Multiomics Reveals Induction of Neuroblastoma SK-N-BE(2)C Cell Death by Mitochondrial Division Inhibitor 1 through Multiple Effects.

Mitochondrial division inhibitor 1 (Mdivi-1) is a well-known synthetic compound aimed at inhibiting dynamin-related protein 1 (Drp1) to suppress mitochondrial fission, making it a valuable tool for studying mitochondrial dynamics. However, its specific effects beyond Drp1 inhibition remain to be confirmed. In this study, we employed integrative proteomics and phosphoproteomics to delve into the molecular responses induced by Mdivi-1 in SK-N-BE(2)C cells. A total of 3070 proteins and 1945 phosphorylation sites were identified, with 880 of them represented as phosphoproteins. Among these, 266 proteins and 97 phosphorylation sites were found to be sensitive to the Mdivi-1 treatment. Functional enrichment analysis unveiled their involvement in serine biosynthesis and extrinsic apoptotic signaling pathways. Through targeted metabolomics, we observed that Mdivi-1 enhanced intracellular serine biosynthesis while reducing the production of C24:1-ceramide. Within these regulated phosphoproteins, dynamic dephosphorylation of proteasome subunit alpha type 3 serine 250 (PSMA3-S250) occurred after Mdivi-1 treatment. Further site-directed mutagenesis experiments revealed that the dephosphorylation-deficient mutant PSMA3-S250A exhibited a decreased cell survival. This research confirms that Mdivi-1's inhibition of mitochondrial division leads to various side effects, ultimately influencing cell survival, rather than solely targeting Drp1 inhibition.

Identification of a gut microbiota member that ameliorates DSS-induced colitis in intestinal barrier enhanced Dusp6-deficient mice.

Strengthening the gut epithelial barrier is a potential strategy for management of gut microbiota-associated illnesses. Here, we demonstrate that dual-specificity phosphatase 6 (Dusp6) knockout enhances baseline colon barrier integrity and ameliorates dextran sulfate sodium (DSS)-induced colonic injury. DUSP6 mutation in Caco-2 cells enhances the epithelial feature and increases mitochondrial oxygen consumption, accompanied by altered glucose metabolism and decreased glycolysis. We find that Dusp6-knockout mice are more resistant to DSS-induced dysbiosis, and the cohousing and fecal microbiota transplantation experiments show that the gut/fecal microbiota derived from Dusp6-knockout mice also confers protection against colitis. Further culturomics and mono-colonialization experiments show that one gut microbiota member in the genus Duncaniella confers host protection from DSS-induced injury. We identify Dusp6 deficiency as beneficial for shaping the gut microbiota eubiosis necessary to protect against gut barrier-related diseases.

Multiresolution Imaging Using Bioluminescence Resonance Energy Transfer Identifies Distinct Biodistribution Profiles of Extracellular Vesicles and Exomeres with Redirected Tropism.

Extracellular particles (EPs) including extracellular vesicles (EVs) and exomeres play significant roles in diseases and therapeutic applications. However, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a suitable method. Therefore, a bioluminescence resonance energy transfer (BRET)-based reporter, PalmGRET, is created to enable pan-EP labeling ranging from exomeres (<50 nm) to small (<200 nm) and medium and large (>200 nm) EVs. PalmGRET emits robust, sustained signals and allows the visualization, tracking, and quantification of the EPs from whole animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, BRET, and fluorescence. Using PalmGRET, it is shown that EPs released by lung metastatic hepatocellular carcinoma (HCC) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. It is further demonstrated that gene knockdown of lung-tropic membrane proteins, solute carrier organic anion transporter family member 2A1, alanine aminopeptidase/Cd13, and chloride intracellular channel 1 decreases HCC-EP distribution to the lungs and yields distinct biodistribution profiles. It is anticipated that EP-specific imaging, quantitative assays, and detailed in vivo characterization are a starting point for more accurate and comprehensive in vivo models of EP biology and therapeutic design.

ZNF322A-mediated protein phosphorylation induces autophagosome formation through modulation of IRS1-AKT glucose uptake and HSP-elicited UPR in lung cancer.

