Purpose: Biomarkers aiding treatment optimization in metastatic castration-resistant prostate cancer (mCRPC) are scarce. Presence or absence of androgen receptor (AR) splice variants, AR-V7 and ARv567es, in mCRPC patient circulating tumor cells (CTCs) may be associated with taxane treatment outcomes. Experimental design: A novel digital droplet PCR (ddPCR) assay assessed AR splice variant expression in CTCs from patients receiving docetaxel or cabazitaxel in TAXYNERGY (NCT01718353). Patient outcomes were examined according to AR splice variant expression, including prostate-specific antigen (PSA)50 response and progression-free survival (PFS). Results: Of 54 evaluable patients, 36 (67%) were AR-V7+, 42 (78%) were ARv567es+, 29 (54%) were double positive and 5 (9%) were double negative. PSA50 response rates at any time were numerically higher for AR-V7- vs AR-V7+ (78% vs 58%; p=0.23) and for ARv567es- vs ARv567es+ (92% vs 57%; p=0.04) patients. When AR-V mRNA status was correlated with change in nuclear AR from Cycle 1 Day 1 to Day 8 (n=24), AR-V7+ patients (n=16) had a 0.4% decrease vs a 12.9% and 26.7% decrease in AR-V7-/ARv567es- (n=3) and AR-V7-/ ARv567es+ (n=5) patients, respectively, suggesting a dominant role for AR-V7 over ARv567es. Median PFS was 12.02 vs 8.48 months for AR-V7- vs AR-V7+ (HR=0.38; p=0.01), and 12.71 vs 7.29 months for ARv567es- vs ARv567es+ (HR=0.37; p=0.02). For AR-V7+, AR-V7-/ARv567es+, and AR-V7-/ ARv567es- patients, median PFS was 8.48, 11.17 and 16.62 months, respectively (p=0.0013 for trend). Conclusions: While detection of CTC-specific AR-V7 and ARv567es by ddPCR both influenced taxane outcomes, AR-V7 primarily mediated the prognostic impact. Absence of both variants was associated with the best response and PFS with taxane treatment.
Tumor cells must overcome apoptosis to survive throughout metastatic dissemination and distal organ colonization. Here we show in the Polyoma Middle T mammary tumor model that N-cadherin expression causes Slug upregulation, which in turn promotes carcinoma cell survival. Slug was dramatically upregulated in metastases relative to primary tumors. Consistent with a role in metastasis, Slug knockdown in carcinoma cells suppressed lung colonization by decreasing cell survival at metastatic sites, but had no effect on tumor cell invasion or extravasation. In support of this idea, Slug inhibition by shRNA, sensitized tumor cells to apoptosis by DNA damage, resulting in caspase-3 and PARP cleavage. The pro-survival effect of Slug was found to be caused by direct repression of the pro-apoptotic gene, Puma, by Slug. Consistent with a pivotal role for a Slug-Puma axis in metastasis, inhibition of Puma by RNA interference in Slug-knockdown cells rescued lung colonization, whereas Puma overexpression in control tumor cells suppressed lung metastasis. The survival function of the Slug-Puma axis was confirmed in human breast cancer cells, where Slug knockdown increased Puma expression and inhibited lung colonization. This study demonstrates a pivotal role for Slug in carcinoma cell survival, implying that disruption of the Slug-Puma axis may impinge on the survival of metastatic cells.
