sulindac and Brain-Neoplasms

Tumor cells acquire metastasis-associated (MA) phenotypes following genetic alterations in them which cause deregulation of different signaling pathways. Earlier, we reported that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the genetic salient features of triple-negative breast cancer (TNBC), and WP signaling is associated with metastasis in TNBC. Using cBioPortal, here we found that collective % of alteration(s) in WP genes, CTNNB1, APC and DVL1 among breast-invasive-carcinomas was 21% as compared to 56% in PAM50 Basal. To understand the functional relevance of WP in the biology of heterogeneous/metastasizing TNBC cells, we undertook this comprehensive study using 15 cell lines in which we examined the role of WP in the context of integrin-dependent MA-phenotypes. Directional movement of tumor cells was observed by confocal immunofluorescence microscopy and quantitative confocal-video-microscopy while matrigel-invasion was studied by MMP7-specific casein-zymography. WntC59, XAV939, sulindac sulfide and beta-catenin siRNA (1) inhibited fibronectin-directed migration, (2) decreased podia-parameters and motility-descriptors, (3) altered filamentous-actin, (4) decreased matrigel-invasion and (5) inhibited cell proliferation as well as 3D clonogenic growth. Sulindac sulfide and beta-catenin siRNA decreased beta-catenin/active-beta-catenin and MMP7. LWnt3ACM-stimulated proliferation, clonogenicity, fibronectin-directed migration and matrigel-invasion were perturbed by WP-modulators, sulindac sulfide and GDC-0941. We studied a direct involvement of WP in metastasis by stimulating brain-metastasis-specific MDA-MB231BR cells to demonstrate that LWnt3ACM-stimulated proliferation, clonogenicity and migration were blocked following sulindac sulfide, GDC-0941 and beta-catenin knockdown. We present the first evidence showing a direct functional relationship between WP activation and integrin-dependent MA-phenotypes. By proving the functional relationship between WP activation and MA-phenotypes, our data mechanistically explains (1) why different components of WP are upregulated in TNBC, (2) how WP activation is associated with metastasis and (3) how integrin-dependent MA-phenotypes can be regulated by mitigating the WP.

Topics: Adenomatous Polyposis Coli Protein; beta Catenin; Brain Neoplasms; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dishevelled Proteins; Female; Fibronectins; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 3-Ring; Humans; Indazoles; Integrins; Matrix Metalloproteinase 7; Microscopy, Confocal; Microscopy, Fluorescence; Microscopy, Video; Mutation; Phenotype; RNA Interference; RNA, Small Interfering; Sulfonamides; Sulindac; Triple Negative Breast Neoplasms; Up-Regulation; Wnt Proteins; Wnt Signaling Pathway

Nonsteroidal anti-inflammatory drug-activated gene, NAG-1, a transforming growth factor-β member, is involved in tumor progression and development. The association between NAG-1 expression and development and progression of glioma has not been well defined. Glioblastoma cell lines have lower basal expression of NAG-1 than other gliomas and normal astrocytes. Most primary human gliomas have very low levels of NAG-1 expression. NAG-1 basal expression appeared to inversely correlate with tumor grade in glioma. Aberrant promoter hypermethylation is a common mechanism for silencing of tumor suppressor genes in cancer cells. In glioblastoma cell lines, NAG-1 expression was increased by the demethylating agent, 5-aza-2'-deoxycytidine. To investigate whether the NAG-1 gene was silenced by hypermethylation in glioblastoma, we examined DNA methylation status using genomic bisulfite sequencing. The NAG-1 promoter was densely methylated in several glioblastoma cell lines as well as in primary oligodendroglioma tumor samples, which have low basal expression of NAG-1. DNA methylation at two specific sites (-53 and +55 CpG sites) in the NAG-1 promoter was strongly associated with low NAG-1 expression. The methylation of the NAG-1 promoter at the -53 site blocks Egr-1 binding and thereby suppresses Nag-1 induction. Treatment of cells with low basal NAG-1 expression with NAG-1 inducer also did not increase NAG-1. Incubation with a demethylation chemical increased Nag-1 basal expression and subsequent incubation with a NAG-1 inducer increased NAG-1 expression. We concluded from these data that methylation of specific promoter sequences causes transcriptional silencing of the NAG-1 locus in glioma and may ultimately contribute to tumor progression.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Azacitidine; Brain Neoplasms; Cell Growth Processes; Cell Line, Tumor; Decitabine; DNA Methylation; Early Growth Response Protein 1; Gene Expression Regulation, Neoplastic; Gene Silencing; Glioblastoma; Growth Differentiation Factor 15; Humans; Hydroxamic Acids; Promoter Regions, Genetic; Sulindac; Transfection

