O-GlcNAcylation of STAT5 controls tyrosine phosphorylation and oncogenic transcription in STAT5-dependent malignancies.

The signal transducer and activator of transcription 5 (STAT5) regulates differentiation, survival, proliferation and transformation of hematopoietic cells. Upon cytokine stimulation, STAT5 tyrosine phosphorylation (pYSTAT5) is transient, while in diverse neoplastic cells persistent overexpression and enhanced pYSTAT5 are frequently found. Post-translational modifications might contribute to enhanced STAT5 activation in the context of transformation, but the strength and duration of pYSTAT5 are incompletely understood. We found that O-GlcNAcylation and tyrosine phosphorylation act together to trigger pYSTAT5 levels and oncogenic transcription in neoplastic cells. The expression of a mutated hyperactive gain-of-function (GOF) STAT5 without O-GlcNAcylation resulted in decreased tyrosine phosphorylation, oligomerization and transactivation potential and complete loss of oncogenic transformation capacity. The lack of O-GlcNAcylation diminished phospho-ERK and phospho-AKT levels. Our data show that O-GlcNAcylation of STAT5 is an important process that contributes to oncogenic transcription through enhanced STAT5 tyrosine phosphorylation and oligomerization driving myeloid transformation. O-GlcNAcylation of STAT5 could be required for nutrient sensing and metabolism of cancer cells.

HDAC1 and HDAC2 integrate the expression of p53 mutants in pancreatic cancer.

Mutation of p53 is a frequent genetic lesion in pancreatic cancer being an unmet clinical challenge. Mutants of p53 have lost the tumour-suppressive functions of wild type p53. In addition, p53 mutants exert tumour-promoting functions, qualifying them as important therapeutic targets. Here, we show that the class I histone deacetylases HDAC1 and HDAC2 contribute to maintain the expression of p53 mutants in human and genetically defined murine pancreatic cancer cells. Our data reveal that the inhibition of these HDACs with small molecule HDAC inhibitors (HDACi), as well as the specific genetic elimination of HDAC1 and HDAC2, reduce the expression of mutant p53 mRNA and protein levels. We further show that HDAC1, HDAC2 and MYC directly bind to the TP53 gene and that MYC recruitment drops upon HDAC inhibitor treatment. Therefore, our results illustrate a previously unrecognized class I HDAC-dependent control of the TP53 gene and provide evidence for a contribution of MYC. A combined approach targeting HDAC1/HDAC2 and MYC may present a novel and molecularly defined strategy to target mutant p53 in pancreatic cancer.

Antagonism between granulocytic maturation and deacetylase inhibitor-induced apoptosis in acute promyelocytic leukaemia cells.

BACKGROUND: Transcriptional repression is a key mechanism driving leukaemogenesis. In acute promyelocytic leukaemia (APL), the fusion protein promyelocytic leukaemia-retinoic acid receptor-α fusion (PML-RARα) recruits transcriptional repressors to myeloid differentiation genes. All-trans-retinoic acid (ATRA) induces the proteasomal degradation of PML-RARα and granulocytic differentiation. Histone deacetylases (HDACs) fall into four classes (I-IV) and contribute to the transcription block caused by PML-RARα. METHODS: Immunoblot, flow cytometry, and May-Grünwald-Giemsa staining were used to analyze differentiation and induction of apoptosis. RESULTS: A PML-RARα- and ATRA-dependent differentiation programme induces granulocytic maturation associated with an accumulation of the myeloid transcription factor CCAAT/enhancer binding protein (C/EBP)ɛ and of the surface protein CD11b. While this process protects APL cells from inhibitors of class I HDAC activity, inhibition of all Zinc-dependent HDACs (classes I, II, and IV) with the pan-HDACi (histone deacetylase inhibitor(s)) LBH589 induces apoptosis of immature and differentiated APL cells. LBH589 can eliminate C/EBPɛ and the mitochondrial apoptosis regulator B-cell lymphoma (BCL)-xL in immature and differentiated NB4 cells. Thus, BCL-xL and C/EBPɛ are newly identified molecular markers for the efficacy of HDACi against APL cells. CONCLUSIONS: Our results could explain the therapeutic limitations occurring with ATRA and class I HDACi combinations. Pro-apoptotic effects caused by pan-HDAC inhibition are not blunted by ATRA-induced differentiation and may provide a clinically interesting alternative.

p53-dependent and p53-independent anticancer effects of different histone deacetylase inhibitors.