BACKGROUND: ZNF322A is an oncogenic transcription factor that belongs to the Cys2His2-type zinc-finger protein family. Accumulating evidence suggests that ZNF322A may contribute to the tumorigenesis of lung cancer, however, the ZNF322A-mediated downstream signaling pathways remain unknown. METHODS: To uncover ZNF322A-mediated functional network, we applied phosphopeptide enrichment and isobaric labeling strategies with mass spectrometry-based proteomics using A549 lung cancer cells, and analyzed the differentially expressed proteins of phosphoproteomic and proteomic profiles to determine ZNF322A-modulated pathways. RESULTS: ZNF322A highlighted a previously unidentified insulin signaling, heat stress, and signal attenuation at the post-translational level. Consistently, protein-phosphoprotein-kinase interaction network analysis revealed phosphorylation of IRS1 and HSP27 were altered upon ZNF322A-silenced lung cancer cells. Thus, we further investigated the molecular regulation of ZNF322A, and found the inhibitory transcriptional regulation of ZNF322A on PIM3, which was able to phosphorylate IRS1 at serine1101 in order to manipulate glucose uptake via the PI3K/AKT/mTOR signaling pathway. Moreover, ZNF322A also affects the unfolded protein response by phosphorylation of HSP27S82 and eIF2aS51, and triggers autophagosome formation in lung cancer cells. CONCLUSIONS: These findings not only give new information about the molecular regulation of the cellular proteins through ZNF322A at the post-translational level, but also provides a resource for the study of lung cancer therapy.

Phosphoproteome Analysis Reveals Dynamic Heat Shock Protein 27 Phosphorylation in Tanshinone IIA-Induced Cell Death.

Gastric cancer is one of the most common types of cancer worldwide. Nevertheless, effective therapeutic strategies have not yet been discovered. Several studies have shown that tanshinone IIA (TIIA), which is extracted from the traditional herbal medicine plant Danshen (Salvia miltiorrhiza), has potential activity against many kinds of cancer. Our previous research demonstrated that TIIA can induce cell death in gastric cancer. However, the exact signaling pathway response is still unclear. Post-translational modification (PTM) plays a significant role in a wide range of physiological processes in cancer, via regulation of both signal transduction cascades and many cellular pathways. Here, we integrated multilayer omics-transcriptomics and dynamic phosphoproteomics-to elucidate the regulatory networks triggered by TIIA in gastric cancer. We identified the phosphorylation of heat shock protein 27 (HSP27) at serine 82 in response to TIIA, which caused reactive oxygen species (ROS) production and unfolded protein response (UPR). Moreover, the accumulation of cellular stress increased the expression of heat shock factor 1 (HSF1). In addition, the downstream targets of HSF1, which were involved in heat shock stress and apoptosis, were also activated in TIIA-treated cells. In conclusion, this study performs a multiomic approach to clarify a comprehensive TIIA-responsive network leading to cell death in gastric cancer.

Combinatorial targeting of MTHFD2 and PAICS in purine synthesis as a novel therapeutic strategy.

MYCN-amplified (MNA) neuroblastoma is an aggressive neural crest-derived pediatric cancer. However, MYCN is indispensable for development and transcriptionally regulates extensive network of genes. Integrating anti-MYCN ChIP-seq and gene expression profiles of neuroblastoma patients revealed the metabolic enzymes, MTHFD2 and PAICS, required for one-carbon metabolism and purine biosynthesis were concomitantly upregulated, which were more susceptible to metastatic neuroblastoma. Moreover, we found that MYCN mediated the folate cycle via MTHFD2, which contributed one-carbon unit to enhance purine synthesis, and further regulated nucleotide production by PAICS in response to cancer progression. Dual knockdown of the MYCN-targeted gene pair, MTHFD2 and PAICS, in MNA neuroblastoma cells synergically reduced cell proliferation, colony formation, migration ability, and DNA synthesis. By systematically screening the compound perturbagens, the gene expression levels of MTHFD2 and PAICS were specifically suppressed by anisomycin and apicidin across cell lines, and our co-treatment results also displayed synergistic inhibition of MNA neuroblastoma cell proliferation. Collectively, targeting a combination of MYCN-targeted genes that interrupts the interconnection of metabolic pathways may overcome drug toxicity and improve the efficacy of current therapeutic agents in MNA neuroblastoma.

Quantitative Proteomics of Th-MYCN Transgenic Mice Reveals Aurora Kinase Inhibitor Altered Metabolic Pathways and Enhanced ACADM To Suppress Neuroblastoma Progression.

