Spatial transcriptomics unravels palmitoylation and zonation-dependent gene regulation by AEG-1 in mouse liver.

Obesity-induced metabolic dysfunction-associated steatohepatitis (MASH) leads to hepatocellular carcinoma (HCC). Astrocyte-elevated gene-1/Metadherin (AEG-1/MTDH) plays a key role in promoting MASH and HCC. AEG-1 is palmitoylated at residue cysteine 75 (Cys75) and a knock-in mouse representing mutated Cys75 to serine (AEG-1-C75S) showed activation of MASH- and HCC-promoting gene signature when compared to wild-type littermates (AEG-1-WT). The liver consists of three zones, periportal, mid-lobular, and pericentral, and zone-specific dysregulated gene expression impairs metabolic homeostasis in the liver, contributing to MASH and HCC. Here, to elucidate how palmitoylation influences AEG-1-mediated gene regulation in regard to hepatic zonation, we performed spatial transcriptomics (ST) in the livers of AEG-1-WT and AEG-1-C75S littermates. ST identified six different clusters in livers and using zone- and cell-type-specific markers we attributed specific zones and cell types to specific clusters. Ingenuity Pathway Analysis (IPA) of differentially expressed genes in each cluster unraveled activation of pro-inflammatory and MASH- and HCC-promoting pathways, mainly in periportal and pericentral hepatocytes, in AEG-1-C75S liver compared to AEG-1-WT. Interestingly, in AEG-1-C75S liver, the mid-lobular zone exhibited widespread inhibition of xenobiotic metabolism pathways and inhibition of PXR/RXR and LXR/RXR activation, versus AEG-1-WT. In conclusion, AEG-1-C75S mutant exhibited zone-specific differential gene expression, which might contribute to metabolic dysfunction and dysregulated drug metabolism leading to MASH and HCC.

Melanoma differentiation associated gene-9/syndecan binding protein promotes hepatocellular carcinoma.

BACKGROUND AND AIMS: The oncogene Melanoma differentiation associated gene-9/syndecan binding protein (MDA-9/SDCBP) is overexpressed in many cancers, promoting aggressive, metastatic disease. However, the role of MDA-9 in regulating hepatocellular carcinoma (HCC) has not been well studied. APPROACH AND RESULTS: To unravel the function of MDA-9 in HCC, we generated and characterized a transgenic mouse with hepatocyte-specific overexpression of MDA-9 (Alb/MDA-9). Compared with wild-type (WT) littermates, Alb/MDA-9 mice demonstrated significantly higher incidence of N-nitrosodiethylamine/phenobarbital-induced HCC, with marked activation and infiltration of macrophages. RNA sequencing (RNA-seq) in naive WT and Alb/MDA-9 hepatocytes identified activation of signaling pathways associated with invasion, angiogenesis, and inflammation, especially NF-κB and integrin-linked kinase signaling pathways. In nonparenchymal cells purified from naive livers, single-cell RNA-seq showed activation of Kupffer cells and macrophages in Alb/MDA-9 mice versus WT mice. A robust increase in the expression of Secreted phosphoprotein 1 (Spp1/osteopontin) was observed upon overexpression of MDA-9. Inhibition of NF-κB pathway blocked MDA-9-induced Spp1 induction, and knock down of Spp1 resulted in inhibition of MDA-9-induced macrophage migration, as well as angiogenesis. CONCLUSIONS: Alb/MDA-9 is a mouse model with MDA-9 overexpression in any tissue type. Our findings unravel an HCC-promoting role of MDA-9 mediated by NF-κB and Spp1 and support the rationale of using MDA-9 inhibitors as a potential treatment for aggressive HCC.

Isolation and Culture of Mouse Hepatocytes and Kupffer Cells (KCs).

