TGFBR2-dependent alterations of exosomal cargo and functions in DNA mismatch repair-deficient HCT116 colorectal cancer cells

Background: Colorectal cancers (CRCs) that lack DNA mismatch repair function exhibit the microsatellite unstable (MSI) phenotype and are characterized by the accumulation of frameshift mutations at short repetitive DNA sequences (microsatellites). These tumors recurrently show inactivating frameshift mutations in the tumor suppressor Transforming Growth Factor Beta Receptor Type 2 (TGFBR2) thereby abrogating downstream signaling. How altered TGFBR2 signaling affects exosome-mediated communication between MSI tumor cells and their environment has not been resolved. Here, we report on molecular alterations of exosomes shed by MSI cells and the biological response evoked in recipient cells. Methods: Exosomes were isolated and characterized by electron microscopy, nanoparticle tracking, and western blot analysis. TGFBR2-dependent effects on the cargo and functions of exosomes were studied in a MSI CRC model cell line enabling reconstituted and inducible TGFBR2 expression and signaling. Microsatellite frameshift mutations in exosomal and cellular DNA were examined by PCR-based DNA fragment analysis and exosomal protein profiles were identified by mass spectrometry. Uptake of fluorescent-labeled exosomes by hepatoma recipient cells was monitored by confocal microscopy. TGFBR2-dependent exosomal effects on secreted cytokine levels of recipient cells were analyzed by Luminex technology and ELISA. Results: Frameshift mutation patterns in microsatellite stretches of TGFBR2 and other MSI target genes were found to be reflected in the cargo of MSI CRC-derived exosomes. At the proteome level, reconstituted TGFBR2 expression and signaling uncovered two protein subsets exclusively occurring in exosomes derived from TGFBR2-deficient (14 proteins) or TGFBR2-proficient (five proteins) MSI donor cells. Uptake of these exosomes by recipient cells caused increased secretion (2–6 fold) of specific cytokines (Interleukin-4, Stem Cell Factor, Platelet-derived Growth Factor-B), depending on the TGFBR2 expression status of the tumor cell. Conclusion: Our results indicate that the coding MSI phenotype of DNA mismatch repair-deficient CRC cells is maintained in their exosomal DNA. Moreover, we uncovered that a recurrent MSI tumor driver mutation like TGFBR2 can reprogram the protein content of MSI cell-derived exosomes and in turn modulate the cytokine secretion profile of recipient cells. Apart from its diagnostic potential, these TGFBR2-dependent exosomal molecular and proteomic signatures might help to understand the signaling routes used by MSI tumors. Graphical Abstract Fricke et al. uncovered coding microsatellite instability-associated mutations of colorectal tumor driver genes like TGFBR2 in MSI tumor cellderived exosomes. Depending on the TGFBR2 expression status of their donor cells, shed exosomes show distinct proteomic signatures and promote altered cytokine secretion profiles in recipient cells

Author Correction: Mutations in PYCR1 cause cutis laxa with progeroid features (Nature Genetics, (2009), 41, 9, (1016-1021), 10.1038/ng.413)

peer reviewedIn the version of this article initially published, there was an omission in the Acknowledgements section for support to Hülya Kayserili. The Acknowledgements should have ended with the text “and a grant from TUBITAK SBAG-108S418 to H.K.”

Mutations in PYCR1 cause cutis laxa with progeroid features.

Autosomal recessive cutis laxa (ARCL) describes a group of syndromal disorders that are often associated with a progeroid appearance, lax and wrinkled skin, osteopenia and mental retardation. Homozygosity mapping in several kindreds with ARCL identified a candidate region on chromosome 17q25. By high-throughput sequencing of the entire candidate region, we detected disease-causing mutations in the gene PYCR1. We found that the gene product, an enzyme involved in proline metabolism, localizes to mitochondria. Altered mitochondrial morphology, membrane potential and increased apoptosis rate upon oxidative stress were evident in fibroblasts from affected individuals. Knockdown of the orthologous genes in Xenopus and zebrafish led to epidermal hypoplasia and blistering that was accompanied by a massive increase of apoptosis. Our findings link mutations in PYCR1 to altered mitochondrial function and progeroid changes in connective tissues