Antiproliferative Signals Research Articles

Acquired genetic and functional alterations associated with transforming growth factor β type I resistance in Hep3B human hepatocellular carcinoma cell line

During the neoplastic process tumour cells frequently acquire resistance to the antiproliferative signals of transforming growth factor-β (TGF-β). Here we examined a human hepatocellular carcinoma cell line (Hep3B-TS) sensitive to TGF-β signalling, and a derivative line (Hep3B-TR) rendered resistant to TGF-β by stepwise exposure to TGF-β1. Comprehensive molecular cytogenetic analysis revealed that the acquisition of TGF-β-resistance by Hep3B-TR cells was due to loss of TGF-β receptor 2 (TGFβRII) gene. As demonstrated by spectral karyotyping and array-based comparative genomic hybridization, and in difference to Hep3B-TS cells, which have three rearranged and two normal copies of chromosome 3 that harbour the TGFβRII gene, Hep3B-TR cells have four rearranged and one apparently normal chromosome 3, which nonetheless underwent a critical microdeletion at the site of TGFβRII gene. Gene expression analysis using an oligonucleotide microarray of 21,397 genes showed that Hep3B-TR differentially expressed 307 genes, out of which 197 and 110 were up- and down-regulated, respectively, compared to Hep3B-TS. Six of differentially expressed genes were identified as downstream targets of the tumour necrosis factor (TNF) gene, suggesting that loss of TGFβRII triggered activation of the TNF pathway known to be regulated by TGF-β1 network. On the functional level, the TGF-β-resistant Hep3B-TR cells displayed significantly enhanced capacity for anchorage independent growth and cell migration in vitro, and also increased tumorigenicity in vivo and in vitro and in vivo tumorigenicity compared with parental sensitive cells.

Negative Regulation of the EGFR-MAPK Cascade by Actin-MAL-Mediated Mig6/Errfi-1 Induction

We analyzed the G-actin-regulated transcriptome by gene expression analysis using previously characterized actin-binding drugs. We found many known MAL/MRTF-dependent target genes of serum response factor (SRF), as well as additional directly regulated genes. Surprisingly, several putative antiproliferative target genes were identified, including mig6/errfi-1, a negative regulator of the EGFR family. Mig6 induction occurred through actin-MAL-SRF signaling, and MAL was inducibly recruited to and activated a mig6 promoter element. Upregulation of Mig6 by lipid agonists such as LPA and S1P or actin drugs involved MAL and correlated with decreased activation of EGFR, MAPK/Erk, and c-fos. Mig6 depletion restored EGFR signaling and provided a proliferative advantage. Overexpression of MAL exhibited strong antiproliferative effects requiring the domains for SRF binding and transactivation, which supports antagonistic functions of MAL on growth-promoting signals. Our results show the existence of negatively acting transcriptional networks between pro- and antiproliferative signaling pathways toward SRF.

P21 in cancer: intricate networks and multiple activities

One of the main engines that drives cellular transformation is the loss of proper control of the mammalian cell cycle. The cyclin-dependent kinase inhibitor p21 (also known as p21WAF1/Cip1) promotes cell cycle arrest in response to many stimuli. It is well positioned to function as both a sensor and an effector of multiple anti-proliferative signals. This Review focuses on recent advances in our understanding of the regulation of p21 and its biological functions with emphasis on its p53-independent tumour suppressor activities and paradoxical tumour-promoting activities, and their implications in cancer.