Nonalcoholic steatohepatitis (NASH) is characterized by accumulation of lipids in the hepatocytes (steatosis) and chronic inflammation. Liver resident macrophages (Kupffer cells) play a pivotal role in inducing inflammation. Cross-talk between hepatocytes and Kupffer cells (KCs) regulate both steatosis and inflammation during the pathogenesis of NASH. Isolated hepatocytes and KC serve as important tools to study mechanistic events during NASH in an in vitro setting. Because mice and humans share identical genes, primary mouse hepatocytes and KC are valuable ex vivo models for NASH studies. However, isolation of mouse liver cells is challenging and requires specific technical procedure and skills. Here, we elaborate a method for effective isolation of both primary hepatocytes and KC from adult liver of the same mouse. This protocol can be used for isolation of liver cells from both wild-type (WT) and genetically-engineered mice. The principle of the method is based on a two-step collagenase perfusion technique in which the liver is washed by perfusion, liver cells are segregated by collagenase treatment, and hepatocytes and KC are then purified and cultured. We optimized this protocol in terms of reproducibility, yield of different population of liver cells, and viability.

Mouse Bone Marrow Cell Isolation and Macrophage Differentiation.

The rapid increase in the incidence of obesity contributes to a parallel increase in nonalcoholic steatohepatitis (NASH). Monocyte-derived macrophages, recruited from the bone marrow to the liver, promote NASH-related inflammation and fibrosis. In addition, adipose tissue macrophages (ATMs) release pro-inflammatory cytokines (PICs) which stimulate adipose tissue lipolysis liberating free fatty acids (FFAs) that can accumulate in the liver as triglycerides (TGs), thereby inducing steatosis. As such, bone marrow-derived macrophages (BMDMs) function as an essential tool to study the pathogenesis of NASH. BMDMs are primary bone marrow-derived cells which are differentiated into macrophages in vitro in the presence of growth factors. Macrophage colony-stimulating factor (M-CSF) is required for the proliferation and differentiation of committed myeloid progenitors into cells of the macrophage/monocyte lineage. Here, we describe a protocol for the isolation of mouse bone marrow cells and subsequent macrophage differentiation in which bone marrow cells are cultured in the presence of M-CSF, supplemented either by conditioned medium from L929 cells or in purified form. The efficiency of the differentiation is confirmed by immunofluorescent staining of macrophage surface antigen F4/80. The BMDMs serve as an excellent ex vivo model for a variety of studies, including hepatocyte-macrophage and adipocyte-macrophage cross-talk regulating NASH.

Dissecting the Balance Between Metabolic and Oncogenic Functions of Astrocyte-Elevated Gene-1/Metadherin.

Obesity is an enormous global health problem, and obesity-induced nonalcoholic steatohepatitis (NASH) is contributing to a rising incidence and mortality for hepatocellular carcinoma (HCC). Increase in de novo lipogenesis and decrease in fatty acid ß-oxidation (FAO) underlie hepatic lipid accumulation in NASH. Astrocyte-elevated gene-1/metadherin (AEG-1) overexpression contributes to both NASH and HCC. AEG-1 harbors an LXXLL motif through which it blocks activation of peroxisome proliferator activated receptor α (PPARα), a key regulator of FAO. To better understand the role of LXXLL motif in mediating AEG-1 function, using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, we generated a mouse model (AEG-1-L24K/L25H) in which the LXXLL motif in AEG-1 was mutated to LXXKH. We observed increased activation of PPARα in AEG-1-L24K/L25H livers providing partial protection from high-fat diet-induced steatosis. Interestingly, even with equal gene dosage levels, compared with AEG-1-wild-type livers, AEG-1-L24K/L25H livers exhibited increase in levels of lipogenic enzymes, mitogenic activity and inflammation, which are attributes observed when AEG-1 is overexpressed. These findings indicate that while LXXLL motif favors steatotic activity of AEG-1, it keeps in check inflammatory and oncogenic functions, thus maintaining a homeostasis in AEG-1 function. AEG-1 is being increasingly appreciated as a viable target for ameliorating NASH and NASH-HCC, and as such, in-depth understanding of the functions and molecular attributes of this molecule is essential. Conclusion: The present study unravels the unique role of the LXXLL motif in mediating the balance between the metabolic and oncogenic functions of AEG-1.