BACKGROUND: Histone deacetylase inhibitors (HDACi) are promising antineoplastic agents, but their precise mechanisms of actions are not well understood. In particular, the relevance of p53 for HDACi-induced effects has not been fully elucidated. We investigated the anticancer effects of four structurally distinct HDACi, vorinostat, entinostat, apicidin and valproic acid, using isogenic HCT-116 colon cancer cell lines differing in p53 status. METHODS: Effects were assessed by MTT assay, flow-cytometric analyses of propidium iodide uptake, mitochondrial depolarisation and cell-cycle distribution, as well as by gene expression profiling. RESULTS: Vorinostat was equally effective in p53 wild-type and null cells, whereas entinostat was less effective in p53 null cells. Histone deacetylase inhibitors treatment suppressed the expression of MDM2 and increased the abundance of p53. Combination treatments showed that vorinostat enhanced the cytotoxic activity of TRAIL and bortezomib, independent of the cellular p53 status. Investigations into the effects of an inhibitor of the sirtuin class of HDAC, tenovin-1, revealed that tenovin-1-mediated cell death hinged on p53. CONCLUSION: These results demonstrate that vorinostat activates p53, but does not require p53 for inducing its anticancer action. Yet they also demonstrate that entinostat-induced cytotoxic effects partially depend on p53, indicating that different HDACi have a different requirement for p53.

SIAH proteins: critical roles in leukemogenesis.

The delicate balance between the synthesis and the degradation of proteins ensures cellular homeostasis. Proteases act in an irreversible manner and therefore have to be strictly regulated. The ubiquitin-proteasome system (UPS) is a major pathway for the proteolytic degradation of cellular proteins. As dysregulation of the UPS is observed in most cancers including leukemia, the UPS is a valid target for therapeutic intervention strategies. Ubiquitin-ligases selectively bind substrates to target them for poly-ubiquitinylation and proteasomal degradation. Therefore, pharmacological modulation of these proteins could allow a specific level of control. Increasing evidence accumulates that ubiquitin-ligases termed mammalian seven in absentia homologs (SIAHs) are not only critical for the pathogenesis of solid tumors but also for leukemogenesis. However, the relevance and therapeutic potential of SIAH-dependent processes has not been fully elucidated. Here, we summarize functions of SIAH ubiquitin-ligases in leukemias, how they select leukemia-relevant substrates for proteasomal degradation, and how the expression and activity of SIAH1 and SIAH2 can be modulated in vivo. We also discuss that epigenetic drugs belonging to the group of histone deacetylase inhibitors induce SIAH-dependent proteasomal degradation to accelerate the turnover of leukemogenic proteins. In addition, our review highlights potential areas for future research on SIAH proteins.

SIAH ubiquitin ligases target the nonreceptor tyrosine kinase ACK1 for ubiquitinylation and proteasomal degradation.

