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Purpose: To investigate the autocrine/endocrine role of Id1-induced insulin-like growth factor-II (IGF-II) in esophageal cancer, and evaluate the potential of IGF-II- and IGF-type I receptor (IGF-IR)-targeted therapies.Experimental Design: Antibody array-based screening was used to identify differentially secreted growth factors from Id1-overexpressing esophageal cancer cells. In vitro and in vivo assays were performed to confirm the induction of IGF-II by Id1, and to study the autocrine and endocrine effects of IGF-II in promoting esophageal cancer progression. Human esophageal cancer tissue microarray was analyzed for overexpression of IGF-II and its correlation with that of Id1 and phosphorylated AKT (p-AKT). The efficacy of intratumorally injected IGF-II antibody and intraperitoneally injected cixutumumab (fully human monoclonal IGF-IR antibody) was evaluated using in vivo tumor xenograft and experimental metastasis models.Results: Id1 overexpression induced IGF-II secretion, which promoted cancer cell proliferation, survival, and invasion by activating AKT in an autocrine manner. Overexpression of IGF-II was found in 21 of 35 (60%) esophageal cancer tissues and was associated with upregulation of Id1 and p-AKT. IGF-II secreted by Id1-overexpressing esophageal cancer xenograft could instigate the growth of distant esophageal tumors, as well as promote metastasis of circulating cancer cells. Targeting IGF-II and IGF-IR had significant suppressive effects on tumor growth and metastasis in mice. Cixutumumab treatment enhanced the chemosensitivity of tumor xenografts to fluorouracil and cisplatin.Conclusions: The Id1–IGF-II–IGF-IR–AKT signaling cascade plays an important role in esophageal cancer progression. Blockade of IGF-II/IGF-IR signaling has therapeutic potential in the management of esophageal cancer. Clin Cancer Res; 20(10); 2651–62. ©2014 AACR.

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Supplementary Table from Anti-HIV Drug Elvitegravir Suppresses Cancer Metastasis via Increased Proteasomal Degradation of m6A Methyltransferase METTL3

PDF file - 1419KB Supplementary Fig S1. Human growth factor antibody array analysis of the conditioned media from HKESC-3-CON and HKESC-3-Id1 cells. Supplementary Fig S2. Effect of knockdown Id1 expression on IGF2 level using a second RNAi sequence. Supplementary Fig S3. Effect of ectopic Id1 expression on IGF2 level in multiple cancer types. Supplementary Fig S4. Effect of Id1 manipulation on IGF2 mRNA expression in ESCC cells. Supplementary Fig S5. IGF2, Id1, and p-AKT expressions in human esophageal cancer. Supplementary Fig S6. Western blot analysis of Id1 and IGF2 expressions in ESCC cell lines. Supplementary Fig S7. Effects of Id1 knockdown on autocrine stimulation of esophageal cancer cell proliferation, migration, and survival. Supplementary Fig S8. Ki-67 and TUNEL staining of tumor xenografts from three groups of mice inoculated with KYSE150-CON-shCON, KYSE150-Id1-shCON, and KYSE150-Id1- shIGF2 cells, respectively. Supplementary Fig S9. Ki-67 and TUNEL staining in "responder" tumors of different groups of mice bearing KYSE150-CON-shCON, KYSE150-Id1-shCON, and KYSE150-Id1- shIGF2 "instigating" tumors, respectively. Supplementary Fig S10. Anti-tumor effects of IGF2-neutralizing antibody in vivo. Supplementary Fig S11. Immunohistochemical analysis of tumor xenografts treated with cixutumumab or the isotype IgG. Supplementary Fig S12. TUNEL staining of tumor xenografts from two groups of mice inoculated with KYSE150 (A) and KYSE270 (B) respectively. Supplementary Fig S13. Evaluation of toxic effects of cixutumumab in nude mice. A, comparison of body weight of cixutumumab-treated mice and isotype IgG-treated control mice. B, histological examination of lung, liver and kidney specimens (H&E stained). Supplementary Fig S14. Increased thymidylate synthase expression in FR cells was inhibited by cixutumumab.

PDF file - 8KB, Immunohistochemical analysis of expression levels of Id1, IGF2, and p-AKT in human ESCC specimens.

Supplementary Data from Anti-HIV Drug Elvitegravir Suppresses Cancer Metastasis via Increased Proteasomal Degradation of m6A Methyltransferase METTL3

As one of the hallmarks of cancer, metabolic reprogramming leads to cancer progression, and targeting glycolytic enzymes could be useful strategies for cancer therapy. By screening a small molecule library consisting of 1320 FDA-approved drugs, we found that penfluridol, an antipsychotic drug used to treat schizophrenia, could inhibit glycolysis and induce apoptosis in esophageal squamous cell carcinoma (ESCC). Gene profiling and Ingenuity Pathway Analysis suggested the important role of AMPK in action mechanism of penfluridol. By using drug affinity responsive target stability (DARTS) technology and proteomics, we identified phosphofructokinase, liver type (PFKL), a key enzyme in glycolysis, as a direct target of penfluridol. Penfluridol could not exhibit its anticancer property in PFKL-deficient cancer cells, illustrating that PFKL is essential for the bioactivity of penfluridol. High PFKL expression is correlated with advanced stages and poor survival of ESCC patients, and silencing of PFKL significantly suppressed tumor growth. Mechanistically, direct binding of penfluridol and PFKL inhibits glucose consumption, lactate and ATP production, leads to nuclear translocation of FOXO3a and subsequent transcriptional activation of BIM in an AMPK-dependent manner. Taken together, PFKL is a potential prognostic biomarker and therapeutic target in ESCC, and penfluridol may be a new therapeutic option for management of this lethal disease.

