PTPLAD1 Regulates PHB-Raf Interaction to Orchestrate Epithelial-Mesenchymal and Mitofusion-Fission Transitions in Colorectal Cancer.

Colorectal cancer (CRC) remains one of the leading causes of cancer-related death worldwide. The poor prognosis of this malignancy is attributed mainly to the persistent activation of cancer signaling for metastasis. Here, we showed that protein tyrosine phosphatase-like A domain containing 1 (PTPLAD1) is down-regulated in highly metastatic CRC cells and negatively associated with poor survival of CRC patients. Systematic analysis reveals that epithelial-to-mesenchymal transition (EMT) and mitochondrial fusion-to-fission (MFT) transition are two critical features for CRC patients with low expression of PTPLAD1. PTPLAD1 overexpression suppresses the metastasis of CRC in vivo and in vitro by inhibiting the Raf/ERK signaling-mediated EMT and mitofission. Mechanically, PTPLAD1 binds with PHB via its middle fragment (141-178 amino acids) and induces dephosphorylation of PHB-Y259 to disrupt the interaction of PHB-Raf, resulting in the inactivation of Raf/ERK signaling. Our results unveil a novel mechanism in which Raf/ERK signaling activated in metastatic CRC induces EMT and mitochondrial fission simultaneously, which can be suppressed by PTPLAD1. This finding may provide a new paradigm for developing more effective treatment strategies for CRC.

LAFEM: A Scoring Model to Evaluate Functional Landscape of Lysine Acetylome.

Protein lysine acetylation is a critical post-translational modification involved in a wide range of biological processes. To date, about 20,000 acetylation sites of Homo sapiens were identified through mass spectrometry-based proteomic technology, but more than 95% of them have unclear functional annotations because of the lack of existing prioritization strategy to assess the functional importance of the acetylation sites on large scale. Hence, we established a lysine acetylation functional evaluating model (LAFEM) by considering eight critical features surrounding lysine acetylation site to high-throughput estimate the functional importance of given acetylation sites. This was achieved by selecting one of the random forest models with the best performance in 10-fold cross-validation on undersampled training dataset. The global analysis demonstrated that the molecular environment of acetylation sites with high acetylation functional scores (AFSs) mainly had the features of larger solvent-accessible surface area, stronger hydrogen bonding-donating abilities, near motif and domain, higher homology, and disordered degree. Importantly, LAFEM performed well in validation dataset and acetylome, showing good accuracy to screen out fitness directly relevant acetylation sites and assisting to explain the core reason for the difference between biological models from the perspective of acetylome. We further used cellular experiments to confirm that, in nuclear casein kinase and cyclin-dependent kinase substrate 1, acetyl-K35 with higher AFS was more important than acetyl-K9 with lower AFS in the proliferation of A549 cells. LAFEM provides a prioritization strategy to large scale discover the fitness directly relevant acetylation sites, which constitutes an unprecedented resource for better understanding of functional acetylome.

Identification of thrombotic biomarkers in orthopedic surgery patients by plasma proteomics.

BACKGROUND: Due to the poor specificity of D-dimer, more accurate thrombus biomarkers are clinically needed to improve the diagnostic power of VTE. METHODS: The plasma samples were classified into low-risk group (n = 6) and high-risk group (n = 6) according to the Caprini Thrombosis Risk Assessment Scale score. Data-independent acquisition mass spectrometry (DIA-MS) was performed to identify the proteins in the 12 plasma samples. Bioinformatics analysis including volcano plot, heatmap, KEGG pathways and chord diagram analysis were drawn to analyze the significantly differentially expressed proteins (DEPs) between the two groups. Then, another 26 plasma samples were collected to verify the key proteins as potential biomarkers of VTE in orthopedic surgery patients. RESULTS: A total of 371 proteins were identified by DIA-MS in 12 plasma samples. Volcano plotting showed that there were 30 DEPs. KEGG pathway enrichment analysis revealed that the DEPs were majorly involved in the blood coagulation pathway. The chord diagram analysis demonstrated that proteins SAA1, VWF, FLNA, ACTB, VINC, F13B, F13A and IPSP in the DEPs were significantly related to blood coagulation. VWF and F13B were selected for validation experiments. ELISA test showed that, as compared with those in the low-risk group, the level of VWF in the high-risk sera was significantly increased. CONCLUSIONS: The level of VWF in the high-risk group of thrombosis after orthopedic surgery was significantly higher than that in the low-risk group of preoperative thrombosis, suggesting that VWF may be used as a potential thrombus biomarker in orthopedic surgery patients.

