Michael Meister

STIMULATION OF PHOSPHOLIPASE C-BETA(2) BY RECOMBINANT GUANINE-NUCLEOTIDE-BINDING PROTEIN BETA-GAMMA DIMERS PRODUCED IN A BACULOVIRUS/INSECT CELL EXPRESSION SYSTEM - REQUIREMENT OF GAMMA-SUBUNIT ISOPRENYLATION FOR STIMULATION OF PHOSPHOLIPASE-C

Recombinant wild-type beta 1 gamma 1 dimers of signal-transducing guanine nucleotide-binding proteins (G proteins) and beta 1 gamma 1 dimers carrying a mutation known to block gamma-subunit isoprenylation (beta 1 gamma 1 C71S) were expressed in baculovirus-infected insect cells. Both wild-type and mutant beta 1 gamma 1 dimers were found in soluble fractions of infected cells upon subcellular fractionation. Anion exchange chromatographic and metabolic-radiolabeling studies revealed that the soluble beta 1 gamma 1 preparation contained approximately equal amounts of non-isoprenylated and isoprenylated beta 1 gamma 1 dimers. Soluble wild-type and mutant beta 1 gamma 1 dimers and native beta 1 gamma 1 dimers purified from bovine retina were reconstituted with recombinant phospholipase C-beta 2. Only isoprenylated beta 1 gamma 1 dimers were capable of stimulating phospholipase C-beta 2. The results show that gamma-subunit isoprenylation and/or additional post-translational processing of the protein are required for beta gamma subunit stimulation of phospholipase C.

Expression, characterization and purification of soluble G-protein βγ dimers composed of defined subunits in baculovirus-infected insect cells

Recombinant β1γ2 dimers of signal‐transducing guanine nucleotide‐binding proteins (G‐proteins) carrying a mutation known to block isoprenylation of the γ2 subunit were expressed as a soluble protein in baculovirus‐infected insect cells. The soluble βγ dimer was analyzed by sucrose density gradient centrifugation and purified to near homogeneity in the absence of detergents. The sedimentation velocity studies gave an s 20,w value of 4.1 ± 0.4 S. The two subunits segregated as a dimer upon sucrose density gradient centrifugation and purification by sequential ion exchange and hydroxylapatite chromatography. The results show that baculovirus‐infected insect cells can be employed for high level production of pure G‐protein βγ dimers suitable for functional and structural characterization.

Kupffer Cell-Derived Tnf Triggers Cholangiocellular Tumorigenesis through JNK due to Chronic Mitochondrial Dysfunction and ROS

Intrahepatic cholangiocarcinoma (ICC) is a highly malignant, heterogeneous cancer with poor treatment options. We found that mitochondrial dysfunction and oxidative stress trigger a niche favoring cholangiocellular overgrowth and tumorigenesis. Liver damage, reactive oxygen species (ROS) and paracrine tumor necrosis factor (Tnf) from Kupffer cells caused JNK-mediated cholangiocellular proliferation and oncogenic transformation. Anti-oxidant treatment, Kupffer cell depletion, Tnfr1 deletion, or JNK inhibition reduced cholangiocellular pre-neoplastic lesions. Liver-specific JNK1/2 deletion led to tumor reduction and enhanced survival in Akt/Notch- or p53/Kras-induced ICC models. In human ICC, high Tnf expression near ICC lesions, cholangiocellular JNK-phosphorylation, and ROS accumulation in surrounding hepatocytes are present. Thus, Kupffer cell-derived Tnf favors cholangiocellular proliferation/differentiation and carcinogenesis. Targeting the ROS/Tnf/JNK axis may provide opportunities for ICC therapy.

Dickkopf-3, a tissue-derived modulator of local T cell responses

The adaptive immune system protects organisms from harmful environmental insults. In parallel, regulatory mechanisms control immune responses in order to assure preservation of organ integrity. Yet, molecules involved in the control of T-cell responses in peripheral tissues are poorly characterized. Here, we investigated the function of Dickkopf-3 in the modulation of local T-cell reactivity. Dkk3 is a secreted, mainly tissue-derived protein with highest expression in organs considered as immune-privileged such as the eye, embryo, placenta, and brain. While T-cell development and activation status in naïve Dkk3-deficient mice was comparable to littermate controls, we found that Dkk3 contributes to the immunosuppressive microenvironment that protects transplanted, class-I mismatched embryoid bodies from T-cell-mediated rejection. Moreover, genetic deletion or antibody-mediated neutralization of Dkk3 led to an exacerbated experimental autoimmune encephalomyelitis (EAE). This phenotype was accompanied by a change of T-cell polarization displayed by an increase of IFNγ-producing T cells within the central nervous system. In the wild-type situation, Dkk3 expression in the brain was up-regulated during the course of EAE in an IFNγ-dependent manner. In turn, Dkk3 decreased IFNγ activity and served as part of a negative feedback mechanism. Thus, our findings suggest that Dkk3 functions as a tissue-derived modulator of local CD4+ and CD8+ T-cell responses.

