Abstract Introduction: Recently our laboratory has shown that Serum Deprived Mesenchymal Stem Cells (SD-MSCs) have the surprising ability to survive for long periods (more than 60 days) by using autophagy. In addition, these SD-MSCs aid in tumor survival/growth both by paracrine effects of the secretome and by miRNAs production that act as post-transcriptional regulators and affect the regulation of protein-coding. There has been an explosion of interest in understanding the role of secreted microRNA by MSCs, presumably through exosomes. Exosomes are 50-90 nm vesicles that are secreted by various types of cells and made up of a double membrane of phospholipids that contain proteins, mRNA, and miRNA. It has been shown that exosomes secreted by cells affect a recipient cell by modifying its protein translation, thus, inducing a cascade of signaling events. In this study we characterize the exosomal fraction of non-apoptotic secretome of SD-MSCs. Materials and Methods: MSCs were cultured in α-MEM without serum for 30 days. The culture supernatant was centrifuged to remove floating large debris and concentrated 120 times using an ultrafiltration cellulose membrane (cutoff 1kDa) mounted on a N2 positive pressure system (Amicon). Concentrated supernatant was centrifuged 1 hour at 100,000g to collect the exosome fraction. miRNA contained in exosomes was extracted using miRVana kit (Ambion) and screened for 704 different miRNA using a RT miRNA PCR array (SABiosciences). Proteins from exosomes were extracted using RIPA buffer. The trypsin digest of the Rapigest(tm)-resuspended pellet was analyzed using both nanoLC MALDI-TOF/TOF and nanoRSLC-ESI-LTQ-OrbiTrap MS/MS analyses. Results: Our results show that at least 110 different miRNAs are expressed in exosomes from serum deprived MSCs including miR-34a, miR-199, miR-23 and let-7 series. These play a role in tumor survival/angiogenesis. Interestingly, we observed an enrichment expression of “passenger” miRNA (miRNA* or miRNA-3p/-5p). Proteomics revealed the presence of at least 281 different proteins from serum deprived-MSCs exosomes including but not limited to growth factors (IGF-2, PAI-1), growth-factors binding proteins (IGFBP-2, -3, -5, -7), transcription factor (AEBP1), integrins (α5, α11, β1), Ras family members (Rab10, Rap1A, Rap1B) and metalloproteinase inhibitors (TIMP1, TIMP2). The identified proteins map to pathways involved in cell growth and proliferation, cell death, cell-to-cell signaling and metabolism. Conclusion: Our study revealed the complexity of exosomes secreted by serum deprived MSCs. The miRNAs and proteins detected could act as indirect post-transcriptional regulators (miRNA) or as direct signaling pathway regulators (growth factors/integrins). This cell to cell communication through exosomes could reveal a novel mechanism for paracrine regulation of stromal cells in solid tumors. Citation Format: Patrice Penfornis, Krishna C. Vallabhaneni, Francois Guillonneau, Griffin Orr, Santosh Dhule, Radhika Pochampally. Exosomal transfer of microRNA and growth factors by human Mesenchymal Stem Cells support to breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2605. doi:10.1158/1538-7445.AM2013-2605
Les triples helices d'ADN ont recemment fait l'objet d'importantes etudes de caracterisation in vitro en raison de l'outil potentiel qu'elles representent dans le cadre de la therapie genique. Cependant, peu de donnees existent sur leur devenir ou sur leur role in vivo. Dans le but d'identifier des proteines impliquees dans la reconnaissance d'acides nucleiques capables de former des triple helices, nous avons mis au point une methode de recherche systematique de proteines affines pour la structure en triple-helice d'ADN. Les outils de la proteomique que sont l'electrophorese bidimensionnelle et la spectrometrie de masse ont ete combinees a la detection par blot. La methode implique une separation electrophoretique des proteines selon leur point isoelectrique et leur masse moleculaire, suivie d'une detection selective des proteines affines pour la triple helice d'ADN utilisee comme sonde. Plusieurs proteines issues d'extraits nucleaires de cellule HeLa, separees puis detectees en southwestern blot et suffisamment abondantes (colorees au bleu de Coomassie), ont ete extraites et identifiees par spectrometrie de masse apres digestion trypsique. Les resultats apportes par cette etude demontrent que plusieurs proteines a domaines RRM/RBD et les proteines a domaines KH, une majorite est impliquee dans la prise en charge des acides nucleiques et leur repartition (hnRNP A, K, E, L, I, helicases DEAD/H) alors que d'autres seraient impliquees dans la reparation et la recombinaison de l'ADN (TLS, nuclease FEN-1). L'interaction de ces proteines avec la triple-helice doit etre confirmee par d'autres methodes, et les roles explores a l'aide de proteines recombinantes. La technique developpee ici pourrait etre appliquee a la recherche de proteines specifiques de structures particulieres d'ADN ou de toute autre molecule d'interet.