MEST promotes lung cancer invasion and metastasis by interacting with VCP to activate NF-κB signaling.

BACKGROUND: Cell invasion is a hallmark of metastatic cancer, leading to unfavorable clinical outcomes. In this study, we established two highly invasive lung cancer cell models (A549-i8 and H1299-i8) and identified mesoderm-specific transcript (MEST) as a novel invasive regulator of lung cancer. We aim to characterize its biological function and clinical significance in lung cancer metastasis. METHODS: Transwell invasion assay was performed to establish high-invasive lung cancer cell model. Immunohistochemistry (IHC) was used to detect MEST expression in tumor tissues. Mass spectrometry and bioinformatic analyses were used to identify MEST-regulated proteins and binding partners. Co-immunoprecipitation assay was performed to detect the interaction of MEST and VCP. The biological functions of MEST were investigated in vitro and in vivo. Immunofluorescence staining was conducted to explore the colocalization of MEST and VCP. RESULTS: MEST overexpression promoted metastasis of lung cancer cells in vivo and in vitro by activating NF-κB signaling. MEST increased the interaction between VCP and IκBα, which accelerated IκBα degradation and NF-κB activation. Such acceleration was abrogated by VCP silencing, indicating that MEST is an upstream activator of the VCP/IκBα/NF-κB signaling pathway. Furthermore, high expressions of MEST and VCP were associated with poor survival of lung cancer patients. CONCLUSION: Collectively, these results demonstrate that MEST plays an important role in driving invasion and metastasis of lung cancer by interacting with VCP to coordinate the IκBα/NF-κB pathway. Targeting the MEST/VCP/IκBα/NF-κB signaling pathway may be a promising strategy to treat lung cancer.

Curcumol Overcomes TRAIL Resistance of Non-Small Cell Lung Cancer by Targeting NRH:Quinone Oxidoreductase 2 (NQO2).

Resistance to tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) of cancer cell remains a key obstacle for clinical cancer therapies. To overcome TRAIL resistance, this study identifies curcumol as a novel safe sensitizer from a food-source compound library, which exhibits synergistic lethal effects in combination with TRAIL on non-small cell lung cancer (NSCLC). SILAC-based cellular thermal shift profiling identifies NRH:quinone oxidoreductase 2 (NQO2) as the key target of curcumol. Mechanistically, curcumol directly targets NQO2 to cause reactive oxygen species (ROS) generation, which triggers endoplasmic reticulum (ER) stress-C/EBP homologous protein (CHOP) death receptor (DR5) signaling, sensitizing NSCLC cell to TRAIL-induced apoptosis. Molecular docking analysis and surface plasmon resonance assay demonstrate that Phe178 in NQO2 is a critical site for curcumol binding. Mutation of Phe178 completely abolishes the function of NQO2 and augments the TRAIL sensitization. This study characterizes the functional role of NQO2 in TRAIL resistance and the sensitizing function of curcumol by directly targeting NQO2, highlighting the potential of using curcumol as an NQO2 inhibitor for clinical treatment of TRAIL-resistant cancers.

Comparative Proteome Identifies Complement Component 3-Mediated Immune Response as Key Difference of Colon Adenocarcinoma and Rectal Adenocarcinoma.

Colorectal cancer (CRC) is one of the most lethal diseases with high morbidity and mortality worldwide. Clinically, tumors located in colon and rectum have diverse prognosis and therapeutic outcome. Here, we performed data mining derived from 20 CRC patient samples to compare proteomic difference between colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ). We found that differential expressed proteins (DEPs) upregulated in COAD were mainly enriched in immune response, moreover, higher immune scores were found in COAD than READ, as calculated by The Cancer Genome Atlas (TCGA) data. To identify the core protein of DEPs with high prognostic value for COAD, we performed topological overlap matrix (TOM) to investigate the hub proteins using 77 immune-relevant DEPs, and identified complement component 3 (C3) as the core protein in the immune-relevant DEPs matrix between the COAD and READ. Moreover, we found that C3 was up-regulated in COAD, and its expression was negatively associated with overall survival of COAD patients but not READ. In conclusion, we identified C3-mediated immune response as key feature to distinguish COAD and READ, and highlighted C3 as potential biomarker with high prognostic value for clinical application, which provided new clue for precise treatment of COAD.