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A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness

A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness Inactivation of p53 contributes significantly to the dismal prognosis of breast tumors, most notably triple-negative breast cancers (TNBCs). How the relief from p53 tumor suppressive functions results in tumor cell aggressive behavior is only partially elucidated. In an attempt to shed light on the implication of microRNAs in this context, we discovered a new signaling axis involving p53, miR-30a and ZEB2. By an in silico approach we identified miR-30a as a putative p53 target and observed that in breast tumors reduced miR-30a expression correlated with p53 inactivation, lymph node positivity and poor prognosis. We demonstrate that p53 binds the MIR30A promoter and induces the transcription of both miRNA strands 5p and 3p. Both miR-30a-5p and -3p showed the capacity of targeting ZEB2, a transcription factor involved in epithelial–mesenchymal transition (EMT), tumor cell migration and drug resistance. Intriguingly, we found that p53 does restrain ZEB2 expression via miR-30a. Finally, we provide evidence that the new p53/miR-30a/ZEB2 axis controls tumor cell invasion and distal spreading and impinges upon miR-200c expression. Overall, this study highlights the existence of a novel axis linking p53 to EMT via miR-30a, and adds support to the notion that miRNAs represent key elements of the complex network whereby p53 inactivation affects TNBC clinical behavior. Introduction Breast cancer (BC) is the most common cancer among women. Despite significant advances in early diagnosis and treatment, metastatic spread still represents a major cause of Edited by S. Fulda death for BC patients. BCs are typically classified into These authors contributed equally: Alessandra di Gennaro, Valentina hormone receptor positive (HR; estrogen receptor and/or Damiano. progesterone receptor), HER2/ERBB2/NEU-positive or triple-negative tumors (TNBC, negative for hormonal and These authors contributed equally: Manuela Santarosa, Roberta Maestro. HER2 receptors) according to their receptor status, as assessed by immunohistochemistry. In 2000, Perou et al. [1] Electronic supplementary material The online version of this article (https://doi.org/10.1038/s41418-018-0103-x) contains supplementary suggested a molecular classification of BC into four major material, which is available to authorized users. * Manuela Santarosa Unit of Cancer Epidemiology, CRO Aviano National Cancer msantarosa@cro.it Institute, Aviano (PN) via F. Gallini 2, Aviano 33081 PN, Italy * Roberta Maestro Medical Oncology Unit, CRO Aviano National Cancer Institute, rmaestro@cro.it via F. Gallini 2, Aviano 33081 PN, Italy Molecular Cell Biology Department, Institute of Biology, Leiden Oncogenetics and Functional Oncogenomics Unit, CRO Aviano University, Leiden 2333CC, The Netherlands National Cancer Institute, via F. Gallini 2, Aviano 33081 PN, Italy Ateneo Vita-Salute, Department of Pathology, IRCCS Scientific Pathology Unit, CRO Aviano National Cancer Institute, Aviano Institute San Raffaele, Milan 20132, Italy (PN), via F. Gallini 2, Aviano 33081 PN, Italy 1234567890();,: 1234567890();,: 2166 A. di Gennaro et al. Table 1 Differentially expressed miRNAs predicted to be p53- subgroups based on the transcriptional profile. These four regulated in TP53-mutated and TP53 wild-type breast cancers molecular BC subtypes overlap only in part with the con- miRNA predicted to Transactivation LOG2 fold P-value ventional receptor classification: luminal A and luminal B, a a b be regulated by p53 score change including most of HR-positive tumors; HER2-positive tumors; and basal-like BC, grossly corresponding to miR-146a 3 1.2 2.1E−07 TNBC [1]. Among the different BC subtypes, TNBC/basal- let-7i 3 0.5 1.6E−06 like tumors feature a particularly aggressive behavior: miR-671 4 0.6 1.9E−06 compared to the other BC subtypes, TNBC patients tend to miR-30a 3 -0.8 8.5E−06 relapse earlier and have higher recurrence rates in the first miR-138-2 3 3.1 1.9E−04 years after diagnosis [2]. In fact, in the absence of an miR-138-1 4 3.7 3.6E−04 approved target therapy for TNBC, radiotherapy and che- miR-15b 3 0.8 4.8E−04 motherapy still represent the mainstay of treatment [3]. miR-615 3 0.8 5.6E−04 Unfortunately, primary or secondary resistance often miR-9-2 3 2.4 2.0E−03 occurs, which contributes to the dismal prognosis of these miR-196a-2 3 0.6 9.2E−03 tumors [3]. miR-181a-1 3 0.3 1.0E−02 The inactivation of the tumor suppressor p53 is thought miR-191 4 0.4 1.3E−02 to play a major role in the aggressiveness of TNBC by miR-328 4 0.3 3.4E−02 promoting metastatic spreading, resistance to therapy and miR-490 3 2.1 9.4E−02 relapse [4]. In TNBC/basal-like BC, TP53 alterations miR-194-1 3 0.2 1.1E−01 involve over 80% of the tumors and are mostly represented miR-302b 4 ND 2.3E−01 by disrupting mutations (gene deletions or insertions). miR-34a 3 0.2 2.4E−01 Instead, only 19% of HR-positive/luminal tumors present miR-135a-2 3 –1.6 2.6E−01 TP53 alterations (12% of luminal A, 29% of luminal B) that are primarily missense mutations [5]. These facts support miR-153-2 3 –0.3 3.9E−01 the notion that p53 contributes to TNBC/basal-like BC miR-1-1 3 –3.0 4.5E−01 mostly through loss of tumor suppressive functions, rather miR-124-3 4 1.2 5.0E−01 than through gain of oncogenic activities (gain-of-function miR-100 3 0.1 7.2E−01 p53 mutations). miR-29b-2 3 0.0 8.3E−01 Loss of function of p53 results in the abolition of p53- Transactivation score as calculated by Gowrisankar and Jegga [15] mediated checkpoints and stress responses, and recent evi- Data are reported as LOG2 fold change between TP53-mutated (82 dence points to a role of microRNAs (miRNAs) in these cases) and wild-type (163 cases) breast cancers (TCGA series; strands contexts [6–8]. miRNAs are small, non-coding RNAs that, from the same miRNA were jointly analyzed) through base pairing with target messenger RNA (mRNA) The top 13 miRNAs were differentially expressed (p < 0.05) in TP53- mutated vs wild-type breast cancers molecules, regulate gene expression by inducing either mRNA degradation or inhibition of translation [9, 10]. p53 has been described to regulate the expression of a number of responsive elements developed by Gowrisankar and Jegga miRNAs that mediate p53 control over several biological [15]. The algorithm identified 23 miRNAs as high con- processes including cell cycle, epithelial–mesenchymal fidence p53 targets (score ≥ 3). The interrogation of the transition (EMT) and cell plasticity, survival and metabo- publicly available TCGA (The Cancer Genome Atlas) BC lism [6,11–14]. On these grounds we sought to investigate dataset (at http://tcga-data.nci.nih.gov/tcga/findArchives. in deeper detail the contribution of miRNAs as mediators of htm [16]) highlighted 13/23 miRNAs as significantly p53 tumor suppressive functions in the context of TNBC/ modulated in TP53-mutated (including missense mutations, basal-like tumors. deletions and insertions) compared to TP53 wild-type BC (Table 1). Among these, miR-30a stood out as it was the only miRNA to be significantly downregulated in TP53- Results mutated tumors. The presence of several putative p53- responsive elements in the promoter region of miR-30a was miR-30a is downregulated in TP53-inactivated TNBC also confirmed by the MatInspector tool [17]. This finding and correlates with poor outcome was suggestive of a potential control of p53 over miR-30a gene expression. To investigate the possible contribution of a p53/miRNA To address the actual existence of a p53/miR-30a inter- pathway in the pathogenesis and aggressive behavior of BC, play in the context of BC we further interrogated the TCGA we took advantage of the in silico predictor of p53- database for miR-30a expression. In the biogenesis of a A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness 2167 Fig. 1 miR-30a-5p and miR-30a-3p expression in breast cancers of the In-house series: d low levels of either miRNA strand are associated TCGA series (a–c) and of the in-house series (d–f). TCGA series: a with lymph node positivity. Data are reported as LOG2 of miRNA the expression levels of miR-30a-5p and miR-30a-3p (expressed as relative levels; p values were calculated by two-tailed t-test; *p < 0.05, LOG2 RPM) are lower in breast tumors (T) compared to matched **p < 0.01. e, f Kaplan–Meier analysis showing overall survival in the normal breast tissues (N; 12 cases). b, c miR-30a-5p and miR-30a-3p TNBC patients classified according to miR-30a-5p (e) and miR-30a-3p levels are lower in the 82 BCs carrying TP53 mutations (missense, (f) median levels. Survival curves are truncated at 84 months. In (a–d) nonsense, frameshift; TP53 mut) compared to the 163 TP53 wild-type lines within the boxes mark the median, boundaries represent the 25th tumors (TP53 WT) (b) and in TNBC (27 cases) compared to hormone and the 75th percentiles and whiskers below and above the boxes receptor-positive tumors (HR, 204 cases) (c). The p values were indicate the 5th and 95th percentiles calculated by Mann–Whitney rank sum test; *p < 0.05, **p < 0.01. miRNA, the primary miRNA (pri-miRNA) transcribed from examples in which both strands give rise to mature miRNAs the miRNA gene is first processed into a precursor miRNA [9, 10]. Analysis of miR-30a expression revealed that this (pre-miRNA) to then form a guide miRNA, which regulates was indeed the case for miR-30a. In fact, both miR-30a-5p target mRNA expression [9]. The other strand, named and miR-30a-3p were expressed in normal breast tissues, passenger strand, is usually degraded, but there are and both were significantly downregulated in BC (p < 0.01, 2168 A. di Gennaro et al. Table 2 Triple-negative breast cancers cases: distribution according to miR-30a (5p and 3p) expression, demographics and clinical pathological features miR-30a-5p miR-30a-3p a a a a Total Low High P-value Low High P-value (N=59) (N=29) (N=30) (N=29) (N=30) c c Median follow-up 63.2 61.0 65.4 0.87 51.3 67.7 0.06 (months) c c Median age at 51 49 52 0.50 52 50 0.13 diagnosis (yrs) No. (%) No. (%) No. (%) No. (%) No. (%) Tumor size d d T1 28 (52.8) 12 (42.9) 16 (64.0) 0.09 11 (39.3) 17 (68.0) 0.06 T2 22 (41.5) 13 (46.4) 9 (36.0) 14 (50.0) 8 (32.0) T3–T4 3 (5.7) 3 (10.7) 0 (0.0) 3 (10.7) 0 (0.0) Lymph nodes d d N0 28 (47.5) 10 (35.7) 18 (75.0) 0.01 9 (32.1) 19 (79.2) <0.001 N+ 24 (40.7) 18 (64.3) 6 (25.0) 19 (67.9) 5 (20.8) Metastasis d d M0 54 (91.5) 28 (96.6) 26 (86.7) 0.35 27 (93.1) 27 (90.0) 1.00 M+ 5 (8.5) 1 (3.5) 4 (13.3) 2 (6.9) 4 (10.0) TNM stage d d I 19 (32.2) 8 (27.6) 11 (36.7) 0.80 7 (24.1) 12 (40.0) 0.43 II 25 (42.4) 13 (44.8) 12 (40.0) 13 (44.8) 12 (40.0) III–IV 15 (25.4) 8 (27.6) 7 (23.3) 9 (31.0) 6 (20.0) Tumor grade d d G1–G2 3 (5.1) 1 (3.6) 2 (6.7) 1.00 0 (0.0) 3 (10.4) 0.24 G3 55 (93.2) 27 (96.4) 28 (93.3) 29 (100.0) 26 (89.7) Radiation treatment d d No 23 (45.1) 10 (38.5) 13 (52.0) 0.40 11 (45.8) 12 (55.6) 1.00 Yes 28 (54.9) 16 (61.5) 12 (48.0) 13 (54.2) 15 (44.4) Pharmacological treatment d d No 9 (16.1) 3 (11.1) 6 (20.7) 0.47 4 (14.8) 5 (17.2) 1.00 Yes 47 (83.9) 24 (88.9) 23 (79.3) 23 (85.2) 24 (82.8) Drugs d d Anthracycline 21 (47.7) 14 (60.9) 7 (33.3) 0.23 11 (52.4) 10 (43.5) 0.73 Anthracycline/Taxanes 11 (25.0) 4 (17.4) 7 (33.3) 4 (19.0) 7 (30.4) CMF 12 (27.3) 5 (21.7) 7 (33.3) 6 (28.6) 6 (26.1) CMF cyclophosphamide, methotrexate and 5-fluorouracil The median value was used as cut-off The sum does not add up to the total because of some missing values Mann–Whitney–Wilcoxon test Fisher’s exact test Fig. 1a), with a high degree of reciprocal correlation (r = difference in miR-30a expression observed between TNBC 0.80, p < 0.01). Interestingly, the expression of both miR- and HR-positive tumors was most likely attributable to p53, 30a strands was significantly lower in TP53-inactivated as the statistical difference between the two subtypes was BCs compared to TP53 wild-type BCs, irrespective of the lost when corrected for TP53 gene status (Supplementary type of mutation (Fig. 1b; Supplementary Figure S1a). Figure S1b). Moreover, miR-30a