Ingrid J. Apel

Matrix Metalloproteinases Regulate Neovascularization by Acting as Pericellular Fibrinolysins

During angiogenesis, endothelial cells penetrate fibrin barriers via undefined proteolytic mechanisms. We demonstrate that the fibrinolytic plasminogen activator (PA)-plasminogen system is not required for this process, since tissues isolated from PA- or plasminogen-deficient mice successfully neovascularize fibrin gels. By contrast, neovessel formation, in vitro and in vivo, is dependent on fibrinolytic, endothelial cell-derived matrix metalloproteinases (MMP). MMPs directly regulate this process as invasion-incompetent cells penetrate fibrin barriers when transfected with the most potent fibrinolytic metalloproteinase identified in endothelium, membrane type-1 MMP (MT1-MMP). Membrane display of MT1-MMP is required, as invasion-incompetent cells expressing a fibrinolytically active, transmembrane-deleted form of MT1-MMP remain noninvasive. These observations identify a PA-independent fibrinolytic pathway wherein tethered MMPs function as pericellular fibrinolysins during the neovascularization process.

Cleavage of NIK by the API2-MALT1 Fusion Oncoprotein Leads to Noncanonical NF-κB Activation

An oncogenic fusion protein promotes lymphomagenesis by activating a noncanonical signaling pathway. Proper regulation of nuclear factor κB (NF-κB) transcriptional activity is required for normal lymphocyte function, and deregulated NF-κB signaling can facilitate lymphomagenesis. We demonstrate that the API2-MALT1 fusion oncoprotein created by the recurrent t(11;18)(q21;q21) in mucosa-associated lymphoid tissue (MALT) lymphoma induces proteolytic cleavage of NF-κB–inducing kinase (NIK) at arginine 325. NIK cleavage requires the concerted actions of both fusion partners and generates a C-terminal NIK fragment that retains kinase activity and is resistant to proteasomal degradation. The resulting deregulated NIK activity is associated with constitutive noncanonical NF-κB signaling, enhanced B cell adhesion, and apoptosis resistance. Our study reveals the gain-of-function proteolytic activity of a fusion oncoprotein and highlights the importance of the noncanonical NF-κB pathway in B lymphoproliferative disease.

Characterization of radish mitochondrial atpA: influence of nuclear background on transcription of atpA-associated sequences and relationship with male sterility

We have previously shown that the mitochondrial gene atpA, encoding the α subunit of F1 ATP synthase, is associated with DNA rearrangements and nuclear-specific transcript patterns in the male-sterile cytoplasm of Ogura radish. Here we present a detailed characterization of this gene from both the normal (fertile) and Ogura (male-sterile) cytoplasms of radish to determine if it is involved in Ogura cytoplasmic male sterility. The normal and Ogura radish atpA loci are virtually identical for 3.8 kb, including a 507 codon open reading frame whose product is approximately 92% identical to other plant ATPA polypeptides. Rearrangement breakpoints have been identified 613 bp 5′ and 1663 bp 3′ to the atpA coding region. The 5′ rearrangement breakpoint is located within a repeated sequence that has been associated with other rearrangement events in radish mitochondria. The previously identified transcript difference results from transcription originating upstream of this rearrangement site. Although the presence of this transcript is affected by nuclear background, analyses in several different sterile and fertile nuclear backgrounds indicate that the presence of this transcript is not strictly correlated with male sterility. In addition, normal levels of ATPA polypeptide are present in sterile plants containing the Ogura cytoplasm.

BET Bromodomain Inhibitors Enhance Efficacy and Disrupt Resistance to AR Antagonists in the Treatment of Prostate Cancer.