Lung adenocarcinoma (LAC) is one of the most common pulmonary adenocarcinomas with a high peak of mortality, and metastasis is the main culprit of LAC deaths. microRNAs play important role in cancer metastasis, and thus are regarded as potential diagnostic and prognostic markers for human cancers. However, many miRNAs exhibit dual roles in diverse cellular contexts. Here, we revealed that hsa‐miR‐335, a previously reported tumor suppressor, exhibited an oncogenic role in LAC. Overexpression of miR‐335 enhanced the abilities of A549 and H1299 cells to invade and migrate by regulating epithelial‐mesenchymal transition, while inhibition of miR‐335 exhibited an opposite effect in vitro and in vivo. Mechanically, miR‐335 inhibited the expression of Copine‐1 (CPNE1), an NF‐κB suppressor, through interacting with its mRNA 3′UTR, while mutating the binding sites abolished this inhibitory effect. This finding not only highlights the suppressive effect of CPNE1 on cell motility, but also provides new insight into miR‐335 in promoting LAC metastasis., Hsa‐miR‐335, a previously reported tumor suppressor in multiple cancers, exhibits an oncogenic role in lung adenocarcinoma. miR‐335 increases the invasion and migration of cancer cells by regulating epithelial‐mesenchymal transition, while inhibition of miR‐335 exhibits opposite effect in vitro and in vivo. Mechanically, miR‐335 inhibits the expression of Copine‐1 (CPNE1), an NF‐κB suppressor, through interacting with its mRNA 3′UTR, while mutating the binding sites abolished this inhibitory effect. This finding not only highlights the suppressive effect of CPNE1 on cell motility, but also provides new insight into miR‐335 in promoting lung adenocarcinoma metastasis.

Background Colorectal cancer (CRC) is one of the most malignant cancer worldwide, and the limited efficacy of existing treatments is the leading cause of death in patients with CRC. Thus, novel drugs for CRC treatment are urgently needed. Methods We screened an FDA-approved small-molecule library upon HCT116 cells, and identified lomitapide as a novel CRC anticancer compound. Then we confirmed the activities of lomitapide on CRC cells by WST-1 assay, colony formation, and flow cytometry. RNA sequencing and GO analysis were used to investigate the mechanisms underlying the anticancer effects of lomitapide. LiP-SMap was introduced to search for the potential targets of lomitapide. The in vivo experiment was conducted to confirm the therapeutic efficiency and safety of lomitapide as an anticancer agent. Results Lomitapide exhibited remarkable antitumor properties in vitro and in vivo, while activated autophagy is characterized by GO analysis as a key biological process in lomitapide-induced CRC repression. Moreover, lomitapide stimulated mitochondrial dysfunction-mediated AMPK activation, resulting in increased AMPK phosphorylation and enhanced Beclin1/Atg14/Vps34 interactions, provoking autophagy induction. LiP-SMap analysis showed that PP2A was the direct target of lomitapide, and the bioactivity of lomitapide was attenuated in PP2A-deficient cells, suggesting that the anticancer effect of lomitapide occurs in a PP2A-dependent manner. Conclusions Our results indicate that lomitapide activates AMPK-regulated autophagy to inhibit the proliferation and tumorigenesis of CRC cells by directly targeting PP2A, and can be a novel therapeutic agent for the treatment of CRC patients.

In recent years, the development of high-throughput omics technology has greatly promoted the development of biomedicine. However, the poor reproducibility of omics techniques limits its application. It is necessary to use standard reference materials of complex RNAs or proteins to test and calibrate the accuracy and reproducibility of omics workflows. However, the transcriptome and proteome of most cell lines shift during culturing, which limits their applicability to serve as standard samples. In this study, we demonstrated that the human hepatocellular cell line MHCC97H has a very stable transcriptome (R2=0.966-0.995) and proteome (R2=0.934-0.976 for DDA, R2=0.942-0.986 for DIA) after 9 subculturing generations, which allows this stable standard sample to be stably produced on an industrial scale for several decades. Moreover, this stability was maintained across labs and platforms. In sum, our results justified a omics standard reference material and reference datasets for transcriptomic and proteomics research. This helps to further standardize the workflow and data quality of omics techniques and thus promotes the application of omics technology in precision medicine.

Acquired resistance to BRAF inhibitor vemurafenib is frequently observed in metastatic colorectal cancer (CRC), and it is a thorny issue that results in treatment failure. As adaptive responses for vemurafenib treatment, a series of cellular bypasses are response for the adaptive feedback reactivation of ERK signaling, which warrant further investigation.We identified ARF1 (ADP-ribosylation factor 1) as a novel regulator of both vemurafenib resistance and cancer metastasis, its molecular mechanism and potential inhibitor were investigated in this study.DIA-based quantitative proteomics and RNA-seq were performed to systematic analyze the profiling of vemurafenib-resistant RKO cells (RKO-VR) and highly invasive RKO cells (RKO-I8), respectively. Co‑immunoprecipitation assay was performed to detect the interaction of ARF1 and IQGAP1 (IQ-domain GTPase activating protein 1). An ELISA-based drug screen system on FDA-approved drug library was established to screen the compounds against the interaction of ARF1-IQGAP1.The biological functions of ARF1 and LY2835219 were determined by transwell, western blotting, Annexin V-FITC/PI staining and in vivo experimental metastasis assays.We found that ARF1 strongly interacted with IQGAP1 to activate ERK signaling in VR and I8 CRC cells. Deletion of IQGAP1 or inactivation of ARF1 (ARF-T48S) restored the invasive ability induced by ARF1. As ARF1-IQGAP1 interaction is essential for ERK activation, we screened LY2835219 as novel inhibitor of ARF1-IQGAP1 interaction, which inactivated ERK signaling and suppressed CRC metastasis and vemurafenib-resistance in vitro and in vivo with no observed side effect. Furthermore, LY2835219 in combined treatment with vemurafenib exerted significantly inhibitory effect on ARF1-mediated cancer metastasis than used independently.This study uncovers that ARF1-IQGAP1 interaction-mediated ERK signaling reactivation is critical for vemurafenib resistance and cancer metastasis, and that LY2835219 is a promising therapeutic agent for CRC both as a single agent and in combination with vemurafenib.

Post-translational modifications (PTMs) of Cyclin-dependent kinase 5 (CDK5) have emerged as important regulatory mechanisms that modulate cancer development in patients. Though CDK5 is an atypical member of the cyclin-dependent kinase family, its aberrant expression links to cell proliferation, DNA damage response, apoptosis, migration and angiogenesis in cancer. Current studies suggested that, new PTMs on CDK5, including S-nitrosylation, sumoylation, and acetylation, serve as molecular switches to control the kinase activity of CDK5 in the cell. However, a majority of these modifications and their biological significance in cancer remain uncharacterized. In this review, we discussed the role of PTMs on CDK5-mediated signaling cascade, and their possible mechanisms of action in malignant tumors, as well as the challenges and future perspectives in this field. On the basis of the newly identified regulatory signaling pathways of CDK5 related to PTMs, researchers have investigated the cancer therapeutic potential of chemical compounds, small-molecule inhibitors, and competitive peptides by targeting CDK5 and its PTMs. Results of these preclinical studies demonstrated that targeting PTMs of CDK5 yields promising antitumor effects and that clinical translation of these therapeutic strategies is warranted.