downregulation was more dramatic in To investigate a possible role of miR-30a in the TNBC compared to HR-positive tumors (Fig. 1c). The aggressive behavior of TNBC, we then interrogated an in- A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness 2169 house cohort of 59 consecutive TNBCs with full clinical used as a positive control in PCR reactions. Quantitative history (Table 2): the expression of miR-30a, particularly PCR-ChIP confirmed a strong enrichment of the amplicons miR-30a-3p, inversely correlated with lymph node posi- encompassing the two p53BS in anti-p53 ChIP compared to tivity (Fig. 1d), shorter disease-free (data not shown) and the background (control IgG-ChIP, Fig. 2f). The identifi- overall survival (Fig. 1e, f). Taken together, these results cation of functional binding sites for p53 in the MIR30A suggested a concerted action of p53 and miR-30a in the promoter region compellingly demonstrated that miR-30a is control of TNBC clinical outcome. a direct transcriptional target of p53. miR-30a is a direct transcriptional target of p53 miR-30a-5p and miR-30a-3p target ZEB2 Based on the data accumulated, we sought to investigate in Having collected evidence that indicates that p53 directly detail the p53/miR-30a interplay. In keeping with the in controls miR-30a and that p53 inactivation results in miR- silico predictions of a p53-mediated control of miR-30a, we 30a downregulation, we then sought to deepen how this observed that modulation of p53 expression in tumor cell interplay impinged upon the aggressive behavior of TNBC. lines affected miR-30a transcription. Specifically, ectopic To gain insights on the pathways regulated by the p53/miR- expression of p53 in TP53-null cells (MDA157) or small 30a axis, we interrogated several in silico prediction tools interfering RNA (siRNA)-mediated downregulation of p53 (microRNA Data Integration Portal-mirDIP [18]). Compu- in TP53 wild-type cells (HCT116 and MCF7) associated tational analyses identified several miR-30a targets, among with a concordant variation in the expression of both miR- which include SNAI1, SNAI2 and ZEB2. miR-30a is 30a strands (Fig. 2a, b). The finding that modulation of p53 known to participate to the control of EMT and cell plas- affected miR-30a expression at the level of primary tran- ticity/stemness and we confirmed in our cell models the script (pri-miRNA; Fig. 2c, d) further supported the notion ability of miR-30a to impinge upon these phenotypes of a control of p53 over miR-30a transcription. (Supplementary Figure S3a-d). SNAI1 and SNAI2 have To validate this hypothesis we generated a MIR30A been previously reported to be regulated by miR-30a reporter plasmid in which the MIR30A promoter, either wild [19–21]. Instead, the ability of miR-30a to target ZEB2 was type or mutagenized in the two p53 binding sites with the a new finding (Fig. 3a). Intriguingly, both miR-30a strands highest prediction score according to MatInspector (p53BS, were predicted to bind ZEB2 mRNA. miR30-1 and miR30-2), was cloned upstream of the luci- ZEB2 is a transcription factor that plays a crucial role in ferase gene (Supplementary Figure S2a). Both p53BS single the control of cell plasticity and in the orchestration of the site (30mut1 and 30mut2) and p53BS dual site mutant EMT phenomena that occur during embryogenesis [22]. In reporters (30mut1/2 with both the predicted p53BS muta- analogy with its embryonic function, ZEB2 overexpression genized) were generated. Reporter assays indicated that has also been shown to promote EMT in tumors, including silencing of p53 (Supplementary Figure S2b) affected the BC [23–25]. Accordingly, ZEB2 sustained the cell motility activity of the MIR30A wild-type reporter, while it had of TNBC cell models. In fact, ZEB2 downregulation sig- negligible effects on the reporters in which either one or nificantly reduced in vitro migration of MDA231, both p53BS were destroyed (Fig. 2e comparison black vs MDA157, BT549, Hs578T and HCC1395 TNBC cell lines gray matched columns). Moreover, in p53 proficient cells (Supplementary Figure S4a). Intriguingly, even a reduction the luciferase activity was significantly reduced in p53BS of expression to 50% sufficed to impact on cell motility. mutated reporters compared to the wild-type one (Fig. 2e Since ZEB2 is also expressed by stromal cells [26], to comparison between black columns); this difference was specifically address the involvement of ZEB2 in human erased after silencing of p53 (Fig. 2e comparison between tumor cells we sought to use an in situ approach. Immu- gray columns). Overall these data added support to the nohistochemical staining revealed that BCs, particularly the notion that miR-30a is a direct transcriptional target of p53. TNBC/basal-like subset, did express the ZEB2 protein To ultimately demonstrate that p53 actually sits on the (Supplementary Figure S4b and c). MIR30A promoter, we performed chromatin immunopreci- Based on these findings, we explored in vitro whether pitation (ChIP) experiments. HCT116 cell lysates were miR-30a affected ZEB2 expression. Modulation of miR-30a immunoprecipitated using a p53-specific antibody and the expression in BC cell lines inversely correlated with ZEB2 regions encompassing the two putative p53BS elements on levels: ectopic expression of miR-30a (5p, 3p or both the MIR30A promoter (miR30-1 and miR30-2) were strands, 5p/3p) resulted in ZEB2 decrease (Fig. 3b and amplified and quantified by quantitative PCR (qPCR). Pre- Supplementary Figure S5a); conversely, anti-miR-mediated immune IgG isotype antibodies were used in a mock inhibition of miR-30a elicited an increase in ZEB2 levels immunoprecipitation as a negative control/background sig- (Fig. 3c and Supplementary Figure S5b). nal; the p53 binding region of the p21 promoter was instead 2170 A. di Gennaro et al. To ascertain whether ZEB2 was a direct target of miR- untranslated region (UTR) sequence of ZEB2, either wild 30a-5p and miR-30a-3p, reporter assays were performed type (WT) or mutated in the seeds for the two miR-30a using luciferase reporter constructs carrying the 3’ strands (5pMUT, 3pMUT; Fig. 3a). Transfection of miR- A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness 2171 Fig. 2 p53 regulates miR-30a-5p and miR-30a-3p expression. a 30a actually affected tumor spreading via ZEB2. To this Ectopic p53 expression in MDA157 results in an augment of mature end, we ectopically expressed miR-30a-5p, miR-30a-3p or miR-30a-5p and miR-30a-3p levels. The immunoblot on the right the control miRNA (miRCTR) in MDA231 cells silenced shows p53 expression in MDA157 engineered cells. GAPDH was used (siRNA) for ZEB2 or in MDA231 engineered to over- as a loading control. b p53 silencing in HCT116 results in a decrement of both miR-30a-5p and miR-30a-3p. The extent of p53 silencing is express ZEB2. Either miR-30a strand inhibited migration of shown in the right panel. c Ectopic p53 induces an increase of pri-miR- ZEB2-proficient but not ZEB2-deficient MDA231 cells. 30a levels in MDA157. d Decrease of pri-miR-30a levels in p53- Moreover, ZEB2 overexpression counteracted the inhibi- depleted HCT116 and MCF7 cells. a–d Gray columns represent p53- tory effect of miR30a-5p and -3p (Fig. 4a and Supple- modulated samples; black columns represent control cells. e p53 regulates the MIR30A promoter. Silencing of p53 results in a decre- mentary Figure S7a-d). ment of the MIR30A promoter activity (30wt). The mutagenesis of the In addition, anti-miR-mediated block of miR-30a affec- two p53 binding sites, singularly (30mut1, 30mut2) or in combination ted cell motility (Fig. 4b). Anti-miR-30a-5p and anti-miR- (30mut1/2), abrogates this effect. Results represent the mean value of 30a-3p induced an increase in cell migration of comparable three independent experiments ± SD. f p53 binds the miR-30a pro- moter. Chromatin immunoprecipitation was performed with the DO-1 extent. Noteworthy, both anti-miRNAs elicited ZEB2 anti-p53 monoclonal antibody on HCT116 genomic DNA. Isotype- upregulation. In contrast, SNAI1, which is reported to be matched pre-immune mouse IgG was used as a negative control. The targeted by miR-30a-5p [20, 21], was upregulated only in immunoprecipitated chromatin was assayed for the enrichment of the response to the cognate anti-miR-30a-5p (Supplementary target MIR30A promoter (miR30-1 and 2, the regions encompassing the two p53BS) by qPCR. The p53 binding region of the p21 promoter Figure S7e-f). Thus, the modulation in cell motility induced and an irrelevant genomic region (CTR neg) [59] were used as positive by anti-miR-30a-3p in this experimental system correlated and negative control, respectively. Data are reported as fold enrich- only with ZEB2 upregulation. This supports a specific role ment over control samples (immunoprecipitation with pre-immune for ZEB2 in the control exerted by miR-30a over cell IgG) p values were calculated by two-tailed t-test; *p < 0.05, **p < 0.01. motility. To validate these concepts in vivo, we performed xeno- transplant experiments using the zebrafish embryo as a 30a-5p and miR-30a-3p significantly inhibited the expres- model, whose transparency enables direct visualization of sion of the ZEB2 3’UTR wild-type reporter (WT), while it fluorophore-labeled tumor cells [29, 30]. MDA231 and had no effect on the reporter in which the cognate miR-30a MDA157 cells, either silenced for ZEB2 (si1-ZEB2, si2- seeds were mutagenized. The combination of the two ZEB2) or ectopically expressing miR-30a (5p/3p), were miRNAs showed a quasi-additive effect (5pMUT, 3pMUT; injected in the yolk sac of zebrafish embryos to monitor Fig. 3d and Supplementary Figure S5c). tumor cell spreading and in the cardinal vein (duct of Overall, these data indicated that miR-30a (5p and 3p Cuvier) to monitor tail fin invasion [31]. Both strands) exerts an epigenetic control over ZEB2 mRNA. To ZEB2 silencing and miR-30a induction resulted in reduced address whether p53 entered into the miR-30a/ZEB2 tumor cell migration (Fig. 4c, d and Supplementary equation, we ectopically expressed p53 in MDA157. This Figure S8a-b) and halved tail fin invasion (Fig. 4e, f and cell line was selected because it is p53 null and because of Supplementary Figure S8c), indicating a diminished tumor its negligible expression of miR-200c [27], a possible cell extravasation and distal dissemination. confounding factor. In fact, miR-200c is a p53-regulated Overall, these results corroborate the concept that the miRNA previously reported to be connected to ZEB2 via miR-30a/ZEB2 axis, which is under the control of p53, is reciprocal feedback loop [13, 28]. The augment of miR-30a involved in tumor cell invasion and distal spreading. induced by ectopic p53 was paralleled by a reduction in ZEB2 protein levels (Fig. 3e). The existence of a p53 The p53/miR-30a/ZEB2 axis impinges upon miR- control on ZEB2 via miR-30a was confirmed by the finding 200c expression that anti-miR-mediated interference of miR-30a abrogated the ability of p53 to affect ZEB2 (Fig. 3e, Supplementary Finally, in the light of the previously reported negative Figure S6a-d). Taken together, these data indicate that, control of ZEB1 and ZEB2 over miR-200 [28, 32, 33], we beside the previously reported p53-miR-34a-SNAI1 axis predicted that miR-30a, by repressing ZEB2, would in turn [11, 14], p53 exerts a control over EMT also through the affect miR-200c. new route involving miR-30a and ZEB2. In accord with this hypothesis, ectopic expression of miR- 30a (5p/3p) resulted in an upregulation of miR-200c that was The miR-30a/ZEB2 axis controls TNBC tumor paralleled by a marked ZEB2 downregulation (Fig. 5a, Sup- spreading plementary Figure S9a and b). Instead, no major and repro- ducible variations in ZEB1 were observed. These results To address the biological implication of the p53/miR-30a/ support a role for the miR-30a/ZEB2 axis in the control of ZEB2 axis in TNBC biology, we ascertained whether miR- miR-200c. Accordingly, silencing of ZEB2, which yielded an 2172 A. di Gennaro et al. augment of miR-200c of similar extent to that induced by BC (Fig. 5c) was somehow corroborated by the finding that miR-30a, nullified