Activated Cdc42-associated kinase 1 (ACK1) is a nonreceptor tyrosine kinase linked to cellular transformation. The aberrant regulation of ACK1 promotes tumor progression and metastasis. Therefore, ACK1 is regarded as a valid target in cancer therapy. Seven in absentia homolog (SIAH) ubiquitin ligases facilitate substrate ubiquitinylation that targets proteins to the proteasomal degradation pathway. Here we report that ACK1 and SIAH1 from Homo sapiens interact in a yeast two-hybrid screen. Protein-protein interaction studies and protein degradation analyses using deletion and point mutants of ACK1 verify that SIAH1 and the related SIAH2 interact with ACK1. The association between SIAHs and ACK1 depends on the integrity of a highly conserved SIAH-binding motif located in the far C-terminus of ACK1. Furthermore, we demonstrate that the interaction of ACK1 with SIAH1 and the induction of proteasomal degradation of ACK1 by SIAH1 are independent of ACK1's kinase activity. Chemical inhibitors blocking proteasomal activity corroborate that SIAH1 and SIAH2 destabilize the ACK1 protein by inducing its proteasomal turnover. This mechanism apparently differs from the lysosomal pathway targeting ACK1 after stimulation with the epidermal growth factor. Our data also show that ACK1, but not ACK1 mutants lacking the SIAH binding motif, has a discernable negative effect on SIAH levels. Additionally, knockdown approaches targeting the SIAH2 mRNA uncover specifically that the induction of SIAH2 expression, by hormonally-induced estrogen receptor (ER) activation, decreases the levels of ACK1 in luminal human breast cancer cells. Collectively, our data provide novel insights into the molecular mechanisms modulating ACK1 and they position SIAH ubiquitin ligases as negative regulators of ACK1 in transformed cells.

Ubiquitin conjugase UBCH8 targets active FMS-like tyrosine kinase 3 for proteasomal degradation.

The class III receptor tyrosine kinase FMS-like tyrosine kinase 3 (FLT3) regulates normal hematopoiesis and immunological functions. Nonetheless, constitutively active mutant FLT3 (FLT3-ITD) causally contributes to transformation and is associated with poor prognosis of acute myeloid leukemia (AML) patients. Histone deacetylase inhibitors (HDACi) can counteract deregulated gene expression profiles and decrease oncoprotein stability, which renders them candidate drugs for AML treatment. However, these drugs have pleiotropic effects and it is often unclear how they correct oncogenic transcriptomes and proteomes. We report here that treatment of AML cells with the HDACi LBH589 induces the ubiquitin-conjugating enzyme UBCH8 and degradation of FLT3-ITD. Gain- and loss-of-function approaches show that UBCH8 and the ubiquitin-ligase SIAH1 physically interact with and target FLT3-ITD for proteasomal degradation. These ubiquitinylating enzymes though have a significantly lesser effect on wild-type FLT3. Furthermore, physiological and pharmacological stimulation of FLT3 phosphorylation, inhibition of FLT3-ITD autophosphorylation and analysis of kinase-inactive FLT3-ITD revealed that tyrosine phosphorylation determines degradation of FLT3 and FLT3-ITD by the proteasome. These results provide novel insights into antileukemic activities of HDACi and position UBCH8, which have been implicated primarily in processes in the nucleus, as a previously unrecognized important modulator of FLT3-ITD stability and leukemic cell survival.

Inhibitors of HDACs--effective drugs against cancer?

Alterations in genomic and non-genomic mechanisms can disturb homeostasis and cause severe human diseases. Histone deacetylases (HDACs) are epigenetic regulators which catalyze the removal of acetyl moieties from histones and non-histone proteins. Aberrant histone deacetylation, due to increased HDAC activity and expression, often correlates with pathological gene repression and neoplastic transformation. Therefore, intense efforts have been made to find small molecule inhibitors of HDACs (HDACIs). Such compounds indeed alter cellular signaling networks relevant for tumorigenesis, and several HDACIs are currently tested in clinical trials against different types of cancer. Although HDACs share a conserved deacetylase domain and an at least similar mechanism of catalysis, isoenzyme-specific HDACIs could be identified and certain HDACIs even evoke degradation of HDACs. Here, we summarize molecular actions of HDACs and of different classes of HDACIs. In addition, we review data obtained in clinical studies involving HDACIs and we discuss how such agents might be beneficial for the treatment of cancer.

Cross talk between stimulated NF-kappaB and the tumor suppressor p53.