Abstract Transforming growth factor-β (TGFβ) signaling is initiated by the type I, II TGFβ receptor (TβRI/TβRII) complex. Here we report the formation of an alternative complex between TβRI and the orphan GPR50, belonging to the G protein-coupled receptor super-family. The interaction of GPR50 with TβRI induces spontaneous TβRI-dependent Smad and non-Smad signaling by stabilizing the active TβRI conformation and competing for the binding of the negative regulator FKBP12 to TβRI. GPR50 overexpression in MDA-MB-231 cells mimics the anti-proliferative effect of TβRI and decreases tumor growth in a xenograft mouse model. Inversely, targeted deletion of GPR50 in the MMTV/Neu spontaneous mammary cancer model shows decreased survival after tumor onset and increased tumor growth. Low GPR50 expression is associated with poor survival prognosis in human breast cancer irrespective of the breast cancer subtype. This describes a previously unappreciated spontaneous TGFβ-independent activation mode of TβRI and identifies GPR50 as a TβRI co-receptor with potential impact on cancer development.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract p27Kip1 (p27) is a cyclin-CDK inhibitor and negative regulator of cell proliferation. p27 also controls other cellular processes including migration and cytoplasmic p27 can act as an oncogene. Furthermore, cytoplasmic p27 promotes invasion and metastasis, in part by promoting epithelial to mesenchymal transition. Herein, we find that p27 promotes cell invasion by binding to and regulating the activity of Cortactin, a critical regulator of invadopodia formation. p27 localizes to invadopodia and limits their number and activity. p27 promotes the interaction of Cortactin with PAK1. In turn, PAK1 promotes invadopodia turnover by phosphorylating Cortactin, and expression of Cortactin mutants for PAK-targeted sites abolishes p27's effect on invadopodia dynamics. Thus, in absence of p27, cells exhibit increased invadopodia stability due to impaired PAK1-Cortactin interaction, but their invasive capacity is reduced compared to wild-type cells. Overall, we find that p27 directly promotes cell invasion by facilitating invadopodia turnover via the Rac1/PAK1/Cortactin pathway. https://doi.org/10.7554/eLife.22207.001 eLife digest When animals develop from embryos to adults, or try to heal wounds later in life, their cells have to move. Moving means that the cells must invade into their surroundings, a dense network of proteins called the extracellular matrix. The cell first attaches to the extracellular matrix; degrades it; and then moves into the newly opened space. Cells have developed specialized structures called invadosomes to enable all these steps. Invadosomes are never static, they first assemble where cells interact with extracellular matrix, they then release proteins that loosen the matrix, and finally disassemble again to allow cells to move. Invadosomes in cancer cells often become overactive, and can allow the tumor cells to spread throughout the body. A lot of different proteins are involved in controlling how and when cells move. p27 is a well-known protein usually found in a cell's nucleus along with the cell's DNA. Inside the nucleus, p27 suppresses tumor growth by stopping cells from dividing. However, often in cancer cells p27 moves outside of the cell's nucleus where it contributes to cell movement via an unknown mechanism. To answer how p27 controls cell invasion, Jeannot et al. used a biochemical technique to uncover which proteins p27 binds to when it is outside of the nucleus. One of its interaction partners was called Cortactin. This protein is known to be an important building block of invadosomes, and is involved in both the assembly and disassembly of these structures. In further experiments, Jeannot studied mouse cells with or without p27 and human cancer cells that can be grown in the laboratory. The experiments revealed that p27 promotes an enzyme called PAK1 to also bind to Cortactin. PAK1 then modified Cortactin, causing whole invadosomes to disassemble, which in turn allowed cells to de-attach from the matrix and move forward. In contrast, cells lacking p27 had more stable invadosomes, attached more strongly to the matrix and were better at degrading it, but could not invade as well as cells with p27. Overall these experiments showed a new way that p27 promotes cell invasion. The next steps will include finding out exactly how the modification