the ability of this miRNA to induce miR- the miR-30a was positively correlated to miR-200c in both 200c (Fig. 5a, b, Supplementary Figure S10a and b). The the TCGA and in the in-house TNBC series (Supplementary interplay between the miR-30a/ZEB2 axis and miR-200c in Figure S11a-d). A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness 2173 Fig. 3 miR-30a-5p and miR-30a-3p inhibit ZEB2 and mediate the p53 control of p53 over these phenotypes, thus contributing to control over ZEB2. a Alignment of ZEB2 3’UTR with miR-30a-5p the aggressive behavior of p53-inactivated breast cancers. and miR-30a-3p (Targetscan). Nucleotides mutagenized to disrupt the Specifically, we show that miR-30a is a direct p53 tran- miRNA/mRNA base pairing are underlined. b Immunoblots showing scriptional target that is downregulated in response to p53 the modulation of ZEB2 in response to miR-30a (5p, 3p or both) ectopic expression and c after anti-miR-mediated inhibition of miR- inactivation. Intriguingly, a study conducted on a mela- 30a. ZEB2 relative levels, normalized over Tubulin (loading control), noma cell line has recently suggested that p53 may repress are reported below. d miR-30a targets the 3′UTR of ZEB2: luciferase miR-30a by binding the MIR30A promoter to a more activity of the ZEB2 3’UTR reporter, wild-type (WT) or mutated in the proximal region than the one here described [47]. miR-30a-5p and -3p binding sites (5pMUT and 3pMUT), was mea- sured 48 h post transfection of MDA231 cells with the indicated Although we cannot exclude p53 may exert on miR-30a miRNAs. miRCTR was set as a reference. CMV-Renilla was used for different effects depending on the cell context, our data normalization. Results represent the mean value ± SD of three compellingly demonstrate that there is a positive func- experiments. The asterisks (*) indicate the comparisons of miR-30a vs tional interplay between p53 and miR-30a, both in human miRCTR that are statistically significant (p < 0.05). e miR-30a-5p and miR-30a-3p expression in MDA157 stably transduced with p53 BC and ininvitrocellmodels. Inkeeping with our (pLenti-p53+) or pLenti-GFP (pLenti-p53−), in the absence (anti- findings, miR-30a was among the miRNAs positively miR-30a−) or presence (anti-miR-30a+) of an anti-miRNA targeting modulated in TP53 wild-type vs TP53-null HCT116 colon miR-30a. Immunoblots for ZEB2, p53 and Vinculin (loading control) cancer cells [48]. are shown on the right. Numbers below the blot indicate ZEB2 relative levels normalized over Vinculin Moreover, we provide evidence that ZEB2 is one important effector of this p53/miR-30a pathway. We demonstrate that the reduction in miR-30a expression eli- Discussion cited by p53 inactivation results in an alleviation of miR- 30a-mediated targeting of ZEB2, which correlates with Several lines of evidence demonstrate that inactivation of increased cell plasticity, migration and in vivo dissemina- p53, the tumor suppressor most frequently involved in tion. Thus, our results of a connection between p53 and TNBC, affects phenotypes such as EMT and cell plasticity/ EMT via miR-30a, with ZEB2 as a novel actor in this play, stemness that contribute to tumor progression and poor indicate that the p53/miR-30a/ZEB2 axis contributes to the response to therapies [5, 7, 34–37]. As a transcription fac- poor outcome of TNBC. tor, p53 controls the expression of several genes involved in Physiologically, the ZEB family of transcription factors these phenotypes, including miRNAs [6, 13, 14]. play a pivotal role in neural crest formation and migration In an attempt to elucidate the mechanisms whereby p53 during embryo development, a process involving EMT [49]. inactivation contributes to the aggressive behavior of BC In recent years, ZEB factors have gained attention for their via miRNA, we identified a novel axis involving p53, pro-oncogenic functions. As reported for other EMT tran- miR-30a and ZEB2. In particular, we found that the scription factors, the expression of ZEB proteins in tumor expression of miR-30a (both miR-30a-5p and miR-30a-3p) cells contributes to the shift from an epithelial towards a was significantly reduced in human BC samples carrying more mesenchymal phenotype and concurrently confers TP53 gene alterations, primarily TNBC, and inversely resistance to DNA damage, apoptosis and premature correlated with patients’ survival. This phenomenon was senescence [32, 50]. attributable to loss of wild-type p53 activity, rather than to Overexpression of ZEB proteins has been shown to con- gain of oncogenic functions [38], as miR-30a down- tribute to several cancer types [51, 52] and, in particular, it is regulation was observed not only in tumors with missense considered an unfavorable factor in BC [25, 53]. ZEB pro- p53 mutations encoding a dysfunctional full-length pro- teins have been previously linked to p53 via miR-200 [13], a tein, but also in tumors carrying alterations resulting in p53 family of p53-regulated miRNAs. In fact, miR-200s establish protein loss. with ZEB a double-negative feedback loop [28, 32, 33, 54]. The miR-30 family, which includes five members Our results indicate that miR-30a complements this equation. (miR-30a, -30b, -30c, -30d, -30e), has been implicated in In fact, miR-30a and miR-200c levels are positively correlated the pathogenesis of different tumor types [39]. Although in human BC and miR-30a-induced downmodulation of they share the same seed sequence, the various members ZEB2 results in miR-200c overexpression. differ for compensatory sequences which account for their Overall, this study demonstrates the existence of a specificity and for their diverse and sometime opposite novel axis, p53/miR-30a/ZEB2, that links p53 inactiva- roles in the regulation of cell proliferation, EMT and tion to EMT and BC aggressiveness (Fig. 5c), and adds apoptosis [19–21, 40–45]. As about miR-30a, loss of support to the notion that the control of p53 over the miR-30a-5p has been reported to favor tumor dissemina- various tumoral phenotypes relies on convergent and tion and chemoresistance by promoting EMT [19, 20, 40, integrated circuits, in which miRNAs appear as emerging 42, 43, 46]. We propose that miR-30a mediates the and pivotal nodes. 2174 A. di Gennaro et al. A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness 2175 Fig. 4 miR-30a (5p/3p) overexpression results in a reduced BC cell Lentiviral delivery, carried out as previously described migration in vitro and in a zebrafish xeno-transplantation model. a In [33], was used to generate stable cell models. MDA157 vitro cell migration assay of MDA231 engineered to ectopically cells stably expressing p53 were generated by using the express miR-30a-5p, miR-30a-3p or control miRNA (miRCTR) in a pLenti6/V5-p53_wt p53 vector (Addgene plasmid # 22945 ZEB2-proficient (siCTR) or ZEB2-deficient (si1-ZEB2 and si2-ZEB2) context (left panel). The right panel shows the migration capacity of [56]). pLenti6GFP was used as a control. MDA231 cells MDA231 engineered to stably overexpress ZEB2 or GFP, used as a were engineered to overexpress ZEB2 by transducting the negative control. b In vitro cell migration assay of MDA231 trans- pLJM-ZEB2 lentiviral vector or pLJM1-EGFP (Addgene # fected with anti-miR-30a-5p (A-miR-30a-5p), anti-miR-30a-5p (A- 19319 [57]) as a control. To generate pLJM-ZEB2, ZEB2 miR-30a-5p) or control anti-miRNA (A-CTR). The percentage of transmigrated cells was measured at 7 h post seeding. Data represent coding sequence was amplified from TOPO-Blunt-ZEB2 the mean of three independent experiments; *p < 0.05. c Scatter plot vector (HsCD000347714; Harvard Medical School, Boston) representing cell dissemination in zebrafish embryos of mCHERRY with primers containing AgeI and BstBI sites and cloned MDA231 transiently silenced for ZEB2 (si1-ZEB2 and si2-ZEB2) or into pLJM1-EGFP. engineered to stably express miR-30a (5p/3p). Cells transfected with an empty vector were used as a control (ctr). Cells were implanted in ZEB2-silenced (sh1-ZEB2, sh2-ZEB2 or sh5-ZEB2) and the yolk sac of 2-day-old embryos (fli1:EGFP strain). Embryos were control cells (shGFP) (MDA231, MDA157, HBL100, automatically imaged at 6 dpi. Dots represent single cells; colors Hs578T, BT549 and HCC1395) were generated by using identify each microinjected embryo; x-axis indicates the migration lentiviral plasmids obtained from a modified version of from the injection point (0,0) toward the head (positive values) or the tail (negative values); n indicates the number of embryos analyzed. d pRSI9 DECIPHER vector (Cellecta) in which an AgeI site Spreading distance of MDA231 and MDA157 calculated from data was introduced by mutagenesis. Individual sequences for represented in (c) and in Supplementary Figure S8b, respectively; *p < shZEB2 and shGFP (Supplementary Table S1) were cloned 0.05. e Representative images of zebrafishes injected with in the AgeIand EcoRI sites. MDA231 cells at 6 dpi. Cells were injected into the blood circulation (duct of Cuvier) of 2-day-old zebrafish embryos. Scale bar = 100 µm. f The MIR200C promoter is notoriously constitutively Percentages of embryos that show caudal micrometastatic colonization methylated in the TNBC cell lines and several cell divisions after injection with MDA231 or MDA157 cells engineered with are needed to achieve appreciable reactivation by deme- control vector (ctr), miR-30a (5p/3p) or silenced for ZEB2 (si1-ZEB2 thylating agents [33, 58]. Thus, stable cell models were and si2-ZEB2). The percentage of ctr embryos showing metastasis was arbitrarily set to 100. Data are shown at 1, 4 and 6 dpi. Figures needed to address the hypothesis of a miR-30a/ZEB2- represent the results of two independent experiments mediated activation of miR-200c. To this end, MDA231, MDA157 and HBL100 were engineered to express miR-30a (5p/3p) or silenced for ZEB2 via lentiviral delivery. Con- Materials and methods stitutive miR-30a (5p/3p) overexpression was achieved by lentiviral infection with pLenti6-miR-30a-(5p/3p) or pLen- Cell models ti6GFP vectors. MIR30A (miR-30a (5p/3p)) genomic region, amplified from MCF7 genomic DNA with primers The human breast cancer cell lines MDA-MB-157, MDA- containing XhoI and NotI sites (Supplementary Table S1), MB-231, MDA-MB-436 (here referred as MDA157, was initially cloned in pLNCX2-vector. The MIR30A MDA231 and MDA436), Hs578T, BT549, HCC1395 and fragment was then cleaved with BglII and ClaI restriction HCT116 colorectal cancer cell lines were obtained from the enzymes and inserted into BamHI and BstBI restriction sites ATCC, and HBL100 from Interlab Cell Line Collection- of pLenti6GFP. Genova. All cell lines, periodically authenticated by short tandem repeat profiling and tested mycoplasma-negative, TCGA dataset were cultured as previously described [55]. siRNAs for p53 (HSS110905, HSS186390, HSS186391; A dataset of 249 BC samples comprising clin- ThermoFisher Scientific) and non-targeting siRNA (12935- icopathological information, miRNA-seq data and TP53 100; ThermoFisher Scientific) were transfected using Lipo- mutational status was retrieved (on December 2014) from fectamine 3000 (ThermoFisher Scientific). Ambion Pre-miR TCGA portal (http://tcga-data.nci.nih.gov/tcga/ miRNA precursors specific either for the 5p or the 3p strand findArchives.htm [16]). The set included 204 BC hormo- (Life Technologies, ThermoFisher Scientific), anti-miRNAs nal receptor positive, 27 TNBC and 13 expressing HER2 (anti-miR) and relative controls (Life Technologies, Ther- but lacking hormonal receptors (5 cases were unknown). moFisher Scientific) were transfected with the siPORT Besides, 163 BCs presented wild-type TP53 and 82 carried NeoFX Transfection reagent (ThermoFisher Scientific) TP53 mutations (4 were unknown). miRNAs data of paired according to the manufacturer’s instructions. Two ON- tumor and normal tissues were collected for 12 cases. Target-plus siRNAs for ZEB2 (J-006914-22, J-006914-23; miRNA-seq data and clinicopathological records of further Dharmacon) and a non-targeting siRNA were transfected 63 TNBC were downloaded from TCGA data portal. Col- using the DharmaFECT reagent 4 (ThermoFisher