Highlights d Systematic identification of colon cancer-associated proteins and phosphosites d Proteomics-supported neoantigens and cancer/testis antigens in 78% of the tumors d Rb phosphorylation is an oncogenic driver and a putative target in colon cancer d Glycolysis inhibition may render MSI tumors more sensitive to checkpoint blockade

Mutant KRAS colorectal cancer (CRC) cells release protein-laden exosomes that can alter the tumor microenvironment. To test whether exosomal RNAs also contribute to changes in gene expression in recipient cells, and whether mutant KRAS might regulate the composition of secreted microRNAs (miRNAs), we compared small RNAs of cells and matched exosomes from isogenic CRC cell lines differing only in KRAS status. We show that exosomal profiles are distinct from cellular profiles, and mutant exosomes cluster separately from wild-type KRAS exosomes. miR-10b was selectively increased in wild-type exosomes, while miR-100 was increased in mutant exosomes. Neutral sphingomyelinase inhibition caused accumulation of miR-100 only in mutant cells, suggesting KRAS-dependent miRNA export. In Transwell co-culture experiments, mutant donor cells conferred miR-100-mediated target repression in wild-type-recipient cells. These findings suggest that extracellular miRNAs can function in target cells and uncover a potential new mode of action for mutant KRAS in CRC.DOI: http://dx.doi.org/10.7554/eLife.07197.001

No abstract

No abstract

SUMMARY The integration of mass spectrometry-based proteomics with next-generation DNA and RNA sequencing profiles tumors more comprehensively. Here this “proteogenomics” approach was applied to 122 treatment-naive primary breast cancers accrued to preserve post-translational modifications, including protein phosphorylation and acetylation. Proteogenomics challenged standard breast cancer diagnoses, provided detailed analysis of the ERBB2 amplicon, defined tumor subsets that could benefit from immune checkpoint therapy, and allowed more accurate assessment of Rb status for prediction of CDK4/6 inhibitor responsiveness. Phosphoproteomics profiles uncovered novel associations between tumor suppressor loss and targetable kinases.Acetylproteome analysis highlighted acetylation on key nuclear proteins involved in the DNA damage response and revealed cross-talk between cytoplasmic and mitochondrial acetylation and metabolism. Our results underscore the potential of proteogenomics for clinical investigation of breast cancer through more accurate annotation of targetable pathways and biological features of this remarkably heterogeneous malignancy.

Recent studies have shown that circular RNAs (circRNAs) are abundant, widely expressed in mammals, and can display cell-type specific expression. However, how production of circRNAs is regulated and their precise biological function remains largely unknown. To study how circRNAs might be regulated during colorectal cancer progression, we used three isogenic colon cancer cell lines that differ only in KRAS mutation status. Cellular RNAs from the parental DLD-1 cells that contain both wild-type and G13D mutant KRAS alleles and isogenically-matched derivative cell lines, DKO-1 (mutant KRAS allele only) and DKs-8 (wild-type KRAS allele only) were analyzed using RNA-Seq. We developed a bioinformatics pipeline to identify and evaluate circRNA candidates from RNA-Seq data. Hundreds of high-quality circRNA candidates were identified in each cell line. Remarkably, circRNAs were significantly down-regulated at a global level in DLD-1 and DKO-1 cells compared to DKs-8 cells, indicating a widespread effect of mutant KRAS on circRNA abundance. This finding was confirmed in two independent colon cancer cell lines HCT116 (KRAS mutant) and HKe3 (KRAS WT). In all three cell lines, circRNAs were also found in secreted extracellular-vesicles, and circRNAs were more abundant in exosomes than cells. Our results suggest that circRNAs may serve as promising cancer biomarkers.

MicroRNAs (miRNAs) are regulators of gene expression in plants and animals. The biogenesis of miRNAs is precisely controlled to secure normal development of organisms. Here we report that TOUGH (TGH) is a component of the DCL1-HYL1-SERRATE complex that processes primary transcripts of miRNAs [i.e., primary miRNAs (pri-miRNAs)] into miRNAs in Arabidopsis. Lack of TGH impairs multiple DCL activities in vitro and reduces the accumulation of miRNAs and siRNAs in vivo. TGH is an RNA-binding protein, binds pri-miRNAs and precursor miRNAs in vivo, and contributes to pri-miRNA-HYL1 interaction. These results indicate that TGH might regulate abundance of miRNAs through promoting DCL1 cleavage efficiency and/or recruitment of pri-miRNAs.S mall RNAs, including microRNAs (miRNAs) and siRNAs, are sequence-specific regulators of gene expression in plants and animals (1). miRNAs are derived from imperfect stem-loop transcripts, called primary miRNAs (pri-miRNAs), which are predominately produced by DNA-dependent RNA polymerase II, whereas siRNAs are processed from perfect or near-perfect long dsRNAs (2). After generation, miRNA and siRNA are loaded into an RNA-induced silencing complex containing the Argonaute protein to guide posttranscriptional or transcriptional gene silencing (1).In animals, pri-miRNAs are first processed to precursor miRNAs (pre-miRNAs) in the nucleus by the microprocessor containing Drosha and a dsRNA-binding protein DGCR8 (1). The resulting pre-miRNAs are then processed by Dicer in the cytoplasm to produce mature miRNAs (1). It has emerged that the activities of Drosha and Dicer are controlled to regulate miRNA expression in response to developmental and environmental signals (3). In Arabidopsis, DCL1, a dsRNA-binding protein, HYL1, and a zinc finger protein, SERRATE (SE), form a complex to process pri-miRNAs in the nucleus to pre-miRNAs and then to mature miRNAs (4-6). The accumulation of miRNAs in Arabidopsis also requires DDL, which was proposed to stabilize pri-miRNAs and to facilitate their processing (7). Recently, two cap-binding proteins, CBP80/ABH1 and CBP20, were found to be required for pre-mRNA splicing and primiRNA processing (8, 9). Plants also encode several classes of endogenous siRNAs, including the natural antisense transcriptderived siRNA, siRNA derived from repetitive DNA sequences (rasiRNA), and transacting siRNA (ta-siRNA) (10). In Arabidopsis, the generation of these siRNAs from long dsRNAs involves DCL1 homologues DCL2, DCL3, and DCL4, which produce 22-nt, 24-nt, and 21-nt siRNAs, respectively (11-13).In this report, we show that TOUGH (TGH) is an important factor for miRNA and siRNA biogenesis. Loss-of-function TGH in tgh-1 reduces the activity of multiple DCLs in vitro and the accumulation of miRNA and siRNAs in vivo. In the miRNA pathway, TGH associates with the DCL1 complex and binds primiRNAs and pre-miRNAs. TGH is required for the efficient in vivo interaction between pri-miRNA and HYL1. These data suggest that TGH assists DCLs to efficiently process and/or recruit the prec...

Highlights d Proteogenomic characterization reveals the functional impact of genomic alterations d Phosphoproteomics uncovers putative therapeutic targets downstream of KRAS d Multiomics links endothelial cell remodeling and glycolysis to immune exclusion d Proteomics and glycoproteomics reveal candidates for early detection or intervention