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

Apoptotic Signaling Pathways in Glioblastoma and Therapeutic Implications

Glioblastoma multiforme (GBM) is the most hostile type of brain cancer. Its aggressiveness is due to increased invasion, migration, proliferation, angiogenesis, and a decreased apoptosis. In this review, we discuss the role of key regulators of apoptosis in GBM and glioblastoma stem cells. Given their importance in the etiology and pathogenesis of GBM, these signaling molecules may represent potential therapeutic targets

Immunological and Functional Characterization of RhoGDI3 and Its Molecular Targets RhoG and RhoB in Human Pancreatic Cancerous and Normal Cells

<div><p>RhoGDI proteins have been implicated in several human cancers; changes in their expression levels have shown pro- or anti-tumorigenic effects. Pancreatic Ductal Adenocarcinoma (PDAC) is a complex pathology, with poor prognosis, and most patients die shortly after diagnosis. Efforts have been focused on understanding the role of RhoGDI's in PDAC, specially, RhoGDI1 and RhoGDI2. However, the role of RhoGDI3 has not been studied in relation to cancer or to PDAC. Here, we characterized the expression and functionality of RhoGDI3 and its target GTPases, RhoG and RhoB in pancreatic cell lines from both normal pancreatic tissue and tissue in late stages of PDAC, and compared them to human biopsies. Through immunofluorescences, pulldown assays and subcellular fractionation, we found a reduction in RhoGDI3 expression in the late stages of PDAC, and this reduction correlates with tumor progression and aggressiveness. Despite the reduction in the expression of RhoGDI3 in PDAC, we found that RhoB was underexpressed while RhoG was overexpressed, suggesting that cancerous cells preserve their capacity to activate this pathway, thus these cells may be more eager to response to the stimuli needed to proliferate and become invasive unlike normal cells. Surprisingly, we found nuclear localization of RhoGDI3 in non-cancerous pancreatic cell line and normal pancreatic tissue biopsies, which could open the possibility of novel nuclear functions for this protein, impacting gene expression regulation and cellular homeostasis.</p></div

Cancerous and non-cancerous pancreatic cell lines show different expression patterns of RhoGDI3 protein.

<p>(A) Immunofluorescence microscopy analysis of RhoGDI3 protein (green); 58 kDa protein, Golgi apparatus marker (Red) and Nuclei (DAPI, blue) was performed on hTERT-HPNE (upper panel), BxPC3 (middle panel) and PANC-1 (bottom panel) cells lines. (B) Representative Immunoblot using antibodies anti-RhoGDI3, anti-RhoGDI2 and anti-GAPDH were used as loading control. Total lysates from hTERT-HPNE, BxPC3 and PANC-1 cell lines were analyzed. (C) Densitometric analysis of the bands detected in the Western blots of RhoGDI3 (n = 3) of protein extracts from all three cell lines, the data was normalized to GAPDH. Densitometric analysis was determined with Image Lab software. Values are means ± SEM, **P<0.005 (Anova-test). Scale bar 20μm.</p

Nuclear localization of RhoGDI3 in rhEGF treated hTERT-HPNE cells.

<p>Subcellular fractionation was performed after cells were treated with rhEGF (marked above the images as 0, 2 and 10 rhEGF Min). Nuclear (N) and cytosolic (C) fractions from hTERT-HPNE (A), BxPC3 (B) and PANC-1 (C) cell lines were obtained and analyzed by immunoblotting, using anti-RhoGDI3, anti-RhoG, anti-RhoB antibodies. Anti-histone H3 antibody was used as a nuclear control and anti-Aldolase B antibody was used as a cytosol control. 20 μg of cell lysates were loaded.</p

The expression of RhoG and RhoB proteins is altered in cancerous pancreatic cell lines.

<p>Immunofluorescence microscopy analysis of RhoGDI3 protein (green); RhoG (A) and RhoB (B) (Red) and Nuclei (DAPI, blue) of hTERT-HPNE (upper panel), BxPC3 (middle panel) and PANC-1 (bottom panel) cells lines. Representative Immunoblot using antibodies anti-RhoG (C), anti RhoB (D), GAPDH was used as loading control. Total lysates from hTERT-HPNE, BxPC3 and PANC-1 cell lines were analyzed. Total amount of RhoG (E) and RhoB (F) proteins was normalized to GAPDH (n = 3). Immunoblot densitometric analysis was performed with Image Lab software. Values are means ± SEM, **P<0.005, *P<0.005 (Anova-test). Scale bar 10μm.</p

Nuclear localization of RhoGDI3 in normal pancreatic tissue.

