Pranlukast Antagonizes CD49f and Reduces Sternness in Triple-Negative Breast Cancer Cells

Introduction: Cancer stem cells (CSCs) drive the initiation, maintenance, and therapy response of breast tumors. CD49f is expressed in breast CSCs and functions in the maintenance of stemness. Thus, blockade of CD49f is a potential therapeutic approach for targeting breast CSCs. In the present study, we aimed to repurpose drugs as CD49f antagonists. Materials and Methods: We performed consensus molecular docking using a subdomain of CD49f that is critical for heterodimerization and a collection of pharmochemicals clini-cally tested. Molecular dynamics simulations were employed to further characterize drug-target binding. Using MDA-MB-231 cells, we evaluated the effects of potential CD49f antagonists on 1) cell adhesion to laminin; 2) mammosphere formation; and 3) cell viability. We analyzed the effects of the drug with better CSC-selectivity on the activation of CD49f-downstream signaling by Western blot (WB) and co-immunoprecipitation. Expressions of the stem cell markers CD44 and SOX2 were analyzed by flow cytometry and WB, respectively. Transactivation of SOX2 promoter was evaluated by luciferase reporter assays. Changes in the number of CSCs were assessed by limiting-dilution xenotransplantation. Results: Pranlukast, a drug used to treat asthma, bound to CD49f in silico and inhibited the adhesion of CD49f+ MDA-MB-231 cells to laminin, indicating that it antagonizes CD49f-containing integrins. Molecular dynamics analysis showed that pranlukast binding induces con-formational changes in CD49f that affect its interaction with β1-integrin subunit and constrained the conformational dynamics of the heterodimer. Pranlukast decreased the clonogenicity of breast cancer cells on mammosphere formation assay but had no impact on the viability of bulk tumor cells. Brief exposure of MDA-MB-231 cells to pranlukast altered CD49f-dependent signaling, reducing focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K) activation. Further, pranlukast-treated cells showed decreased CD44 and SOX2 expression, SOX2 promoter transacti-vation, and in vivo tumorigenicity, supporting that this drug reduces the frequency of CSC. Conclusion: Our results support the function of pranlukast as a CD49f antagonist that reduces the CSC population in triple-negative breast cancer cells. The pharmacokinetics and toxicology of this drug have already been established, rendering a potential adjuvant therapy for breast cancer patients

Evaluación del efecto del extracto dializable de leucocitos (DLE), en la encefalomielitis autoinmune experimental (EAE)

Tesis (Maestría en Ciencias en Inmunología), Instituto Politécnico Nacional, SEPI, ENCB, 2009, 1 archivo PDF, (106 páginas). tesis.ipn.m

Inhibins Tune the Thymocyte Selection Process by Regulating Thymic Stromal Cell Differentiation

Inhibins and Activins are members of the TGF-β superfamily that regulate the differentiation of several cell types. These ligands were initially identified as hormones that regulate the hypothalamus-pituitary-gonadal axis; however, increasing evidence has demonstrated that they are key regulators in the immune system. We have previously demonstrated that Inhibins are the main Activin ligands expressed in the murine thymus and that they regulate thymocyte differentiation, promoting the DN3-DN4 transition and the selection of SP thymocytes. As Inhibins are mainly produced by thymic stromal cells, which also express Activin receptors and Smad proteins, we hypothesized that Inhibins might play a role in stromal cell differentiation and function. Here, we demonstrate that, in the absence of Inhibins, thymic conventional dendritic cells display reduced levels of MHC Class II (MHCII) and CD86. In addition, the ratio between cTECs and mTECs was affected, indicating that mTEC differentiation was favoured and cTEC diminished in the absence of Inhibins. These changes appeared to impact thymocyte selection leading to a decreased selection of CD4SP thymocytes and increased generation of natural regulatory T cells. These findings demonstrate that Inhibins tune the T cell selection process by regulating both thymocyte and stromal cell differentiation

A Key Role for Inhibins in Dendritic Cell Maturation and Function.

Inhibins are members of the TGFβ superfamily, which regulate many cellular processes including differentiation, proliferation, survival and apoptosis. Although initially described as hormones regulating the hypothalamus-pituitary-gonadal axis, based on their ability to antagonize Activins, our group has recently reported that they play a role in thymocyte differentiation and survival, as well as in thymic stromal cell maturation and nTreg generation. Here, we used Inhibin knock out mice (Inhα-/-) to investigate the role of Inhibins in peripheral dendritic cell maturation and function. We first demonstrated that LPS treated Inhα+/+ bone marrow derived dendritic cells (BMDC) were capable to produce significant levels of Inhibin A. Interestingly, Inhα-/- BMDC showed reduced MHCII and CD86 upregulation and increased PD-L1 expression in response to LPS compared to Inhα+/+, which correlated with reduced ability to induce proliferation of allogeneic T cells. The "semi-mature" phenotype displayed by Inhα-/- mBMDC correlated with increased levels of IL-10 and slightly decreased IL-6 production after LPS stimulation. In addition, Inhα-/- mBMDC showed impaired migration towards CCL19 and CCL21, assessed by in vitro chemotaxis and in vivo competitive homing experiments, despite their normal CCR7 expression. Furthermore, in vivo LPS-induced DC maturation was also diminished in Inhα-/- mice, specially within the LC (CD207+ CD11b+ CD103-) subpopulation. Finally, analysis of delayed type hypersensitivity responses in Inhα-/- mice, showed reduced ear swelling as a result of reduced cellular infiltration in the skin, correlating with impaired homing of CD207+ DCs to the draining lymph nodes. In summary, our data demonstrate for the first time that Inhibins play a key role in peripheral DC maturation and function, regulating the balance between immunity and tolerance

Neural Crest Cells Isolated from the Bone Marrow of Transgenic Mice Express JCV T-Antigen.

