Comprehensive screening of genes resistant to an anticancer drug in esophageal squamous cell carcinoma

Inhibitors of double minute 2 protein (MDM2)-tumor protein 53 (TP53) interaction are predicted to be effective in tumors in which the TP53 gene is wild type, by preventing TP53 protein degradation. One such setting is represented by the frequent CDKN2A deletion in human cancer that, through inactivation of p14ARF, activates MDM2 protein, which in turn degrades TP53 tumor suppressor. Here we used piggyBac (PB) transposon insertional mutagenesis to anticipate resistance mechanisms occurring during treatment with the MDM2-TP53 inhibitor HDM201. Constitutive PB mutagenesis in Arf −/− mice provided a collection of spontaneous tumors with characterized insertional genetic landscapes. Tumors were allografted in large cohorts of mice to assess the pharmacologic effects of HDM201. Sixteen out of 21 allograft models were sensitive to HDM201 but ultimately relapsed under treatment. A comparison of tumors with acquired resistance to HDM201 and untreated tumors identified 87 genes that were differentially and significantly targeted by the PB transposon. Resistant tumors displayed a complex clonality pattern suggesting the emergence of several resistant subclones. Among the most frequent alterations conferring resistance, we observed somatic and insertional loss-of-function mutations in transformation-related protein 53 (Trp53) in 54% of tumors and transposonmediated gain-of-function alterations in B-cell lymphoma-extra large (Bcl-xL), Mdm4, and two TP53 family members, resulting in expression of the TP53 dominant negative truncations ΔNTrp63 and ΔNTrp73. Enhanced BCL-xL and MDM4 protein expression was confirmed in resistant tumors, as well as in HDM201-resistant patient-derived tumor xenografts. Interestingly, concomitant inhibition of MDM2 and BCL-xL demonstrated significant synergy in p53 wild-type cell lines in vitro. Collectively, our findings identify several potential mechanisms by which TP53 wild-type tumors may escape MDM2-targeted therapy.drug resistance | MDM2 inhibitor | piggyBac screen | cancer A mong the genes most commonly altered in human cancer, regardless of tumor type, are tumor protein 53 (TP53) tumor suppressor (1) and CDKN2A (INK4a/ARF) (2). The latter gene encodes two tumor suppressor proteins: p16INK4a (3), an inhibitor of cyclin D-dependent kinases that, at least in part, regulates the function of the retinoblastoma protein (RB), and p19ARF (4), a negative regulator of double minute 2 protein (MDM2) function that activates TP53, thereby inducing cell cycle arrest or apoptosis. TP53 protein levels are regulated through MDM2-mediated degradation.Toward the goal of reactivating TP53 in cells harboring inactivating upstream pathway alterations, compounds inhibiting the interaction between MDM2 and TP53, preventing TP53 degradation, have been discovered. Such agents induce TP53 reactivation in tumors in which the TP53 gene is wild type (5-8).Although preclinical studies of such MDM2 inhibitors have demonstrated significant antitumor activity, tumors commonly relapse (9, 10).Rapid emergence of resistance is...

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“…In several studies, this method also has been used to characterize resistance mechanisms in vitro (17)(18)(19) and in mice…”

mentioning

confidence: 99%

Resistance mechanisms to TP53-MDM2 inhibition identified by in vivo piggyBac transposon mutagenesis screen in an Arf ^−/− mouse model

Chapeau

Gembarska

Durand

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…We established transposon-tagged cells in TE4 and TE15, human well-differentiated esophageal squamous cell carcinoma cell lines provided by the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan) (6). Puromycin-resistant genes in transposons were also inserted into the transposon-tagged cells for puromycin selection.…”

Section: Methodsmentioning

confidence: 99%

“…The base sequences of the transposons' insertion sites were detected using Splinkerette polymerase chain reaction (PCR), TOPO cloning, and Sanger sequencing performed in accordance with the protocol of our previous study (6). Insertion sites were aligned using the BLAST function of the National Library of Medicine (http://blast.ncbi.nlm.nih.…”

