Assessment of CXC ligand 12-mediated calcium signalling and its regulators in basal-like breast cancer cells.

CXC ligand (L)12 is a chemokine implicated in the migration, invasion and metastasis of cancer cells via interaction with its receptors CXC chemokine receptor (CXCR)4 and CXCR7. In the present study, CXCL12-mediated Ca2+ signalling was compared with two basal-like breast cancer cell lines, MDA-MB-231 and MDA-MB-468, which demonstrate distinct metastatic potential. CXCL12 treatment induced Ca2+ responses in the more metastatic MDA-MB-231 cells but not in the less metastatic MDA-MB-468 cells. Assessment of mRNA levels of CXCL12 receptors and their potential modulators in both cell lines revealed that CXCR4 and CXCR7 levels were increased in MDA-MB-231 cells compared with MDA-MB-468 cells. Cluster of differentiation (CD)24, the negative regulator of CXCL12 responses, demonstrated increased expression in MDA-MB-468 cells compared with MDA-MB-231 cells, and the two cell lines expressed comparable levels of hypoxia-inducible factor (HIF)2α, a CXCR4 regulator. Induction of epithelial-mesenchymal transition (EMT) by epidermal growth factor exhibited opposite effects on CXCR4 mRNA levels compared with hypoxia-induced EMT. Neither EMT inducer exhibited an effect on CXCR7 expression, however hypoxia increased HIF2α expression levels in MDA-MB-468 cells. Analysis of the gene expression profiles of breast tumours revealed that the highest expression levels of CXCR4 and CXCR7 were in the Claudin-Low molecular subtype, which is markedly associated with EMT features.

Oncosis and apoptosis induction by activation of an overexpressed ion channel in breast cancer cells.

The critical role of calcium signalling in processes related to cancer cell proliferation and invasion has seen a focus on pharmacological inhibition of overexpressed ion channels in specific cancer subtypes as a potential therapeutic approach. However, despite the critical role of calcium in cell death pathways, pharmacological activation of overexpressed ion channels has not been extensively evaluated in breast cancer. Here we define the overexpression of transient receptor potential vanilloid 4 (TRPV4) in a subgroup of breast cancers of the basal molecular subtype. We also report that pharmacological activation of TRPV4 with GSK1016790A reduced viability of two basal breast cancer cell lines with pronounced endogenous overexpression of TRPV4, MDA-MB-468 and HCC1569. Pharmacological activation of TRPV4 produced pronounced cell death through two mechanisms: apoptosis and oncosis in MDA-MB-468 cells. Apoptosis was associated with PARP-1 cleavage and oncosis was associated with a rapid decline in intracellular ATP levels, which was a consequence of, rather than the cause of, the intracellular ion increase. TRPV4 activation also resulted in reduced tumour growth in vivo. These studies define a novel therapeutic strategy for breast cancers that overexpress specific calcium permeable plasmalemmal ion channels with available selective pharmacological activators.

The MYB proto-oncogene suppresses monocytic differentiation of acute myeloid leukemia cells via transcriptional activation of its target gene GFI1.

The MYB gene is a master regulator of hematopoiesis and contributes to leukemogenesis in several species including humans. Although it is clear that MYB can promote proliferation, suppress apoptosis and block differentiation, the identities of the MYB target genes that mediate these effects have only been partially elucidated. Several studies, including our own, have collectively identified substantial numbers of MYB target genes, including candidates for each of these activities; however, functional validation, particularly in the case of differentiation suppression, has lagged well behind. Here we show that GFI1, which encodes an important regulator of hematopoietic stem cell (HSC) function and granulocytic differentiation, is a direct target of MYB in myeloid leukemia cells. Chromatin immunoprecipitation and reporter studies identified a functional MYB-binding site in the promoter region of GFI, whereas ectopic expression and small hairpin RNA-mediated knockdown of MYB resulted in concomitant increases and decreases, respectively, in GFI1 expression. We also demonstrate that GFI1, like MYB, can block the induced monocytic differentiation of a human acute myeloid leukemia cell line, and most importantly, that GFI1 is essential for MYB's ability to block monocytic differentiation. Thus, we have identified a target of MYB that is a likely mediator of its myeloid differentiation-blocking activity, and which may also be involved in MYB's activities in regulating normal HSC function and myeloid differentiation.

