Phospholipid scramblase activity is involved in the collapse of phospholipid (PL) asymmetry at the plasma membrane leading to externalization of phosphatidylserine. This activity is crucial for ...initiation of the blood coagulation cascade and for recognition/elimination of apoptotic cells by macrophages. Efforts to identify gene products associated with this activity led to the characterization of PL scramblase (PLSCR) and XKR family members which contribute to phosphatidylserine exposure in response to apoptotic stimuli. Meanwhile, TMEM16 family members were identified to externalize phosphatidylserine in response to elevated calcium in Scott syndrome platelets, which is critical for activation of the coagulation cascade. Herein, we report their mechanisms of gene regulation, molecular functions independent of their scrambling activity, and their potential roles in pathogenic conditions.
Brain metastasis cancer-associated fibroblasts (bmCAFs) are emerging as crucial players in the development of breast cancer brain metastasis (BCBM), but our understanding of the underlying molecular ...mechanisms is limited. In this study, we aim to elucidate the pathological contributions of fucosylation (the post-translational modification of proteins by the dietary sugar L-fucose) to tumor-stromal interactions that drive the development of BCBM. Here, we report that patient-derived bmCAFs secrete high levels of polio virus receptor (PVR), which enhance the invasive capacity of BC cells. Mechanistically, we find that HIF1α transcriptionally upregulates fucosyltransferase 11, which fucosylates PVR, triggering its secretion from bmCAFs. Global phosphoproteomic analysis of BC cells followed by functional verification identifies cell-cell junction and actin cytoskeletal signaling as modulated by bmCAF-secreted, -fucosylated PVR. Our findings delineate a hypoxia- and fucosylation-regulated mechanism by which bmCAFs contribute to the invasiveness of BCBM in the brain.
Display omitted
•Brain met-CAFs exhibit high level of secreted, fucosylated polio virus receptor (sfPVR)•Hypoxia stimulates fucosyltransferase 11-mediated fucosylation and secretion of PVR•sfPVR alters cell-cell junctions and actin cytoskeleton dynamics, driving BC cell invasion•sfPVR/FUT11 expression by bmCAFs enhances BC invasion in the brain
Adhikari et al. report that low oxygen levels trigger secretion of fucosylated polio virus receptor from breast cancer brain metastasis (BCBM)-associated fibroblasts, which potently drives BC invasion in the brain, highlighting a potential therapeutic target and biomarker for BCBM.
A primary goal of the US National Cancer Institute's Ras initiative at the Frederick National Laboratory for Cancer Research is to develop methods to quantify RAS signaling to facilitate development ...of novel cancer therapeutics. We use targeted proteomics technologies to develop a community resource consisting of 256 validated multiple reaction monitoring (MRM)-based, multiplexed assays for quantifying protein expression and phosphorylation through the receptor tyrosine kinase, MAPK, and AKT signaling networks. As proof of concept, we quantify the response of melanoma (A375 and SK-MEL-2) and colorectal cancer (HCT-116 and HT-29) cell lines to BRAF inhibition by PLX4720. These assays replace over 60 western blots with quantitative mass-spectrometry-based assays of high molecular specificity and quantitative precision, showing the value of these methods for pharmacodynamic measurements and mechanism-of-action studies. Methods, fit-for-purpose validation, and results are publicly available as a resource for the community at assays.cancer.gov.
Display omitted
•Quantitative protein assays are required to understand cancer signaling networks•We develop a suite of multiplexed mass-spectrometry-based assays•The assays offer specific and precise quantification of key networks and PTMs•The assays provide a resource for mechanism-of-action and pharmacodynamic measurements
A lack of quantitative, multiplexable assays for phosphosignaling limits comprehensive investigation of aberrant signaling in cancer and evaluation of novel treatments. To alleviate this limitation, we sought to develop assays by using targeted mass spectrometry for quantifying protein expression and phosphorylation through the receptor tyrosine kinase, MAPK, and AKT signaling networks. The resulting assays provide a resource for replacing over 60 western blots in examining cancer signaling and tumor biology with high molecular specificity and quantitative rigor.
