Role of CARMA3 and MALT1 in mediating 2D proliferation and 3D spheroid growth.

Custom NanoString codeset for evaluating 72 NFkB regulated genes.

Ang II-dependant activation on NFkB in additional AGTR1+ breast cancer cell lines (Hs578T and Hs606T).

AGTR1 expression in breast cancer specimens from METABRIC and TCGA, and in breast cancer cell lines.

Role of Bcl10 in mediating cell migration and invasion of additional AGTR1+ breast cancer cell lines (Hs578T and Hs606T).

Supplementary Materials and Methods

Constitutive and Ang II-induced NFkB activity in BT549 and ZR75-AGTR1 cells; dependence on AGTR1 and the canonical IKK complex.

Candidate pro-angiogenic factors secreted from Ang II-stimulated AGTR1+ breast cancer cells.

In this study, a genetically diverse panel of 43 mouse strains was exposed to ammonia, and genome-wide association mapping was performed employing a single-nucleotide polymorphism (SNP) assembly. Transcriptomic analysis was used to help resolve the genetic determinants of ammonia-induced acute lung injury. The encoded proteins were prioritized based on molecular function, nonsynonymous SNP within a functional domain or SNP within the promoter region that altered expression. This integrative functional approach revealed 14 candidate genes that included Aatf, Avil, Cep162, Hrh4, Lama3, Plcb4, and Ube2cbp, which had significant SNP associations, and Aff1, Bcar3, Cntn4, Kcnq5, Prdm10, Ptcd3, and Snx19, which had suggestive SNP associations. Of these genes, Bcar3, Cep162, Hrh4, Kcnq5, and Lama3 are particularly noteworthy and had pathophysiological roles that could be associated with acute lung injury in several ways.

Antigen receptor-dependent (AgR-dependent) stimulation of the NF-κB transcription factor in lymphocytes is a required event during adaptive immune response, but dysregulated activation of this signaling pathway can lead to lymphoma. AgR stimulation promotes assembly of the CARMA1-BCL10-MALT1 complex, wherein MALT1 acts as (a) a scaffold to recruit components of the canonical NF-κB machinery, and (b) a protease to cleave and inactivate specific substrates, including negative regulators of NF-κB. In multiple lymphoma subtypes, malignant B cells hijack AgR signaling pathways to promote their own growth and survival, and inhibiting MALT1 reduces the viability and growth of these tumors. As such, MALT1 has emerged as a potential pharmaceutical target. Here, we identified G protein-coupled receptor kinase 2 (GRK2) as a new MALT1-interacting protein. We demonstrated that GRK2 binds the death domain of MALT1 and inhibits MALT1 scaffolding and proteolytic activities. We found that lower GRK2 levels in activated B cell-type diffuse large B cell lymphoma (ABC-DLBCL) are associated with reduced survival, and that GRK2 knockdown enhances ABC-DLBCL tumor growth in vitro and in vivo. Together, our findings suggest that GRK2 can function as a tumor suppressor by inhibiting MALT1 and provide a roadmap for developing new strategies to inhibit MALT1-dependent lymphomagenesis.