Role and potential for therapeutic targeting of MYB in leukemia.

The Myb protein was first identified as an oncogene that causes leukemia in chickens. Since then, it has been widely associated with different types of cancers and studied in detail in myeloid leukemias. However, despite these studies, its role in the induction, pathogenesis and maintenance of AML, and other blood disorders, is still not well understood. Recent efforts to uncover its plethora of transcriptional targets have provided key insights into understanding its mechanism of action. This review evaluates our current knowledge of the role of Myb in leukemia, with a particular focus on AML, from the vast literature spanning three decades, highlighting key studies that have influenced our understanding. We discuss recent insights into its role in leukemogenesis and how these could be exploited for the therapeutic targeting of Myb, its associated co-regulators or its target genes, in order to improve outcomes in the treatment of a wide range of hematopoietic malignancies.

PG545, a dual heparanase and angiogenesis inhibitor, induces potent anti-tumour and anti-metastatic efficacy in preclinical models.

BACKGROUND: PG545 is a heparan sulfate (HS) mimetic that inhibits tumour angiogenesis by sequestering angiogenic growth factors in the extracellular matrix (ECM), thus limiting subsequent binding to receptors. Importantly, PG545 also inhibits heparanase, the only endoglycosidase which cleaves HS chains in the ECM. The aim of the study was to assess PG545 in various solid tumour and metastasis models. METHODS: The anti-angiogenic, anti-tumour and anti-metastatic properties of PG545 were assessed using in vivo angiogenesis, solid tumour and metastasis models. Pharmacokinetic (PK) data were also generated in tumour-bearing mice to gain an understanding of optimal dosing schedules and regimens. RESULTS: PG545 was shown to inhibit angiogenesis in vivo and induce anti-tumour or anti-metastatic effects in murine models of breast, prostate, liver, lung, colon, head and neck cancers and melanoma. Enhanced anti-tumour activity was also noted when used in combination with sorafenib in a liver cancer model. PK data revealed that the half-life of PG545 was relatively long, with pharmacologically relevant concentrations of radiolabeled PG545 observed in liver tumours. CONCLUSION: PG545 is a new anti-angiogenic clinical candidate for cancer therapy. The anti-metastatic property of PG545, likely due to the inhibition of heparanase, may prove to be a critical attribute as the compound enters phase I clinical trials.

A functional SUMO-interacting motif in the transactivation domain of c-Myb regulates its myeloid transforming ability.

c-Myb is an essential hematopoietic transcription factor that controls proliferation and differentiation of progenitors during blood cell development. Whereas sumoylation of the C-terminal regulatory domain (CRD) is known to have a major impact on the activity of c-Myb, no role for noncovalent binding of small ubiquitin-like modifier (SUMO) to c-Myb has been described. Based on the consensus SUMO-interacting motif (SIM), we identified and examined putative SIMs in human c-Myb. Interaction and reporter assays showed that the SIM in the in the transactivation domain of c-Myb (V(267)NIV) is functional. This motif is necessary for c-Myb to be able to interact noncovalently with SUMO, preferentially SUMO2/3. Destroying the SUMO-binding properties by mutation resulted in a large increase in the transactivation potential of c-Myb. Mutational analysis and overexpression of conjugation-defective SUMO argued against intramolecular repression caused by sumoylated CRD and in favor of SUMO-dependent repression in trans. Using both a myeloid cell line-based assay and a primary hematopoietic cell assay, we addressed the transforming abilities of SUMO binding and conjugation mutants. Interestingly, only loss of SUMO binding, and not SUMO conjugation, enhanced the myeloid transformational potential of c-Myb. c-Myb with the SIM mutated conferred a higher proliferative ability than the wild-type and caused an effective differentiation block. This establishes SUMO binding as a mechanism involved in modulating the transactivation activity of c-Myb, and responsible for keeping the transforming potential of the oncoprotein in check.