Gene regulation and deregulation by MAL/MRTF coactivators

Proteins of the MRTF family play a key role in regulated gene expression downstream of Rho family GTPases. MAL/MKL1/MRTF-A is a transcriptional coactivator of serum response factor (SRF) and is bound and inhibited by G-actin in the non-induced state. In acute megakaryocytic leukaemia in infants, a recurrent translocation t(1;22) results in the OTT-MAL/RBM15-MKL1 fusion oncoprotein. How it contributes to the malignancy is unknown. We tested by biochemical analysis whether OTT-MAL is functionally deregulated. We showed that OTT-MAL is a constitutive activator of SRF and target gene expression. This requires the SRF binding motif and the MAL-derived transactivation domain. OTT-MAL localises to the nucleus and is not regulated by upstream signalling. OTT-MAL deregulation reflects its independence from control by G-actin, which fails to interact with OTT-MAL in co-immunoprecipitation experiments. OTT-MAL also caused a delayed induction of the MAL-independent, TCF-dependent target genes c-fos and egr-1, and the MAPK/Erk pathway. In addition, RBPJ/CBF-1 regulated gene expression was activated by OTT-MAL but not by MAL. Our data suggest that the deregulated activation of MAL-dependent and independent promoters results in tissue-specific functions of OTT-MAL. When tested in heterologous tissue culture systems, however, we observed strong anti-proliferative effects of OTT-MAL. Similarly, overexpression of MAL exhibited antiproliferative and pro-apoptotic effects requiring transcription through SRF. To gain insight into the molecular mechanisms involved, we performed gene expression analysis. By using a combination of actin binding drugs, which specifically interfere with the actin-MAL complex, we identified on a genome wide basis 210 genes primarily regulated by G-actin. We found many known MAL-dependent SRF target genes, as well as novel directly regulated genes. Several putative antiproliferative target genes were newly identified. We showed that a group of MAL regulated genes negatively interferes with the EGFR-MAPK pathway, thereby reducing proliferative signalling. Our results show the existence of negatively acting transcriptional networks between pro- and antiproliferative signalling pathways towards subsets of SRF target genes.

Palmitoylation of Cytoskeleton Associated Protein 4 by DHHC2 Regulates Antiproliferative Factor-mediated Signaling

Previously, we identified cytoskeleton-associated protein 4 (CKAP4) as a major substrate of the palmitoyl acyltransferase, DHHC2, using a novel proteomic method called palmitoyl-cysteine identification, capture and analysis (PICA). CKAP4 is a reversibly palmitoylated and phosphorylated protein that links the ER to the cytoskeleton. It is also a high-affinity receptor for antiproliferative factor (APF), a small sialoglycopeptide secreted from bladder epithelial cells of patients with interstitial cystitis (IC). The role of DHHC2-mediated palmitoylation of CKAP4 in the antiproliferative response of HeLa and normal bladder epithelial cells to APF was investigated. Our data show that siRNA-mediated knockdown of DHHC2 and consequent suppression of CKAP4 palmitoylation inhibited the ability of APF to regulate cellular proliferation and blocked APF-induced changes in the expression of E-cadherin, vimentin, and ZO-1 (genes known to play a role in cellular proliferation and tumorigenesis). Immunocytochemistry revealed that CKAP4 palmitoylation by DHHC2 is required for its trafficking from the ER to the plasma membrane and for its nuclear localization. These data suggest an important role for DHHC2-mediated palmitoylation of CKAP4 in IC and in opposing cancer-related cellular behaviors and support the idea that DHHC2 is a tumor suppressor.

Protein Kinase A-mediated Gating of Neuregulin-dependent ErbB2-ErbB3 Activation Underlies the Synergistic Action of cAMP on Schwann Cell Proliferation

In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) enhances the action of neuregulin, the most potent known mitogen for SCs, by synergistically increasing the activation of two crucial signaling pathways: ERK and Akt. However, the underlying mechanism of cross-talk between neuregulin and cAMP signaling remains mostly undefined. Here, we report that the activation of protein kinase A (PKA), but not that of exchange protein activated by cAMP (EPAC), enhances S-phase entry of SCs by synergistically enhancing the ligand-dependent tyrosine phosphorylation/activation of the neuregulin co-receptor, ErbB2-ErbB3. The role of PKA in neuregulin-ErbB signaling was confirmed using PKA inhibitors, pathway-selective cAMP analogs, and natural ligands stimulating PKA activity in SCs, such as adenosine and epinephrine. Two basic observations defined the synergistic action of PKA as "gating" for neuregulin-ErbB signaling: 1) the activation of PKA was not sufficient to induce S-phase entry or the activation of either ErbB2 or ErbB3; and 2) the presence of neuregulin was strictly required to ignite ErbB activation and thereby ERK and Akt signaling. However, PKA directly phosphorylated ErbB2 on Thr-686, a highly conserved intracellular regulatory site that was required for the PKA-mediated synergistic enhancement of neuregulin-induced ErbB2-ErbB3 activation and proliferation in SCs. The gating action of PKA on neuregulin-induced ErbB2-ErbB3 activation has important biological significance, because it insures signal amplification into the ERK and Akt pathways without compromising either the neuregulin dependence or the high specificity of ErbB signaling pathways.

