Unravelling a p73-regulated network: The role of a novel p73-dependent target, MIR3158, in cancer cell migration and invasiveness

Accepted Manuscript Unravelling a p73-regulated network: the role of a novel p73-dependent target, MIR3158, in cancer cell migration and invasiveness Sotiris Galtsidis, Stella Logotheti, Athanasia Pavlopoulou, Christos P. Zampetidis, Georgia Papachristopoulou, Andreas Scorilas, Borek Vojtesek, Vassilis Gorgoulis, Vassilis Zoumpourlis PII:

S0304-3835(16)30741-8

DOI:

10.1016/j.canlet.2016.11.036

Reference:

CAN 13140

To appear in:

Cancer Letters

Received Date: 25 October 2016 Revised Date:

25 November 2016

Accepted Date: 28 November 2016

Please cite this article as: S. Galtsidis, S. Logotheti, A. Pavlopoulou, C.P. Zampetidis, G. Papachristopoulou, A. Scorilas, B. Vojtesek, V. Gorgoulis, V. Zoumpourlis, Unravelling a p73-regulated network: the role of a novel p73-dependent target, MIR3158, in cancer cell migration and invasiveness, Cancer Letters (2017), doi: 10.1016/j.canlet.2016.11.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Unravelling a p73-regulated network: the role of a novel p73-dependent target,

1

RI PT

MIR3158, in cancer cell migration and invasiveness

1

Sotiris Galtsidisa, , Stella Logothetia, , Athanasia Pavlopouloub, Christos P. Zampetidisc, Georgia Papachristopouloud, Andreas Scorilasd, Borek Vojteseke, Vassilis Gorgoulisc, Vassilis Zoumpourlisa,*

Biomedical Applications Unit, Institute of Biology, Medicinal Chemistry and Biotechnology, National

SC

a

Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, 116 35 Athens, Greece. Centre of Systems Biology, Biomedical Research Foundation, Academy of Athens, 4 Soranou Efesiou str.,

M AN U

b

11527 Athens, Greece. c

Laboratory of Histology-Embryology, Molecular Carcinogenesis Group, Medical School, National and

Kapodistrian University of Athens, Athens, Greece.

Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian

TE D

d

University of Athens, Athens, Greece e

Regional Center for Applied and Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech

EP

Republic

AC C

Running title: Function of a p73-regulated network

* To whom correspondence should be addressed: National Hellenic Research Foundation, Institute of Biology, Medicinal Chemistry and Biotechnology, Unit of Biomedical Applications , 48 Vas Constantinou Ave, 116 35 Athens, Greece. Tel: 0030210-7273730; FAX: 0030210-7273677; e-mail: [email protected]

1

These authors contributed equally in this work.

1

ACCEPTED MANUSCRIPT Abstract The transcription factor p73 is homologous to the well-known tumor-suppressor p53. The p73-regulated networks are of significant clinical interest, because they may substitute for impaired p53-regulated networks which are commonly perturbed in cancer. Herein, we aimed to characterize a p73-regulated network that

demonstrate

that

p73

regulates

a

network

underlying

cell

RI PT

mediates cell migration and restores anti-oncogenic responses in p53-mutant cancer cells. In this study, we migration,

which

consists

of

MIR34A/MIR3158/vimentin/β-catenin/lef1. The p73 isoforms transactivate the miRNA components (MIR34A/MIR3158) of this network, which in turn, downregulate their EMT-related mRNA co-targets

SC

(vimentin/β-catenin/lef1) to decrease cell-migration. Modulation of this network, by increasing the level of the

M AN U

novel p73-dependent target MIR3158, was found to induce anti-oncogenic/anti-invasive responses in p53mutant cancer cells. Taken together, a p73-regulated, MIR3158-containing, network restores anti-invasive phenotypes in p53-mutant cancer cells; this property could be exploited towards the development of

Keywords

TE D

anticancer therapeutics.

AC C

Abbreviations

EP

p73, MIR3158, MIR34A, epithelial-mesenchymal transition, anticancer targeting

TSS: Transcription Start Site; ChIP: Chromatin immunoprecipitation; RE: responsive element; qPCR: quantitative real-time polymerase chain reaction; EMT: epithelial-mesenchymal transition.

