ORIGINAL RESEARCH published: 12 August 2020 doi: 10.3389/fonc.2020.01415 Hsa-miR-155-5p Up-Regulation in Breast Cancer and Its Relevance for Treatment With Poly[ADP-Ribose] Edited by: Polymerase 1 (PARP-1) Inhibitors Ivan Garcia-Bassets, University of California, San Diego, Barbara Pasculli 1 , Raffaela Barbano 1† , Andrea Fontana 2† , Tommaso Biagini 3† , United States Maria Pia Di Viesti 1‡ , Michelina Rendina 1 , Vanna Maria Valori 4 , Maria Morritti 4 , Reviewed by: Sara Bravaccini 5 , Sara Ravaioli 5 , Evaristo Maiello 4 , Paolo Graziano 6 , Roberto Murgo 7 , Carolyn M. Klinge, Massimiliano Copetti 2 , Tommaso Mazza 3 , Vito Michele Fazio 1 , Manel Esteller 8,9,10,11 and University of Louisville, United States Paola Parrella 1* Radka Kaneva, 1 Medical University-Sofia, Bulgaria Fondazione IRCCS Casa Sollievo Della Sofferenza, Laboratorio di Oncologia, San Giovanni Rotondo, Italy, 2 Fondazione IRCCS Casa Sollievo Della Sofferenza, UO di Biostatistica, San Giovanni Rotondo, Italy, 3 Fondazione IRCCS Casa Sollievo *Correspondence: Della Sofferenza, Laboratory of Bioinformatics Unit, San Giovanni Rotondo, Italy, 4 Fondazione IRCCS Casa Sollievo Della Paola Parrella Sofferenza, UO di Oncologia, San Giovanni Rotondo, Italy, 5 Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori pparrella@operapadrepio.it (IRST) IRCCS, Biosciences Laboratory, Meldola, Italy, 6 Fondazione IRCCS Casa Sollievo Della Sofferenza, UO di Anatomia † These Patologica, San Giovanni Rotondo, Italy, 7 Fondazione IRCCS Casa Sollievo Della Sofferenza, UO di Chirurgia Senologica, authors have contributed San Giovanni Rotondo, Italy, 8 Josep Carreras Leukaemia Research Institute (IJC), Badalona, Spain, 9 Centro de Investigación equally to this work Biomédica en Red Cáncer (CIBERONC), Madrid, Spain, 10 Physiological Sciences Department, School of Medicine and ‡ Present address: Health Sciences, University of Barcelona, Barcelona, Catalonia, Spain, 11 Institució Catalana de Recerca i Estudis Avançats Maria Pia Di Viesti, (ICREA), Barcelona, Catalonia, Spain Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana,” University of Salerno, Baronissi, Italy 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 Specialty section: are associated with unfavorable prognostic factors in two independent breast cancer This article was submitted to Women’s Cancer, cohorts (CSS cohort, n = 283; and TCGA-BRCA dataset, n = 1,095). Consistently, a section of the journal miR-155-5p results as differentially expressed in the breast cancer subgroups identified Frontiers in Oncology by the surrogate molecular classification in the CSS cohort and the PAM50 classifier Received: 26 March 2020 Accepted: 06 July 2020 in TCGA-BRCA dataset, with the TNBC and HER2-amplified tumors carrying the Published: 12 August 2020 highest levels. Since the analysis of TCGA-BC dataset also demonstrated a significant Citation: association between miR-155-5p levels and the presence of mutations in homologous Pasculli B, Barbano R, Fontana A, recombination (HR) genes, we hypothesized that miR-155-5p might affect cell response Biagini T, Di Viesti MP, Rendina M, Valori VM, Morritti M, Bravaccini S, to the PARP-1 inhibitor Olaparib. As expected, miR-155-5p ectopic overexpression Ravaioli S, Maiello E, Graziano P, followed by Olaparib administration resulted in a greater reduction of cell viability as Murgo R, Copetti M, Mazza T, Fazio VM, Esteller M and Parrella P compared to Olaparib administration alone, suggesting that miR-155-5p might induce (2020) Hsa-miR-155-5p a synthetic lethal effect in cancer cells when coupled with PARP-1-inhibition. Overall, Up-Regulation in Breast Cancer and our data point to a role of miR-155-5p in homologous recombination deficiency and Its Relevance for Treatment With Poly[ADP-Ribose] Polymerase 1 suggest miR-155-5p might be useful in predicting response to PARP1 inhibitors in the (PARP-1) Inhibitors. clinical setting. Front. Oncol. 10:1415. doi: 10.3389/fonc.2020.01415 Keywords: breast cancer, hsa-miR-155-5p, homologous recombination, PARP-1 inhibitors, Olaparib Frontiers in Oncology | www.frontiersin.org 1 August 2020 | Volume 10 | Article 1415 Pasculli et al. miR-155-5p Relevance for Breast Cancer INTRODUCTION whereas lower levels characterize the Luminal A and Luminal B tumors. Moreover, high miR-155-5p expression was found in Breast cancer (BC) is the most frequent cancer among women breast cancer tumors from the TCGA dataset carrying mutations worldwide, accounting for 13% of all cancer-related deaths (1). in HR genes. Lastly, we propose that miR-155-5p might play Breast cancer is a heterogeneous disease that includes different a role in the response to the poly(ADP-ribose) polymerase- histological and molecular entities, clinical presentations, and 1 (PARP-1) inhibitor Olaparib (AZD2281) which have been behaviors, which vary in prognosis and response to therapy. To receiving much attention as promising therapeutic strategy date, Oestrogen Receptor (ER), Progesterone Receptor (PgR), beyond BRCA-mutant tumors, such as those with BRCAness and human epidermal growth factor receptor 2 (HER2), and Ki67, homologous recombination deficiency (HRD). together with age, tumor size, histological grade, and lymph node engagement still represent the most reliable markers that provide prognostic information (2). Lately, microarray- MATERIALS AND METHODS based gene expression studies have identified intrinsic breast Study Design cancer subtypes of biological and, more importantly, clinical We evaluated hsa-miR-155-5p expression in a retrospective relevance consisting of two oestrogen receptor positive (ER+) (prospectively collected) cohort of 283 breast cancer cases subtypes, characterized by a relatively low (luminal A) and with a median follow-up of 81-months (“Casa Sollievo high (luminal B) expression of proliferation-related genes, a della Sofferenza”, hereafter CSS, cohort). The study was subtype enriched for HER2-amplified tumors (HER2-enriched), conducted according to the REporting of tumor MARKer a subtype characterized by the absence of ER, PgR expression Studies (REMARK) guidelines (27). Breast cancer samples and HER2 amplification (Basal-Like) and a subtype ER +/– were collected at the Breast-Unit, IRCCS “Casa Sollievo della and negative for, PgR, HER2, claudin 3, claudin 4, claudin Sofferenza.” Upon receipt from surgery, tissues from the bulk 7, and E-cadherin (Claudin-low) (3–6). This information has of the tumor were sampled by the pathologist, then snap-frozen helped develop risk scores based on differential breast cancer in liquid nitrogen and stored at −80◦ C. For legal reasons, only molecular profiles that are currently entering the clinical practice one tumor specimen (∼50–100 mg of frozen tissue in weight) to identify low-risk breast cancer patients who may avoid could be collected from each patient. Only women older than adjuvant treatment (7). Nevertheless, even these classification 18 years