Autophagic Genes Research Articles

Therapeutic Effects of Crocin Nanoparticles Alone or in Combination with Doxorubicin against Hepatocellular Carcinoma In vitro.

Crocin (CRO), the primary antioxidant in saffron, is known for its anticancer properties. However, its effectiveness in topical therapy is limited due to low bioavailability, poor absorption, and low physicochemical stability. This study aimed to prepare crocin nanoparticles (CRO-NPs) to enhance their pharmaceutical efficacy and evaluate the synergistic effects of Cro-NPs with doxorubicin (DOX) chemotherapy on two cell lines: human hepatocellular carcinoma cells (HepG2) and non-cancerous cells (WI38). CRO-NPs were prepared using the emulsion diffusion technique and characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), Zeta potential, and Fourier transform infrared spectroscopy (FT-IR). Cell proliferation inhibition was assessed using the MTT assay for DOX, CRO, CRO-NPs, and DOX+CRO-NPs. Apoptosis and cell cycle were evaluated by flow cytometry, and changes in the expression of apoptotic gene (P53) and autophagic genes (ATG5 & LC3) were analyzed using real-time polymerase chain reaction. TEM and SEM revealed that CRO-NPs exhibited a relatively spherical shape with an average size of 9.3 nm, and zeta potential analysis indicated better stability of CRO-NPs compared to native CRO. Significantly higher antitumor effects of CRO-NPs were observed against HepG2 cells (IC50 = 1.1 mg/ml and 0.57 mg/ml) compared to native CRO (IC50 = 6.1 mg/ml and 3.2 mg/ml) after 24 and 48 hours, respectively. Annexin-V assay on HepG2 cells indicated increased apoptotic rates across all treatments, with the highest percentage observed in CRO-NPs, accompanied by cell cycle arrest at the G2/M phase. Furthermore, gene expression analysis showed upregulation of P53, ATG5, and LC3 genes in DOX/CRO-NPs co-treatment compared to individual treatments. In contrast, WI38 cells exhibited greater sensitivity to DOX toxicity but showed no adverse response to CRONPs. Although more in vivo studies in animal models are required to corroborate these results, our findings suggest that CRO-NPs can be a potential new anticancer agent for hepatocellular carcinoma. Moreover, they have a synergistic effect with DOX against HepG2 cells and mitigate the toxicity of DOX on normal WI38 cells.

Phenolic Contents, Antioxidant Activities, LCMS Profiles of Mespilus germanica Leaf Extract and Effects on mRNA Transcription Levels of Apoptotic, Autophagic, and Necrotic Genes in MCF7 and A549 Cancer Cell Lines.

Cancer, defined by the continuous, uncontrollable proliferation of cells in the human body, is a disease with a rapidly increasing incidence and mortality rate. Scientists are looking for novel ways to cure and prevent this sneaky disease because of the toxicity of contemporary chemotherapy and the cancer cells' resilience to anticancer drugs. Determining the effect of herbal medicines, which do not have as harmful side effects as synthetic drugs, on cancer cell lines is an essential preliminary study in the production of effective drugs against cancer. In this study, the phenolic acid profile, antioxidant capacity, and cytotoxicity of the medicinal plant Mespilus germanica (MG) leaf extract were determined, and its effects on the expression of some apoptotic, necrotic, and autophagic pathway genes of MCF7 (Human breast cancer line) and A549 (Human lung cancer line) and healthy HDF (Human Dermal Fibroblasts) cells were investigated for the first time. The LCMS device detected many important phenolic compounds previously reported to act against cancer cells in Mespilus germanica leaf extract. DPPH and total phenolic content showed high antioxidant capacity. The cytotoxicity of MG was determined by the MTT method. The levels of mRNA transcription for Atg5, Atg3, Rıpk1, Bcl2, Bax, Apaf1, Caspase-8, Caspase-7, Caspase-3, and Caspase-9, as well as the expression patterns of the DNA damage markers P53 and Parp-1 genes, were assessed. MG leaf extract did not cause significant toxicity against healthy HDF cells. However, it had a cytotoxic effect on A549 and MCF7 cancer cell lines, increasing the transcription levels of essential genes involved in cell death mechanisms. This research is the first to analyze the phenolic components and antioxidant capabilities of leaf extracts from Mespilus germanica. Additionally, it investigates the impact of these extracts on crucial genes involved in cell death pathways of A549 lung cancer, MCF7 breast cancer, and non-cancerous HDF (Human Dermal Fibroblasts) cells.

