C.P. Sustained JAK1/STAT3 signalling is usually maintained by DNA methyltransferase DNMT1. Consistently, in human lung and head and neck carcinomas, STAT3 acetylation and phosphorylation are inversely correlated Ecteinascidin-Analog-1 with SHP-1 expression. Combined inhibition of DNMT activities and JAK signalling, and methylations29. Here using combination of three-dimensional model of organotypic invasion assays of head and neck and breast tumours, and using and models of breast carcinomas, we demonstrate that an epigenetic switch initiates and maintains the proinvasive phenotype of CAF. We show that Ecteinascidin-Analog-1 LIF induces constitutive activation of the JAK1/STAT3 signalling pathway by post-translational regulation of STAT3 acetylation by p300, in fibroblasts. Acetylated STAT3 leads to an epigenetic-dependent loss of expression of the SHP-1 tyrosine phosphatase, which is a negative regulator of the JAK/STAT pathway. Silencing of SHP-1 by promoter methylation leads to sustained constitutive phosphorylation of the JAK1 kinase and the STAT3 transcription factor that maintain the contractile Ecteinascidin-Analog-1 and proinvasive fibroblasts abilities. Blockage of both JAK signalling and DNA methyltransferase activities both and results in long-term proinvasive phenotypic reversion of CAF. Finally, we corroborate our and findings through immunhistological analysis of STAT3 activity and SHP-1 expression in both head and neck and lung human carcinoma biopsies. Therefore, we conclude that in human carcinomas from different origins, LIF induces a sustained proinvasive activation of CAF through an epigenetic-dependent loss of SHP-1 phosphatase. Results Epigenetic mechanisms sustain the proinvasive CAF phenotype We first assessed whether, similar to CAF isolated from head and neck, lung and breast human carcinomas (HN-CAF, Lu-CAF and Br-CAF, respectively), the long-term LIF or TGF-activated human dermal fibroblasts (hDF_LIF or hDF_TGF) constitutively retain their proinvasive properties. hDF were stimulated for 7 days in culture using LIF or TGF in the presence or absence of a LIF blocking antibody. After 15- 30- and 60-day culture in low serum concentration (Fig. 1a), the hDF proinvasive ability was assessed using a three-dimensional SCC12 cell organotypic invasion assay30. In these conditions LIF induced the sustained fibroblast proinvasive phenotype (Fig. 1b) and, constitutively, the JAK1/STAT3 signalling pathway (Supplementary Fig. 1a) as observed with CAF compared with primary hDF (Supplementary Fig. 1b). Interestingly, TGF, the major and CAF activator, relied on LIF to constitutively activate the proinvasive ability of hDF (Fig. 1b) and the JAK1/STAT3 signalling pathway (Supplementary Fig. 1a). These results indicate that a 7-day LIF stimulation is sufficient to confer a long-term proinvasive CAF-like phenotype to hDF. CAF secrete procarcinogenic factors, including interleukin-6 (IL6)-family cytokines2 that may sustain constitutive activation of JAK1 kinase via an autocrine regulatory loop. Therefore, we investigated whether media conditioned (CM) by CAF, hDF_LIF and hDF_TGF can activate JAK1/STAT3 signalling in hDF. Stimulation of hDF by long-term CAF, LIF- and TGF-activated hDF CM failed to promote JAK1/STAT3 phosphorylation (Fig. 1c) and collagen gel contraction (Fig. 1d), while short-term TGF-activated CM (hDF-TGF-6?h), in which LIF is detectable16, promoted both STAT3 phosphorylation and gel contraction (Fig. 1c,d). Accordingly, in hDF_LIF, abrogation of GP130 receptor or addition of a specific LIF blocking Ecteinascidin-Analog-1 antibody failed to alter STAT3 phosphorylation, which contrasts with the abrogation of JAK1 expression (Supplementary Fig. 1c,d). Taken together, these data demonstrate that LIF confers permanent proinvasive conversion to hDF that is independent of a Cd8a LIF autocrine signalling regulatory loops but dependent on JAK1 activity. We thus hypothesized that epigenetic modifications might be involved in the constitutive activation of CAF and hDF_LIF. To test this idea, HN-CAF-dependent three-dimensional matrix gel contraction assays were performed in the presence of 45 small-molecule inhibitors targeting the epigenetic and DNA-modifier cell machinery (Fig. 1e, Supplementary Fig. 1e and Supplementary Table 1). For each tested molecule, the screen was performed at optimized concentrations (Supplementary Table 1); dimethylsulphoxide was used as.