Preclinical Analysis of the Combined Activity of SGN-40, Anti-CD40 Monoclonal Antibody, with Rituximab in Non-Hodgkin Lymphoma.

SGN-40 is a humanized monoclonal antibody against CD40, a TNF receptor family member expressed in non-Hodgkin lymphoma (NHL), multiple myeloma, and several carcinomas. SGN-40 is cytotoxic to NHL cell lines via activation of proapoptotic signal transduction pathways and mediates antibody dependent cellular cytotoxicity (ADCC) effector function activity. In this study we examined the anti-tumor activity of SGN-40 in combination with the anti-CD20 antibody, rituximab, in lymphoma cell line models. Cell proliferation data (3H-thymidine incorporation assay) was generated for SGN-40 and rituximab alone and in combination for three NHL cell lines (Ramos, RL, and SU-DHL-4) and combination index (CI) analyses performed. SGN-40 was reproducibly synergistic with rituximab in Ramos cells and additive in the RL and SU-DHL-4 cell lines in this assay. This suggested that different anti-proliferative signaling events are activated by these antibodies, which produce a greater anti-tumor effect when combined. To better understand the combined activity of SGN-40 and rituximab, the signal transduction pathways activated by each antibody were examined. In Ramos cells SGN-40 signaling caused the degradation of pro-survival BCL-6 oncoprotein and upregulation of TAp63α, a proapoptic p53 family member, while only BCL-6 degradation was triggered in the RL and SU-DHL-4 cell lines. In contrast, rituximab signaling degraded BCL-6 protein in only one cell line (SU-DHL-4), and did not upregulate TAp63α expression in any of the cell lines examined. To further define the combined activity of SGN-40 with rituximab the effector function activity of both antibodies were examined in vitro. ADCC assays in the WIL2-S and Raji cell lines both showed a greater percent cell lysis in the presence of both SGN-40 and rituximab compared to either drug alone. Next, the SGN-40 and rituximab were tested in subcutaneous mouse models of NHL to evaluate this combination in vivo. In a Ramos model, SGN-40 and rituximab (dosed at 4.0mg/kg, q4dx4, ip) had significantly greater anti-tumor response when combined compared to the equivalent dose of either antibody alone. The anti-tumor response achieved with dual dosing of SGN-40 and rituximab was greater than the response expected if the combination was additive. Our data suggests that the improved efficacy of SGN-40, rituximab combination therapy in vivo is due to distinct apoptotic signaling pathways activated by these two antibodies in addition to augmented effector function activity. The combination of SGN-40 and rituximab is currently being studied in clinical trials of NHL.

HDAC4 represses p21WAF1/Cip1 expression in human cancer cells through a Sp1-dependent, p53-independent mechanism

Cancer cells have complex, unique characteristics that distinguish them from normal cells, such as increased growth rates and evasion of anti-proliferative signals. Global inhibition of class I and II histone deacetylases (HDACs) stops cancer cell proliferation in vitro and has proven effective against cancer in clinical trials, at least in part, through transcriptional reactivation of the p21(WAF1/Cip1)gene. The HDACs that regulate p21(WAF1/Cip1) are not fully identified. Using small interfering RNAs, we found that HDAC4 participates in the repression of p21(WAF1/Cip1) through Sp1/Sp3-, but not p53-binding sites. HDAC4 interacts with Sp1, binds and reduces histone H3 acetylation at the Sp1/Sp3 binding site-rich p21(WAF1/Cip1) proximal promoter, suggesting a key role for Sp1 in HDAC4-mediated repression of p21(WAF1/Cip1). Induction of p21(WAF1/Cip1) mediated by silencing of HDAC4 arrested cancer cell growth in vitro and inhibited tumor growth in an in vivo human glioblastoma model. Thus, HDAC4 could be a useful target for new anti-cancer therapies based on selective inhibition of specific HDACs.