Covalent inhibitors with an electrophilic warhead have received considerable attention due to their remarkable pharmacological properties. However, the electrophilic warhead in covalent drugs is often an α, β-unsaturated amide, and the targets are mainly cysteine or lysine residues. Thus, the development of novel electrophiles that can target other amino acids is highly desirable. Ynamide, a useful and versatile building block, is commonly employed in the construction of various compounds in organic synthesis. The performance of this functional group in a proteome-wide environment has been studied here for the first time, and it has been shown that it can efficiently modify carboxyl residues in situ and in vitro. Upon incorporation of this ynamide warhead into the pharmacophores of kinase inhibitors, the resulting compound showed moderate inhibition against the EGFR L858R mutant but not against EGFR WT. This novel electrophilic group can be used in the development of new types of covalent inhibitors.

Harmful algal blooms (HABs) are symptomatic of ecosystem imbalance, leading to major worldwide marine natural disasters, and seriously threaten the human health. Some HAB algae's exceptional genome size prohibited the genomic investigations on molecular mechanisms, for example, Prorocentrum. This study performed translatome sequencing (RNC-seq) for Prorocentrum donghaiense to assemble the translatome reference sequences on appropriate cost to enable the global molecular study at translatome and proteome levels. By analyzing the translatome and proteome of P. donghaiense in phosphor-rich, phosphor-deficient, and phosphor-restored media, we found massive up-regulation of energy and material production pathways in phosphor-rich conditions that enables autoactivation of translation, which is the key to its exponential growth in HABs. To break down the autoactivation, we demonstrated that mild translation delay using very low concentrations of cycloheximide efficiently controls the blooming without harming other aquatic organisms and humans. Our result provides a novel hint for controlling HABs and demonstrated the RNC-seq as an economic strategy on investigating functions of organisms with large and unknown genomes.

The unreasonable misuse of antibiotics has led to the emergence of large-scale drug-resistant bacteria, seriously threatening human health. Compared with drug-sensitive bacteria, resistant bacteria are difficult to clear by host immunity. To fully explore the adaptive mechanism of resistant bacteria to the iron-restricted environment, we performed data-independent acquisition-based quantitative proteomics on ciprofloxacin (CIP)-resistant (CIP-R) Staphylococcus aureus in the presence or absence of iron. On bioinformatics analysis, CIP-R bacteria showed stronger amino acid synthesis and energy storage ability. Notably, CIP-R bacteria increased virulence by upregulating the expression of many virulence-related proteins and enhancing the synthesis of virulence-related amino acids under iron-restricted stress. This study will help us to further explain the adaptive mechanisms that lead to bacterial resistance to antibiotics depending on the host environment and provide insights into the development of novel drugs for the treatment of drug-resistant bacterial infections.

Trans-Cinnamaldehyde (TC) is a widely used food additive, known for its sterilization, disinfection, and antiseptic properties. However, its antibacterial mechanism is not completely understood. In this study, quantitative proteomics was performed to investigate differentially expressed proteins (DEPs) in Escherichia coli in response to TC treatment. Bioinformatics analysis suggested aldehyde toxicity, acid stress, oxidative stress, interference of carbohydrate metabolism, energy metabolism, and protein translation as the bactericidal mechanism. E. coli BW25113ΔyqhD, ΔgldA, ΔbetB, ΔtktB, ΔgadA, ΔgadB, ΔgadC, and Δrmf were used to investigate the functions of DEPs through biochemical methods. The present study revealed that TC exerts its antibacterial effects by inducing the toxicity of its aldehyde group producing acid stress. These findings will contribute to the application of TC in the antibacterial field.

Esophageal cancer (EC) is one of the most lethal cancers in the world, and its morbidity and mortality rates rank among the top ten in China. Currently, surgical resection, radiotherapy and chemotherapy are the primary clinical treatments for esophageal cancer. However, outcomes are still unsatisfactory due to the limited efficacy and severe adverse effects of conventional treatments. As a new type of approach, targeted therapies have been confirmed to play an important role in the treatment of esophageal cancer; these include cetuximab and bevacizumab, which target epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF), respectively. In addition, other drugs targeting surface antigens and signaling pathways or acting on immune checkpoints have been continuously developed. For example, trastuzumab, a monoclonal antibody targeting human epidermal growth factor receptor 2 (HER-2), has been approved by the Food and Drug Administration (FDA) as a first-line treatment of HER-2-positive cancer. Moreover, the PD-L1 inhibitor pembrolizumab has been approved as a highly efficient drug for patients with PD-L1-positive or advanced esophageal squamous cell carcinoma (ESCC). These novel drugs can be used alone or in combination with other treatment strategies to further improve the treatment efficacy and prognosis of cancer patients. Nevertheless, adverse events, optimal dosages and effective combinations still need further investigation. In this review, we expound an outline of the latest advances in targeted therapies of esophageal cancer and the mechanisms of relevant drugs, discuss their efficacy and safety, and provide a clinical rationale for precision medicine in esophageal cancer.

A great number of non-coding RNAs (ncRNAs) account for the majority of the genome. The translation of these ncRNAs has been noted but seriously underestimated due to both technological and theoretical limitations. Based on the development of ribosome profiling (Ribo-seq), full length translating RNA analysis (RNC-seq) and mass spectrometry technology, more and more ncRNAs are being found to be translated in different organism, and some of them can produce functional peptides. While recently, not only individual new functional proteins, but also a new proteome have been experimentally discovered to be encoded by endogenous lncRNAs and circRNAs. These new proteins are of biological significance, suggesting the connection of the translation of ncRNAs to human physiology and diseases. Therefore, an in-depth and systematic understanding of the coding capabilities of ncRNAs is necessary for basic biology and medicine. In this review, we summarize the advances in the field of discovering this new proteome, i.e. "ncRNA-coded" proteins.