Multifunctional Role of Astrocyte Elevated Gene-1 (AEG-1) in Cancer: Focus on Drug Resistance.

Cancer development results from the acquisition of numerous genetic and epigenetic alterations in cancer cells themselves, as well as continuous changes in their microenvironment. The plasticity of cancer cells allows them to continuously adapt to selective pressures brought forth by exogenous environmental stresses, the internal milieu of the tumor and cancer treatment itself. Resistance to treatment, either inherent or acquired after the commencement of treatment, is a major obstacle an oncologist confronts in an endeavor to efficiently manage the disease. Resistance to chemotherapy, chemoresistance, is an important hallmark of aggressive cancers, and driver oncogene-induced signaling pathways and molecular abnormalities create the platform for chemoresistance. The oncogene Astrocyte elevated gene-1/Metadherin (AEG-1/MTDH) is overexpressed in a diverse array of cancers, and its overexpression promotes all the hallmarks of cancer, such as proliferation, invasion, metastasis, angiogenesis and chemoresistance. The present review provides a comprehensive description of the molecular mechanism by which AEG-1 promotes tumorigenesis, with a special emphasis on its ability to regulate chemoresistance.

Non-Coding RNAs: Regulating Disease Progression and Therapy Resistance in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC), the primary liver cancer arising from hepatocytes, is a universal health problem and one of the most common malignant tumors. Surgery followed by chemotherapy as well as tyrosine kinase inhibitors (TKIs), such as sorafenib, are primary treatment procedures for HCC, but recurrence of disease because of therapy resistance results in high mortality. It is necessary to identify novel regulators of HCC for developing effective targeted therapies that can significantly interfere with progression of the disease process. Non-coding RNAs (ncRNAs) are an abundant group of versatile RNA transcripts that do not translate into proteins, rather serve as potentially functional RNAs. The role of ncRNAs in regulating diverse aspects of the carcinogenesis process are gradually being elucidated. Recent advances in RNA sequencing technology have identified a plethora of ncRNAs regulating all aspects of hepatocarcinogenesis process and serving as potential prognostic or diagnostic biomarkers. The present review provides a comprehensive description of the biological roles of ncRNAs in disease process and therapy resistance, and potential clinical application of these ncRNAs in HCC.

Anticancer activity of a 1,4-dihydropyridine in DMBA-induced mouse skin tumor model.

Antitumor potential of a 1,4-dihydropyridine derivative (DHP-8) has been successfully studied previously in a number of cancer cell lines including the human melanoma cells, A375. In order to validate its anticancer activity, DMBA induced tumor in Swiss Albino mice was considered for this study. DMBA causes skin carcinoma in murine systems and is an important in vivo model for evaluating the efficacy of any new chemical entity against skin cancer. Topical administration of DHP-8 at the dose rate of 33.3 and 50.0 mg/kg body weight showed a significant reduction in tumor parameters. It also prevented the progression and differentiation of squamous cell carcinoma, as evidenced from histopathological studies. Immunohistochemical analysis for the expression of Ki67 indicated that it also reduced cancer cell proliferation. Additionally, it induced apoptosis in the tumor cells by activation of Caspase3. Our results indicated that DHP-8 efficiently attenuated DMBA induced tumor progression and it could be a potent therapeutic agent for skin cancer treatment.

Probing the mechanism of SIRT1 activation by a 1,4-dihydropyridine.

