Phosphorylation of eIF4E by MNKs supports protein synthesis, cell cycle progression and proliferation in prostate cancer cells.

Deregulation of the phosphatidyl inositol trisphosphate kinase/AKT/mammalian target of rapamycin (mTOR) and RAS/mitogen-activated protein kinase (MAPK)/MNK pathways frequently occurs in human prostate carcinomas (PCas) and leads to aberrant modulation of messenger RNA (mRNA) translation. We have investigated the relative contribution of these pathways to translational regulation and proliferation of PCa cells. MNK-dependent phosphorylation of eIF4E is elevated in DU145 cells, which have low basal levels of AKT/mTOR activity due to the expression of the tumor suppressor PTEN. In contrast, eIF4E phosphorylation is low in PC3 and LNCaP cells with mutated PTEN and constitutively active AKT/mTOR pathway, but it can be strongly induced through inhibition of mTOR activity by rapamycin or serum depletion. Remarkably, we found that inhibition of MNKs strongly reduced the polysomal recruitment of terminal oligopyrimidine messenger RNAs (TOP mRNAs), which are known targets of mTOR-dependent translational control. Pull-down assays of the eIF4F complex indicated that translation initiation was differently affected by inhibition of MNKs and mTOR. In addition, concomitant treatment with MNK inhibitor and rapamycin exerted additive effects on polysomal recruitment of TOP mRNAs and protein synthesis. The MNK inhibitor was more effective than rapamycin in blocking proliferation of PTEN-expressing cells, whereas combination of the two inhibitors suppressed cell cycle progression in both cell lines. Microarray analysis showed that MNK affected translation of mRNAs involved in cell cycle progression. Thus, our results indicate that a balance between the activity of the AKT/mTOR and the MAPK/MNK pathway in PCa cells maintains a defined translational level of specific mRNAs required for ribosome biogenesis, cell proliferation and stress response and might confer to these cells the ability to overcome negative insults.

AML-associated translocation products block vitamin D(3)-induced differentiation by sequestering the vitamin D(3) receptor.

Acute myeloid leukemia (AML)-associated chromosomal translocations result in formation of chimeric transcription factors, such as PML/RARalpha, PLZF/RARalpha, and AML-1/ETO, of which the components are involved in regulation of transcription by chromatin modeling through histone acetylation/deacetylation. The leukemic differentiation block is attributed to deregulated transcription caused by these chimeric fusion proteins, which aberrantly recruit histone-deacetylase (HDAC) activity. One essential differentiation pathway blocked by the leukemic fusion proteins is the vitamin (Vit) D(3) signaling. Here we investigated the mechanisms by which the leukemic fusion proteins interfere with VitD(3)-induced differentiation. The VitD(3)-receptor (VDR) is, like the retinoid receptors RAR, retinoid X receptor, and the thyroid hormone receptor (TR), a ligand-inducible transcription factor. In the absence of ligand, the transcriptional activity of TR and RAR is silenced by recruitment of HDAC activity through binding to corepressors. In the presence of ligand, TR and RAR activate transcription by releasing HDAC activity and by recruiting histone-acetyltransferase activity. Here we report that VDR binds corepressors in a ligand-dependent manner and that inhibition of HDAC activity increases VitD(3) sensitivity of HL-60 cells. Nevertheless, the inhibition of HDAC activity is unable to overcome the block of VitD(3)-induced differentiation caused by PLZF/RARalpha expression. Here we demonstrate that the expression of the translocation products PML/RARalpha and PLZF/RARalpha impairs the localization of VDR in the nucleus by binding to VDR. Furthermore, the overexpression of VDR in U937 cells expressing AML-related translocation products completely abolishes the block of VitD(3)-induced differentiation. Taken together these data indicate that the AML-associated translocation products block differentiation not only by interfering with chromatin-modeling but also by sequestering factors involved in the differentiation signaling pathways, such as VDR in the VitD(3)-induced differentiation.

Inhibition of interleukin-8 (CXCL8/IL-8) responses by repertaxin, a new inhibitor of the chemokine receptors CXCR1 and CXCR2.

Repertaxin is a new non-competitive allosteric blocker of interleukin-8 (CXCL8/IL-8) receptors (CXCR1/R2), which by locking CXCR1/R2 in an inactive conformation prevents receptor signaling and human polymorphonuclear leukocyte (PMN) chemotaxis. Given the unique mode of action of repertaxin it was important to examine the ability of repertaxin to inhibit a wide range of biological activities induced by CXCL8 in human leukocytes. Our results show that repertaxin potently and selectively blocked PMN adhesion to fibrinogen and CD11b up-regulation induced by CXCL8. Reduction of CXCL8-mediated PMN adhesion by repertaxin was paralleled by inhibition of PMN activation including secondary and tertiary granule release and pro-inflammatory cytokine production, whereas PMN phagocytosis of Escherichia coli bacteria was unaffected. Repertaxin also selectively blocked CXCL8-induced T lymphocyte and natural killer (NK) cell migration. These data suggest that repertaxin is a potent and specific inhibitor of a wide range of CXCL8-mediated activities related to leukocyte recruitment and functional activation in inflammatory sites.

