Identification of<i>cis-</i>and<i>trans</i>-acting elements regulating calretinin expression in mesothelioma cells

Kresoja‐Rakic, Jelena; Kapaklikaya, Esra; Ziltener, Gabriela; Dalcher, Damian; Santoro, Raffaella; Christensen, Brock C.; Johnson, Kevin C.; Schwaller, Beat; Weder, Walter; Stahel, Rolf A.; Felley‐Bosco, Emanuela

doi:10.18632/oncotarget.7114

Cited by 16 publications

(29 citation statements)

References 53 publications

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“…Changes in mesothelin, calretinin and podoplanin gene expression levels were also evaluated in tumor samples (Figure 1 E). Differences observed in vivo for selected mRNAs involved either in differentiation ( HES1, RUNX2 ), EMT ( GREM1, CTGF ), senescence ( PUMA, LOX, PAI1 ), drug transport ( SLC22A4, ABCC1 ), cell cycle ( CALB2 ( 20 ), CCND1, CDKN2A ), response to oxidative stress or hypoxia ( HO-1, CAIX, GLUT1 ), or MPM-associated surface proteins ( PDPN, THY1, CD276, TNFRSF10B ) were weakly ( R 2 = 0.2641) but significantly correlated with differences observed in primary cultures in patient P236 (Figure 1 F). This supports the idea of using primary cultures for the functional investigation of mechanisms underlying tumor evolution.…”

Section: Resultsmentioning

confidence: 99%

Live-Cell Mesothelioma Biobank to Explore Mechanisms of Tumor Progression

et al. 2018

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Experimental models closely representing in vivo conditions allow investigating mechanisms of resistance. Our aims were to establish a live-cell biobank of malignant pleural mesothelioma (MPM) samples and to obtain proof of principle that primary culture chemoresistant models, mimicking tumor progression observed in patients, can be obtained in vitro, providing a useful tool to investigate underlying mechanisms. Primary mesothelioma cultures were established from 235 samples between 2007 and 2014. Of two MPM patients, primary cultures obtained at different time points: at initial diagnosis, after neoadjuvant treatment at surgery and/or after tumor recurrence, were deeply investigated. Cells and corresponding tumor tissue were characterized by mesothelial protein and gene expression analysis. In addition, primary cultures from chemo naive patients were exposed to increasing doses of cisplatin/pemetrexed during three months and compared with non-treated cells in a cytotoxicity assay, and by selected profiling of senescence markers. In vitro chemoresistance in the primary mesothelioma cell cultures was associated with increased Thy1 (CD90) expression. Thy1 expression in MPM samples was significantly associated with poor overall survival in the TCGA MPM cohort. Our results illustrate that the establishment of a large live-cell MPM biobank contributes to a better understanding of therapy resistance observed in vivo, which eventually may lead to a more logical approach for developing new treatment strategies.

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Section: Resultsmentioning

confidence: 99%

Live-Cell Mesothelioma Biobank to Explore Mechanisms of Tumor Progression

et al. 2018

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“…siRNA-mediated down-regulation of NRF-1 causes a decrease in CR expression levels indicating that NRF-1 acts as a positive regulator of CR expression (14). Moreover, the strong correlation between CALB2 mRNA and CR protein expression levels in MM cells is indicative of a control at the transcriptional level [ 14 ]. In colon cancer cells, two butyrate-responsive elements (BRE) embracing the TATA box of the CALB2 gene function as butyrate-sensitive repressors of CR expression, while the same sequence has no effect in cells of mesothelial origin, e.g.…”

Section: Introductionmentioning

confidence: 99%

Regulation of calretinin in malignant mesothelioma is mediated by septin 7 binding to the CALB2 promoter