2

ACCEPTED MANUSCRIPT 1. Introduction The p73 gene belongs to the p53 family of transcription factors and is homologous to the cardinal tumorsuppressor p53, which is the most frequently mutated gene in cancer. The p53-regulated networks are often impaired in cancer [1]; thus, their re-activation poses as the “holy grail” of targeted cancer therapeutics [2].

RI PT

The p73 has attracted increasing attention, because it frequently acts as p53’s surrogate and restores antioncogenic responses in cells with defective p53-regulated pathways [3]. To this end, the p73-regulated pathways and/or networks are of particular interest, since they provide alternative means of restoring defective p53-regulated pathways and/or networks in cancer cells [4-6]. Activating p73-regulated networks that mediate

SC

anti-oncogenic responses, especially in a p53-independent manner, may be proven to be an

M AN U

alternative/substitute to the “holy grail” in targeted cancer therapeutics. For instance, John and colleagues have shown that manipulation of a p73-regulated, miRNA-containing circuit, which is conserved between mouse and human, can induce anti-oncogenic responses in p53-mutant cancer cells [5]. It was recently shown that p53 suppresses factors that are implicated in epithelial-mesenchymal transition (EMT) signaling [7], i.e. a biological process by which an epithelial cell acquires mesenchymal characteristics

TE D

and becomes pluripotent, more motile and invasive, via alterations of several crucial factors (N-cadherin, vimentin, Snail1, Snail2, Twist, EF1/ZEB1, SIP1/ZEB2, β-catenin etc.). Cancer cells re-activate EMT-factors to become more aggressive [8]. Therefore, by inhibiting these factors, p53 inhibits cancer progression. The

EP

p53’s effect on EMT-factors is mediated by miRNAs (i.e. 16-27-nucleotide long RNA molecules which bind to complementary regions in the 3’ UTR of downstream target mRNAs to induce either mRNA degradation or

AC C

inhibition of translation [9, 10]); the miRNA genes themselves are regulated by transcription factors (TFs). In this way, functional network modules [11] are formed, which may often have the general pattern “TF/miRNA/target genes” [12]. The effect of p53 on cell migration seems to follow, at least in part, this network pattern. In particular, p53 transactivates miRNA genes (i.e. miR-34a/miR-200), which in turn, target EMT-factors (such as Snail1 and ZEB1/ZEB2) and inhibit cell migration and invasiveness (Fig. 1a) [13]. The p73 gene synthesizes two main classes of isoforms: the full-length TAp73 isoforms, which have an intact N-terminal transactivation domain and are typically transactivation-competent, and the DNp73 isoforms, which lack part of or the entire transactivation domain and are usually transactivation-incompetent. The

3

ACCEPTED MANUSCRIPT TAp73 C-terminal splice variants (designated as α, β, γ, δ, ε, ζ, η, η*, η1 and θ) are anti-oncogenic and frequently shown to mimic or substitute for p53 [14]. Similar to p53, TAp73 isoforms suppress EMT-factors and cell migration [14-17]. However, TAp73-regulated networks underlying these latter processes have not been identified yet (Fig. 1a).

RI PT

In this study, we aimed to unveil functional networks of p73/miRNA/EMT-factors that mediate tumor cell migration and invasiveness. We hypothesized that manipulation of miRNA components of this p73-regulated network could restore anti-oncogenic responses in p53-mutated cancer cells. In this way, we identified, for the first time, the previously uncharacterized MIR3158 as a p73-dependent target, as well as a p73-regulated

SC

functional network, consisting of MIR34A/MIR3158/vimentin/β-catenin/lef1, mediating cell migration. Notably, exogenous overexpression of the newly identified MIR3158 component was found to have an anti-

M AN U

oncogenic and anti-invasive outcome on p53-mutant cancer cells.