of age in were included in the study. All methods were systems do not account for all the reported pathological and carried out following the international Helsinki Declaration clinical heterogeneity of breast cancer. (7th rev, 2013, EU Directive 2004/23/EC) and Italian (D. Lgs. MicroRNAs (miRNAs) are small endogenous non coding 30/06/2003, n. 196) regulations for research on patients. The RNAs that fine-tune gene expression by post-transcriptional experimental procedures of this study were approved by the silencing of target mRNAs. Among the plethora of miRNAs Ethical Committee of the IRCCS “Casa Sollievo della Sofferenza” that have been linked to human cancers, miR-155-5p (hsa-miR- (Prot. N◦ 140/CE). Prior written informed consent was obtained 155-5p) stands out as prominent oncomiR showing frequent from all patients in agreement with the experimental protocol overexpression in several hematological and solid tumors (8–12). approved by the Ethical Committee. All patients received In breast cancer, miR-155-5p has been found mostly upregulated, either breast-conserving surgery or total mastectomy, plus and associated with high-grade tumors, advanced stages, and sentinel node biopsy, or complete axillary dissection. The post- lymph node metastases as well as worse disease-free and overall surgery treatments were performed according to the following survival (13–22). At functional level, miR-155-5p validated guidelines: AIOM (Associazione Italiana Oncologia Medica), St target genes potentially place miR-155-5p within several cancer- Gallen, National Comprehensive Cancer Network (NCCN) and related pathways encompassing cell proliferation, block of American Society of Clinical Oncology (ASCO). Progression was differentiation, epithelial-mesenchymal transition (EMT), and defined as evidence of loco-regional (recurrence) and/or distant even DNA damage repair (DDR) (12, 23–26). However, these disease over 4 months from diagnosis and after curative-intent relationships between miR-155-5p and breast cancer clinical surgical treatment. Clinical data were collected at each of the markers, such as ER and PgR status and tumor subtype, scheduled follow-up times. along with its causal role in breast cancer development remain controversial, likely due to limited patient sample sizes and Clinicopathological Data discrepancies among studies in terms of methodologies and Pathological assessment consists of evaluating the histological experimental models. type, grade and stage. ER, PgR, Ki-67 labeling index, and Herein, we report the results from the expression analysis of HER2 status were determined by immunohistochemistry (IHC). hsa-miR-155-5p we performed in a large cohort of 283 breast Hormone receptor positive BCs, referred to as cases expressing cancer patients with a complete median follow-up of 81 months oestrogen (ER) or progesterone (PgR) receptors in ≥1% that likely enabled us to define robust relationships between miR- of neoplastic cells by international guidelines (28), and 155-5p and clinical parameters. We confirm that miR-155-5p HER2-amplified BCs were established according to standard expression is associated with unfavorable prognostic indicators recommendations (29). Of the 283 patients, 106 (38%) were in both our cohort and the TCGA-breast cancer dataset (TCGA- lymph node negative, and 177 (62%) lymph node positive. BRCA). We also found that higher miR-155-5p levels correlate Supplemental Table 1 summarizes descriptive statistics for the with the basal-like subgroup followed by HER2-enriched tumors, 283 cases from the CSS cohort analysed in this study. The median Frontiers in Oncology | www.frontiersin.org 2 August 2020 | Volume 10 | Article 1415 Pasculli et al. miR-155-5p Relevance for Breast Cancer age of the study population was 59 years (range, 29–89), and Statistical and Bioinformatics Analyses the median tumor size was 2.5 cm (range, 0.5–11.0). Metastases Patients’ baseline characteristics were reported as median along at diagnosis were found in 15 cases whereas among non- with interquartile range (IQR, i.e., first-third quartiles) or metastatic patients (N = 268), 55 experienced disease progression frequencies and percentages for continuous and categorical (Incidence Rate, IR 3.25 events per 100 PY), and 30 of them (IR variables, respectively. Comparisons between miR-155-5p levels 1.65 events per 100 PY) died for the disease. and clinical pathological characteristics were assessed by Pearson correlation with continuous variables and by the two-sample t- test (or ANOVA model as appropriate) or Mann–Whitney U-test Cell Lines, Culture Conditions, and (or Kruskal–Wallis test when appropriate). In case of ANOVA Reagents model, pairwise comparisons were assessed from statistical In vitro assays were performed in a series of four breast contrasts (defined within the model) and computed p-values cancer cell lines, encompassing the molecular subtypes of TNBC, were adjusted following the step-up Bonferroni method due to which were as follows: MDA-MB-231 (claudin-low), MDA-MB- Hochberg. In the case of the Kruskal–Wallis test, Dunn’s post- 436 (basal-like), MDA-MB-468 (basal-like), and MDA-MB-453 hoc tests were carried out on each pair of groups and p-values (LAR (Luminal-Androgen-Receptor) subtype). MDA-MB-436 were adjusted using the Bonferroni method. The assumption cell line was used as experimental model of BRCA1-mutant cells of normality distribution was checked by means of Q–Q plots as carrying the pathogenic variant 5396+1G>A in the splice and Shapiro–Wilks test. As the distribution of miR-155-5p donor site of exon 20 resulting in a BRCA1 truncated protein expression levels in the CSS cohort was log-normal, all statistical (30). MDA-MB-436 (HTB-130, lot#63048503, p19), MDA-MB- analyses that involved this cohort were performed on their 468 (HTB-132, lot#63226339, p340), and MDA-MB-453 (HTB- log-transformed values. Comparisons between distributions of 131, lot#62959336, p348) cell lines were purchased from ATCC categorical variables were assessed by Chi-Square test. Time-to- (American Type Culture Collection). MDA-MB-231 (Cat No. event analysis was performed in patients without metastases at 92020424, lot#11D011, p40) cell line was purchased from ECACC diagnosis by univariable proportional hazards Cox regression (European Collection of Authenticated Cell Cultures). Culture models. Overall Survival (OS) was defined as the time between conditions were as follows: MDA-MB-231, MDA-MB-468, and the enrollment date and cancer-related death. Progression Free MDA-MB-453 were maintained in MEM w/o L-Gln, 10% FBS, Survival (PFS) was defined as the time between the enrollment 2 mM L-Gln, 1XNEAA, whereas MDA-MB-436 were maintained date and the tumor progression. Metastasis Free Survival (MFS) in MEM w/o L-Gln 10% FBS, 2 mM L-Gln, 1XNEAA, 5 mM was defined as the time between the enrollment date and the NaP∗ , Human Insulin 10 µg/mL, GSH 16 µg/Ml. All cell lines development