Capillary Sprout Endothelial Cells Exhibit a CD36 low Phenotype : Regulation by Shear Stress and Vascular Endothelial Growth Factor-Induced Mechanism for Attenuating Anti-Proliferative Thrombospondin-1 Signaling

Endothelial cells acquire distinctive molecular signatures in their transformation to an angiogenic phenotype that are indicative of changes in cell behavior and function. Using a rat mesentery model of inflammation-induced angiogenesis and a panel of known endothelial markers (CD31, VE-cadherin, BS-I lectin), we identified a capillary sprout-specific endothelial phenotype that is characterized by the marked down-regulation of CD36, a receptor for the anti-angiogenic molecule thrombospondin-1 (TSP-1). TSP-1/CD36 interactions were shown to regulate angiogenesis in this model as application of TSP-1 inhibited angiogenesis and blockade of both TSP-1 and CD36 accelerated angiogenesis. Vascular endothelial growth factor, which was up-regulated in the in vivo model, elicited a dose- and time-dependent down-regulation of CD36 (ie, to a CD36 low phenotype) in cultured human umbilical vein endothelial cells. Human umbilical vein endothelial cells that had been conditioned to a CD36 low phenotype with VEGF were found to be refractory to anti-proliferative TSP-1 signaling via a CD36-dependent mechanism. The loss of exposure to wall shear stress, which occurs in vivo when previously quiescent cells begin to sprout, also generated a CD36 low phenotype. Ultimately, our results identified the regulation of endothelial cell CD36 expression as a novel mechanism through which VEGF stimulates and sustains capillary sprouting in the presence of TSP-1. Additionally, CD36 was shown to function as a potential molecular linkage through which wall shear stress may regulate both microvessel sprouting and quiescence.

Der Transforming Growth Factor-Beta (TGF-β)-Signalweg und seine Bedeutung beim malignen Melanom

Transforming growth factor-β is a potent growth inhibitor for normal melanocytes. This function is lost in the course of tumorigenesis, since several melanoma cell lines are only slightly or not at all inhibited by TGF-β. In melanoma, the transduction of antiproliferative signals by TGF-β is often abolished, and the autocrine production of TGF-β increased. By this way, several TGF-β-driven paracrine effects, such as extracellular matrix remodeling, neoangiogenesis, and immunosuppression induce local tumor growth and metastasis. The interaction of Smads, the major TGF-β signaling proteins, with other signaling systems such as the mitogen-activated protein kinases, the SKI/SnoN proteins, and the protein kinase C family, possibly contributes to the escape of melanoma cells from TGF-β growth control. Therefore, the clarification of the molecular interactions of the TGF-β signaling pathway may further promote the development of new treatment concepts for melanoma.

Mammary epithelial cell transformation is associated with deregulation of the vitamin D pathway

The vitamin D endocrine system mediates anti-proliferative and pro-differentiating signaling in multiple epithelial tissues, including mammary gland and breast tumors. The vitamin D metabolite 1alpha,25(OH)2D3 mediates growth inhibitory signaling via activation of the vitamin D receptor (VDR), a ligand dependent transcription factor. 1alpha,25(OH)(2)D3 is synthesized from 25(OH)D3 (the major circulating form of the vitamin) by the mitochondrial enzyme CYP27b1 in renal and other tissues. Human mammary epithelial (HME) cells express VDR and CYP27b1 and undergo growth inhibition when exposed to physiological concentrations of 25(OH)D3, suggesting that autocrine or paracrine vitamin D signaling contributes to maintenance of differentiation and quiescence in the mammary epithelium. In the current studies we tested the hypothesis that cancer cells would exhibit reduced sensitivity to vitamin D mediated negative growth regulation. We used a series of progressively transformed HME cell lines expressing known oncogenic manipulations to study the effects of transformation per se on the vitamin D pathway. We report that mRNA and protein levels of VDR and CYP27b1 were reduced greater than 70% upon stable introduction of known oncogenes (SV40 T antigens and H-rasV12) into HME cells. Oncogenic transformation was also associated with reduced 1alpha,25(OH)2D3 synthesis, and cellular sensitivity to growth inhibition by 1alpha,25(OH)2D3 and 25(OH)D3 was decreased approximately 100-fold in transformed cells. These studies provide evidence that disruption of the vitamin D signaling pathway occurs early in the cancer development process.

