Sabine Müller-Brüsselbach

Compensatory PI3-kinase/Akt/mTor activation regulates imatinib resistance development.

BCR/ABL-kinase mutations frequently mediate clinical resistance to the selective tyrosine kinase inhibitor Imatinib mesylate (IM, Gleevec®). However, mechanisms that promote survival of BCR/ABL-positive cells before clinically overt IM resistance occurs have poorly been defined so far. Here, we demonstrate that IM-treatment activated the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTor)-pathway in BCR/ABL-positive LAMA-cells and primary leukemia cells in vitro, as well as in a chronic phase CML patient in vivo. In fact, PI3K/Akt-activation critically mediated survival during the early phase of IM resistance development before manifestation of BCR/ABL-dependent strong IM resistance such as through a kinase mutation. Accordingly, inhibition of IM-induced Akt activation using mTor inhibitors and Akt-specific siRNA effectively antagonized development of incipient IM-resistance in vitro. In contrast, IM-resistant chronic myeloid leukemia (CML) patients with BCR/ABL kinase mutations (n=15), and IM-refractory BCR/ABL-positive acute lymphatic leukemia patients (n=2) displayed inconsistent and kinase mutation-independent autonomous patterns of Akt-pathway activation, and mTor-inhibition overcame IM resistance only if Akt was strongly activated. Together, an IM-induced compensatory Akt/mTor activation may represent a novel mechanism for the persistence of BCR/ABL-positive cells in IM-treated patients. Treatment with mTor inhibitors may thus be particularly effective in IM-sensitive patients, whereas Akt-pathway activation variably contributes to clinically overt IM resistance.

Deregulation of tumor angiogenesis and blockade of tumor growth in PPARβ-deficient mice

The peroxisome proliferator‐activated receptor‐β (PPARβ) has been implicated in tumorigenesis, but its precise role remains unclear. Here, we show that the growth of syngeneic Pparb wild‐type tumors is impaired in Pparb−/− mice, concomitant with a diminished blood flow and an abundance of hyperplastic microvascular structures. Matrigel plugs containing pro‐angiogenic growth factors harbor increased numbers of morphologically immature, proliferating endothelial cells in Pparb−/− mice, and retroviral transduction of Pparb triggers microvessel maturation. We have identified the Cdkn1c gene encoding the cell cycle inhibitor p57Kip2 as a PPARβ target gene and a mediator of the PPARβ‐mediated inhibition of cell proliferation, which provides a possible mechanistic explanation for the observed tumor endothelial hyperplasia and deregulation of tumor angiogenesis in Pparb−/− mice. Our data point to an unexpected essential role for PPARβ in constraining tumor endothelial cell proliferation to allow for the formation of functional tumor microvessels.

Mixed‐polarization phenotype of ascites‐associated macrophages in human ovarian carcinoma: Correlation of CD163 expression, cytokine levels and early relapse

Ovarian cancer is typically accompanied by the occurrence of malignant ascites containing large number of macrophages. It has been suggested that these tumor‐associated macrophages (TAMs) are skewed to alternative polarization (M2) and thereby play an essential role in therapy resistance and metastatic spread. In our study, we have investigated the nature, regulation and clinical correlations of TAM polarization in serous ovarian cancer. Macrophage polarization markers on TAMs and ascites cytokine levels were analyzed for 30 patients and associated with relapse‐free survival (RFS) in a prospective study with 20 evaluable patients. Surface expression of the M2 marker CD163 on TAMs was inversely associated with RFS (p < 0.01). However, global gene expression profiles determined for 17 of these patients revealed a mixed‐polarization phenotype unrelated to the M1/M2 classification. CD163 surface expression also correlated with the ascites levels of IL‐6 and IL‐10 (p < 0.05), both cytokines induced CD163 expression, and their ascites levels showed a clear inverse association with RFS (p < 0.01). These findings define a subgroup of patients with high CD163 expression, high IL‐6 and/or IL‐10 levels and poor clinical outcome.

Genomewide Analyses Define Different Modes of Transcriptional Regulation by Peroxisome Proliferator-Activated Receptor-β/δ (PPARβ/δ)

