capture assay to detect GPC3 enriched exosomes. Our preliminary assessment shows that GPC3 positive exosomes can be used for HCC detection and prediction of treatment outcomes.

Elevated S-adenosylhomocysteine induces adipocyte dysfunction to promote alcohol-associated liver steatosis.

It has been previously shown that chronic ethanol administration-induced increase in adipose tissue lipolysis and reduction in the secretion of protective adipokines collectively contribute to alcohol-associated liver disease (ALD) pathogenesis. Further studies have revealed that increased adipose S-adenosylhomocysteine (SAH) levels generate methylation defects that promote lipolysis. Here, we hypothesized that increased intracellular SAH alone causes additional related pathological changes in adipose tissue as seen with alcohol administration. To test this, we used 3-deazaadenosine (DZA), which selectively elevates intracellular SAH levels by blocking its hydrolysis. Fully differentiated 3T3-L1 adipocytes were treated in vitro for 48 h with DZA and analysed for lipolysis, adipokine release and differentiation status. DZA treatment enhanced adipocyte lipolysis, as judged by lower levels of intracellular triglycerides, reduced lipid droplet sizes and higher levels of glycerol and free fatty acids released into the culture medium. These findings coincided with activation of both adipose triglyceride lipase and hormone sensitive lipase. DZA treatment also significantly reduced adipocyte differentiation factors, impaired adiponectin and leptin secretion but increased release of pro-inflammatory cytokines, IL-6, TNF and MCP-1. Together, our results demonstrate that elevation of intracellular SAH alone by DZA treatment of 3T3-L1 adipocytes induces lipolysis and dysregulates adipokine secretion. Selective elevation of intracellular SAH by DZA treatment mimics ethanol's effects and induces adipose dysfunction. We conclude that alcohol-induced elevations in adipose SAH levels contribute to the pathogenesis and progression of ALD.

COVID-19 and Cancer Comorbidity: Therapeutic Opportunities and Challenges.

The coronavirus disease 2019 (COVID-19) is a viral disease caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that affects the respiratory system of infected individuals. COVID-19 spreads between humans through respiratory droplets produced when an infected person coughs or sneezes. The COVID-19 outbreak originated in Wuhan, China at the end of 2019. As of 29 Sept 2020, over 235 countries, areas or territories across the globe reported a total of 33,441,919 confirmed cases, and 1,003,497 confirmed deaths due to COVID-19. Individuals of all ages are at risk for infection, but in most cases disease severity is associated with age and pre-existing diseases that compromise immunity, like cancer. Numerous reports suggest that people with cancer can be at higher risk of severe illness and related deaths from COVID-19. Therefore, managing cancer care under this pandemic is challenging and requires a collaborative multidisciplinary approach for optimal care of cancer patients in hospital settings. In this comprehensive review, we discuss the impact of the COVID-19 pandemic on cancer patients, their care, and treatment. Further, this review covers the SARS-CoV-2 pandemic, genome characterization, COVID-19 pathophysiology, and associated signaling pathways in cancer, and the choice of anticancer agents as repurposed drugs for treating COVID-19.

Exosomes: Novel Players of Therapy Resistance in Neuroblastoma.

Neuroblastoma is a solid tumor (a lump or mass), often found in the small glands on top of the kidneys, and most commonly affects infants and young children. Among neuroblastomas, high-risk neuroblastomas are very aggressive and resistant to most kinds of intensive treatment. Immunotherapy, which uses the immune system to fight against cancer, has shown great promise in treating many types of cancer. However, high-risk neuroblastoma is often resistant to this approach as well. Recent studies revealed that small vesicles known as exosomes, which are envelopes, could deliver a cargo of small RNA molecules and provide communication between neuroblastoma cells and the surrounding cells and trigger metastasis and resistance to immunotherapy. In this chapter, we describe the role of exosomes and small RNA molecules in the metastasis and regression of neuroblastoma and the potential therapeutic approaches to combat this menace.

