ProNGF promotes brain metastasis through TrkA/EphA2 induced Src activation in triple negative breast cancer cells.

BACKGROUND: Triple-Negative Breast Cancer is particularly aggressive, and its metastasis to the brain has a significant psychological impact on patients' quality of life, in addition to reducing survival. The development of brain metastases is particularly harmful in triple-negative breast cancer (TNBC). To date, the mechanisms that induce brain metastasis in TNBC are poorly understood. METHODS: Using a human blood-brain barrier (BBB) in vitro model, an in vitro 3D organotypic extracellular matrix, an ex vivo mouse brain slices co-culture and in an in vivo xenograft experiment, key step of brain metastasis were recapitulated to study TNBC behaviors. RESULTS: In this study, we demonstrated for the first time the involvement of the precursor of Nerve Growth Factor (proNGF) in the development of brain metastasis. More importantly, our results showed that proNGF acts through TrkA independent of its phosphorylation to induce brain metastasis in TNBC. In addition, we found that proNGF induces BBB transmigration through the TrkA/EphA2 signaling complex. More importantly, our results showed that combinatorial inhibition of TrkA and EphA2 decreased TBNC brain metastasis in a preclinical model. CONCLUSIONS: These disruptive findings provide new insights into the mechanisms underlying brain metastasis with proNGF as a driver of brain metastasis of TNBC and identify TrkA/EphA2 complex as a potential therapeutic target.

Mitochondrial adaptation decreases drug sensitivity of persistent triple negative breast cancer cells surviving combinatory and sequential chemotherapy.

Triple negative breast cancer (TNBC) is an aggressive malignancy for which chemotherapy remains the standard treatment. However, between 3 and 5 years after chemotherapy, about half patients will relapse and it is essential to identify vulnerabilities of cancer cells surviving neoadujuvant therapy. In this study, we established persistent TNBC cell models after treating MDA-MB-231 and SUM159-PT TNBC cell lines with epirubicin and cyclophosphamide, and then with paclitaxel, for a total of 18 weeks. The resulting chemo-persistent cell lines were more proliferative, both in vitro and in xenografted mice. Interestingly, MDA-MB-231 persistent cells became less sensitive to chemotherapeutic drugs, whereas SUM159-PT persistent cells kept similar sensitivity compared to control cells. The reduced sensitivity to chemotherapy in MDA-MB-231 persistent cells was found to be associated with an increased oxidative phosphorylation (OXPHOS) and modified levels of tricarboxylic acid cycle (TCA) intermediates. Integration of data from proteomics and metabolomics demonstrated TCA cycle among the most upregulated pathways in MDA-MB-231 persistent cells. The absence of glucose and pyruvate impeded OXPHOS in persistent cells, while the absence of glutamine did not. In contrast, OXPHOS was not modified in control cells independently of TCA substrates, indicating that MDA-MB-231 persistent cells evolved towards a more pyruvate dependent profile. Finally, the inhibition of pyruvate entry into mitochondria with UK-5099 reduced OXPHOS and re-sensitized persistent cells to therapeutic agents. Together, these findings suggest that targeting mitochondrial pyruvate metabolism may help to overcome mitochondrial adaptation of chemo-persistent TNBC.

M-CSF directs myeloid and NK cell differentiation to protect from CMV after hematopoietic cell transplantation.

Therapies reconstituting autologous antiviral immunocompetence may represent an important prophylaxis and treatment for immunosuppressed individuals. Following hematopoietic cell transplantation (HCT), patients are susceptible to Herpesviridae including cytomegalovirus (CMV). We show in a murine model of HCT that macrophage colony-stimulating factor (M-CSF) promoted rapid antiviral activity and protection from viremia caused by murine CMV. M-CSF given at transplantation stimulated sequential myeloid and natural killer (NK) cell differentiation culminating in increased NK cell numbers, production of granzyme B and interferon-γ. This depended upon M-CSF-induced myelopoiesis leading to IL15Rα-mediated presentation of IL-15 on monocytes, augmented by type I interferons from plasmacytoid dendritic cells. Demonstrating relevance to human HCT, M-CSF induced myelomonocytic IL15Rα expression and numbers of functional NK cells in G-CSF-mobilized hematopoietic stem and progenitor cells. Together, M-CSF-induced myelopoiesis triggered an integrated differentiation of myeloid and NK cells to protect HCT recipients from CMV. Thus, our results identify a rationale for the therapeutic use of M-CSF to rapidly reconstitute antiviral activity in immunocompromised individuals, which may provide a general paradigm to boost innate antiviral immunocompetence using host-directed therapies.