Der Transforming Growth Factor-Beta (TGF-β)-Signalweg und seine Bedeutung beim malignen Melanom

Der Transforming Growth Factor-β (TGF-β) stellt einen potenten Wachstumsinhibitor bei normalen Melanozyten dar. Diese Funktion scheint im Verlauf der Tumorgenese zunehmend verloren zu gehen, da viele Melanomzellen durch TGF-β nur sehr schwach oder gar nicht gehemmt werden. Beim Melanom wird der antiproliferative Signalweg des TGF-β häufig aufgehoben, und die Produktion des TGF-β autokrin hochreguliert. Somit kommt es zu einer Reihe parakriner Effekte, wie des extrazellulären Matrixumbaus, der Neoangiogenese, und der Immunsuppression, die letztendlich zum lokalen Tumorwachstum und zur Metastasierung führen. Die Wechselwirkungen der TGF-β-signalübertragenden Smad Proteine mit anderen Signalsystemen, wie der mitogenaktivierten Proteinkinasen, des SKI/SnoN und des Proteinkinase-C-Systems, tragen möglicherweise zum Entweichen der Melanomzellen von der TGF-β-Wachstumskontrolle bei. Die Abklärung der molekularen Interaktionen des TGF-β-Signalweges wird möglicherweise zu der Entwicklung neurer Konzepte für die Therapie des malignen Melanoms beitragen.

Differential cytotoxicity of novel somatostatin and dopamine chimeric compounds on bronchopulmonary and small intestinal neuroendocrine tumor cell lines.

Survival rates for gastrointestinal (GI) and bronchopulmonary (BP) neuroendocrine tumors (NETs) have not significantly altered (overall 67%, 5-year survival) in 30 years (1973-2004), whereas the incidence has increased ( approximately 1000%) in the same time frame. No effective or specific antineoplastic agent is available for treatment, although somatostatin analogs inhibit tumor secretion. Given the coexistence of somatostatin and dopamine regulatory receptors on NET cells, the antiproliferative efficacy as well as the signaling and transcriptional targets of their ligands were evaluated. The cytotoxic effects of 12 somatostatin/dopamine compounds were evaluated in 3 NET cell lines, and real-time polymerase chain reaction and enzyme-linked immunoadsorbent assay studies were performed to delineate antiproliferative signaling pathways. The atypical BP-NET, NCI-H720, was most sensitive to the sst(5) analog BIM23206 (half-maximal concentration, 2.4 pM) and demonstrated similar sensitivity to lanreotide and the sst(2) analog BIM23120. The typical BP-NET, NCI-H727, was most sensitive to BIM23120 (0.7 nM) and to the pan-somatostatin receptor analog (BIM23A779). The GI-NET, KRJ-I, was most sensitive to sst(2,5) analogs lanreotide (1 nM) and BIM23244 (7.4 nM). Lanreotide activated extracellular signal regulated kinase-1/2 phosphorylation and p21(WAF1/CIP1) transcription, but inhibited Ki-67 transcription. NCI-H720 was most sensitive to the sst(2,5)- and D(2)-selective compound BIM23A761 (4.2 nM), as was NCI-H727 (5.5 nM). KRJ-I did not respond to any chimeric analog. BIM23A761 activated c-Jun N-terminal kinase signaling and caused inhibition of Ki-67 transcription. P21(WAF1/CIP1) transcription was activated only in NCI-H727 cells. The different responses of each individual cell line suggested that NETs from different locations arising from different neuroendocrine cells may require cell-specific antiproliferative agents based on the unique receptor profile of individual lesions.

Enterotoxin preconditioning restores calcium-sensing receptor-mediated cytostasis in colon cancer cells

Guanylyl cyclase C (GCC), the receptor for diarrheagenic bacterial heat-stable enterotoxins (STs), inhibits colorectal cancer cell proliferation by co-opting Ca(2+) as the intracellular messenger. Similarly, extracellular Ca(2+) (Ca(2+)(o)) opposes proliferation and induces terminal differentiation in intestinal epithelial cells. In that context, human colon cancer cells develop a phenotype characterized by insensitivity to cytostasis imposed by Ca(2+)(o). Here, preconditioning with ST, mediated by GCC signaling through cyclic nucleotide-gated channels, restored Ca(2+)(o)-dependent cytostasis, reflecting posttranscriptional regulation of calcium-sensing receptors (CaRs). ST-induced GCC signaling deployed CaRs to the surface of human colon cancer cells, whereas elimination of GCC signaling in mice nearly abolished CaR expression in enterocytes. Moreover, ST-induced Ca(2+)(o)-dependent cytostasis was abrogated by CaR-specific antisense oligonucleotides. Importantly, following ST preconditioning, newly expressed CaRs at the cell surface represented tumor cell receptor targets for antiproliferative signaling by CaR agonists. Since expression of the endogenous paracrine hormones for GCC is uniformly lost early in carcinogenesis, these observations offer a mechanistic explanation for the Ca(2+)(o)-resistant phenotype of colon cancer cells. Restoration of antitumorigenic CaR signaling by GCC ligand replacement therapy represents a previously unrecognized paradigm for the prevention and treatment of human colorectal cancer employing dietary Ca(2+) supplementation.

