Primary tumours also prime metastatic niches by releasing extracellular vesicles (EVs)such as exosomesinto the circulation

Primary tumours also prime metastatic niches by releasing extracellular vesicles (EVs)such as exosomesinto the circulation. mediate intercellular transfer of mutp53s invasive/migratory gain-of-function by increasing RCP-dependent integrin recycling in other tumour cells. This process depends on mutp53s ability Gefitinib (Iressa) to control production of the sialomucin, podocalyxin, and activity of the Rab35 GTPase which interacts with podocalyxin to influence its sorting to exosomes. Exosomes from mutp53-expressing tumour cells also influence integrin trafficking in normal fibroblasts to promote deposition of a highly pro-invasive extracellular matrix (ECM), and quantitative second harmonic generation microscopy indicates that this ECM displays a characteristic orthogonal morphology. Gefitinib (Iressa) The lung ECM of mice possessing mutp53-driven pancreatic adenocarcinomas also displays increased orthogonal characteristics which precedes metastasis, indicating that mutp53 can influence the microenvironment Gefitinib (Iressa) in distant organs in a way that can support invasive growth. Introduction Loss of wild-type p53 function is a key watershed in tumour initiation and progression. This occurs through loss of p53 expression or mutations that generate p53 proteins defective in wild-type function. A gain-of-function for mutant p53 (ref. 1) (mutp53) first became apparent following the construction of a mouse model of Li-Fraumeni syndrome2. In this animal, wild-type Retn p53 was replaced with mutp53 alleles (p53R270H and p53R172H) and this led to the spontaneous growth of tumours with more aggressive phenotypes than was observed in p53 null mice. The ability of mutp53 to drive metastasis was then demonstrated using autochthonous mouse models of pancreatic cancer3, and cells isolated from mutp53 pancreatic tumours are more invasive than their p53 null counterparts4, indicating that mutp53s pro-metastatic gain-of-function is associated with increased cell migration5,6. The way in which integrin receptors for the ECM are trafficked through the endosomal pathway and returned, or recycled, to the plasma membrane is key to the migratory behaviour of cancer cells7,8. The Rab11 effector, Rab-coupling protein (RCP), controls integrin recycling, and it is now clear that mutant p53s can drive invasive migration by promoting RCP-dependent integrin recycling6. The characteristics of the tumour ECM is closely correlated with disease progression, resistance to therapy, and poor prognosis, and there is now much interest in targeting the ECM and its receptors as an anti-cancer strategy9. The ECM within tumours is deposited primarily by fibroblastic cells (carcinoma-associated fibroblasts (CAFs)) and this is controlled by autocrine and paracrine pathways which relay signals between malignant cells and CAFs10. Furthermore, ECM proteins are assembled and extensively re-modelled following secretion, and the way that integrins are trafficked through the endosomal system can control this11,12. Finally, secreted factors, such as lysyl oxidase, can act directly on the ECM to introduce cross-links which alter ECM organisation and stiffness in way that promotes local invasiveness13. The ECM of target organs also contributes to metastasis, and cells in the primary tumour can influence this by releasing factors into the circulation. For instance, lysyl oxidase not only influences the ECM of primary tumours in the breast but also primes bone marrow niches to enable metastatic seeding14. Primary tumours also prime metastatic niches by releasing extracellular vesicles (EVs)such as exosomesinto the circulation. Exosomes released by melanomas can influence differentiation of bone marrow-derived stem cells to promote their mobilisation to tissuessuch as the lungwhere they contribute to deposition of ECM proteins15. More recently exosomes from pancreatic adenocarcinoma cells were shown to promote TGF secretion from Kupffer cells which led to fibronectin production by liver stellate cells16. However, despite studies outlining how certain factors, such as oncogenic proteins and microRNAs might be transmitted between cells, the molecular players that mediate the pro-metastatic effects of oncogenes are not yet clear. Here we report that primary tumours expressing mutp53s with pro-metastatic gain-of-function can evoke pro-invasive alterations to the ECM in a metastatic target organ, and we provide the molecular details of how this occurs. Results Mutp53 promotes release of diffusible pro-invasive factor(s) Organotypic plugs of acid-extracted type I collagen in which the ECM has been preconditioned by human fibroblasts recapitulate key characteristics of the stromal microenvironment17. When plated onto organotypic plugs preconditioned with telomerase-immortalised human fibroblasts (TIFs), H1229 non-small cell lung carcinoma cells (which do not express p53) (H1299-p53?/?) were poorly invasive, with most cells residing in the upper portion of the plug 10 days after plating (Fig.?1a, d). By contrast, H1299 cells expressing the conformational gain-of-function mutant of p53, p53R273H (H1299-p53R273H) invaded extensively into organotypic plugs (Fig.?1b, d). Open in a separate window Fig. 1 Mutant p53 promotes release of diffusible factors to foster tumour cell invasion in an organotypic microenvironment. Organotypic plugs were generated allowing acid-extracted rat tail collagen to polymerise in the presence of telomerase-immortalised human dermal fibroblasts (TIFs). Fibroblast-containing plugs were conditioned for 2 days to allow TIFs to deposit and remodel the ECM. Preconditioned plugs were overlaid Gefitinib (Iressa) with H1299-p53?/??(a) or H1299-p53R273H (b) cells and placed onto grids in independent.