Next-generation antiandrogen therapies, such as enzalutamide and abiraterone, have had a profound impact on the management of metastatic castration-resistant prostate cancer (mCRPC). However, mCRPC patients invariably develop resistance to these agents. Here, a series of clonal cell lines were developed from enzalutamide-resistant prostate tumor xenografts to study the molecular mechanism of resistance and test their oncogenic potential under various treatment conditions. Androgen receptor (AR) signaling was maintained in these cell lines, which acquired potential resistance mechanisms, including expression of AR-variant 7 (AR-v7) and glucocorticoid receptor. BET bromodomain inhibitors were shown previously to attenuate AR signaling in mCRPC; here, we demonstrate the efficacy of bromodomain and extraterminal (BET) inhibitors in enzalutamide-resistant prostate cancer models. AR antagonists, enzalutamide, and ARN509 exhibit enhanced prostate tumor growth inhibition when combined with BET inhibitors, JQ1 and OTX015, respectively. Taken together, these data provide a compelling preclinical rationale to combine BET inhibitors with AR antagonists to subvert resistance mechanisms. Implications: Therapeutic combinations of BET inhibitors and AR antagonists may enhance the clinical efficacy in the treatment of mCRPC. Visual Overview: http://mcr.aacrjournals.org/content/14/4/324/F1.large.jpg. Mol Cancer Res; 14(4); 324–31. ©2016 AACR. Visual Overview

Thrombin-dependent NF-κB Activation and Monocyte/Endothelial Adhesion Are Mediated by the CARMA3·Bcl10·MALT1 Signalosome

Thrombin is a potent modulator of endothelial function and, through stimulation of NF-κB, induces endothelial expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). These cell surface adhesion molecules recruit inflammatory cells to the vessel wall and thereby participate in the development of atherosclerosis, which is increasingly recognized as an inflammatory condition. The principal receptor for thrombin on endothelial cells is protease-activated receptor-1 (PAR-1), a member of the G protein-coupled receptor superfamily. Although it is known that PAR-1 signaling to NF-κB depends on initial PKC activation, the subsequent steps leading to stimulation of the canonical NF-κB machinery have remained unclear. Here, we demonstrate that a complex of proteins containing CARMA3, Bcl10, and MALT1 links PAR-1 activation to stimulation of the IκB kinase complex. IκB kinase in turn phosphorylates IκB, leading to its degradation and the release of active NF-κB. Further, we find that although this CARMA3·Bcl10·MALT1 signalosome shares features with a CARMA1-containing signalosome found in lymphocytes, there are significant differences in how the signalosomes communicate with their cognate receptors. Specifically, whereas the CARMA1-containing lymphocyte complex relies on 3-phosphoinositide-dependent protein kinase 1 for assembly and activation, the CARMA3-containing endothelial signalosome functions completely independent of 3-phosphoinositide-dependent protein kinase 1 and instead relies on β-arrestin 2 for assembly. Finally, we show that thrombin-dependent adhesion of monocytes to endothelial cells requires an intact endothelial CARMA3·Bcl10·MALT1 signalosome, underscoring the importance of the signalosome in mediating one of the most significant pro-atherogenic effects of thrombin.

The role of coxI-associated repeated sequences in plant mitochondrial DNA rearrangements and radish cytoplasmic male sterility

SummaryThe gene coxI, encoding subunit I of mitochondrial cytochrome c oxidase, has been characterized from the normal (fertile) and Ogura (male-sterile) cytoplasms of radish to determine if a previously identified mitochondrial DNA rearrangement, and its associated transcriptional differences, could play a role in Ogura cytoplasmic male sterility (CMS). The normal and Ogura loci are virtually identical for 2.8 kb, including a 527-codon open reading frame whose product is approximately 95% identical to other plant COXI polypeptides. A rearrangement 120 bp 5′ to the coding region results in different 5′ transcript termini for the two genes. A comparison of several crucifer mitochondrial DNAs indicates that this rearrangement also occurs in the normal radish cytoplasm and is, therefore, not involved in Ogura CMS. Sequences present at the coxI locus belong to at least two different dispersed repeat families, members of which are also associated with other rearranged genes in radish.

Development of Peptidomimetic Inhibitors of the ERG Gene Fusion Product in Prostate Cancer

Transcription factors play a key role in the development of diverse cancers, and therapeutically targeting them has remained a challenge. In prostate cancer, the gene encoding the transcription factor ERG is recurrently rearranged and plays a critical role in prostate oncogenesis. Here, we identified a series of peptides that interact specifically with the DNA binding domain of ERG. ERG inhibitory peptides (EIPs) and derived peptidomimetics bound ERG with high affinity and specificity, leading to proteolytic degradation of the ERG protein. The EIPs attenuated ERG-mediated transcription, chromatin recruitment, protein-protein interactions, cell invasion and proliferation, and tumor growth. Thus, peptidomimetic targeting of transcription factor fusion products may provide a promising therapeutic strategy for prostate cancer as well as other malignancies.

