Andrew R. Mackay

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.

bcl‐2 over‐expression enhances NF‐κB activity and induces mmp‐9 transcription in human MCF7ADR breast‐cancer cells

bcl‐2 expression is often associated with poor prognosis in several types of tumors; however, the role of this molecule in breast cancer is still controversial. We found earlier that over‐expression of bcl‐2 in a human breast‐cancer cell line (MCF7ADR) enhances its tumorigenicity and metastatic potential by inducing metastasis‐associated properties such as increased secretion of the matrix metalloproteinase‐9 (mmp‐9). In the present study, we investigated the effect of bcl‐2 over‐expression on the activity of the transcription factor NF‐κB, an important regulator of genes involved in tumor progression and invasion. Transient transfection experiments indicate that over‐expression of bcl‐2 in the MCF7ADR cell line, enhances NF‐κB‐dependent transcriptional activity. Mobility‐shift analysis revealed an increase of NF‐κB DNA‐binding in bcl‐2‐over‐expressing clones that correlated with lower levels of the NF‐κB cytoplasmic inhibitor IκBα. Moreover, point mutations of 2 highly conserved residues within the BH1 and BH2 domains that abrogate the interaction of bcl‐2 with bax, or deletion of the N‐terminal BH4 domain, completely eliminate the ability of this molecule to up‐regulate NF‐κB‐dependent transactivation. Since mmp‐9 is a NF‐κB‐regulated gene, we also investigated whether bcl‐2 over‐expression up‐regulated mmp‐9 transcription. We found that induction of mmp‐9 mRNA correlates with the activation of an mmp‐9‐promoter‐reporter‐gene construct in transient transfection assay, and a mutation of the (−600)mmp‐9‐NF‐κB binding element abolishes this effect. The overall data indicate that bcl‐2‐mediated regulation of NF‐κB‐transcription‐factor activity may represent an important mechanism for the promotion of malignant behavior in MCF‐7ADR cells. Int. J. Cancer 86:188–196, 2000.

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.

EGF- and cell-cycle-regulated STAG1/PMEPA1/ERG1.2 belongs to a conserved gene family and is overexpressed and amplified in breast and ovarian cancer.

The abnormal activation of the epidermal growth factor (EGF) pathway is one of the most common findings in human cancer, and a number of molecular devices of laboratory and clinical relevance have been designed to block this transduction pathway. Because of the large number of cellular events that might be regulated through the activation of the four EGF receptor family members, it is possible that screening methodologies for the identification of new molecular targets working downstream of these pathways may provide new tools for cancer diagnosis and potentially prevention and therapy. In searching for EGF target genes, we have identified ERG1.2, the mouse homolog of the solid tumor‐associated gene STAG1. Both in humans and in mice, it belongs to a new gene family that can give origin to several protein isoforms through alternative splicing and/or multiple translation starts. Sequence analysis and experimental data suggest that ERG1.2 is likely to function as a membrane‐bound protein interacting with downstream signaling molecules through WW‐ and SH3‐binding domains. ERG1.2 is a cell‐cycle–regulated gene, and both ERG1.2 and STAG1 are induced by EGF and other growth factors at the transcript and protein levels. Finally, we have demonstrated that, besides prostate cancer and renal cell carcinoma, STAG1 was also overexpressed in breast and ovarian cancer cell lines and in breast primary tumors. Although in most cases STAG1 overexpression is probably due to the abnormal activation of the EGF pathway, we have also demonstrated genetic amplification and rearrangement of its locus in one breast cancer cell line and one primary ovarian cancer, suggesting that STAG1 might be a direct molecular target in the carcinogenetic process. Thus its overexpression might be regarded not only as a tumor marker but also as a potentially pathogenetic event.

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.

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.