Characterization of fibroblast growth factor receptor 2 overexpression in the human breast cancer cell line SUM-52PE

Statement of findings The fibroblast growth factor receptor (FGFR)2 gene has been shown to be amplified in 5-10% of breast cancer patients. A breast cancer cell line developed in our laboratory, SUM-52PE, was shown to have a 12-fold amplification of the FGFR2 gene, and FGFR2 message was found to be overexpressed 40-fold in SUM-52PE cells as compared with normal human mammary epithelial (HME) cells. Both human breast cancer (HBC) cell lines and HME cells expressed two FGFR2 isoforms, whereas SUM-52PE cells overexpressed those two isoforms, as well as several unique FGFR2 polypeptides. SUM-52PE cells expressed exclusively FGFR2-IIIb isoforms, which are high-affinity receptors for fibroblast growth factor (FGF)-1 and FGF-7. Differences were identified in the expression of the extracellular Ig-like domains, acid box and carboxyl termini, and several variants not previously reported were isolated from these cells.http://deepblue.lib.umich.edu/bitstream/2027.42/135727/1/13058_1999_Article_73.pd

Combination of roflumilast with a beta-2 adrenergic receptor agonist inhibits proinflammatory and profibrotic mediator release from human lung fibroblasts

<p>Abstract</p> <p>Background</p> <p>Small airway narrowing is an important pathology which impacts lung function in chronic obstructive pulmonary disease (COPD). The accumulation of fibroblasts and myofibroblasts contribute to inflammation, remodeling and fibrosis by production and release of mediators such as cytokines, profibrotic factors and extracellular matrix proteins. This study investigated the effects of the phosphodiesterase 4 inhibitor roflumilast, combined with the long acting β₂adrenergic agonist indacaterol, both approved therapeutics for COPD, on fibroblast functions that contribute to inflammation and airway fibrosis.</p> <p>Methods</p> <p>The effects of roflumilast and indacaterol treatment were characterized on transforming growth factor β1 (TGFβ1)-treated normal human lung fibroblasts (NHLF). NHLF were evaluated for expression of the profibrotic mediators endothelin-1 (ET-1) and connective tissue growth factor (CTGF), expression of the myofibroblast marker alpha smooth muscle actin, and fibronectin (FN) secretion. Tumor necrosis factor-α (TNF-α) was used to induce secretion of chemokine C-X-C motif ligand 10 (CXCL10), chemokine C-C motif ligand 5 (CCL5) and granulocyte macrophage colony-stimulating factor (GM-CSF) from NHLF and drug inhibition was assessed.</p> <p>Results</p> <p>Evaluation of roflumilast (1-10 μM) showed no significant inhibition alone on TGFβ1-induced ET-1 and CTGF mRNA transcripts, ET-1 and FN protein production, alpha smooth muscle expression, or TNF-α-induced secretion of CXCL10, CCL5 and GM-CSF. A concentration-dependent inhibition of ET-1 and CTGF was shown with indacaterol treatment, and a submaximal concentration was chosen for combination studies. When indacaterol (0.1 nM) was added to roflumilast, significant inhibition was seen on all inflammatory and fibrotic mediators evaluated, which was superior to the inhibition seen with either drug alone. Roflumilast plus indacaterol combination treatment resulted in significantly elevated phosphorylation of the transcription factor cAMP response element-binding protein (CREB), an effect that was protein kinase A-dependent. Inhibition of protein kinase A was also found to reverse the inhibition of indacaterol and roflumilast on CTGF.</p> <p>Conclusions</p> <p>These results demonstrate that addition of roflumilast to a LABA inhibits primary fibroblast/myofibroblast function and therapeutically this may impact lung fibroblast proinflammatory and profibrotic mediator release which contributes to small airway remodeling and airway obstruction in COPD.</p

GS-5759, a Bifunctional b 2 -Adrenoceptor Agonist and Phosphodiesterase 4 Inhibitor for Chronic Obstructive Pulmonary Disease with a Unique Mode of Action: Effects on Gene Expression in Human Airway Epithelial Cells s

