Accumulating evidence illustrates a fundamental role for mitochondria in lung alveolar type 2 epithelial cell (AEC2) dysfunction in the pathogenesis of idiopathic pulmonary fibrosis. However, the role of mitochondrial fusion in AEC2 function and lung fibrosis development remains unknown. Here we report that the absence of the mitochondrial fusion proteins mitofusin1 (MFN1) and mitofusin2 (MFN2) in murine AEC2 cells leads to morbidity and mortality associated with spontaneous lung fibrosis. We uncover a crucial role for MFN1 and MFN2 in the production of surfactant lipids with MFN1 and MFN2 regulating the synthesis of phospholipids and cholesterol in AEC2 cells. Loss of MFN1, MFN2 or inhibiting lipid synthesis via fatty acid synthase deficiency in AEC2 cells exacerbates bleomycin-induced lung fibrosis. We propose a tenet that mitochondrial fusion and lipid metabolism are tightly linked to regulate AEC2 cell injury and subsequent fibrotic remodeling in the lung.

BackgroundProtein phosphorylation regulated by plant hormone is involved in the coordination of fundamental plant development. Brassinosteroids (BRs), a group of phytohormones, regulated phosphorylation dynamics remains to be delineated in plants. In this study, we performed a mass spectrometry (MS)-based phosphoproteomics to conduct a global and dynamic phosphoproteome profiling across five time points of BR treatment in the period between 5 min and 12 h. MS coupling with phosphopeptide enrichment techniques has become the powerful tool for profiling protein phosphorylation. However, MS-based methods tend to have data consistency and coverage issues. To address these issues, bioinformatics approaches were used to complement the non-detected proteins and recover the dynamics of phosphorylation events.ResultsA total of 1104 unique phosphorylated peptides from 739 unique phosphoproteins were identified. The time-dependent gene ontology (GO) analysis shows the transition of biological processes from signaling transduction to morphogenesis and stress response. The protein-protein interaction analysis found that most of identified phosphoproteins have strongly connections with known BR signaling components. The analysis by using Motif-X was performed to identify 15 enriched motifs, 11 of which correspond to 6 known kinase families. To uncover the dynamic activities of kinases, the enriched motifs were combined with phosphorylation profiles and revealed that the substrates of casein kinase 2 and mitogen-activated protein kinase were significantly phosphorylated and dephosphorylated at initial time of BR treatment, respectively. The time-dependent kinase-substrate interaction networks were constructed and showed many substrates are the downstream of other signals, such as auxin and ABA signaling. While comparing BR responsive phosphoproteome and gene expression data, we found most of phosphorylation changes were not led by gene expression changes. Our results suggested many downstream proteins of BR signaling are induced by phosphorylation via various kinases, not through transcriptional regulation.ConclusionsThrough a large-scale dynamic profile of phosphoproteome coupled with bioinformatics, a complicated kinase-centered network related to BR-regulated growth was deciphered. The phosphoproteins and phosphosites identified in our study provide a useful dataset for revealing signaling networks of BR regulation, and also expanded our knowledge of protein phosphorylation modification in plants as well as further deal to solve the plant growth problems.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1753-4) contains supplementary material, which is available to authorized users.