1 0 3 6 jci.org Volume 130 Number 2 February 2020 Introduction Diffuse large B cell lymphoma (DLBCL) is the most frequent subtype of non-Hodgkin lymphoma (NHL), accounting for 30%–40% of newly diagnosed cases (1). With more than 20,000 deaths from lymphoma occurring each year in the United States (2), new approaches to diagnosis, prognosis, and treatment are needed. Although the molecular and genetic features that drive aggressive behavior of B cell lymphomas are not fully defined, gene expression profiling studies have identified at least 2 distinct molecular subtypes of DLBCL, termed germinal center B cell (GCB) and activated B cell (ABC) (3). ABC-DLBCL exhibits an inferior outcome following standard R-CHOP chemotherapy, with a 3-year progression-free survival of approximately 40% compared with 75% for non-ABC cases, and is associated with constitutive canonical NF-κB activation (4, 5). A loss-of-function RNA interference screen revealed that the majority of ABC-DLBCL cell lines rely on components of the B cell receptor–dependent (BCR-dependent) NF-κB signaling pathway for growth and survival (6). Stimulation of the B or T cell antigen receptor (AgR) results in assembly and activation of the CBM complex, which is composed of the scaffolding protein CARMA1 (also known as CARD11), the adaptor protein BCL10, and the protease MALT1 (7, 8). Loss of any component of this complex is toxic for ABC-DLBCL cells, indicating that it plays a critical role in the molecular pathogenesis of this tumor (6). MALT1 functions as the essential downstream effector molecule of the CBM complex by carrying out 2 important functions. First, MALT1 acts as a scaffold to recruit downstream signaling proteins, most notably the ubiquitin ligase TNF receptor–associated factor 6 (TRAF6). In turn, TRAF6 directs an array of polyubiquitinations that promote stimulation of the IκB kinase (IKK) complex (9–11). IKK then phosphorylates the inhibitor of NF-κB (IκB), thereby targeting it for proteasomal degradation and freeing NF-κB subunits to translocate into the nucleus and alter target gene expression (12). Second, MALT1 acts as protease to enzymatically cleave and inactivate multiple substrates, including several negative regulators of canonical NF-κB signaling (13, 14). Thus, MALT1 protease activity is thought to amplify and sustain NF-κB activation by clearing proteins that dampen this pathway. For example, activated MALT1 protease cleaves the NF-κB family member RELB (15). Since RELB is an inhibitor of canonical RELA-dependent transcriptional activity, MALT1-dependent cleavage of RELB results in enhanced expression of canonical NF-κB gene targets. MALT1 also cleaves several other substrates, Antigen receptor–dependent (AgR-dependent) stimulation of the NF-κB transcription factor in lymphocytes is a required event during adaptive immune response, but dysregulated activation of this signaling pathway can lead to lymphoma. AgR stimulation promotes assembly of the CARMA1-BCL10-MALT1 complex, wherein MALT1 acts as (a) a scaffold to recruit components of the canonical NF-κB machinery and (b) a protease to cleave and inactivate specific substrates, including negative regulators of NF-κB. In multiple lymphoma subtypes, malignant B cells hijack AgR signaling pathways to promote their own growth and survival, and inhibiting MALT1 reduces the viability and growth of these tumors. As such, MALT1 has emerged as a potential pharmaceutical target. Here, we identified G protein–coupled receptor kinase 2 (GRK2) as a new MALT1-interacting protein. We demonstrated that GRK2 binds the death domain of MALT1 and inhibits MALT1 scaffolding and proteolytic activities. We found that lower GRK2 levels in activated B cell–type diffuse large B cell lymphoma (ABCDLBCL) are associated with reduced survival, and that GRK2 knockdown enhances ABC-DLBCL tumor growth in vitro and in vivo. Together, our findings suggest that GRK2 can function as a tumor suppressor by inhibiting MALT1 and provide a roadmap for developing new strategies to inhibit MALT1-dependent lymphomagenesis. GRK2 suppresses lymphomagenesis by inhibiting the MALT1 proto-oncoprotein

Protease-activated receptor 1 (PAR1), a thrombin-responsive G protein-coupled receptor (GPCR), is implicated in promoting metastasis in multiple tumor types, including both sarcomas and carcinomas, but the molecular mechanisms responsible remain largely unknown. We previously discovered that PAR1 stimulation in endothelial cells leads to activation of NF-κB, mediated by a protein complex comprised of CARMA3, Bcl10, and the MALT1 effector protein (CBM complex). Given the strong association between NF-κB and metastasis, we hypothesized that this CBM complex could play a critical role in the PAR1-driven metastatic progression of specific solid tumors. In support of our hypothesis, we demonstrate that PAR1 stimulation results in NF-κB activation in both osteosarcoma and breast cancer, which is suppressed by siRNA-mediated MALT1 knockdown, suggesting that an intact CBM complex is required for the response in both tumor cell types. We identify several metastasis-associated genes that are upregulated in a MALT1-dependent manner after PAR1 stimulation in cancer cells, including those encoding the matrix remodeling protein, MMP9, and the cytokines, IL-1β and IL-8. Further, exogenous expression of PAR1 in MCF7 breast cancer cells confers highly invasive and metastatic behavior which can be blocked by CRISPR/Cas9-mediated MALT1 knockout. Importantly, we find that PAR1 stimulation induces MALT1 protease activity in both osteosarcoma and breast cancer cells, an activity that is mechanistically linked to NF-κB activation and potentially other responses associated with aggressive phenotype. Several small molecule MALT1 protease inhibitors have recently been described that could therefore represent promising new therapeutics for the prevention and/or treatment of PAR1-driven tumor metastasis.