Scientific). lected miRNA-seq data (level 3) included the calculated 2176 A. di Gennaro et al. Fig. 5 The p53/miR-30a/ZEB2 axis impinges upon miR-200c. a miR-200c levels in MDA231, MDA157 and HBL100 cell lines engineered to express miR-30a (5p/3p) or silenced for ZEB2 (sh1-ZEB2; sh2-ZEB2). Control vectors (pLenti6GFP and shGFP) yielded similar values and are here represented once as ctr. miR-200c levels in HBL100 ctr were set to 1. b miR-200c levels in MDA231 proficient (shGFP) or deficient (sh1-ZEB2, sh2-ZEB2) for ZEB2 expression, in the absence (ctr, pLenti6GFP) or presence (miR- 30a-5p/3p) of ectopic miR-30a; *p < 0.05. c A unifying model of the new p53/miR-30a/ZEB2 axis (highlighted in bold) involved in TNBC expression for all reads aligned to a specific miRNA Analyses reported in Table 1 were performed by using data reported as RPM (reads per million miRNA mapped). from mirnas.quantification files; in the analyses of miR-30a Comparisons were performed on LOG2 of RPM values. isoforms the isoform.quantification files were used. A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness 2177 Patients and samples transfected at 25 nM final concentration. Reporter assays were performed 48 h after transfection using the Dual- Formalin-fixed, paraffin-embedded (FFPE) specimens of 59 luciferase assay system (Promega). Transfection efficiency TNBCs were retrospectively collected at the CRO Aviano was normalized by calculating the Luciferase/Renilla National Cancer Institute biobank (2000–2010). Cases were activity ratio. All experiments were done in triplicate and selected based on the following criteria: naive for che- data confirmed in at least three independent experiments. motherapy and radiotherapy, tumor cellularity greater than 70%, suitability of the material for molecular analyses. Western blot analyses Informed consent was obtained and use of patient samples was approved by the Institutional Review Board. Clin- Protein extraction and western blot were performed as icopathological and follow-up data were retrieved from previously described [55]. Detailed description of the used clinical records (Table 2). antibodies is reported in Supplementary Table S2. Immu- noreactivity was detected with anti-mouse and anti-rabbit RNA extraction and qRT-PCR secondary antibodies horseradish peroxidase labeled (Per- kinElmer) using Western Lightning Chemiluminescence Total RNA was isolated from FFPE tumors samples using Reagent Plus (PerkinElmer). Images were captured and the Recover All Total Nucleic Acid Isolation Kit (Ther- analyzed using the Chemidoc XRS+ system (Bio-Rad). moFisher Scientific). The miRNeasy Mini Kit (Qiagen) was Expression levels were quantified using the ImageLab used to isolate total RNA from cell lines. Complementary imaging software (Bio-Rad). Results were confirmed in at DNA was generated by using SuperScriptIII-Reverse least three independent experiments. Transcriptase (Applied Biosystems, ThermoFisher Scien- tific) for pri-miRNA detection and by TaqMan MicroRNA Chromatin immunoprecipitation Reverse Transcription Kit (Life Technologies, Thermo- Fisher Scientific) for miRNA analyses. Pri-miR-30a, miR- Chromatin crosslinking was performed according to the NAs (miR-30a-5p, miR-30a-3p, miR-200c) and RNU48 protocol developed by P.J. Farnham (available online at: and RNU6B (reference genes) were then amplified by http://farnham.genomecenter.ucdavis.edu/pdf/FarnhamLa quantitative reverse transcription-PCR (qRT-PCR) using bChIP%20Protocol.pdf). TaqMan-specific kits (Life Technologies, ThermoFisher For ChIP, 4 µg of DO-1 anti-p53 monoclonal antibody or Scientific). Relative expression levels were normalized to isotype-matched pre-immune mouse IgG, as a negative controls (geometric mean of the reference genes) by using control, were used. Quantitative real-time PCRs with the the comparative Ct (ΔΔCt) method and the Bio-Rad CFX EvaGreen dye technology (Bio-Rad) was used to quantify manager software. All experiments were done in triplicate the DNA in ChIP samples. Analysis of ChIP data was and confirmed in at least three independent experiments. carried out using the fold enrichment method normalized to mock (IgG) control for each sample (ThermoFisher Scien- Dual-Luciferase reporter assay tific, https://tools.thermofisher.com/content/sfs/brochures/ Step-by-Step-Guide-to-Successful-ChIP-Assays.pdf). The miR-30a promoter region was amplified from genomic Details about oligonucleotides and antibodies are reported DNA extracted from MCF7 cells and cloned into the pGL3 in Supplementary Table S1 and S2, respectively. The oli- basic Luciferase vector (Promega). The 3’UTR of ZEB2 gonucleotides used for positive and negative controls were was amplified from genomic DNA extracted from MDA231 as previously described [59]. The results were confirmed in TM and cloned in pMIR-REPORT Luciferase vector (Ther- two independent experiments. moFisher Scientific). The p53 binding sites identified on the miR-30a promoter by the MatInspector software (Geno- Migration assays matix Software GmbH, Munich, Germany) and the miR- 30a-5p and miR-30a-3p binding sites on the 3’UTR of Migration assays were performed on several cell models ZEB2 were modified by site-direct mutagenesis (Quik- modulated for ZEB2 and/or miR-30a expression, namely: ChangeTM Site-Directed Mutagenesis Kit, Stratagene). The MDA231, MDA157, BT549, Hs578T and HCC1395 stably primers used for amplification and mutagenesis are reported silenced for ZEB2 via lentiviral delivery; ZEB2-silenced/ in Supplementary Table S1. miR-30a overexpressing MDA231 cell models, generated Reporter plasmids were transiently transfected in the by first transfecting ZEB2-specific siRNAs or non-targeting indicated cell lines using the Lipofectamine 3000 reagent siRNA (ThermoFisher Scientific, 25 nM) and then (24 h (ThermoFisher Scientific); pCMV-Renilla or PGK-Renilla later) by further transfecting pre-miR-30a-5p, pre-miR-30a-3p were used for normalization. siRNA and pre-miRNA were or pre-miR control (Ambion, 5 nM); MDA231 cells 2178 A. di Gennaro et al. engineered to stably express ZEB2 or control, transfected with EGFP embryos. The fraction of embryos exhibiting pre-miR-30a-5p, pre-miR-30a-3p or pre-miRNA control micrometastastatic colonization of the caudal fin (>10 cells) (Ambion, 5 nM); MDA231 cells transfected with anti- was calculated at 1, 4 and 6 dpi (days post injection), as miRNAs and relative control (ThermoFisher Scientific, previously described [31]. Data are representative of two 10 nM). At 48 h post transfection, cells were collected for independent experiments with at least 24 embryos per subsequent analyses. group. All experiments were performed twice. Migration assays were performed as described in Spes- sotto et al. [60]. Briefly, cells were trypsinized, collected Statistical analyses and fluorescently labeled with Fast DiI dye solution (Molecular Probes, Inc.) for 10 min at 37 °C in 5% CO . For miRNA expression analysis tumor samples were cate- Cells were then washed in serum-free medium and seeded gorized according to the median expression value into (10 cells/insert) in serum-free medium on the top side of “low” (expression levels<median) and “high” (expression Fluoroblok inserts (Corning). Medium containing 10% of levels≥median). The Mann–Whitney–Wilcoxon test and fetal bovine serum was used as chemoattractant in the lower Fisher’s exact test were used to assess associations between chamber (bottom side). Fluorescence intensity at 576 nm of miRNA expression and selected prognostic factors (age at top (nonmigrated cells) and bottom (transmigrated cells) diagnosis, tumor size, lymph nodes status, metastasis, TNM side of the well was measured at the indicated time points (tumor, node, metastasis) stage, tumor grade and treat- (tx, 7 or 24 h) using a microplate reader (Infinite ments). Survival analyses were conducted considering the M1000PRO, TECAN). The percentage of transmigrated time from diagnosis to the date of the event (death, relapse cells was determined as follows: 100×(FB −FB )/FT or last follow-up). Overall and disease-free survivals were tx t0 t0 where FB is the fluorescence intensity of bottom side at estimated using the Kaplan–Meier method and differences tx the indicated time point; FB is the fluorescence intensity of between curves were evaluated using the log-rank test. t0 bottom side at the time zero; FT is fluorescence intensity Differences in miRNA expression levels between groups t0 of top side at the time zero. All experiments were performed were assessed by using Mann–Whitney rank sum test for in triplicate and data confirmed in at least three independent TCGA dataset (values not normally distributed) and by experiments. t-test for the in-house series (values normally distributed and equal variance between groups). Statistical analyses for Zebrafish in vivo experiments in vitro experiments were performed using two-tailed t-test. Correlation between miRNA levels was evaluated by cal- To address the role of the mir-30a/ZEB2 axis in vivo, culating Spearman’s correlation coefficient (r). Statistical experiments were performed using the zebrafish embryo analyses were performed with SAS 9.4 (SAS Institute Inc.) model. We choose this model because, beside meeting the and SigmaPlot (Systat Software Inc.). 3R recommendations of using animals with a reduced ner- Acknowledgements The authors are grateful to Giovanna Zerial for vous system development, its transparency allows an her experimental contribution. This study was supported by: Asso- effective and real-time assessment of tumor cell growth and ciazione Italiana per la Ricerca sul Cancro (AIRC), Italian Ministry of migration. Health, Associazione Via di Natale, Fondazione Umberto Veronesi, The transgenic zebrafish line Tg (fli1:EGFP), expressing Fondazione CRO Onlus, Banca Popolare FriulAdria, CRO 5X1000. enhanced green fluorescent protein (EGFP) in endothelial cells in wild-type background, was used for in vivo evalua- Compliance with ethical standards tion of tumor cell dissemination and micrometastatization. Conflict of interest The authors declare that they have no conflict of Zebrafish and embryos were raised, staged and maintained interest. according to standard procedures (http://ZFIN.org)incom- pliance with the local animal welfare regulations. Open Access This article is licensed under a Creative Commons BC cells, engineered as specified in the text and made Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as fluorescent following infection with the mCHERRY lenti- long as you give appropriate credit to the original author(s) and the viral vector (pCMV-mCherry-bc-puro-Kl201), were injec- source, provide a link to the Creative Commons license, and indicate if ted in the yolk sac and analyzed as previously described changes were made. The images or other third party material in this [29]. Tumor dissemination was measured as “spreading article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not distance per object” representing the mean cell migration included in the article’s Creative Commons license and your intended for each embryo. use is not permitted by statutory regulation or exceeds the permitted To investigate the ability of engineered cells to extra- use, you will need to obtain permission directly from the copyright vasate and form distal metastasis, mCHERRY-positive BC holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. cells were injected into the duct of Cuvier of 2-day-old fli1: A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness 2179 References 20. Kumarswamy R, Mudduluru G, Ceppi P, Muppala S, Kozlowski M, Niklinski J, et al. MicroRNA-30a inhibits epithelial-to- mesenchymal transition by targeting Snai1 and is downregulated 1. Perou CM, Sørlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees in non-small cell lung cancer. Int J Cancer. 2012;130:2044–53. CA, et al. 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References (64)

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Springer Journals
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Copyright © 2018 by ADMC Associazione Differenziamento e Morte Cellulare
Subject
Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Stem Cells; Apoptosis; Cell Cycle Analysis
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1350-9047
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1476-5403
DOI
10.1038/s41418-018-0103-x
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Abstract

Inactivation of p53 contributes significantly to the dismal prognosis of breast tumors, most notably triple-negative breast cancers (TNBCs). How the relief from p53 tumor suppressive functions results in tumor cell aggressive behavior is only partially elucidated. In an attempt to shed light on the implication of microRNAs in this context, we discovered a new signaling axis involving p53, miR-30a and ZEB2. By an in silico approach we identified miR-30a as a putative p53 target and observed that in breast tumors reduced miR-30a expression correlated with p53 inactivation, lymph node positivity and poor prognosis. We demonstrate that p53 binds the MIR30A promoter and induces the transcription of both miRNA strands 5p and 3p. Both miR-30a-5p and -3p showed the capacity of targeting ZEB2, a transcription factor involved in epithelial–mesenchymal transition (EMT), tumor cell migration and drug resistance. Intriguingly, we found that p53 does restrain ZEB2 expression via miR-30a. Finally, we provide evidence that the new p53/miR-30a/ZEB2 axis controls tumor cell invasion and distal spreading and impinges upon miR-200c expression. Overall, this study highlights the existence of a novel axis linking p53 to EMT via miR-30a, and adds support to the notion that miRNAs represent key elements of the complex network whereby p53 inactivation affects TNBC clinical behavior. Introduction Breast cancer (BC) is the most common cancer among women. Despite significant advances in early diagnosis and treatment, metastatic spread still represents a major cause of Edited by S. Fulda death for BC patients. BCs are typically classified into These authors contributed equally: Alessandra di Gennaro, Valentina hormone receptor positive (HR; estrogen receptor and/or Damiano. progesterone receptor), HER2/ERBB2/NEU-positive or triple-negative tumors (TNBC, negative for hormonal and These authors contributed equally: Manuela Santarosa, Roberta Maestro. HER2 receptors) according to their receptor status, as assessed by immunohistochemistry. In 2000, Perou et al. [1] Electronic supplementary material The online version of this article (https://doi.org/10.1038/s41418-018-0103-x) contains supplementary suggested a molecular classification of BC into four major material, which is available to authorized users. * Manuela Santarosa Unit of Cancer Epidemiology, CRO Aviano National Cancer msantarosa@cro.it Institute, Aviano (PN) via F. Gallini 2, Aviano 33081 PN, Italy * Roberta Maestro Medical Oncology Unit, CRO Aviano National Cancer Institute, rmaestro@cro.it via F. Gallini 2, Aviano 33081 PN, Italy Molecular Cell Biology Department, Institute of Biology, Leiden Oncogenetics and Functional Oncogenomics Unit, CRO Aviano University, Leiden 2333CC, The Netherlands National Cancer Institute, via F. Gallini 2, Aviano 33081 PN, Italy Ateneo Vita-Salute, Department of Pathology, IRCCS Scientific Pathology Unit, CRO Aviano National Cancer Institute, Aviano Institute San Raffaele, Milan 20132, Italy (PN), via F. Gallini 2, Aviano 33081 PN, Italy 1234567890();,: 1234567890();,: 2166 A. di Gennaro et al. Table 1 Differentially expressed miRNAs predicted to be p53- subgroups based on the transcriptional profile. These four regulated in TP53-mutated and TP53 wild-type breast cancers molecular BC subtypes overlap only in part with the con- miRNA predicted to Transactivation LOG2 fold P-value ventional receptor classification: luminal A and luminal B, a a b be regulated by p53 score change including most of HR-positive tumors; HER2-positive tumors; and basal-like BC, grossly corresponding to miR-146a 3 1.2 2.1E−07 TNBC [1]. Among the different BC subtypes, TNBC/basal- let-7i 3 0.5 1.6E−06 like tumors feature a particularly aggressive behavior: miR-671 4 0.6 1.9E−06 compared to the other BC subtypes, TNBC patients tend to miR-30a 3 -0.8 8.5E−06 relapse earlier and have higher recurrence rates in the first miR-138-2 3 3.1 1.9E−04 years after diagnosis [2]. In fact, in the absence of an miR-138-1 4 3.7 3.6E−04 approved target therapy for TNBC, radiotherapy and che- miR-15b 3 0.8 4.8E−04 motherapy still represent the mainstay of treatment [3]. miR-615 3 0.8 5.6E−04 Unfortunately, primary or secondary resistance often miR-9-2 3 2.4 2.0E−03 occurs, which contributes to the dismal prognosis of these miR-196a-2 3 0.6 9.2E−03 tumors [3]. miR-181a-1 3 0.3 1.0E−02 The inactivation of the tumor suppressor p53 is thought miR-191 4 0.4 1.3E−02 to play a major role in the aggressiveness of TNBC by miR-328 4 0.3 3.4E−02 promoting metastatic spreading, resistance to therapy and miR-490 3 2.1 9.4E−02 relapse [4]. In TNBC/basal-like BC, TP53 alterations miR-194-1 3 0.2 1.1E−01 involve over 80% of the tumors and are mostly represented miR-302b 4 ND 2.3E−01 by disrupting mutations (gene deletions or insertions). miR-34a 3 0.2 2.4E−01 Instead, only 19% of HR-positive/luminal tumors present miR-135a-2 3 –1.6 2.6E−01 TP53 alterations (12% of luminal A, 29% of luminal B) that are primarily missense mutations [5]. These facts support miR-153-2 3 –0.3 3.9E−01 the notion that p53 contributes to TNBC/basal-like BC miR-1-1 3 –3.0 4.5E−01 mostly through loss of tumor suppressive functions, rather miR-124-3 4 1.2 5.0E−01 than through gain of oncogenic activities (gain-of-function miR-100 3 0.1 7.2E−01 p53 mutations). miR-29b-2 3 0.0 8.3E−01 Loss of function of p53 results in the abolition of p53- Transactivation score as calculated by Gowrisankar and Jegga [15] mediated checkpoints and stress responses, and recent evi- Data are reported as LOG2 fold change between TP53-mutated (82 dence points to a role of microRNAs (miRNAs) in these cases) and wild-type (163 cases) breast cancers (TCGA series; strands contexts [6–8]. miRNAs are small, non-coding RNAs that, from the same miRNA were jointly analyzed) through base pairing with target messenger RNA (mRNA) The top 13 miRNAs were differentially expressed (p < 0.05) in TP53- mutated vs wild-type breast cancers molecules, regulate gene expression by inducing either mRNA degradation or inhibition of translation [9, 10]. p53 has been described to regulate the expression of a number of responsive elements developed by Gowrisankar and Jegga miRNAs that mediate p53 control over several biological [15]. The algorithm identified 23 miRNAs as high con- processes including cell cycle, epithelial–mesenchymal fidence p53 targets (score ≥ 3). The interrogation of the transition (EMT) and cell plasticity, survival and metabo- publicly available TCGA (The Cancer Genome Atlas) BC lism [6,11–14]. On these grounds we sought to investigate dataset (at http://tcga-data.nci.nih.gov/tcga/findArchives. in deeper detail the contribution of miRNAs as mediators of htm [16]) highlighted 13/23 miRNAs as significantly p53 tumor suppressive functions in the context of TNBC/ modulated in TP53-mutated (including missense mutations, basal-like tumors. deletions and insertions) compared to TP53 wild-type BC (Table 1). Among these, miR-30a stood out as it was the only miRNA to be significantly downregulated in TP53- Results mutated tumors. The presence of several putative p53- responsive elements in the promoter region of miR-30a was miR-30a is downregulated in TP53-inactivated TNBC also confirmed by the MatInspector tool [17]. This finding and correlates with poor outcome was suggestive of a potential control of p53 over miR-30a gene expression. To investigate the possible contribution of a p53/miRNA To address the actual existence of a p53/miR-30a inter- pathway in the pathogenesis and aggressive behavior of BC, play in the context of BC we further interrogated the TCGA we took advantage of the in silico predictor of p53- database for miR-30a expression. In the biogenesis of a A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness 2167 Fig. 1 miR-30a-5p and miR-30a-3p expression in breast cancers of the In-house series: d low levels of either miRNA strand are associated TCGA series (a–c) and of the in-house series (d–f). TCGA series: a with lymph node positivity. Data are reported as LOG2 of miRNA the expression levels of miR-30a-5p and miR-30a-3p (expressed as relative levels; p values were calculated by two-tailed t-test; *p < 0.05, LOG2 RPM) are lower in breast tumors (T) compared to matched **p < 0.01. e, f Kaplan–Meier analysis showing overall survival in the normal breast tissues (N; 12 cases). b, c miR-30a-5p and miR-30a-3p TNBC patients classified according to miR-30a-5p (e) and miR-30a-3p levels are lower in the 82 BCs carrying TP53 mutations (missense, (f) median levels. Survival curves are truncated at 84 months. In (a–d) nonsense, frameshift; TP53 mut) compared to the 163 TP53 wild-type lines within the boxes mark the median, boundaries represent the 25th tumors (TP53 WT) (b) and in TNBC (27 cases) compared to hormone and the 75th percentiles and whiskers below and above the boxes receptor-positive tumors (HR, 204 cases) (c). The p values were indicate the 5th and 95th percentiles calculated by Mann–Whitney rank sum test; *p < 0.05, **p < 0.01. miRNA, the primary miRNA (pri-miRNA) transcribed from examples in which both strands give rise to mature miRNAs the miRNA gene is first processed into a precursor miRNA [9, 10]. Analysis of miR-30a expression revealed that this (pre-miRNA) to then form a guide miRNA, which regulates was indeed the case for miR-30a. In fact, both miR-30a-5p target mRNA expression [9]. The other strand, named and miR-30a-3p were expressed in normal breast tissues, passenger strand, is usually degraded, but there are and both were significantly downregulated in BC (p < 0.01, 2168 A. di Gennaro et al. Table 2 Triple-negative breast cancers cases: distribution according to miR-30a (5p and 3p) expression, demographics and clinical pathological features miR-30a-5p miR-30a-3p a a a a Total Low High P-value Low High P-value (N=59) (N=29) (N=30) (N=29) (N=30) c c Median follow-up 63.2 61.0 65.4 0.87 51.3 67.7 0.06 (months) c c Median age at 51 49 52 0.50 52 50 0.13 diagnosis (yrs) No. (%) No. (%) No. (%) No. (%) No. (%) Tumor size d d T1 28 (52.8) 12 (42.9) 16 (64.0) 0.09 11 (39.3) 17 (68.0) 0.06 T2 22 (41.5) 13 (46.4) 9 (36.0) 14 (50.0) 8 (32.0) T3–T4 3 (5.7) 3 (10.7) 0 (0.0) 3 (10.7) 0 (0.0) Lymph nodes d d N0 28 (47.5) 10 (35.7) 18 (75.0) 0.01 9 (32.1) 19 (79.2) <0.001 N+ 24 (40.7) 18 (64.3) 6 (25.0) 19 (67.9) 5 (20.8) Metastasis d d M0 54 (91.5) 28 (96.6) 26 (86.7) 0.35 27 (93.1) 27 (90.0) 1.00 M+ 5 (8.5) 1 (3.5) 4 (13.3) 2 (6.9) 4 (10.0) TNM stage d d I 19 (32.2) 8 (27.6) 11 (36.7) 0.80 7 (24.1) 12 (40.0) 0.43 II 25 (42.4) 13 (44.8) 12 (40.0) 13 (44.8) 12 (40.0) III–IV 15 (25.4) 8 (27.6) 7 (23.3) 9 (31.0) 6 (20.0) Tumor grade d d G1–G2 3 (5.1) 1 (3.6) 2 (6.7) 1.00 0 (0.0) 3 (10.4) 0.24 G3 55 (93.2) 27 (96.4) 28 (93.3) 29 (100.0) 26 (89.7) Radiation treatment d d No 23 (45.1) 10 (38.5) 13 (52.0) 0.40 11 (45.8) 12 (55.6) 1.00 Yes 28 (54.9) 16 (61.5) 12 (48.0) 13 (54.2) 15 (44.4) Pharmacological treatment d d No 9 (16.1) 3 (11.1) 6 (20.7) 0.47 4 (14.8) 5 (17.2) 1.00 Yes 47 (83.9) 24 (88.9) 23 (79.3) 23 (85.2) 24 (82.8) Drugs d d Anthracycline 21 (47.7) 14 (60.9) 7 (33.3) 0.23 11 (52.4) 10 (43.5) 0.73 Anthracycline/Taxanes 11 (25.0) 4 (17.4) 7 (33.3) 4 (19.0) 7 (30.4) CMF 12 (27.3) 5 (21.7) 7 (33.3) 6 (28.6) 6 (26.1) CMF cyclophosphamide, methotrexate and 5-fluorouracil The median value was used as cut-off The sum does not add up to the total because of some missing values Mann–Whitney–Wilcoxon test Fisher’s exact test Fig. 1a), with a high degree of reciprocal correlation (r = difference in miR-30a expression observed between TNBC 0.80, p < 0.01). Interestingly, the expression of both miR- and HR-positive tumors was most likely attributable to p53, 30a strands was significantly lower in TP53-inactivated as the statistical difference between the two subtypes was BCs compared to TP53 wild-type BCs, irrespective of