Amino Acids Regulate Cisplatin Insensitivity in Neuroblastoma.

Neuroblastoma are pediatric, extracranial malignancies showing alarming survival prognosis outcomes due to their resilience to current aggressive treatment regimens, including chemotherapies with cisplatin (CDDP) provided in the first line of therapy regimens. Metabolic deregulation supports tumor cell survival in drug-treated conditions. However, metabolic pathways underlying cisplatin-resistance are least studied in neuroblastoma. Our metabolomics analysis revealed that cisplatin-insensitive cells alter their metabolism; especially, the metabolism of amino acids was upregulated in cisplatin-insensitive cells compared to the cisplatin-sensitive neuroblastoma cell line. A significant increase in amino acid levels in cisplatin-insensitive cells led us to hypothesize that the mechanisms upregulating intracellular amino acid pools facilitate insensitivity in neuroblastoma. We hereby report that amino acid depletion reduces cell survival and cisplatin-insensitivity in neuroblastoma cells. Since cells regulate their amino acids levels through processes, such as autophagy, we evaluated the effects of hydroxychloroquine (HCQ), a terminal autophagy inhibitor, on the survival and amino acid metabolism of cisplatin-insensitive neuroblastoma cells. Our results demonstrate that combining HCQ with CDDP abrogated the amino acid metabolism in cisplatin-insensitive cells and sensitized neuroblastoma cells to sub-lethal doses of cisplatin. Our results suggest that targeting of amino acid replenishing mechanisms could be considered as a potential approach in developing combination therapies for treating neuroblastomas.

miR-15a-5p, miR-15b-5p, and miR-16-5p inhibit tumor progression by directly targeting MYCN in neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid malignancy in children. Despite current aggressive treatment regimens, the prognosis for high-risk NB patients remains poor, with the survival of less than 40%. Amplification/stabilization of MYCN oncogene, in NB is associated with a high risk of recurrence. Thus, there is an urgent need for novel therapeutics. The deregulated expression of microRNA (miR) is reported in NB; nonetheless, its effect on MYCN regulation is poorly understood. First, we identified that miR-15a-5p, miR-15b-5p, and miR-16-5p (hereafter miR-15a, miR-15b or miR-16) were down-regulated in patient-derived xenografts (PDX) with high MYCN expression. MiR targeting sequences on MYCN mRNA were predicted using online databases such as TargetScan and miR database. The R2 database, containing 105 NB patients, showed an inverse correlation between MYCN mRNA and deleted in lymphocytic leukemia (DLEU) 2, a host gene of miR-15. Moreover, overexpression of miR-15a, miR-15b or miR-16 significantly reduced the levels of MYCN mRNA and N-Myc protein. Conversely, inhibiting miR dramatically enhanced MYCN mRNA and N-Myc protein levels, as well as increasing mRNA half-life in NB cells. By performing immunoprecipitation assays of argonaute-2 (Ago2), a core component of the RNA-induced silencing complex, we showed that miR-15a, miR-15b and miR-16 interact with MYCN mRNA. Luciferase reporter assays showed that miR-15a, miR-15b and miR-16 bind with 3'UTR of MYCN mRNA, resulting in MYCN suppression. Moreover, induced expression of miR-15a, miR-15b and miR-16 significantly reduced the proliferation, migration, and invasion of NB cells. Finally, transplanting miR-15a-, miR-15b- and miR-16-expressing NB cells into NSG mice repressed tumor formation and MYCN expression. These data suggest that miR-15a, miR-15b and miR-16 exert a tumor-suppressive function in NB by targeting MYCN. Therefore, these miRs could be considered as potential targets for NB treatment.

Long non-coding RNAs and nuclear factor-κB crosstalk in cancer and other human diseases.