) and propranolol, were agonist-dependent, being significantly lower for GS-5759 than b2A. Collectively, these data can be explained by &quot;forced proximity,&quot; bivalent binding where the pharmacophore in GS-5759 responsible for PDE4 inhibition also interacts with a nonallosteric domain within the b 2 -adrenoceptor that enhances the affinity of b2A for the orthosteric site. Microarray analyses revealed that, after 2-hour exposure, GS-5759 increased the expression of .3500 genes in BEAS-2B cells that were highly rank-order correlated with gene expression changes produced by indacaterol and GSK 256066 in combination (Ind/GSK). Moreover, the line of regression began close to the origin with a slope of 0.88, indicating that the magnitude of most gene expression changes produced by Ind/GSK was quantitatively replicated by GS-5759. Thus, GS-5759 is a novel compound exhibiting dual b 2 -adrenoceptor agonism and PDE4 inhibition with potential to interact on target tissues in a synergistic manner. Such polypharmacological behavior may be particularly effective in chronic obstructive pulmonary disease and other complex disorders where multiple processes interact to promote disease pathogenesis and progression

PI3Kδ inhibitor idelalisib in combination with BTK inhibitor ONO/GS-4059 in diffuse large B cell lymphoma with acquired resistance to PI3Kδ and BTK inhibitors

<div><p>Activated B-cell-like diffuse large B-cell lymphoma relies on B-cell receptor signaling to drive proliferation and survival. Downstream of the B-cell receptor, the key signaling kinases Bruton’s tyrosine kinase and phosphoinositide 3-kinase δ offer opportunities for therapeutic intervention by agents such as ibrutinib, ONO/GS-4059, and idelalisib. Combination therapy with such targeted agents could provide enhanced efficacy due to complimentary mechanisms of action. In this study, we describe both the additive interaction of and resistance mechanisms to idelalisib and ONO/GS-4059 in a model of activated B-cell-like diffuse large B-cell lymphoma. Significant tumor regression was observed with a combination of PI3Kδ and Bruton’s tyrosine kinase inhibitors in the mouse TMD8 xenograft. Acquired resistance to idelalisib in the TMD8 cell line occurred by loss of phosphatase and tensin homolog and phosphoinositide 3-kinase pathway upregulation, but not by mutation of <i>PIK3CD</i>. Sensitivity to idelalisib could be restored by combining idelalisib and ONO/GS-4059. Further evaluation of targeted inhibitors revealed that the combination of idelalisib and the phosphoinositide-dependent kinase-1 inhibitor GSK2334470 or the AKT inhibitor MK-2206 could partially overcome resistance. Characterization of acquired Bruton’s tyrosine kinase inhibitor resistance revealed a novel tumor necrosis factor alpha induced protein 3 mutation (<i>TNFAIP3</i> Q143*), which led to a loss of A20 protein, and increased p-IκBα. The combination of idelalisib and ONO/GS-4059 partially restored sensitivity in this resistant line. Additionally, a mutation in Bruton’s tyrosine kinase at C481F was identified as a mechanism of resistance. The combination activity observed with idelalisib and ONO/GS-4059, taken together with the ability to overcome resistance, could lead to a new therapeutic option in activated B-cell-like diffuse large B-cell lymphoma. A clinical trial is currently underway to evaluate the combination of idelalisib and ONO/GS-4059 (NCT02457598).</p></div

TMD8 as a model for evaluation of PI3Kδ and BTK inhibition.

<p>(A) Cell viability with isoform-specific PI3K inhibitors was assessed by CellTiter Glo after 96 hours, mean ± SEM, n = 4. (B) PI3K isoform expression in TMD8 cells using Simple Western, concentration (pg/μL) quantitated by recombinant protein standards, representative experiment shown of n = 3. (C) Cell viability of TMD8 was assessed with idelalisib in combination with ONO/GS-4059 at fixed concentrations (3, 6, or 12 nM) or with ONO/GS-4059 in combination with idelalisib at fixed concentrations (20, 100 or 600 nM) after 96 hours, mean ± SEM, n = 8. (D) Cells were treated with idelalisib (420 nM), ONO/GS-4059 (320 nM) or in combination for 48 hours and apoptosis was assessed by FITC Annexin V staining, and measured by flow cytometry, mean values ± SD, n = 4. (E) Cells were treated with idelalisib (420 nM), ONO/GS-4059 (320 nM) or in combination for 2 and 24 hours; lysates were analyzed by western blot (p-AKT S473) and Simple Western, representative experiment shown of n = 3.</p

PI3K upregulation in acquired resistance is sensitive to idelalisib and AKT or PDK1 inhibitor combinations.