2. Methods and Materials

TE D

2.1. Cell Culture

Human osteosarcoma Saos2 cells, Saos2-TAp73α, Saos2-TAp73β, Saos2-TAp73γ and Saos-p53 inducible clones (kindly provided by Dr. Vousden); human breast cancer cell lines MCF12A, BT-20, SKBR3, MDA-

EP

MB-231, MDA-MB-468 (American Type Culture Collection, Manassas, VA 20110, U.S.A.) were cultured as previously described [18]. Isoform expression in Saos2-TAp73α, Saos2-TAp73β, Saos2-TAp73γ and Saos-

AC C

p53 inducible clones was activated upon treatment with 2.0-2.5µg/mL doxycycline for 48h. Parental Saos2,

MDA-MB-231 and MDA-MB-468 cell lines were cultured with or without doxocycline to measure the expression levels of hsa-miR-34a-5p and hsa-miR-3158-5p in order to exclude any possible effect of doxocycline. Q-PCR results showed no significant differences in the expression levels of the abovementioned miRNAs (data not shown) when each cell line cultured in the presence of doxocycline was compared to its corresponding control (without doxocycline).

4

ACCEPTED MANUSCRIPT 2.2. Patients and tissues Frozen primary breast tumors from a cohort of 22 patients (age range 23-81 years, mean 48.6 years) were obtained from “Agios Savvas” Cancer Hospital of Athens. Eight (8) tumors were histologically classified as benign and eight as malignant (grade I and grade III). Patients received no neoadjuvant chemotherapy or

RI PT

hormonal therapy pre-operatively. The study protocol was approved by the Hospital’s Scientific Committee and the associating Bioethics committee of the Medical School of Athens. All the patients provided written, informed consent in accordance with the Declaration of Helsinki.

SC

2.3. RNA isolation, cDNA synthesis and qPCR

Total mRNA and miRNA extraction, corresponding cDNA synthesis and qPCR were performed as previously

M AN U

described [18]. The sequences of the qPCR primers used are listed in Supplementary Table S1. Expression of hsa-miR374a-5p and hsa-miR148b-3p was used as normalization control [18].

2.4. Cell lysates preparation and Western blot analysis

TE D

Total cell lysates were prepared and quantified, and Western blots were performed as previously described [19]. The primary antibodies against vimentin (diluted 1:1000), Zeb1 (diluted 1:1000) and β-actin (diluted 1:1000) were used (Santa Cruz Biotechnology, Santa Cruz, CA, USA; cat. no’s sc-32322, sc-25388, sc-47778,

EP

respectively). Snail1 primary antibody (cat. no. ab180714) and HA-tag antibody were used in a 1:1000 dilution each (Abcam, Cambridge, UK). The secondary antibodies (Millipore) required according to each

AC C

antibody’s datasheet were used in a 1:3000 dilution. Relative expression of proteins was analyzed using ImageJ software.

2.5. miRNA arrays

miRNA array analysis was conducted at Exiqon Services, Vedbæk, Denmark, following a previously described Exiqon proprietary protocol [18].

2.6. Plasmid constructs The constructs used in this study are described in Supplementary Methods. 5

ACCEPTED MANUSCRIPT

2.7. ChIP assays Saos inducible p73 clones (Saos TAp73α, β, γ) were treated with doxocycline for 48 hours in order to express the corresponding isoforms. Untreated clones were used as corresponding negative controls. The ChIP

RI PT

protocol that was applied has been previously described [20]. Immunoprecipitation was performed using 2µg of HA-tag antibody (Abcam, Cambridge, UK). Chromatin incubated with antibody against GAPDH (Abcam, Cambridge, UK) was used as a negative immunoprecipitation control, whereas input was used as a positive qPCR control. The analysis of the results was conducted as previously described [21]. The pelleted DNA was

SC

resuspended in 10µL of nuclease free water and amplified by qPCR, using the following primers that are specific to the promoter region of the human MIR3158 gene: forward, 5’-TAGTGTGGTGCCGGTGATTC-3’

M AN U

and reverse, 5’-TTCAAGCTGCCACTGGC-3’. As a negative control of TAp73-specific binding on MIR3158 promoter, we used primers against a 420bp long DNA region which included the 81bp long pre-miRNAencoding region of the MIR3158 gene plus 339bps downstream this region, i.e. forward primer: 5’

TE D

TCAGGCCGGTCCTGCAGA 3, reverse primer: 5’ CACTTCAGACAGGGGTGCCT 3’.

