Identification of Chemical Inhibitors of ß-Catenin-Driven Liver Tumorigenesis in Zebrafish.

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers. The search for targeted treatments has been hampered by the lack of relevant animal models for the genetically diverse subsets of HCC, including the 20-40% of HCCs that are defined by activating mutations in the gene encoding ß-catenin. To address this chemotherapeutic challenge, we created and characterized transgenic zebrafish expressing hepatocyte-specific activated ß-catenin. By 2 months post fertilization (mpf), 33% of transgenic zebrafish developed HCC in their livers, and 78% and 80% of transgenic zebrafish showed HCC at 6 and 12 mpf, respectively. As expected for a malignant process, transgenic zebrafish showed significantly decreased mean adult survival compared to non-transgenic control siblings. Using this novel transgenic model, we screened for druggable pathways that mediate ß-catenin-induced liver growth and identified two c-Jun N-terminal kinase (JNK) inhibitors and two antidepressants (one tricyclic antidepressant, amitriptyline, and one selective serotonin reuptake inhibitor) that suppressed this phenotype. We further found that activated ß-catenin was associated with JNK pathway hyperactivation in zebrafish and in human HCC. In zebrafish larvae, JNK inhibition decreased liver size specifically in the presence of activated ß-catenin. The ß-catenin-specific growth-inhibitory effect of targeting JNK was conserved in human liver cancer cells. Our other class of hits, antidepressants, has been used in patient treatment for decades, raising the exciting possibility that these drugs could potentially be repurposed for cancer treatment. In support of this proposal, we found that amitriptyline decreased tumor burden in a mouse HCC model. Our studies implicate JNK inhibitors and antidepressants as potential therapeutics for ß-catenin-induced liver tumors.

Monocytes promote liver carcinogenesis in an oncogene-specific manner.

BACKGROUND & AIMS: The leukocyte composition of tumors is heterogeneous, as is the involvement of each leukocyte subset in promoting or restraining tumorigenesis. This heterogeneity reflects the tissue of origin, tumor stage, and the functional state of leukocyte activation, but its biological roots remain poorly understood. Since tumorigenesis is driven by various genetic events, we assessed the role of driver genes in shaping the profiles and the roles of leukocytes in tumorigenesis. METHODS: Mouse liver tumors were induced by hepatic overexpression of either MYC or the combination of myristoylated AKT and NRAS(V12) oncogenes via hydrodynamic transfection. A comparative, flow cytometry- and histology-based immunophenotyping of liver-infiltrating leukocytes was performed at various stages of liver tumorigenesis. The roles of the most abundant leukocyte subsets in tumorigenesis were addressed by immunodepletion. The contribution of liver injury was assessed by comparing the injury-inducing hydrodynamic transfection model to a model in which MYC is an inducible transgene. RESULTS: Myristoylated AKT and NRAS(V12) promoted a marked recruitment of CD11b(+)Ly6G(hi)Ly6C(int) neutrophils and CD11b(+)Ly6G(-)Ly6C(hi) monocytes to the liver, but their immunodepletion did not alter tumorigenesis. In contrast, despite minimal invasion by monocytes/neutrophils during MYC-driven tumorigenesis, immunodepletion of these cells reduced MYC tumor burden and extended survival. MYC-driven tumor initiation was augmented specifically by Ly6C+ monocytes and their ability to promote liver injury. CONCLUSIONS: Our results demonstrate that leukocyte profiles do not necessarily predict their involvement in tumorigenesis, the functional role of leukocytes can be shaped by oncogenes, and that monocyte-dependent tissue injury selectively cooperates with MYC during tumorigenesis.

TREM1 activation of myeloid cells promotes antitumor immunity.

Myeloid cells in the tumor microenvironment (TME) can exist in immunosuppressive and immunostimulatory states that impede or promote antitumor immunity, respectively. Blocking suppressive myeloid cells or increasing stimulatory cells to enhance antitumor immune responses is an area of interest for therapeutic intervention. Triggering receptor expressed on myeloid cells-1 (TREM1) is a proinflammatory receptor that amplifies immune responses. TREM1 is expressed on neutrophils, subsets of monocytes and tissue macrophages, and suppressive myeloid populations in the TME, including tumor-associated neutrophils, monocytes, and tumor-associated macrophages. Depletion or inhibition of immunosuppressive myeloid cells, or stimulation by TREM1-mediated inflammatory signaling, could be used to promote an immunostimulatory TME. We developed PY159, an afucosylated humanized anti-TREM1 monoclonal antibody with enhanced FcγR binding. PY159 is a TREM1 agonist that induces signaling, leading to up-regulation of costimulatory molecules on monocytes and macrophages, production of proinflammatory cytokines and chemokines, and enhancement of T cell activation in vitro. An antibody against mouse TREM1, PY159m, promoted antitumor efficacy in syngeneic mouse tumor models. These results suggest that PY159-mediated agonism of TREM1 on tumoral myeloid cells can promote a proinflammatory TME and offer a promising strategy for immunotherapy.