of distant metastases. For patients who did not were grown in a 5% CO2 humidified incubator at 37 ◦ C. All cell develop the event of interest, the endpoints (i.e., OS, PFS, and lines were mycoplasma tested by Hoechst DNA staining and PCR MFS) were defined as the time between the enrollment date by using N-GARDe Mycoplasma PCR kit (Euroclone). and the date of the last available follow-up control. Incidence The PARP-1 inhibitor Olaparib (AZD2281) was kindly rates (IR, i.e., mortality and disease progression rates) were provided by AstraZeneca, and dissolved in DMSO as a stock reported as number of events per 100 person-years (PY). A two- solution of 10 mg/mL. sided p < 0.05 was considered for statistical significance. All statistical analyses were performed using SAS Release 9.4 (SAS Institute, Cary, NC, USA) and plots were produced using R RNA Isolation and Quantitative Reverse Foundation for Statistical Computing version 3.6. The PRISM Transcription Polymerase Chain Reaction software (version 5, GraphPad, Inc.) was used to make graphs and (RT-qPCR) Analysis statistical analysis of in vitro assays. Single nucleotide variations Total RNA from tissues and cells was isolated according to (missense, frameshift, nonsense, and splicing SNV), copy number standard TRIzolTM protocol (Invitrogen, Thermo Fisher Sc). variations (CNV), gene, and miRNA expression data and clinical RNA samples from CSS cohort were selected as previously information concerning the TCGA-BRCA cohort, divided into described (31). To assess miR-155-5p expression levels in our tumor without synchronous metastases and healthy individuals, cohort of breast cancers, we applied a quantification method were retrieved from the NCI Genomic Data Commons (GDC, with standard curve. Briefly, for both miR-155-5p (ID 002623, https://portal.gdc.cancer.gov/) data portal as pre-processed raw Thermo Fisher Sc) and RNU48 (ID 001006, Thermo Fisher files. Germline point mutations in BRCA1 and BRCA2 genes Sc) endogenous control, standard curves were constructed by were retrieved from cBioPortal (TCGA PanCancer Atlas subset, plotting the threshold cycle (Ct) values against logarithm10 of https://www.cbioportal.org/). Individuals were classified into the copy number and fitting by linear least square regression. Prediction Analysis of Microarray 50 (PAM50) subgroups by For each sample, real-time PCR reactions were run in triplicate means of the R/Bioconductor TCGAbiolinks package. Files and on ABI PRISM 7900 HT Sequence Detection System (Thermo data were manipulated using the R Foundation for Statistical Fis Sc). For tissue samples, miR-155-5p expression levels were Computing version 3.6. Clinicopathological characteristics of determined as the ratio of the miR-155-5p copy number to the these individuals were summarized in Supplemental Table 2. RNU48 copy number and then multiplied by 1000 for easier The median age of the study population was 59 years (range, 27– tabulation [i.e., (hsa-miR-155-5p/RNU48) × 1,000] (31). 90) and the median follow up was 12 months (IQR 3–35 months). Frontiers in Oncology | www.frontiersin.org 3 August 2020 | Volume 10 | Article 1415 Pasculli et al. miR-155-5p Relevance for Breast Cancer Of the 1,095 patients, 991 (90%) were alive, and 104 (10%) died and transfection procedures, experimental results are presented for the disease. as comparison between miR-155 overexpressing/downregulated cells and control c(-) cells, treated with Olaparib or vehicle Determination of Short-Term Olaparib IC50 (DMSO). Data are shown as mean ± SD of n = 4 biological (IC5072 h ) by PrestoBlueTM Viability Reagent replicates (Student’s t-test, ∗ p < 0.05, ∗∗ p < 0.01; ∗∗∗ p < 0.001). BC cells were seeded at 14,000 cells per well onto 96-well- To assess the extent of miR-155-5p overexpression/inhibition plates and treated with a series of 2-fold Olaparib serial dilutions by RT-qPCR, adherent cells were harvested by both 96-well- ranging from 230 to 1.8 µM, or vehicle (DMSO), in a final plates and 6-well-plates, and processed for RNA isolation volume of 90 µL. For each treatment and vehicle condition, as described above. For each cell line, miR-155-5p levels six technical replicates were prepared. Moreover, for each cell in mimic/inhibitor-transfected cells were calculated as fold line, determination of Olaparib IC5072h was repeated in three change (2−11CT ) to correspondent negative control c(–)- independent 96-well-plates at different times. Cell viability was transfected cells. then evaluated after 72 h of treatment by PrestoBlue viability Western Blotting Analysis reagent (Thermo Fisher Sc). Briefly, 10 µL of the ready-to-use Whole protein extracts from 6-well-plate seeded-cells were PrestoBlue Reagent were added directly to the medium. The obtained by RIPA buffer (Tris-HCl 10 mM pH 7.5, NaCl 140 mM, 96-well-plates were incubated at 37◦ C for 2 h, and fluorescence Sodium deoxycholate 1%, sodium orthovanadate 1 mM, SDS was measured on a plate reader (Synergy HT Microplate Reader, 0.1%, Sodium fluoride 1 mM, EDTA 1 mM, Triton X-100 1%, BioTek) at 560 nm. Cell viability was calculated as the percentage 1X Protease inhibitor), resolved by SDS-PAGE, and transferred of cells inhibited by the treatment as measured by the ratio onto PVDF membranes. Primary antibodies for the bone-fide in fluorescence between Olaparib-treated and vehicle-treated miR-155-5p target C/EBPβ (sc150, Santa Cruz Biotechnology) (DMSO) cells. The percentage of viable cells was plotted against and normalizer Vinculin (#13901, Cell Signaling Tech) were Log concentrations of Olaparib. Individual data points on dose- detected by species-specific secondary HRP-linked antibodies, response curves are shown as mean ± SD of n = 3 biological and revealed by using Pierce ECL 2 Substrate (Thermo Fisher replicates. The Olaparib IC5072h for BC cell lines, i.e., the Sc.). Image acquisition was performed on ChemiDoc XRS (Bio- concentration of Olaparib causing a 50% reduction in cell Rad, Richmond, CA). viability in a time-interval of 72 h, were as followed: MDA- MB-436 IC5072h 25.8 µM (95%CI: 24.52–27.15), MDA-MB-231 IC5072h 56.8 µM (95%CI: 53.71–59.98), MDA-MB-468 IC5072h RESULTS 33.81 µM (95%CI: 31.24–36.60), and MDA-MB-453 IC5072h Surrogate Molecular Classification of the 175 µM (95%CI: 160.8–190.6). CSS Cohort Several genome-wide expression analyses have tried to identify In vitro Transient Transfection of clinically relevant molecular breast cancer subtypes (3–6). miR-155-5p Mimic/Inhibitor and Olaparib Among them, the PAM50 (4, 5) distinguishes five intrinsic Treatment subtypes: luminal A, luminal B, basal-like, HER2-enriched, and To achieve transient overexpression or inhibition of miR-155- normal breast-like, characterized by different clinical behaviors. 5p before Olaparib treatment, BC cells were first seeded onto However, due to a lack of reimbursement, multigene tests are not 96-well-plates at 14,000 cells/well and 6-well-plates at 4.1 × readily available for all patients in many countries including Italy. 