of Cortactin causes the invadosomes to disassemble. Furthermore, it will be important to study whether forcing p27 back into the nucleus can reduce the spread of cancer cells in the body. https://doi.org/10.7554/eLife.22207.002 Introduction p27Kip1 is a cell cycle inhibitor that binds to a broad range of cyclin-CDK complexes (Besson et al., 2008). p27-mediated cyclin-CDK inhibition involves the N-terminal extremity (aa 28–89) of the protein that allows interaction with both cyclin and CDK subunits (Ou et al., 2012; Besson et al., 2008). The critical role of p27 as a negative regulator of cell proliferation is underscored by the phenotype of p27 knockout mice, which exhibit a hyperproliferative phenotype in multiple tissues and are more susceptible to tumor development than wild-type animals (Fero et al., 1996; Kiyokawa et al., 1996; Nakayama et al., 1996; Besson et al., 2006; Fero et al., 1998). While the tumor suppressive role of p27 is confirmed by the frequent loss of p27 expression in many types of cancers, p27 is also mislocalized in a fraction of tumors due to activation of various signaling pathways that cause its nuclear export and/or cytoplasmic retention (Chu et al., 2008). Cytoplasmic p27 has been associated with decreased patient survival, high tumor grade and metastasis in several tumor types including breast carcinomas, acute myeloid leukemia, glioblastoma, melanoma and non-small cell lung carcinomas (Lin et al., 2016; Chu et al., 2008; Liang et al., 2002; Yang et al., 2011; Min et al., 2004; Cheng et al., 2015). The possibility that cytoplasmic p27 may confer a pro-tumorigenic advantage was confirmed in a mouse model expressing a mutant form of p27 that cannot bind cyclin-CDK complexes (p27CK-) (Besson et al., 2007; Serres et al., 2011). These mice are more susceptible than p27-null and wild-type mice to both spontaneous and induced tumor development, thus uncovering an oncogenic role for p27 in vivo (Serres et al., 2011; Besson et al., 2007). It now appears that p27 is a multifunctional protein involved in the regulation of multiple cellular processes, some of which are regulated by p27 in a CDK-independent manner (Besson et al., 2008; Sharma and Pledger, 2016). Indeed, p27 has been implicated in the control of cell migration, transcriptional repression, autophagy, stem cell specification and differentiation, cytokinesis, and apoptosis (Besson et al., 2008; Sharma and Pledger, 2016; Baldassarre et al., 2005; Besson et al., 2004b, 2007; Pippa et al., 2012; Serres et al., 2012; Nickeleit et al., 2008; Liang et al., 2007; Li et al., 2012; Nguyen et al., 2006; Jeannot et al., 2015). This functional versatility may stem from the intrinsically disordered structure of p27, which folds upon binding to other proteins, allowing p27 to interact with a wide variety of partners (Lacy et al., 2004; Galea et al., 2008). While these various functions participate in homeostasis in normal cells and tissues, they may be co-opted by tumor cells to promote oncogenesis. Metastasis, the process whereby cells from a primary tumor disseminate throughout the body and form secondary tumors at distant sites, is the leading cause of cancer related death (Sethi and Kang, 2011). It requires tumor cells to migrate and invade through neighboring tissues, enter the blood flow and then extravasate to disseminate and form new tumors in host tissues (Sethi and Kang, 2011). Regulation of cell motility was the first CDK-independent role ascribed to p27 (Nagahara et al., 1998; Denicourt et al., 2007; Besson et al., 2004b). In the cytosol, p27 can interact with RhoA, preventing RhoA activation by guanine-nucleotide exchange factors (GEFs) and thereby modulating actin cytoskeleton dynamics and migration (Besson et al., 2004a, 2004b). Consequently, mouse embryo fibroblasts (MEFs) lacking p27 have more RhoA activity, increased numbers of stress fibers and focal adhesions and exhibit a defect in migration (Besson et al., 2004b). This pathway is important for proper migration of bone marrow macrophages and developing cortical neurons and for cancer cell migration and invasion in vivo (Godin et al., 2012; Nguyen et al., 2006; Papakonstanti et al., 2007; Gui et al., 2014; Wu et al., 2006; See et al., 2010; Larrea et al., 2009; Jin et al., 2013). p27 can also regulate cell migration by controlling microtubule stability through Stathmin or directly by binding to microtubules and promoting microtubule polymerization (Baldassarre et al., 2005; Godin et al., 2012). In a 3D environment, cells adopt different strategies to migrate and invade through surrounding matrix (Petrie and Yamada, 2016; Sethi and Kang, 2011). The mode of migration is influenced, at least in part, by the activities of Rho GTPases: RhoA activity favors