BAP1 missense mutations in cancer: friend or foe?

BRCA-associated protein-1 (BAP1) is mutated in several cancers and a few therapies targeting BAP1 loss-of-function mutations have been proposed, some of them being already tested in clinical trials. However, most of the missense mutations have not been functionally characterized, although such information is essential for successful patient stratification

Decreased activity of inducible nitric oxide synthase type 2 and modulation of the expression of glutathione S-transferase alpha, bcl-2, and metallothioneins during the differentiation of CaCo-2 cells.

Reactive oxygen species modulate the cell growth of a wide variety of mammalian cells. To determine whether oxidative metabolism is altered during the differentiation process, we studied the expression of pro- and antioxidant proteins in proliferating and differentiated CaCo-2 cells, a human colon adenocarcinoma cell line. Nitric oxide synthase type 2 (iNOS) produces nitric oxide (NO). Depending on its rate of synthesis, NO may either promote cellular and DNA damage or reduce the ability of other free radicals to induce cell injury. Using Western and Northern blot analysis and arginine conversion assay, we demonstrate that the expression of iNOS decreases when cells undergo differentiation. This biological event entails a diminished production of NO metabolites and correlates with the loss of activation of soluble guanylate cyclase activity. In differentiated cells, a 2-fold down-regulation of the nuclear factor kappa B activity was observed, suggesting that nuclear factor kappa B could be one of the iNOS gene regulatory factors in the CaCo-2 model. In parallel, we studied the expression of other antioxidant proteins including glutathione S-transferase alpha (GST alpha), bcl-2, and the metallothioneins (MTs). We show that the protein levels of GST alpha and MT increase during the differentiation of CaCo-2 cells, whereas bcl-2 levels decrease. Our investigation indicates that the expression of iNOS, GST alpha, bcl-2, and MT is associated with the enterocytic differentiation. The shift in the expression of specific antioxidant genes during CaCo-2 cell differentiation may occur to avoid alterations in the cell redox potential

Polarized distribution of inducible nitric oxide synthase regulates activity in intestinal epithelial cells

Inducible nitric oxide synthase (iNOS) functions as a homodimer. In cell extracts, iNOS molecules partition both in cytosolic and particulate fractions, indicating that iNOS exists as soluble and membrane associated forms. In this study, iNOS features were investigated in human intestinal epithelial cells stimulated with cytokines and in duodenum from mice exposed to flagellin. Our experiments indicate that iNOS is mainly associated with the particulate fraction of cell extracts. Confocal microscopy showed a preferential localization of iNOS at the apical pole of intestinal epithelial cells. In particulate fractions, iNOS dimers were more abundant than in the cytosolic fraction. Similar observations were seen in mouse duodenum samples. These results suggest that, in epithelial cells, iNOS activity is regulated by localization-dependent processes.Facultad de Ciencias Exacta

Stem Cell Factor-Based Identification and Functional Properties of In Vitro-Selected Subpopulations of Malignant Mesothelioma Cells

Malignant mesothelioma (MM) is an aggressive neoplasm characterized by a poor patient survival rate, because of rapid tumor recurrence following first-line therapy. Cancer stem cells (CSCs) are assumed to be responsible for initiating tumorigenesis and driving relapse after therapeutic interventions. CSC-enriched MM cell subpopulations were identified by an OCT4/SOX2 reporter approach and were characterized by (1) increased resistance to cisplatin, (2) increased sensitivity toward the FAK inhibitor VS-6063 in vitro, and (3) a higher tumor-initiating capacity in vivo in orthotopic xenograft and allograft mouse models. Overexpression of NF2 (neurofibromatosis 2, merlin), a tumor suppressor often mutated or lost in MM, did not affect proliferation and viability of CSC-enriched MM populations but robustly decreased the viability of reporter-negative cells. In contrast, downregulation of calretinin strongly decreased proliferation and viability of both populations. In summary, we have enriched and characterized a small MM cell subpopulation that bears the expected CSC characteristics

Stem cell factor-based identification and functional properties of in vitro-selected subpopulations of malignant mesothelioma cells

Malignant mesothelioma (MM) is an aggressive neoplasm characterized by a poor patient survival rate, because of rapid tumor recurrence following first-line therapy. Cancer stem cells (CSCs) are assumed to be responsible for initiating tumorigenesis and driving relapse after therapeutic interventions. CSC-enriched MM cell subpopulations were identified by an OCT4/SOX2 reporter approach and were characterized by (1) increased resistance to cisplatin, (2) increased sensitivity toward the FAK inhibitor VS-6063 in vitro, and (3) a higher tumor-initiating capacity in vivo in orthotopic xenograft and allograft mouse models. Overexpression of NF2 (neurofibromatosis 2, merlin), a tumor suppressor often mutated or lost in MM, did not affect proliferation and viability of CSC-enriched MM populations but robustly decreased the viability of reporter-negative cells. In contrast, downregulation of calretinin strongly decreased proliferation and viability of both populations. In summary, we have enriched and characterized a small MM cell subpopulation that bears the expected CSC characteristics

Prevalence of BRCA-1 associated protein 1 germline mutation in sporadic malignant pleural mesothelioma cases

