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NF639 cells original population vs EGCG-resistant population

ABSTRACT: Overexpression of the epidermal growth factor receptor family member Her-2/neu in breast cancer leads to autophosphorylation of the receptor, and induction of multiple downstream signaling pathways including Akt kinase to NF-kappaB cascade that is associated with poor prognosis. Previously, we demonstrated green tea polyhenol epigallocatechin 3-gallate (EGCG) inhibits growth of NF639 Her-2/neu-driven breast cancer cells via reducing receptor autophosphorylation, and downstream Akt and NF-kappaB activities (Pianetti et al., 2002). Interestingly, we noted that upon prolonged culture in the presence of EGCG some cells developed resistance to the polyphenol. Here we report that resistant cells have lost tyrosine phosphorylation on the Her-2/neu receptor. Surprisingly, they displayed elevated NF-kappaB activity, and inhibition of this activity sensitized cells to EGCG. Data from microarray analysis of the original and resistant NF639 populations of cells were subjected to Gene Set Enrichment Analysis (GSEA) pathway analysis, which revealed that the mitogen activated protein kinase (MAPK) pathway was activated in the resistant cells. Treatment of the resistant cells with a combination of EGCG and the MAPK inhibitor U0216 dramatically reduced colony growth and mesenchymal phenotype. Thus, activation of the MAPK pathway mediates resistance to EGCG. Our studies suggest that gene expression profiling of drug resistant cells may provide a mechanism of determining effective systemic therapies for treatment of these advanced cancers. Keywords: Drug resistance

ORGANISM(S): Mus musculus

PROVIDER: GSE4140 | GEO | 2006/06/01

SECONDARY ACCESSION(S): PRJNA94987

REPOSITORIES: GEO

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Project description:Overexpression of the epidermal growth factor receptor family member Her-2/neu in breast cancer leads to autophosphorylation of the receptor, and induction of multiple downstream signaling pathways including Akt kinase to NF-ï«B cascade that is associated with poor prognosis. Previously, we demonstrated green tea polyhenol epigallocatechin 3-gallate (EGCG) inhibits growth of NF639 Her-2/neu-driven breast cancer cells via reducing receptor autophosphorylation, and downstream Akt and NF-ï«B activities (Pianetti et al., 2002). Interestingly, we noted that upon prolonged culture in the presence of EGCG some cells developed resistance to the polyphenol. Here we report that resistant cells have lost tyrosine phosphorylation on the Her-2/neu receptor. Surprisingly, they displayed elevated NF-ï«B activity, and inhibition of this activity sensitized cells to EGCG. Data from microarray analysis of the original and resistant NF639 populations of cells were subjected to Gene Set Enrichment Analysis (GSEA) pathway analysis, which revealed that the mitogen activated protein kinase (MAPK) pathway was activated in the resistant cells. Treatment of the resistant cells with a combination of EGCG and the MAPK inhibitor U0216 dramatically reduced colony growth and mesenchymal phenotype. Thus, activation of the MAPK pathway mediates resistance to EGCG. Our studies suggest that gene expression profiling of drug resistant cells may provide a mechanism of determining effective systemic therapies for treatment of these advanced cancers. Experiment Overall Design: Total RNA from original NF639 cells and EGCG resistant NF639 cells were harvested using the UltraspecII RNA isolation kit (Biotecx), following the manufacturerâs instruction. The RNA samples were submitted to Boston University Microarray Resource for microarray hybridization using Affymetrix Mouse 430A 2.0 chips. RNAs from two independent experiments were analyzed and data pooled for computational analysis.

Project description:Classical Hodgkin lymphoma (cHL) is one of the most common malignant lymphomas. It is characterized by the presence of rare Hodgkin and Reed/Sternberg (HRS) cells embedded in an extensive inflammatory infiltrate. Constitutive activation of nuclear factor-kappaB (NF-kappaB) in HRS cells which transcriptionally regulates expression of multiple anti-apoptotic factors and pro-inflammatory cytokines plays a central role in the pathogenesis of cHL (1, 2). In non-stimulated condition, NF-kappaB proteins are rendered inactive by binding to their inhibitors (IkappaB s), which sequester them in the cytoplasm. Stimulation of multiple receptors activates the IkappaB kinase (IKK) complex that phosphorylates IkappaB at two specific serine residues, followed by its ubiquitination and proteasomal degradation, thereby releasing NF-kappaB proteins and allowing their nuclear translocation (3). Recently, two studies provided further insights into the molecular mechanisms of IKK activation upon TNF stimulation (4, 5). Activation of the IKK complex and subsequent NF-kappaB activation requires Lys63 polyubiquitination of RIP1, a kinase which is recruited to the receptor upon TNF stimulation. IKK-ï§ï (NEMO), the regulatory subunit of the IKK complex, specifically recognizes these Lys63-linked polyubiquitins attached to RIP1 and thereby activates IKK and NF-kappaB (4, 5). A20 is an ubiquitin-modifying enzyme that inhibits NF-kappaB activation in succession of tumor necrosis factor (TNF) receptor and Toll-like receptor induced signals (6-8). This enzyme removes Lys63 linked ubiquitin chains from RIP1 and adds Lys48 polyubiquitins to RIP1, thereby targeting this factor for proteasomal degradation, thus explaining the molecular mechanism of NF-kappaB inhibition by A20 (6). A20 likely inhibits NF-kappaB acitivity also by additional means, including interaction with TRAF1 and TRAF2 (9). SNP 6.0 array (Affymetrix) analyses were performed according to the manufacturer's directions on DNA extracted from three Hodgkin cell lines (L1236, HDLM-2, U-HO1), HapMap samples included in the Genotyping Console Software 3.0 were used as references.

Project description:Mitochondrial DNA-depleted human skin fibroblasts (HSF rho0) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NF-kappaB and STAT3 signaling pathways and by decreased activity of the mitochondrial death pathway, compared to the parent rho+ HSF. In contrast, the extrinsic TRAIL/TRAIL-Receptor-mediated death pathway remained highly active, and exogenous TRAIL induced higher levels of apoptosis in rho0 cells compared to rho+ HSF. Global gene expression analysis using microarray and quantitative RT-PCR demonstrated that expression levels of many growth factors and their adaptor proteins (FGF13, HGF, IGFBP4, IGFBP6, IGFL2), cytokines (IL6, IL17B, IL18, IL19, IL28B) and cytokine receptors (IL1R1, IL21R, IL31RA) were substantially decreased after mitochondrial depletion. Some of these genes were targets of NF-kappaB and STAT3, and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation induced expression of several NF-kappaB/STAT3 target genes, including IL1A, IL1B, IL6, PTGS2/COX2 and MMP12, in rho+ HSF, but this response was substantially decreased in rho0 HSF. Suppression of the IKK-NF-kappaB pathway by the small molecular inhibitor BMS-345541 and of the JAK2-STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated rho+ HSF. Inhibitory antibodies against IL6, the main activator of JAK2-STAT3 pathway, added into the cell media, also increased TRAIL-induced apoptosis in rho+ HSF. However, NF-kappaB activation was partially lost in mitochondrial DNA-depleted HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-kappaB targets, further suppressing IL6-JAK2-STAT3, that in concert with NF-kappaB, regulated protection against TRAIL-induced apoptosis. There are 12 total samples, 3 biological replicates each of HSF rho+ and rho0 cells that were not irradiated (control=C) or irradiated (alpha=A). Cells were harvested at 4 hours after treatment.

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NF639 cells original population vs EGCG-resistant population

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