Rationale: Effective targeting therapies are limited in Hepatocellular carcinoma (HCC) clinic. Characterization of tumor suppressor genes (TSGs) and elucidation their signaling cascades could shed light on new strategies for developing targeting therapies for HCC. Methods: We checked genome-wide DNA copy number variation (CNV) of HCC samples, focusing on deleted genes for TSG candidates. Clinical data, in vitro and in vivo data were collected to validate the tumor suppressor functions. Results: Focal deletion of GATA4 gene locus was the most prominent feature across all liver cancer samples. Ectopic expression of GATA4 resulted in senescence of HCC cell lines. Mechanistically, GATA4 exerted tumor suppressive role by orchestrating the assembly of a tumor suppressor enhancing module: GATA4 directly bound and potently inhibited the mRNA transcription activity of β-catenin; meanwhile, β-catenin was recruited by GATA4 to promoter regions and facilitated transcription of GATA4 target genes, which were TSGs per se. Expression of GATA4 was effective to shrink GATA4-deficient HCC tumors in vivo. We also showed that β-catenin inhibitor was capable of shrinking GATA4-deficient tumors. Conclusions: Our study unveiled a previously unnoticed tumor suppressor enhancing module assembled by ectopically expressed GATA4 in HCC cells and denoted a therapeutic opportunity for GATA4 deficient HCC patients. Our study also presented an interesting case that an oncogenic transcription factor conditionally functioned as a tumor suppressor when recruited by a TSG transcription factor.

Graphical abstract, Highlights • It is the first study to identify neoandrographolide as a novel inhibitor of Rab5. • Neoandrographolide can occupy the surface groove of Rab5 for inhibiting its activity. • Neoandrographolide exhibits anticancer effect on lung cancer by targeting Rab5., Rab5 is a small GTPase that plays a crucial role in oncogenic signal transduction, which was considered as an attractive target for cancer therapy. Rapid GDP/GTP exchange in the packet of Rab5 sustains its high activity for promoting cancer progression. However, Rab5 currently remains undruggable due to the lack of specific inhibitor. Herein, we reported the discovery of a novel Rab5 inhibitor, neoandrographolide (NAP), by using high-throughput virtual screening with a natural product library containing 7459 compounds, which can occupy the surface groove of Rab5, competing with GDP/GTP for the binding. Ser34 is the most important residue in the groove of Rab5, as it forms most hydrogen-bond interactions with GDP/GTP or NAP, and in silico mutation of Ser34 decreased the stabilization of Rab5. Moreover, fluorescence titration experiment and isothermal titration calorimetry (ITC) assay revealed a direct binding between NAP and Rab5. Biochemical and cell-based assays showed that NAP treatment not only diminished the activity of Rab5, but also suppressed cell growth of cancer cell. This finding firstly identifies NAP as a novel inhibitor of Rab5, which directly binds with Rab5 by occupying the GDP/GTP binding groove to suppress its functions, highlighting a great potential of NAP to be developed as a chemotherapeutic agent in cancer therapy.

Antibiotic resistance is increasingly becoming a serious challenge to public health. The regulation of metabolism by post-translational modifications (PTMs) has been widely studied; however, the comprehensive mechanism underlying the regulation of acetylation in bacterial resistance against antibiotics is unknown. Herein, with Escherichia coli as the model, we performed quantitative analysis of the acetylated proteome of wild-type sensitive strain (WT) and ampicillin- (Re-Amp), kanamycin- (Re-Kan), and polymyxin B-resistant (Re-Pol) strains. Based on bioinformatics analysis combined with biochemical validations, we found that a common regulatory mechanism exists between the different resistant strains. Acetylation negatively regulates bacterial metabolism to maintain antibiotic resistance, but positively regulates bacterial motility. Further analyses revealed that key enzymes in various metabolic pathways were differentially acetylated. Particularly, pyruvate kinase (PykF), a key glycolytic enzyme regulating bacterial metabolism, and its acetylated form were highly expressed in the three resistant types and were identified as reversibly acetylated by the deacetylase CobB and the acetyl-transferase PatZ, and also could be acetylated by non-enzyme AcP in vitro. Further, the deacetylation of Lys413 of PykF increased the enzyme activity by changing the conformation of ATP binding site of PykF, resulting in an increase in energy production, which in turn increased the sensitivity of drug-resistant strains to antibiotics. This study provides novel insights for understanding bacterial resistance and lays the foundation for future research on regulation of acetylation in antibiotic-resistant strains.ImportanceThe misuse of antibiotics has resulted in an emergence of a large number of antibiotic-resistant strains, which seriously threaten human health. Bacterial metabolism is tightly controlled by protein post-translational modifications, especially acetylation. However, the comprehensive mechanism underlying regulation of acetylation in bacterial resistance remains unexplored. Here, acetylation was found to positively regulate bacterial motility and negatively regulate energy metabolism, which was common in all the different antibiotic-resistant strains. Moreover, the acetylation and deacetylation process of PykF was uncovered, and deacetylation of the Lys 413 of PykF was found to contribute to bacterial sensitivity to antibiotics. This study provides a new direction for research on development of bacterial resistance through post-translational modifications and provides a theoretical basis for the development of antibacterial drugs.

Accurate full-length sequencing of a purified unknown protein is still challenging nowadays due to the error-prone mass-spectrometry (MS)-based methods.

Generation of a promising antioxidative reagent with superior biocompatibility is urgently needed to remedy spinal cord injuries (SCI), repair the damaged neurons and restrain the secondary injuries caused by inflammation-induced oxidative stress. Inhibitory elements in the injury sites and necessitous inherent neural regeneration ability were major challenges for functional recovery after spinal cord injuries. We here developed a highly bioactive iridium complex (IrFPHtz) with enhanced antioxidative activities and improved SCI therapeutic efficacy. Both in vivo and in vitro, IrFPHtz has exhibited neuroprotective and anti-inflammatory properties. Mechanically, IrFPHtz directly targets SOD1 and upregulates the expression of SOD1 to eliminate the excess Reactive Oxygen Species (ROS) production induced by SCI, and thus protecting neuron cells from further damage. As a result, IrFPHtz safeguarded the neurons and myelin sheaths against trauma, lessened glial scar conformations and facilitated the repair of neurons and long axon expansion in the glial scar. Furthermore, IrFPHtz significantly ameliorated the behavioral functions of SCI mice and promoted a satisfactory curative effect. Therefore, this study sheds light on a novel method for SCI treatment using IrFPHtz as a potential drug and implicates the clinical significance of metal complexes in diseases featuring with upregulated ROS species.