The PG500 series: novel heparan sulfate mimetics as potent angiogenesis and heparanase inhibitors for cancer therapy.

Heparan sulfate mimetics, which we have called the PG500 series, have been developed to target the inhibition of both angiogenesis and heparanase activity. This series extends the technology underpinning PI-88, a mixture of highly sulfated oligosaccharides which reached Phase III clinical development for hepatocellular carcinoma. Advances in the chemistry of the PG500 series provide numerous advantages over PI-88. These new compounds are fully sulfated, single entity oligosaccharides attached to a lipophilic moiety, which have been optimized for drug development. The rational design of these compounds has led to vast improvements in potency compared to PI-88, based on in vitro angiogenesis assays and in vivo tumor models. Based on these and other data, PG545 has been selected as the lead clinical candidate for oncology and is currently undergoing formal preclinical development as a novel treatment for advanced cancer.

Intestinal adenoma formation and MYC activation are regulated by cooperation between MYB and Wnt signaling.

Aberrant Wnt signaling mediated by mutations affecting APC (adenomatous polyposis coli) or beta-catenin initiates the majority of human colorectal cancers (CRC) and drives tumorigenesis through the activation of specific genes such as MYC. We report here a novel association whereby another oncogenic transcription factor, MYB/c-Myb, is necessary for intestinal adenoma development directed by activated Wnt signaling. APC(Min/+) mice in which c-myb is haploinsufficient survive longer than wild-type APC(Min/+) animals due to a delay in adenoma formation. Intestinal adenomas from APC(Min/+) mice were assessed and found to have high levels of c-myc gene expression. We explored the relationship between activated Wnt signaling and MYB in regulating MYC and found activated beta-catenin in combination with MYB induces robust upregulation of MYC promoter activity, as well as endogenous MYC mRNA and protein expression, in human cells. This cooperation occurred through independent binding of MYB and beta-catenin to the MYC promoter. These data highlight a cooperative function for MYB in the context of activated Wnt signaling and provide a molecular basis for the expression of MYC in CRC.

Repression of Gadd45alpha by activated FLT3 and GM-CSF receptor mutants contributes to growth, survival and blocked differentiation.

The tumor suppressor Gadd45alpha was earlier shown to be a repressed target of sustained receptor-mediated ERK1/2 signaling. We have identified Gadd45alpha as a downregulated gene in response to constitutive signaling from two FLT3 mutants (FLT3-ITD and FLT3-TKD) commonly found in AML, and a leukemogenic GM-CSF receptor trans-membrane mutant (GMR-V449E). GADD45A mRNA downregulation is also associated with FLT3-ITD(+) AML. Sustained ERK1/2 signaling contributes significantly to receptor-mediated downregulation of Gadd45alpha mRNA in FDB1 cells expressing activated receptor mutants, and in the FLT3-ITD(+) cell line MV4;11. Knockdown of Gadd45alpha with shRNA led to increased growth and survival of FDB1 cells and enforced expression of Gadd45alpha in FDB1 cells expressing FLT3-ITD or GMR-V449E resulted in reduced growth and viability. Gadd45alpha overexpression in FLT3-ITD(+) AML cell lines also resulted in reduced growth associated with increased apoptosis and G(1)/S cell cycle arrest. Overexpression of Gadd45alpha in FDB1 cells expressing GMR-V449E was sufficient to induce changes associated with myeloid differentiation suggesting Gadd45alpha downregulation contributes to the maintenance of receptor-induced myeloid differentiation block. Thus, we show that ERK1/2-mediated downregulation of Gadd45alpha by sustained receptor signaling contributes to growth, survival and arrested differentiation in AML.

Functional expression cloning reveals proapoptotic role for protein phosphatase 4.

