Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in &lt;em&gt;In Vitro&lt;/em&gt; Cancer Models

Sciarrillo, Rocco; Wojtuszkiewicz, Anna; Kooi, Irsan; Gomez, Valentina; Boggi, Ugo; Jansen, Gerrit; Kaspers, Gertjan J. L.; Cloos, Jacqueline; Giovannetti, Elisa

doi:10.3791/54714

Cited by 18 publications

(27 citation statements)

References 35 publications

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“…SRB assay. Cell growth inhibition was performed with the sulforhodamine B assay (SRB), as previously described in (21). Briefly, cells in exponential growth phase were harvested by trypsinization, counted with the Coulter Counter (Z Series, Beckman, Indianapolis, USA) and plated at concentrations of 3000 cells per well in a 96-well flat bottom plate (Greiner Bio-One GmbH, Frickenhausen, Germany).…”

Section: Methodsmentioning

confidence: 99%

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A mechanical memory of pancreatic cancer cells

Carnevale

Capula

Giovannetti

et al. 2019

Preprint

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Cells sense and respond to mechanical stimuli in healthy and pathological conditions. Although the major mechanisms underlying cellular mechanotransduction have been described, it remains largely unclear how cells store information on past mechanical cues over time. Such mechanical memory is extremely relevant in the onset of metastasis in which cancer cells migrate through tissues of different stiffness, e.g. from a stiffer tumor microenvironment to softer metastatic sites as commonly occurs for pancreatic cancer. Here, we used micropillar-based traction force microscopy to show that Suit-2.28 pancreatic cancer cells mechanically primed on a stiff matrix exerted higher traction forces even when transferred to a soft secondary matrix, as compared to soft-primed cells. This mechanical memory effect was mediated by the Yes-associated protein (YAP) and the microRNA-21 (miR-21) that are two mechanosensors initially identified as long-term memory keepers in mesenchymal stem cells. Soft-primed cells showed (i) a lower YAP nuclear translocation when transferred to a stiff secondary matrix and (ii) a loss of rigidity sensing through YAP, as compared to stiffprimed cells. The mechanical adaptation resulted in a differential expression of miR-21, inversely proportional to the priming rigidity. The long-term mechanical memory retained by miR-21 unveiled a previously unidentified mechanical modulation of drug resistance by past matrix stiffness. The higher expression of miR-21 in soft-primed cells correlated with the increased resistance to gemcitabine, as compared to stiff-primed and nonprimed pancreatic cancer cells. pancreatic cancer | cell mechanics | chemoresistance | gemcitabine Correspondence: stefano.

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Section: Methodsmentioning

confidence: 99%

“…10, 100, 1000 nM for 36 hours exposure) for soft-, stiff-primed and non-primed Suit-2.28 cells ('Soft-primed', 'Stiff-primed', and 'Plastic', respectively) at passage P4 (see Fig. 1D), as quantified by sulforhodamine B (SRB) assay (21). Values represent mean±SD of six separate tests.…”

Section: S O F T S O F T S T I F F S O F T S O F T S T I F F S T I F mentioning

confidence: 99%

A mechanical memory of pancreatic cancer cells

Carnevale

Capula

Giovannetti

et al. 2019

Preprint

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“…The sequencing reaction yielded 22.3 ± 5.1 (average ± SD) million passing-filter raw reads/sample (raw data are deposited in the GEO database with accession number: GSE133499). The bioinformatic pipeline for data analysis was described previously [37] and summarized in the Supplemental Methods.…”

Section: Rna Sequencingmentioning

confidence: 99%

“…Ψ is computed as the proportion of reads supporting the inclusion of the exon in question divided by the sum of reads supporting the inclusion and skipping of this exon. Subsequently, it compares the average Ψ values of GC-sensitive specimens with that of GC-resistant samples by computing the Inclusion Level Difference (∆Ψ) and the corresponding p-value and false discovery rate (FDR) for each splice event [37]. We found, in total, 994 significant differential splicing events (FDR < 0.05, Figure 2B and Supplemental Data S2) affecting 762 genes.…”

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“…This suggests that leukemic cells of GC-resistant patients carry a more profound splicing dysregulation which could be exploited for therapeutic purposes.Building on these data, the aim of the current study is to characterize the global alternative splicing profiles associated with ex vivo GC resistance in childhood ALL. Specifically, we investigated differential splicing profiles in 38 primary childhood ALL samples by using RNA sequencing [37]. Finally, we tested whether resistant cell lines and primary specimens can be sensitized to GC treatment by using SF3B modulators.…”

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Glucocorticoid Resistant Pediatric Acute Lymphoblastic Leukemia Samples Display Altered Splicing Profile and Vulnerability to Spliceosome Modulation

