Supplementary MaterialsSupplementary Information 41467_2018_3224_MOESM1_ESM. Mutp53-reprogammed TAMs favor anti-inflammatory immunosuppression with increased activity of TGF-. These findings, associated with poor survival in colon cancer patients, strongly support a microenvironmental GOF role for mutp53 in actively engaging the immune system to promote cancer progression and metastasis. Introduction Exosomes are small spherical packages and one of the vesicle types released by cells into the extracellular environment. Exosomes convey information to neighboring or remote cells by delivering RNAs and Romidepsin inhibition proteins thus affecting signaling pathways in various physiological and pathological conditions including cancer1,2. The production of exosomes and the molecular cargo they carry are affected Rabbit Polyclonal to SGK (phospho-Ser422) by external signals such as oxidative stress and ionizing radiation3,4. Therefore, p53, a cellular stress responsive transcription factor, Romidepsin inhibition plays a major role in exosome machinery and release while under microenvironmental stress. For instance, p53-dependent regulation of TSAP6 was reported to govern exosome secretion and content5,6. Mutations in the gene (encoding for the p53 protein) are one of the most frequent genetic alterations in human cancer7C9. Besides the abrogation of the wild-type (WT) p53-mediated tumor suppression, a distinct set of missense mutations was reported to endow mutant p53 (mutp53) proteins with novel activities termed gain-of-function (GOF). Romidepsin inhibition Such GOF activities dramatically alter tumor cell characteristics, primarily through their interactions with other cellular proteins and regulation of cancer cell transcriptional programs10C13. On a?cellular level, increased mutp53 protein stability leads to a substantial intracellular mutp53 accumulation in cancer cells, further disrupting cellular homeostasis and creating oncogenic stress14,15. Thus, cancer cells appear to be addicted to high levels of mutp53 for their survival and oncogenic properties. In this study, we hypothesized that in addition to its cell-autonomous GOF mechanisms, mutp53 might affect microenvironmental conditions by facilitating the release of exosomes stemming from mutp53-dependent cellular stress. In most solid cancers, a major component of the tumor stroma are macrophages referred to as tumor-associated macrophages (TAMs)16 that are mostly derived from peripheral blood monocytes recruited into the tumor mass17C20. In recent years, TAMs have been extensively studied and proposed as a significant contributing factor to tumor progression. The communication between tumor cells and macrophages was suggested to be mediated via exosomal transfer where packaged proteins and microRNAs (miRs) were reported to immunomodulate the macrophages at the receiving end21C23. In this study, we discovered a microenvironmental GOF mechanism for mutant p53 by driving exosome-based communication between tumor and immune cells forming a distinct sub-population of tumor supportive macrophages. Our findings identify miR-1246 as a unique cargo of mutp53-derived exosomes potentially amenable for therapeutic and diagnostic applications in colon cancer. Results Tumor cells harboring mutp53 reprogram?macrophages We investigated the mechanism by which tumor cells harboring specific missense mutations in the gene (mutp53) might reprogram neighboring macrophages. In the initial human cell co-culture experiment, both cultures were separated by a membrane allowing the transport of molecules and particles less than 0.4?m in size. The macrophage culture originated from CD14+ primary human monocytes (Supplementary Fig.?1a, b), which were activated by three different stimulatory cytokine cocktails to derive either M0 macrophages (not polarized), M1 macrophages (classically activated), or M2 macrophages (alternatively activated). Polarization patterns were validated by conducting a gene expression array for M1 and M2 polarized primary macrophages (Supplementary Table?1). For the carcinoma cell compartment of the co-culture, we selected several cellular models where mutp53 was either expressed (the R248W mutant in HCT116 cells), induced (the V157F, R175H, R273H or R249S in H358 Romidepsin inhibition cells), or knocked-down (the R273H in HT29 cells) Romidepsin inhibition (Supplementary Fig.?1c). We monitored the effect of mutp53 on the co-cultured macrophages using a set of cytokines previously reported to be altered in the TAM equilibrium24. After being exposed to tumor cells that harbor mutp53, M0 and.