N6-methyladenosine (m6A) methylation is an abundant modification in eukaryotic mRNAs. Accumulating evidence suggests a role for RNA m6A methylation in various aspects of cancer biology. In this study, we aimed to explore the biological role of RNA m6A modification in tumor metastasis and to identify novel therapeutic strategies for esophageal squamous cell carcinoma (ESCC). Integration of genome-wide CRISPR/Cas9 functional screening with highly invasive and metastatic ESCC subline models led to the identification of METTL3, the catalytic subunit of the N6-adenosine-methyltransferase complex, as a promoter of cancer metastasis. METTL3 expression was upregulated in ESCC tumors and metastatic tissues. In vitro and in vivo experiments indicated that METTL3 increased m6A in EGR1 mRNA and enhanced its stability in a YTHDF3-dependent manner, activating EGR1/Snail signaling. Investigation into the regulation of METTL3 expression found that KAT2A increased H3K27 acetylation levels in the METTL3 promoter region and activated transcription of METTL3, whereas SIRT2 exerted the opposite effects. Molecular docking and computational screening in a Food and Drug Administration–approved compound library consisting of 1,443 small molecules identified compounds targeting METTL3 to suppress cancer metastasis. Elvitegravir, originally developed to treat human immunodeficiency virus (HIV) infection, suppressed metastasis by directly targeting METTL3 and enhancing its STUB1-mediated proteasomal degradation. Overall, RNA m6A modifications are important in cancer metastasis, and targeting METTL3 with elvitegravir has therapeutic potential for treating ESCC. Significance: This study finds that METTL3 promotes cancer metastasis by activating EGR1/Snail signaling in an m6A-dependent manner, revealing vulnerability to METTL3 blockade in esophageal squamous cell carcinoma.

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.

Gastrointestinal cancer is a leading cause of cancer-related mortality and remains a major challenge for cancer treatment. Despite the combined administration of modern surgical techniques and chemoradiotherapy (CRT), the overall 5-year survival rate of gastrointestinal cancer patients in advanced stage disease is less than 15%, due to rapid disease progression, metastasis, and CRT resistance. A better understanding of the mechanisms underlying cancer progression and optimized treatment strategies for gastrointestinal cancer are urgently needed. With increasing evidence highlighting the protective role of immune responses in cancer initiation and progression, immunotherapy has become a hot research topic in the integrative management of gastrointestinal cancer. Here, an overview of the molecular understanding of colorectal cancer, esophageal cancer and gastric cancer is provided. Subsequently, recently developed immunotherapy strategies, including immune checkpoint inhibitors, chimeric antigen receptor T cell therapies, tumor vaccines and therapies targeting other immune cells, have been described. Finally, the underlying mechanisms, fundamental research and clinical trials of each agent are discussed. Overall, this review summarizes recent advances and future directions for immunotherapy for patients with gastrointestinal malignancies.

It has been a long debate whether the 98% ‘non-coding’ fraction of human genome can encode functional proteins besides short peptides. With full-length translating mRNA sequencing and ribosome profiling, we found that up to 3330 long non-coding RNAs (lncRNAs) were bound to ribosomes with active translation elongation. With shotgun proteomics, 308 lncRNA-encoded new proteins were detected. A total of 207 unique peptides of these new proteins were verified by multiple reaction monitoring (MRM) and/or parallel reaction monitoring (PRM); and 10 new proteins were verified by immunoblotting. We found that these new proteins deviated from the canonical proteins with various physical and chemical properties, and emerged mostly in primates during evolution. We further deduced the protein functions by the assays of translation efficiency, RNA folding and intracellular localizations. As the new protein UBAP1-AST6 is localized in the nucleoli and is preferentially expressed by lung cancer cell lines, we biologically verified that it has a function associated with cell proliferation. In sum, we experimentally evidenced a hidden human functional proteome encoded by purported lncRNAs, suggesting a resource for annotating new human proteins.

Lung cancer is the leading cause of cancer-related deaths worldwide. Tumor suppressor genes remain to be systemically identified for lung cancer. Through the genome-wide screening of tumor-suppressive transcription factors, we demonstrate here that GATA4 functions as an essential tumor suppressor in lung cancer in vitro and in vivo. Ectopic GATA4 expression results in lung cancer cell senescence. Mechanistically, GATA4 upregulates multiple miRNAs targeting TGFB2 mRNA and causes ensuing WNT7B downregulation and eventually triggers cell senescence. Decreased GATA4 level in clinical specimens negatively correlates with WNT7B or TGF-β2 level and is significantly associated with poor prognosis. TGFBR1 inhibitors show synergy with existing therapeutics in treating GATA4-deficient lung cancers in genetically engineered mouse model as well as patient-derived xenograft (PDX) mouse models. Collectively, our work demonstrates that GATA4 functions as a tumor suppressor in lung cancer and targeting the TGF-β signaling provides a potential way for the treatment of GATA4-deficient lung cancer., The tumor suppressor GATA4 is frequently epigenetically silenced in lung cancer. In this study, Gao et al. demonstrate that GATA4 regulates the expression of TGFBR2 and that TGFRB1 inhibitors can synergise with chemotherapeutics to inhibit the growth of GATA4-deficient tumors in mice.

Drug resistance in bacteria is becoming a significant threat to global public health, and the development of novel and efficient antibacterial compounds is urgently needed. Recently, rhodium complexes have attracted attention as antimicrobial agents, yet their antibacterial mechanism remains unknown. In this study, we observed that the dirhodium (II) complex Rh2Ac4 inhibited Streptococcus. pneumoniae growth without significant cytotoxic side-effects on host cells in vitro. We subsequently investigated the antibacterial mechanism of Rh2Ac4 using iTRAQ-based proteomics combined with cellular and biochemical assays. Bioinformatics analysis on the proteomic alterations demonstrated that six molecular functional groups, including metal ion binding and twelve metabolic pathways, were significantly affected after treatment with Rh2Ac4. The interaction network analysis of metal ion binding proteins suggested that Rh2Ac4 decreased the protein expression levels of SPD_1652, SPD_1590 and Gap, which are associated with haem uptake/metabolism. Cellular and biochemical assays further confirmed that Rh2Ac4 could be taken up by bacteria via the PiuABCD haem-uptake system. The structurally similar Rh complex may compete with Fe-haem to decrease Fe-uptake via the PiuABCD system, disrupting iron metabolism to exert its antibacterial activity against S. pneumoniae. These data indicate that Rh2Ac4 is a promising new drug for the treatment of S. pneumoniae infections.