The regulation of the pleiotropic transcription factor, nuclear factor-κB (NF-κB) by miRNAs and proteins is extensively studied. More recently, the NF-κB signaling was also reported to be regulated by several long non-coding RNAs (lncRNAs) that constitute the major portion of the noncoding component of the human genome. The common NF-κB associated lncRNAs include NKILA, HOTAIR, MALAT1, ANRIL, Lethe, MIR31HG, and PACER. The lncRNA and NF-κB signaling crosstalk during cancer and other diseases such as cardiomyopathy, celiac disease, cerebral infarction, chronic kidney disease, diabetes mellitus, Kawasaki disease, pregnancy loss, and rheumatoid arthritis. Some NF-κB related lncRNAs can affect gene expression without modulating NF-κB signaling. Most of the lncRNAs with a potential to modulate NF-κB signaling are regulated by NF-κB itself suggesting a feedback regulation. The discovery of lncRNAs have provided a new type of regulation for the NF-κB signaling and thus could be explored for therapeutic interventions. The manner in which lncRNA and NF-κB crosstalk affects human pathophysiology is discussed in this review. The challenges associated with the therapeutic interventions of this crosstalk are also discussed.

The role of exosomes and MYC in therapy resistance of acute myeloid leukemia: Challenges and opportunities.

Acute myeloid leukemia (AML) is caused by abnormal production of white blood cells, red blood cells or platelets. The leukemia cells communicate with their microenvironment through nano-vesicle exosomes that are 30-100 nm in diameter. These nano-vesicles are released from body fluids upon fusion of an endocytic compartment with the cell membrane. Exosomes function as cargo to deliver signaling molecules to distant cells. This allows cross-talk between hematopoietic cells and other distant target cell environments. Exosomes support leukemia growth by acting as messengers between tumor cells and the microenvironment as well as inducing oncogenic factors such as c-Myc. Exosomes have also been used as biomarkers in the clinical diagnosis of leukemia. Glycogen synthase kinase-3 (GSK-3) and protein phosphatase 2A (PP2A) are two crucial signaling molecules involved in the AML pathogenesis and MYC stability. GSK-3 is a serine/threonine protein kinase that coordinates with over 40 different proteins during physiological/pathological conditions in blood cells. The dysregulation in GSK-3 has been reported during hematological malignancies. GSK-3 acts as a tumor suppressor by targeting c-MYC, MCL-1 and ß-catenin. Conversely, GSK-3 can also act as tumor promoter in some instances. The pharmacological modulators of GSK-3 such as ABT-869, 6-Bromoindirubin-3'-oxime (BIO), GS-87 and LY2090314 have shown promise in the treatment of hematological malignancy. PP2A is a heterotrimeric serine/threonine phosphatase involved in the regulation of hematological malignancy. PP2A-activating drugs (PADs) can effectively antagonize leukemogenesis. The discovery of exosomes, kinase inhibitors and phosphatase activators have provided new hope to the leukemia patients. This review discusses the role of exosomes, GSK-3 and PP2A in the pathogenesis of leukemia. We provide evidence from both preclinical and clinical studies.

The Transcribed-Ultra Conserved Regions: Novel Non-Coding RNA Players in Neuroblastoma Progression.

The Transcribed-Ultra Conserved Regions (T-UCRs) are a class of novel non-coding RNAs that arise from the dark matter of the genome. T-UCRs are highly conserved between mouse, rat, and human genomes, which might indicate a definitive role for these elements in health and disease. The growing body of evidence suggests that T-UCRs contribute to oncogenic pathways. Neuroblastoma is a type of childhood cancer that is challenging to treat. The role of non-coding RNAs in the pathogenesis of neuroblastoma, in particular for cancer development, progression, and therapy resistance, has been documented. Exosmic non-coding RNAs are also involved in shaping the biology of the tumor microenvironment in neuroblastoma. In recent years, the involvement of T-UCRs in a wide variety of pathways in neuroblastoma has been discovered. Here, we present an overview of the involvement of T-UCRs in various cellular pathways, such as DNA damage response, proliferation, chemotherapy response, MYCN (v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian)) amplification, gene copy number, and immune response, as well as correlate it to patient survival in neuroblastoma.