an amoeboid migration, whereas Rac1 activity promotes mesenchymal migration (Sahai and Marshall, 2003; Vial et al., 2003). Accordingly, cytoplasmic p27 promotes mesenchymal migration via the inhibition of RhoA activity, while cells lacking p27 tend to adopt an amoeboid mode of migration (Gui et al., 2014; Belletti et al., 2010). More recently, p27 was reported to promote epithelial to mesenchymal transition by binding to JAK2, promoting STAT3 activation and the upregulation of Twist1 (Zhao et al., 2015). Here, we describe a novel mechanism by which p27 directly regulates invadopodia formation and cell invasion through extracellular matrix (ECM) via Cortactin. Invadosomes (designating both invadopodia and podosomes) are thought to allow cells to coordinate ECM degradation with migration within the tissue microenvironment (Murphy and Courtneidge, 2011; Linder et al., 2011; Di Martino et al., 2016). Cortactin plays a key role in the formation of actin protrusive structures such as lamellipodia and invadosomes (Murphy and Courtneidge, 2011; Kirkbride et al., 2011; MacGrath and Koleske, 2012). Cortactin has been involved in all steps of the invadosome lifecycle, from assembly, maturation, proteolytic activity and disassembly (MacGrath and Koleske, 2012; Murphy and Courtneidge, 2011; Moshfegh et al., 2014). All these steps appear to be regulated by phosphorylation events on Cortactin (Oser et al., 2009; Moshfegh et al., 2014; Murphy and Courtneidge, 2011). Overall, Cortactin is a scaffold protein composed of an N-terminal acidic domain, followed by several actin-binding repeats allowing its interaction with F-actin, a Pro-rich region and a SH3 domain (MacGrath and Koleske, 2012; Kirkbride et al., 2011). Phosphorylation of Cortactin by Src and Abl family tyrosine kinases has two effects: first, this releases the actin severing protein Cofilin from an inhibitory interaction with Cortactin and generates actin barbed ends; second, Arp2/3, N-WASP and Nck1 are recruited onto Cortactin, promoting the polymerization of branched actin (Oser et al., 2009; Weaver et al., 2002; Oser et al., 2010). Dephosphorylation of Cortactin then allows the inhibition of Cofilin activity and stabilizes the invadopodia (Oser et al., 2009). Cortactin also plays a key role in matrix metalloproteinase secretion in mature invadopodia (Clark et al., 2007). Finally, invadopodia disassembly is induced by sequential activation of the Rac-GEF Trio, Rac1 and p21-Activated Kinase-1 (PAK1) pathway and presumably by PAK1-mediated phosphorylation of Cortactin on Ser113, since a Ser113 to Ala mutant blocked invadopodia disassembly (Moshfegh et al., 2014). How PAK-phosphorylated Cortactin mediates invadopodia disassembly is still unclear but could be due, at least in part, to a decreased affinity of Cortactin phosphorylated within its F-actin binding repeats (on S113, S150 and/or S282) for F-actin, potentially destabilizing the structure (Webb et al., 2006, 2005). We found that p27 binds to Cortactin and localizes to invadopodia following serum or growth factor stimulation and promotes cell invasion. Paradoxically, p27-null cells have more invadopodia and degrade gelatin more efficiently, but exhibit impaired invasive capacity. In fact, we found that p27 promotes the recruitment of PAK1 to Cortactin and invadopodia turnover and this is dependent on phosphorylation of S113/S150/S282 of Cortactin, the sites targeted by PAK kinases. Thus, in absence of p27, the dynamics of invadopodia is altered and prevents efficient invasion through ECM. Altogether, we have identified a novel mechanism by which p27 directly controls cell invasion that could contribute to the increased invasive and metastatic capacity of tumors where p27 is mislocalized in the cytoplasm. Results p27 interacts with Cortactin and localizes to invadopodia While the role of p27 in the regulation of cell migration is firmly established, how p27 is targeted to specific locations in the cytoplasm to control motility and invasion remains unclear (Gui et al., 2014; Belletti et al., 2010; Besson et al., 2004a, 2004b; Godin et al., 2012; Nguyen et al., 2006; Papakonstanti et al., 2007; Wu et al., 2006; See et al., 2010; Larrea et al., 2009; Jin et al., 2013). We recently identified Cortactin in a proteomic screen in which protein arrays (Protoarray, ThermoFisher Scientific) were probed with recombinant human p27, indicating that the two proteins interact directly. We confirmed the binding of p27 to Cortactin in HEK 293 cells overexpressing p27 and Myc-tagged Cortactin (Figure 1A), as well as on endogenous proteins in Hela cells and MEFs immortalized with the human papilloma virus E6 protein (Figure 1B) (Serres et al., 2011). The interaction domain of Cortactin on p27 was mapped