Antonietta R. Farina

Transforming growth factor-β1 enhances the invasiveness of human MDA-MB-231 breast cancer cells by up-regulating urokinase activity

Transforming growth factor‐beta (TGFβ1) enhances human MDA‐MB‐231 breast tumour cell invasion of reconstituted basement membrane in vitro but does not inhibit proliferation of this cell line. In contrast to basal invasion, which is plasmin‐, urokinase (uPA)‐, tissue‐type plasminogen activator (t‐PA)‐, matrix metalloproteinase (MMP)‐9‐ and TIMP‐1‐inhibitable MMP‐dependent, TGFβ1 enhanced‐invasion is dependent upon plasmin and uPA activity but does not appear to involve t‐PA‐, MMP9‐ or TIMP‐1‐inhibitable MMPs, as judged by inhibitor studies. Enhanced invasion is associated with increased u‐PA, UPAR, PAI‐1, MT‐MMP‐1, MMP‐9 and TIMP‐1 expression; with reduced t‐PA, MMP‐1 and MMP‐3 expression; and with the induction of membrane MMP‐9 association. The net result of these changes includes increased secreted, but not membrane‐associated, uPA levels and activity and reduced secreted levels of plasmin and APMA‐activatable gelatinolytic, collagenolytic and caseinolytic MMP activity but no change in membrane‐associated gelatinolytic activity, despite increased MT‐MMP‐1 expression and MMP‐9 membrane association. TGFβ1 does not induce MMP‐2 expression. Our data indicate that TGFβ1 can promote the malignant behaviour of MDA‐MB‐231 cells refractory to TGFβ1‐mediated proliferation control by enhancing their invasive capacity. We suggest that this results from the action of a uPA/plasmin‐dependent mechanism resulting from stimulation of uPA expression, secretion and subsequent activity, despite elevated PAI‐1 inhibitor levels. Int. J. Cancer 75:721–730, 1998.© 1998 Wiley‐Liss, Inc.

Activation of MMP-2 by human GCT23 giant cell tumour cells induced by osteopontin, bone sialoprotein and GRGDSP peptides is RGD and cell shape change dependent

We show that osteopontin (OPN), bone sialoprotein (BSP) and GRGDSP peptides, in solution, induce activation of metalloproteinase‐2 (MMP‐2) secreted by human GCT23 giant cell tumour cells. Activation of MMP‐2 is RGD sequence dependent, possibly involves anti‐αVβ3 integrins, is preceded by a change from spread to rounded cell morphology and is mimicked by the actin depolymerising agent cytochalasin B. Cells that had spread on OPN, BSP and GRGDSP substrata failed to activate MMP‐2, but subsequent addition of soluble GRGDSP induced rounding and MMP‐2 activation. Activation induced by GRGDSP and cytochalasin B was cell mediated, inhibited by EDTA, tissue inhibitor of metalloproteinase‐2 (TIMP‐2) and carboxyl terminal MMP‐2 consistent with a role for membrane type (MT)‐MMP but did not involve urokinase, plasmin or thrombin activity. Activation induced by GRGDSP and cytochalasin B, but not cell rounding, was inhibited by herbimycin A, cycloheximide and actinomycin D, suggesting a role for tyrosine kinases, protein and RNA synthesis, but was not associated with changes in mRNA for MT‐MMP‐1, MMP‐1, MMP‐2, TIMP‐1 or TIMP‐2. GRGDSP and cytochalasin B enhanced levels of membrane‐associated pro‐ and active form MMP‐1 and MMP‐2 but not MT‐MMP‐1, stimulated cell surface MMP‐1 staining and induced that of MT‐MMP‐1, MMP‐2 and TIMP‐2. This was consistent with the possible relocation of constitutive MT‐MMP‐1 to the cell surface as a prerequisite for subsequent cell surface MMP‐2/TIMP‐2/MT‐MMP‐1 complex formation and to the potential induction of conditions favourable for reciprocal cell surface MMP‐1/MMP‐2 activation. Our data provide a novel insight into interactions between RGD containing bone matrices, GCT cells and MMPs of potential relevance to GCT pathology. Int. J. Cancer 77:82–93, 1998.© 1998 Wiley‐Liss, Inc.

Gelatinase B/MMP-9 in Tumour Pathogenesis and Progression

Since its original identification as a leukocyte gelatinase/type V collagenase and tumour type IV collagenase, gelatinase B/matrix metalloproteinase (MMP)-9 is now recognised as playing a central role in many aspects of tumour progression. In this review, we relate current concepts concerning the many ways in which gelatinase B/MMP-9 influences tumour biology. Following a brief outline of the gelatinase B/MMP-9 gene and protein, we analyse the role(s) of gelatinase B/MMP-9 in different phases of the tumorigenic process, and compare the importance of gelatinase B/MMP-9 source in the carcinogenic process. What becomes apparent is the importance of inflammatory cell-derived gelatinase B/MMP-9 in tumour promotion, early progression and triggering of the “angiogenic switch”, the integral relationship between inflammatory, stromal and tumour components with respect to gelatinase B/MMP-9 production and activation, and the fundamental role for gelatinase B/MMP-9 in the formation and maintenance of tumour stem cell and metastatic niches. It is also apparent that gelatinase B/MMP-9 plays important tumour suppressing functions, producing endogenous angiogenesis inhibitors, promoting inflammatory anti-tumour activity, and inducing apoptosis. The fundamental roles of gelatinase B/MMP-9 in cancer biology underpins the need for specific therapeutic inhibitors of gelatinase B/MMP-9 function, the use of which must take into account and substitute for tumour-suppressing gelatinase B/MMP-9 activity and also limit inhibition of physiological gelatinase B/MMP-9 function.

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.

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.

Multiple components are required for sequence recognition of the AP1 site in the gibbon ape leukemia virus enhancer.

At least two subunits contributed to the formation in vitro of a specific complex binding to the AP1 consensus sequence (TGAGTCA) in the gibbon ape leukemia virus (GALV) enhancer in MLA144 cells. This complex can be dissociated on a monomeric GALV oligonucleotide affinity column. One protein, termed the core protein, was retained on the oligonucleotide affinity column. The second protein flowed through the oligonucleotide affinity column and, when alone, did not bind to DNA; however, when present with the core protein, it bound strongly and very specifically to the GALV sequence. MLA144 cells contained only trace amounts of c-fos and c-jun by immunoblot analysis, suggesting that the proteins specifically binding to the GALV AP1 site were distinct from c-fos and c-jun. In addition to the major complex that recognized the GALV element, MLA144 cells contained a minor complex that is chromatographically different from and antigenically related to c-fos. The factor in the flowthrough complemented a human T-cell nuclear extract (Jurkat cell line), which, when alone, had no assayable complex that specifically bound to the GALV enhancer; this complementation gave rise to a specific complex similar to that seen in MLA144 cells. Together, these results suggest that the GALV enhancer can interact with multicomponent protein complexes in a cell-line-specific manner.

Autoimmune antigen Ku is enriched on oligonucleotide columns distinct from those containing the octamer binding protein DNA consensus sequence

During purification of the AP1 complex from the T cell line MLA144 we enriched for a complex which bound to an oligonucleotide column containing the AP1 DNA consensus sequence and co‐eluted with a fraction required for AP1 binding activity. This complex although co‐eluting with AP1 binding activity had previously been determined to be non‐specific in its DNA binding properties. Further investigation determined that the complex was a heterodimer of 85 and 70 kDa which was antigenically related to the autoimmune antigen Ku. It is important to be aware of the abundance and avidity of the Ku complex to bind oligonucleotide columns when purifying sequence specific binding proteins.

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.