Lucia Cappabianca

Thioredoxin stimulates MMP‐9 expression, de‐regulates the MMP‐9/TIMP‐1 equilibrium and promotes MMP‐9 dependent invasion in human MDA‐MB‐231 breast cancer cells

Increased expression of thioredoxin (Trx)‐1 and matrix metalloproteinase (MMP)‐9 associates with malignant breast cancer progression. Here, we describe a functional relationship between Trx‐1 and MMP‐9 in promoting MDA‐MB‐231 breast cancer cell invasive behaviour. Trx‐1 overexpression stimulated MMP‐9 expression, de‐regulated the MMP‐9/TIMP‐1 equilibrium and augmented MMP‐9 involvement in a more invasive phenotype. Trx‐1 augmented MMP‐9 transcription through NF‐κB, AP‐1 and SP1 elements; stimulated p50/p65 NF‐κB activity and recruitment to the MMP‐9 promoter; and facilitated MMP‐9 promoter‐accessibility to NF‐κB by preventing HDAC recruitment and maintaining MMP‐9 promoter histone acetylation. Our data provide a functional basis for Trx‐1 and MMP‐9 association in malignant breast cancer and identify Trx‐1 and NF‐κB as potentially druggable targets for reducing MMP‐9 involvement in malignant behaviour.

TrkAIII. A novel hypoxia-regulated alternative TrkA splice variant of potential physiological and pathological importance.

Nerve growth factor receptor TrkA is critical for development and maturation of central and peripheral nervous systems, regulating proliferation, differentiation and apoptosis. In cancer, TrkA frequently exhibits suppressor activity in non-mutated form and oncogenic activity upon mutation. Our identification of a novel hypoxia-regulated alternative TrkAIII splice variant, expressed by neural crest-derived neuroblastic tumors, that exhibits neuroblastoma tumor promoting activity, adds significantly to our understanding of potential TrkA involvement in cancer. Our observation that hypoxia, which characterises the tumor micro-environment, stimulates alternative TrkAIII splicing, provides a way by which TrkA tumor suppressing signals may convert to tumor promoting signals during progression and is consistent with conservation and pathological subversion by neural crest-derived neuroblastic tumors of a mechanism of potential physiological importance to normal neural stem/neural crest progenitors.

Nature of the Accessible Chromatin at a Glucocorticoid-Responsive Enhancer

ABSTRACT To gain a better understanding of the nature of active chromatin in mammals, we have characterized in living cells the various chromatin modification events triggered by the glucocorticoid receptor (GR) at the rat tyrosine aminotransferase gene. GR promotes a local remodeling at a glucocorticoid-responsive unit (GRU) located 2.5 kb upstream of the transcription start site, creating nuclease hypersensitivity that encompasses 450 bp of DNA. Nucleosomes at the GRU occupy multiple frames that are remodeled without nucleosome repositioning, showing that nucleosome positioning is not the key determinant of chromatin accessibility at this locus. Remodeling affects nucleosomes and adjacent linker sequences, enhancing accessibility at both regions. This is associated with decreased interaction of both the linker histone H1 and the core histone H3 with DNA. Thus, our results indicate that nucleosome and linker histone removal rather than nucleosome repositioning is associated with GR-triggered accessibility. Interestingly, GR induces hyperacetylation of histones H3 and H4, but this is not sufficient either for remodeling or for transcriptional activation. Finally, our data favor the coexistence of several chromatin states within the population, which may account for the previously encountered difficulties in characterizing unambiguously the active chromatin structure in living cells.

RETINOIC ACID-ENHANCED INVASION THROUGH RECONSTITUTED BASEMENT MEMBRANE BY HUMAN SK-N-SH NEUROBLASTOMA CELLS INVOLVES MEMBRANE-ASSOCIATED TISSUE-TYPE PLASMINOGEN ACTIVATOR

Al‐trans retinoic acid (RA) enhanced human, S‐type, SK‐N‐SH neuroblastoma cell invasion of reconstituted basement membrane in vitro but did not induce terminal differentiation of this cell line. In contrast to basal invasion, which was urokinase (uPA)‐ and plasmin‐dependent, RA‐enhanced invasion was dependent on tissue‐type plasminogen activator (t‐PA) and plasmin activity. Neither basal nor RA‐enhanced invasion involved TIMP‐2 inhibitable metalloproteinases. Enhanced invasion was associated with the induction of t‐PA expression, increased expression of the putative t‐PA receptor amphoterin, increased association of t‐PA with cell membranes and increased net membrane‐associated PA activity. Enhanced invasion was not associated with significant changes in the expression of uPA or its membrane receptor UPAR; plasminogen activator inhibitors PAI‐1 and PAI‐2; metalloproteinases MMP‐1, MMP‐2, MMP‐3, MMP‐9 and membrane type MMP1; or tissue inhibitors of metalloproteinases TIMP‐1 and TIMP‐2. RA stimulated the association of t‐PA with the external cell membrane surface, which could be inhibited by heparin sulphate but not by mannose sugars or chelators of divalent cations, consistent with a role for amphoterin. Our data indicate that RA can promote the malignant behavior of S‐type neuroblastoma cells refractory to RA‐mediated terminal differentiation by enhancing their basement membrane invasive capacity. We suggest that this results from the action of a novel, RA‐regulated mechanism involving stimulation of t‐PA expression and its association with the cell membrane leading to increased PA‐dependent matrix degradation. Int. J. Cancer 73:740–748, 1997.

