Using quantitative real-time PCR (Number?1B) and immunoblotting (IB) methods (Number?1C), we detected high c-Met, CD44, and CD44v6 expression both in the mRNA and protein levels in DAOY and UW228 cell lines, and much less (c-Met) or no (CD44/CD44v6) expression in D341 and D425 cell lines
Using quantitative real-time PCR (Number?1B) and immunoblotting (IB) methods (Number?1C), we detected high c-Met, CD44, and CD44v6 expression both in the mRNA and protein levels in DAOY and UW228 cell lines, and much less (c-Met) or no (CD44/CD44v6) expression in D341 and D425 cell lines. the c-Met ligand HGF could drive dissemination of MB cells expressing high levels of c-Met, and identified downstream effector mechanisms of this process. We detected variable c-Met expression in different established human being MB cell lines, and we found that in lines expressing high c-Met levels, HGF advertised cell dissemination and invasiveness. Specifically, HGF-induced c-Met activation enhanced the capability of the individual cells to migrate inside a JNK-dependent manner. Additionally, we recognized the Ser/Thr kinase MAP4K4 like a novel driver of c-Met-induced invasive cell dissemination. This increased invasive motility was due to MAP4K4 control of F-actin dynamics in constructions required for migration and invasion. Therefore, MAP4K4 couples growth element signaling to actin cytoskeleton rules in tumor cells, suggesting that MAP4K4 could present a encouraging novel target to be evaluated for treating growth factor-induced dissemination of MB tumors of different subgroups and of additional human cancers. Electronic supplementary material The online version of this article (doi:10.1186/s40064-015-0784-2) contains supplementary material, which is available to authorized users. two- and three-dimensional (2D/3D) motility assays combined with live-cell imaging and biochemical approaches to investigate and characterize potentially druggable mediators of HGF-c-Met-induced MB cell dissemination. Results c-Met and its co-receptor CD44 are highly expressed inside a subset of MB tumors and patient derived cell lines To determine the potential medical relevance of c-Met in larger cohorts of MB, we compared the mRNA manifestation levels of c-Met in the Gilbertson, the Kool and the Delattre datasets available through the R2 platform for visualization and analysis of the microarray data. As control, we used nine cerebellum samples of individuals aged between 23 and 50?years. We found that the median mRNA level of c-Met and its ligand HGF in MB tumors from these three different main sample cohorts were clearly below that of normal human being cerebellum (Number?1A). However, a sub-population of MB tumors averaging 17.5% (Figure?1A, c-Met high) showed significantly increased c-Met manifestation. Moreover, the same datasets exposed high mRNA manifestation of the c-Met co-receptor CD44 (Orian-Rousseau et al. 2002) in all MB tumor samples. By analyzing 103 main MB tumors of the Northcott 103 dataset (Northcott et al. 2011), Onvani explained the association of c-Met with the SHH subgroup (Onvani et al. 2012). We confirmed this getting using the 285 tumors of the MAGIC dataset (Northcott et al. 2012b) (Additional file 1: Number S1A). An analogous but less designated association was also observed for HGF (Additional file 1: Number S1B), but not for CD44 (Additional file 1: Number S1C). Using quantitative real-time ML167 PCR (Number?1B) and immunoblotting (IB) methods (Number?1C), we detected high c-Met, CD44, and CD44v6 expression both in the mRNA and protein levels in DAOY and Rabbit Polyclonal to FOXD3 UW228 cell lines, and much less (c-Met) or no (CD44/CD44v6) expression in D341 and D425 cell lines. Interestingly, three bands were recognized in the anti-CD44v6 blot (Number?1C, arrowheads), suggesting the presence of different CD44 isoforms with integrated v6 variable ML167 region. DAOY cells are sensitive to sonic hedgehog (Gotschel et al. 2013) and considered a SHH-like MB cell collection, whereas D341 is considered a group 3 cell collection (Snuderl et al. 2013). We confirmed surface manifestation of c-Met, CD44, and CD44v6 on DAOY (Number?1D) and UW228 cell lines (not shown) by circulation cytometry. This analysis exposed that >90% of DAOY cells indicated c-Met, 100% indicated CD44, while only approximately 40% indicated the CD44v6 isoform. We consequently continued our studies by focusing specifically on c-Met and by studying what effects c-Met activation by its ligand HGF may have on cell migration and invasion and which effector pathways are needed to mediate the c-Met reactions. Open in a separate windows Number 1 Manifestation of c-Met in medulloblastoma medical samples and cell ML167 lines. (A) Manifestation ML167 analysis of c-Met, HGF and CD44 in three different MB tumor selections (ntotal?=?195) and in normal adult cerebellum (n?=?9). (B) Comparative quantitative real-time PCR manifestation analysis of c-Met, CD44 and CD44v6 in founded MB cell lines and adult cerebellum sample. (C) Manifestation and activation analysis of the c-Met pathway, CD44, and CD44v6 by immunoblotting (IB) in four different MB cell lines using the antibodies indicated to.