by pull-down assays using various GST-p27 fusion proteins and Myc-Cortactin in HEK 293 cells. Cortactin bound to the p27CK- mutant, that cannot interact with cyclins and CDKs (Besson et al., 2006, 2007), and to the C-terminal half (aa 88–198) of p27 but not the N-terminal half (aa 1–87) of the protein that contains the cyclin and CDK interaction domains (Figure 1C and D). The Cortactin interaction domain on p27 was narrowed down to the last 8 aa of p27, as a p27CK- 1–190 mutant did not interact with Cortactin any longer (Figure 1E). A p27CK- 1–197 mutant, lacking only the C-terminal threonine residue, still bound to Cortactin (Figure 1E), suggesting that phosphorylation on T198 is not needed for binding to Cortactin. Figure 1 Download asset Open asset p27 binds to Cortactin. (A–B) Co-immunoprecipitation of p27 and Cortactin: (A) p27 was immunoprecipitated using rabbit anti-p27 (C19) antibodies from HEK293 lysates transfected with plasmids encoding p27, Myc-Cortactin (Myc-Cort) or both. (B) Immunoprecipitation of endogenous Cortactin using rabbit anti-Cortactin (H191) antibodies from HeLa or E6 MEF lysates, beads alone were used as control. (A–B) Co-immunoprecipitated proteins were detected by immunoblot with mouse anti-c-Myc (9E10) (A) or mouse anti-p27 (F-8) antibodies (B). Immunoprecipitated proteins were visualized by reprobing the membrane with mouse anti-p27 (F-8) (A) or with rabbit anti-Cortactin (H-191) antibodies and anti-rabbit Ig light-chain secondary antibodies (B). Immunoblots of extracts show the level of proteins in each condition. (C–F) Pull-down assays: HEK293 cells were transfected with Myc-Cortactin (C–E) or various deletion mutants of Myc-Cortactin (F) (ΔABR6, ΔABR5-6, ΔABR4-6, ΔABR3-6, ΔABR, ΔABR/NTA or ΔSH3, described in the schematic representation of Cortactin, bottom panel). NTA: N-Terminal acidic domain; ABR: actin binding repeat; Helix: helical domain; SH3: Src-homology three domain. Lysates were subjected to pull-down assays using GST, GST-p27 or GST-p27CK- (C), or GST, GST-p27, GST-p27 NT (1-87) and GST-p27 CT (88-198) (D), or GST, GST-p27CK-, GST-p27CK- (1-197) and GST-p27CK- (1-190) (E) or GST and GST-p27 (F). The amounts of Myc-Cortactin bound to the beads and of transfected protein present in the extracts were detected by immunoblot using mouse anti c-Myc (9E10) antibodies. The amounts of GST or GST p27 and mutants used in the assays were visualized by Coomassie staining of the gels. (A–F) All panels show representative results of at least three independent experiments. https://doi.org/10.7554/eLife.22207.003 Similarly, pull-down assays using full-length GST-p27 were performed on HEK 293 cell lysates expressing various Myc-tagged Cortactin truncation mutants to map the p27 interaction domain on Cortactin (Katsube et al., 2004). These experiments revealed that p27 interacts with the N-terminal half of Cortactin, within the actin binding repeats, as a mutant Cortactin lacking the actin binding repeats 3 to 6 (ΔABR3-6) did not bind p27 (Figure 1F). Since Cortactin is a key component of invadopodia, we determined if p27 could colocalize with Cortactin in these structures. To remove most of soluble p27 prior to fixation and immunostaining, p27+/+E6 MEFs were permeabilized with digitonin, as described previously (Serres et al., 2012). In these conditions, p27 could be readily observed in invadopodia where it colocalized with Tks5, a commonly used invadopodia marker (Seals et al., 2005), or Cortactin (Figure 2A and B, respectively). The same approach was used to confirm p27 localization to invadopodia in human A549 lung adenocarcinoma and A375 melanoma cell lines (Figure 2—figure supplement 1). Figure 2 with 2 supplements see all Download asset Open asset p27 colocalizes with Cortactin in invadopodia. (A–B) p27+/+ MEFs were seeded on gelatin-coated coverslips for 24 hr. Cells were permeabilized with digitonin prior to fixation. Cells were labeled with mouse anti p27 (SX53G8.5) in red (A–B) and rabbit anti-Tks5 (M-300) (A) or rabbit anti-Cortactin (H-191) (B) in green. Images were acquired using a 60x objective and images displayed are cropped areas. Graphs displaying the fluorescence intensity (arbitrary unit) under the arrows in the enlarged panels were generated with the NIS Element software. Scale bars: 20 μm. (C) p27+/+ E6 MEFs were starved overnight in DMEM-0.1% FCS and then stimulated with growth medium for the indicated times. Cell lysates were subjected to immunoprecipitation using rabbit anti-p27 (C–19). Immunoprecipitated proteins and corresponding cell extracts were immunoblotted with rabbit anti-Cortactin (H–191) and mouse anti-p27 (F–8) antibodies. β-actin was used as loading control. The graph represents the mean fold change in amounts of