Extracellular vesicles (EVs) include the exosomes (30-100 nm) that are produced through the endocytic pathway via the multivesicular bodies and the ectosomes (100-1000 nm) that are released through the budding of the plasma membrane. Despite the differences in the mode of biogenesis and size, reliable markers that can distinguish between exosomes and ectosomes are non-existent. Moreover, the precise functional differences between exosomes and ectosomes remains poorly characterised. Here, using label-free quantitative proteomics, we highlight proteins that could be exploited as markers to discriminate between exosomes and ectosomes. For the first time, a global proteogenomics analysis unveiled the secretion of mutant proteins that are implicated in cancer progression through tumor-derived EVs. Follow up integrated bioinformatics analysis highlighted the enrichment of oncogenic cargo in exosomes and ectosomes. Interestingly, exosomes induced significant cell proliferation and migration in recipient cells compared to ectosomes confirming the oncogenic nature of exosomes. These findings ascertain that cancer cells facilitate oncogenesis by the secretion of mutant and oncoproteins into the tumor microenvironment via exosomes and ectosomes. The integrative proteogenomics approach utilized in this study has the potential to identify disease biomarker candidates which can be later assayed in liquid biopsies obtained from cancer patients.

The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs. Milk-derived EVs orally administered to mice implanted with colorectal and breast cancer cells reduce the primary tumor burden. Intriguingly, despite the reduction in primary tumor growth, milk-derived EVs accelerate metastasis in breast and pancreatic cancer mouse models. Proteomic and biochemical analysis reveal the induction of senescence and epithelial-to-mesenchymal transition in cancer cells upon treatment with milk-derived EVs. Timing of EV administration is critical as oral administration after resection of the primary tumor reverses the pro-metastatic effects of milk-derived EVs in breast cancer models. Taken together, our study provides context-based and opposing roles of milk-derived EVs as metastasis inducers and suppressors.

The TIM22 complex mediates the import of hydrophobic carrier proteins into the mitochondrial inner membrane. While the TIM22 machinery has been well characterised in yeast, the human complex remains poorly characterised. Here, we identify Tim29 (C19orf52) as a novel, metazoan-specific subunit of the human TIM22 complex. The protein is integrated into the mitochondrial inner membrane with it’s C-terminus exposed to the intermembrane space. Tim29 is required for the stability of the TIM22 complex and functions in the assembly of hTim22. Furthermore, Tim29 contacts the Translocase of the Outer Mitochondrial Membrane, TOM complex, enabling a mechanism for transport of hydrophobic carrier substrates across the aqueous intermembrane space. Identification of Tim29 highlights the significance of analysing mitochondrial import systems across phylogenetic boundaries, which can reveal novel components and mechanisms in higher organisms.DOI: http://dx.doi.org/10.7554/eLife.17463.001

No abstract

No abstract

Introduction Large scale sequencing studies have identified that 93% of the colorectal cancer (CRC) patients carry at least one mutation in genes implicated in Wnt signalling pathway. Notably, majority of the CRC patients (88%) carry either APC or b-catenin mutations that can activate the Wnt signalling pathway. Recent evidences suggest that Wnt/b-catenin signalling activity is regulated by CDH17 in hepatocellular carcinoma. As CDH17 is exclusively expressed in the intestine and overexpressed in CRC, we hypothesised that CDH17 could be utilised as a therapeutic target to treat CRC patients. Material and methods RNA interference-based stable knockdowns were established in a panel of CRC cells with varying mutations in APC and b-catenin. Wnt signalling activity of the cells were measured by TOPflash assay. Apoptosis studies were performed using fluorescence activated cell sorting. Cells were further subjected to immunoprecipitations with anti-CDH17 and anti-b-catenin antibodies followed by label-free quantitative proteomics analysis. A monoclonal antibody was developed to block CDH17 and sensitise CRC cells to chemotherapeutic drugs. Results and discussions Knockdown of CDH17 in CRC cells downregulated b-catenin and attenuated Wnt signalling activity irrespective of APC and/or b-catenin mutations. Furthermore, CDH17 silencing induced apoptosis and sensitised CRC cells to the chemotherapeutic drugs 5-Fluorouracil. Immunoprecipitations using anti-CDH17 and anti-b-catenin antibodies followed by label-free quantitative proteomics analysis highlighted no direct interaction between CDH17 and b-catenin hence implying an indirect regulation of b-catenin expression and Wnt signalling pathway by CDH17. The analysis revealed Ecadherin and FAT1 as common interactors of CDH17 and bcatenin. Quantitative proteomic analysis of cell lysates revealed the upregulation of FAT1, a negative regulator of Wnt signalling pathway, upon knockdown of CDH17. Monoclonal antibodies developed against CDH17 were able to increase apoptosis and sensitivity of CRC cells to 5-Fluorouracil. Conclusion Overall, these findings suggest that CDH17 can attenuate Wnt signalling pathway and induce apoptosis irrespective of the APC and b-catenin mutational status. As Wnt signalling pathway is aberrated in 93% of CRC patients, the membrane protein CDH17 can be exploited as therapeutic target to treat CRC.