Macrophage-Colony-Stimulating Factor Receptor Enhances Prostate Cancer Cell Growth and Aggressiveness In Vitro and In Vivo and Increases Osteopontin Expression.

Prostate cancer is a major public health concern and one of the most prevalent forms of cancer worldwide. The definition of altered signaling pathways implicated in this complex disease is thus essential. In this context, abnormal expression of the receptor of Macrophage Colony-Stimulating Factor-1 (M-CSF or CSF-1) has been described in prostate cancer cells. Yet, outcomes of this expression remain unknown. Using mouse and human prostate cancer cell lines, this study has investigated the functionality of the wild-type CSF-1 receptor in prostate tumor cells and identified molecular mechanisms underlying its ligand-induced activation. Here, we showed that upon CSF-1 binding, the receptor autophosphorylates and activates multiple signaling pathways in prostate tumor cells. Biological experiments demonstrated that the CSF-1R/CSF-1 axis conferred significant advantages in cell growth and cell invasion in vitro. Mouse xenograft experiments showed that CSF-1R expression promoted the aggressiveness of prostate tumor cells. In particular, we demonstrated that the ligand-activated CSF-1R increased the expression of spp1 transcript encoding for osteopontin, a key player in cancer development and metastasis. Therefore, this study highlights that the CSF-1 receptor is fully functional in a prostate cancer cell and may be a potential therapeutic target for the treatment of prostate cancer.

Enhancement of Breast Cancer Cell Aggressiveness by lncRNA H19 and its Mir-675 Derivative: Insight into Shared and Different Actions.

Breast cancer is a major public health problem and the leading world cause of women death by cancer. Both the recurrence and mortality of breast cancer are mainly caused by the formation of metastasis. The long non-coding RNA H19, the precursor of miR-675, is involved in breast cancer development. The aim of this work was to determine the implication but, also, the relative contribution of H19 and miR-675 to the enhancement of breast cancer metastatic potential. We showed that both H19 and miR-675 increase the invasive capacities of breast cancer cells in xenografted transgenic zebrafish models. In vitro, H19 and miR-675 enhance the cell migration and invasion, as well as colony formation. H19 seems to induce the epithelial-to-mesenchymal transition (EMT), with a decreased expression of epithelial markers and an increased expression of mesenchymal markers. Interestingly, miR-675 simultaneously increases the expression of both epithelial and mesenchymal markers, suggesting the induction of a hybrid phenotype or mesenchymal-to-epithelial transition (MET). Finally, we demonstrated for the first time that miR-675, like its precursor H19, increases the stemness properties of breast cancer cells. Altogether, our data suggest that H19 and miR-675 could enhance the aggressiveness of breast cancer cells through both common and different mechanisms.

s-SHIP Promoter Expression Identifies Mouse Mammary Cancer Stem Cells.

During normal mammary gland development, s-SHIP promoter expression marks a distinct type of mammary stem cells, at two different stages, puberty and early mid-pregnancy. To determine whether s-SHIP is a marker of mammary cancer stem cells (CSCs), we generated bitransgenic mice by crossing the C3(1)-SV40 T-antigen transgenic mouse model of breast cancer, and a transgenic mouse (11.5kb-GFP) expressing green fluorescent protein from the s-SHIP promoter. Here we show that in mammary tumors originating in these bitransgenic mice, s-SHIP promoter expression enriches a rare cell population with CSC activity as demonstrated by sphere-forming assays in vitro and limiting dilution transplantation in vivo. These s-SHIP-positive CSCs are characterized by lower expression of Delta-like non-canonical Notch ligand 1 (DLK1), a negative regulator of the Notch pathway. Inactivation of Dlk1 in s-SHIP-negative tumor cells increases their tumorigenic potential, suggesting a role for DLK1 in mammary cancer stemness.

Synuclein gamma expression enhances radiation resistance of breast cancer cells.