Receptor-interacting protein kinase 1 confers autophagic promotion of gasdermin E-mediated pyroptosis in aristolochic acid-induced acute kidney injury

Aristolochic acid (AA) exposure is a severe public health concern worldwide. AAs damage the kidney with an inevitable acute phase that is similar to acute kidney injury (AKI). Gasdermin E (GSDME) is abundant in the kidney; thus; it-mediated pyroptosis might be essential in connecting cell death and inflammation and promoting AAs-AKI. However, the role and exact mechanism of pyroptosis in AAs-AKI have not been investigated. In this study, aristolochic acid I (AAI) was used to establish AKI models. The expression and translocation results showed GSDME-mediated pyroptosis in AAI-AKI. Knocking down GSDME attenuated AAI-induced cell death and transcription of proinflammatory cytokines. Mechanistic research inhibiting caspase (casp) 3, casp 8, and casp 9 with specific chemical antagonists demonstrated that GSDME was activated by cleaved casp 3. Furthermore, the kinase activity of upstream receptor-interacting protein kinase 1 (RIPK1) was significantly elevated, and inhibiting RIPK1 with specific inhibitors markedly improved AAI-induced cell damage. In addition, the level of autophagy was obviously increased. Pretreatment with a specific autophagic inhibitor (3-methyladenine) or knockdown of autophagic genes (Atg5 or Atg7) evidently reduced the activity of RIPK1 and downstream apoptosis and pyroptosis, thus attenuating AA-induced cell injury, which suggested that RIPK1 was a novel link conferring autophagic promotion of pyroptosis. These findings reveal GSDME-mediated pyroptosis for the first time in AAI-induced AKI, propose its novel role in the transcription of cytokines, and demonstrate that autophagy promotes pyroptosis via the RIPK1-dependent apoptotic pathway. This study promotes the understanding of the toxic effects and exact mechanisms of AAs. This will contribute to evaluating the environmental risk of AA exposure and might provide potential therapeutic targets for AA-AKI.

Effect of impaired autophagic flux on breast carcinogenesis through the enhanced LC3-p62-NRF2 feedback loop.

6 Background: Breast cancer affects more women worldwide than any other type of cancer. Although some genes have been identified as potential causes, the exact molecular mechanism of the disease is still unknown. In our study, we aimed to investigate the role of NRF2 and the autophagy markers p62 and LC3 in breast cancer cell lines that have different Tp53 status using a new compound called 6,6'dihydroxythiobinupharidine (DTBN). In our previous research, we have shown that DTBN modulates key cellular signal transduction pathways that are relevant to disease biology, including cancer. Furthermore, we analyzed online databases to determine the correlation between NRF2, p62, LC3, and Tp53 in breast cancer patients. Methods: To evaluate the effect of DTBN on NRF2, p62 and LC3 in different types of breast cancer cell lines with varying TP53 status, we conducted molecular studies by inhibiting NRF2 activity. We synthesized the correlation between NRF2, p62, LC3 and TP53 expression and clinical parameters by analyzing TCGA and GEO datasets. Results: Our findings suggest that DTBN induces cell death and autophagy in all breast cancer cell lines, regardless of their Tp53 status. However, they also led us to hypothesize that NRF2 may hinder the sensitivity of cancer cells to DTBN-induced cell death and autophagy. To test this hypothesis, we inhibited NRF2 activity using an NRF2 inhibitor called brusatol. The results of the study indicate that the inhibition of NRF2 significantly increased cell death and autophagy in DTBN-treated cells. We observed a decrease in the expression of the autophagy marker p62, while an increase in the expression of another autophagy marker LC3. These findings suggest that the interaction between NRF2, LC3 and p62, which is activated in response to DTBN treatment, may serve as a potential anticancer drug target. We have validated the positive correlation between NRF2 and the autophagic gene p62 and LC3, in different Tp53 status breast cancer cell lines by analyzing TCGA and GEO data. Conclusions: Our study shows that high levels of Nrf2, LC3 and p62 expression are significantly associated with increased breast cancer cell proliferation and migration. This implies that patients with breast cancer have lower overall survival and a higher rate of recurrence. Our findings highlight the role of DTBN-induced NRF2 and LC3 expression and reduced p62 expression in breast cancer cells with varying Tp53 status. These results may represent a paradigm for better understanding the cancer cell response to therapies and designing more efficient combined anticancer therapies targeting NRF2, LC3 p62, and Tp53.