Protection against malaria by immunization with non-attenuated sporozoites under single-dose piperaquine-tetraphosphate chemoprophylaxis

Experimental whole-parasite immunization through concurrent administration of infectious Plasmodium sporozoites with drugs that prevent pathogenic blood-stage infection represents the current benchmark in malaria vaccine development. Key questions concerning translation remain, including the requirement for single-dose drug regimens that can reliably prevent breakthrough infections. We assessed the feasibility and efficacy of immunization with single-dose piperaquine chemoprophylaxis and concurrent sporozoite administration (PPQ-CPS) in the murine P. berghei ANKA/C57BL/6 infection model. We demonstrate that PPQ-CPS is protective with an efficacy comparable to previous findings using whole-parasite immunization under chloroquine chemoprophylaxis. PPQ-CPS immunization resulted in an expansion of intrahepatic and intrasplenic effector memory CD8(+) T cells. In summary, PPQ-CPS appears to be a safe and efficacious immunization regimen in the rodent malaria model and may thus become an important improvement regarding the translation of whole-parasite immunization toward a human malaria vaccine.

Tubular Dickkopf-3 promotes the development of renal atrophy and fibrosis

Renal tubular atrophy and interstitial fibrosis are common hallmarks of etiologically different progressive chronic kidney diseases (CKD) that eventually result in organ failure. Even though these pathological manifestations constitute a major public health problem, diagnostic tests, as well as therapeutic options, are currently limited. Members of the dickkopf (DKK) family, DKK1 and -2, have been associated with inhibition of Wnt signaling and organ fibrosis. Here, we identify DKK3 as a stress-induced, tubular epithelia-derived, secreted glycoprotein that mediates kidney fibrosis. Genetic as well as antibody-mediated abrogation of DKK3 led to reduced tubular atrophy and decreased interstitial matrix accumulation in two mouse models of renal fibrosis. This was facilitated by an amplified, antifibrogenic, inflammatory T cell response and diminished canonical Wnt/β-catenin signaling in stressed tubular epithelial cells. Moreover, in humans, urinary DKK3 levels specifically correlated with the extent of tubular atrophy and interstitial fibrosis in different glomerular and tubulointerstitial diseases. In summary, our data suggest that DKK3 constitutes an immunosuppressive and a profibrotic epithelial protein that might serve as a potential therapeutic target and diagnostic marker in renal fibrosis.

SMARCA4 and SMARCA2 deficiency in non-small cell lung cancer: immunohistochemical survey of 316 consecutive specimens.

The chromatin remodeling switch sucrose nonfermentable (SWI/SNF) complex has been increasingly implicated in the pathogenesis and dedifferentiation of neoplasms from several organs with prognostic and potential therapeutic implications. We herein investigated the expression of the SWI/SNF complex catalytic subunits SMARCA4 (BRG1) and SMARCA2 (BRM) in 316 consecutive non-small cell lung cancer (NSCLC) specimens on tissue microarrays (171 adenocarcinomas [ADCAs], 130 squamous cell carcinomas [SCCs], 9 adenosquamous carcinomas, and 6 large cell carcinomas) excluding undifferentiated/giant cell or rhabdoid carcinomas. Complete loss of SMARCA4 was observed in 8 (5.5%) of 146 evaluable pulmonary ADCAs and 6 (5.2%) of 115 evaluable pulmonary SCCs, whereas 9 (6.4%) of 140 ADCAs and 2 (1.7%) of 117 SCCs showed SMARCA2 loss. Two of 6 large cell carcinomas were SMARCA2 deficient. Concurrent loss of both markers was observed in 4 cases (2 ADCAs and 2 SCCs). Of 15 ADCAs with loss of either or both markers, 12 (80%) were TTF1 negative. In conclusion, SMARCA4 and SMARCA2 deficiency is observed in 5.1% and 4.8% of NSCLC, respectively. SMARCB1 expression was intact in all cases. The presence of differentiated histology (glandular or squamous) is a novel aspect among SWI/SNF-deficient carcinomas which in other organs generally are associated with undifferentiated/rhabdoid morphology. The predominance of TTF1 negativity among SWI/SNF-deficient pulmonary ADCA (80%) underlines the need to include these 2 markers in the evaluation of TTF1-negative ADCA of putative pulmonary origin. Given the recently documented potential of SMARCA4 loss as a predictor of chemosensitivity to platinum-based chemotherapy in NSCLC, recognition of the clinicopathological features of SMARCA4-deficient NSCLC in routine surgical pathology practice is recommended.