OBJECTIVE: 23% of mesothelioma tumor specimens have a mutation in the BRCA1-associated protein 1 (BAP1) gene and germline BAP1 mutations predispose to malignant pleural mesothelioma (MPM). Our aim was to investigate germline BAP1 mutations in sporadic MPM patients. MATERIALS AND METHODS: Exonic DNA from peripheral blood leucocytes of 78 MPM patients was screened for germline BAP1 mutation. RESULTS: One out of 78 patients showed a germline synonymous mutation in exon 11. In all other patients wild-type sequence without any single-nucleotide polymorphisms was detected. CONCLUSIONS: Taking into account previous similar screenings, the prevalence of germline BAP1 mutations in sporadic MPM patients can be estimated around 1-2%, suggesting a minor role of germline BAP1 mutation in the pathogenesis of sporadic MPM

Development of a matrix-based technology platform for the high throughput analysis of 3D cell cultures

The screening of large cell libraries is an important process in pharmaceutical discovery and R&D, e.g. to define drug targets or develop effective medicines. The goal of this project is the implementation of a screening platform based on 3D cultivation of primary human mesothelioma cells encapsulated in alginate hydrogels. To this end, new hydrogel compositions will be designed, tested and finally utilized in the Nanoliter Reactor (NLR) cultivation system that enables high throughput analysis of 3D cell cultures

Genomic landscape of pleural and peritoneal mesothelioma tumours

BACKGROUND Malignant pleural and peritoneal mesotheliomas are rare malignancies with unacceptable poor prognoses and limited treatment options. The genomic landscape is mainly characterised by the loss of tumour suppressor genes and mutations in DNA repair genes. Currently, data from next-generation sequencing (NGS) of mesothelioma tumours is restricted to a limited number of cases; moreover, data comparing molecular features of mesothelioma from the pleural and peritoneal origin with NGS are lacking. METHODS We analysed 1113 pleural mesothelioma and 355 peritoneal mesothelioma samples. All tumours were sequenced with the FoundationOne® or FoundationOne®CDx assay for detection of substitutions, insertion-deletions, copy-number alterations and selected rearrangements in at least 324 cancer genes. RESULTS This analysis revealed alterations in 19 genes with an overall prevalence of at least 2%. Alterations in BAP1, CDKN2A, CDKN2B, NF2, MTAP, TP53 and SETD2 occurred with a prevalence of at least 10%. Peritoneal, compared to pleural mesothelioma, was characterised by a lower prevalence of alterations in CDKN2A, CDKN2B and MTAP. Moreover, we could define four distinct subgroups according to alterations in BAP1 and CDKN2A/B. Alterations in Hedgehog pathway-related genes (PTCH1/2 and SUFU) and Hippo pathway-related gene (NF2) as well as KRAS, EGFR, PDGFRA/B, ERBB2 and FGFR3 were detected in both cohorts. CONCLUSION Here, we report the molecular aberrations from the largest cohort of patients with mesothelioma. This analysis identified a proportion of patients with targetable alterations and suggests that molecular profiling can identify new treatment options for patients with mesothelioma

Searching for targets for the systemic therapy of mesothelioma

Increasing knowledge about the molecular characteristics of mesothelioma had led to the identification of novel potential targets for systemic therapy. This review elaborates on the rationale behind targeted approaches that have been and are undergoing exploration in mesothelioma and summarizes available clinical results and ongoing efforts to improve the systemic therapy of mesotheliom

Immuno-chemotherapy reduces recurrence of malignant pleural mesothelioma: an experimental setting

Objective: To assess the effect of immuno-chemotherapy on the extent of local tumour recurrence in an established rat model of malignant pleural mesothelioma (MPM). Methods: Six days after subpleural inoculation of a syngeneic MPM cell line Interleukin-45 (IL-45), left-sided pneumonectomy and resection of the tumour nodule was performed. Animals were randomised into four treatment groups for intrapleural therapy: control (n=6), 500μg cytosine phosphate guanosine oligodeoxynucleotide (CpG-ODN) (n=6), cisplatin-fibrin (n=6), cisplatin-fibrin+500μg CpG (n=6). Six days later the volume of tumour recurrence was assessed, which was the primary endpoint. Secondary endpoints were quantification of the ratio host/tumour cells in the local recurrence and cytokine expression profile in the tumour tissue by real time quantitative PCR (qPCR). T lymphocyte subpopulations in the tumour recurrence tissue were evaluated by immunohistochemistry. Treatment-related toxicity was monitored by measuring blood chemistry and complete blood count. Results: The volume of tumour recurrence was significantly reduced from 610mm3 in the control group to 11.7mm3 in the cisplatin-fibrin group (p=0.004) and to 21.8mm3 in the cisplatin-fibrin+CpG group (p=0.004). Pro-inflammatory cytokines (Interferon-γ (IFN-γ), Interleukin-6 (IL-6), Interleukin-12 (IL-12)) were increased after treatment with cisplatin-fibrin+CpG in comparison to cisplatin-fibrin alone but differences were not statistically significant. We found a higher ratio of host/tumour cells in the cisplatin-fibrin+CpG group (45/55%) compared to the cisplatin-fibrin group (27/73%). In comparison to the control group, animals treated with cisplatin-fibrin+CpG showed a higher number of CD8+ T-cells in the tumour tissue. No significant treatment-related toxicity was observed. Conclusions: Adjuvant treatment with chemotherapy or immuno-chemotherapy leads to significant reduction of mesothelioma recurrence after surgery in this rat MPM model. Immuno-chemotherapy resulted in an increased recruitment of inflammatory cells to the site of tumourigenesis and elicited higher level of tumour growth inhibiting cytokine