Autophagic genes and antioxidant status during doxorubicin therapy under temperature-stressed conditions in breast cancer cells

ObjectivesAutophagy as a cellular event swings between apoptosis induction and cellular maintenance during chemotherapy. The interplay between autophagy and reactive oxygen species (ROS) remains obscure in cancer progression and treatment. This study aimed to determine the combination effect of chemotherapy and hyperthermia on cancer cell proliferation, autophagy signaling, and oxidative stress status. MethodsTo estimate the autophagic genes' role and antioxidant capacity involvement during chemotherapy, human breast cancer cell lines (MCF7 and CAL51) were exposed to a high temperature at 42 °C and treated with doxorubicin (DOX). The MTT assays were performed to determine the cell survival levels, and the autophagic gene expression levels (ATG5, LC3A, LC3B, and Beclin1) were analyzed. The intracellular TAS (total antioxidant status) and SOD (superoxide dismutase) activities were determined. ResultsThe combined effects of high temperature with chemotherapy significantly reduced cell viability and cell survival compared to cells treated with the chemotherapeutic agent DOX alone. The cell's autophagic gene activities were significantly increased after exposure to 42 °C temperature and DOX-based chemotherapy compared to the cells treated with DOX alone. It was observed that TAS and SOD activities were increased in the cell lines exposed to high temperatures with DOX compared to the cells treated with DOX alone. Increased autophagic gene expression levels and cell death were observed in response to the high temperature and DOX treatment in breast cancer cells. ConclusionThe study may provide a plausible route and treatment plan for the individual adoption of cancer chemotherapy and be developed as part of the personalized medicine protocol.

Delving into Autophagy: Utilizing Natural Bioactive Compounds to Combat Alzheimer's Disease

Abstract: Autophagy involves breaking down entire cell components, including organelles and macromolecules found in the cytoplasm of eukaryotic cells, especially proteins with extended lifespans. Pharmacological, therapeutic, and herbal methods are crucial throughout this deteriorating phase. Autophagy is a widespread and historically conserved process that occurs in all eukaryotic cells. The significance lies in cell malfunction impacting the autophagy process, which is associated with various significant conditions such as neurological and metabolic disorders in the brain. The role of various autophagic genes is also important in the positive regulation of autophagy. This research will provide a concise summary of various forms of autophagy, their molecular processes, their relationships to neuronal health, and the function of natural chemicals in the enhancement of autophagy. However, the focus of this work is on different ways to encourage autophagy. It is possible to treat metabolic neurodegenerative illnesses by triggering this process with a range of herbal and natural substances. In this article, these topics are explored and debated.

Selagibenzophenone B and Its Derivatives: SelB-1, a Dual Topoisomerase I/II Inhibitor Identified through In Vitro and In Silico Analyses.

The development of multitargeted drugs represents an innovative approach to cancer treatment, aiming to enhance drug effectiveness while minimizing side effects. Herein, we sought to elucidate the inhibitory effect of selagibenzophenone B derivatives on the survival of cancer cells and dual topoisomerase I/II enzyme activity. Results demonstrated that among the compounds, SelB-1 selectively inhibited the proliferation and migration of prostate cancer cells while exhibiting minimal effects on healthy cells. Furthermore, SelB-1 showed a dual inhibitory effect on topoisomerases. Computational analyses mirrored the results from enzyme inhibition assays, demonstrating the compound's strong binding affinity to the catalytic sites of the topoisomerases. To our surprise, SelB-1 did not induce apoptosis in prostate cancer cells; instead, it induced autophagic gene expression and lipid peroxidation while reducing GSH levels, which might be associated with ferroptotic death mechanisms. To summarize, the findings suggest that SelB-1 possesses the potential to serve as a dual topoisomerase inhibitor and can be further developed as a promising candidate for prostate cancer treatment.

Screening and Mechanism Study of Three Antagonistic Drugs, Oxysophoridine, Rutin, and Phellodendrine, against Zearalenone-Induced Reproductive Toxicity in Ovine Oocytes.

Zearalenone (ZEN) is a common fungal toxin with reproductive toxicity in various grains. It poses a serious threat to ovine and other animal husbandry industries, as well as human reproductive health. Therefore, investigating the mechanism of toxicity and screening antagonistic drugs are of great importance. In this study, based on the natural compound library and previous Smart-seq2 results, antioxidant and anti-apoptotic drugs were selected for screening as potential antagonistic drugs. Three natural plant compounds (oxysophoridine, rutin, and phellodendrine) were screened for their ability to counteract the reproductive toxicity of ZEN on ovine oocytes in vitro using quantitative polymerase chain reaction (qPCR) and reactive oxygen species detection. The compounds exhibited varying pharmacological effects, notably impacting the expression of antioxidant (GPX, SOD1, and SOD2), autophagic (ATG3, ULK2, and LC3), and apoptotic (CAS3, CAS8, and CAS9) genes. Oxysophoridine promoted GPX, SOD1, ULK2, and LC3 expression, while inhibiting CAS3 and CAS8 expression. Rutin promoted SOD2 and ATG3 expression, and inhibited CAS3 and CAS9 expression. Phellodendrine promoted SOD2 and ATG3 expression, and inhibited CAS9 expression. However, all compounds promoted the expression of genes related to cell cycle, spindle checkpoint, oocyte maturation, and cumulus expansion factors. Although the three drugs had different regulatory mechanisms in enhancing antioxidant capacity, enhancing autophagy, and inhibiting cell apoptosis, they all maintained a stable intracellular environment and a normal cell cycle, promoted oocyte maturation and release of cumulus expansion factors, and, ultimately, counteracted ZEN reproductive toxicity to promote the in vitro maturation of ovine oocytes. This study identified three drugs that antagonize the reproductive toxicity of ZEN on ovine oocytes, and compared their mechanisms of action, providing data support and a theoretical basis for their subsequent application in the ovine breeding industry, reducing losses in the breeding industry, screening of ZEN reproductive toxicity antagonists and various toxin antagonists, improving the study of ZEN reproductive toxicity mechanisms, and even protection of human reproductive health.