Combination of Mesothelin and CEA Significantly Improves the Differentiation between Malignant Pleural Mesothelioma, Benign Asbestos Disease, and Lung Cancer

Introduction: Soluble mesothelin-related peptides (SMRP) have been reported as potential markers for the diagnosis of malignant pleural mesothelioma (MPM). We wondered, whether a combination with a carcinoembryonic antigen (CEA) test might improve the relatively low diagnostic yield of the SMRP test. Methods: In a retrospective study, SMRP (mesothelin) and CEA serum concentrations were measured, using commercially available kits, in 93 previously untreated MPM patients, 75 patients with benign asbestos disease, and 139 patients suffering from lung cancer (LC). Results: The differentiation between MPM, LC, and benign asbestos disease could be improved by applying the ratio mesothelin/CEA. Whereas CEA expression was found to be low in MPM, most LC patients had elevated CEA serum levels. The area under curve (AUC) of the receiver operator characteristics curve for mesothelin alone was found to be only 0.708. For mesothelin/CEA the AUC of the receiver operator characteristics curve increased to 0.978. The sensitivity was 93% (69%) at 95% (100%) specificity for the differentiation between MPM and LC. Comparison of MPM and benign asbestos disease showed that the AUC was 0.887 and the sensitivity 56% (47%) at 95% (100%) specificity. In contrast, the AUC for the mesothelin test alone was only 0.715, and for the CEA test alone it was 0.16. An average increment in sensitivity of 38% (range, 16%–63%) could be achieved by the quotient mesothelin/CEA compared with the sensitivity of mesothelin alone. Conclusion: The diagnostic yield of the mesothelin test can be considerably improved when combined with a CEA test with regard to the differential diagnosis between MPM and LC and between MPM and benign asbestos disease.

Epigenetically Regulated Chromosome 14q32 miRNA Cluster Induces Metastasis and Predicts Poor Prognosis in Lung Adenocarcinoma Patients

Most lung cancer deaths are related to metastases, which indicates the necessity of detecting and inhibiting tumor cell dissemination. Here, we aimed to identify miRNAs involved in metastasis of lung adenocarcinoma as prognostic biomarkers and therapeutic targets. To that end, lymph node metastasis–associated miRNAs were identified in The Cancer Genome Atlas lung adenocarcinoma patient cohort (sequencing data; n = 449) and subsequently validated by qRT-PCR in an independent clinical cohort (n = 108). Overexpression of miRNAs located on chromosome 14q32 was associated with metastasis in lung adenocarcinoma patients. Importantly, Kaplan–Meier analysis and log-rank test revealed that higher expression levels of individual 14q32 miRNAs (mir-539, mir-323b, and mir-487a) associated with worse disease-free survival of never-smoker patients. Epigenetic analysis including DNA methylation microarray data and bisulfite sequencing validation demonstrated that the induction of 14q32 cluster correlated with genomic hypomethylation of the 14q32 locus. CRISPR activation technology, applied for the first time to functionally study the increase of clustered miRNA levels in a coordinated manner, showed that simultaneous overexpression of 14q32 miRNAs promoted tumor cell migratory and invasive properties. Analysis of individual miRNAs by mimic transfection further illustrated that miR-323b-3p, miR-487a-3p, and miR-539-5p significantly contributed to the invasive phenotype through the indirect regulation of different target genes. In conclusion, overexpression of 14q32 miRNAs, associated with the respective genomic hypomethylation, promotes metastasis and correlates with poor patient prognosis in lung adenocarcinoma. Implications: This study points to chromosome 14q32 miRNAs as promising targets to inhibit tumor cell dissemination and to predict patient prognosis in lung adenocarcinoma. Mol Cancer Res; 16(3); 390–402. ©2018 AACR.

Self-Antigen Presentation by Keratinocytes in the Inflamed Adult Skin Modulates T-Cell Auto-Reactivity

Keratinocytes have a pivotal role in the regulation of immune responses, but the impact of antigen presentation by these cells is still poorly understood, particularly in a situation where the antigen will be presented only in adult life. Here, we generated a transgenic mouse model in which keratinocytes exclusively present a myelin basic protein (MBP) peptide covalently linked to the major histocompatibility complex class II β-chain, solely under inflammatory conditions. In these mice, inflammation caused by epicutaneous contact sensitizer treatment resulted in keratinocyte-mediated expansion of MBP-specific CD4(+) T cells in the skin. Moreover, repeated contact sensitizer application preceding a systemic MBP immunization reduced the reactivity of the respective CD4(+) T cells and lowered the symptoms of the resulting experimental autoimmune encephalomyelitis. This downregulation was CD4(+) T-cell-mediated and dependent on the presence of the immune modulator Dickkopf-3. Thus, presentation of a neo self-antigen by keratinocytes in the inflamed, adult skin can modulate CD4(+) T-cell auto-aggression at a distal organ.