<p>(A) Protein lysates were generated from TMD8^IDELA-S and TMD8^IDELA-R cells, and analyzed by western blot (p-AKT S473, p-AKT T308, AKT, p-S6RP S235/236, S6RP, p-GSK3β S9, GSK3β) and Simple Western (PTEN). (B) Cell viability of TMD8^IDELA-S (left) and TMD8^IDELA-R (right) cells treated with idelalisib, MK-2206 or combination of idelalisib and MK-2206 (1 μM), 96 hours CellTiterGlo assay, mean ± SEM, n = 4. Combination curve is normalized to the single agent alone. (C) Cell viability of TMD8^IDELA-S (left) and TMD8^IDELA-R (right) cells treated with idelalisib, GSK2334470 or combination of idelalisib and GSK2334470 (3 μM), 96 hours CellTiterGlo assay, mean ± SEM, n = 4. Combination curve is normalized to the single agent alone. (D) TMD8^IDELA-R cells were treated with vehicle, idelalisib (1 μM), MK-2206 (1 μM), and idelalisib plus MK-2206, or (E) Vehicle, idelalisib (1 μM), GSK2334470 (1 μM), and idelalisib plus GSK2334470 for 48 hours. Apoptosis was assessed by 7AAD and PE annexin V staining, and analyzed by flow cytometry, mean ± SD, n = 3. (F) TMD8^IDELA-R cells were treated with vehicle, idelalisib (1 μM), MK-2206 (1 μM), or idelalisib plus MK-2206 and (G) Vehicle, idelalisib (1 μM), GSK2334470 (1 μM), or idelalisib plus GSK2334470 for 2 hours. Protein lysates were generated and analyzed by western blot, representative experiment of n = 3. TMD8^IDELA-S vehicle control was included as a reference on the same membrane.</p

Combination of idelalisib and ONO/GS-4059 can overcome TMD8 acquired idelalisib resistance.

<p>(A) Cell viability of vehicle control line (TMD8^IDELA-S) or idelalisib resistant line (TMD8^IDELA-R) in response to idelalisib treatment, 96 hour CellTiterGlo assay, mean ± SEM, n = 4. (B) Cell viability of TMD8^IDELA-S (top graph) or TMD8^IDELA-R cells (bottom graph) treated with idelalisib, ONO/GS-4059 or ONO/GS-4059 in combination with idelalisib (1 μM), n = 4, mean ± SEM. Combination curve is normalized to the single agent alone. (C) Protein lysates from TMD8^IDELA-S (vehicle treated) and TMD8^IDELA-R treated with vehicle, idelalisib (420 nM), ONO/GS-4059 (320 nM) or in combination for 2 hours were generated and analyzed in a single run using Simple Western, representative image of a single run of n = 3. (D) Quantification of Fig 3C showing AUC normalization to actin, and values normalized to TMD8^IDELA-S DMSO treated control for all treatment groups from a single run.</p

Loss of A20 and BTK C481F mutation as mechanisms of BTK inhibitor resistance in TMD8.

<p>(A) Cell viability of BTK inhibitor (BTKi)-sensitive (TMD8^BTKi-S) and BTKi-resistant (TMD8^BTKi-R) cells to ibrutinib, 96 hour CellTiterGlo assay, mean ± SEM, n = 4. (B) Protein lysates were generated for TMD8 (DMS0 control), TMD8^A20-Q143*and TMD8^BTK-C481F cell lines, and protein expression of A20, p-IκBα, total IκBα, p-BTK and total BTK was analyzed using Simple Western. (C) Cell viability of BTKi-resistant TMD8^A20-Q143* (left) or BTKi-resistant TMD8^BTK-C481F (right) treated with ONO/GS-4059, idelalisib, or a combination of idelalisib plus ONO/GS-4059 (50 nM), mean ± SEM, n = 4. (D) Lysates of TMD8 DMSO control and TMD8^A20-Q143* lines were analyzed by western blot (p-ERK 1/2, p-AKT S473, AKT) and Simple Western (p-BTK, p-IκBα S32, IκBα). Cells were treated with idelalisib (420 nM), ONO/GS-4059 (320 nM) or in combination for 24 hours. (E) Quantification of p-ERK1/2 and p-IκBα S32 blot results from Fig 5D. Values are normalized to actin and graphed as percent of DMSO vehicle control. Simple Western and western blot images are representative experiments from n = 2–3 experiments.</p