JC virus (JCV), a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML). In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs) isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when cultured under neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro. These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow-derived stem cells in the pathogenesis of JCV-related diseases and the opportunities for the use of this model in development of therapeutic strategies

Inhα^-/- mice exhibit impaired DTH response induced by OVA challenge.

<p>Mice were immunized with 100ng OVA + 10μg LPS in the back. After 7 days mice were challenged with 100ng of OVA in the ear. A. H&E staining of ear sections at 24h after challenge from a representative experiment is shown. B. Ear thickness (top), expressed as relative increment between OVA-ear thickness divided by PBS-ear thickness, and quantification of infiltrating cells (bottom) in challenged ears after 24h. Graphs represent mean ± SEM of 2 independent experiments <b>(n = 3).</b> C. Percentages and numbers of cDC subsets in dLN expressed as relative increment between challenged ears divided by PBS ears. Graphs represent mean ± SEM of 2 independent experiments (n <b>=</b> 3). Asterisks denote statistical significance *p≤0.05; **p≤0.01; ***p≤0.001.</p

Inhα^-/- BMDC produce increased IL-10 after LPS stimulation.

<p>A) Time course of cytokines from supernatants of BMDC cultures were quantified by ELISA (Inhibin A and Activin A) and CBA (IL-6, IL-10, TNF and CCL2). Graphs represent mean ± SEM of at least 3 independent experiments and 2 independent experiments for Inhibin A. b) Time course of IL-10 and IL-6 production by Inhα^+/+ (left) and Inhα^-/- (right) BMDCs in the presence of exogenous recombinant Inhibin A (150pg/ml) at 0h, 6h, 12h and 18h post stimulation with LPS. Data represent the mean ± SEM of 3 independent experiments (n = 3 mice). Statistical significance was determined by using a two-tailed unpaired Student t-Test *p≤0.05; **p≤0.01; ***p≤0.001.</p

LPS-stimulated Inhα^-/- BMDC have impaired migration towards CCL19 and CCL21.

<p>A. Chemotaxis towards CCL19 and CCL21 were performed with Inhα^+/+ and Inhα^-/- unstimulated or 24h LPS-stimulated BMDCs. Graphs show the chemotaxis index expressed as mean ± SEM (n = 4 independent experiments). B. Mix of Inhα^+/+ (GFP⁺) and Inhα^-/- (CTV⁺) BMDC (1:1) were inoculated in a C57BL/6 footpad and 48 hours later dLN were analyzed for the presence of BMDC. Graph represents the ratio of the mix inoculated and the ratio obtained in the dLN (mean ± SEM of 2 independent experiments; n = 5). C. CCR7 surface expression in BMDC. MFI was determined and reported as relative expression compared to Inhα^+/+ mice. Bar graphs show mean ± SEM of 3 independent experiments. D) Calcium flux analysis of Inhα^+/+ and Inhα^-/- mBMDC stimulated with 300ng/ml of CCL19 and CCL21. E) Time course of Erk phosphorylation in response to CCR7 ligands in Inhα^+/+ and Inhα^-/- mBMDC analyzed by flow cytometry. MFI was determined and reported as relative expression compared to unstimulated cells. Statistical significance was determined by a two-tailed unpaired Student t-Test. *p≤0.05; **p≤0.01.</p

LPS-induced DC maturation <i>in vivo</i> is impaired in Inhα^-/- mice.

<p>Inhα^+/+ and Inhα^-/- 3-week-old mice were inoculated intradermal with LPS (1μg) in left ear and PBS in right ear as control. After 6, 18 and 72 h, dLN and ear epidermal layers were obtained to evaluate DC maturation. A. Time course of MHCII, CD80, CD86 and PD-L1 expression in cDCs (CD11c⁺ MHCII⁺). B. Confocal microscopy micrographs of epidermal sheets stained for MHCII (green) and CD80 (red). C. MHCII and CD80 relative expression in LC from epidermal sheets. Graphs represent relative increment of LPS-ear versus PBS-ear mean ± SEM of 5 independent experiments. Statistical significance was determined by using a two tailed unpaired Student t-Test *p≤0.05; **p≤0.01; ***p≤0.001.</p

BMDC Inhα^-/- showed poor allogeneic CD4⁺ CD25^- Foxp3^- T cells stimulation capacity.

<p><b>Unstimulated and 24h LPS-stimulated</b> CD11c⁺ cells were sorted at day 6 of culture and were co-cultivated with CD4⁺ CD25^- Foxp3^- T cells from Foxp3-GFP KI Balb/c. At day 5, proliferation of T cells was measured by CTV dilution. A. Representative dot plot of CTV dilution from Inhα^+/+ BMDC (left) and Inhα^-/- BMDC (right). B. Graphs of divided T cells at 1:10 ratio represent mean ± SEM of 4 independent experiments. Statistical significance was determined by a two-tailed unpaired Student t-Test. *p≤0.05.</p