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Modulation of Biliary Cancer Chemo-Resistance Through MicroRNA-Mediated Rewiring of the Expansion of CD133+ Cells.

ABSTRACT: BACKGROUND AND AIMS:Changes in single microRNA (miRNA) expression have been associated with chemo-resistance in biliary tract cancers (BTCs). However, a global assessment of the dynamic role of the microRNome has never been performed to identify potential therapeutic targets that are functionally relevant in the BTC cell response to chemotherapy. APPROACH AND RESULTS:High-throughput screening (HTS) of 997 locked nucleic acid miRNA inhibitors was performed in six cholangiocarcinoma cell lines treated with cisplatin and gemcitabine (CG) seeking changes in cell viability. Validation experiments were performed with mirVana probes. MicroRNA and gene expression was assessed by TaqMan assay, RNA-sequencing, and in situ hybridization in four independent cohorts of human BTCs. Knockout of microRNA was achieved by CRISPR-CAS9 in CCLP cells (MIR1249KO) and tested for effects on chemotherapy sensitivity in vitro and in vivo. HTS revealed that MIR1249 inhibition enhanced chemotherapy sensitivity across all cell lines. MIR1249 expression was increased in 41% of cases in human BTCs. In validation experiments, MIR1249 inhibition did not alter cell viability in untreated or dimethyl sulfoxide-treated cells; however, it did increase the CG effect. MIR1249 expression was increased in CD133+ biliary cancer cells freshly isolated from the stem cell niche of human BTCs as well as in CD133+ chemo-resistant CCLP cells. MIR1249 modulated the chemotherapy-induced enrichment of CD133+ cells by controlling their clonal expansion through the Wnt-regulator FZD8. MIR1249KO cells had impaired expansion of the CD133+ subclone and its enrichment after chemotherapy, reduced expression of cancer stem cell markers, and increased chemosensitivity. MIR1249KO xenograft BTC models showed tumor shrinkage after exposure to weekly CG, whereas wild-type models showed only stable disease over treatment. CONCLUSIONS:MIR1249 mediates resistance to CG in BTCs and may be tested as a target for therapeutics.

SUBMITTER: Carotenuto P

PROVIDER: S-EPMC7590111 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Modulation of Biliary Cancer Chemo-Resistance Through MicroRNA-Mediated Rewiring of the Expansion of CD133+ Cells.

Carotenuto Pietro P Hedayat Somaieh S Fassan Matteo M Cardinale Vincenzo V Lampis Andrea A Guzzardo Vincenza V Vicentini Caterina C Scarpa Aldo A Cascione Luciano L Costantini Daniele D Carpino Guido G Alvaro Domenico D Ghidini Michele M Trevisani Francesco F Te Poele Robert R Salati Massimiliano M Ventura Sofia S Vlachogiannis Georgios G Hahne Jens C JC Boulter Luke L Forbes Stuart J SJ Guest Rachel V RV Cillo Umberto U Said-Huntingford Ian I Begum Ruwaida R Smyth Elizabeth E Michalarea Vasiliki V Cunningham David D Rimassa Lorenza L Santoro Armando A Roncalli Massimo M Kirkin Vladimir V Clarke Paul P Workman Paul P Valeri Nicola N Braconi Chiara C

Hepatology (Baltimore, Md.) 20200910 3

<h4>Background and aims</h4>Changes in single microRNA (miRNA) expression have been associated with chemo-resistance in biliary tract cancers (BTCs). However, a global assessment of the dynamic role of the microRNome has never been performed to identify potential therapeutic targets that are functionally relevant in the BTC cell response to chemotherapy.<h4>Approach and results</h4>High-throughput screening (HTS) of 997 locked nucleic acid miRNA inhibitors was performed in six cholangiocarcinoma ...[more]

PMID: 31879968

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Project description:Glioblastoma (GBM) is the commonest brain tumour in adults. A sub-population of cells within these tumours, known as cancer stem cells (CSCs), is thought to mediate their chemo-/radiotherapy resistance. CD133 is a cell surface marker that is used to identify and isolate GBM CSCs. However, its functional significance, as well as the relevant microenvironment in which to study CD133, have so far remained unknown. Here, we examined the effect of hypoxia on the expression of CD133 and on that of the hypoxia-related factors HIF-1? and HIF-2?, and the potential functional significance of CD133 expression on the acquisition of chemo-resistance by GBM cells.CD133, HIF-1?, HIF-2?, VEFG and (control) HPRT mRNA expression analyses were carried out on GBM cells (U251, U87 and SNB19; 2D or 3D cultures) under both normoxic and hypoxic conditions, using qRT-PCR. siRNA was used to downregulate CD133, HIF-1? and HIF-2? expression in the GBM cells, which was confirmed by flow cytometry and qRT-PCR, respectively. Drug sensitivity-related IC50 values were established using an Alamar Blue cell viability assay in conjunction with the Graphpad prism software tool.We found that the expression of CD133 was upregulated under hypoxic conditions in both the 2D and 3D GBM cell culture models. In addition, an increased resistance to cisplatin, temozolomide and etoposide was observed in the GBM cells cultured under hypoxic conditions compared to normoxic conditions. siRNA-mediated knockdown of either HIF-1? or HIF-2? resulted in a reduced CD133 expression, with HIF-2? having a more long-term effect. We also found that HIF-2? downregulation sensitized the GBM cells to cisplatin to a greater extent than HIF-1?, whereas CD133 knockdown had a more marked effect on cisplatin sensitisation than knockdown of either one of the HIFs, suggesting the existence of a HIF-independent cisplatin resistance mechanism mediated by CD133. This same mechanism does not seem to be involved in temozolomide resistance, since we found that HIF-1? downregulation, but not HIF-2? or CD133 downregulation, sensitized GBM cells to temozolomide.From our data we conclude that the mechanisms underlying hypoxia-induced CD133-mediated cisplatin resistance may be instrumental for the design of new GBM treatment strategies.

Dataset Information

Modulation of Biliary Cancer Chemo-Resistance Through MicroRNA-Mediated Rewiring of the Expansion of CD133+ Cells.

Publications

Modulation of Biliary Cancer Chemo-Resistance Through MicroRNA-Mediated Rewiring of the Expansion of CD133+ Cells.

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