Long non-coding RNAs are emerging targets of phytochemicals for cancer and other chronic diseases.

The long non-coding RNAs (lncRNAs) are the crucial regulators of human chronic diseases. Therefore, approaches such as antisense oligonucleotides, RNAi technology, and small molecule inhibitors have been used for the therapeutic targeting of lncRNAs. During the last decade, phytochemicals and nutraceuticals have been explored for their potential against lncRNAs. The common lncRNAs known to be modulated by phytochemicals include ROR, PVT1, HOTAIR, MALAT1, H19, MEG3, PCAT29, PANDAR, NEAT1, and GAS5. The phytochemicals such as curcumin, resveratrol, sulforaphane, berberine, EGCG, and gambogic acid have been examined against lncRNAs. In some cases, formulation of phytochemicals has also been used. The disease models where phytochemicals have been demonstrated to modulate lncRNAs expression include cancer, rheumatoid arthritis, osteoarthritis, and nonalcoholic fatty liver disease. The regulation of lncRNAs by phytochemicals can affect multi-steps of tumor development. When administered in combination with the conventional drugs, phytochemicals can also produce synergistic effects on lncRNAs leading to the sensitization of cancer cells. Phytochemicals target lncRNAs either directly or indirectly by affecting a wide variety of upstream molecules. However, the potential of phytochemicals against lncRNAs has been demonstrated mostly by preclinical studies in cancer models. How the modulation of lncRNAs by phytochemicals produce therapeutic effects on cancer and other chronic diseases is discussed in this review.

Targeting IκappaB kinases for cancer therapy.

The inhibitory kappa B kinases (IKKs) and IKK related kinases are crucial regulators of the pro-inflammatory transcription factor, nuclear factor kappa B (NF-κB). The dysregulation in the activities of these kinases has been reported in several cancer types. These kinases are known to regulate survival, proliferation, invasion, angiogenesis, and metastasis of cancer cells. Thus, IKK and IKK related kinases have emerged as an attractive target for the development of cancer therapeutics. Several IKK inhibitors have been developed, few of which have advanced to the clinic. These inhibitors target IKK either directly or indirectly by modulating the activities of other signaling molecules. Some inhibitors suppress IKK activity by disrupting the protein-protein interaction in the IKK complex. The inhibition of IKK has also been shown to enhance the efficacy of conventional chemotherapeutic agents. Because IKK and NF-κB are the key components of innate immunity, suppressing IKK is associated with the risk of immune suppression. Furthermore, IKK inhibitors may hit other signaling molecules and thus may produce off-target effects. Recent studies suggest that multiple cytoplasmic and nuclear proteins distinct from NF-κB and inhibitory κB are also substrates of IKK. In this review, we discuss the utility of IKK inhibitors for cancer therapy. The limitations associated with the intervention of IKK are also discussed.

Chaperone-Mediated Autophagy Promotes Beclin1 Degradation in Persistently Infected Hepatitis C Virus Cell Culture.

Liver cirrhosis is an independent risk factor for hepatocellular carcinoma (HCC). The mechanisms that contribute to HCC development in the cirrhotic microenvironment are unknown. We found that HCC grown in the highly stressed cirrhotic microenvironment undergoes autophagy switching from a protective state characterized by high macroautophagy with low chaperone-mediated autophagy (CMA) to an HCC-promoting state characterized by low macroautophagy with high CMA. This study examined how the stress response executes oncogenic cell programming through autophagy switching using hepatitis C virus cell culture. Protein kinase R-like endoplasmic reticulum kinase expression increased to high levels in hepatitis C virus culture. Protein kinase R-like endoplasmic reticulum kinase-dependent activation of nuclear factor erythroid 2-related factor (Nrf2) led to increased transcription of the cytoprotective genes: heat shock cognate 70 kDa protein and lysosome-associated membrane protein 2A (LAMP2A) and precipitated the induction of CMA. CMA selectively targeted beclin1 degradation, leading to accumulation of the autophagy flux protein p62 due to impaired autophagosome-endosome fusion. This impaired autophagosome-endosome fusion due to beclin1 degradation inhibited endocytosis and degradation of epidermal growth factor receptor. Silencing Nrf2 and LAMP2A reduced cell viability, suggesting that the stress response activates CMA as a compensatory mechanism of cell survival. We report a novel mechanism through which stress response triggers oncogenic Nrf2 signaling that promotes autophagy switching to favor cell survival.