Whiteaker et al. describe a suite of mass-spectrometry-based assays for quantification of protein expression and phosphorylation in receptor tyrosine kinase, AKT, and MAP-kinase networks. The assays provide a resource for replacing over 60 commonly used cancer signaling and tumor biology western blots with high molecular specificity and quantitative rigor.
Aberrant splice variants provide a potentially rich source of novel pathogenic drivers and biomarkers. Using the example of clear cell renal cell carcinoma, we describe a robust and widely applicable ...pipeline to identify disease-specific aberrant splice variants and explore associations with underlying disease biology and clinical outcomes.
Alternative mRNA splicing can be dysregulated in cancer, resulting in the generation of aberrant splice variants (SVs). Given the paucity of actionable genomic mutations in clear cell renal cell carcinoma (ccRCC), aberrant SVs may be an avenue to novel mechanisms of pathogenesis.
To identify and characterize aberrant SVs enriched in ccRCC.
Using RNA-seq data from the Cancer Cell Line Encyclopedia, we identified neojunctions uniquely expressed in ccRCC. Candidate SVs were then checked for expression across normal tissue in the Genotype-Tissue Expression Project and primary tumor tissue from The Cancer Genome Atlas (TCGA), Clinical Proteomic Tumor Analysis Consortium (CPTAC), and our institutional Total Cancer Care database.
Clinicopathologic, genomic, and survival data were available for all cohorts. Epigenetic data were available for the TCGA and CPTAC cohorts. Proteomic data were available for the CPTAC cohort. The association of aberrant SV expression with these variables was examined using the Kruskal-Wallis test, pairwise t test, Spearman correlation test, and Cox regression analysis.
Our pipeline identified 16 ccRCC-enriched SVs. EGFR, HPCAL1-SV and RNASET2-SV expression was negatively correlated with gene-specific CpG methylation. We derived a survival risk score based primarily on the expression of five SVs (RNASET2, FGD1, PDZD2, COBLL1, and PTPN14), which was consistent and applicable across multiple cohorts on multivariate analysis. The splicing factor RBM4, which modulates splicing of HIF-1α, exhibited significantly lower expression at the protein level in the high-risk group, as defined by our SV-based score.
We describe 16 aberrant SVs enriched in ccRCC, many of which are associated with disease biology and/or clinical outcomes. This study provides a novel strategy for identifying and characterizing disease-specific aberrant SVs.
We describe a method to identify disease targets and biomarkers using transcriptomic analysis beyond somatic mutations or gene expression. Kidney tumors express unique splice variants that may provide additional prognostic information following surgery.
To better understand the signaling complexity of AXL, a member of the tumor-associated macrophage (TAM) receptor tyrosine kinase family, we created a physical and functional map of AXL signaling ...interactions, phosphorylation events, and target-engagement of three AXL tyrosine kinase inhibitors (TKI). We assessed AXL protein complexes using proximity-dependent biotinylation (BioID), effects of AXL TKI on global phosphoproteins using mass spectrometry, and target engagement of AXL TKI using activity-based protein profiling. BioID identifies AXL-interacting proteins that are mostly involved in cell adhesion/migration. Global phosphoproteomics show that AXL inhibition decreases phosphorylation of peptides involved in phosphatidylinositol-mediated signaling and cell adhesion/migration. Comparison of three AXL inhibitors reveals that TKI RXDX-106 inhibits pAXL, pAKT, and migration/invasion of these cells without reducing their viability, while bemcentinib exerts AXL-independent phenotypic effects on viability. Proteomic characterization of these TKIs demonstrates that they inhibit diverse targets in addition to AXL, with bemcentinib having the most off-targets. AXL and EGFR TKI cotreatment did not reverse resistance in cell line models of erlotinib resistance. However, a unique vulnerability was identified in one resistant clone, wherein combination of bemcentinib and erlotinib inhibited cell viability and signaling. We also show that AXL is overexpressed in approximately 30% to 40% of nonsmall but rarely in small cell lung cancer. Cell lines have a wide range of AXL expression, with basal activation detected rarely.
Our study defines mechanisms of action of AXL in lung cancers which can be used to establish assays to measure drug targetable active AXL complexes in patient tissues and inform the strategy for targeting it's signaling as an anticancer therapy.
PDF