104 cells/well. Next, BC cell lines were transfected by using Consequently, the use of immunohistochemistry (IHC) based 50 nM mirVana miR-155-5p mimic (MC12601, Thermo Fisher biomarkers such as Ki-67 together with HER2 status has been Sc.) or mirVana miR-155-5p inhibitor (MH12601, Thermo Fisher proposed to distinguish ER-positive breast cancer cases at high Sc.), and correspondent negative controls (mirVanaTM miRNA risk of disease progression (i.e., bona fide Luminal B tumors) Mimic, Negative Control #1 and mirVanaTM miRNA Inhibitor from those at low risk of disease progression (i.e., bona fide Negative Control #1, respectively, Thermo Fisher Sc.) according Luminal A tumors) (7). Instead, it is well established that HER2- to Lipofectamine RNAiMAX forward transfection protocol amplification or intense overexpression (IHC 3+) is associated (Thermo Fisher Sc). For each condition, eight and two technical with worse prognosis in ER-positive BC patients. It is more replicates were prepared in each 96-well and 6-well-plate. difficult to classify the risk of ER-positive HER2-negative tumors After 24 h from transfection, cell lines were treated with because the prognostic performance of Ki-67 IHC staining shows correspondent Olaparib IC5072h , except for MDA-MB-453 cell a high variability among studies and laboratories (7). In our line, which was treated with a lower Olaparib dose (i.e., cohort, we evaluated two possible Ki-67 cut-offs: a 20% cut- IC4072 h, 60 µM) because showing higher sensitivity, in terms off, as proposed by AIOM Guidelines (https://www.aiom.it/wp- of cell tolerance, to liposomes-mediated transfection. Next, cell content/uploads/2018/11/2018_LG_AIOM_Mammella.pdf), and viability upon miR-155 modulation followed by 72-h exposure the median cut off value of Ki-67 calculated in our study to Olaparib was measured by PrestoBlueTM viability reagent as population (30%). As shown in Supplemental Table 3, only described above. a Ki-67 cut-off of <30% was able to distinguish patients at To evaluate the true effects of miR-155-5p and Olaparib low risk from patients at higher risk of disease progression combination on cell viability and rule out those related to vehicle- (PFS HR = 0.38; 95%CI = 0.16–0.90; P = 0.029), metastases Frontiers in Oncology | www.frontiersin.org 4 August 2020 | Volume 10 | Article 1415 Pasculli et al. miR-155-5p Relevance for Breast Cancer development (MFS HR = 0.34; 95%CI = 0.14–0.79; P = miR-155-5p across the surrogate molecular classification groups 0.012) and death (OS HR = 0.11; 95%CI = 0.01–0.87; P of the CSS cohort (n = 262) and the PAM50 subgroups for = 0.037) within the ER-positive subgroup. Thus, we defined the TCGA-BRCA dataset (n = 505). In both cohorts, miR- Luminal A (LUMA) tumors as ER-positive/PgR positive, and 155-5p was differentially expressed among the subgroups (CSS HER2-negative with a low Ki-67 assessment (<30%), and cohort P = 0.0008; TCGA-BRCA dataset P = 0.0005). In Luminal B (LUMB) tumors as ER/PgR positive either HER2- particular, higher miR-155 expression was found in HER2- positive or with a Ki-67 ≥ 30%. Overall, 94 cases (36%) amplified and TNBC subgroups, as compared with both Luminal were classified as Luminal A, 98 cases (37%) as Luminal B A (P = 0.012 and 0.013, respectively) and Luminal B (both along with 34 HER2-amplified cases (13%), and 36 Triple P = 0.0219) in the CSS cohort (Figure 1A). Accordingly, in Negative Breast Cancer cases (TNBC) (14%). The 21 remaining the TCGA-BRCA dataset, the highest level of miR-155 was cases were not classified because HER2 or Ki-67 statuses found in the basal-like subgroup followed by HER2-enriched were unknown. tumors, whereas lower levels were detected in the Luminal A and Luminal B tumors. Pairwise comparison among groups miR-155-5p Expression Is Associated With evidenced statistically significant differences in the Basal-like Unfavorable Prognostic Indicators in Both subtype as compared with both Luminal A (P = 0.0001) CSS Cohort and TCGA-BRCA Dataset and Luminal B subtypes (P = 0.0001), whereas the HER2- The expression of miR-155-5p [(hsa-miR-155-5p/RNU48) × enriched subgroup showed significant differences only with the 1,000] was evaluated in 283 primary breast tumors. As shown Luminal A subtype (P = 0.0001; Figure 1B). No significant in Supplemental Table 4, miR-155 expression was associated associations were found between miR-155 expression and HER2 with unfavorable prognostic indicators including high tumor amplification/overexpression in neither CSS cohort nor TCGA- grade (overall P = 0.0007; G2 vs. G3 P = 0.0053), reduced BRCA dataset. expression of ER (r = −0.243; P = 0.0002) and PgR (r = When we sought to determine putative associations of −0.240; P < 0.0001), and high Ki-67 expression (r = 0.215; P miR-155 with clinical outcomes, no statistically significant = 0.0005). Overall, these data suggest that miR-155 increased associations between miR-155 levels and patients’ survival expression is associated with hormone receptor independency, emerged from univariable Cox Regression analyses, in either high proliferation rate, and poor differentiation. However, no the total population or molecular subgroups of both CSS significant association was found with the pathological stage, cohort and TCGA-BRCA dataset. Moreover, no association was lymph node status, and metastases development. These results found in the subgroups of CSS patients stratified according to were confirmed by the analysis of the TCGA-BRCA dataset therapeutic regimens (chemotherapy, radiotherapy, and anti- (Supplemental Table 5). Then, we evaluated the expression of HER2 monoclonal antibodies; data not shown). 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. Frontiers in Oncology | www.frontiersin.org 5 August 2020 | Volume 10 | Article 1415 Pasculli et al. miR-155-5p Relevance for Breast Cancer miR-155-5p Is Differentially Expressed in (mutHR). Overall, 3,144 mutations were reported of which 91% Breast Cancer From TCGA-BRCA Dataset were CNV and 9% were SNP or indels (Supplemental Table 6). The list of SNPs and indel mutations, together with their putative According to the Mutational Status of functional relevance, is reported in Supplemental Table 7. As Homologous Recombination Genes shown in Figure 2A, miR-155 was increased in tumor samples miR-155-5p has been found to be epigenetically repressed by (Median 302.94, IQR = 184.62–536.70) as compared with wild type BRCA1 through its association with HDAC2 at controls (n = 104) (Median = 129.96, IQR = 103.57–179.24; MIR155 gene promoter and such a regulation is impaired by P = 0.0001). Higher miR-155 expression was found in the the R1699Q variant of BRCA1 (32). This, together with the mutHR subgroup (Median = 446.600, IQR = 198.82–596.07) findings that high levels of miR-155-5p associate with the Basal as compared with the wtHR subgroup (Median = 246.55, IQR Like phenotype, a