Exercise-Induced Laminar Blood Flow Maintains Vascular Function by Enhanced Endothelial Homeostasis

PURPOSE: Laminar blood flow is known to play a critical role in maintaining endothelial homeostasis in blood vessels. It exerts a shear stress on the endothelial cells (ECs), which is essential for the regulation of various vascular functions. In this study, the effects of laminar blood flow-induced enhanced endothelial homeostasis on the maintenance of vascular function were investigated.METHODS: Human umbilical vein endothelial cells (HUVECs) were employed to study gene expression associated with vessel dilation and angiogenesis in response to varying shear stress levels. To assess angiogenesis, experimental mice participated in voluntary wheel running for 16 weeks. To evaluate the vascular autophagic function, the six weeks old C57BL/6 mice underwent an hour of forced treadmill running for 10 weeks. The resulting gene expression was evaluated through western blotting and quantitative real-time polymerase chain reaction (qRT-PCR).RESULTS: Disturbed flow resulting from partial ligation surgery in the mice’s carotid artery led to a decrease in angiogenic capacity compared to the laminar flow observed in ex vivo sprouting assays. Cultured HUVECs exhibited a significant increase in VEGF expression when exposed to increased durations of 20 dyne/cm² laminar shear stress (LSS). Following a 10-week treadmill exercise regimen, ECs in the carotid arteries of mice showed a significant increase in the expression of autophagic genes, such as LC3II, Atg3, and Atg7, which was not observed in smooth muscle cells (SMCs). LSS was found to boost the expression of both total and phosphorylated eNOS, genes involved in vascular dilation regulation, specifically in ECs.CONCLUSIONS: These findings suggest that laminar blood flow sustains vascular function, such as angiogenic capacity, through the enhancement of endothelial homeostasis.

CTCF and BORIS-mediated autophagy regulation via alternative splicing of BNIP3L in breast cancer

Autophagy is a pivotal regulatory and catabolic process, induced under various stressful conditions, including hypoxia. However, little is known about alternative splicing of autophagy genes in the hypoxic landscape in breast cancer. Our research unravels the hitherto unreported alternative splicing of BNIP3L, a crucial hypoxia-induced autophagic gene. We showed that BNIP3L, under hypoxic condition, forms two isoforms, a full-length isoform (BNIP3L-F) and a shorter isoform lacking exon 1 (BNIP3L-Δ1). The hypoxia-induced BNIP3L-F promotes autophagy, while under normoxia, the BNIP3L-Δ1 inhibits autophagy. We discovered a novel dimension of hypoxia-mediated epigenetic modification that regulates the alternative splicing of BNIP3L. Here, we showed differential DNA methylation of BNIP3L intron 1, causing reciprocal binding of epigenetic factor CTCF and its paralogue BORIS. Additionally, we highlighted the role of CTCF, and BORIS impacting autophagy in breast cancer. The differential binding of CTCF and BORIS results in alternative splicing of BNIP3L forming BNIP3L-F and BNIP3L-Δ1, respectively. The binding of CTCF on unmethylated BNIP3L intron 1 under hypoxia results in RNA Pol-II pause and inclusion of exon 1, promoting BNIP3L-F and autophagy. Interestingly, the binding of BORIS on methylated BNIP3L intron 1 under normoxia also results in RNA Pol-II pause but leads to the exclusion of exon 1 from BNIP3L mRNA. Finally, we reported the critical role of BORIS-mediated RNA Pol-II pause, which subsequently recruits SRSF6, redirecting the proximal splice-site selection, promoting BNIP3L-Δ1, and inhibiting autophagy. Our study provides novel insights into the potential avenues for breast cancer therapy by targeting autophagy regulation, specifically under hypoxic condition.