the lost when corrected for TP53 gene status (Supplementary type of mutation (Fig. 1b; Supplementary Figure S1a). Figure S1b). Moreover, miR-30a downregulation was more dramatic in To investigate a possible role of miR-30a in the TNBC compared to HR-positive tumors (Fig. 1c). The aggressive behavior of TNBC, we then interrogated an in- A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness 2169 house cohort of 59 consecutive TNBCs with full clinical used as a positive control in PCR reactions. Quantitative history (Table 2): the expression of miR-30a, particularly PCR-ChIP confirmed a strong enrichment of the amplicons miR-30a-3p, inversely correlated with lymph node posi- encompassing the two p53BS in anti-p53 ChIP compared to tivity (Fig. 1d), shorter disease-free (data not shown) and the background (control IgG-ChIP, Fig. 2f). The identifi- overall survival (Fig. 1e, f). Taken together, these results cation of functional binding sites for p53 in the MIR30A suggested a concerted action of p53 and miR-30a in the promoter region compellingly demonstrated that miR-30a is control of TNBC clinical outcome. a direct transcriptional target of p53. miR-30a is a direct transcriptional target of p53 miR-30a-5p and miR-30a-3p target ZEB2 Based on the data accumulated, we sought to investigate in Having collected evidence that indicates that p53 directly detail the p53/miR-30a interplay. In keeping with the in controls miR-30a and that p53 inactivation results in miR- silico predictions of a p53-mediated control of miR-30a, we 30a downregulation, we then sought to deepen how this observed that modulation of p53 expression in tumor cell interplay impinged upon the aggressive behavior of TNBC. lines affected miR-30a transcription. Specifically, ectopic To gain insights on the pathways regulated by the p53/miR- expression of p53 in TP53-null cells (MDA157) or small 30a axis, we interrogated several in silico prediction tools interfering RNA (siRNA)-mediated downregulation of p53 (microRNA Data Integration Portal-mirDIP [18]). Compu- in TP53 wild-type cells (HCT116 and MCF7) associated tational analyses identified several miR-30a targets, among with a concordant variation in the expression of both miR- which include SNAI1, SNAI2 and ZEB2. miR-30a is 30a strands (Fig. 2a, b). The finding that modulation of p53 known to participate to the control of EMT and cell plas- affected miR-30a expression at the level of primary tran- ticity/stemness and we confirmed in our cell models the script (pri-miRNA; Fig. 2c, d) further supported the notion ability of miR-30a to impinge upon these phenotypes of a control of p53 over miR-30a transcription. (Supplementary Figure S3a-d). SNAI1 and SNAI2 have To validate this hypothesis we generated a MIR30A been previously reported to be regulated by miR-30a reporter plasmid in which the MIR30A promoter, either wild [19–21]. Instead, the ability of miR-30a to target ZEB2 was type or mutagenized in the two p53 binding sites with the a new finding (Fig. 3a). Intriguingly, both miR-30a strands highest prediction score according to MatInspector (p53BS, were predicted to bind ZEB2 mRNA. miR30-1 and miR30-2), was cloned upstream of the luci- ZEB2 is a transcription factor that plays a crucial role in ferase gene (Supplementary Figure S2a). Both p53BS single the control of cell plasticity and in the orchestration of the site (30mut1 and 30mut2) and p53BS dual site mutant EMT phenomena that occur during embryogenesis [22]. In reporters (30mut1/2 with both the predicted p53BS muta- analogy with its embryonic function, ZEB2 overexpression genized) were generated. Reporter assays indicated that has also been shown to promote EMT in tumors, including silencing of p53 (Supplementary Figure S2b) affected the BC [23–25]. Accordingly, ZEB2 sustained the cell motility activity of the MIR30A wild-type reporter, while it had of TNBC cell models. In fact, ZEB2 downregulation sig- negligible effects on the reporters in which either one or nificantly reduced in vitro migration of MDA231, both p53BS were destroyed (Fig. 2e comparison black vs MDA157, BT549, Hs578T and HCC1395 TNBC cell lines gray matched columns). Moreover, in p53 proficient cells (Supplementary Figure S4a). Intriguingly, even a reduction the luciferase activity was significantly reduced in p53BS of expression to 50% sufficed to impact on cell motility. mutated reporters compared to the wild-type one (Fig. 2e Since ZEB2 is also expressed by stromal cells [26], to comparison between black columns); this difference was specifically address the involvement of ZEB2 in human erased after silencing of p53 (Fig. 2e comparison between tumor cells we sought to use an in situ approach. Immu- gray columns). Overall these data added support to the nohistochemical staining revealed that BCs, particularly the notion that miR-30a is a direct transcriptional target of p53. TNBC/basal-like subset, did express the ZEB2 protein To ultimately demonstrate that p53 actually sits on the (Supplementary Figure S4b and c). MIR30A promoter, we performed chromatin immunopreci- Based on these findings, we explored in vitro whether pitation (ChIP) experiments. HCT116 cell lysates were miR-30a affected ZEB2 expression. Modulation of miR-30a immunoprecipitated using a p53-specific antibody and the expression in BC cell lines inversely correlated with ZEB2 regions encompassing the two putative p53BS elements on levels: ectopic expression of miR-30a (5p, 3p or both the MIR30A promoter (miR30-1 and miR30-2) were strands, 5p/3p) resulted in ZEB2 decrease (Fig. 3b and amplified and quantified by quantitative PCR (qPCR). Pre- Supplementary Figure S5a); conversely, anti-miR-mediated immune IgG isotype antibodies were used in a mock inhibition of miR-30a elicited an increase in ZEB2 levels immunoprecipitation as a negative control/background sig- (Fig. 3c and Supplementary Figure S5b). nal; the p53 binding region of the p21 promoter was instead 2170 A. di Gennaro et al. To ascertain whether ZEB2 was a direct target of miR- untranslated region (UTR) sequence of ZEB2, either wild 30a-5p and miR-30a-3p, reporter assays were performed type (WT) or mutated in the seeds for the two miR-30a using luciferase reporter constructs carrying the 3’ strands (5pMUT, 3pMUT; Fig. 3a). Transfection of miR- A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness 2171 Fig. 2 p53 regulates miR-30a-5p and miR-30a-3p expression. a 30a actually affected tumor spreading via ZEB2. To this Ectopic p53 expression in MDA157 results in an augment of mature end, we ectopically expressed miR-30a-5p, miR-30a-3p or miR-30a-5p and miR-30a-3p levels. The immunoblot on the right the control miRNA (miRCTR) in MDA231 cells silenced shows p53 expression in MDA157 engineered cells. GAPDH was used (siRNA) for ZEB2 or in MDA231 engineered to over- as a loading control. b p53 silencing in HCT116 results in a decrement of both miR-30a-5p and miR-30a-3p. The extent of p53 silencing is express ZEB2. Either miR-30a strand inhibited migration of shown in the right panel. c Ectopic p53 induces an increase of pri-miR- ZEB2-proficient but not ZEB2-deficient MDA231 cells. 30a levels in MDA157. d Decrease of pri-miR-30a levels in p53- Moreover, ZEB2 overexpression counteracted the inhibi- depleted HCT116 and MCF7 cells. a–d Gray columns represent p53- tory effect of miR30a-5p and -3p (Fig. 4a and Supple- modulated samples; black columns represent control cells. e p53 regulates the MIR30A promoter. Silencing of p53 results in a decre- mentary Figure S7a-d). ment of the MIR30A promoter activity (30wt). The mutagenesis of the In addition, anti-miR-mediated block of miR-30a affec- two p53 binding sites, singularly (30mut1, 30mut2) or in combination ted cell motility (Fig. 4b). Anti-miR-30a-5p and anti-miR- (30mut1/2), abrogates this effect. Results represent the mean value of 30a-3p induced an increase in cell migration of comparable three independent experiments ± SD. f p53 binds the miR-30a pro- moter. Chromatin immunoprecipitation was performed with the DO-1 extent. Noteworthy, both anti-miRNAs elicited ZEB2 anti-p53 monoclonal antibody on HCT116 genomic DNA. Isotype- upregulation. In contrast, SNAI1, which is reported to be matched pre-immune mouse IgG was used as a negative control. The targeted by miR-30a-5p [20, 21], was upregulated only in immunoprecipitated chromatin was assayed for the enrichment of the response to the cognate anti-miR-30a-5p (Supplementary target MIR30A promoter (miR30-1 and 2, the regions encompassing the two p53BS) by qPCR. The p53 binding region of the p21 promoter Figure S7e-f). Thus, the modulation in cell motility induced and an irrelevant genomic region (CTR neg) [59] were used as positive by anti-miR-30a-3p in this experimental system correlated and negative control, respectively. Data are reported as fold enrich- only with ZEB2 upregulation. This supports a specific role ment over control samples (immunoprecipitation with pre-immune for ZEB2 in the control exerted by miR-30a over cell IgG) p values were calculated by two-tailed t-test; *p < 0.05, **p < 0.01. motility. To validate these concepts in vivo, we performed xeno- transplant experiments using the zebrafish embryo as a 30a-5p and miR-30a-3p significantly inhibited the expres- model, whose transparency enables direct visualization of sion of the ZEB2 3’UTR wild-type reporter (WT), while it fluorophore-labeled tumor cells [29, 30]. MDA231 and had no effect on the reporter in which the cognate miR-30a MDA157 cells, either silenced for ZEB2 (si1-ZEB2, si2- seeds were mutagenized. The combination of the two ZEB2) or ectopically expressing miR-30a (5p/3p), were miRNAs showed a quasi-additive effect (5pMUT, 3pMUT; injected in the yolk sac of zebrafish embryos to monitor Fig. 3d and Supplementary Figure S5c). tumor cell spreading and in the cardinal vein (duct of Overall, these data indicated that miR-30a (5p and 3p Cuvier) to monitor tail fin invasion [31]. Both strands) exerts an epigenetic control over ZEB2 mRNA. To ZEB2 silencing and miR-30a induction resulted in reduced address whether p53 entered into the miR-30a/ZEB2 tumor cell migration (Fig. 4c, d and Supplementary equation, we ectopically expressed p53 in MDA157. This Figure S8a-b) and halved tail fin invasion (Fig. 4e, f and cell line was selected because it is p53 null and because of Supplementary Figure S8c), indicating a diminished tumor its negligible expression of miR-200c [27], a possible cell extravasation and distal dissemination. confounding factor. In fact, miR-200c is a p53-regulated Overall, these results corroborate the concept that the miRNA previously reported to be connected to ZEB2 via miR-30a/ZEB2 axis, which is under the control of p53, is reciprocal feedback loop [13, 28]. The augment of miR-30a involved in tumor cell invasion and distal spreading. induced by ectopic p53 was paralleled by a reduction in ZEB2 protein levels (Fig. 3e). The existence of a p53 The p53/miR-30a/ZEB2 axis impinges upon miR- control on ZEB2 via miR-30a was confirmed by the finding 200c expression that anti-miR-mediated interference of miR-30a abrogated the ability of p53 to affect ZEB2 (Fig. 3e, Supplementary Finally, in the light of the previously reported negative Figure S6a-d). Taken together, these data indicate that, control of ZEB1 and ZEB2 over miR-200 [28, 32, 33], we beside the previously reported p53-miR-34a-SNAI1 axis predicted that miR-30a, by repressing ZEB2, would in turn [11, 14], p53 exerts a control over EMT also through the affect miR-200c. new route involving miR-30a and ZEB2. In accord with this hypothesis, ectopic expression of miR- 30a (5p/3p) resulted in an upregulation of miR-200c that was The miR-30a/ZEB2 axis controls TNBC tumor paralleled by a marked ZEB2 downregulation (Fig. 5a, Sup- spreading plementary Figure S9a and b). Instead, no major and repro- ducible variations in ZEB1 were observed. These results To address the biological implication of the p53/miR-30a/ support a role for the miR-30a/ZEB2 axis in the control of ZEB2 axis in TNBC biology, we ascertained whether miR- miR-200c. Accordingly, silencing of ZEB2, which yielded an 2172 A. di