The CCN3 gene coding for an extracellular adhesion-related protein is transcriptionally activated by the p53 tumor suppressor

The CCN3 protein (Nov, Nephroblastoma overexpressed) is a member of the CCN family (Cyr61, CTGF, Nov) of growth regulators and exerts antiproliferative properties. We show here that the tumor suppressor p53 transcriptionally upregulates the CCN3 gene. p53 is an important transcription factor contributing to cell cycle arrest and apoptosis after cell damage through the regulation of numerous target genes. We show that CCN3 mRNA and protein are upregulated following p53 expression. A DNA binding-deficient p53 mutant fails to regulate CCN3. CCN3 protein is located in the perinuclear space after induction and is also exported to the extracellular matrix. Furthermore, the CCN3 promoter is inducible by p53 and the response element is located in the first exon of the CCN3 gene. Chromatin immunoprecipitations show that p53 binds to the CCN3 promoter in vivo. As CCN3 was shown to inhibit cell growth, transcriptional induction by p53 may serve as an antiproliferative signal in the extracellular matrix. Furthermore, CCN3 depletion was also reported to reduce collagen type IV-dependent adhesion of melanocytes. Thus, elevated levels of CCN3 protein regulated by p53 might influence cell adhesion.

Molecular cloning and expression analysis of interferon-inducible transmembrane protein 1 in large yellow croaker Pseudosciaena crocea

In human cells, interferon-inducible transmembrane protein 1 (IFITM1) is a component of protein complexes involved in homotypic adhesion and the transduction of antiproliferative signals. Here, we reported the cloning of an IFITM1 homologue from the spleen of large yellow croaker Pseudosciaena crocea (LycIFITM1). The complete cDNA of LycIFITM1 is 734 nucleotides (nt) encoding a protein of 124 amino acids (aa), with a putative molecular weight of 13.6 kDa. The deduced LycIFITM1 protein is significantly homologous to interferon-inducible transmembrane proteins (IFITMs) in mammals and fish, and has the typical structural features of IFITMs, including two transmembrane domains (residues 43–63 and 90–112, respectively) and one intracellular domain between them (residues 64–89), as well as one conserved protein kinase C (PKC) phosphorylation site (residues 65–67, SIK). Phylogenetic analysis showed that LycIFITM1 formed a cluster with fish IFITM, reflecting a relative distant evolutionary relationship from mammals. LycIFITM1 gene was constitutively expressed in various tissues examined, such as gills, intestine, liver, kidney, heart, spleen, muscle and blood. Upon induction with poly(I:C), LycIFITM1 gene expression was obviously up-regulated in gills, kidney, heart and spleen at 24 h after stimulation, suggesting that LycIFITM1 may be involved in the immune response induced by poly(I:C). Time course analysis using real-time PCR showed that the mRNA levels of LycIFITM1 in spleen and kidney were quickly up-regulated by poly(I:C) and reached the peak at 24 h post-induction (48.7- and 280.4-fold mRNA increases in spleen and kidney, respectively). The results suggest that the IFITM1 homologue from large yellow croaker may represent a novel member of IFITMs family in fish.

Telomeres, senescence, and hematopoietic stem cells

The replicative lifespan of normal somatic cells is restricted by the erosion of telomeres, which are protective caps at the ends of linear chromosomes. The loss of telomeres induces antiproliferative signals that eventually lead to cellular senescence. The enzyme complex telomerase can maintain telomeres, but its expression is confined to highly proliferative cells such as stem cells and tumor cells. The immense regenerative capacity of the hematopoietic system is provided by a distinct type of adult stem cell: hematopoietic stem cells (HSCs). Although blood cells have to be produced continuously throughout life, the HSC pool seems not to be spared by aging processes. Indeed, limited expression of telomerase is not sufficient to prevent telomere shortening in these cells, which is thought ultimately to limit their proliferative capacity. In this review, we discuss the relevance of telomere maintenance for the hematopoietic stem cell compartment and consider potential functions of telomerase in this context. We also present possible clinical applications of telomere manipulation in HSCs and new insights affecting the aging of the hematopoietic stem cell pool and replicative exhaustion.