Neuroblastoma is a neural crest-derived embryonal tumor and accounts for about 15% of all cancer deaths in children. MYCN amplification is associated with aggressive and advanced stage of high-risk neuroblastoma, which remains difficult to treat and exhibits poor survival under current multimodality treatment. Here, we analyzed the transcriptomic profiles of neuroblastoma patients and showed that aurora kinases lead to poor survival and had positive correlation with MYCN amplification and high-risk disease. Further, pan-aurora kinase inhibitor (tozasertib) treatment not only induces cell-cycle arrest and suppresses cell proliferation, migration, and invasion ability in MYCN-amplified (MNA) neuroblastoma cell lines, but also inhibits tumor growth and prolongs animal survival in Th-MYCN transgenic mice. Moreover, we performed quantitative proteomics and identified 150 differentially expressed proteins after tozasertib treatment in the Th-MYCN mouse model. The functional and network-based enrichment revealed that tozasertib alters metabolic processes and identified a mitochondrial flavoenzyme in fatty acid ß-oxidation, ACADM, which is correlated with aurora kinases and neuroblastoma patient survival. Our findings indicate that the aurora kinase inhibitor could cause metabolic imbalance, possibly by disturbing carbohydrate and fatty acid metabolic pathways, and ACADM may be a potential target in MNA neuroblastoma.

MCM2-regulated functional networks in lung cancer by multi-dimensional proteomic approach.

DNA replication control is vital for maintaining genome stability and the cell cycle, perhaps most notably during cell division. Malignancies often exhibit defective minichromosome maintenance protein 2 (MCM2), a cancer proliferation biomarker that serves as a licensing factor in the initiation of DNA replication. MCM2 is also known to be one of the ATPase active sites that facilitates conformational changes and drives DNA unwinding at the origin of DNA replication. However, the biological networks of MCM2 in lung cancer cells via protein phosphorylation remain unmapped. The RNA-seq datasets from The Cancer Genome Atlas (TCGA) revealed that MCM2 overexpression is correlated with poor survival rate in lung cancer patients. To uncover MCM2-regulated functional networks in lung cancer, we performed multi-dimensional proteomic approach by integrating analysis of the phosphoproteome and proteome, and identified a total of 2361 phosphorylation sites on 753 phosphoproteins, and 4672 proteins. We found that the deregulation of MCM2 is involved in lung cancer cell proliferation, the cell cycle, and migration. Furthermore, HMGA1S99 phosphorylation was found to be differentially expressed under MCM2 perturbation in opposite directions, and plays an important role in regulating lung cancer cell proliferation. This study therefore enhances our capacity to therapeutically target cancer-specific phosphoproteins.

Quantitative proteomics in lung cancer.

Lung cancer is the most common cause of cancer-related death worldwide, less than 7% of patients survive 10 years following diagnosis across all stages of lung cancer. Late stage of diagnosis and lack of effective and personalized medicine reflect the need for a better understanding of the mechanisms that underlie lung cancer progression. Quantitative proteomics provides the relative different protein abundance in normal and cancer patients which offers the information for molecular interactions, signaling pathways, and biomarker identification. Here we introduce both theoretical and practical applications in the use of quantitative proteomics approaches, with principles of current technologies and methodologies including gel-based, label free, stable isotope labeling as well as targeted proteomics. Predictive markers of drug resistance, candidate biomarkers for diagnosis, and prognostic markers in lung cancer have also been discovered and analyzed by quantitative proteomic analysis. Moreover, construction of protein networks enables to provide an opportunity to interpret disease pathway and improve our understanding in cancer therapeutic strategies, allowing the discovery of molecular markers and new therapeutic targets for lung cancer.

Novel Utilization of Terminators in the Design of Biologically Adjustable Synthetic Filters.

Terminators, which signal the end of transcription processes, are typically placed behind the last coding sequence of an operon to prevent interference between transcript units in most biologically synthetic systems. Here, we seek to extend the usability of terminators in genetic system design by using terminators as regulatory genetic parts. Terminators with different impacts on their upstream and downstream genes are characterized in detail via dynamic modeling to predict the behavior of the overall genetic system. Some nonlinear effects of terminators observed in our terminator measurements potentially facilitate regulation of gene expression. Through dynamic modeling in silico, we find that such genetic systems may behave like genetic filters. In agreement with the simulations, we successfully implement genetic high-pass and bandpass filters in vivo, demonstrating the potential of using terminators as regulatory parts. The genetic bandpass filter in this work is implemented through the interdependence between genetic parts, in which the termination efficiency of a terminator varies with the strength of the upstream promoter. This design strategy for a bandpass filter requires fewer base pairs than the conventional strategy of concatenating high-pass and low-pass filters. Our results show that this novel utilization of terminators as regulatory parts may provide a new perspective for efficient design of genetic circuits. We believe that further exploration of the complicated dynamics of terminators is important in the development of synthetic biology.