Cortactin co-precipitated with p27 in each condition compared to time zero from five independent experiments. These differences were not statistically significant. https://doi.org/10.7554/eLife.22207.004 Figure 2—source data 1 Quantification of co-immunoprecipitation between p27 and Cortactin in MEF E6 (Figure 2C) and HeLa cells (Figure 2—figure supplement 2). https://doi.org/10.7554/eLife.22207.005 Download elife-22207-fig2-data1-v2.xlsx Figure 2—source data 2 Statistical analyses for Figure 2C and Figure 2—figure supplement 2. https://doi.org/10.7554/eLife.22207.006 Download elife-22207-fig2-data2-v2.pzf Invadopodium and podosome formation is stimulated by growth factors (Murphy and Courtneidge, 2011). Growth factor stimulation causes the degradation of p27 as cells enter the cell cycle but also the relocalization of a fraction of p27 in the cytoplasm between 1 hr and 6 hr post-stimulation, dependent on the phoshorylation of p27 on Ser10 (Besson et al., 2006, 2004b; Boehm et al., 2002; Ishida et al., 2002; Rodier et al., 2001; Connor et al., 2003). Consistent with the translocation of p27 in the cytoplasm and the formation of invadopodia after serum or growth factor stimulation, an increased association between p27 and Cortactin by co-immunoprecipitation at 1 hr and 3 hr post-stimulation in p27+/+E6 MEFs (Figure 2C) and in Hela cells (Figure 2—figure supplement 2) was observed in five and three independent experiments, respectively. However, due to variability in signal intensities among independent experiments, these differences were not statistically significant. Together, our data indicate that a fraction of cytoplasmic p27 localizes to invadopodia after growth factor stimulation, where it binds to Cortactin. p27 regulates invadopodia formation and promotes invasion Invadopodia and podosomes are capable of both adhering to and degrading ECM (Murphy and Courtneidge, 2011; Di Martino et al., 2016). Fibroblasts do not normally form invadosomes unless transformed by Src (Murphy and Courtneidge, 2011). We first verified that the E6 immortalized MEF lines used in our study were forming functional, matrix degrading invadopodia. For this, p27+/+, p27−/− and p27CK-/CK- E6 MEFs were seeded on fluorescent gelatin and Tks5 immunostaining indicated that these cells could all form Tks5-containing structures that efficiently degraded gelatin (Figure 3—figure supplement 1). p27 colocalized with Tks5 at sites of gelatin degradation in MEFs and A549 cells, suggesting that p27 can be present at functional invadopodia (Figure 3—figure supplement 2). Since p27 binds to Cortactin and can localize to invadopodia, we determined if p27 status influenced the ability of cells to form invadopodia. Counting of the number of cells forming invadopodia using Tks5 immunostaining (Figure 3A) revealed that while cells expressing p27 or p27CK- rarely formed invadopodia (5.83% and 8.68%, respectively), nearly half of MEFs lacking p27 had invadopodia (44.66%) (Figure 3B). In keeping with the number of invadopodia forming cells, measurement of the area of fluorescent gelatin degraded per cell showed that p27−/− cells had a dramatically increased capacity to degrade ECM compared to p27+/+ and p27CK-/CK- MEFs (Figure 3C and D). Figure 3 with 2 supplements see all Download asset Open asset p27 regulates invadopodia formation and matrix degradation. (A) p27+/+, p27CK−/CK− and p27−/− immortalized MEFs were seeded on Oregon green-gelatin (gelatin-A488) for 16 hr. Cells were stained with rabbit anti-Tks5 (M-300) to visualize invadopodia. (B) The percentage of cells forming invadopodia was determined in a minimum of 15 fields, representing a minimum of 330 cells per genotype, for each experiment. The graph shows the mean of 3 independent experiments. (C) Cells were seeded as in (A). Tks5 staining shows invadopodia (red) and areas of degraded fluorescent gelatin indicate invadopodia activity (green). (D) The areas of degraded gelatin were measured in at least 15 fields per genotype in each experiment. The graph shows the mean of 3 independent experiments. (E–G) p27−/− E6 MEFs were infected with either empty vector or p27, p27CK− or p27CK− 1–190 vectors and then seeded on Gelatin-A488 for 48 hr. (E) p27 levels after retroviral infection were determined by immunoblot with rabbit anti-p27 (N-20) antibodies; Grb2 was used as loading control. (F–G) After Tks5 staining, invadopodia forming cells (F) were quantified as in B and area of degraded gelatin (G) as in D. Scale bars: 50 μm; 'ns' not significant; ****p<0.0001. In A and C, images were acquired using a 40x objective and images displayed are cropped areas. https://doi.org/10.7554/eLife.22207.009 Figure 3—source data 1 Quantification of cells with invadopodia (Figure 3B); quantification