common feature of BRCA- mutated tumors, = 148.43–419.62; P < 0.0001). The fact that no statistically spurred us to investigate whether miR-155-5p may be associated significant differences were found for the germBRCA subgroup with Homologous Recombination Deficiency (HRD), and thus (Median = 446.60, IQR = 186.68–656.97) as compared with both represents a typical trait of BRCAness. To test this hypothesis, wt (P = 0.134) and mutHR (P = 1.00), might be ascribed to the we selected those breast cancer cases for which the mutational low number of germMut cases (n = 26; Figure 2A). Furthermore, status of 24 genes involved in the Homologous recombination the distribution of PAM50 subtypes differed significantly among pathway (Table 1) was available (n = 1,095 plus 104 normal the groups, with Basal-like and HER2-amplified tumors being controls) from the TCGA-BRCA dataset. Of those tumors, 801 more frequent in mutHR (83 and 55, respectively) as compared carried at least one mutation (indel, missense or CNV) in one with wt (8 and 1, respectively; P = 0.0001). In the mutHR of the homologous recombination genes. Information about the subgroup (Figure 2B), miR-155 was differentially expressed expression levels of miR155-5p was available for 793 out of 801 among the intrinsic molecular subtypes with higher expression mutated tumors and 291 out of the 295 cases without mutation in the Basal-Like (Median = 660.07, IQR = 296.79–1,262.98) in these genes (wt). Of the 793 mutated cases, 26 patients carried and HER2-enriched (Median = 423.58, IQR = 251.10–653.04) germline mutations in BRCA1 or BRCA2 (germMut), while 763 tumors as compared with Luminal subtypes (LUMA Median = carried somatic mutations in homologous recombination genes 252.87, IQR = 178.77–435.31, and LUMB Median = 292.89, IQR TABLE 1 | Summary of mutations detected in the 24 homologous recombination genes evaluated in the TCGA-BRCA dataset. Gene symbol Ensembl ID Cytoband Copy Number Variation Frameshift mutation SNP Deletions Amplifications Deletion Insertion Missense Non sense Splice site ARID1A ENSG00000117713 1p36.11 172 19 10 3 13 14 3 ATM ENSG00000149311 11q22.3 157 34 1 0 15 5 2 ATRX ENSG00000085224 Xq21.1 21 18 2 0 39 3 1 BAP1 ENSG00000163930 3p21.1 140 11 1 1 9 1 0 BARD1 ENSG00000138376 2q35 47 36 0 0 0 0 0 BLM ENSG00000197299 15q26.1 14 92 1 0 15 1 0 BRCA1 ENSG00000012048 17q21.31 89 40 4 0 12 5 2 BRCA2 ENSG00000139618 13q13.1 86 73 5 0 18 3 1 BRIP1 ENSG00000136492 17q23.2 11 233 0 1 9 1 1 CHEK1 ENSG00000149554 11q24.2 157 25 0 0 2 0 0 CHEK2 ENSG00000183765 22q12.1 24 39 0 0 6 1 0 FANCA ENSG00000187741 16q24.3 64 40 0 0 8 0 0 FANCD2 ENSG00000144554 3p25.3 25 97 0 1 8 2 0 FANCE ENSG00000112039 6p21.31 31 66 0 0 3 0 0 FANCF ENSG00000183161 11p14.3 24 56 0 1 2 0 0 FANCG ENSG00000221829 9p13.3 18 56 0 0 3 0 0 FANCL ENSG00000115392 2p16.1 7 59 0 0 1 0 1 MRE11 ENSG00000020922 11q21 89 54 0 0 0 0 0 NBN ENSG00000104320 8q21.3 19 150 0 0 5 1 0 PALB2 ENSG00000083093 16p12.2 12 42 0 0 5 1 0 RAD50 ENSG00000113522 5q31.1 37 31 0 1 8 1 0 RAD51 ENSG00000051180 15q15.1 100 21 0 0 2 0 0 RAD51B ENSG00000182185 14q24.1 89 52 0 0 2 0 1 WRN ENSG00000165392 8p12 51 39 1 0 8 0 0 Frontiers in Oncology | www.frontiersin.org 6 August 2020 | Volume 10 | Article 1415 Pasculli et al. miR-155-5p Relevance for Breast Cancer 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 PrestoBlueTM Reagent. The percentage of viable cells was plotted against Log-transformed Olaparib concentrations. Data are presented as mean (±SD) of three independent experiments. = 211.76–511.78; P < 0.0001; Figure 1B). Overall, these data somatic) high-grade ovarian cancer (33). More recently, results indicate that miR-155 expression may represent a hallmark of from the OlympiAD Phase 3 clinical trial indicate that Olaparib homologous recombination deficiency (HRD). is also effective in BRCA1 or BRCA2 mutated HER2-negative metastatic breast cancer (34, 35). Our findings that high levels miR-155-5p May Indirectly Affect Cell of miR-155-5p associate with the presence of mutations at HR Response to PARP-1 Inhibitor Olaparib genes prompted us to investigate whether miR-155-5p may affect PARP-1 inhibitor Olaparib has been initially approved as the response to the PARP inhibitor Olaparib to some extent. monotherapy for the maintenance treatment of adult patients To this aim, we selected four breast cancer cell lines of TNBC, with platinum-sensitive relapsed BRCA-mutated (germline or including the BRCA1 mutant cell line MDA-MB-436 (basal-like), Frontiers in Oncology | www.frontiersin.org 7 August 2020 | Volume 10 | Article 1415 Pasculli et al. miR-155-5p Relevance for Breast Cancer and the BRCA1 wild type lines with BRCA1 allelic loss but IC5072h , with the exception of MDA-MB-453 cell line, for normal transcript levels MDA-MB-468 (basal-like), MDA-MB- which we chose a lower Olaparib dose (i.e., IC4072 h, 60 µM) 231 (claudin-low) and MDA-MB-453 (LAR-subtype). We did because showing reduced tolerance to transfection procedures find that miR-155-5p endogenous levels were significantly compared to the other cell lines. The ectopic overexpression elevated in mutBRCA1 MDA-MB-436 cells compared to the of miR-155-5p itself induced a very significant decrease in other cell lines (Figure 3A), suggesting the role of this BRCA1 cell viability in all cell lines compared to control (c(–)) cells. pathogenic variant in abrogating negative regulation of miR- Moreover, when we treated both miR-155-5p overexpressing 155-5p (30, 32). Interestingly, we also observed that, when we and control cells with Olaparib compound, we did observe a tested a wide range (see section Materials and Methods) of further reduction of cell viability, indicating that Olaparib and Olaparib concentrations that reduced cell viability significantly miR-155-5p may cooperate, and severely impair the survival in a short time-interval of 72 h (IC5072 ), both basal-like TNBC of cancer cells (Figures 4A–C). Nevertheless, no alterations cell lines encountered a 50% reduction of viability upon Olaparib in Olaparib cell sensitivity were observed upon miR-155-5p treatment at similar doses independently of BRCA1 status inhibition in MDA-MB-436 (Figure 4D), suggesting that miR- (MDA-MB-436, 25.80 µM, 95%CI = 24.52–27.15; MDA-MB- 155-5p regulatory role may be necessary but not sufficient 468 33.81 µM 95%CI = 31.24–36.60) (Figure 3B). Next, to for determining Olaparib sensitivity. The efficacy, as well test the hypothesis that miR-155-5p might affect response to as functionality of miR-155-5p overexpression/inhibition, was Olaparib, we ectopically overexpressed miR-155-5p in the less- checked by both RT-qPCR and western blot analyses of responsive wt BRCA1 cell lines (MDA-MB-453, MDA-MB- its bone-fide target C/EBPβ protein levels (Figures 5A–D, 231, and MDA-MB-468), and treated them at correspondent Supplemental Figure 1). FIGURE 4 | miR-155-5p effects on cell sensitivity to Olaparib. Cell viability was assessed by