Functional expression cloning strategies are highly suitable for the analysis of the molecular control of apoptosis. This approach has two critical advantages. Firstly, it eliminates prior assumptions about the properties of the proteins involved, and, secondly, it selectively targets proteins that are causally involved in apoptosis control and which affect the crucial cellular decision between survival and death. The application of this strategy to the isolation of cDNAs conferring resistance to dexamethasone and gamma-irradiation resulted in the isolation of a partial cDNA for the catalytic subunit of protein phosphatase 4 (PP4). Cells transfected with this partial cDNA in an expression vector downregulated PP4 and were resistant to both dexamethasone and UV radiation, as demonstrated by both membrane integrity and colony-forming assays. These observations suggest that PP4 plays an important proapoptotic role in T lymphocytes.

Mutation screening of the c-MYB negative regulatory domain in acute and chronic myeloid leukaemia.

Over-expression of the c-myb gene and expression of activated forms of myb are known to transform haemopoietic cells, particularly cells of the myeloid lineage. Truncations or mutations that disrupt the negative regulatory domain (NRD) of the Myb protein confer an increased ability to transform cells. Although it has proved difficult to link mutations in c-MYB to human leukaemia, no studies investigating the presence of mutations within the c-MYB NRD have been reported. Therefore, we have performed mutational analysis of this region, using polymerase chain reaction-single-stranded conformation polymorphism and sequence analysis, in 26 patients with acute or chronic myeloid leukaemia. No mutations were detected, indicating that mutation of this region of the Myb protein is not common in the pathogenesis or progression of these diseases.

Identification and validation of candidate Myb target genes.

While a considerable number of candidate Myb target genes have been reported to date, most of these are likely to play little or no role in transformation by myb oncogenes. Here we have used a conditionally myb-transformed myeloid cell line (ERMYB) to further examine Myb regulation of one candidate target gene--c-myc--that has the potential to affect cell proliferation. It was found that the major influence on c-myc expression was the presence of cytokine (GM-CSF) rather than Myb activity. We also describe the application of PCR-based subtractive hybridization and low-density cDNA array screening, in conjunction with the ERMYB line, to the identification of additional Myb target genes. Preliminary identification of a number of candidates is reported; these include myeloperoxidase, which is known to have essential Myb-binding sites in its regulatory region.

A model for assembly and activation of the GM-CSF, IL-3 and IL-5 receptors: insights from activated mutants of the common beta subunit.

Granulocyte-macrophage colony stimulating factor (GM-CSF), Interleukin-3 (IL-3) and Interleukin-5 (IL-5) have overlapping, pleiotropic effects on hematopoietic cells, including neutrophils, eosinophils, monocytes and early progenitor cells. The high-affinity receptors for human GM-CSF, IL-3, and IL-5 share a common beta-subunit (hbeta(c)), which is essential for signalling and plays a major role in recruiting intracellular signalling molecules. While activation of the cytoplasmic tyrosine kinase JAK2 appears to be the initiating event for signalling, the immediate events that trigger this are still unclear. We have isolated a number of activated mutants of hbeta(c), which can be grouped into classes defined by their state of receptor phosphorylation, their requirement for alpha subunit as a cofactor, and their activities in primary cells and cell lines. We discuss these findings with regard to the stoichiometry, activation, and signalling of the normal GM-CSF/IL-3/IL-5 receptor complexes. Specifically, this work has implications for the role of the ligand-specific alpha-subunits in initiating the signalling through the beta-subunit, the role of beta subunit dimerization as a receptor trigger, and the function of receptor tyrosine phosphorylation in generating growth and survival signals. Based on the properties of the activated mutants and the recent structures of erythropoietin receptor (Epo-R) complexes, we propose a model in which (1) activation of hbeta(c) can occur via alternative states that differ with respect to stoichiometry and subunit assembly, but which all mediate proliferative responses, and (2) each of the different classes of activated mutants mimics one of these alternative states.

The role of disulfide-linked dimerization in interleukin-3 receptor signaling and biological activity.

