MiR-29 and MiR-140 regulate TRAIL-induced drug tolerance in lung cancer

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has chemotherapeutic potential as a regulator of an extrinsic apoptotic ligand, but its effect as a drug is limited by innate and acquired resistance. Recent findings suggest that an intermediate drug tolerance could mediate acquired resistance, which has made the main obstacle for limited utility of TRAIL as an anti-cancer therapeutics. We propose miRNA-dependent epigenetic modification drives the drug tolerant state in TRAIL-induced drug tolerant (TDT). Transcriptomic analysis revealed miR-29 target gene activation in TDT cells, showing oncogenic signature in lung cancer. Also, the restored TRAIL-sensitivity was associated with miR-29ac and 140-5p expressions, which is known as tumor suppressor by suppressing oncogenic protein RSK2 (p90 ribosomal S6 kinase), further confirmed in patient samples. Moreover, we extended this finding into 119 lung cancer cell lines from public data set, suggesting a significant correlation between TRAIL-sensitivity and RSK2 mRNA expression. Finally, we found that increased RSK2 mRNA is responsible for NF-κB activation, which we previously showed as a key determinant in both innate and acquired TRAIL-resistance. Our findings support further investigation of miR-29ac and -140-5p inhibition to maintain TRAIL-sensitivity and improve the durability of response to TRAIL in lung cancer.

Keywords: MicroRNAs; RSK2; TRAIL-persistence; lung cancer.

Figure 1.

Downregulation of oncogenic miR-29 and -140-5p causes TRAIL-induced Drug-tolerant (TDT) state. (A) A schematic diagram showing the strategy to generate TRAIL-revertant from the resistant cells. (B) Gene Ontology terms associated genes differentially expressed in TRAIL-resistant cell (H460R) compared with sensitive cell. Left panel is a principal component analysis showing clustering between sensitive and resistant quadruplicate. (C) Heatmap showing upregulated miR-29 target genes in H460R cell. (D) Heatmap of miR-29 target genes upregulated in TRAIL-resistant cells. Kaplan-Meier curve depicting the survival probability in the patients with LUAD associated with (E) miR-29 or (F) miR-140 expression. (G) The restored expression of miR-29 a/c and -140-5p in H460R in response to drug holiday. (H) Cell survival rate using CellTiter-Glo assay. Error bars indicate mean ±SD (n = 4) and the p-values were calculated by one-way ANOVA test (**p < 0.01). (I) Chamber invasion assay of H460R cell reconstituted by miR-29 and/or -140-5p. Error bars show mean ±SD (n = 6) and the p-values were calculated by two-tailed student t-test (* p < 0.05, **p < 0.01).

Figure 2.

MiR-29a/c and -140-5p directly suppressed RSK2 protein expression causing TRAIL-sensitivity (A) A schematic diagram showing seed sequence of miR-29/-140-5p and the target sequence of RPS6KA3 3’UTR. (B) Luciferase reporter assay using 3’UTR of RPS6KA3 or 3’UTR harboring deletion mutations of the miR-29a/c and -140-5p in HEK293 cells over-expressing miR-29a/c or-140-5p. Error bars indicate mean ±SD (n = 3). P-values were calculated by two-tailed student t-test. (C) Western blot analysis indicating suppressed RSK2 protein level in lung cancer cells. Indicated cells were transfected by indicated miRNAs, respectively. After 48h, cells were harvested and subjected to Western blotting with indicated antibodies. (D) Expressional comparison of the miR-29a and -140-5p in lung cancer tissues compared to their adjunct normal tissues. Taqman-based qRT-PCR analysis was applied to analyze expression of the target miRNAs, and the expression of the target miRNAs in each cancer sample (n = 15) was normalized by their paired control sample (n = 15), respectively. P-values were obtained by one-way ANOVA test (* p < 0.05). (E) Correlation analysis of miR-29a/-140-5p and RSK2 protein expression in cancer. Expression of RSK2 protein was quantitated by measuring band intensities using Image J software. The intensity in cancer sample was normalized by paired normal sample. Likewise, relative value for miR-29a or miR-140-5p was calculated by comparing qRT-PCR values between cancer sample (n = 15) and its paired control sample (n = 15). P-value was obtained by one-way ANOVA test (**p < 0.01). (F) Expressional correlation of RPS6KA3 and TRAIL-sensitivity in NSCLC. TRAIL-sensitivity for each cell line was obtained from previous literatures and colored by their sensitivity as indicated. For statistical analysis, cell lines were divided into two groups with median RPS6KA3 expression, annotated by TRAIL-sensitivity from literatures, Wilcoxon rank sum test was applied to obtain significance. Detailed information about mRNA expression of RPS6KA3 gene and TRAIL-sensitivity is described in Material and Methods section. Gene Ontology analysis using upregulated and downregulated genes in H460R cells showing differentially regulated pathways. (*p < 0.05, **p < 0.01)

Figure 3.

RSK2 is responsible for H460 revertant phenotype. (A and B) Western blot analysis showing the alteration of RSK2 expression in H460R revertant cell. (C) Western blot analysis showing increased PARP-1 cleavage by targeting RSK2. H292 TRAIL-sensitive cells were treated with anti-miR-29 / -140 with siRSK2 siRNAs for 48h. Subsequently the cells were treated by TRAIL, followed by performing Western blot analysis as indicated antibodies. (D) Cell survival of the H460R cells in RSK2-dependent manner. The precursor of miR-29a or -140-5p was co-transfected with either empty vector or pcDNA-RPS6KA3 plasmid for 48h, and the cells were subsequently stimulated by TRAIL as indicated. After that, the CellTiter-Glo assay was performed to determine cell survival rate. P-value was calculated by two tailed student t-test (*p < 0.05, **p <0.01). (E) Enhanced TRAIL-sensitivity by pharmacologic inhibition of RSK2 activity. BI-D1870 was treated to H460R as indicated, and cell survival rate was determined by CellTiter-Glo assay. Bars shows mean ±SD (n = 6) and the p-values were calculated by two-tailed student t-test (* p < 0.01, **p < 0.001). (F) The re-sensitized TRAIL-sensitivity upon RSK2 suppression in cells harboring anti-miR-29 and -140-5p. Bars shows mean ±SD (n = 4) and the p-values were calculated by two-tailed student t test (*p < 0.01, **p < 0.001).

Figure 4.

The NF-κB regulation by miRNA-dependent RSK2 suppression: (A) NF-κB promoter measurement using luciferase reporter assay. (B) Restored NF-κB activity by RSK2 overexpression in miR-29/-140-5p expressing A549. The cells were co-transfected by miR-29a/-140-5p and pcDNA-RPS6KA3 plasmid for 48h. Subsequently the promoter activity was measured by NF-κB promoter luciferase assay. Error bars indicate mean ±SD (n = 3) and the p-value was obtained by fisher exact t-test (*p < 0.05, **p < 0.01). (C) The phosphorylation of P65 subunit of NF-κB protein was analyzed by Western blot analysis. (D) Subcellular fractionation assay indicating nuclear localization of p65 depending on RSK2. Enhanced NF-κB activity by miR-29/-140 suppression. The H460 revertant was co-transfected by anti-miR-29/-140 and siRPS6KA3 siRNAs for 48h, and the promoter activities were addressed by (E) luciferase assay and (F) Western blot analysis. P-values were calculated by paired student t-test (*p < 0.05, **p < 0.005, ***p < 0.0005).