PrestoBlueTM 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). Frontiers in Oncology | www.frontiersin.org 8 August 2020 | Volume 10 | Article 1415 Pasculli et al. miR-155-5p Relevance for Breast Cancer Alterations of C/EBPβ Isoforms Abundance was induced specifically by Olaparib administration (Figure 5A). May Mediate the Growth Inhibitory Effect Since the smaller LAP2 isoform is considered to be the most transcriptionally active of the C/EBPβ isoforms, it is likely that its of miR-155-5p Coupled With Olaparib overexpression coupled with LIP downregulation, resulting in a As mentioned above, we evaluated the protein levels of miR- LIP:LAP imbalance, severely impairs cell proliferation. We could 155-5p bona-fide target C/EBPβ (36) by western blot to ensure not appreciate significant differences in MDA-MB-436 cell line, that ectopic modulation of miR-155-5p endogenous levels in the although transfection appeared to be successful, probably due to transfected-cell lines was functional. C/EBPβ (CCAAT/enhancer- high C/EBPβ basal levels in this cell line, as shown by negative binding protein) is a well-known transcription factor playing control (c(–))-transfected cells (Figure 5D). a central role in controlling the growth and differentiation of normal mammary gland, and it is produced in three isoforms through alternative usage of different start codons: DISCUSSION the transcriptional activators liver activating protein 1 and 2 (LAP1 and LAP2), and the transcriptional inhibitor LIP, which miR-155-5p is one of the best conserved and multifunctional can inhibit LAPs-mediated gene activation (36). The analyses miRNA involved in several physiological processes, such as of C/EBPβ protein levels in our cell lines showed a decreased proliferation, cell cycle, apoptosis, and differentiation (8–12). level of the LIP isoform in miR-155-5p overexpressing cells Altered expression of miR-155-5p has been found to be associated compared to control cells, in both Olaparib- and vehicle- with hematopoietic lineage differentiation, immune response, treated cells (Figures 5A–C), demonstrating the functionality inflammation, and tumorigenesis (8–12). Several studies suggest of enforced miR-155-5p overexpression, and likely explaining a role of miR-155-5p in breast cancer development and the decrease in cell viability we observed after miR-155-5p progression. However, its putative role as a biomarker is still overexpression. More interestingly, we noticed that, together controversial, likely due to limited patient sample sizes and with the miR-155-5p-mediated LIP reduction, a marked increase discrepancies among studies in terms of methodologies and in LAP2 isoform in the claudin-low MDA-MB-231 cell line experimental models (13–22). 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 (2−11CT ) 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). Frontiers in Oncology | www.frontiersin.org 9 August 2020 | Volume 10 | Article 1415 Pasculli et al. miR-155-5p Relevance for Breast Cancer The primary aim of our study was the evaluation of miR- components of the homologous recombination apparatus can 155-5p expression in a large cohort of breast cancer cases lead to HRD in sporadic cancers, broadly termed BRCAness (44). with a median follow up of 81 months. Our results were These alterations can occur in the form of germline mutations in then validated in the TCGA-BRCA dataset. Consistently with HRR-associated genes, such as PALB2, FANCM, CHECK2, and previous reports, miR-155-5p expression was associated with RAD51C/D (44) or somatic mutations including ATM, BAP1, negative prognostic factors including reduced expression of CDK12 (44) so that numerous studies are currently testing the hormone receptor, high histological grade, and proliferation possibility to apply mutational signatures to the prediction of index measured by Ki67 expression in both our cohort and PARP-inhibitors responsiveness (45, 46). BRCAness and HRD TCGA-BRCA dataset. Indeed, when we evaluated miR-155-5p also involve methylation of the BRCA-promoter, which appears expression within the subgroups identified by the surrogate to be most common in TNBCs (47, 48). Breast tumors with molecular classification in the CSS cohort, and the intrinsic BRCA1 methylation also show the higher histological grade, like molecular classification in TCGA-BRCA dataset, the highest that of BRCA1-mutated tumors (49). Additional contributors to levels of expression were found in the TNBC and Basal- HRD involve copy number alterations (CNA), and early research like subgroups followed by HER2-amplified tumors and the has revealed that the basal-like subtype of breast cancer has lowest expression in the Luminal subgroups. Overall, these higher numbers of gains/losses, while the Luminal B subtype has data support the oncogenic role of miR-155-5p in deregulating a more frequent high-level DNA amplification (50). cell proliferation and differentiation within the hormone- Thus far, a few miRNAs have emerged as determining independent breast cancer subtypes. No associations were the BRCAness phenotype and, therefore, the response to found with lymph node status and metastases development PARP inhibition-based therapy (51, 52). miR-155-5p has been (Supplemental Tables 4, 5), as well as miR-155-5p did not previously found to increase the tumor mutation load and correlate with patient’s survival in univariable Cox regression promote the so-called “mutator phenotype” in inflammation- analyses in both cohorts. These data rule against the role of driven tumors due to the combined targeting of cell-cycle miR155 as prognostic biomarkers in breast cancer. Nevertheless, regulators and DNA repair enzymes (25, 26). Moreover, the small number of TNBC (n = 34) and HER2 (n = 36) miR-155-5p expression has been found to be epigenetically cases in our cohort, and the absence of information about controlled by BRCA1 through the recruitment of histone PFS and MFS in the publicly available TCGA-BRCA dataset deacetylase 2 (HDAC2) at the MIR155 gene promoter (32) cannot completely exclude such a role in these breast cancer that ultimately leads to transcriptional silencing. In human cell subgroups. Thus, this result should be further evaluated in lines, BRCA1-deficient cells showed 50-fold higher miR-155- retrospective or prospective studies with a specific focus on 5p levels compared with those with functional BRCA1. Finally, these subgroups. the transient overexpression of BRCA1 protein reduces the Breast cancers with loss-of-function mutations in BRCA1 expression of miR-155-5p and, in clinical samples, miR-155- or BRCA2 genes are deficient in the HR (Homologous 5p levels were 2- to 6-fold higher in BRCA1-mutant tumors Recombination) pathway that manages the repair of DNA DSBs compared to wt BRCA1 tumors. (Double Strand Breaks); hence, they are exquisitely sensitive For these reasons, we wondered whether miR-155-5p might to poly(ADP ribose) polymerase (PARP) inhibitors (37). This correlate with defects in HR genes and represent a putative trait sensitivity to PARP inhibition derives from the synthetic lethality of BRCAness in the breast cancer of Triple Negative subtype of cells with defective homologous recombination-mediated (TNBC). Overall, we found that defects in the homologous DNA repair to toxic replication intermediates generated on recombination genes are a common feature of breast cancer, with chromatin by PARP “trapping” (38). Preclinical studies and 73% of cases in the TCGA-BRCA dataset carrying at least one clinical trials in breast, ovarian cancer, and other cancers germline or somatic mutation in HR genes. Almost all Basal have shown the PARP inhibitors efficacy in BRCA1- and Like and HER2-associated tumors were HR mutated. When we BRCA2-mutant patients (38–40). This led in 2014 to the tested the putative association of miR-155-5p with the presence approval by drug regulatory agencies of the PARP inhibitor of mutations in Homologous Recombination genes, we were not Olaparib for the treatment of patients with recurrent ovarian surprised to find that miR-155-5p did show higher expression cancer and BRCAs mutations (41). Encouraging results with levels in the mutHR subgroup rather than in the wtHR subgroups Olaparib have also been obtained in patients with metastatic in the TCGA Breast cancer dataset. breast cancer-bearing germline BRCAs mutations (40–42). The Next, we assessed the capability of miR-155-5p to alter the randomized, phase 3 OlympiAD trial showed that among response to the first-in-class PARP inhibitor Olaparib (AZD2281) germline BRCA mutated patients affected by ER-positive HER2- of four BC cell lines, differing in both molecular subtype and negative metastatic TNBC, median progression-free survival was BRCA1 status. As expected, the MDA-MB-436 cells, representing significantly longer with oral Olaparib monotherapy than with a BRCA1-mutant basal-like TNBC cell line, showed a higher standard chemotherapy (34). This has finally led to Olaparib sensitivity to a short-term (72 h) treatment with Olaparib alone FDA-approval in January 2018 for gBRCA1/2+ HER2–breast (IC5072h 25.80 µM, 95%CI = 24.52–27.15) compared to the cancers in the metastatic setting (35). other cell lines. In addition, we also observed that, independently Although germline BRCA (germBRCA) mutations still remain of BRCA-mutational status, the second basal-like TNBC cell the best clinical biomarkers for response to PARP inhibitor line MDA-MB-468, carrying normal levels of BRCA1 transcript therapy (34, 43), genetic and epigenetic inactivation of other despite allelic loss, and low expression of ATM, did show a Frontiers in Oncology | www.frontiersin.org 10 August 2020 | Volume 10 | Article 1415 Pasculli et al. miR-155-5p Relevance for Breast Cancer high sensitivity to Olaparib (IC5072h 33.81 µM, 95%CI = 31.24– and the understanding of the BRCAness as well as considering 36.60), consistent with previous findings supporting this cell’s the specific BC subtype context warrants further investigation. BRCAness phenotype (49), and confirming that HR deficiency In conclusion, the analysis of two independent breast cancer might be exploited to get a cancer cell response beyond BRCA- cohorts corroborates the oncogenic role for miR-155-5p. Indeed, mutations. Then, when we enforced miR-155-5p overexpression increased miR-155-5p expression was associated with loss of in MDA-MB-231, MDA-MB-468, and MDA-MB-453 cell lines, hormone receptor, reduced differentiation, and high proliferation we observed a significant decrease in cell viability following miR- rate. Consistently, miR-155-5p was differentially expressed in 155-5p overexpression alone in all cell lines. Interestingly, miR- breast cancer molecular subgroups, with the highest expression 155-5p-induced overexpression, followed by Olaparib exposure, in basal-like and HER2-related tumors. In addition, higher resulted in an increased sensitivity to PARP inhibition and a miR-155-5p expression was found in breast cancer cases from further reduction in cell viability. the TCGA dataset carrying mutations in HR genes. Lastly, we In order to determine the functionality of modulated miR- propose that a novel mechanism of synthetic lethality mediated 155-5p in our in vitro models, we analysed the protein expression by PARP inhibition and miR-155-5p may promote cancer cell levels of the transcriptional factor C/EBPβ, known to be one death, and suggest miR-155-5p as an additional trait of the of bone-fide targets of miR-155-5p that plays an important BRCAness phenotype. role in regulating the balance between cell differentiation and Currently, several scoring systems aimed to identify HRD proliferation in the mammary gland, according to the differential tumors are under investigation. These systems showed some expression pattern of its isoforms. In particular, C/EBPβ has been predictive value in metastatic breast cancer patients treated recognized as core factor of the TGFβ cytostatic program, being with platinum derivatives (61). By using a high-depth whole- essential for the induction of p15INK4b and the repression of c- genome sequencing approach, Davies H. et al. developed a MYC (53), and mediator of Ras-induced senescence (54). The tool, they called HRDetect, as a predictor of BRCA1 and association of C/EBPβ defects with breast cancer progression BRCA2 deficiency based on mutational signatures (62). Such has been mostly attributed to aberrant levels of LIP isoform a technology was able to identify six different mutational that, once elevated, may antagonize the transcriptional activities signatures and classify BRCA1/2-deficient tumors correctly of LAPs and thus confer a growth advantage to cancer cells with 98.7% sensitivity. In addition, in a cohort of 560 (55–58). Consistently with these data, ectopic overexpression individuals with BC, HRDetect identified 22 tumors with of miR-155-5p in our cell lines showed to reduce the levels somatic loss-of-BRCA1 or BRCA2 and 47 tumors with functional of the inhibitory LIP isoform in miR-155-5p-overexpressing BRCA1/2 deficiency, none of which had mutations detected cells with respect to control-transfected cells in both Olaparib- with standard analysis (61). However, to date, HRDetect and vehicle-treated cells, thus demonstrating the functionality has not been correlated yet with therapeutic responses to of synthetic oligos, and likely explaining the decrease in cell PARPi. Another recent study evaluated a novel gene expression viability we observed after miR-155-5p mimic transfection signature-generating algorithm to predict therapeutic response (Figures 5A–C). Interestingly, we could detect a significant to PARPi (63). Our results indicate that, together with increase in the C/EBPβ LAP2 isoform protein level as a specific mutation and gene expression signatures, miRNA expression pattern of Olaparib treatment compared to vehicle in claudin- analysis may aid