Targeting TREM2 on tumor-associated macrophages enhances immunotherapy.

Converting checkpoint inhibitor (CPI)-resistant individuals to being responsive requires identifying suppressive mechanisms. We identify TREM2+ tumor-associated macrophages (TAMs) as being correlated with exhausted CD8+ tumor-infiltrating lymphocytes (TILs) in mouse syngeneic tumor models and human solid tumors of multiple histological types. Fc domain-enhanced anti-TREM2 monoclonal antibody (mAb) therapy promotes anti-tumor immunity by elimination and modulation of TAM populations, which leads to enhanced CD8+ TIL infiltration and effector function. TREM2+ TAMs are most enriched in individuals with ovarian cancer, where TREM2 expression corresponds to disease grade accompanied by worse recurrence-free survival. In an aggressive orthotopic ovarian cancer model, anti-TREM2 mAb therapy drives potent anti-tumor immunity. These results highlight TREM2 as a highly attractive target for immunotherapy modulation in individuals who are refractory to CPI therapy and likely have a TAM-rich tumor microenvironment.

Detection of Parkin (PARK2) and DJ1 (PARK7) mutations in early-onset Parkinson disease: Parkin mutation frequency depends on ethnic origin of patients.

Mutations in the Parkin (PARK2) and the DJ1 (PARK7) gene cause early-onset Parkinson disease (EOPD). We tested 75 Serbian EOPD patients for mutations in both genes by conventional mutational screening (SSCP/dHPLC/sequencing) to detect small sequence alterations and by gene dosage studies (quantitative PCR) to reveal deletions or multiplications of one or more exons. A compound heterozygous Parkin mutation (exon deletion and point mutation; [c.836_972del]+[c.1411C>T]; +1 is first nucleotide of GenBank AB009973.1) was identified in a patient who showed a relatively benign course after a disease onset at 41 years. Another case had a heterozygous exon deletion in DJ1 ([c.253_322del]+[?]) and presented with an age at onset of 45 years and a rapid disease course. In conclusion, Parkin mutations are surprisingly rare in our Serbian EOPD sample, suggesting that the mutation rate depends on the ethnic origin of the patients. Although DJ1 mutations appear to be rare, we confirm their role in EOPD and demonstrate the importance of gene dosage studies.

TNFα-induced apoptosis enabled by CCN1/CYR61: pathways of reactive oxygen species generation and cytochrome c release.

Although TNFα is a strong inducer of apoptosis, its cytotoxicity in most normal cells in vitro requires blockade of NFκB signaling or inhibition of de novo protein synthesis, typically by the addition of cycloheximide. However, several members of CCN (CYR61/CTGF/NOV) family of extracellular matrix proteins enable TNFα-dependent apoptosis in vitro without inhibiting NFκB or de novo protein synthesis, and CCN1 (CYR61) is essential for optimal TNFα cytotoxicity in vivo. Previous studies showed that CCN1 unmasks the cytotoxicity of TNFα by binding integrins α(v)ß(5), α(6)ß(1), and the cell surface heparan sulfate proteoglycan syndecan 4 to induce the accumulation of a high level of reactive oxygen species (ROS), leading to a biphasic activation of JNK necessary for apoptosis. Here we show for the first time that CCN1 interacts with the low density lipoprotein receptor-related protein 1 (LRP1) in a protein complex, and that binding to LRP1 is critical for CCN1-induced ROS generation and apoptotic synergism with TNFα. We also found that neutral sphingomyelinase 1 (nSMase1), which contributes to CCN1-induced ROS generation, is required for CCN1/TNFα-induced apoptosis. Furthermore, CCN1 promotes the activation of p53 and p38 MAPK, which mediate enhanced cytochrome c release to amplify the cytotoxicity of TNFα. By contrast, LRP1, nSMase1, p53, and p38 MAPK are not required when TNFα-dependent apoptosis is facilitated by the presence of cycloheximide, indicating that they function in the CCN1 signaling pathway that converges with TNFα-induced signaling events. Since CCN1/CYR61 is a physiological regulator of TNFα cytotoxicity at least in some contexts, these findings may reveal important mediators of TNFα-induced apoptosis in vivo and identify potential therapeutic targets for thwarting TNFα-dependent tissue damage.

Fas-mediated apoptosis is regulated by the extracellular matrix protein CCN1 (CYR61) in vitro and in vivo.