Stable isotope labeling with amino acids in cell culture (SILAC) is a robust proteomics method with advantages such as reproducibility and easy handling. This method is popular for the analysis of mammalian cells. However, amino acid conversion in bacteria decreases the labeling efficiency and quantification accuracy, limiting the application of SILAC in bacterial proteomics to auxotrophic bacteria or to single labeling with lysine. In this study, we found that adding high concentrations of isotope-labeled (heavy) and natural (light) amino acids into SILAC minimal medium can efficiently inhibit the complicated amino acid conversions. This simple and straightforward strategy facilitated complete incorporation of amino acids into the bacterial proteome with good accuracy. High labeling efficiency can be achieved in different bacteria by slightly modifying the supplementation of amino acids in culture media, promoting the widespread application of SILAC technique in bacterial proteomics. SIGNIFICANCE: Amino acid conversion in bacteria decreases labeling efficiency, limiting the application of Stable isotope labeling with amino acids in cell culture (SILAC) in bacterial proteomics to auxotrophic bacteria or single labeling with lysine. In this study, we found that high concentrations of isotope-labeled (heavy) and natural (light) amino acids facilitate full incorporation of amino acids into the bacterial proteome with good reproducibility. This improved double labeling SILAC technique using medium supplemented with high concentrations of amino acids is suitable for quantitative proteomics research on both gram-positive and -negative bacteria, facilitating the broad application of quantitative proteomics in bacterial studies.

Rationale: Dysregulated microRNA (miRNA) expressions in cancer can contribute to chemoresistance. This study aims to identify miRNAs that are associated with fluorouracil (5-FU) chemoresistance in esophageal squamous cell carcinoma (ESCC). The potential of miR-29c as a novel diagnostic, prognostic and treatment-predictive marker in ESCC, and its mechanisms and therapeutic implication in overcoming 5-FU chemoresistance were explored. Methods: The miRNA profiles of an ESCC cell model with acquired chemoresistance to 5-FU were analyzed using a Taqman miRNA microarray to identify novel miRNAs associated with 5-FU chemoresistance. Quantitative real-time PCR was used to determine miR-29c expression in tissue and serum samples of patients. Bioinformatics, gain- and loss-of-function experiments, and luciferase reporter assay were performed to validate F-box only protein 31 (FBXO31) as a direct target of miR-29c, and to identify potential transcription factor binding events that control miR-29c expression. The potential of systemic miR-29c oligonucleotide-based therapy in overcoming 5-FU chemoresistance was evaluated in tumor xenograft model. Results: MiR-29c, under the regulatory control of STAT5A, was frequently downregulated in tumor and serum samples of patients with ESCC, and the expression level was correlated with overall survival. Functional studies showed that miR-29c could override 5-FU chemoresistance in vitro and in vivo by directly interacting with the 3'UTR of FBXO31, leading to repression of FBXO31 expression and downstream activation of p38 MAPK. Systemically administered miR-29c dramatically improved response of 5-FU chemoresistant ESCC xenografts in vivo. Conclusions: MiR-29c modulates chemoresistance by interacting with FBXO31, and is a promising non-invasive biomarker and therapeutic target in ESCC.

Harmful algal blooms (HABs) are symptomatic of ecosystem imbalance, leading to major worldwide marine natural disasters, and seriously threaten the human health. Some HAB algae's exceptional genome size prohibited the genomic investigations on molecular mechanisms, for example

The unreasonable misuse of antibiotics has led to the emergence of large-scale drug-resistant bacteria, seriously threatening human health. Compared with drug-sensitive bacteria, resistant bacteria are difficult to clear by host immunity. To fully explore the adaptive mechanism of resistant bacteria to the iron-restricted environment, we performed data-independent acquisition-based quantitative proteomics on ciprofloxacin (CIP)-resistant (CIP-R)

Keloid is a benign tumor characterized by persistent inflammation, increased fibroblast proliferation, and abnormal deposition of collagen in the wound. The etiology of keloid is unclear. Here, we explored the phospho-signaling changes in human keloid fibroblasts via phosphoproteome mass spectrometry analysis. We found that comparative phosphoproteomics could statistically distinguish keloid from control fibroblasts. Differentially expressed phosphoproteins could predict the activation of known keloid-relevant upstream regulators including transforming growth factor-β1, interleukin (IL)-4, and IL-5. With multiple bioinformatics analyses, phosphorylated FLNA, TLN1, and VCL were significantly enriched in terms of calcium homeostasis and platelet aggregation. We biologically verified that keloid fibroblasts had a higher level of Ca2+ influx than the control fibroblasts upon ionomycin stimulation. Via co-cultivation analysis, we found that human keloid fibroblasts could directly promote platelet aggregation. As suggested by PhosphoPath and gene set enrichment analysis, pFLNA was centered as the top phosphoproteins associated with keloid phenotypes. We validated that pFLNA was upregulated both in keloid fibroblasts and keloid tissue section, implicating its biomarker potential. In conclusion, we reported the first phosphoproteome on keloid fibroblasts, based on which we revealed that keloid fibroblasts had aberrant calcium homeostasis and could directly induce platelet aggregation.