et al. 2018

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BackgroundThe calcium-binding protein calretinin (gene name: CALB2) is currently considered as the most sensitive and specific marker for the diagnosis of malignant mesothelioma (MM). MM is a very aggressive tumor strongly linked to asbestos exposure and with no existing cure so far. The mechanisms of calretinin regulation, as well as its distinct function in MM are still poorly understood.MethodsWe searched for transcription factors binding to the CALB2 promoter and modulating calretinin expression. For this, DNA-binding assays followed by peptide shotgun-mass spectroscopy analyses were used. CALB2 promoter activity was assessed by dual-luciferase reporter assays. Furthermore, we analyzed the effects of CALB2 promoter-binding proteins by lentiviral-mediated overexpression or down-regulation of identified proteins in MM cells. The modulation of expression of such proteins by butyrate was determined by subsequent Western blot analysis. Immunohistochemical analysis of embryonic mouse lung tissue served to verify the simultaneous co-expression of calretinin and proteins interacting with the CALB2 promoter during early development. Finally, direct interactions of calretinin with target proteins were evidenced by co-immunoprecipitation experiments.ResultsSeptin 7 was identified as a butyrate-dependent transcription factor binding to a CALB2 promoter region containing butyrate-responsive elements (BRE) resulting in decreased calretinin expression. Accordingly, septin 7 overexpression decreased calretinin expression levels in MM cells. The regulation was found to operate bi-directionally, i.e. calretinin overexpression also decreased septin 7 levels. During murine embryonic development calretinin and septin 7 were found to be co-expressed in embryonic mesenchyme and undifferentiated mesothelial cells. In MM cells, calretinin and septin 7 colocalized during cytokinesis in distinct regions of the cleavage furrow and in the midbody region of mitotic cells. Co-immunoprecipitation experiments revealed this co-localization to be the result of a direct interaction between calretinin and septin 7.ConclusionsOur results demonstrate septin 7 not only serving as a “cytoskeletal” protein, but also as a transcription factor repressing calretinin expression. The negative regulation of calretinin by septin 7 and vice versa sheds new light on mechanisms possibly implicated in MM formation and identifies these proteins as transcriptional regulators and putative targets for MM therapy.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4385-7) contains supplementary material, which is available to authorized users.

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“…Total protein extracts were obtained by lysing the cells with hot Laemmli buffer (60 mmol/L Tris-HCl pH 6.8, 100 mmol/L DTT, 5% glycerol, 1.7% SDS) and passed through syringes (26G; ref. 18). Spheroids were collected 48 hours posttreatment.…”

Section: Protein Extraction and Western Blottingmentioning

confidence: 99%

Functional Genomic Screen in Mesothelioma Reveals that Loss of Function of BRCA1-Associated Protein 1 Induces Chemoresistance to Ribonucleotide Reductase Inhibition

Okonska

Rao

et al. 2020

Molecular Cancer Therapeutics

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Loss of function of BRCA1-associated protein 1 (BAP1) is observed in about 50% of malignant pleural mesothelioma (MPM) cases. The aim of this study was to investigate whether this aspect could be exploited for targeted therapy. A genetically engineered model was established expressing either functional or nonfunctional BAP1, and whole-genome siRNA synthetic lethality screens were performed assessing differentially impaired survival between the two cell lines. The whole-genome siRNA screen unexpectedly revealed 11 hits (FDR < 0.05) that were more cytotoxic to BAP1proficient cells. Two actionable targets, ribonucleotide reductase (RNR) catalytic subunit M1 (RRM1) and RNR regulatory subunit M2 (RRM2), were validated. In line with the screen results, primary mesothelioma (BAP1 þ/À) overexpressing BAP1 C91A (catalytically dead mutant) was more resistant to RNR inhibition, while BAP1 knockdown in the BAP1-proficient cell lines rescued the cells from their vulnerability to RNR depletion. Gemcitabine and hydroxyurea were more cytotoxic in BAP1-proficient cell line-derived spheroids compared with BAP1 deficient. Upregulation of RRM2 upon gemcitabine and hydroxyurea treatment was more profound in BAP1 mut/del cell lines. Increased lethality mediated by RNR inhibition was observed in NCI-H2452 cells reconstituted with BAP1-WT but not with BAP1 C91A. Upregulation of RRM2 in NCI-H2452-BAP1 WT spheroids was modest compared with control or C91A mutant. Together, we found that BAP1 is involved in the regulation of RNR levels during replication stress. Our observations reveal a potential clinical application where BAP1 status could serve as predictive or stratification biomarker for RNR inhibition-based therapy in MPM.

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Identification ofcis-andtrans-acting elements regulating calretinin expression in mesothelioma cells

Cited by 16 publications

References 53 publications

Live-Cell Mesothelioma Biobank to Explore Mechanisms of Tumor Progression

Live-Cell Mesothelioma Biobank to Explore Mechanisms of Tumor Progression

Regulation of calretinin in malignant mesothelioma is mediated by septin 7 binding to the CALB2 promoter

Functional Genomic Screen in Mesothelioma Reveals that Loss of Function of BRCA1-Associated Protein 1 Induces Chemoresistance to Ribonucleotide Reductase Inhibition

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