Neuronal autosis is Na+/K+-ATPase alpha 3-dependent and involved in hypoxic-ischemic neuronal death

Macroautophagy (hereafter called autophagy) is an essential physiological process of degradation of organelles and long-lived proteins. The discovery of autosis, a Na+/K+-ATPase (ATP1)-dependent type of autophagic cell death with specific morphological and biochemical features, has strongly contributed to the acceptance of a pro-death role of autophagy. However, the occurrence and relevance of autosis in neurons has never been clearly investigated, whereas we previously provided evidence that autophagy mechanisms could be involved in neuronal death in different in vitro and in vivo rodent models of hypoxia-ischemia (HI) and that morphological features of autosis were observed in dying neurons following rat perinatal cerebral HI. In the present study, we demonstrated that neuronal autosis could occur in primary cortical neurons using two different stimulations enhancing autophagy flux and neuronal death: a neurotoxic concentration of Tat-BECN1 (an autophagy-inducing peptide) and a hypoxic/excitotoxic stimulus (mimicking neuronal death induced by cerebral HI). Both stimulations induce autophagic neuronal death (dependent on canonical autophagic genes and independent on apoptotic, necroptotic or ferroptotic pathways) with all morphological and biochemical (ATP1a-dependent) features of autosis. However, we demonstrated that autosis is not dependent on the ubiquitous subunit ATP1a1 in neurons, as in dividing cell types, but on the neuronal specific ATP1a3 subunit. We also provided evidence that, in different in vitro and in vivo models where autosis is induced, ATP1a3-BECN1 interaction is increased and prevented by cardiac glycosides treatment. Interestingly, an increase in ATP1a3-BECN1 interaction is also detected in dying neurons in the autoptic brains of human newborns with severe hypoxic-ischemic encephalopathy (HIE). Altogether, these results suggest that ATP1a3-BECN1-dependent autosis could play an important role in neuronal death in HI conditions, paving the way for the development of new neuroprotective strategies in hypoxic-ischemic conditions including in severe case of human HIE.

TFE3–SLC36A1 axis promotes resistance to glucose starvation in kidney cancer cells

Higher demand for nutrients including glucose is characteristic of cancer. "Starving cancer" has been pursued to curb tumor progression. An ,intriguing regime is to inhibit glucose transporter GLUT1 in cancer cells. In addition, during cancer progression, cancer cells may suffer from insufficient glucose supply. Yet cancer cells can somehow tolerate glucose starvation. Uncovering the underlying mechanisms shall not only shed insight into cancer progression but also benefit cancer therapy. TFE3 is a transcription factor known to activate autophagic genes. Physiological TFE3 activity is regulated by phosphorylation-triggered translocation responsive to nutrient status. We recently reported TFE3 constitutively localizes to the cell nucleus and promotes cell proliferation in kidney cancer even under nutrient replete condition. Whether and how TFE3 responds to glucose starvation remain unclear. In this study, we show TFE3 promotes kidney cancer cell resistance to glucose starvation by exposing cells to physiologically relevant glucose concentration. We find glucose starvation triggers TFE3 protein stabilization through increasing its O-GlcNAcylation. Furthermore, through an unbiased functional genomic study, we identify SLC36A1, a lysosomal amino acid transporter, as a TFE3 target gene sensitive to TFE3 protein level. We find SLC36A1 is overexpressed in kidney cancer, which promotes mTOR activity and kidney cancer cell proliferation. Importantly, SLC36A1 level is induced by glucose starvation through TFE3, which enhances cellular resistance to glucose starvation. Suppressing TFE3 or SLC36A1 significantly increases cellular sensitivity to GLUT1 inhibitor in kidney cancer cells. Collectively, we uncover a functional TFE3-SLC36A1 axis that responds to glucose starvation and enhances starvation tolerance in kidney cancer.

Abstract 5481: Spatial multiplex proteins profiling of tumor microenvironment to decipher de novo metastasis in nasopharyngeal carcinoma

