Translational regulation of the mRNA encoding the ubiquitin peptidase USP1 involved in the DNA damage response as a determinant of Cisplatin resistance

Abstract

Cisplatin (cis-diaminedichloroplatin (II), CDDP) is part of the standard therapy for a number of solid tumors including Non-Small-Cell Lung Cancer (NSCLC). The initial response observed is in most cases only transient and tumors quickly become refractory to the drug. Tumor cell resistance to CDDP relies on multiple mechanisms, some of which still remain unknown. In search for such mechanisms, we examined the impact of CDDP on mRNA translation in a sensitive and in a matched resistant NSCLC cell line. We identified a set of genes whose mRNAs are differentially translated in CDDP resistant vs. sensitive cells. The translation of the mRNA encoding the Ubiquitin-Specific Peptidase 1 (USP1), a Ubiquitin peptidase with important function in multiple DNA repair pathways, is inhibited by CDDP exposure in the sensitive cells, but not in the resistant cells. This lack of down-regulation of USP1 expression at the translational level plays a primary role in CDDP resistance since inhibition of USP1 expression or activity by siRNA or the small molecule inhibitor ML323, respectively is sufficient to re-sensitize resistant cells to CDDP. We involved the USP1 mRNA translation as a major mechanism of CDDP resistance in NSCLC cells and suggest that USP1 could be evaluated as a candidate predictive marker and as a therapeutic target to overcome CDDP resistance. More generally, our results indicate that analysis of gene expression at the level of mRNA translation is a useful approach to identify new determinants of CDDP resistance.

Keywords: 5′ untranslated region; cisplatin resistance; deubiquitination; lung cancer; monoubiquitination; translation.

Figure 1.

The CDDP-induced translational regulation of USP1 mRNA is lost in the resistant cell line (A) A549 and A549-R cells survival was measure after 48 hours exposure to CDDP using a colorimetric proliferation assay. The IC50 of CDDP determined by non-linear fitting of the absorbance is 30 µM for the A549 cell line and 90 µM for the A549-R cells. Error bars represent standard error of the mean. (B) Cell extracts from CDDP- or vehicle-treated A549 and A549-R cells were separated on a sucrose density gradient and collected into 22 fractions across the gradient. mRNA was isolated from individual fractions 8 to 22 and qPCR was performed using primers specific to USP1 mRNA. The percentage of USP1 mRNA in each fraction was plotted. Error bars represent standard error of the mean. (C) Western blot detection of USP1 protein expression in A549 and A549-R cells treated with CDDP as indicated. GADPH detection is used as a loading control. Band quantification and USP1/GAPDH ratio are shown underneath blots. (D) Western blot detection of USP1 and TP53 proteins in A549 and A549-R cells treated with cycloheximide (CHX) for the time indicated. β-Actin and GAPDH were used as loading controls. Bands were quantified and the percentage of signal relative to untreated conditions are shown underneath blots.

Figure 2.

siRNA-mediated silencing of USP1 partially re-sensitize A549-R cells to CDDP (A) RNAs from A549 and A549-R transfected with USP1-targetting or control siRNA were isolated. qPCR was performed to assess USP1 mRNA levels. Error bars represent standard error of the mean. (B) CDDP dose-response of A549 and A549-R cells transfected with control or USP1-targetting siRNA as indicated. Drug treatment was started 24 h after transfection.  Cell survival was measure after 48 h exposure to CDDP using a colorimetric proliferation assay. The IC50 of CDDP was determined by non-linear fitting of the absorbance. Error bars represent standard error of the mean.

Figure 3.

The USP1/UAF1 complex small molecule inhibitor ML33 restore CDDP sensitivity in A549-R resistant cells (A) Western blot analysis of PCNA and FANCD2 monoubiquitination. Protein extract were prepared from A549 and A549-R cells treated with a combination of CDDP (100 µM) and ML323 (100 µM) as indicated. Western blot analyses were performed with antibodies against PCNA (top panel) and FANCD2 (bottom panel). Β-tubulin detection was used as a loading control. (B) CDDP dose-response of A549 (top panel) and A549-R cells (bottom panel) co-treated with ML323 30 µM or 100 µM as indicated. Drug treatment was started 24 h after cell plating.  Cell survival was measure after 48 h exposure to the drug(s) using a colorimetric proliferation assay. The IC50 of CDDP was determined by non-linear fitting of the absorbance. Error bars represent standard error of the mean.

Figure 4.

Alternative USP1 mRNA 5′-UTR in the resistant A549-R cells (A) Schematic representation of the 5′-region of USP1 transcript. Exons are represented as boxes. Intronic sequences are represented by solid lines. Red dotted line represents alternative splicing. Putative promoters are denoted by black arrows and the translation start site by a black stars. Position of the primers used in the PCR analysis are shown (colored arrows). (B) Total RNAs from A549 and A549-R cells treated with CDDP 30 µM or with vehicle for 16 h were analyzed by semi-quantitative RT-PCR using primers as depicted in Figure 4A. Band quantification is shown underneath blots.