in evaluating the competency of homologous low MDA-MB-231 cells (Figure 5A). We might speculate that recombination, and thus eventually increase the ability to the decreased cell viability achieved in this cell line by miR- predict response to PARPi, in order to ultimately identify 155-5p overexpression and Olaparib combination was a result additional breast cancer patients eligible for PARP-inhibition of an enforced cell growth inhibitory effect exerted by LAP2 including regimens. regulated genes and PARP inhibition. Moreover, since increased LAP2 was observed specifically in Olaparib-treated cells, we cannot rule out that the specific Olaparib action may go beyond PARP enzymes inhibition, and stimulate additional pathways DATA AVAILABILITY STATEMENT toward growth inhibition. Hence, these data raise the question of The datasets presented in this study can be found in online whether increasing instead of inhibiting miR-155-5p in different repositories. The names of the repository/repositories cancer settings may enhance PARPi efficacy. Indeed, previous and accession number(s) can be found in the reports have already demonstrated the capability of miR-155- article/Supplementary Material. 5p of boosting the anticancer immune response by targeting anti-inflammatory factors and immune checkpoints, including CTLA4 and PD-L1 [reviewed in (59)], something that may be of relevance for putative PARPi and Immunocheckpoints Inhibitors ETHICS STATEMENT (ICI) combined therapies (60). However, our results as well as the in vitro evidence that The studies involving human participants were reviewed and inhibition of miR-155-5p in BRCA1mut MDA-MB-436 cell line approved by Fondazione IRCCS Casa Sollievo della Sofferenza. did not affect the higher sensitivity to PARP inhibition support The patients/participants provided their written informed the idea that several mechanisms underlie sensitivity to PARPi, consent to participate in this study. Frontiers in Oncology | www.frontiersin.org 11 August 2020 | Volume 10 | Article 1415 Pasculli et al. miR-155-5p Relevance for Breast Cancer AUTHOR CONTRIBUTIONS SUPPLEMENTARY MATERIAL BP, RB, and PP: substantial contribution to conception and The Supplementary Material for this article can be found design of the study. BP, RB, MD, MR, SB, and SR: performed online at: https://www.frontiersin.org/articles/10.3389/fonc. analytical procedures. AF and MC: performed statistical analyses. 2020.01415/full#supplementary-material TB and TM: performed bioinformatics analyses. VV, MM, EM, Supplemental Figure 1 | miR-155-5p expression levels following enforced and RM: collected clinical follow up data and reviewed the overexpression/inhibition of miR-155-5p. miR-155-5p levels were quantified by manuscript. PG: pathological evaluation of specimens. PP, BP, RT-qPCR. Relative expression levels of miR-155-5p mimic/inhibitor-transfected cells were normalized to correspondent negative control c(–)-transfected cells RB, MD, AF, MC, TB, and TM: analysis and interpretation of (2−11CT ) to rule out effects due to transfection procedures. Normalized data are data. PP, BP, RB, AF, MC, and TM: manuscript writing. VF presented as fold increase over the expression levels of MDA-MB-231 and and ME: critical review of the manuscript. All authors: reviewed represent the mean (±SD) of four independent experiments. the manuscript. Supplemental Table 1 | Clinicopathological characteristics of the CSS patient cohort (n = 283). FUNDING Supplemental Table 2 | Clinicopathological characteristics of the TCGA-BRCA dataset (n = 1,095). Associazione Italiana Ricerca sul Cancro (AIRC) Project Code: Supplemental Table 3 | Univariable Cox Regression survival analysis evaluating 16747. Italian Ministry of Health (MoH) TRANSCAN Joint two different KI-67 cut-off to estimate di risk of disease progression, metastases Transnational Call (JTC) 2013 co-funded by the European development, and death in ER positive breast cancer patients. Regional Development Fund, A way of making Europe RRC- Supplemental Table 4 | Associations between tumor clinicopathological 2014-2354565; Italian Ministry of Health (MoH) Ricerca characteristics and miR-155-5p expression in the CSS-cohort. Corrente Program and 5 × 1,000 voluntary contributions. Supplemental Table 5 | Associations between tumor clinicopathological characteristics and miR-155-5p expression in the TCGA-BRCA dataset. ACKNOWLEDGMENTS Supplemental Table 6 | Number of SNPs and indel detected according to gene and variant type. We thank AstraZeneca for providing Olaparib (AZD2281) free of Supplemental Table 7 | List of SNPs and indel mutation detected in homologous charge under a grant agreement (NCR-16-12037). recombination genes and their putative functional consequences. REFERENCES leukemia: miR-155 upregulation independently identifies high-risk patients. J Clin Oncol. (2013) 31:2086–93. doi: 10.1200/JCO.2012.45.6228 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global 10. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, cancer statistics 2018: GLOBOCAN estimates of incidence and mortality et al. A microRNA expression signature of human solid tumors defines worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 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Expression of the CCAAT/enhancer-binding proteins C/EBPa, C/EBPb and C/EBPd Conflict of Interest: The authors declare that the research was conducted in the in breast cancer: correlations with clinicopathologic parameters absence of any commercial or financial relationships that could be construed as a and cell-cycle regulatory proteins. Breast Cancer Res Treat. (2003) potential conflict of interest. 79:175–85. doi: 10.1023/A:1023929504884 58. Raught B, Gingras AC, James A, Medina D, Sonenberg N, Rosen, Copyright © 2020 Pasculli, Barbano, Fontana, Biagini, Di Viesti, Rendina, Valori, JM. Expression of a translationally regulated, dominant-negative Morritti, Bravaccini, Ravaioli, Maiello, Graziano, Murgo, Copetti, Mazza, Fazio, CCAAT/enhancer-binding protein b isoform and up-regulation of the Esteller and Parrella. This is an open-access article distributed under the terms eukaryotic translation initiation factor 2a are correlated with neoplastic of the Creative Commons Attribution License (CC BY). The use, distribution or transformation of mammary epithelial cells. Cancer Res. (1996) 56:4382–6. reproduction in other forums is permitted, provided the original author(s) and the 59. Michaille, JJ, Awad H, Fortman EC, Efanov AA, Tili A. miR-155 expression copyright owner(s) are credited and that the original publication in this journal in antitumor immunity: the higher the better? Genes Chromosomes Cancer. is cited, in accordance with accepted academic practice. No use, distribution or (2019) 58:208–18. doi: 10.1002/gcc.22698 reproduction is permitted which does not comply with these terms. Frontiers in Oncology | www.frontiersin.org 14 August 2020 | Volume 10 | Article 1415