Hsa-miR-155-5p Up-Regulation in Breast Cancer and Its Relevance for Treatment With Poly[ADP-Ribose] Polymerase 1 (PARP-1) Inhibitors

Abstract

miR-155-5p is a well-known oncogenic microRNA, showing frequent overexpression in human malignancies, including breast cancer. Here, we show that high miR-155-5p levels are associated with unfavorable prognostic factors in two independent breast cancer cohorts (CSS cohort, n = 283; and TCGA-BRCA dataset, n = 1,095). Consistently, miR-155-5p results as differentially expressed in the breast cancer subgroups identified by the surrogate molecular classification in the CSS cohort and the PAM50 classifier in TCGA-BRCA dataset, with the TNBC and HER2-amplified tumors carrying the highest levels. Since the analysis of TCGA-BC dataset also demonstrated a significant association between miR-155-5p levels and the presence of mutations in homologous recombination (HR) genes, we hypothesized that miR-155-5p might affect cell response to the PARP-1 inhibitor Olaparib. As expected, miR-155-5p ectopic overexpression followed by Olaparib administration resulted in a greater reduction of cell viability as compared to Olaparib administration alone, suggesting that miR-155-5p might induce a synthetic lethal effect in cancer cells when coupled with PARP-1-inhibition. Overall, our data point to a role of miR-155-5p in homologous recombination deficiency and suggest miR-155-5p might be useful in predicting response to PARP1 inhibitors in the clinical setting.

Keywords: Olaparib; PARP-1 inhibitors; breast cancer; homologous recombination; hsa-miR-155-5p.

Figure 1

miR-155-5p is differentially expressed within the molecular breast cancer subgroups. (A) miR-155-5p expression within the surrogate molecular classification in CSS-cohort. High miR-155-5p expression was found in TNBC and HER2-amplified subgroups as compared with LUMA and LUMB tumors. (B) miR-155-5p expression within molecular subgroups identified by PAM50 in TCGA-BRCA dataset. High miR-155-5p levels were found in the Basal-like subgroup as compared with both Luminal A and Luminal B tumors, and in HER2-enriched subgroup as compared with Luminal A subgroup.

Figure 2

miR-155-5p expression is associated with mutations in HR genes and basal-like phenotype. (A) Higher miR-155-5p levels were detected in tumors carrying mutations in HR genes. Statistically significant differences were found among controls with either germline BRCA1/2 mutated tumors (germBRCA), HR mutated (mutHR) or non HR mutated tumors (wt). High miR-155-5p expression was found in both germBRCA and mutHR tumors as compared with the wild type (wt) subgroups. (B) In mutHR tumors, miR-155-5p is differentially expressed according to PAM50 classification. Higher miR-155-5p levels were found in Basal-like and HER2 tumors as compared with the Luminal A subtype, and in the HER2-amplified tumors as compared with Luminal A subtype.

Figure 3

(A) miR-155-5p endogenous expression levels are significantly increased in the BRCA1mut MDA-MB-436 cell line as compared to the other wtBRCA-TNBC cell lines. miR-155-5p was quantified by RT-qPCR and normalized using RNU48 as endogenous control. Data are presented as fold increase over the expression levels of MDA-MB-231 and were derived from 6 biological replicates (Student's t-test, **p < 0.01). (B) Olaparib similarly affects cell viability of basal-like TNBC cell lines independently of BRCA-status. Growth curves of MDA-MB-231, MDA-MB-436, MDA-MB-468, and MDA-MB-453 (TNBC cell lines) were construed following 72 h of treatment with a range of 2-fold serial dilutions of Olaparib compound (230–1.8 μM) and cell viability assessment by PrestoBlue^TM Reagent. The percentage of viable cells was plotted against Log-transformed Olaparib concentrations. Data are presented as mean (±SD) of three independent experiments.

Figure 4

miR-155-5p effects on cell sensitivity to Olaparib. Cell viability was assessed by PrestoBlue^TM reagent after induced overexpression in (A) MDA-MB-231, (B) MDA-MB-468, and (C) MDA-MB-453 or inhibition in (D) MDA-MB-436 of miR-155-5p by mirVana miR-155-5p mimic or inhibitor followed by Olaparib administration for 72 h. Data are presented as percentage of viable cells calculated in each condition (Olaparib vs. Vehicle) with respect to vehicle-treated c(–)-transfected cells, and represent the mean (±SD) of four independent experiments (Student's t-test, ***p < 0.001, ns = not significant).

Figure 5

Evaluation of efficacy and functionality of miR-155-5p overexpression/inhibition. miR-155-5p expression levels and western blot analyses of C/EBPβ protein levels in (A) MDA-MB-231, (B) MDA-MB-468, (C) MDA-MB-453, and (D) MDA-MB-436. miR-155-5p expression levels are presented as fold increase (2^−ΔΔCT) with respect to correspondent negative control c(–)-transfected cells, in the presence of Olaparib or vehicle, and represent the mean (±SD) of four independent experiments (Student's t-test, *p < 0.05, **p < 0.01; ***p < 0.001).