Chondrosarcoma (CHS) is the second most common bone malignancy with limited therapeutic approaches. Our previous study has found that Yes associated protein 1 (YAP1) is downregulated in CHS cells treated with bromodomain and extraterminal domain (BET) inhibitor JQ1. However, the precise role of YAP1 in CHS is largely unknown. Herein, we found that YAP1 expression was upregulated in CHS tissues, and positively correlated with its grading score. Loss of YAP1 inhibited CHS proliferation and induced cellular senescence, while expression of YAP1 mutants revealed YAP1/TEA domain family member (TEAD)-dependent negative regulation of p21 and subsequent cellular senescence. These results were validated by in vivo experiments using stable shYAP1 cell lines. Mechanistically, negative regulation of p21 by YAP1 occurred post-transcriptionally via Dicer-regulated miRNA networks, specifically, the miR-17 family. Furthermore, we demonstrated that sequential targeting of YAP1 and p21 enhanced the elimination of JQ1-induced senescent cells in a Bcl-2-like 1 (Bcl-XL)/Caspase-3 dependent manner. Altogether, we unveil a novel role of YAP1 signaling in mediating CHS cell senescence and propose a one-two punch approach that sequentially targets the YAP1/p21 axis to eliminate senescent cells.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors with poor survival. It is urgent to search for new efficient drugs with good stability and safety for clinical therapy. This study aims to identify potential anticancer drugs from a compound library consisting of 429 natural products. Echinatin, a compound isolated from the Chinese herb Glycyrrhiza uralensis Fisch, was found to markedly induce apoptosis and inhibit proliferation and colony-formation ability in ESCC. Confocal fluorescence microscopy data showed that echinatin significantly induced autophagy in ESCC cells, and autophagy inhibitor bafilomycinA1 attenuated the suppressive effects of echinatin on cell viability and apoptosis. Mechanistically, RNA sequencing coupled with bioinformatics analysis and a series of functional assays revealed that echinatin induced apoptosis and autophagy through inactivation of AKT/mTOR signaling pathway, whereas constitutive activation of AKT significantly abrogated these effects. Furthermore, we demonstrated that echinatin had a significant antitumor effect in the tumor xenograft model and markedly suppressed cell migration and invasion abilities of ESCC cells in a dose-dependent manner. Our findings provide the first evidence that echinatin could be a novel therapeutic strategy for treating ESCC.

Bacterial resistance has become a serious threat to human health. In particular, the gradual development of resistance to polymyxins, the last line of defense for human infections, is a major issue. Secreted proteins contribute to the interactions between bacteria and the environment. In this study, we compared the secretomes of polymyxin B-sensitive and -resistant Escherichia coli strains by data-independent acquisition mass spectrometry. In total, 87 differentially expressed secreted proteins were identified in polymyxin B-resistant E. coli compared to the sensitive strain. A GO enrichment analysis indicated that the differentially expressed proteins were involved in biological processes, including bacterial-type flagellum-dependent cell motility, ion transport, carbohydrate derivative biosynthetic process, cellular response to stimulus, organelle organization, and cell wall organization or biogenesis. The differentially expressed secreted proteins in polymyxin B-resistant bacteria were enriched for multiple pathways, suggesting that the resistance phenotype depends on complex regulatory mechanisms. A potential biomarker or drug target (YebV) was found in polymyxin B-resistant E. coli. This work clarifies the secretome changes associated with the acquisition of polymyxin resistance and may contribute to drug development.

Lung cancer is the most frequent cancer worldwide with a poor prognosis. Identification of novel cancer targets and useful therapeutic strategies without toxicity are urgently needed. In this study, we screened natural products for anticancer bioactivity in a library consisting of 429 small molecules. We demonstrated for the first time that daurisoline, a constituent of Rhizoma Menispermi, repressed lung cancer cell proliferation by inducing cell cycle arrest at the G1 phase. Furthermore, daurisoline was found not only to suppress the growth of lung tumor xenografts in animals without obvious side effects, but also to inhibit cell migration and invasion. Mechanistically, quantitative proteomics and bioinformatics analyses, Western blotting and qRT-PCR confirmed that daurisoline exerted its anticancer effects by inhibiting the expression levels of β-catenin and its downstream targets c-myc and cyclin D1. Furthermore, our data from Drug Affinity Responsive Target Stability (DARTS), isothermal titration calorimetry (ITC) and a series of functional assays demonstrated that daurisoline could target HSP90 directly and disrupt its interaction with β-catenin, therefore increasing the ubiquitin-mediated proteasomal degradation of β-catenin. This study reveals that daurisoline could be a promising therapeutic strategy for the treatment of lung cancer.

Purpose: Clinical success of precision medicine is severely limited by de novo or acquired drug resistance. It remains a clinically unmet need to treat these patients. Tumor suppressor genes (TSGs) play a critical role in tumorigenesis and impact the therapeutic effect of various treatments. Experimental Design: Using clinical data, in vitro cell line data and in vivo mouse model data, we revealed the tumor suppressive role of Clusterin in lung cancer. We also delineated the signaling cascade elicited by loss of function of CLU in NSCLC cells and tested precision medicine for CLU deficient lung cancers. Results: CLU is a potent and clinically relevant TSG in lung cancer. Mechanistically, CLU inhibits TGFBR1 to recruit TRAF6/TAB2/TAK1 complex and thus inhibits activation of TAK1- NF-κB signaling axis. Lung cancer cells with loss of function of CLU show exquisite sensitivity to TAK1 inhibitors. Importantly, we show that a significant portion of Kras mutation positive NSCLC patients are concurrently deficient of CLU and that TAK1 kinase inhibitor synergizes with existing drugs to treat this portion of lung cancers patients. Conclusions: Combinational treatment with TAK1 inhibitor and MEK1/2 inhibitor effectively shrank Kras mutation positive and CLU deficient NSCLC tumors. Moreover, we put forward a concept that loss of function of a TSG rewires signaling network and thereby creates an Achilles' heel in tumor cells which could be exploited in precision medicine.

Metastasis is the main factor of treatment failure in cancer patients, but the underlying mechanism remains to be elucidated and effective new treatment strategies are urgently needed. This study aims to explore novel key metastasis-related microRNAs (miRNAs) in esophageal squamous cell carcinoma (ESCC). By comparing miRNA profiles of the highly metastatic ESCC cell sublines, we established through serial in vivo selection with the parental cells, we found that the expression level of miR-515-3p was lower in ESCC tumor tissues than adjacent normal tissues, further decreased in metastatic tumors, and moreover, markedly associated with advanced stage, metastasis and patient survival. The in vitro and in vivo assays suggested that miR-515-3p could increase the expression of the epithelial markers as well as decrease the expression of the mesenchymal markers, and more importantly, suppress invasion and metastasis of ESCC cells. Mechanistically, we revealed that miR-515-3p directly regulated vimentin and matrix metalloproteinase-3 (MMP3) expression by binding to the coding sequence and 3′untranslated region, respectively. In addition, the data from whole-genome methylation sequencing and methylation-specific PCR indicated that the CpG island within miR-515-3p promoter was markedly hypermethylated in ESCC cell lines and ESCC tumor tissues, which may lead to deregulation of miR-515-3p expression in ESCC. Furthermore, our preclinical experiment provides solid evidence that systemic delivery of miR-515-3p oligonucleotide obviously suppressed the metastasis of ESCC cells in nude mice. Taken together, this study demonstrates that miR-515-3p suppresses tumor metastasis and thus represents a promising prognostic biomarker and therapeutic strategy in ESCC.