Section: Methodsmentioning

confidence: 99%

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Downregulation of cytochrome c oxidase 1 induced radioresistance in esophageal squamous cell carcinoma

et al. 2017

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Abstract. Comprehensive gene screening with transposons is a novel procedure for the systematic identification of resistant genes. The present study aimed to use this technique to identify candidate radioresistant genes in esophageal squamous cell carcinoma. A transposon is a base sequence that can translocate to another location in the genome at random. By inserting the cytomegalovirus promotor as a transcriptional activator in the transposon, the following gene in the new location becomes overexpressed and the gene located at the transposon insertion site is downregulated. Consequently, various transposon-tagged cells, which have differentially overexpressed or downregulated genes using the transposon method can be obtained. Following the irradiation of transposon-tagged cells, candidate radioresistant genes can be selected in order to detect the location of the transposon in the cells that have survived. A total of 11 genes were detected as candidate radioresistant genes. Cytochrome c oxidase 1 (MT-CO1), an enzyme involved in apoptosis through the activation of the caspase cascade, was one of the candidate genes identified. The relative expression level of MT-CO1 was 0.12 in MT-CO1-downregulated cells which was significantly lower compared with the expression level in parent TE4 cells (P<0.001). The survival rate was 28.7% in MT-CO1-downregulated cells and 10.5% in parent TE4 cells 9 days following 5-Gy irradiation. The activity of cytochrome c and caspase-3 following irradiation was significantly lower in the MT-CO1-downregulated radioresistant cells compared with in TE4 cells. In conclusion, the novel gene screening technique demonstrated to be useful for detecting candidate radioresistant genes in esophageal squamous cell carcinoma. The results of the present study revealed that the downregulation of MT-CO1 induced radioresistance occurs by inhibiting the activation of the caspase cascade in radioresistant esophageal cancer cells.

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“…Superbugs (i.e., bacteria which are resistant to nearly all antibiotics) have evolved due to the abuse, misuse, and negligence of antibiotics. They are capable of transferring their resistance by mechanisms as plasmid [2], transposon [3], and integrons [4]. This means that just one superbug can render an infection nearly untreatable.…”

Section: Introductionmentioning

confidence: 99%

Biodegradable Chitosan Matrix Composite Reinforced with Titanium Dioxide for Biocidal Applications

Calero¹,

Berríos²,

Suárez³

2019

Renewable and Sustainable Composites

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Access to drinkable water is increasingly difficult in developing nations. According to the World Health Organization, more than 1.2 billion people lack access to this resource. The scientific community has risen to the challenge by developing innovating methods to purify water. In this context, a chitosan/titanium dioxide biocomposite represents an appealing, economically viable solution to remove oils, heavy metals, and pathogen colonies from contaminated waters. The present chapter reports the study of a chitosan film matrix impregnated with nanoparticles, which was capable of expressing antibacterial properties when exposed to UV light. Several techniques allowed characterizing the biocomposite uniformity, corroborating the crystal structure and assessing the bonding type of this biocomposite mixture. Antibacterial tests with Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) were completed via growth curve analysis and the Kirby-Bauer technique. The results of this encompassing study revealed that bacterial growth was reduced by more than 50%. Optical microscopy, Fourier-transform infrared spectroscopy, and X-ray diffraction helped to characterize this biocomposite film. This chapter discusses how a novel and biodegradable film represents a better antibacterial material that is able to eliminate pathogens cost-effectively.

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Comprehensive screening of genes resistant to an anticancer drug in esophageal squamous cell carcinoma

Cited by 16 publications

References 21 publications

Resistance mechanisms to TP53-MDM2 inhibition identified by in vivo piggyBac transposon mutagenesis screen in an Arf ^−/− mouse model

Resistance mechanisms to TP53-MDM2 inhibition identified by in vivo piggyBac transposon mutagenesis screen in an Arf ^−/− mouse model

Downregulation of cytochrome c oxidase 1 induced radioresistance in esophageal squamous cell carcinoma

Biodegradable Chitosan Matrix Composite Reinforced with Titanium Dioxide for Biocidal Applications

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Comprehensive screening of genes resistant to an anticancer drug in esophageal squamous cell carcinoma

Cited by 16 publications

References 21 publications

Resistance mechanisms to TP53-MDM2 inhibition identified by in vivo piggyBac transposon mutagenesis screen in an Arf −/− mouse model

Resistance mechanisms to TP53-MDM2 inhibition identified by in vivo piggyBac transposon mutagenesis screen in an Arf −/− mouse model

Downregulation of cytochrome c oxidase 1 induced radioresistance in esophageal squamous cell carcinoma

Biodegradable Chitosan Matrix Composite Reinforced with Titanium Dioxide for Biocidal Applications

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Product

Resources

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Resistance mechanisms to TP53-MDM2 inhibition identified by in vivo piggyBac transposon mutagenesis screen in an Arf ^−/− mouse model

Resistance mechanisms to TP53-MDM2 inhibition identified by in vivo piggyBac transposon mutagenesis screen in an Arf ^−/− mouse model