BackgroundTanshinone IIA (TIIA) is a diterpene quinone extracted from the plant Danshen (Salvia miltiorrhiza) used in traditional Chinese herbal medicine. It has been reported to have anti-tumor potential against several kinds of cancer, including gastric cancer. In most solid tumors, a metabolic switch to glucose is a hallmark of cancer cells, which do this to provide nutrients for cell proliferation. However, the mechanism associated with glucose metabolism by which TIIA acts on gastric cancer cells remains to be elucidated.ResultsWe found that TIIA treatment is able to significantly inhibit cell growth and the proliferation of gastric cancer in a dose-dependent manner. Using next-generation sequencing-based RNA-seq transcriptomics and quantitative proteomics-isobaric tags for relative and absolute quantification (iTRAQ), we characterized the mechanism of TIIA regulation in gastric cancer cell line AGS. In total, 16,603 unique transcripts and 102 proteins were identified. After enrichment analysis, we found that TIIA regulated genes are involved in carbohydrate metabolism, the cell cycle, apoptosis, DNA damage and cytoskeleton reorganization. Our proteomics data revealed the downregulation of intracellular ATP levels, glucose-6-phosphate isomerase and L-lactate dehydrogenase B chains by TIIA, which might work with disorders of glucose metabolism and extracellular lactate levels to suppress cell proliferation. The up-regulation of p53 and down-regulation of AKT was shown in TIIA- treated cells, which indicates the transformation of oncogenes. Severe DNA damage, cell cycle arrest at the G2/M transition and apoptosis with cytoskeleton reorganization were detected in TIIA-treated gastric cancer cells.ConclusionsCombining transcriptomics and proteomics results, we propose that TIIA treatment could lead cell stresses, including nutrient deficiency and DNA damage, by inhibiting the glucose metabolism of cancer cells. This study provides an insight into how the TIIA regulatory metabolism in gastric cancer cells suppresses cell growth, and may help improve the development of cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1230-0) contains supplementary material, which is available to authorized users.

Carbonic anhydrase IX (CA9) expression level has been considered as a poor prognostic factor in hepatocellular carcinoma (HCC) patients. However, the judging criteria of CA9 level is hard to define for potential clinical applications. Unlike CA9 expression level, CA9 polymorphism is poorly documented in HCC. Here, we found that people carry A allele at CA9 rs1048638, a 3′UTR SNP, has higher risk of HCC. rs1048638-CA correlates with advanced stages, larger tumor sizes, more vascular invasion, and shorter survival of HCC patients. A allele at CA9 rs1048638 impairs miR-34a, a tumor suppressor miRNA in HCC, binding to CA9 3′UTR and desensitizes CA9 mRNA to miR-34a-dependent RNA degradation. CA9 expression levels were also correlated with miR-34a levels and rs1048638 genotypes in HCC patients. rs1048638 influences HCC risk and progression through effects on miR-34a-targeted CA9 expression in HCC. In conclusion, genetic variations of the CA9 3′UTR play important roles in regulating CA9 expression and cancer progression, which is a novel determinant and target for HCC metastasis and prognosis.

BackgroundRas is frequently mutated in a variety of human cancers, including lung cancer, leading to constitutive activation of MAPK signaling. Despite decades of research focused on the Ras oncogene, Ras-targeted phosphorylation events and signaling pathways have not been described on a proteome-wide scale.Methodology/Principal FindingsBy functional phosphoproteomics, we studied the molecular mechanics of oncogenic Ras signaling using a pathway-based approach. We identified Ras-regulated phosphorylation events (n = 77) using label-free comparative proteomics analysis of immortalized human bronchial epithelial cells with and without the expression of oncogenic Ras. Many were newly identified as potential targets of the Ras signaling pathway. A majority (∼60%) of the Ras-targeted events consisted of a [pSer/Thr]-Pro motif, indicating the involvement of proline-directed kinases. By integrating the phosphorylated signatures into the Pathway Interaction Database, we further inferred Ras-regulated pathways, including MAPK signaling and other novel cascades, in governing diverse functions such as gene expression, apoptosis, cell growth, and RNA processing. Comparisons of Ras-regulated phosphorylation events, pathways, and related kinases in lung cancer-derived cells supported a role of oncogenic Ras signaling in lung adenocarcinoma A549 and H322 cells, but not in large cell carcinoma H1299 cells.Conclusions/SignificanceThis study reveals phosphorylation events, signaling networks, and molecular functions that are regulated by oncogenic Ras. The results observed in this study may aid to extend our knowledge on Ras signaling in lung cancer.