Sciarrillo

Wojtuszkiewicz

Kooi

et al. 2020

Cancers

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Cancers 2020, 12, 723 3 of 27 Whereas spliceosome mutations were infrequent in ALL [32], BCP-ALL cells did display global aberrant splicing when compared with non-malignant controls [33,34]. Interestingly, we previously showed that aberrant splicing of folylpolyglutamate synthetase (FPGS), a key determinant of methotrexate (MTX) efficacy, is associated with MTX resistance in childhood ALL [35,36]. Notably, high levels of one particular aberration, i.e., FPGS intron 8 partial retention, were also associated with increased resistance to GCs. This suggests that leukemic cells of GC-resistant patients carry a more profound splicing dysregulation which could be exploited for therapeutic purposes.Building on these data, the aim of the current study is to characterize the global alternative splicing profiles associated with ex vivo GC resistance in childhood ALL. Specifically, we investigated differential splicing profiles in 38 primary childhood ALL samples by using RNA sequencing [37]. Finally, we tested whether resistant cell lines and primary specimens can be sensitized to GC treatment by using SF3B modulators. Results Differential Splicing Landscape Associated with GC Resistance in Pediatric ALLIn order to evaluate whether GC resistance is associated with specific splicing patterns in childhood ALL, we used RNA sequencing to profile transcriptomes of specimens obtained from 36 newly diagnosed and two relapsed pediatric ALL patients ( Figure 1A). This study cohort was well characterized with respect to clinical features and ex vivo drug resistance, including Dex and Pred (Supplemental Data S1). The patient specimens were classified either as GC-sensitive (N = 15) or GC-resistant (N = 23) based on ex vivo Dex and Pred LC 50 values according to the previously established cut-offs of 0.01 µg/mL and 0.1 µg/mL, respectively [9] ( Figure 1B). Furthermore, we determined the immunophenotype and genetic profile of the samples, including mutations in GR and recurrent genetic alterations associated with ALL [21,38] (Figure 1C), which allowed us to account for possible confounders in the analysis.The global differential splicing profiles of GC-sensitive and GC-resistant samples were determined using the rMATS algorithm (Figure 2A). This software identifies sequencing reads which support a certain splice event (e.g., the inclusion or skipping of a certain exon in a gene of interest) and calculates the inclusion levels (or Percentage Spliced-In, Ψ). Ψ is computed as the proportion of reads supporting the inclusion of the exon in question divided by the sum of reads supporting the inclusion and skipping of this exon. Subsequently, it compares the average Ψ values of GC-sensitive specimens with that of GC-resistant samples by computing the Inclusion Level Difference (∆Ψ) and the corresponding p-value and false discovery rate (FDR) for each splice event [37]. We found, in total, 994 significant differential splicing events (FDR < 0.05, Figure 2B and Supplemental Data S2) affecting 762 genes. Hierarchical clustering ( Figure 2C) and ...

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1,2,4‐Oxadiazole Topsentin Analogs with Antiproliferative Activity against Pancreatic Cancer Cells, Targeting GSK3β Kinase

et al. 2020

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A new series of topsentin analogs, in which the central imidazole ring of the natural lead was replaced by a 1,2,4‐oxadiazole moiety, was efficiently synthesized. All derivatives were pre‐screened for antiproliferative activity against the National Cancer Institute (NCI‐60) cell lines panel. The five most potent compounds were further investigated in various pancreatic ductal adenocarcinoma (PDAC) cell lines, including SUIT‐2, Capan‐1, and Panc‐1 cells, eliciting EC50 values in the micromolar and sub‐micromolar range, associated with significant reduction of cell migration. These remarkable results might be explained by the effects of these new topsentin analogues on epithelial‐to‐mesenchymal transition markers, including SNAIL‐1/2 and metalloproteinase‐9. Moreover, flow cytometric analysis after Annexin V‐FITC and propidium iodide staining demonstrated that these derivatives enhanced apoptosis of PDAC cells. Keeping with these data, the PathScan intracellular signaling and ELISA array revealed cleavage of caspase‐3 and PARP and a significant inhibition of GSK3β phosphorylation, suggesting this kinase as a potential downstream target of our novel compounds. This was further supported by a specific assay for the evaluation of GSK3β activity, showing IC50 values for the most active compounds against this enzyme in the micromolar range.

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Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in <em>In Vitro</em> Cancer Models

Cited by 18 publications

References 35 publications

A mechanical memory of pancreatic cancer cells

A mechanical memory of pancreatic cancer cells

Glucocorticoid Resistant Pediatric Acute Lymphoblastic Leukemia Samples Display Altered Splicing Profile and Vulnerability to Spliceosome Modulation

1,2,4‐Oxadiazole Topsentin Analogs with Antiproliferative Activity against Pancreatic Cancer Cells, Targeting GSK3β Kinase

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