Resistance to therapy is a major obstacle for the effective treatment of cancer. Expression of synuclein-gamma (SNCG) has been associated with poor prognosis and resistance to therapy. While reports on SNCG overexpression contributing to chemoresistance exist, limited information is available on the relationship between SNCG and radioresistance of cancer cells. Here we investigated the role of SNCG in radiation resistance in breast cancer cells. siRNA mediated knockdown of SNCG (siSNCG) markedly reduced SNCG protein level compared to scrambled siRNA (siScr) treatment. Furthermore, siSNCG treatment sensitized Estrogen Receptor-positive breast cancer cells (MCF7 and T47D) to ionizing radiation at 4 to 12 Gy as evidenced by the significant increase of apoptotic or senescent cells and reduction in clonogenic cell survival in siSNCG treated cells compared to siScr treated cells. On the other hand, we established an in vitro model of SNCG ectopic expression by using a triple-negative breast cancer cell line (SUM159PT) to further investigate the radioprotective effect of SNCG. We showed that ectopic expression of SNCG significantly decreased apoptosis of SUM159PT cells and enhanced clonogenic cell survival after radiation treatment. At the molecular level, after irradiation, the p53 pathway was less activated when SNCG was present. Conversely, p21Waf1/Cip1 expression was upregulated in SNCG-expressing cells. When p21 was down-regulated by siRNA, radiosensitivity of SNCG-expressing SUM159PT cells was dramatically increased. This suggested a possible connection between p21 and SNCG in radioresistance in these cells. In conclusion, our data provide for the first time experimental evidence for the role of SNCG in the radioresistance of breast cancer cells.

s-SHIP expression identifies a subset of murine basal prostate cells as neonatal stem cells.

Isolation of prostate stem cells (PSCs) is crucial for understanding their biology during normal development and tumorigenesis. In this aim, we used a transgenic mouse model expressing GFP from the stem cell-specific s-SHIP promoter to mark putative stem cells during postnatal prostate development. Here we show that cells identified by GFP expression are present transiently during early prostate development and localize to the basal cell layer of the epithelium. These prostate GFP+ cells are a subpopulation of the Lin- CD24+ Sca-1+ CD49f+ cells and are capable of self-renewal together with enhanced growth potential in sphere-forming assay in vitro, a phenotype consistent with that of a PSC population. Transplantation assays of prostate GFP+ cells demonstrate reconstitution of prostate ducts containing both basal and luminal cells in renal grafts. Altogether, these results demonstrate that s-SHIP promoter expression is a new marker for neonatal basal prostate cells exhibiting stem cell properties that enables PSCs in situ identification and isolation via a single consistent parameter. Transcriptional profiling of these GFP+ neonatal stem cells showed an increased expression of several components of the Wnt signaling pathway. It also identified stem cell regulators with potential applications for further analyses of normal and cancer stem cells.

H19 non coding RNA-derived miR-675 enhances tumorigenesis and metastasis of breast cancer cells by downregulating c-Cbl and Cbl-b.

H19 is a long non-coding RNA precursor of miR-675 microRNA. H19 is increasingly described to play key roles in the progression and metastasis of cancers from different tissue origins. We have previously shown that the H19 gene is activated by growth factors and increases breast cancer cell invasion. In this study, we established H19/miR-675 ectopic expression models of MDA-MB-231 breast cancer cells to further investigate the underlying mechanisms of H19 oncogenic action. We showed that overexpression of H19/miR-675 enhanced the aggressive phenotype of breast cancer cells including increased cell proliferation and migration in vitro, and increased tumor growth and metastasis in vivo. Moreover, we identified ubiquitin ligase E3 family (c-Cbl and Cbl-b) as direct targets of miR-675 in breast cancer cells. Using a luciferase assay, we demonstrated that H19, through its microRNA, decreased both c-Cbl and Cbl-b expression in all breast cancer cell lines tested. Thus, by directly binding c-Cbl and Cbl-b mRNA, miR-675 increased the stability and the activation of EGFR and c-Met, leading to sustained activation of Akt and Erk as well as enhanced cell proliferation and migration. Our data describe a novel mechanism of protumoral action of H19 in breast cancer.

Enrichment of Human Stem-Like Prostate Cells with s-SHIP Promoter Activity Uncovers a Role in Stemness for the Long Noncoding RNA H19.