The API2–MALT1 fusion exploits TNFR pathway-associated RIP1 ubiquitination to promote oncogenic NF-κB signaling

The API2-MALT1 fusion oncoprotein is created by the recurrent t(11;18)(q21;q21) chromosomal translocation in mucosa-associated lymphoid tissue (MALT) lymphoma. We identified receptor interacting protein-1 (RIP1) as a novel API2–MALT1-associated protein, and demonstrate that RIP1 is required for API2–MALT1 to stimulate canonical nuclear factor kappa B (NF-κB). API2–MALT1 promotes ubiquitination of RIP1 at lysine (K) 377, which is necessary for full NF-κB activation. Furthermore, we found that TNF receptor-associated factor 2 (TRAF2) recruitment is required for API2–MALT1 to induce RIP1 ubiquitination, NF-κB activation and cellular transformation. Although both TRAF2 and RIP1 interact with the API2 moiety of API2–MALT1, this moiety alone is insufficient to induce RIP1 ubiquitination or activate NF-κB, indicating that API2–MALT1-dependent RIP1 ubiquitination represents a gain of function requiring the concerted actions of both the API2 and MALT1 moieties of the fusion. Intriguingly, constitutive RIP1 ubiquitination was recently demonstrated in several solid tumors, and now our study implicates RIP1 ubiquitination as a critical component of API2–MALT1-dependent lymphomagenesis.

Bcl10 Mediates Angiotensin II–Induced Cardiac Damage and Electrical Remodeling

Angiotensin (Ang) II is a potent mediator of both hypertension and cardiac damage; however, the mechanisms by which this occur remain unclear. B-cell lymphoma/leukemia 10 (Bcl10) is a member of the CBM signalosome, which links Ang II and nuclear factor-&kgr;B signaling. We hypothesized that Bcl10 is pivotal in the pathogenesis of Ang II–induced cardiac damage. Ang II infusion in mice lacking Bcl10 resulted in reduced cardiac fibrosis, less cellular infiltration, and improved arrhythmogenic electric remodeling, despite a similar degree of hypertension or cardiac hypertrophy. Adoptive transfer of bone marrow (BM), whereby Bcl10 knockout or wildtype BM was transferred to their opposite genotype recipients, revealed the dual importance of Bcl10 within both cardiac and immune cells. Loss of Bcl10 in cardiac cells resulted in reduced expression of genes important for the adhesion and recruitment of immune cells. In vitro experiments demonstrated that adhesion of monocytes to Ang II–treated endothelial cells also required Bcl10. Additionally, Bcl10 deficiency in macrophages reduced their intrinsic migratory ability. To address the role of BM-derived fibroblasts in the formation of cardiac fibrosis, we explored whether Bcl10 is also important for the infiltration of BM-derived (myo)fibroblasts into the heart. The transfer of green fluorescent protein positive wildtype BM into Bcl10 knockout recipient mice revealed a reduced number of noncardiac (myo)fibroblasts compared with those wildtype recipients. Our results demonstrate the significant role of Bcl10 in multiple cell types important for the generation of Ang II–induced cardiac damage and electric remodeling and may provide a new avenue for therapeutic intervention.

A technique for the determination of protein concentration by neutron activation analysis of silver binding

A method for the quantitative determination of small amounts of protein samples was developed employing neutron activation analysis. Current methods of protein concentration determination are severely limited as a result of differences in the specific characteristics of each protein. Silver binding has been used as a sensitive colorimetric method to indicate the presence of protein. However, silver-protein complexes can have a variety of absorbance spectra unique to each protein, which complicate the analysis. Various amounts of specific proteins were equilibrated in an excess of silver nitrate prior to the reduction of the silver by the addition of NaBH4, HCHO, and NaOH. The protein-silver complex was rapidly separated from the unbound silver by centrifugation chromatography and the amount of bound silver was determined by INAA. The amount of silver was proportional to the amount of protein present in each sample. When the silver was not reduced prior to removal of the unbound silver by chromatography, only negligible amounts of silver remained bound to the protein. The stoichiometry of bound silver to protein on a molar basis showed relatively small differences for the proteins that were examined. This ratio was found to depend on the conditions of the binding and reduction of the silver. The results suggest that the binding of silver is not specific to any charged or polar groups on these proteins and may, therefore, provide a means of determination of the concentration of protein that has general application for all proteins.