Although Fas ligand (FasL) is primarily expressed by lymphoid cells, its receptor Fas (CD95/Apo-1) is broadly expressed in numerous nonlymphoid tissues and can mediate apoptosis of parenchymal cells upon injury and infiltration of inflammatory cells. Here we show that CCN1 (CYR61) and CCN2 (CTGF), matricellular proteins upregulated at sites of inflammation and wound repair, synergize with FasL to induce apoptosis by elevating cellular levels of reactive oxygen species (ROS). CCN1 acts through engagement of integrin alpha(6)beta(1) and cell surface heparan sulfate proteoglycans, leading to ROS-dependent hyperactivation of p38 mitogen-activated protein kinase in the presence of FasL to enhance mitochondrial cytochrome c release. We show that CCN1 activates neutral sphingomyelinase, which functions as a key source of CCN1-induced ROS critical for synergism with FasL. Furthermore, Fas-dependent hepatic apoptosis induced by an agonistic monoclonal anti-Fas antibody or intragastric administration of alcohol is severely blunted in knock-in mice expressing an apoptosis-defective Ccn1 allele. These results demonstrate that CCN1 is a physiologic regulator of Fas-mediated apoptosis and that the extracellular matrix microenvironment can modulate Fas-dependent apoptosis through CCN1 expression.

Matrix protein CCN1 is critical for prostate carcinoma cell proliferation and TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) plays an important role in immune surveillance and preferentially induces apoptosis in cancer cells over normal cells, suggesting its potential in cancer therapy. However, the molecular basis for its selective killing of cancer cells is not well understood. Recent studies have identified the CCN family of integrin-binding matricellular proteins as important regulators of cell behavior, including cell adhesion, proliferation, migration, differentiation, and survival. We show here that CCN1 (CYR61) supports the adhesion of prostatic carcinoma cells as an adhesion substrate through integrins and heparan sulfate proteoglycans. Knockdown of CCN1 expression in PC-3 and DU-145 androgen-independent prostate cancer cells strongly inhibited their proliferation without causing apoptosis, indicating that CCN1 promotes their growth. However, CCN1 also significantly enhances TRAIL-induced apoptosis through interaction with integrins alphavbeta3 and alpha6beta4 and the cell-surface heparan sulfate proteoglycan syndecan-4, acting through a protein kinase Calpha-dependent mechanism without requiring de novo protein synthesis. Knockdown of CCN1 expression in PC-3, DU-145, and LNCaP cells severely blunted their sensitivity to TRAIL, an effect that was reversed by exogenously added CCN1 protein. These findings reveal a functional dichotomy for CCN1 in prostate carcinoma cells, because it contributes to both cell proliferation and TRAIL-induced cell death and suggest that CCN1 expression status may be an important parameter in assessing the efficacy of TRAIL-dependent cancer therapy.

Targeted mutagenesis of the angiogenic protein CCN1 (CYR61). Selective inactivation of integrin alpha6beta1-heparan sulfate proteoglycan coreceptor-mediated cellular functions.

The matricellular protein CCN1 (CYR61) regulates multiple cellular processes and plays essential roles in embryonic vascular development. A ligand of several integrin receptors, CCN1 acts through integrin alpha6beta1 and heparan sulfate proteoglycans (HSPGs) to promote specific functions in fibroblasts, smooth muscle cells, and endothelial cells. We have previously identified a novel alpha6beta1 binding site, T1, in domain III of CCN1. Here we uncover two novel 16-residue sequences, H1 and H2, in domain IV that can support alpha6beta1- and HSPGs-dependent cell adhesion, suggesting that these sequences contain closely juxtaposed or overlapping sites for interaction with alpha6beta1 and HSPGs. Furthermore, fibroblast adhesion to the H1 and H2 peptides is sufficient to induce prolonged MAPK activation, whereas adhesion to T1 induces transient MAPK activation. To dissect the roles of these sites in CCN1 function, we have created mutants disrupted in T1, H1, and H2 or in all three sites in the context of full-length CCN1. We show that the T1 and H1/H2 sites are functionally non-equivalent, and disruption of these sites differentially affected cell adhesion, migration, mitogen-activated protein kinase activation, and regulation of gene expression. Disruption of all three sites completely abolished alpha6beta1-HSPG-mediated cellular activities. All mutants disrupting T1, H1, and H2 fully retain alphavbeta3-mediated pro-angiogenic activities, indicating that these mutants are biologically active and are defective only in alpha6beta1-HSPG-mediated functions. Together, these findings identify and dissect the differential roles of the three sites (T1, H1, H2) required for alpha6beta1-HSPG-dependent CCN1 activities and provide a strategy to investigate these alpha6beta1-HSPG-specific activities in vivo.