Understanding normal and cancer stem cells should provide insights into the origin of prostate cancer and their mechanisms of resistance to current treatment strategies. In this study, we isolated and characterized stem-like cells present in the immortalized human prostate cell line, RWPE-1. We used a reporter system with green fluorescent protein (GFP) driven by the promoter of s-SHIP (for stem-SH2-domain-containing 5'-inositol phosphatase) whose stem cell-specific expression has been previously shown. We observed that s-SHIP-GFP-expressing RWPE-1 cells showed stem cell characteristics such as increased expression of stem cell surface markers (CD44, CD166, TROP2) and pluripotency transcription factors (Oct4, Sox2), and enhanced sphere-forming capacity and resistance to arsenite-induced cell death. Concomitant increased expression of the long noncoding RNA H19 was observed, which prompted us to investigate a putative role in stemness for this oncofetal gene. Targeted suppression of H19 with siRNA decreased Oct4 and Sox2 gene expression and colony-forming potential in RWPE-1 cells. Conversely, overexpression of H19 significantly increased gene expression of these two transcription factors and the sphere-forming capacity of RWPE-1 cells. Analysis of H19 expression in various prostate and mammary human cell lines revealed similarities with Sox2 expression, suggesting that a functional relationship may exist between H19 and Sox2. Collectively, we provide the first evidence that s-SHIP-GFP promoter reporter offers a unique marker for the enrichment of human stem-like cell populations and highlight a role in stemness for the long noncoding RNA H19.

Anti-apoptotic role of caspase-cleaved GAB1 adaptor protein in hepatocyte growth factor/scatter factor-MET receptor protein signaling.

The GRB2-associated binder 1 (GAB1) docking/scaffold protein is a key mediator of the MET-tyrosine kinase receptor activated by hepatocyte growth factor/scatter factor (HGF/SF). Activated MET promotes recruitment and tyrosine phosphorylation of GAB1, which in turn recruits multiple proteins and mediates MET signaling leading to cell survival, motility, and morphogenesis. We previously reported that, without its ligand, MET is a functional caspase target during apoptosis, allowing the generation of a p40-MET fragment that amplifies apoptosis. In this study we established that GAB1 is also a functional caspase target by evidencing a caspase-cleaved p35-GAB1 fragment that contains the MET binding domain. GAB1 is cleaved by caspases before MET, and the resulting p35-GAB1 fragment is phosphorylated by MET upon HGF/SF binding and can interact with a subset of GAB1 partners, PI3K, and GRB2 but not with SHP2. This p35-GAB1 fragment favors cell survival by maintaining HGF/SF-induced MET activation of AKT and by hindering p40-MET pro-apoptotic function. These data demonstrate an anti-apoptotic role of caspase-cleaved GAB1 in HGF/SF-MET signaling.

Macrophage differentiation of myeloid progenitor cells in response to M-CSF is regulated by the dual-specificity phosphatase DUSP5.

M-CSF regulates the production, survival, and function of monocytes and macrophages. The MAPKs ERK1/2 are key elements for signal integration downstream of the M-CSFR, and their sustained activation is essential for macrophage differentiation. In this study, we sought to isolate genes whose induction by M-CSF is dependent on persistent MAPK activation, thereby being possibly involved in the commitment of myeloid progenitors to macrophage differentiation. Following SSH between cDNA libraries from FD-Fms cells stimulated by M-CSF for 8 h in the presence or the absence of the MEK inhibitor U0126, we isolated DUSP5. DUSP5 expression is induced by M-CSF in various myeloid cells and acts as a specific negative-feedback regulator of ERK1/2. In FD-Fms cells that proliferate and differentiate toward macrophages in response to M-CSF, overexpression of DUSP5 increased M-CSF-dependent proliferation and strongly decreased differentiation. Similarly, overexpression of DUSP5 in the multipotent EGER-Fms cells not only significantly increased M-CSF-induced proliferation and prevented macrophage differentiation but also favored granulocytic differentiation. Altogether, experiments demonstrated that DUSP5 is implicated in M-CSF signaling and suggested that it may influence myeloid cell fate.

The biological role of interferon-inducible P204 protein in the development of the mononuclear phagocyte system.