Abstract The angiotensin II receptor AGTR1, which mediates vasoconstrictive and inflammatory signaling in vascular disease, is overexpressed aberrantly in some breast cancers. In this study, we established the significance of an AGTR1-responsive NFκB signaling pathway in this breast cancer subset. We documented that AGTR1 overexpression occurred in the luminal A and B subtypes of breast cancer, was mutually exclusive of HER2 expression, and correlated with aggressive features that include increased lymph node metastasis, reduced responsiveness to neoadjuvant therapy, and reduced overall survival. Mechanistically, AGTR1 overexpression directed both ligand-independent and ligand-dependent activation of NFκB, mediated by a signaling pathway that requires the triad of CARMA3, Bcl10, and MALT1 (CBM signalosome). Activation of this pathway drove cancer cell–intrinsic responses that include proliferation, migration, and invasion. In addition, CBM-dependent activation of NFκB elicited cancer cell–extrinsic effects, impacting endothelial cells of the tumor microenvironment to promote tumor angiogenesis. CBM/NFκB signaling in AGTR1+ breast cancer therefore conspires to promote aggressive behavior through pleiotropic effects. Overall, our results point to the prognostic and therapeutic value of identifying AGTR1 overexpression in a subset of HER2-negative breast cancers, and they provide a mechanistic rationale to explore the repurposing of drugs that target angiotensin II–dependent NFκB signaling pathways to improve the treatment of this breast cancer subset. Significance: These findings offer a mechanistic rationale to explore the repurposing of drugs that target angiotensin action to improve the treatment of AGTR1-expressing breast cancers. Cancer Res; 78(5); 1225–40. ©2017 AACR.

Microvascular endothelial cells maintain a tight barrier to prevent passage of plasma and circulating immune cells into the extravascular tissue compartment, yet endothelial cells respond rapidly to vasoactive substances, including thrombin, allowing transient paracellular permeability. This response is a cornerstone of acute inflammation, but the mechanisms responsible are still incompletely understood. Here, we demonstrate that thrombin triggers MALT1 to proteolytically cleave cylindromatosis (CYLD). Fragmentation of CYLD results in microtubule disruption and a cascade of events leading to endothelial cell retraction and an acute permeability response. This finding reveals an unexpected role for the MALT1 protease, which previously has been viewed mostly as a driver of pro-inflammatory NF-κB signaling in lymphocytes. Thus, MALT1 not only promotes immune cell activation but also acutely regulates endothelial cell biology, actions that together facilitate tissue inflammation. Pharmacologic inhibition of MALT1 may therefore have synergistic impact by targeting multiple disparate steps in the overall inflammatory response.

Abstract Angiotensin II (Ang II) is a potent vasoconstrictor and vascular pro-inflammatory mediator, known classically for its role in promoting arterial dysfunction. Many of these pathogenic effects result from activation of NF-κB in response to stimulation of the type-1 Ang II receptor (AGTR1), a G protein-coupled receptor. We previously discovered that a complex composed of three proteins, CARMA3, Bcl10 and MALT1 (CBM signalosome) mediates AGTR1-dependent activation of NF-κB in vascular cells. Despite its principal role in vascular pathobiology, we recently found that AGTR1 is aberrantly overexpressed in 10-20% of breast cancers (characterized as Luminal, ER+, PR+, Her2-). In this context, AGTR1 promotes cellular invasion and in vivo tumorigenesis, but the mechanisms driving this effect of AGTR1 have remained unclear. We hypothesized that stimulation of AGTR1 in this subset of breast tumors induces CBM-dependent NF-κB activation, thus contributing to tumorigenesis. To develop experimental models for studying the impact of AGTR1 in breast cancer (BC), we screened a panel of BC cell lines and found overexpression of endogenous AGTR1 in BT549 cells, among a few others, but no expression in most lines. To complement the BT549 line, we stably over-expressed exogenous AGTR1 in ZR75 cells (an AGTR1-, luminal line) to produce the ZR75-AGTR1 line. All BC lines tested, irrespective of AGTR1 status, expressed CARMA3, Bcl10 and MALT1. We found that Ang II treatment induces time-dependent phosphorylation of IκB, indicating NF-κB activation, in both BT549 and ZR75-AGTR1 cells, but not in control ZR75 cells. We then employed an NF-κB reporter assay to generate further evidence of NF-κB activation in ZR75-AGTR1 cells. We also demonstrated induction of several endogenous NF-κB target genes using real-time Q-PCR. Importantly, Ang II-dependent NF-κB activation is effectively blocked by pretreatment with losartan (AGTR1 antagonist) or with IKK-VI (IKKβ inhibitor), as well as by siRNA-mediated knockdown of Bcl10 or MALT1. These data indicate that Ang II dependent NF-κB activation in these cells is dependent on activation of AGTR1, proceeds through the canonical NF-κB machinery, and requires the CBM signalosome. Moreover, we showed that AGTR1 activation triggers a dramatic increase in migration and invasion of both BT549 and ZR75-AGTR1 cells, and that this response is completely blocked by siRNA-mediated Bcl10 knockdown. These results demonstrate for the first time that AGTR1 stimulation leads to NF-κB activation in a subset of breast cancer cells, and link this response to aggressive phenotypic behavior. These studies have significant clinical implications since AGTR1 antagonists, currently used for treatment of hypertension, might be effectively re-deployed for treatment of breast cancers that harbor AGTR1 overexpression. Inhibition of CBM-dependent signaling may also represent a novel treatment approach. Citation Format: Prasanna Ekambaram, Nathaniel Hubel, Jia-Ying Lee, Linda Klei, Vincent Concel, Philip Delekta, Scott Tomlins, Linda McAllister-Lucas, Peter Lucas. The CARMA3-Bcl10-MALT1 signalosome mediates NF-κB activation and cellular invasion in AGTR1-positive breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5109. doi:10.1158/1538-7445.AM2015-5109