Gennaro et al. augment of miR-200c of similar extent to that induced by BC (Fig. 5c) was somehow corroborated by the finding that miR-30a, nullified the ability of this miRNA to induce miR- the miR-30a was positively correlated to miR-200c in both 200c (Fig. 5a, b, Supplementary Figure S10a and b). The the TCGA and in the in-house TNBC series (Supplementary interplay between the miR-30a/ZEB2 axis and miR-200c in Figure S11a-d). A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness 2173 Fig. 3 miR-30a-5p and miR-30a-3p inhibit ZEB2 and mediate the p53 control of p53 over these phenotypes, thus contributing to control over ZEB2. a Alignment of ZEB2 3’UTR with miR-30a-5p the aggressive behavior of p53-inactivated breast cancers. and miR-30a-3p (Targetscan). Nucleotides mutagenized to disrupt the Specifically, we show that miR-30a is a direct p53 tran- miRNA/mRNA base pairing are underlined. b Immunoblots showing scriptional target that is downregulated in response to p53 the modulation of ZEB2 in response to miR-30a (5p, 3p or both) ectopic expression and c after anti-miR-mediated inhibition of miR- inactivation. Intriguingly, a study conducted on a mela- 30a. ZEB2 relative levels, normalized over Tubulin (loading control), noma cell line has recently suggested that p53 may repress are reported below. d miR-30a targets the 3′UTR of ZEB2: luciferase miR-30a by binding the MIR30A promoter to a more activity of the ZEB2 3’UTR reporter, wild-type (WT) or mutated in the proximal region than the one here described [47]. miR-30a-5p and -3p binding sites (5pMUT and 3pMUT), was mea- sured 48 h post transfection of MDA231 cells with the indicated Although we cannot exclude p53 may exert on miR-30a miRNAs. miRCTR was set as a reference. CMV-Renilla was used for different effects depending on the cell context, our data normalization. Results represent the mean value ± SD of three compellingly demonstrate that there is a positive func- experiments. The asterisks (*) indicate the comparisons of miR-30a vs tional interplay between p53 and miR-30a, both in human miRCTR that are statistically significant (p < 0.05). e miR-30a-5p and miR-30a-3p expression in MDA157 stably transduced with p53 BC and ininvitrocellmodels. Inkeeping with our (pLenti-p53+) or pLenti-GFP (pLenti-p53−), in the absence (anti- findings, miR-30a was among the miRNAs positively miR-30a−) or presence (anti-miR-30a+) of an anti-miRNA targeting modulated in TP53 wild-type vs TP53-null HCT116 colon miR-30a. Immunoblots for ZEB2, p53 and Vinculin (loading control) cancer cells [48]. are shown on the right. Numbers below the blot indicate ZEB2 relative levels normalized over Vinculin Moreover, we provide evidence that ZEB2 is one important effector of this p53/miR-30a pathway. We demonstrate that the reduction in miR-30a expression eli- Discussion cited by p53 inactivation results in an alleviation of miR- 30a-mediated targeting of ZEB2, which correlates with Several lines of evidence demonstrate that inactivation of increased cell plasticity, migration and in vivo dissemina- p53, the tumor suppressor most frequently involved in tion. Thus, our results of a connection between p53 and TNBC, affects phenotypes such as EMT and cell plasticity/ EMT via miR-30a, with ZEB2 as a novel actor in this play, stemness that contribute to tumor progression and poor indicate that the p53/miR-30a/ZEB2 axis contributes to the response to therapies [5, 7, 34–37]. As a transcription fac- poor outcome of TNBC. tor, p53 controls the expression of several genes involved in Physiologically, the ZEB family of transcription factors these phenotypes, including miRNAs [6, 13, 14]. play a pivotal role in neural crest formation and migration In an attempt to elucidate the mechanisms whereby p53 during embryo development, a process involving EMT [49]. inactivation contributes to the aggressive behavior of BC In recent years, ZEB factors have gained attention for their via miRNA, we identified a novel axis involving p53, pro-oncogenic functions. As reported for other EMT tran- miR-30a and ZEB2. In particular, we found that the scription factors, the expression of ZEB proteins in tumor expression of miR-30a (both miR-30a-5p and miR-30a-3p) cells contributes to the shift from an epithelial towards a was significantly reduced in human BC samples carrying more mesenchymal phenotype and concurrently confers TP53 gene alterations, primarily TNBC, and inversely resistance to DNA damage, apoptosis and premature correlated with patients’ survival. This phenomenon was senescence [32, 50]. attributable to loss of wild-type p53 activity, rather than to Overexpression of ZEB proteins has been shown to con- gain of oncogenic functions [38], as miR-30a down- tribute to several cancer types [51, 52] and, in particular, it is regulation was observed not only in tumors with missense considered an unfavorable factor in BC [25, 53]. ZEB pro- p53 mutations encoding a dysfunctional full-length pro- teins have been previously linked to p53 via miR-200 [13], a tein, but also in tumors carrying alterations resulting in p53 family of p53-regulated miRNAs. In fact, miR-200s establish protein loss. with ZEB a double-negative feedback loop [28, 32, 33, 54]. The miR-30 family, which includes five members Our results indicate that miR-30a complements this equation. (miR-30a, -30b, -30c, -30d, -30e), has been implicated in In fact, miR-30a and miR-200c levels are positively correlated the pathogenesis of different tumor types [39]. Although in human BC and miR-30a-induced downmodulation of they share the same seed sequence, the various members ZEB2 results in miR-200c overexpression. differ for compensatory sequences which account for their Overall, this study demonstrates the existence of a specificity and for their diverse and sometime opposite novel axis, p53/miR-30a/ZEB2, that links p53 inactiva- roles in the regulation of cell proliferation, EMT and tion to EMT and BC aggressiveness (Fig. 5c), and adds apoptosis [19–21, 40–45]. As about miR-30a, loss of support to the notion that the control of p53 over the miR-30a-5p has been reported to favor tumor dissemina- various tumoral phenotypes relies on convergent and tion and chemoresistance by promoting EMT [19, 20, 40, integrated circuits, in which miRNAs appear as emerging 42, 43, 46]. We propose that miR-30a mediates the and pivotal nodes. 2174 A. di Gennaro et al. A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness 2175 Fig. 4 miR-30a (5p/3p) overexpression results in a reduced BC cell Lentiviral delivery, carried out as previously described migration in vitro and in a zebrafish xeno-transplantation model. a In [33], was used to generate stable cell models. MDA157 vitro cell migration assay of MDA231 engineered to ectopically cells stably expressing p53 were generated by using the express miR-30a-5p, miR-30a-3p or control miRNA (miRCTR) in a pLenti6/V5-p53_wt p53 vector (Addgene plasmid # 22945 ZEB2-proficient (siCTR) or ZEB2-deficient (si1-ZEB2 and si2-ZEB2) context (left panel). The right panel shows the migration capacity of [56]). pLenti6GFP was used as a control. MDA231 cells MDA231 engineered to stably overexpress ZEB2 or GFP, used as a were engineered to overexpress ZEB2 by transducting the negative control. b In vitro cell migration assay of MDA231 trans- pLJM-ZEB2 lentiviral vector or pLJM1-EGFP (Addgene # fected with anti-miR-30a-5p (A-miR-30a-5p), anti-miR-30a-5p (A- 19319 [57]) as a control. To generate pLJM-ZEB2, ZEB2 miR-30a-5p) or control anti-miRNA (A-CTR). The percentage of transmigrated cells was measured at 7 h post seeding. Data represent coding sequence was amplified from TOPO-Blunt-ZEB2 the mean of three independent experiments; *p < 0.05. c Scatter plot vector (HsCD000347714; Harvard Medical School, Boston) representing cell dissemination in zebrafish embryos of mCHERRY with primers containing AgeI and BstBI sites and cloned MDA231 transiently silenced for ZEB2 (si1-ZEB2 and si2-ZEB2) or into pLJM1-EGFP. engineered to stably express miR-30a (5p/3p). Cells transfected with an empty vector were used as a control (ctr). Cells were implanted in ZEB2-silenced (sh1-ZEB2, sh2-ZEB2 or sh5-ZEB2) and the yolk sac of 2-day-old embryos (fli1:EGFP strain). Embryos were control cells (shGFP) (MDA231, MDA157, HBL100, automatically imaged at 6 dpi. Dots represent single cells; colors Hs578T, BT549 and HCC1395) were generated by using identify each microinjected embryo; x-axis indicates the migration lentiviral plasmids obtained from a modified version of from the injection point (0,0) toward the head (positive values) or the tail (negative values); n indicates the number of embryos analyzed. d pRSI9 DECIPHER vector (Cellecta) in which an AgeI site Spreading distance of MDA231 and MDA157 calculated from data was introduced by mutagenesis. Individual sequences for represented in (c) and in Supplementary Figure S8b, respectively; *p < shZEB2 and shGFP (Supplementary Table S1) were cloned 0.05. e Representative images of zebrafishes injected with in the AgeIand EcoRI sites. MDA231 cells at 6 dpi. Cells were injected into the blood circulation (duct of Cuvier) of 2-day-old zebrafish embryos. Scale bar = 100 µm. f The MIR200C promoter is notoriously constitutively Percentages of embryos that show caudal micrometastatic colonization methylated in the TNBC cell lines and several cell divisions after injection with MDA231 or MDA157 cells engineered with are needed to achieve appreciable reactivation by deme- control vector (ctr), miR-30a (5p/3p) or silenced for ZEB2 (si1-ZEB2 thylating agents [33, 58]. Thus, stable cell models were and si2-ZEB2). The percentage of ctr embryos showing metastasis was arbitrarily set to 100. Data are shown at 1, 4 and 6 dpi. Figures needed to address the hypothesis of a miR-30a/ZEB2- represent the results of two independent experiments mediated activation of miR-200c. To this end, MDA231, MDA157 and HBL100 were engineered to express miR-30a (5p/3p) or silenced for ZEB2 via lentiviral delivery. Con- Materials and methods stitutive miR-30a (5p/3p) overexpression was achieved by lentiviral infection with pLenti6-miR-30a-(5p/3p) or pLen- Cell models ti6GFP vectors. MIR30A (miR-30a (5p/3p)) genomic region, amplified from MCF7 genomic DNA with primers The human breast cancer cell lines MDA-MB-157, MDA- containing XhoI and NotI sites (Supplementary Table S1), MB-231, MDA-MB-436 (here referred as MDA157, was initially cloned in pLNCX2-vector. The MIR30A MDA231 and MDA436), Hs578T, BT549, HCC1395 and fragment was then cleaved with BglII and ClaI restriction HCT116 colorectal cancer cell lines were obtained from the enzymes and inserted into BamHI and BstBI restriction sites ATCC, and HBL100 from Interlab Cell Line Collection- of pLenti6GFP. Genova. All cell lines, periodically authenticated by short tandem repeat profiling and tested mycoplasma-negative, TCGA dataset were cultured as previously described [55]. siRNAs for p53 (HSS110905, HSS186390, HSS186391; A dataset of 249 BC samples comprising clin- ThermoFisher Scientific) and non-targeting siRNA (12935- icopathological information, miRNA-seq data and TP53 100; ThermoFisher Scientific) were transfected using Lipo- mutational status was retrieved (on December 2014) from fectamine 3000 (ThermoFisher Scientific). Ambion Pre-miR TCGA portal (http://tcga-data.nci.nih.gov/tcga/ miRNA precursors specific either for the 5p or the 3p strand findArchives.htm [16]). The set included 204 BC hormo- (Life Technologies, ThermoFisher Scientific), anti-miRNAs nal receptor positive, 27 TNBC and 13 expressing HER2 (anti-miR) and relative controls (Life Technologies, Ther- but lacking hormonal receptors (5 cases were unknown). moFisher Scientific) were transfected with the siPORT Besides, 163 BCs presented wild-type TP53 and 82 carried NeoFX Transfection reagent (ThermoFisher Scientific) TP53 mutations (4 were unknown). miRNAs data of paired according to the manufacturer’s instructions. Two ON- tumor and normal tissues were collected for 12 cases. Target-plus siRNAs for ZEB2 (J-006914-22, J-006914-23; miRNA-seq data and clinicopathological records of further Dharmacon) and a