Alternative TrkAIII splicing: a potential regulated tumor-promoting switch and therapeutic target in neuroblastoma

An association between elevated tyrosine kinase receptor (Trk)-A expression and better prognosis; the absence of mutation-activated TrkA oncogenes; the induction of apoptosis, growth arrest, morphological differentiation and inhibition of xenograft growth; and angiogenesis by TrkA gene transduction, provide the basis for the current concept of an exclusively tumor-suppressor role for TrkA in the aggressive pediatric tumor, neuroblastoma. This concept, however, has recently been challenged by the discovery of a novel hypoxia-regulated alternative TrkAIII splice variant, initial data for which suggest predominant expression in advanced-stage neuroblastoma. TrkAIII exhibits neuroblastoma xenograft tumor-promoting activity associated with the induction of a more angiogenic and stress-resistant neuroblastoma phenotype and antagonises nerve growth factor/TrkAI antioncogenic signaling. In this short review, the authors integrate this novel information into a modified concept that places alternative TrkA splicing as a potential pivotal regulator of neuroblastoma behavior and identifies the TrkAIII alternative splice variant as a potential biomarker of patient prognosis and novel therapeutic target.

The neuroblastoma tumour-suppressor TrkAI and its oncogenic alternative TrkAIII splice variant exhibit geldanamycin-sensitive interactions with Hsp90 in human neuroblastoma cells.

Hsp90 chaperones stabilize many tyrosine kinases including several oncogenes, which are inhibited or induced to degrade by the Hsp90 inhibitor geldanamycin (GA). As a consequence, GA has been developed for future chemotherapeutic use in several tumour types including neuroblastoma (NB). Alternative splicing of the neurotrophin receptor tyrosine kinase TrkA may have a pivotal function in regulating NB behaviour, with reports suggesting that tumour-suppressing signals from TrkA may be converted to oncogenic signals by stress-regulated alternative TrkAIII splicing. Within this context, it is important to know whether Hsp90 interacts with TrkA variants in NB cells and how GA influences this. Here, we report that both TrkAI and TrkAIII are Hsp90 clients in human NB cells. TrkAI exhibits GA-sensitive interaction with Hsp90 required for receptor endoplasmic reticulum export, maturation, cell surface stabilization and ligand-mediated activation, whereas TrkAIII exhibits GA-sensitive interactions with Hsp90 required for spontaneous activity and to a lesser extent stability. We show that GA inhibits proliferation and induces apoptosis of TrkAI expressing NB cells, whereas TrkAIII reduces the sensitivity of NB cells to GA-induced elimination. Our data suggest that GA-sensitive interactions with Hsp90 are critical for both TrkAI tumour suppressor and TrkAIII oncogenic function in NB and that TrkAIII expression exerts a negative impact on GA-induced NB cell eradication, which can be counteracted by a novel TrkAIII-specific peptide nucleic acid inhibitor.

The alternative TrkAIII splice variant targets the centrosome and promotes genetic instability.

ABSTRACT The hypoxia-regulated alternative TrkAIII splice variant expressed by human neuroblastomas exhibits oncogenic potential, driven by in-frame exon 6 and 7 alternative splicing, leading to omission of the receptor extracellular immunoglobulin C1 domain and several N-glycosylation sites. Here, we show that the TrkAIII oncogene promotes genetic instability by interacting with and exhibiting catalytic activity at the centrosome. This function depends upon intracellular TrkAIII accumulation and spontaneous interphase-restricted activation, in cytoplasmic tyrosine kinase (tk) domain orientation, predominantly within structures that closely associate with the fully assembled endoplasmic reticulum intermediate compartment and Golgi network. This facilitates TrkAIII tk-mediated binding of γ-tubulin, which is regulated by endogenous protein tyrosine phosphatases and geldanamycin-sensitive interaction with Hsp90, paving the way for TrkAIII recruitment to the centrosome. At the centrosome, TrkAIII differentially phosphorylates several centrosome-associated components, increases centrosome interaction with polo kinase 4, and decreases centrosome interaction with separase, the net results of which are centrosome amplification and increased genetic instability. The data characterize TrkAIII as a novel internal membrane-associated centrosome kinase, unveiling an important alternative mechanism to “classical” cell surface oncogenic receptor tk signaling through which stress-regulated alternative TrkAIII splicing influences the oncogenic process.