Background: Previously we detected impaired lung function in gene targeted Sod3(-/-) mice and associated two functional SOD3 single nucleotide polymorphisms to decreased forced expiratory volume in 1s and maximal expiratory flow at 25% volume in children. Decreased lung SOD3 levels and activity were detected in JF1/Msf mice (poor lung function) compared to C3H/HeJ mice (higher lung function) with the greatest difference during postnatal (P) days 14- P28 ie. alveogenesis phase. In this work we studied the global lung transcript profile in Sod3(-/-) mice during alveogenesis. Methods: Lung transcript levels were measured by microarray analysis comparing female P14 and P28 Sod3 (-/-) with matched S od3 (+/+) mice (n=4-16). Significantly altered transcripts (≥2-fold decreased or increased) were analyzed for enriched pathways/categories using Database for Annotation, Visualization, and Integrated Discovery (DAVID), Gene Ontogeny (GO) molecular function, GO biological process, GO cell component, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Results: The GO/KEGG categories for decreased transcripts at P14 (n=1793) and P28 (n= 1047) in S od3 (-/-) mouse lung included Wnt signaling, developmental process, respiratory system development, lung development, MTOR, MAPK, and VEGF signaling. Noteworthy transcripts in these categories include Sftpd, Sftpa1, Wnt4, Wnt1 Wisp1 , Chi3l1, Klf15, Fgfbp1, Spry4, Stk3, and Fgfr4 . Discussion: Our preliminary data indicates altered regulation of key lung developmental pathways during the peak alveogenesis phase in mice lacking SOD3 that may have contributed in impaired lung function development in Sod3(-/-) mice.

In this study, a genetically diverse panel of 43 mouse strains was exposed to phosgene and genome-wide association mapping performed using a high-density single nucleotide polymorphism (SNP) assembly. Transcriptomic analysis was also used to improve the genetic resolution in the identification of genetic determinants of phosgene-induced acute lung injury (ALI). We prioritized the identified genes based on whether the encoded protein was previously associated with lung injury or contained a nonsynonymous SNP within a functional domain. Candidates were selected that contained a promoter SNP that could alter a putative transcription factor binding site and had variable expression by transcriptomic analyses. The latter two criteria also required that ≥10% of mice carried the minor allele and that this allele could account for ≥10% of the phenotypic difference noted between the strains at the phenotypic extremes. This integrative, functional approach revealed 14 candidate genes that included Atp1a1, Alox5, Galnt11, Hrh1, Mbd4, Phactr2, Plxnd1, Ptprt, Reln, and Zfand4, which had significant SNP associations, and Itga9, Man1a2, Mapk14, and Vwf, which had suggestive SNP associations. Of the genes with significant SNP associations, Atp1a1, Alox5, Plxnd1, Ptprt, and Zfand4 could be associated with ALI in several ways. Using a competitive electrophoretic mobility shift analysis, Atp1a1 promoter (rs215053185) oligonucleotide containing the minor G allele formed a major distinct faster-migrating complex. In addition, a gene with a suggestive SNP association, Itga9, is linked to transforming growth factor β1 signaling, which previously has been associated with the susceptibility to ALI in mice.