Background: Colorectal cancer (CRC) is a high incidence of malignant tumors that lacks highly effective and targeted drugs and thus it is in urgent need of finding new specific molecular targets. Methods and Results: In this study, by using WST-1 (Highly water-soluble tetrazolium salt-1) and colony formation assays, we found that C20orf27 (chromosome 20 open reading frame 27), a functionally unknown protein, enhanced the growth and proliferation of CRC cells. The nude mouse tumor formation experiments verified that C20orf27 promoted the growth of CRC. Signal pathway analysis identified the TGF&beta, R-TAK1-NFĸB cascade as a mediator in C20orf27-induced CRC progression. Inhibition experiments using NFĸB inhibitors reversed this progression. Co-immunoprecipitation showed that C20orf27 promoted the activation of the TGF&beta, R-TAK1-NFĸB pathway by interacting with PP1c (the catalytic subunit of type 1 phosphatase). Conclusions: Our results firstly characterized the functional role and molecular mechanism of C20orf27 in driving CRC growth and proliferation through the TGF&beta, R-TAK1-NFĸB pathway, suggesting its potential as a novel CRC candidate therapeutic target and tumor marker.

Mitochondria are involved in many crucial cellular processes. Maintaining healthy mitochondria is essential for cellular homeostasis. Parkin-dependent mitophagy plays an important role in selectively eliminating damaged mitochondria in mammalian cells. However, mechanisms of Parkin-dependent mitophagy remain elusive. In this research, we performed data-independent acquisition-based quantitative mitochondrial proteomics to study the proteomic alterations of carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced Parkin-mediated mitophagy. We identified 222 differentially expressed proteins, with 76 upregulations and 146 downregulations, which were potentially involved in mitophagy. We then demonstrated that annexin A7 (ANXA7), a calcium-dependent phospholipid-binding protein, can translocate to impaired mitochondria upon CCCP treatment, where it played a pivotal part in the process of Parkin-dependent mitophagy via interacting with BASP1. As a mitochondrial uncoupling agent, CCCP indirectly regulated ANXA7 and BASP1 to induce Parkin-dependent mitophagy.

Iron is an essential element for almost all bacteria. The iron ATP-binding cassette (ABC) transporters located on the cell membrane affects bacterial virulence and infection. Although a variety of Fe3+-transporters have been found in bacteria, their evolutionary processes are rarely studied. Pneumococcal iron ABC transporter (PitA), a highly conserved Fe3+-transporter in most pathogenic bacteria, influences the capsule formation and virulence of bacteria. However, multiple sequence alignment revealed that PitA is expressed in four different variants in bacteria, and the structural complexity of these variants increases progressively. To more efficiently import Fe3+ ions into bacterial cells, bacteria have evolved a fused PitA from two separately expressed PitA-1 (SPD_0227) and PitA-2 (SPD_0226) proteins. Further biochemical characterization indicated that both PitA-1 and PitA-2 have weaker Fe3+-binding ability than their protein complex. More importantly, Glutathione S-Transferase (GST) pull-down and isothermal titration calorimetry (ITC) detection showed that PitA-1 and PitA-2 interact with each other via Tyr111-Leu37, Asn112-Gln38, Asn103-Leu33, and Asn103-Thr34. Further molecular dynamics (MD) simulations demonstrated that this interaction in full-length PitA is stronger than that in the two individual proteins. Deletion of PitA family genes could lead to decrease in the ability of iron acquisition and of adhesion and invasion of S. pneumoniae. Our study revealed the evolving state and molecular mechanism of Fe3+-transporter PitAs in bacteria and provided important information for understanding the iron transportation mechanism in bacteria and designing new antibacterial drugs.

Scope: Colorectal cancer (CRC) is one of the most common cancers worldwide with poor survival and limited therapeutic options, and there is an urgent need to develop novel therapeutic agents with good treatment efficiency and low toxicity. This study aims to examine the anticancer bioactivity of liensinine, a constituent of Nelumbo nucifera Gaertn, in CRC and investigate the action mechanisms involved. Methods and results: Liensinine was found to induce apoptosis and exert a significant inhibitory effect on the proliferation and colony-forming ability of CRC cells in a dose-dependent manner without any observed cytotoxicity on normal colorectal epithelial cells. Mechanistically, our data from quantitative proteomics, western blot analysis and flow cytometry analyses demonstrated that exposure of CRC cells to liensinine caused cell cycle arrest, mitochondrial dysfunction and apoptosis, accompanied by the activation of the JNK signaling pathway. Furthermore, animal experiments showed that liensinine markedly suppressed the growth of CRC tumor xenografts in nude mice by reducing the Ki-67 proliferation index, but did not damage the vital organs of the animals. Conclusion: This study demonstrated for the first time that liensinine, a food-source natural product, could be a novel therapeutic strategy for treating CRC without obvious side effects.

Preeclampsia (PE) is a placenta disease, featured by hypertension, proteinuria, and other multiorgan dysfunctions, and its etiology is unclear. We and others have shown that intensive endoplasmic reticulum (ER) stress and unfolded protein response (UPR) occur in the PE placenta. In this study, we isolated detergent-insoluble proteins (DIPs) from human placenta tissues, which were enriched with protein aggregates, to characterize the placenta UPR in PE. With data-independent acquisition (DIA) mass spectrometry, we identified 2066 DIPs across all normal (n = 10) and PE (n = 10) placenta samples, among which 110 and 108 DIPs were significantly up- and down-regulated in PE, respectively. Per clustering analysis, differential DIPs could generally distinguish PE from normal placentas. We verified the MS quantitation of endoglin and vimentin by immunoblotting. In addition, we observed that PE placenta tissues have remarkably more endoglin in the cytoplasm. Furthermore, we found that DIPs were evenly distributed across different chromosomes and could be enriched in diversified gene ontology terms, while differential DIPs avoided to distribute on X-chromosome. Significantly up-regulated DIPs in PE were focused on the top functions of lipid metabolism, while 23 of these DIPs could form the top network regulating cellular movement, development, growth, and proliferation. Our results implicate that human PE placentas have disease-relevant differential DIPs, which reflect aberrantly aggregated proteins of placental tissues. The mass spectrometry proteomics data have been deposited to ProteomeXchange consortium with the data set identifier PXD006654, and iProX database (accession number: IPX0000948000).