EP4 expression in human glioblastoma cells correlates with growth on soft agar. The cyclooxygenase inhibitor sulindac sulfide first altered specificity protein-1 (Sp-1) and early growth response gene-1 expression, then increased the expression of nonsteroidal anti-inflammatory drug-activated gene 1 and activating transcription factor 3, and then decreased EP4 expression. EP4 suppression was dependent on blocking the Sp-1 binding sites in the human EP4 promoter. Mutation in the Sp-1 sites in EP4 altered the promoter activity and abolished sulindac sulfide effects. The inhibitory effect of sulindac sulfide on EP4 expression was reversed by PD98059, a mitogen-activated protein/extracellular signal-regulated kinase kinase-1/extracellular signal-regulated kinase inhibitor. Sp-1 phosphorylation was dependent on sulindac sulfide-induced Erk activation. Chromatin immunoprecipitation assay confirmed that Sp-1 phosphorylation decreases Sp-1 binding to DNA and leads to the suppression of EP4. Inhibition of cell growth on soft agar assay was found to be a highly complex process and seems to require not only the inhibition of cyclooxygenase activity but also increased expression of nonsteroidal anti-inflammatory drug-activated gene 1 and activating transcription factor 3 and suppression of EP4 expression. Our data suggest that the suppression of EP4 expression by sulindac sulfide represents a new mechanism for understanding the tumor suppressor activity.

Topics: Activating Transcription Factor 3; Anti-Inflammatory Agents, Non-Steroidal; Blotting, Western; Brain Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; Chromatin Immunoprecipitation; Colony-Forming Units Assay; Cyclooxygenase Inhibitors; Early Growth Response Protein 1; Flavonoids; Glioblastoma; Humans; Immunoprecipitation; Luciferases; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Phosphorylation; Promoter Regions, Genetic; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP4 Subtype; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Sp1 Transcription Factor; Sulindac; Tumor Cells, Cultured

Exisulind (sulindac sulfone) and two potent derivatives, CP248 and CP461, have been shown previously to cause growth inhibition and apoptosis in several types of human carcinoma cell lines. These and related compounds have not been previously studied with respect to glioma cell lines. In the present study, we found that these three compounds caused marked growth inhibition in four rat glioma and eight human glioma cell lines, with IC50 values of 150, 1, and 0.075 microm, respectively. When studied at these concentrations exisulind and CP461 had no significant effect on the cell cycle profile of glioma cells, but CP248 caused marked arrest in mitosis. Detailed studies of CP248 in the 9L rat gliosarcoma cell line indicated that treatment with 0.075 microM CP248 caused abnormalities in the spindle apparatus and activation of the spindle assembly check point. In interphase glioma cells, CP248 stabilized microtubules (MTs) at low concentrations (0.075 microM) and depolymerized MTs at higher concentrations (0.2-0.4 microM). In NIH 3T3 fibroblasts, 0.1 microM CP248 caused extensive MT depolymerization. CP248 also caused MT depolymerization when added to assembled MTs in vitro, which indicated that it can directly affect MTs, perhaps because it shares certain structural similarities with Colcemid. In glioma cells, the effects of CP248 on MTs were independent of the previously reported effects of this compound on activation of protein kinase G. Therefore, CP248 is a novel MT-active agent that may be useful in the treatment of glioblastoma, and possibly other types of cancer, because of its dual effects on protein kinase G and MTs.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; 3T3 Cells; Animals; Antineoplastic Agents; Apoptosis; Brain Neoplasms; Cell Cycle Proteins; Cell Division; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 2; Cyclic Nucleotide Phosphodiesterases, Type 5; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Glioma; Humans; Immunoenzyme Techniques; In Vitro Techniques; Interphase; Kinesins; Mice; Microtubules; Phosphoproteins; Phosphoric Diester Hydrolases; Rats; Spindle Apparatus; Sulindac; Thymidine