<p>Immunofluorescence microscopy staining of RhoGDI3 (green) and RhoG (red) was carried out on human pancreatic normal (A) and moderate (aggressiveness) PDAC biopsies (C). (B) Magnification and lateral view of the immunofluorescence of RhoGDI3 and RhoG, to evidence nuclear localization in human pancreatic normal tissue. Arrowheads denote the localization of RhoGDI3 and RhoG into the nuclei. Scale bar 10 = μm.</p

Activation of RhoG GTPase in hTERT-HPNE, BxPC3 and PANC-1 pancreatic cell lines.

<p>Cells were starved 6 hours and confronted with rhEGF for the period of 0, 2 and 10 minutes (Marked as 0, 2 and 10). Fluorescence microscopic staining of RhoG (green) was carried out in hTERT-HPNE (A), BxPC3 (B) and PANC-1 (C) cells lines. To show the cytoskeleton reorganization, F-Actin was stained with rhodamine phalloidin. Measurement of RhoG activity was performed using RhoG pulldown assay. Immunoblots for RhoG, Rac-1 and GAPDH proteins for hTERT-HPNE (D), BxPC3 (E) and PANC-1 (F) are shown. To quantify the amount of RhoG-GTP and bound-Rac-1 through the temporal course, densitometric analysis was performed using Image Lab software, hTERT-HPNE (G), BxPC3 (H) and PANC-1 (I). For comparison of RhoG activity, the total amount of RhoG in cell lysates was normalized to total RhoG. GAPDH was used as a protein loading control. ELMO1-GST beads coomassie are shown as beads loading control. Arrowheads denote the localization of RhoG into the peripheral membrane; boxes with number represent the number of cells with this phenotype. Scale bar 100 μm.</p

GTPase RhoB shows differential activation in PDAC cell lines.

<p>Cells were starved for 6 hours and treated with rhEGF for a period of 0, 2 and 10 minutes (Marked as 0, 2 and 10 min). An immunofluorescence microscopy analysis of RhoB (green) was carried out on hTERT-HPNE (A), BxPC3 (B) and PANC-1 (C) cells lines. To show the cytoskeleton reorganization, F-Actin was stained with rhodamine phalloidin. Measurement of RhoB activity was performed using RhoB pulldown assay. Immunoblots for RhoB and GAPDH, as loading control for hTERT-HPNE (D), BxPC3 (E) and PANC-1 (F) cell lines are shown. To quantify the amount of RhoB-GTP, densitometric analysis (n = 3) was performed using Image Lab software for samples of hTERT-HPNE (G), BxPC3 (H) and PANC-1 (I) cell lines. For comparison of RhoB activity, GTP-RhoB was normalized to total RhoB. GAPDH was used as a protein loading control. Coomassie of RBD-GST beads are shown as beads loading control. Arrowheads denote the localization of RhoB into the peripheral membrane; boxes with number represent the quantity of cells per field with this phenotype. Scale bar = 100 μm.</p

TYK2 Variants in B-Acute Lymphoblastic Leukaemia

B-cell precursor acute lymphoblastic leukaemia (B-ALL) is a malignancy of lymphoid progenitor cells with altered genes including the Janus kinase (JAK) gene family. Among them, tyrosine kinase 2 (TYK2) is involved in signal transduction of cytokines such as interferon (IFN) α/β through IFN−α/β receptor alpha chain (IFNAR1). To search for disease-associated TYK2 variants, bone marrow samples from 62 B-ALL patients at diagnosis were analysed by next-generation sequencing. TYK2 variants were found in 16 patients (25.8%): one patient had a novel mutation at the four-point-one, ezrin, radixin, moesin (FERM) domain (S431G) and two patients had the rare variants rs150601734 or rs55882956 (R425H or R832W). To functionally characterise them, they were generated by direct mutagenesis, cloned in expression vectors, and transfected in TYK2-deficient cells. Under high-IFNα doses, the three variants were competent to phosphorylate STAT1/2. While R425H and R832W induced STAT1/2-target genes measured by qPCR, S431G behaved as the kinase-dead form of the protein. None of these variants phosphorylated STAT3 in in vitro kinase assays. Molecular dynamics simulation showed that TYK2/IFNAR1 interaction is not affected by these variants. Finally, qPCR analysis revealed diminished expression of TYK2 in B-ALL patients at diagnosis compared to that in healthy donors, further stressing the tumour immune surveillance role of TYK