BackgroundRegorafenib and other multikinase inhibitors may enhance antitumor efficacy of anti-program cell death-1 (anti-PD1) therapy in hepatocellular carcinoma (HCC). Its immunomodulatory effects, besides anti-angiogenesis, were not clearly defined.MethodsIn vivo antitumor efficacy was tested in multiple syngeneic liver cancer models. Murine bone marrow–derived macrophages (BMDMs) were tested in vitro for modulation of polarization by regorafenib and activation of cocultured T cells. Markers of M1/M2 polarization were measured by quantitative reverse transcription PCR (RT-PCR), arginase activity, flow cytometry, and ELISA. Knockdown of p38 kinase and downstream Creb1/Klf4 signaling on macrophage polarization were confirmed by using knockdown of the upstream MAPK14 kinase, chemical p38 kinase inhibitor, and chromatin immunoprecipitation.ResultsRegorafenib (5 mg/kg/day, corresponding to about half of human clinical dosage) inhibited tumor growth and angiogenesis in vivo similarly to DC-101 (anti-VEGFR2 antibody) but produced higher T cell activation and M1 macrophage polarization, increased the ratio of M1/M2 polarized BMDMs and proliferation/activation of cocultured T cells in vitro, indicating angiogenesis-independent immunomodulatory effects. Suppression of p38 kinase phosphorylation and downstream Creb1/Klf4 activity in BMDMs by regorafenib reversed M2 polarization. Regorafenib enhanced antitumor efficacy of adoptively transferred antigen-specific T cells. Synergistic antitumor efficacy between regorafenib and anti-PD1 was associated with multiple immune-related pathways in the tumor microenvironment.ConclusionRegorafenib may enhance antitumor immunity through modulation of macrophage polarization, independent of its anti-angiogenic effects. Optimization of regorafenib dosage for rational design of combination therapy regimen may improve the therapeutic index in the clinic.

MYCN, an oncogenic transcription factor of the Myc family, is a major driver of neuroblastoma tumorigenesis. Due to the difficulty in drugging MYCN directly, revealing the molecules in MYCN regulatory networks will help to identify effective therapeutic targets for neuroblastoma therapy. Here we perform ChIP-sequencing and small RNA-sequencing of neuroblastoma cells to determine the MYCN-binding sites and MYCN-associated microRNAs, and integrate various types of genomic data to construct MYCN regulatory networks. The overall analysis indicated that MYCN-regulated genes were involved in a wide range of biological processes and could be used as signatures to identify poor-prognosis MYCN-non-amplified patients. Analysis of the MYCN binding sites showed that MYCN principally served as an activator. Using a computational approach, we identified 32 MYCN co-regulators, and some of these findings are supported by previous studies. Moreover, we investigated the interplay between MYCN transcriptional and microRNA post-transcriptional regulations and identified several microRNAs, such as miR-124-3p and miR-93-5p, which may significantly contribute to neuroblastoma pathogenesis. We also found MYCN and its regulated microRNAs acted together to repress the tumor suppressor genes. This work provides a comprehensive view of MYCN regulations for exploring therapeutic targets in neuroblastoma, as well as insights into the mechanism of neuroblastoma tumorigenesis.

Differential coexpression analysis is emerging as a complement to conventional differential gene expression analysis. The identified differential coexpression links can be assembled into a differential coexpression network (DCEN) in response to environmental stresses or genetic changes. Differential coexpression analyses have been successfully used to identify condition-specific modules; however, the structural properties and biological significance of general DCENs have not been well investigated. Here, we analyzed two independent Saccharomyces cerevisiae DCENs constructed from large-scale time-course gene expression profiles in response to different situations. Topological analyses show that DCENs are tree-like networks possessing scale-free characteristics, but not small-world. Functional analyses indicate that differentially coexpressed gene pairs in DCEN tend to link different biological processes, achieving complementary or synergistic effects. Furthermore, the gene pairs lacking common transcription factors are sensitive to perturbation and hence lead to differential coexpression. Based on these observations, we integrated transcriptional regulatory information into DCEN and identified transcription factors that might cause differential coexpression by gain or loss of activation in response to different situations. Collectively, our results not only uncover the unique structural characteristics of DCEN but also provide new insights into interpretation of DCEN to reveal its biological significance and infer the underlying gene regulatory dynamics.