Abstract Background: Nasopharyngeal carcinoma (NPC) has a high incidence rate in Southeast Asia, where more than 90% of NPC cases are associated with Epstein-Barr virus (EBV). Although NPC patients are generally sensitive to radiotherapy and chemotherapy, about 20-30% of patients have treatment failure due to the development of distant metastasis, i.e. de novo metastasis. In this study, we utilized GeoMx Digital Spatial Profiler (DSP), a multiplex and high-throughput sequencing platform designed for spatially- and morphologically resolved protein expression profiling to investigate the intra-tumoral and inter-tumoral heterogeneity of tumor microenvironment (TME) and identify the mechanisms underlying distant failure in NPC. Methods: We utilized GeoMx Protein Assays consisting of 147 proteins from 15 functional modules to compare the difference between NPC cases developing distant failure (DF) (n=21) and stage-, age-, gender- and treatment-matched NPC cases without developing distant failure (noDF) (n=15) within five years of treatment. Two fluorescent morphological markers including PanCK and CD45, were used for delineating the structural architecture of the tissues, identifying cell types, and selecting regions of interest (ROIs), followed by sequencing for protein expression quantification. NanoString GeoMx DSP Analysis Suite was used for quality control and data normalization. The NPC TME heterogeneity was further investigated by the 10X Visum platform for spatial transcriptome. The public single-cell RNA sequencing (scRNA-Seq) and bulk RNA sequencing datasets were obtained for the downstream analyses. Results: By analyzing protein expression profiles, the study revealed the intratumoral and intertumoral heterogeneity associated with EBV latent infection in the TME of NPC. Regions with high EBV latent gene expression exhibited a noticeable reduction in T cell infiltration, indicating an "immune cold" phenotype, whereas regions with low EBV latent gene expression displayed heavy T cell infiltration, indicating an "immune hot" phenotype. Moreover, the study identified a panel of autophagic and anti-apoptotic proteins that were significantly upregulated in the cancer cells of NPC cases developing distant failure after treatment. This observation, supported by the single-cell RNA sequencing datasets, further implicated EBV latent infection for the upregulation of autophagic and anti-apoptotic genes. Notably, the study also demonstrated that the genes upregulated in the immune cells in NPC cases with distant failure were associated with poor progression-free survival in an independent NPC cohort. Conclusions: These findings shed light on the crucial role of EBV latent infection in TME heterogeneity and highlight the potential involvement of autophagy and anti-apoptosis in de novo metastasis in NPC. Citation Format: Larry Ka-Yue Chow, Ying Wang, Lanqi Gong, Siqi Xu, Zhaozheng Hou, Xinyuan Guan, Dora Lai-Wan Kwong, Victor Ho-Fun Lee, Wai To ng, Anne Wing-Mui Lee, Zhonghua Liu, Yunfei Xia, Wei Dai. Spatial multiplex proteins profiling of tumor microenvironment to decipher de novo metastasis in nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5481.

Molecular mechanisms in regulation of autophagy and apoptosis in view of epigenetic regulation of genes and involvement of liquid-liquid phase separation

Cellular physiology is critically regulated by multiple signaling nexuses, among which cell death mechanisms play crucial roles in controlling the homeostatic landscape at the tissue level within an organism. Apoptosis, also known as programmed cell death, can be induced by external and internal stimuli directing the cells to commit suicide in unfavourable conditions. In contrast, stress conditions like nutrient deprivation, infection and hypoxia trigger autophagy, which is lysosome-mediated processing of damaged cellular organelle for recycling of the degraded products, including amino acids. Apparently, apoptosis and autophagy both are catabolic and tumor-suppressive pathways; apoptosis is essential during development and cancer cell death, while autophagy promotes cell survival under stress. Moreover, autophagy plays dual role during cancer development and progression by facilitating the survival of cancer cells under stressed conditions and inducing death in extreme adversity. Despite having two different molecular mechanisms, both apoptosis and autophagy are interconnected by several crosslinking intermediates. Epigenetic modifications, such as DNA methylation, post-translational modification of histone tails, and miRNA play a pivotal role in regulating genes involved in both autophagy and apoptosis. Both autophagic and apoptotic genes can undergo various epigenetic modifications and promote or inhibit these processes under normal and cancerous conditions. Epigenetic modifiers are uniquely important in controlling the signaling pathways regulating autophagy and apoptosis. Therefore, these epigenetic modifiers of both autophagic and apoptotic genes can act as novel therapeutic targets against cancers. Additionally, liquid-liquid phase separation (LLPS) also modulates the aggregation of misfolded proteins and provokes autophagy in the cytosolic environment. This review deals with the molecular mechanisms of both autophagy and apoptosis including crosstalk between them; emphasizing epigenetic regulation, involvement of LLPS therein, and possible therapeutic approaches against cancers.

Identification of m6A-associated autophagy genes in non-alcoholic fatty liver.