Response to histone deacetylase inhibition of novel PML/RARalpha mutants detected in retinoic acid-resistant APL cells.

Resistance to all-trans retinoic acid (ATRA) remains a clinical problem in the treatment of acute promyelocytic leukemia (APL) and provides a model for the development of novel therapies. Molecular alterations in the ligand-binding domain (LBD) of the PML/RARalpha fusion gene that characterizes APL constitute one mechanism of acquired resistance to ATRA. We identified missense mutations in PML/RARalpha from an additional ATRA-resistant patient at relapse and in a novel ATRA-resistant cell line, NB4-MRA1. These cause altered binding to ligand and transcriptional coregulators, leading to a dominant-negative block of transcription. These mutations are in regions of the LBD that appear to be mutational hot spots occurring repeatedly in ATRA-resistant APL patient cells. We evaluated whether histone deacetylase (HDAC) inhibition could overcome the effects of these mutations on ATRA-induced gene expression. Cotreatment with ATRA and TSA restored RARbeta gene expression in NB4-MRA1 cells, whose PML/RARalpha mutation is in helix 12 of the LBD, but not in an APL cell line harboring the patient-derived PML/RARalpha mutation, which was between helix 5 and 6. Furthermore, ATRA combined with TSA increases histone 4 acetylation on the RARbeta promoter only in NB4-MRA1 cells. Consistent with these results, the combined treatment induces differentiation of NB4-MRA1 only. Thus, the ability of an HDAC inhibitor to restore ATRA sensitivity in resistant cells may depend on their specific molecular defects. The variety of PML/RARalpha mutations arising in ATRA-resistant patients begins to explain how APL patients in relapse may differ in response to transcription therapy with HDAC inhibitors.

Targeting of the N-terminal coiled coil oligomerization interface of BCR interferes with the transformation potential of BCR-ABL and increases sensitivity to STI571.

Translocations involving the abl locus on chromosome 9 fuses the tyrosine kinase c-ABL to proteins harboring oligomerization interfaces such as BCR or TEL, enabling these ABL-fusion proteins (X-ABL) to transform cells and to induce leukemia. The ABL kinase activity is blocked by the ABL kinase inhibitor STI571 which abrogates transformation by X-ABL. To investigate the role of oligomerization for the transformation potential of X-ABL and for the sensitivity to STI571, we constructed ABL chimeras with oligomerization interfaces of proteins involved in leukemia-associated translocations such as BCR, TEL, PML, and PLZF. We assessed the capacity of these chimeras to form high molecular weight (HMW) complexes as compared with p185(BCR-ABL). There was a direct relationship between the size of HMW complexes formed by these chimeras and their capacity to induce factor independence in Ba/F3 cells, whereas there was an inverse relationship between the size of the HMW complexes and the sensitivity to STI571. The targeting of the oligomerization interface of p185(BCR-ABL) by a peptide representing the coiled coil region of BCR reduced its potential to transform fibroblasts and increased sensitivity to STI571. Our results indicate that targeting of the oligomerization interfaces of the X-ABL enhances the effects of STI571 in the treatment of leukemia caused by X-ABL.

Expression of SMRTbeta promotes ligand-induced activation of mutated and wild-type retinoid receptors.

Nuclear receptors are ligand-modulated transcription factors regulated by interactions with corepressors and coactivators, whose functions are not fully understood. Acute promyelocytic leukemia (APL) is characterized by a translocation, t(15;17), that produces a PML/RARalpha fusion oncoprotein, whose abnormal transcriptional function is successfully targeted by pharmacologic levels of all-trans-retinoic acid (ATRA). Mutations in the ligand-binding domain of PML/RARalpha that confer resistance to ATRA have been studied by expression in nonhematopoietic cells, such as Cos-1. Here, we show that ATRA binding and transcriptional activation by the same PML/RARalpha mutant differ markedly between nonhematopoietic and leukemic cell lines. Differential expression of the corepressor isoform silencing mediator for retinoid and thyroid receptors beta (SMRTbeta) correlates with increased ligand binding and transcription by the mutant PML/RARalpha. Transient and stable overexpression of SMRTbeta in hematopoietic cells that only express SMRTalpha increased ATRA binding, ligand-induced transcription, and ATRA-induced cell differentiation. This effect may not be limited to abnormal nuclear receptors, because overexpression of SMRTbeta increased ATRA-induced binding and transcriptional activation of wild-type receptors PML/RARalpha and RARalpha. Our results suggest a novel role for the SMRTbeta isoform whereby its cell-specific expression may influence the binding and transcriptional capacities of nuclear receptors, thus providing new evidence of distinct functions of corepressor isoforms and adding complexity to transcriptional regulation.