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors with essential functions in lipid, glucose and energy homeostasis, cell differentiation, inflammation and metabolic disorders, and represent important drug targets. PPARs heterodimerize with retinoid X receptors (RXRs) and can form transcriptional activator or repressor complexes at specific DNA elements (PPREs). It is believed that the decision between repression and activation is generally governed by a ligand-mediated switch. We have performed genomewide analyses of agonist-treated and PPARβ/δ-depleted human myofibroblasts to test this hypothesis and to identify global principles of PPARβ/δ-mediated gene regulation. Chromatin immunoprecipitation sequencing (ChIP-Seq) of PPARβ/δ, H3K4me3 and RNA polymerase II enrichment sites combined with transcriptional profiling enabled the definition of 112 bona fide PPARβ/δ target genes showing either of three distinct types of transcriptional response: (I) ligand-independent repression by PPARβ/δ; (II) ligand-induced activation and/or derepression by PPARβ/δ; and (III) ligand-independent activation by PPARβ/δ. These data identify PPRE-mediated repression as a major mechanism of transcriptional regulation by PPARβ/δ, but, unexpectedly, also show that only a subset of repressed genes are activated by a ligand-mediated switch. Our results also suggest that the type of transcriptional response by a given target gene is connected to the structure of its associated PPRE(s) and the biological function of its encoded protein. These observations have important implications for understanding the regulatory PPAR network and PPARβ/δ ligand-based drugs.

15-Hydroxyeicosatetraenoic Acid Is a Preferential Peroxisome Proliferator-Activated Receptor β/δ Agonist

Peroxisome proliferator-activated receptor (PPARs) modulate target gene expression in response to unsaturated fatty acid ligands, such as arachidonic acid (AA). Here, we report that the AA metabolite 15-hydroxyeicosatetraenoic acid (15-HETE) activates the ligand-dependent activation domain (AF2) of PPARβ/δ in vivo, competes with synthetic agonists in a PPARβ/δ ligand binding assay in vitro, and triggers the interaction of PPARβ/δ with coactivator peptides. These agonistic effects were also seen with PPARα and PPARγ, but to a significantly weaker extent. We further show that 15-HETE strongly induces the expression of the bona fide PPAR target gene Angptl4 in a PPARβ/δ-dependent manner and, conversely, that inhibition of 15-HETE synthesis reduces PPARβ/δ transcriptional activity. Consistent with its function as an agonistic ligand, 15-HETE triggers profound changes in chromatin-associated PPARβ/δ complexes in vivo, including the recruitment of the coactivator cAMP response element-binding protein binding protein. Both 15R-HETE and 15S-HETE are similarly potent at inducing PPARβ/δ coactivator binding and transcriptional activation, indicating that 15-HETE enantiomers generated by different pathways function as PPARβ/δ agonists.

Ligand-Mediated Regulation of Peroxisome Proliferator-Activated Receptor (PPAR) β/δ: A Comparative Analysis of PPAR-Selective Agonists and All-trans Retinoic Acid

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that modulate target gene expression in response to natural fatty acid ligands and synthetic agonists. It is noteworthy that all trans-retinoic acid (atRA) has recently been reported to act as a ligand for PPARβ/δ, to activate its transcriptional activity, and, in contrast to the “classic” function of atRA, to stimulate cell proliferation (Schug et al., 2007). Here, we report that in contrast to synthetic PPARβ/δ agonists, atRA failed to induce the transcriptional activity of PPARβ/δ using different types of reporter gene assays. Likewise, atRA did not affect the expression of the bona fide PPARβ/δ target genes ADRP and ANGPTL4 but strongly increased expression of the retinoic acid target gene CYP26A under the identical experimental conditions. Consistent with these observations, atRA did not compete with established PPARβ/δ agonists in a ligand binding assay, and atRA did not enable the interaction of PPARβ/δ with a coactivator peptide in a time-resolved fluorescence resonance energy transfer assay in vitro. These results are in sharp contrast to the effect of established PPARβ/δ agonists in both in vitro assays. Taken as a whole, these data strongly suggest that atRA does not function as a ligand of PPARβ/δ in any of the experimental systems tested and that the previously reported atRA effects are more likely to reflect an uncharacterized and less direct mechanism.

Transcriptional Profiling Identifies Functional Interactions of TGFβ and PPARβ/δ Signaling SYNERGISTIC INDUCTION OF ANGPTL4 TRANSCRIPTION

Peroxisome proliferator-activated receptors (PPARs) not only play a key role in regulating metabolic pathways but also modulate inflammatory processes, pointing to a functional interaction between PPAR and cytokine signaling pathways. In this study, we show by genome-wide transcriptional profiling that PPARβ/δ and transforming growth factor-β (TGFβ) pathways functionally interact in human myofibroblasts and that a subset of these genes is cooperatively activated by TGFβ and PPARβ/δ. Using the angiopoietin-like 4 (ANGPTL4) gene as a model, we demonstrate that two enhancer regions cooperate to mediate the observed synergistic response. A TGFβ-responsive enhancer located ∼8 kb upstream of the transcriptional start site is regulated by a mechanism involving SMAD3, ETS1, RUNX, and AP-1 transcription factors that interact with multiple contiguous binding sites. A second enhancer (PPAR-E) consisting of three juxtaposed PPAR response elements is located in the third intron ∼3.5 kb downstream of the transcriptional start site. The PPAR-E is strongly activated by all three PPAR subtypes, with a novel type of PPAR response element motif playing a central role. Although the PPAR-E is not regulated by TGFβ, it interacts with SMAD3, ETS1, RUNX2, and AP-1 in vivo, providing a possible mechanistic explanation for the observed synergism.