Studies had shown that autophagy was closely related to nonalcoholic fat liver disease (NAFLD), while N6-methyladenosine (m6A) was involved in the regulation of autophagy. However, the mechanism of m6A related autophagy in NAFLD was unclear. The NAFLD related datasets were gained via the Gene Expression Omnibus (GEO) database, and we also extracted 232 autophagy-related genes (ARGs) and 37 m6A. First, differentially expressed ARGs (DE-ARGs) and differentially expressed m6A (DE-m6A) were screened out by differential expression analysis. DE-ARGs associated with m6A were sifted out by Pearson correlation analysis, and the m6A-ARGs relationship pairs were acquired. Then, autophagic genes in m6A-ARGs pairs were analyzed for machine learning algorithms to obtain feature genes. Further, we validated the relationship between feature genes and NAFLD through quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB). Finally, the immuno-infiltration analysis was implement, and we also constructed the TF-mRNA and drug-gene networks. There were 19 DE-ARGs and four DE-m6A between NAFLD and normal samples. The three m6A genes and five AGRs formed the m6A-ARGs relationship pairs. Afterwards, genes obtained from machine learning algorithms were intersected to yield three feature genes (TBK1, RAB1A, and GOPC), which showed significant positive correlation with astrocytes, macrophages, smooth muscle, and showed significant negative correlation with epithelial cells, and endothelial cells. Besides, qRT-PCR and WB indicate that TBK1, RAB1A and GOPC significantly upregulated in NAFLD. Ultimately, we found that the TF-mRNA network included FOXP1-GOPC, ATF1-RAB1A and other relationship pairs, and eight therapeutic agents such as R-406 and adavosertib were predicted based on the TBK1. The study investigated the potential molecular mechanisms of m6A related autophagy feature genes (TBK1, RAB1A, and GOPC) in NAFLD through bioinformatic analyses and animal model validation. However, it is critical to note that these findings, although consequential, demonstrate correlations rather than cause-and-effect relationships. As such, more research is required to fully elucidate the underlying mechanisms and validate the clinical relevance of these feature genes.

Pparα activation stimulates autophagic flux through lipid catabolism-independent route.

Autophagy is a cellular process that involves the fusion of autophagosomes and lysosomes to degrade damaged proteins or organelles. Triglycerides are hydrolyzed by autophagy, releasing fatty acids for energy through mitochondrial fatty acid oxidation (FAO). Inhibited mitochondrial FAO induces autophagy, establishing a crosstalk between lipid catabolism and autophagy. Peroxisome proliferator-activated receptor α (PPARα), a transcription factor, stimulates lipid catabolism genes, including fatty acid transport and mitochondrial FAO, while also inducing autophagy through transcriptional regulation of transcription factor EB (TFEB). Therefore, the study explores whether PPARα regulates autophagy through TFEB transcriptional control or mitochondrial FAO. In aquaculture, addressing liver lipid accumulation in fish is crucial. Investigating the link between lipid catabolism and autophagy is significant for devising lipid-lowering strategies and maintaining fish health. The present study investigated the impact of dietary fenofibrate and L-carnitine on autophagy by activating Pparα and enhancing FAO in Nile tilapia (Oreochromis niloticus), respectively. The dietary fenofibrate and L-carnitine reduced liver lipid content and enhanced ATP production, particularly fenofibrate. FAO enhancement by L-carnitine showed no changes in autophagic protein levels and autophagic flux. Moreover, fenofibrate-activated Pparα promoted the expression and nuclear translocation of Tfeb, upregulating autophagic initiation and lysosomal biogenesis genes. Pparα activation exhibited an increasing trend of LC3II protein at the basal autophagy and cumulative p62 protein trends after autophagy inhibition in zebrafish liver cells. These data show that Pparα activation-induced autophagic flux should be independent of lipid catabolism.

NSD1 supports cell growth and regulates autophagy in HPV-negative head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Despite advances in therapeutic management and immunotherapy, the 5-year survival rate for head and neck cancer remains at ~66% of all diagnosed cases. A better definition of drivers of HPV-negative HNSCC that are targetable points of tumor vulnerability could lead to significant clinical advances. NSD1 is a histone methyltransferase that catalyzes histone H3 lysine 36 di-methylation (H3K36me2); mutations inactivating NSD1 have been linked to improved outcomes in HNSCC. In this study, we show that NSD1 induces H3K36me2 levels in HNSCC and that the depletion of NSD1 reduces HNSCC of cell growth in vitro and in vivo. We also find that NSD1 strongly promotes activation of the Akt/mTORC1 signaling pathway. NSD1 depletion in HNSCC induces an autophagic gene program activation, causes accumulation of the p62 and LC3B-II proteins, and decreases the autophagic signaling protein ULK1 at both protein and mRNA levels. Reflecting these signaling defects, the knockdown of NSD1 disrupts autophagic flux in HNSCC cells. Taken together, these data identify positive regulation of Akt/mTORC1 signaling and autophagy as novel NSD1 functions in HNSCC, suggesting that NSD1 may be of value as a therapeutic target in this cancer.

