A small molecule drug promoting miRNA processing induces alternative splicing of MdmX transcript and rescues p53 activity in human cancer cells overexpressing MdmX protein

Abstract

MdmX overexpression contributes to the development of cancer by inhibiting tumor suppressor p53. A switch in the alternative splicing of MdmX transcript, leading to the inclusion of exon 6, has been identified as the primary mechanism responsible for increased MdmX protein levels in human cancers, including melanoma. However, there are no approved drugs, which could translate these new findings into clinical applications. We analyzed the anti-melanoma activity of enoxacin, a fluoroquinolone antibiotic inhibiting the growth of some human cancers in vitro and in vivo by promoting miRNA maturation. We found that enoxacin inhibited the growth and viability of human melanoma cell lines much stronger than a structurally related fluoroquinolone ofloxacin, which only weakly modulates miRNA processing. A microarray analysis identified a set of miRNAs significantly dysregulated in enoxacin-treated A375 melanoma cells. They had the potential to target multiple signaling pathways required for cancer cell growth, among them the RNA splicing. Recent studies showed that interfering with cellular splicing machinery can result in MdmX downregulation in cancer cells. We, therefore, hypothesized that enoxacin could, by modulating miRNAs targeting splicing machinery, activate p53 in melanoma cells overexpressing MdmX. We found that enoxacin and ciprofloxacin, a related fluoroquinolone capable of promoting microRNA processing, but not ofloxacin, strongly activated wild type p53-dependent transcription in A375 melanoma without causing significant DNA damage. On the molecular level, the drugs promoted MdmX exon 6 skipping, leading to a dose-dependent downregulation of MdmX. Not only in melanoma, but also in MCF7 breast carcinoma and A2780 ovarian carcinoma cells overexpressing MdmX.Together, our results suggest that some clinically approved fluoroquinolones could potentially be repurposed as activators of p53 tumor suppressor in cancers overexpressing MdmX oncoprotein and that p53 activation might contribute to the previously reported activity of enoxacin towards human cancer cells.

Targeting the spliceosome for cutaneous squamous cell carcinoma therapy: a role for c-MYC and wild-type p53 in determining the degree of tumour selectivity

“…We reported that a reduction in the level of MDMX acts as a sensor of alterations in the spliceosome independently of DNA-damage signalling [ 49 ]. Similar observations have now been made in other in vitro and in vivo systems [ 50 , 53 , 54 ]. This can involve changes in MDMX mRNA splicing due to weak splice sites that are sensitive to interference with the spliceosome but other mechanisms may also contribute, including enhanced MDMX protein degradation [ 49 , 50 , 53 , 54 ].…”

Dysregulation of Mdm2 and Mdm4 alternative splicing underlies motor neuron death in spinal muscular atrophy

“…However, they are also oncogenes, as their overexpression promotes uncontrolled cell proliferation through excess inhibition of the tumor suppressor activity of p53 in certain cancers (Wade et al 2013). Moreover, enhanced skipping of Mdm4 exon 7 (exon 6 in human MDM4) has been proposed as a candidate therapeutic approach to activate the p53 response in specific cancers (such as melanomas and diffuse large B-cell lymphoma) that retain a functional p53 allele (Dewaele et al 2016;Valianatos et al 2017). Considering the strong synergistic effects that we demonstrated here, induction of both Mdm2 exon 3 and Mdm4 exon 7 skipping has the potential for much more pronounced activation of p53 and increased therapeutic benefit in cancer.…”

CDK9 activity is critical for maintaining MDM4 overexpression in tumor cells

“…Antisense oligonucleotides and specific inhibitors of Cdc-like kinases (CLKs) were shown to promote a switch in MDM4 alternative splicing in cancer cells 40,61 . Clinically used fluoroquinolone antibiotics were recently reported to induce a switch in MDM4 mRNA splicing, and benzimidazole anthelmintics inhibited MDM4 expression in melanoma cells 62,63 , indicating opportunities for drug repositioning.…”