Reverse crosstalk of TGFβ and PPARβ/δ signaling identified by transcriptional profiling

Previous work has provided strong evidence for a role of peroxisome proliferator-activated receptor β/δ (PPARβ/δ) and transforming growth factor-β (TGFβ) in inflammation and tumor stroma function, raising the possibility that both signaling pathways are interconnected. We have addressed this hypothesis by microarray analyses of human diploid fibroblasts induced to myofibroblastic differentiation, which revealed a substantial, mostly reverse crosstalk of both pathways and identified distinct classes of genes. A major class encompasses classical PPAR target genes, including ANGPTL4, CPT1A, ADRP and PDK4. These genes are repressed by TGFβ, which is counteracted by PPARβ/δ activation. This is mediated, at least in part, by the TGFβ-induced recruitment of the corepressor SMRT to PPAR response elements, and its release by PPARβ/δ ligands, indicating that TGFβ and PPARβ/δ signals are integrated by chromatin-associated complexes. A second class represents TGFβ-induced genes that are downregulated by PPARβ/δ agonists, exemplified by CD274 and IL6, which is consistent with the anti-inflammatory properties of PPARβ/δ ligands. Finally, cooperative regulation by both ligands was observed for a minor group of genes, including several regulators of cell proliferation. These observations indicate that PPARβ/δ is able to influence the expression of distinct sets of both TGFβ-repressed and TGFβ-activated genes in both directions.

Induction of PPARβ and prostacyclin (PGI2) synthesis by Raf signaling : failure of PGI2 to activate PPARβ

A role for the nuclear receptor peroxisome proliferator‐activated receptor‐β (PPARβ) in oncogenesis has been suggested by a number of observations but its precise role remains elusive. Prostaglandin I2 (PGI2, prostacyclin), a major arachidonic acid (AA) derived cyclooxygenase (Cox) product, has been proposed as a PPARβ agonist. Here, we show that the 4‐hydroxytamoxifen (4‐OHT) mediated activation of a C‐Raf‐estrogen receptor fusion protein leads to the induction of both the PPARβ and Cox‐2 genes, concomitant with a dramatic increase in PGI2 synthesis. Surprisingly, however, 4‐OHT failed to activate PPARβ transcriptional activity, indicating that PGI2 is insufficient for PPARβ activation. In agreement with this conclusion, the overexpression of ectopic Cox‐2 and PGI2 synthase (PGIS) resulted in massive PGI2 synthesis but did not activate the transcriptional activity of PPARβ. Conversely, inhibition of PGIS blocked PGI2 synthesis but did not affect the AA mediated activation of PPARβ. Our data obtained with four different cell types and different experimental strategies do not support the prevailing opinion that PGI2 plays a significant role in the regulation of PPARβ.

(Z)-2-(2-bromophenyl)-3-{[4-(1-methyl-piperazine)amino]phenyl}acrylonitrile (DG172): an orally bioavailable PPARβ/δ-selective ligand with inverse agonistic properties.

The ligand-regulated nuclear receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a potential pharmacological target due to its role in disease-related biological processes. We used TR-FRET-based competitive ligand binding and coregulator interaction assays to screen 2693 compounds of the Open Chemical Repository of the NCI/NIH Developmental Therapeutics Program for inhibitory PPARβ/δ ligands. One compound, (Z)-3-(4-dimethylamino-phenyl)-2-phenyl-acrylonitrile, was used for a systematic SAR study. This led to the design of derivative 37, (Z)-2-(2-bromophenyl)-3-{[4-(1-methyl-piperazine)amino]phenyl}acrylonitrile (DG172), a novel PPARβ/δ-selective ligand showing high binding affinity (IC(50) = 27 nM) and potent inverse agonistic properties. 37 selectively inhibited the agonist-induced activity of PPARβ/δ, enhanced transcriptional corepressor recruitment, and down-regulated transcription of the PPARβ/δ target gene Angptl4 in mouse myoblasts (IC(50) = 9.5 nM). Importantly, 37 was bioavailable after oral application to mice with peak plasma levels in the concentration range of its maximal inhibitory potency, suggesting that 37 will be an invaluable tool to elucidate the functions and therapeutic potential of PPARβ/δ.