The GATOR2 complex maintains lysosomal-autophagic function by inhibiting the protein degradation of MiT/TFEs

Lysosomes are central to metabolic homeostasis. The microphthalmia bHLH-LZ transcription factors (MiT/TFEs) family members MITF, TFEB, and TFE3 promote the transcription of lysosomal and autophagic genes and are often deregulated in cancer. Here, we show that the GATOR2 complex, an activator of the metabolic regulator TORC1, maintains lysosomal function by protecting MiT/TFEs from proteasomal degradation independent of TORC1, GATOR1, and the RAG GTPase. We determine that in GATOR2 knockout HeLa cells, members of the MiT/TFEs family are ubiquitylated by a trio of E3 ligases and are degraded, resulting in lysosome dysfunction. Additionally, we demonstrate that GATOR2 protects MiT/TFE proteins in pancreatic ductal adenocarcinoma and Xp11 translocation renal cell carcinoma, two cancers that are driven by MiT/TFE hyperactivation. In summary, we find that the GATOR2 complex has independent roles in TORC1 regulation and MiT/TFE protein protection and thus is central to coordinating cellular metabolism with control of the lysosomal-autophagic system.

Regulation of Benzo[a]pyrene-Induced Hepatic Lipid Accumulation through CYP1B1-Induced mTOR-Mediated Lipophagy.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is caused by lipid accumulation within the liver. The pathogenesis underlying its development is poorly understood. Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon and a group 1 carcinogen. The aryl hydrocarbon receptor activation by B[a]P induces cytochrome P450 (CYP) enzymes, contributing to hepatic lipid accumulation. However, the molecular mechanism through which the B[a]P-mediated induction of CYP enzymes causes hepatic lipid accumulation is unknown. This research was conducted to elucidate the role of CYP1B1 in regulating B[a]P-induced lipid accumulation within hepatocytes. B[a]P increased hepatic lipid accumulation, which was mitigated by CYP1B1 knockdown. An increase in the mammalian target of rapamycin (mTOR) by B[a]P was specifically reduced by CYP1B1 knockdown. The reduction of mTOR increased the expression of autophagic flux-related genes and promoted phagolysosome formation. Both the expression and translocation of TFE3, a central regulator of lipophagy, were induced, along with the expression of lipophagy-related genes. Conversely, enhanced mTOR activity reduced TFE3 expression and translocation, which reduced the expression of lipophagy-related genes, diminished phagolysosome production, and increased lipid accumulation. Our results indicate that B[a]P-induced hepatic lipid accumulation is caused by CYP1B1-induced mTOR and the reduction of lipophagy, thereby introducing novel targets and mechanisms to provide insights for understanding B[a]P-induced MASLD.

Autophagic Regulation of Adipogenesis Through TP53INP2: Insights from In Silico and In Vitro Analysis.

Obesity is an epidemic disease associated with multimorbidity resulting in higher mortality risk. The imbalance between energy storage and expenditure is the prime factor in the prognosis of the disease. Specifically, excessive lipid storage through adipogenesis leads to obesity. Adipogenesis is the process that converts preadipocytes into mature adipocytes by regulating major transcription factors like PPARγ and C/EBPα, contributes to lipid storage in adipose tissue. On the contrary, autophagy is a self-degradative process that maintains homeostasis in adipose tissue by regulating adipogenesis and lipolysis. TP53INP2 is a key player that regulates the autophagy process, and it negatively regulates adipogenesis and lipid storage. The gene expression profile GSE93637 was retrieved from the GEO database and analyzed using an integrated bioinformatics approach. The differentially expressed genes (DEGs) were analyzed using R-Bioconductor for TP53INP2 knockdown microarray dataset of 3T3L1 cells, and the DEGs were analyzed for the functional enrichment analysis. Further, the genes involved in the potential biological and molecular functions were evaluated for pathway enrichment analysis by KEGG (Kyoto Encyclopedia of Genes and Genomes). A total of 726 DEGs were found including 391 upregulated and 335 downregulated genes. Further, the functional and pathway enrichment analysis was employed to identify the highly interacting genes, and we identified a total of 56 genes that are highly interacting through a protein-protein interaction network. The DEGs mainly regulate the Peroxisome proliferator-activated receptor (PPAR) signaling pathway, lipolysis, and autophagy. Further, we investigated the associated Hub genes for enriched pathway genes and found the involvement of two autophagic genes ATG7 and sequestosome 1 (p62). In addition, in vitro studies of qRT-PCR (Quantitative real-time polymerase chain reaction) and Western blot analysis revealed that increased autophagy resulted in reduced lipid storage through down-regulation of the adipogenic gene. Moreover, increased expression of autophagic gene TP53INP2 and ATG7 facilitates the down-regulation of p62 and PPARγ gene resulting in lipolysis in mature adipocytes through autophagy. There is no specific treatment to reduce obesity other than a caloric diet and exercise. Hence, this study provides sufficient evidence to conclude that TP53INP2 negatively regulates adipogenesis and increases the degradation of lipids in mature adipocytes which is crucial for reducing obesity. Therefore, it is plausible to consider TP53INP2 as a promising therapeutic target for managing adipogenesis and obesity. However, further studies are necessary to validate their functional and molecular pathway analysis in the regulation of adipogenesis and obesity.