The genetic basis for the underlying individual susceptibility to chlorine-induced acute lung injury is unknown. To uncover the genetic basis and pathophysiological processes that could provide additional homeostatic capacities during lung injury, 40 inbred murine strains were exposed to chlorine, and haplotype association mapping was performed. The identified single-nucleotide polymorphism (SNP) associations were evaluated through transcriptomic and metabolomic profiling. Using ≥ 10% allelic frequency and ≥ 10% phenotype explained as threshold criteria, promoter SNPs that could eliminate putative transcriptional factor recognition sites in candidate genes were assessed by determining transcript levels through microarray and reverse real-time PCR during chlorine exposure. The mean survival time varied by approximately 5-fold among strains, and SNP associations were identified for 13 candidate genes on chromosomes 1, 4, 5, 9, and 15. Microarrays revealed several differentially enriched pathways, including protein transport (decreased more in the sensitive C57BLKS/J lung) and protein catabolic process (increased more in the resistant C57BL/10J lung). Lung metabolomic profiling revealed 95 of the 280 metabolites measured were altered by chlorine exposure, and included alanine, which decreased more in the C57BLKS/J than in the C57BL/10J strain, and glutamine, which increased more in the C57BL/10J than in the C57BLKS/J strain. Genetic associations from haplotype mapping were strengthened by an integrated assessment using transcriptomic and metabolomic profiling. The leading candidate genes associated with increased susceptibility to acute lung injury in mice included Klf4, Sema7a, Tns1, Aacs, and a gene that encodes an amino acid carrier, Slc38a4.

RATIONALE:Because acute lung injury is a sporadic disease produced by heterogeneous precipitating factors, previous genetic analyses are mainly limited to candidate gene case-control studies. OBJECTIVES:To develop a genome-wide strategy in which single nucleotide polymorphism associations are assessed for functional consequences to survival during acute lung injury in mice. METHODS:To identify genes associated with acute lung injury, 40 inbred strains were exposed to acrolein and haplotype association mapping, microarray, and DNA-protein binding were assessed. MEASUREMENTS AND MAIN RESULTS:The mean survival time varied among mouse strains with polar strains differing approximately 2.5-fold. Associations were identified on chromosomes 1, 2, 4, 11, and 12. Seven genes (Acvr1, Cacnb4, Ccdc148, Galnt13, Rfwd2, Rpap2, and Tgfbr3) had single nucleotide polymorphism (SNP) associations within the gene. Because SNP associations may encompass "blocks" of associated variants, functional assessment was performed in 91 genes within ± 1 Mbp of each SNP association. Using 10% or greater allelic frequency and 10% or greater phenotype explained as threshold criteria, 16 genes were assessed by microarray and reverse real-time polymerase chain reaction. Microarray revealed several enriched pathways including transforming growth factor-β signaling. Transcripts for Acvr1, Arhgap15, Cacybp, Rfwd2, and Tgfbr3 differed between the strains with exposure and contained SNPs that could eliminate putative transcriptional factor recognition sites. Ccdc148, Fancl, and Tnn had sequence differences that could produce an amino acid substitution. Mycn and Mgat4a had a promoter SNP or 3'untranslated region SNPs, respectively. Several genes were related and encoded receptors (ACVR1, TGFBR3), transcription factors (MYCN, possibly CCDC148), and ubiquitin-proteasome (RFWD2, FANCL, CACYBP) proteins that can modulate cell signaling. An Acvr1 SNP eliminated a putative ELK1 binding site and diminished DNA-protein binding. CONCLUSIONS:Assessment of genetic associations can be strengthened using a genetic/genomic approach. This approach identified several candidate genes, including Acvr1, associated with increased susceptibility to acute lung injury in mice.

SCOPE:This investigation sought to better understand the metabolic role of the lung and to generate insights into the pathogenesis of acrolein-induced acute lung injury. A respiratory irritant, acrolein is generated by overheating cooking oils or by domestic cooking using biomass fuels, and is in environmental tobacco smoke, a health hazard in the restaurant workplace. METHODS AND RESULTS:Using SM/J (sensitive) and 129X1/SvJ (resistant) inbred mouse strains, the lung metabolome was integrated with the transcriptome profile before and after acrolein exposure. A total of 280 small molecules were identified and mean values (log 2 >0.58 or <-0.58, p<0.05) were considered different for between-strain comparisons or within-strain responses to acrolein treatment. At baseline, 24 small molecules increased and 33 small molecules decreased in the SM/J mouse lung as compared to 129X1/SvJ mouse lung. Notable among the increased compounds was malonylcarnitine. Following acrolein exposure, several molecules indicative of glycolysis and branched chain amino acid metabolism increased similarly in both strains, whereas SM/J mice were less effective in generating metabolites related to fatty acid β-oxidation. CONCLUSION:These findings suggest management of energetic stress varies between these strains, and that the ability to evoke auxiliary energy generating pathways rapidly and effectively may be critical in enhancing survival during acute lung injury in mice.