The NAD+-dependent deacetylase SIRT1 plays important roles in several physiological processes such as transcription, genome stability, stress responses, and aging. Due to its diverse role in metabolisms, SIRT1 has emerged as a potential therapeutic target in many human disorders such as type II diabetes, cardiovascular and neurodegenerative diseases, and cancer. Recent studies have reported that modulation of SIRT1 activity by phenolic activators like resveratrol and some 1,4-dihydropyridines (1,4-DHPs) can inhibit tumor growth by promoting apoptosis in cancer cells. However, the mechanism of SIRT1 activation is still not clear. In this report, we have tried to elucidate the mechanism of SIRT1 activation from studies on its interaction with a synthetic 1,4-DHP derivative (DHP-8; 3,5-diethoxy carbonyl-4-(4-nitrophenyl)-2,6-dimethyl-1,4-dihydropyridine) using molecular modeling, docking, simulation, and free energy analyses. Owing to the absence of full-length human SIRT1 structure, multi-template based modeling approach was opted followed by docking of DHP-8 at its allosteric site. In presence of DHP-8, the overall conformation of SIRT1 was found to be more stable (especially at its substrate binding sites) with a large structural variation at its N-terminal domain while bound to substrate p53 or p53-W. Determination of the MM/PBSA free energy indicated that the binding of DHP-8 to SIRT1 significantly increased the binding affinity of SIRT1 to its substrate p53-W as well as to NAD+. Overall, this study depicts the atomistic detailed mechanism for the direct activation of SIRT1 by a 1,4-DHP. This would serve to develop new SIRT1 activators for future therapeutic perspectives.

Novel 1,4-dihydropyridine induces apoptosis in human cancer cells through overexpression of Sirtuin1.

1,4-Dihydropyridines (1,4-DHPs) are important as a class of heterocyclic compounds that exhibit wide range of biological actions. Many of its derivatives are already characterized as medicinally important drugs and used worldwide. In this study, we have screened some novel Hantzsch 1,4-DHP compounds using both in silico (QSAR and Pharmacophore) and in vitro (cytotoxic screening). 1,4-DHP showed selective cytotoxicity against five human cancerous cell lines; A375, A549, HeLa, HepG2 and SH-SY5Y but limited effect towards normal skin keratinocyte (HaCaT), lung fibroblast (WL-38) and healthy peripheral blood mononuclear cells. In A375 and HepG2 cells, one of the 1,4-DHP derivative (DHP-8) was found to inhibit cell proliferation, and simultaneously increased the apoptotic population as well as mitochondrial membrane depolarization. Furthermore, the mitochondrial signal was triggered with the activation of cleaved Caspase9, Caspase3 and PARP. The treatment with DHP-8 also increased the expression level of SIRT1, subsequently decreasing the level of pAKTser473 and survivin. Reduced pAKTser473 expression led to decrease the phosphorylated inactive form of GSK3ßser9 and as a result, proteasomal degradation of Mcl-1 occurred in both the cell lines. Here, we suggest that the apoptotic effect of DHP-8 in A375 and HepG2 cells was mediated by AKT and survivin pathways through SIRT1 activation. The involvement of DHP-8 in SIRT1 activation was further verified by co-treatment of nicotinamide with DHP-8 in both A375 and HepG2 cells. Overall, this study emphasizes the possible potential and therapeutic role of DHP-8 in skin and liver cancer.

Effect of radiofrequency radiation in cultured mammalian cells: A review.

The use of mobile phone related technologies will continue to increase in the foreseeable future worldwide. This has drawn attention to the probable interaction of radiofrequency electromagnetic radiation with different biological targets. Studies have been conducted on various organisms to evaluate the alleged ill-effect on health. We have therefore attempted to review those work limited to in vitro cultured cells where irradiation conditions were well controlled. Different investigators have studied varied endpoints like DNA damage, cell cycle arrest, reactive oxygen species (ROS) formation, cellular morphology and viability to weigh the genotoxic effect of such radiation by utilizing different frequencies and dose rates under various irradiation conditions that include continuous or pulsed exposures and also amplitude- or frequency-modulated waves. Cells adapt to change in their intra and extracellular environment from different chemical and physical stimuli through organized alterations in gene or protein expression that result in the induction of stress responses. Many studies have focused on such effects for risk estimations. Though the effects of microwave radiation on cells are often not pronounced, some investigators have therefore combined radiofrequency radiation with other physical or chemical agents to observe whether the effects of such agents were augmented or not. Such reports in cultured cellular systems have also included in this review. The findings from different workers have revealed that, effects were dependent on cell type and the endpoint selection. However, contradictory findings were also observed in same cell types with same assay, in such cases the specific absorption rate (SAR) values were significant.