TrkAIII expression in the thymus.

The alternative TrkAIII splice variant is expressed by murine and human thymus. Alternative TrkAIII splicing predominates in postembryonic day E13 (E17 and E18), postnatal murine (3 week and 3 month) and human thymuses, with TrkAIII mRNA expressed by selected thymocyte subsets and thymic epithelial cells (TECs) and a 100 kDa immunoprecipitable TrkAIII-like protein detected in purified thymocyte and whole thymus extracts. FACS and immunohistochemical analysis indicate a non-cell surface localisation for the TrkAIII-like protein in cortical CD4+/CD8+ double positive and, to a lesser extent, single positive thymocyte subsets at the cortex/medulla boundary and in Hassles corpuscles, reticular epithelial and dendritic cells of the thymic medulla. TrkA(I/II) expression, on the other hand, predominates in sub-capsular regions of the thymus. TrkAIII-like immunoreactivity at the cortex/medulla boundary associates with regions of thymocyte proliferation and not apoptosis. A potential role for thymic hypoxia in thymocyte alternative TrkAIII splicing is supported by reversal to TrkAI splicing by normoxic but not hypoxic culture and induction of Jurkat T cell alternative TrkAIII splicing by the hypoxia mimic CoCl2. In contrast, TEC expression of TrkAIII predominates in both normoxic and hypoxic culture conditions. The data support a potential role for TrkAIII in thymic development and function, of particular relevance to intermediate stage CD4+/CD8+ thymocyte subsets and TECs, which potentially reflects a reversible thymocyte and more permanent TEC adaptation to thymic environment. Since intracellular TrkAIII neither binds nor responds to NGF and can impede regular NGF/TrkA signalling (Tacconelli et al., Cancer Cell, 2004), its expression would be expected to provide an alternative and/or impediment to regular NGF/TrkA signalling within the developing and developed thymus of potential functional importance.

Constitutive autotaxin transcription by Nmyc-amplified and non-amplified neuroblastoma cells is regulated by a novel AP-1 and SP-mediated mechanism and abrogated by curcumin

The motility, angiogenesis and metastasis‐stimulating factor Autotaxin (Atx), over expressed by human neuroblastomas (NB), is constitutively expressed by human Nmyc‐amplified SK‐N‐BE and non‐Nmyc‐amplified SH‐SY5Y NB cells. Here, we characterise a novel Atx transcriptional mechanism, utilised by both cell lines, that is restricted to the first 285 bp of the Atx promoter and involves AP‐1 and SP transcription factors, acting through a CRE/AP‐1‐like element at position −142 to −149 and a GAbox at position −227 to −235 relative to the Atx translational start site. This novel transcriptional mechanism can be inhibited by internally initiated SP‐3 and the natural phenol curcumin.

Reconstitution of TIMP-2 expression in SH-SY5Y neuroblastoma cells by 5-azacytidine is mediated transcriptionally by NF-Y through an inverted CCAAT site.

Advanced stage neuroblastomas (NB) exhibit a tissue inhibitor of metalloproteinase (TIMP)-2/matrix metalloproteinase (MMP) imbalance, considered a prerequisite for MMP involvement in tumor progression in vivo. Human SH-SY5Y NB cells exhibit a similar TIMP-2/MMP imbalance that promotes in vitro invasive behavior that is inhibited by exogenous TIMP-2. The DNA methyltransferase inhibitor 5-azacytidine (5-AzaC) redresses this TIMP-2/MMP imbalance, reconstituting TIMP-2 expression, without effecting that of MMP-2, by stimulating TIMP-2 transcription and inhibiting in vitro invasivity of SH-SY5Y cells. 5-AzaC stimulated transcription from a nonmethylated TIMP-2 promoter reporter gene construct consistent with regulation of a TIMP-2 transactivator. Promoter deletion and point-mutation analysis localized this effect to an inverted CCAAT element at position -73. This element bound specific complexes containing NF-YA and NF-YB proteins in SH-SY5Y nuclear extracts, the binding of which was augmented by 5-AzaC in association with enhanced levels of NF-YB protein and the function of which was confirmed by inhibition using dominant-negative NF-YA. The data highlight a novel indirect methylation-mediated mechanism for regulating the TIMP/MMP equilibrium in NB cells, involving repression of TIMP-2 relative to MMP-2 expression, dependent upon suboptimal NF-Y transcription factor function, which can be reversed by methyltransferase inhibition.