PD-L1, inflammation, non-coding RNAs, and neuroblastoma: Immuno-oncology perspective.

Neuroblastoma is the most common pediatric solid tumor of neural crest origin. The current treatment options for neuroblastoma produce severe side effects. Programmed death-ligand 1 (PD-L1), chronic inflammation, and non-coding RNAs are known to play a significant role in the pathogenesis of neuroblastoma. Cancer cells and the surrounding cells in the tumor microenvironment express PD-L1. Programmed death-1 (PD-1) is a co-receptor expressed predominantly by T cells. The binding of PD-1 to its ligands, PD-L1 or PD-L2, is vital for the physiologic regulation of the immune system. Chronic inflammation is involved in the recruitment of leukocytes, production of cytokines and chemokines that in turn, lead to survival, metastasis, and angiogenesis in neuroblastoma tumors. The miRNAs and long non-coding (lnc) RNAs have emerged as a novel class of non-coding RNAs that can regulate neuroblastoma associated cell-signaling pathways. The dysregulation of PD-1/PD-L1, inflammatory pathways, lncRNAs, and miRNAs have been reported in clinical and experimental samples of neuroblastoma. These signaling molecules are currently being evaluated for their potential as the biomarker and therapeutic targets in the management of neuroblastoma. A monoclonal antibody called dinutuximab (Unituxin) that attaches to a carbohydrate molecule GD2, on the surface of many neuroblastoma cells, is being used as an immunotherapy drug for neuroblastoma treatment. Atezolizumab (Tecentriq), an engineered monoclonal antibody against PD-L1, are currently in clinical trial for neuroblastoma patients. The lncRNA/miRNA-based therapeutics is being developed to deliver tumor suppressor lncRNAs/miRNAs or silencing of oncogenic lncRNAs/miRNAs. The focus of this review is to discuss the current knowledge on the immune checkpoint molecules, PD-1/PD-L1 signaling, inflammation, and non-coding RNAs in neuroblastoma.

Activation of PERK-Nrf2 oncogenic signaling promotes Mdm2-mediated Rb degradation in persistently infected HCV culture.

The mechanism of how chronic hepatitis C virus (HCV) infection leads to such a high rate of hepatocellular carcinoma (HCC) is unknown. We found that the PERK axis of endoplasmic reticulum (ER) stress elicited prominent nuclear translocation of Nrf2 in 100% of HCV infected hepatocytes. The sustained nuclear translocation of Nrf2 in chronically infected culture induces Mdm2-mediated retinoblastoma protein (Rb) degradation. Silencing PERK and Nrf2 restored Mdm2-mediated Rb degradation, suggesting that sustained activation of PERK/Nrf2 axis creates oncogenic stress in chronically infected HCV culture model. The activation of Nrf2 and its nuclear translocation were prevented by ER-stress and PERK inhibitors, suggesting that PERK axis is involved in the sustained activation of Nrf2 signaling during chronic HCV infection. Furthermore, we show that HCV clearance induced by interferon-α based antiviral normalized the ER-stress response and prevented nuclear translocation of Nrf2, whereas HCV clearance by DAAs combination does neither. In conclusion, we report here a novel mechanism for how sustained activation of PERK axis of ER-stress during chronic HCV infection activates oncogenic Nrf2 signaling that promotes hepatocyte survival and oncogenesis by inducing Mdm2-mediated Rb degradation.