Cysteine residues 86 and 91 of the beta subunit of the human interleukin (hIL)-3 receptor (hbetac) participate in disulfide-linked receptor subunit heterodimerization. This linkage is essential for receptor tyrosine phosphorylation, since the Cys-86 --> Ala (Mc4) and Cys-91 --> Ala (Mc5) mutations abolished both events. Here, we used these mutants to examine whether disulfide-linked receptor dimerization affects the biological and biochemical activities of the IL-3 receptor. Murine T cells expressing hIL-3Ralpha and Mc4 or Mc5 did not proliferate in hIL-3, whereas cells expressing wild-type hbetac exhibited rapid proliferation. However, a small subpopulation of cells expressing each mutant could be selected for growth in IL-3, and these proliferated similarly to cells expressing wild-type hbetac, despite failing to undergo IL-3-stimulated hbetac tyrosine phosphorylation. The Mc4 and Mc5 mutations substantially reduced, but did not abrogate, IL-3-mediated anti-apoptotic activity in the unselected populations. Moreover, the mutations abolished IL-3-induced JAK2, STAT, and AKT activation in the unselected cells, whereas activation of these molecules in IL-3-selected cells was normal. In contrast, Mc4 and Mc5 showed a limited effect on activation of Erk1 and -2 in unselected cells. These data suggest that whereas disulfide-mediated cross-linking and hbetac tyrosine phosphorylation are normally important for receptor activation, alternative mechanisms can bypass these requirements.

Novel murine myeloid cell lines that exhibit a differentiation switch in response to IL-3 or GM-CSF, or to different constitutively active mutants of the GM-CSF receptor beta subunit.

Several activating mutations have recently been described in the common beta subunit for the human interleukin(IL)-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors (hbetac). Two of these, FIDelta and I374N, result, respectively, in a 37-amino acid duplication and an isoleucine-to-asparagine substitution in the extracellular domain. A third, V449E, leads to valine-to-glutamic acid substitution in the transmembrane domain. Previous studies have shown that when expressed in murine hemopoietic cells in vitro, the extracellular mutants can confer factor independence on only the granulocyte-macrophage lineage while the transmembrane mutant can do so to all cell types of the myeloid and erythroid compartments. To further study the signaling properties of the constitutively active hbetac mutants, we have used novel murine hemopoietic cell lines, which we describe in this report. These lines, FDB1 and FDB2, proliferate in murine IL-3 and undergo granulocyte-macrophage differentiation in response to murine GM-CSF. We find that while the transmembrane mutant, V449E, confers factor-independent proliferation on these cell lines, the extracellular hbetac mutants promote differentiation. Hence, in addition to their ability to confer factor independence on distinct cell types, transmembrane and extracellular activated hbetac mutants deliver distinct signals to the same cell type. Thus, the FDB cell lines, in combination with activated hbetac mutants, constitute a powerful new system to distinguish between signals that determine hemopoietic proliferation or differentiation. (Blood. 2000;95:120-127)

Myeloproliferative disorder and leukaemia in mice induced by different classes of constitutive mutants of the human IL-3/IL-5/GM-CSF receptor common beta subunit.

Several constitutively active mutant forms of the common beta subunit of the human IL-3, IL-5 and GM-CSF receptors (hbetac), which enable it to signal in the absence of ligand, have recently been described. Two of these, V449E and I374N, are amino acid substitutions in the transmembrane and extracellular regions of hbetac, respectively. A third, FIDelta, contains a 37 amino acid duplication in the extracellular domain. We have shown previously that when expressed in primary murine haemopoietic cells, the extracellular mutants confer factor-independence on cells of the neutrophil and monocyte lineages only, whereas V449E does so on all cell types of the myeloid and erythroid compartments. To study the in vivo effects and leukaemic potential of these mutants, we have expressed all three in mice by bone marrow reconstitution using retrovirally infected donor cells. Expression of the extracellular mutants leads to an early onset, chronic myeloproliferative disorder marked by elevations in the neutrophil, monocyte, erythrocyte and platelet lineages. In contrast, expression of V449E leads to an acute leukaemia-like syndrome of anaemia, thrombocytopaenia and blast cell expansion. These data support the possibility that activating mutations in hbetac are involved in haemopoietic disorders in man.