Membrane type 1 (MT1) matrix metalloproteinase (MMP-14)is a membrane-tethered MMP considered to be a major mediator of pericellular proteolysis. MT1-MMP is regulated by a complex array of mechanisms, including processing and endocytosis that determine the pool of active proteases on the plasma membrane. Autocatalytic processing of active MT1-MMP generates an inactive membrane-tethered 44-kDa product (44-MT1) lacking the catalytic domain. This form preserves all other enzyme domains and is retained at the cell surface. Paradoxically, accumulation of the 44-kDa form has been associated with increased enzymatic activity. Here we report that expression of a recom- Membrane type 1 matrix metalloproteinase (MT1-MMP, 2 MMP-14) is a type I-transmembrane protease and a major mediator of pericellular proteolysis. MT1-MMP is responsible for the proteolytic cleavage of multiple pericellular and membrane-associated substrates, including collagens and other extracellular matrix proteins, growth factors, growth factor receptors, cell adhesion proteins and their receptors, cytokines, protease inhibitors, and proteases, just to mention a few (1-5). MT1-MMP is also the major physiological activator of pro-MMP-2 (pro-gelatinase A) on the cell surface (6, 7), a process that further contributes to pericellular proteolysis. As a multifunctional protease, MT1-MMP elicits profound effects on cell behavior and has been implicated in the pathogenesis of various human diseases, including cancer (8 -10), diabetes (11, 12), vascular (13, 14), and connective tissue diseases (2).The importance of MT1-MMP for pericellular proteolysis demands a tight control of its catalytic activity at the cell surface. This is partly achieved by the action of endogenous protease inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), which bind to the active site inhibiting catalysis (15). In addition, by virtue of being a membrane-anchored protease, MT1-MMP developed a unique set of regulatory mechanisms that control enzymatic activity independently of TIMPs. These processes include the targeting of active enzyme to specific plasma membrane locations, endocytosis, and autocatalytic processing (1, 16 -19). Together, these distinct processes determine the level of active enzyme on the cell surface. However, how these processes are integrated to control the pool of active MT1-MMP is not understood.The processing of MT1-MMP is a cell surface event in which the active enzyme is usually autocatalytically cleaved in trans to generate a major membrane-anchored product of ϳ44 kDa (also referred to as the 43-or 45-kDa species in some studies) and a soluble ϳ18-kDa inactive fragment of the catalytic domain (6, 20 -25). The 44-kDa product of MT1-MMP is detected in cultured cells expressing natural MT1-MMP (20, 26 -32) and has been found in platelets (33), human tumors extracts (34 -36), and extracts of arthritic synovial tissues (37). MT1-MMP processing is stimulated by a variety of factors known to stimulate MT1-MMP expression, trafficking, and/or endocytosi...
The membrane type (MT) 6 matrix metalloproteinase (MMP) (MMP25) is a glycosylphosphatidylinositol-anchored matrix metalloproteinase (MMP) that is highly expressed in leukocytes and in some cancer tissues. We previously showed that natural MT6-MMP is expressed on the cell surface as a major reductionsensitive form of M r 120, likely representing enzyme homodimers held by disulfide bridges. Among the membrane type-MMPs, the stem region of MT6-MMP contains three cysteine residues at positions 530, 532, and 534 which may contribute to dimerization. A systematic site-directed mutagenesis study of the Cys residues in the stem region shows that Cys 532 is involved in MT6-MMP dimerization by forming an intermolecular disulfide bond. The mutagenesis data also suggest that Cys 530 and Cys 534 form an intramolecular disulfide bond. The experimental observations on cysteines were also investigated by computational studies of the stem peptide, which validate these proposals. Dimerization is not essential for transport of MT6-MMP to the cell surface, partitioning into lipid rafts or cleavage of ␣-1-proteinase inhibitor. However, monomeric forms of MT6-MMP exhibited enhanced autolysis and metalloprotease-dependent degradation. Collectively, these studies establish the stem region of MT6-MMP as the dimerization interface, an event whose outcome imparts protease stability to the protein.The MMP 3 family of zinc-dependent endopeptidases includes both secreted and membrane-anchored proteases that can hydrolyze multiple substrates at distinct cellular and extracellular locations in physiological and pathological conditions (1). The membrane-anchored MMPs, known as membrane type-MMPs (MT-MMPs) evolved to specifically target substrates at the pericellular space and in the plasma membrane by incorporating two distinct anchoring motifs, namely a transmembrane domain as in the case of MT1-(MMP14), MT2-(MMP15), MT3-(MMP16), and MT5-MMP (MMP24) or a glycosylphosphatidylinositol (GPI) moiety as in the case of MT4-(MMP17) and MT6-MMP (MMP25) (2). The presence of membrane-anchoring domains confers unique properties and regulatory mechanisms to this subset of MMPs, which serve to tightly control the localization and amount of protease at the cell surface. These regulatory mechanisms include targeting to cell-matrix contacts, insertion into membrane microdomains, internalization and recycling, processing, oligomerization, and ectodomain shedding (3, 4). Although there is substantial information on the regulation of transmembrane MT-MMPs by these processes, in particular with MT1-MMP (3-5), little is known about the regulation of GPI-MT-MMPs, MT4-and MT6-MMP.Structurally, the GPI-MT-MMPs comprise the classical domain organization of MMPs including an N-terminal prodomain, a zinc-containing catalytic domain, a hinge region, and a C-terminal hemopexin-like domain (6 -8). Like all members of the MT-MMP family, the GPI-MT-MMPs also contain a stem (stalk) region downstream of the hemopexin-like domain which is followed by the anchoring motif. Howeve...
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