The mononuclear phagocyte system (MPS) is a cell population derived from progenitor cells in the bone marrow, and comprising monocytes, macrophages, osteoclasts, dendritic cells, and microglia. Homeostasis of the MPS and response to physiological stress is under the control of signaling molecules and nuclear factors; among them, macrophage-colony-stimulating factor (M-CSF) controls monocyte/macrophage lineage development. Here we discuss the implication of Ifi204, a M-CSF-responsive gene, in the proliferation and differentiation of monocytes/macrophages. Ifi204 is a member of the interferon-inducible p200 family of proteins, and was found to be an important regulator of differentiation of both skeletal and cardiac muscles and osteogenesis. Ifi204 is expressed at the early stages of differentiation of MPS cells and later in the monocyte/macrophage lineage. IFI16, the closest Ifi protein in human, is expressed all along the the monocytic lineage. In MPS cells, Ifi204 expression is induced by interferons but also by various stimuli, independently of the presence of interferon. Enforced expression of p204 in interleukin-3 (IL3)-dependent FD-Fms cell line strongly decreased both IL3- and M-CSF-dependent proliferation and conversely favored macrophage differentiation of FD-Fms cells in response to M-CSF. Altogether, data enlighten a role of Ifi204 as a regulator of monocyte/macrophage differentiation and make possible a connection with other myeloid regulators.

The interferon-inducible gene, Ifi204, is transcriptionally activated in response to M-CSF, and its expression favors macrophage differentiation in myeloid progenitor cells.

The interferon-inducible (Ifi)204 gene was isolated as a macrophage-colony stimulating factor (M-CSF)-responsive gene using a gene trap approach in the myeloid interleukin-3 (IL-3)-dependent FD-Fms cell line, which differentiates in macrophages in response to M-CSF. Here, we show that Ifi204 was transcriptionally activated in response to M-CSF, and FD-Fms cells decreased their growth and committed toward a macrophage morphology; this induction was abrogated when the differentiation signal of the M-CSF receptor was blocked; the Ifi204 gene was also induced during macrophage differentiation controlled by leukemia inhibitory factor; and the Ifi204 gene is expressed in different mature monocyte/macrophage cells. Finally, we showed that enforced expression of Ifi204 strongly decreased IL-3- and M-CSF-dependent proliferation and conversely, favored macrophage differentiation of FD-Fms cells in response to M-CSF. Altogether, these results demonstrate that the Ifi204 gene is activated during macrophage development and suggest that the Ifi204 protein may act as a regulator of the balance between proliferation and differentiation. Moreover, this study suggests that other members of the Ifi family might act as regulators of hematopoiesis under the control of hemopoietic cytokines.

Macrophage colony-stimulating factor receptor induces tyrosine phosphorylation of SKAP55R adaptor and its association with actin.

The production, survival, and function of monocytes and macrophages are regulated by the macrophage colony-stimulating factor (M-CSF or CSF-1) through its tyrosine kinase receptor. M-CSF receptor activates multiple cytoplasmic pathways in which adaptor and scaffolding proteins play a central role. In this study, we showed that SKAP55-related (SKAP55R) adaptor protein is expressed in myeloid cells and macrophages and is rapidly and transiently tyrosine-phosphorylated in response to M-CSF. M-CSF induced SKAP55R association with other tyrosine-phosphorylated proteins and with actin. When overexpressed in myeloid cells, SKAP55R decreased M-CSF-dependent proliferation without affecting differentiation. Altogether, these results demonstrate that SKAP55R adaptor is implicated in the M-CSF signaling pathway and suggest its role as a negative regulator of growth. Moreover, specific association between SKAP55R and actin support the idea that SKAP55R is implicated in the regulation of actin dynamics under the control of M-CSF.

US11 of herpes simplex virus type 1 interacts with HIPK2 and antagonizes HIPK2-induced cell growth arrest.

Homeodomain-interacting protein kinase 2 (HIPK2) is a nuclear serine/threonine kinase of the subfamily of dual-specificity Yak1-related kinase proteins. HIPK2 was first described as a homeodomain-interacting protein kinase acting as a corepressor for homeodomain transcription factors. More recently, it was reported that HIPK2 plays a role in p53-mediated cellular apoptosis and could also participate in the regulation of the cell cycle. US11 protein of herpes simplex virus type 1 is a multifunctional protein involved in the regulation of several processes related to the survival of cells submitted to environmental stresses by mechanisms that are not fully elucidated. In an attempt to better understand the multiple functions of US11, we identified cellular binding partners of this protein by using the yeast two-hybrid system. We report that US11 interacts with HIPK2 through the PEST domain of HIPK2 and that this interaction occurs also in human cells. This interaction modifies the subcellular distribution of HIPK2 and protects the cell against the HIPK2-induced cell growth arrest.