of degraded gelatin area per cell (Figure 3C); quantification of cells with invadopodia after p27 re-expression (Figure 3F) and quantification of degraded gelatin area per cell after p27 re-expression (Figure 3G). https://doi.org/10.7554/eLife.22207.010 Download elife-22207-fig3-data1-v2.xlsx Figure 3—source data 2 Statistical analyses for Figure 3B,C,F and G. https://doi.org/10.7554/eLife.22207.011 Download elife-22207-fig3-data2-v2.pzf Figure 3—source data 3 Immunoblot scans of Figure 3E. https://doi.org/10.7554/eLife.22207.012 Download elife-22207-fig3-data3-v2.jpg To confirm the involvement of p27 in regulating invadopodia number and proteolytic activity, we retrovirally infected p27−/− E6 MEFs with either empty vector, wild-type (WT) p27, p27CK- or a p27CK- 1–190 truncation mutant (Besson et al., 2004a; Serres et al., 2012) that cannot interact with Cortactin (Figure 1E). Expression levels of p27 in these infected cells were determined by immunoblot (Figure 3E). Quantification of the number of cells forming invadopodia and of the area of gelatin degraded per cell indicated that expression of WT p27 and p27CK- significantly decreased both the number of p27−/− cells forming invadopodia and their capacity to degrade gelatin, while the p27CK- 1–190 mutant had no effect (Figure 3F and G). Thus, our data suggests that p27 limits invadopodia formation and that the domain mediating its interaction with Cortactin is required for this function. The finding that p27 knockout cells more frequently form invadopodia and exhibit increased ECM degradation activity was surprising given the previous reports that p27 promotes migration and invasion (Denicourt et al., 2007; Godin et al., 2012; Nguyen et al., 2006; Papakonstanti et al., 2007; Gui et al., 2014; Wu et al., 2006; See et al., 2010; Larrea et al., 2009; Jin et al., 2013; Zhao et al., 2015; Besson et al., 2004b). We compared the motility of p27+/+, p27−/− and p27CK-/CK- E6 MEFs in 2D scratch wound assays and p27−/− cells had a migration defect (Figure 4A and B), in agreement with our previous findings (Besson et al., 2004b). We next measured the capacity of these MEFs to invade through a layer of Collagen I in transwell inserts. Similar to 2D migration, 3D invasion was reduced in p27−/− MEFs compared to either WT p27 or p27CK- expressing cells (Figure 4C and D), consistent with previous reports (Denicourt et al., 2007; Gui et al., 2014; Wu et al., 2006; See et al., 2010; Jin et al., 2013; Zhao et al., 2015). To confirm the involvement of p27 in regulating invasion, p27−/− E6 MEFs retrovirally infected with either empty vector, p27CK- or p27CK- 1–190 (Figure 4E, left panel) were subjected to transwell invasion assays. While p27CK- restored invasion of p27−/− cells, the p27CK- 1–190 mutant had no effect (Figure 4E, right panel). Taken together our results indicate that although p27 limits the number of invadopodia in MEFs, it also promotes cellular invasion through ECM. On the other hand, while p27-null cells more frequently form invadopodia and exhibit an increased capacity to degrade ECM, their ability to invade through matrix was significantly impaired. Figure 4 Download asset Open asset p27 promotes cell migration and invasion. (A) Representative images of scratch wound migration assays with p27+/+, p27CK−/CK− and p27−/− immortalized MEFs. Dark grey areas show the initial wound masks and dotted lines the migration fronts. Scale bars: 300 μm. (B) Mean cell migration at 12 hr and 24 hr post wounding for each genotype of five independent experiments. Percent of area in which the cells migrated, or wound closing (relative wound density) was calculated with the Incucyte software. 'ns': not significant; ****p<0.0001. (C) Representative images of p27+/+, p27CK−/CK− and p27−/− immortalized MEFs that invaded through a layer of Collagen I in transwell invasion assays and migrated to the bottom side of the transwell membrane after 48 hr. Scale bars: 100 μm. (D) The graph shows the mean number of cells that invaded through Collagen I quantified by XTT staining, expressed relative to p27+/+ cells, of three independent experiments. **p<0.01. (E) p27−/− E6 MEFs were infected with either empty vector, p27CK- or p27CK- 1–190 vectors and used in transwell invasion assays as in (C–D). p27 levels after retroviral infection were determined by immunoblot with mouse anti-p27 (SX53G8.5); β-actin was used as loading control. The graph shows the mean number of cells that invaded through Collagen I quantified by XTT staining, expressed relative to p27−/− cells, of four independent experiments. https://doi.org/10.7554/eLife.22207.015 Figure 4—source data 1 quantification of relative wound density (Figure 4B); quantification of invasion (Figure 4D); and