The TIM22 complex mediates the import of hydrophobic carrier proteins into the mitochondrial inner membrane. While the TIM22 machinery has been well characterised in yeast, the human complex remains poorly characterised. Here, we identify Tim29 (C19orf52) as a novel, metazoan-specific subunit of the human TIM22 complex. The protein is integrated into the mitochondrial inner membrane with it's C-terminus exposed to the intermembrane space. Tim29 is required for the stability of the TIM22 complex and functions in the assembly of hTim22. Furthermore, Tim29 contacts the Translocase of the Outer Mitochondrial Membrane, TOM complex, enabling a mechanism for transport of hydrophobic carrier substrates across the aqueous intermembrane space. Identification of Tim29 highlights the significance of analysing mitochondrial import systems across phylogenetic boundaries, which can reveal novel components and mechanisms in higher organisms.

Neuroblastoma, a paediatric cancer, accounts for 15% of childhood cancer mortality. Even though neuroblastoma is an aggressive cancer, the exact mechanisms by which the cells resist treatment is poorly understood. Here, we hypothesise that neuroblastoma cells have high expression of mesenchymal markers and hence could attribute to the aggressive phenotype. P120ctn is downregulated in epithelial cancers and is known to play a major role in EMT and aggressiveness. In this study, immunohistochemical staining of neuroblastoma patient tissues suggested that p120ctn is highly abundant. Hence, the role of p120ctn and N-Myc in neuroblastoma aggressiveness was investigated by using RNA interference. Amplification of N-Myc oncogene occurs in 20% of neuroblastoma patients and is considered high risk as it correlates with aggressiveness and poor prognosis. Interestingly, knockdown of p120ctn down regulated N-Myc both at mRNA and protein levels. Upon knockdown of p120ctn and N-Myc, the proliferation, invasion and migration of neuroblastoma cells were significantly reduced. Quantitative proteomic and qPCR analysis of the wild type and knockdown cells revealed that p120ctn knockdown cells underwent mesenchymal-to-epithelial transition. Confocal microscopy and Western blotting analysis of subcellular fractionation showed nuclear accumulation of β-catenin upon p120ctn knockdown. Once in the nucleus, β-catenin activated Wnt signalling pathway and up regulated Wnt target genes including C-Myc. Interestingly, down regulation of p120ctn sensitised the neuroblastoma cells to doxorubicin. Currently, there is no published study that explores the role of p120ctn in neuroblastoma. However, these findings are contradictory to scientific literature in the context of the functional role of p120ctn in epithelial cancer. Hence to validate our findings, we established knockdown of p120ctn in epithelial colorectal cancer cells. Consistent with the literature, knockdown of p120ctn induced EMT, proliferation and migration. These results suggest that the role of p120ctn is cell type dependent. Overall, the findings from this study suggest that p120ctn plays a pivotal role in progression of neuroblastoma. Citation Format: Pamali Fonseka, Suresh Mathivanan, Michael Liem, Ishara Atukorala. Dual role of p120ctn in cancer: epithelial vs mesenchymal [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3932. doi:10.1158/1538-7445.AM2017-3932