A cell type-specific constitutive point mutant of the common beta-subunit of the human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 receptors requires the GM-CSF receptor alpha-subunit for activation.

The high affinity receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a cytokine-specific alpha-subunit (hGMRalpha) and a common signal-transducing beta-subunit (hbetac) that is shared with the interleukin-3 and -5 receptors. We have previously identified a constitutively active extracellular point mutant of hbetac, I374N, that can confer factor independence on murine FDC-P1 cells but not BAF-B03 or CTLL-2 cells (Jenkins, B. J., D'Andrea, R. J., and Gonda, T. J. (1995) EMBO J. 14, 4276-4287). This restricted activity suggested the involvement of cell type-specific signaling molecules in the activation of this mutant. We report here that one such molecule is the mouse GMRalpha (mGMRalpha) subunit, since introduction of mGMRalpha, but not hGMRalpha, into BAF-B03 or CTLL-2 cells expressing the I374N mutant conferred factor independence. Experiments utilizing mouse/human chimeric GMRalpha subunits indicated that the species specificity lies in the extracellular domain of GMRalpha. Importantly, the requirement for mGMRalpha correlated with the ability of I374N (but not wild-type hbetac) to constitutively associate with mGMRalpha. Expression of I374N in human factor-dependent UT7 cells also led to factor-independent proliferation, with concomitant up-regulation of hGMRalpha surface expression. Taken together, these findings suggest a critical role for association with GMRalpha in the constitutive activity of I374N.

Molecular cloning and chromosomal mapping of the human homologue of MYB binding protein (P160) 1A (MYBBP1A) to 17p13.3.

We have previously isolated and characterized murine MYB binding protein (p160) 1a, a protein that specifically interacts with the leucine zipper motif within the negative regulatory domain of the c-Myb proto-oncoprotein. We now describe the molecular cloning of the human MYBBP1A cDNA and chromosomal localization to 17p13.3 by fluorescence in situ hybridization analysis. Given the likely presence of a tumor suppressor gene (or genes) within this region of chromosome 17, the position of MYBBP1A was further mapped by radiation hybrid analysis and was found to lie between markers D17S1828 and D17S938. A P1 artificial chromosome clone containing the 5' region of MYBBP1A was isolated and indicates a physical linkage between MYBBP1A and the 15-lipoxygenase gene (ALOX15). A novel, polymorphic (CA)(25) dinucleotide repeat was also isolated from this PAC and may serve as a useful marker for MYBBP1A and this region of chromosome 17.

Dysregulated hematopoiesis and a progressive neurological disorder induced by expression of an activated form of the human common beta chain in transgenic mice.

Previously we described activating mutations of hbetac, the common signaling subunit of the receptors for the hematopoietic and inflammatory cytokines, GM-CSF, IL-3, and IL-5. The activated mutant, hbetacFIDelta, is able to confer growth factor-independent proliferation on the murine myeloid cell line FDC-P1, and on primary committed myeloid progenitors. We have used this activating mutation to study the effects of chronic cytokine receptor stimulation. Transgenic mice were produced carrying the hbetacFIDelta cDNA linked to the constitutive promoter derived from the phosphoglycerate kinase gene, PGK-1. Transgene expression was demonstrated in several tissues and functional activity of the mutant receptor was confirmed in hematopoietic tissues by the presence of granulocyte macrophage and macrophage colony-forming cells (CFU-GM and CFU-M) in the absence of added cytokines. All transgenic mice display a myeloproliferative disorder characterized by splenomegaly, erythrocytosis, and granulocytic and megakaryocytic hyperplasia. This disorder resembles the human disease polycythemia vera, suggesting that activating mutations in hbetac may play a role in the pathogenesis of this myeloproliferative disorder. In addition, these transgenic mice develop a sporadic, progressive neurological disease and display bilateral, symmetrical foci of necrosis in the white matter of brain stem associated with an accumulation of macrophages. Thus, chronic hbetac activation has the potential to contribute to pathological events in the central nervous system.