Structural basis for SH3 domain-mediated high-affinity binding between Mona/Gads and SLP-76.

SH3 domains are protein recognition modules within many adaptors and enzymes. With more than 500 SH3 domains in the human genome, binding selectivity is a key issue in understanding the molecular basis of SH3 domain interactions. The Grb2-like adaptor protein Mona/Gads associates stably with the T-cell receptor signal transducer SLP-76. The crystal structure of a complex between the C-terminal SH3 domain (SH3C) of Mona/Gads and a SLP-76 peptide has now been solved to 1.7 A. The peptide lacks the canonical SH3 domain binding motif P-x-x-P and does not form a frequently observed poly-proline type II helix. Instead, it adopts a clamp-like shape around the circumfence of the SH3C beta-barrel. The central R-x-x-K motif of the peptide forms a 3(10) helix and inserts into a negatively charged double pocket on the SH3C while several other residues complement binding through hydrophobic interactions, creating a short linear SH3C binding epitope of uniquely high affinity. Interestingly, the SH3C displays ion-dependent dimerization in the crystal and in solution, suggesting a novel mechanism for the regulation of SH3 domain functions.

M-CSF induced differentiation of myeloid precursor cells involves activation of PKC-delta and expression of Pkare.

Macrophage-colony stimulating factor (M-CSF) regulates proliferation and differentiation of cells belonging to the monocytic lineage. We investigated the mechanisms of M-CSF differentiation signaling in follicular dendritic cell-P1 cells and analyzed the catalytic activation of different protein kinase C (PKC) isoforms. M-CSF induced rapid catalytic activation of PKC-delta and membrane translocation of the tyrosine phosphorylated form of PKC-delta. Mutation of tyrosine 807 in the M-CSF receptor (Fms) abrogates cell differentiation but not a proliferative response to M-CSF, and FmsY807F failed to activate PKC-delta. We also investigated the downstream signaling pathways from PKC-delta. A cyclic adenosine monophosphate-regulated Ser/Thr kinase gene, protein kinase X (PRKX), has been associated with macrophage differentiation in human cells. We found that M-CSF and PKC-delta induced the expression of the PRKX murine homologue: PKA-related gene. Taken together, our results indicate that PKC-delta functions as a critical mediator of M-CSF-induced differentiation signaling.

Induced expression and association of the Mona/Gads adapter and Gab3 scaffolding protein during monocyte/macrophage differentiation.

Mona/Gads is a Grb2-related, Src homology 3 (SH3) and SH2 domain-containing adapter protein whose expression is restricted to cells of hematopoietic lineage (i.e., monocytes and T lymphocytes). During monocyte/macrophage differentiation, Mona is induced and interacts with the macrophage colony-stimulating factor receptor, M-CSFR (also called Fms), suggesting that Mona could be involved in developmental signaling downstream of the M-CSFR by recruiting additional signaling proteins to the activated receptor. Our present results identify Mona as a specific partner protein for the DOS/Gab family member Gab3 in monocytic/macrophage development. Mona does not interact with Gab2; however, Gab3 also forms a complex with the Mona-related adapter Grb2. Glutathione S-transferase pull-down experiments demonstrate that the Mona and Gab3 interaction utilizes the carboxy-terminal SH3 domain of Mona and the atypical proline-rich domain of Gab3. Mona is known to interact with the phosphorylated Y697 site of the M-CSFR. The M-CSFR mutation Y697F exhibited qualitative and quantitative abnormalities in receptor and Gab3 tyrosine phosphorylation, and Mona induction was greatly reduced. The Y807F M-CSFR mutation is defective in differentiation signaling, but not growth signaling, and also fails to induce Mona protein expression. During M-CSF-stimulated macrophage differentiation of mouse bone marrow cells, Mona and Gab3 expression is coinduced, these proteins interact, and Mona engages in multimolecular complexes. These data suggest that association of Mona and Gab3 plays a specific role in mediating the M-CSFR differentiation signal.