quantification of invasion rescue by p27 re-expression (Figure 4E). https://doi.org/10.7554/eLife.22207.016 Download elife-22207-fig4-data1-v2.xlsx Figure 4—source data 2 Statistical analyses for Figure 4B,D and E. https://doi.org/10.7554/eLife.22207.017 Download elife-22207-fig4-data2-v2.pzf p27 promotes the interaction of Cortactin with PAK1 Interestingly, a similar, seemingly paradoxical finding was recently reported, in which inhibition of Rac1, Trio or PAK1 with siRNAs caused a dramatic increase in invadopodia lifetime and in their capacity to degrade matrix, but this was accompanied by a sharp decrease in cell invasion (Moshfegh et al., 2014). In this study, activation of the Trio/Rac1/PAK1 pathway induced a putative PAK1-mediated phosphorylation of Cortactin and invadopodia disassembly, as expression of a S113A Cortactin mutant blocked this pathway (Moshfegh et al., 2014). In this study, altering the dynamics of invadopodia turnover inhibited invasion (Moshfegh et al., 2014). To find out whether p27-null cells more frequently form invadopodia than p27+/+ cells (Figure 3A and B) due to an increase in their lifetime, we infected p27+/+ and p27−/− MEFs with Tks5-GFP to visualize invadopodia in live cells. Videomicroscopy analyses revealed that while invadopodia lifetime was on average 16.7 min in p27+/+ cells, their mean duration was 65.9 min in p27−/− cells (Figure 5A). Thus, invadopodia appear more stable in cells lacking p27. Figure 5 with 1 supplement see all Download asset Open asset p27 promotes binding of Cortactin to PAK1. (A) Live p27+/+ and p27−/− immortalized MEFs expressing eGFP-Tks5 were imaged by videomicroscopy to measure invadopodia lifetime, using Tks5 as an invadopodia marker. The graph represents the average invadopodia lifetime of 20 invadopodia per genotype per experiment from three independent experim
Abstract Breast cancer-associated fibroblasts (bCAFs) comprise inflammatory CAFs (iCAFs), characterized by the secretion of pro-inflammatory cytokines, and myofibroblastic CAFs (myCAFs), distinguished by their high production of extracellular matrix and their immunosuppressive properties. We previously showed that targeting the anti-apoptotic protein MCL-1 in primary culture of bCAF derived directly from human samples reduces their myofibroblastic characteristics. We herein show by single-cell RNA-sequencing analysis of bCAFs that MCL-1 knock down induces a phenotypic shift from wound-myCAF to IL-iCAF, characterized by the upregulation of genes associated with inflammation as well as angiogenesis-related genes. In vitro, genetic and pharmacologic MCL-1 inhibition increases VEGF secretion by bCAFs, enhancing endothelial cell tubulogenesis. In a chicken chorioallantoic membrane (CAM) model in ovo, co-engraftment of breast cancer cells and bCAFs with reduced MCL-1 expression leads to heightened peritumoral vascular density, driven by VEGF. Mechanistically, the pro-angiogenic phenotype revealed by MCL-1 inhibition is dependent on BAX-BAK activity. It results in NF-κB activation, inhibition of which by a IKKβ inhibitor suppresses the transcription of VEGF and pro-inflammatory factors triggered by MCL-1 inhibition in bCAFs. Chemotherapy induces a downregulation of MCL-1 in bCAFs and it promotes a pro-angiogenic phenotype, counteracted by overexpressed MCL-1. Overall, these findings uncover a novel regulatory function of MCL-1 in determining bCAF subpopulation differentiation and highlight its role in modulating their pro-angiogenic properties, in response to treatment in particular.
Despite the involvement of several serine hydrolases (SHs) in the metabolism of xenobiotics such as dibutyl phthalate (DBP), no study has focused on mapping this enzyme class in zebrafish, a model organism frequently used in ecotoxicology. Here, we survey and identify active SHs in zebrafish larvae and search for biological markers of SH type after exposure to DBP. Zebrafish were exposed to 0, 5, and 100 µg/L DBP from 4 to 120 h post-fertilization. A significant decrease in vitellogenin expression level of about 2-fold compared to the control was found in larvae exposed to 100 µg/L DBP for 120 h. The first comprehensive profiling of active SHs in zebrafish proteome was achieved with an activity-based protein profiling (ABPP) approach. Among 49 SHs identified with high confidence, one was the carboxypeptidase ctsa overexpressed in larvae exposed to 100 µg/L DBP for 120 h. To the best of our knowledge, this is the first time that a carboxypeptidase has been identified as deregulated following exposure to DBP. The overall results indicate that targeted proteomics approaches, such as ABPP, can, therefore, be an asset for understanding the mechanism of action related to xenobiotics in ecotoxicology.