non-targeting siRNA were transfected 63 TNBC were downloaded from TCGA data portal. Col- using the DharmaFECT reagent 4 (ThermoFisher Scientific). lected miRNA-seq data (level 3) included the calculated 2176 A. di Gennaro et al. Fig. 5 The p53/miR-30a/ZEB2 axis impinges upon miR-200c. a miR-200c levels in MDA231, MDA157 and HBL100 cell lines engineered to express miR-30a (5p/3p) or silenced for ZEB2 (sh1-ZEB2; sh2-ZEB2). Control vectors (pLenti6GFP and shGFP) yielded similar values and are here represented once as ctr. miR-200c levels in HBL100 ctr were set to 1. b miR-200c levels in MDA231 proficient (shGFP) or deficient (sh1-ZEB2, sh2-ZEB2) for ZEB2 expression, in the absence (ctr, pLenti6GFP) or presence (miR- 30a-5p/3p) of ectopic miR-30a; *p < 0.05. c A unifying model of the new p53/miR-30a/ZEB2 axis (highlighted in bold) involved in TNBC expression for all reads aligned to a specific miRNA Analyses reported in Table 1 were performed by using data reported as RPM (reads per million miRNA mapped). from mirnas.quantification files; in the analyses of miR-30a Comparisons were performed on LOG2 of RPM values. isoforms the isoform.quantification files were used. A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness 2177 Patients and samples transfected at 25 nM final concentration. Reporter assays were performed 48 h after transfection using the Dual- Formalin-fixed, paraffin-embedded (FFPE) specimens of 59 luciferase assay system (Promega). Transfection efficiency TNBCs were retrospectively collected at the CRO Aviano was normalized by calculating the Luciferase/Renilla National Cancer Institute biobank (2000–2010). Cases were activity ratio. All experiments were done in triplicate and selected based on the following criteria: naive for che- data confirmed in at least three independent experiments. motherapy and radiotherapy, tumor cellularity greater than 70%, suitability of the material for molecular analyses. Western blot analyses Informed consent was obtained and use of patient samples was approved by the Institutional Review Board. Clin- Protein extraction and western blot were performed as icopathological and follow-up data were retrieved from previously described [55]. Detailed description of the used clinical records (Table 2). antibodies is reported in Supplementary Table S2. Immu- noreactivity was detected with anti-mouse and anti-rabbit RNA extraction and qRT-PCR secondary antibodies horseradish peroxidase labeled (Per- kinElmer) using Western Lightning Chemiluminescence Total RNA was isolated from FFPE tumors samples using Reagent Plus (PerkinElmer). Images were captured and the Recover All Total Nucleic Acid Isolation Kit (Ther- analyzed using the Chemidoc XRS+ system (Bio-Rad). moFisher Scientific). The miRNeasy Mini Kit (Qiagen) was Expression levels were quantified using the ImageLab used to isolate total RNA from cell lines. Complementary imaging software (Bio-Rad). Results were confirmed in at DNA was generated by using SuperScriptIII-Reverse least three independent experiments. Transcriptase (Applied Biosystems, ThermoFisher Scien- tific) for pri-miRNA detection and by TaqMan MicroRNA Chromatin immunoprecipitation Reverse Transcription Kit (Life Technologies, Thermo- Fisher Scientific) for miRNA analyses. Pri-miR-30a, miR- Chromatin crosslinking was performed according to the NAs (miR-30a-5p, miR-30a-3p, miR-200c) and RNU48 protocol developed by P.J. Farnham (available online at: and RNU6B (reference genes) were then amplified by http://farnham.genomecenter.ucdavis.edu/pdf/FarnhamLa quantitative reverse transcription-PCR (qRT-PCR) using bChIP%20Protocol.pdf). TaqMan-specific kits (Life Technologies, ThermoFisher For ChIP, 4 µg of DO-1 anti-p53 monoclonal antibody or Scientific). Relative expression levels were normalized to isotype-matched pre-immune mouse IgG, as a negative controls (geometric mean of the reference genes) by using control, were used. Quantitative real-time PCRs with the the comparative Ct (ΔΔCt) method and the Bio-Rad CFX EvaGreen dye technology (Bio-Rad) was used to quantify manager software. All experiments were done in triplicate the DNA in ChIP samples. Analysis of ChIP data was and confirmed in at least three independent experiments. carried out using the fold enrichment method normalized to mock (IgG) control for each sample (ThermoFisher Scien- Dual-Luciferase reporter assay tific, https://tools.thermofisher.com/content/sfs/brochures/ Step-by-Step-Guide-to-Successful-ChIP-Assays.pdf). The miR-30a promoter region was amplified from genomic Details about oligonucleotides and antibodies are reported DNA extracted from MCF7 cells and cloned into the pGL3 in Supplementary Table S1 and S2, respectively. The oli- basic Luciferase vector (Promega). The 3’UTR of ZEB2 gonucleotides used for positive and negative controls were was amplified from genomic DNA extracted from MDA231 as previously described [59]. The results were confirmed in TM and cloned in pMIR-REPORT Luciferase vector (Ther- two independent experiments. moFisher Scientific). The p53 binding sites identified on the miR-30a promoter by the MatInspector software (Geno- Migration assays matix Software GmbH, Munich, Germany) and the miR- 30a-5p and miR-30a-3p binding sites on the 3’UTR of Migration assays were performed on several cell models ZEB2 were modified by site-direct mutagenesis (Quik- modulated for ZEB2 and/or miR-30a expression, namely: ChangeTM Site-Directed Mutagenesis Kit, Stratagene). The MDA231, MDA157, BT549, Hs578T and HCC1395 stably primers used for amplification and mutagenesis are reported silenced for ZEB2 via lentiviral delivery; ZEB2-silenced/ in Supplementary Table S1. miR-30a overexpressing MDA231 cell models, generated Reporter plasmids were transiently transfected in the by first transfecting ZEB2-specific siRNAs or non-targeting indicated cell lines using the Lipofectamine 3000 reagent siRNA (ThermoFisher Scientific, 25 nM) and then (24 h (ThermoFisher Scientific); pCMV-Renilla or PGK-Renilla later) by further transfecting pre-miR-30a-5p, pre-miR-30a-3p were used for normalization. siRNA and pre-miRNA were or pre-miR control (Ambion, 5 nM); MDA231 cells 2178 A. di Gennaro et al. engineered to stably express ZEB2 or control, transfected with EGFP embryos. The fraction of embryos exhibiting pre-miR-30a-5p, pre-miR-30a-3p or pre-miRNA control micrometastastatic colonization of the caudal fin (>10 cells) (Ambion, 5 nM); MDA231 cells transfected with anti- was calculated at 1, 4 and 6 dpi (days post injection), as miRNAs and relative control (ThermoFisher Scientific, previously described [31]. Data are representative of two 10 nM). At 48 h post transfection, cells were collected for independent experiments with at least 24 embryos per subsequent analyses. group. All experiments were performed twice. Migration assays were performed as described in Spes- sotto et al. [60]. Briefly, cells were trypsinized, collected Statistical analyses and fluorescently labeled with Fast DiI dye solution (Molecular Probes, Inc.) for 10 min at 37 °C in 5% CO . For miRNA expression analysis tumor samples were cate- Cells were then washed in serum-free medium and seeded gorized according to the median expression value into (10 cells/insert) in serum-free medium on the top side of “low” (expression levels<median) and “high” (expression Fluoroblok inserts (Corning). Medium containing 10% of levels≥median). The Mann–Whitney–Wilcoxon test and fetal bovine serum was used as chemoattractant in the lower Fisher’s exact test were used to assess associations between chamber (bottom side). Fluorescence intensity at 576 nm of miRNA expression and selected prognostic factors (age at top (nonmigrated cells) and bottom (transmigrated cells) diagnosis, tumor size, lymph nodes status, metastasis, TNM side of the well was measured at the indicated time points (tumor, node, metastasis) stage, tumor grade and treat- (tx, 7 or 24 h) using a microplate reader (Infinite ments). Survival analyses were conducted considering the M1000PRO, TECAN). The percentage of transmigrated time from diagnosis to the date of the event (death, relapse cells was determined as follows: 100×(FB −FB )/FT or last follow-up). Overall and disease-free survivals were tx t0 t0 where FB is the fluorescence intensity of bottom side at estimated using the Kaplan–Meier method and differences tx the indicated time point; FB is the fluorescence intensity of between curves were evaluated using the log-rank test. t0 bottom side at the time zero; FT is fluorescence intensity Differences in miRNA expression levels between groups t0 of top side at the time zero. All experiments were performed were assessed by using Mann–Whitney rank sum test for in triplicate and data confirmed in at least three independent TCGA dataset (values not normally distributed) and by experiments. t-test for the in-house series (values normally distributed and equal variance between groups). Statistical analyses for Zebrafish in vivo experiments in vitro experiments were performed using two-tailed t-test. Correlation between miRNA levels was evaluated by cal- To address the role of the mir-30a/ZEB2 axis in vivo, culating Spearman’s correlation coefficient (r). Statistical experiments were performed using the zebrafish embryo analyses were performed with SAS 9.4 (SAS Institute Inc.) model. We choose this model because, beside meeting the and SigmaPlot (Systat Software Inc.). 3R recommendations of using animals with a reduced ner- Acknowledgements The authors are grateful to Giovanna Zerial for vous system development, its transparency allows an her experimental contribution. This study was supported by: Asso- effective and real-time assessment of tumor cell growth and ciazione Italiana per la Ricerca sul Cancro (AIRC), Italian Ministry of migration. Health, Associazione Via di Natale, Fondazione Umberto Veronesi, The transgenic zebrafish line Tg (fli1:EGFP), expressing Fondazione CRO Onlus, Banca Popolare FriulAdria, CRO 5X1000. enhanced green fluorescent protein (EGFP) in endothelial cells in wild-type background, was used for in vivo evalua- Compliance with ethical standards tion of tumor cell dissemination and micrometastatization. Conflict of interest The authors declare that they have no conflict of Zebrafish and embryos were raised, staged and maintained interest. according to standard procedures (http://ZFIN.org)incom- pliance with the local animal welfare regulations. Open Access This article is licensed under a Creative Commons BC cells, engineered as specified in the text and made Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as fluorescent following infection with the mCHERRY lenti- long as you give appropriate credit to the original author(s) and the viral vector (pCMV-mCherry-bc-puro-Kl201), were injec- source, provide a link to the Creative Commons license, and indicate if ted in the yolk sac and analyzed as previously described changes were made. The images or other third party material in this [29]. Tumor dissemination was measured as “spreading article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not distance per object” representing the mean cell migration included in the article’s Creative Commons license and your intended for each embryo. use is not permitted by statutory regulation or exceeds the permitted To investigate the ability of engineered cells to extra- use, you will need to obtain permission directly from the copyright vasate and form distal metastasis, mCHERRY-positive BC holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. cells were injected into the duct of Cuvier of 2-day-old fli1: A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness 2179 References 20. Kumarswamy R, Mudduluru G, Ceppi P, Muppala S, Kozlowski M, Niklinski J, et al. MicroRNA-30a inhibits epithelial-to- mesenchymal transition by targeting Snai1 and is downregulated 1. Perou CM, Sørlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees in non-small cell lung cancer. Int J Cancer. 2012;130:2044–53. CA, et al. 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Published: Apr 17, 2018

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