High nerve growth factor receptor (p75NTR) expression is a favourable prognostic factor in paediatric B cell precursor‐acute lymphoblastic leukaemia

Nerve growth factor (NGF) plays a pivotal role in cellular survival/death decisions with the low affinity receptor p75NTR predominately transmitting anti-proliferative signals. In spite of its established role in B-cell function and identification as a prognostically favourable marker in a number of malignancies, little is known about the expression pattern and prognostic significance of p75NTR in B cell precursor-acute lymphoblastic leukaemia (BCP-ALL). p75NTR expression was prospectively studied on primary ALL-blasts in a cohort of paediatric patients with common ALL (n = 86) and preB-ALL (n = 34) treated within the Co-operative study group for childhood acute lymphoblastic leukaemia (CoALL) protocol, CoALL06-97. Flow cytometric analysis showed that almost half of the patients expressed no or negligible amounts of p75NTR (<10%). The median expression in patients expressing p75NTR beyond that threshold was 49% (range 11-100%). In patients classified as low-risk at diagnosis, p75NTR expression was significantly higher than in high-risk patients (P = 0.001). Of note, p75NTR expression was lower in the 21 patients who subsequently developed relapse compared with those remaining in remission (P = 0.038). Accordingly, relapse-free survival was significantly better in patients expressing high surface p75NTR (P = 0.041). Thus, in this prospective analysis, high p75NTR expression was a strong prognostic marker that identified a group of paediatric ALL patients with favourable outcome.

Somatostatin/somatostatin receptor signalling: Phosphotyrosine phosphatases

Activation of phosphotyrosine phosphatases (PTPs) by somatostatin receptor (SSTR) represents one of the main intracellular mechanisms involved in the antiproliferative effect of somatostatin (SST) and analogues. Since their molecular cloning, the role of PTPs is emerging as a major regulator of different cell functions including cell proliferation, differentiation, cell to cell interactions, cell matrix adhesion and cell migration. It was demonstrated that PTPs possess high substrate specificity and their activity is tightly regulated. Importantly, different G protein-coupled receptors transduce their biological activities through PTPs. PTPs were identified as down-stream effectors of SSTRs to transduce antiproliferative signals, and so far, three family members (SHP-1, SHP-2 and DEP-1/PTPeta) have been identified as selective SSTR intracellular effectors. Here, the molecular mechanisms leading SSTRs to regulate PTP activity are discussed, focusing on recent data showing a close interplay between PTPs and tyrosine kinases to transduce tumoral cell growth arrest following SST analogs administration.

Toll-like Receptor 3 Expressed by Melanoma Cells as a Target for Therapy?

The immunomodulatory properties of Toll-like receptors (TLR) agonists have inspired their use as experimental adjuvants for vaccination of cancer patients. However, it is now well recognized that TLR expression is not restricted to immune cells but can also be found in many cell types, including those giving rise to tumors. It is therefore mandatory to explore the potential effects of TLR triggering directly on tumor cells. In the present work, we have investigated TLR3 protein expression in melanoma cell lines derived from patients, and analyzed the effects of TLR3 agonists on tumor cell survival. Moreover, we used RNA interference to stably knock down TLR3 expression and study the involvement of this receptor in dsRNA-induced effects on melanoma cells viability. Human melanoma cells can express functional TLR3 protein. Interestingly, the engagement of the receptor by TLR3 agonists can directly inhibit cell proliferation and induce tumor cell death when combined to treatment with either type I IFN or protein synthesis inhibitors. These effects were shown by RNA interference to be largely dependent on TLR3. Moreover, TLR3-mediated cell death involves the activation of caspases and engages both extrinsic and intrinsic apoptotic pathways. TLR3 protein can be expressed in human melanoma cells, where it can deliver proapoptotic and antiproliferative signaling. Altogether, these results suggest that TLR3 agonists represent very promising adjuvants for cancer vaccines not only based on their well-described immunostimulatory properties, but also due to their newly identified cytostatic and cytotoxic effects directly on tumor cells.