Nuclear factor-kappaB (NF-kappaB) and p53 critically determine cancer development and progression. Defining the cross talk between these transcription factors can expand our knowledge on molecular mechanisms of tumorigenesis. Here, we show that induction of replicational stress activates NF-kappaB p65 and triggers its interaction with p53 in the nucleus. Experiments with knockout cells show that p65 and p53 are both required for enhanced NF-kappaB activity during S-phase checkpoint activation involving ataxia-telangiectasia mutated and checkpoint kinase-1. Accordingly, the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) also triggers formation of a transcriptionally active complex containing nuclear p65 and p53 on kappaB response elements. Gene expression analyses revealed that, independent of NF-kappaB activation in the cytosol, TNF-induced NF-kappaB-directed gene expression relies on p53. Hence, p53 is unexpectedly necessary for NF-kappaB-mediated gene expression induced by atypical and classical stimuli. Remarkably, data from gain- and loss-of function approaches argue that anti-apoptotic NF-kappaB p65 activity is constitutively evoked by a p53 hot-spot mutant frequently found in tumors. Our observations suggest explanations for the outstanding question why p53 mutations rather than p53 deletions arise in tumors of various origins.

Histone deacetylase inhibitors and hydroxyurea modulate the cell cycle and cooperatively induce apoptosis.

Therapy resistance represents a major problem for disease management in oncology. Histone deacetylase inhibitors (HDACi) have been shown to modulate the cell cycle, to induce apoptosis and to sensitize cancer cells for other chemotherapeutics. Our study shows that the HDACi valproic acid (VPA) and the ribonucleotide reductase inhibitor hydroxyurea (HU) potentiate the pro-apoptotic effects of each other towards several cancer cell lines. This correlates with the HU-induced degradation of the cyclin-dependent kinase inhibitors (CDKI) p21 and p27, mediated by the proteasome or caspase-3. Moreover, we found that caspase-3 activation is required for VPA-induced apoptosis. Remarkably, p21 and p27 can confer resistance against VPA and HU. Both CDKI interact with caspase-3 and compete with other caspase-3 substrates. Hence, p21 and p27 may contribute to chemotherapy resistance as apoptosis inhibitors. Since the biological effects of VPA and HU could be achieved at concentrations used in current treatment protocols, the combined application of these compounds might be considered as a potential strategy for cancer treatment.

Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells.

Histone deacetylases (HDACs) play important roles in transcriptional regulation and pathogenesis of cancer. Thus, HDAC inhibitors are candidate drugs for differentiation therapy of cancer. Here, we show that the well-tolerated antiepileptic drug valproic acid is a powerful HDAC inhibitor. Valproic acid relieves HDAC-dependent transcriptional repression and causes hyperacetylation of histones in cultured cells and in vivo. Valproic acid inhibits HDAC activity in vitro, most probably by binding to the catalytic center of HDACs. Most importantly, valproic acid induces differentiation of carcinoma cells, transformed hematopoietic progenitor cells and leukemic blasts from acute myeloid leukemia patients. More over, tumor growth and metastasis formation are significantly reduced in animal experiments. Therefore, valproic acid might serve as an effective drug for cancer therapy.

Histone deacetylase as a therapeutic target.

The maintenance of health depends on the coordinated and tightly regulated expression of genetic information. Certain forms of leukemia have become paradigms for the pathogenic role of aberrant repression of differentiation genes. In these acute leukemias, fusion proteins generated by chromosomal translocations no longer function as transcriptional activators, but instead repress target genes by recruiting histone deacetylases (HDACs). The potential benefit of HDAC inhibition has been established by the use of enzyme inhibitors in vitro and in a single reported case of experimental therapy. Because recently identified HDAC inhibitors appear to overcome many drawbacks of early inhibitory compounds in clinical use, the stage is set to test the therapeutic value of HDAC inhibition in leukemias and in other diseases, including solid tumors and aberrant hormonal signaling. This review summarizes the range of diseases expected to respond to HDAC inhibition.