2.8. Luciferase reporter gene assay

In order to test the ability of TAp73 isoforms to transactivate the MIR3158 gene, Saos2 cells were transiently

EP

co-transfected with the MIR3158 RE-luc plasmid and each of the pcDNA3.1-TAp73α, pcDNA3.1-TAp73β and pcDNA3.1-TAp73γ expression plasmids using Effectene (Qiagen, Valencia, CA, USA), according to

AC C

manufacturer’s instructions. To test the ability of miR-3158-5p to target vimentin, Saos2 cells were cotransfected with 3’ UTR vimentin-Luc plasmid and either miR-3158-5p mimic or a negative control miRNA mimic. Then, luciferase assays were performed as previously described [22].

2.9. Stably transfected clones Human MDA-MB-231 and MDA-MB-468 cells grown in 6-well plates were transfected with the pcDNA3.1hsa-MIR3158 plasmid using Effectene, according to the manufacturer’s instructions, in a 50% confluence. Stable transfectants were selected on 1mg/mL geneticin G-418 (Sigma-Aldrich, Seelze, Germany) after twenty days. Two MDA-MB-231 clones stably expressing human MIR3158, namely MDA-MB-231-clone 3 6

ACCEPTED MANUSCRIPT and MDA-MB-231-clone 10; and two MDA-MB-468 clones stably expressing human MIR3158, namely MDA-MB-468-clone 3 and MDA-MB-468-clone 7, were selected for analysis.

2.10. Transfection with miRNA modulators

RI PT

Transfection with miRNA modulators (miR-3158-5p mimic or the negative control; Ambion, Life Technologies, Austin, TX, USA) was performed using a previously described protocol [18].

2.11. Wound healing, MTS proliferation, invasion and colony formation assays

SC

For wound healing assays, cells were grown in six-well plates for 24h. The confluent monolayers were wounded by scraping with a P200 micropipette tip and washed two times with PBS. Following scrapping, cell

M AN U

monolayers were photographed at specific time points. Cell migration was determined by visual assessment of cells migrating into the wound using phase contrast microscopy. All MTS proliferation, invasion and colony formation assays were performed as previously described [18].

TE D

2.12. In vivo tumorigenicity studies

Parental cells, control vector-transfected cells and plasmid-transfected cells were injected subcutaneously in SCID mice following a previously described protocol. The experiments complied with the National Hellenic

EP

Research Foundation’s legislation regarding the welfare of experimental animals [18].

AC C

2.13. Bioinformatics and statistical analyses The in silico analyses performed in this study are presented in detail in Supplementary Methods. Experiments were performed three times, in triplicates each. Statistical significant differences were evaluated using student’s t-test. Where appropriate, a value of p<0.05 is depicted with an asterisk (*) in the corresponding figures.

7

ACCEPTED MANUSCRIPT

3. Results 3.1. TAp73-induced miRNomes inhibit EMT-factors and cell migration In order to identify TAp73-induced miRNAs that inhibit EMT-factors and cell migration, we used a well-

RI PT

established system for studying TAp73 isoforms [18], comprised of doxocycline-inducible Saos2 clones stably transfected with ΤΑp73α, ΤΑp73β or ΤΑp73γ isoforms. Notably, this study system, due to its p53-null background, is specifically advantageous for identifying direct and indirect targets of TAp73. In this way, this system allows to monitor downstream molecular networks programs activated by each TAp73 isoform

SC

specifically, unaffected by p53 interference, which could obscure the overall activity of TAp73 isoforms [18].

M AN U

Expression of TAp73 isoforms in Saos2-ΤΑp73α, Saos2-ΤΑp73β and Saos2-ΤΑp73γ versus their uninduced controls (Fig. 1b), decreased cell migration, consistent with reduction of the protein levels of the representative EMT markers ZEB-1, vimentin and Snail1 [8]. Notably, TAp73β and TAp73γ exerted a higher degree of inhibition on all-three EMT markers’ expression, as opposed to the lesser effect of TAp73α (Fig. 1c). Since the expression levels of TAp73 isoforms upon doxocycline induction (day 2) were comparable for

isoforms (Fig. 1d).

TE D

all-three clones, these differences are attributed to the different quality, rather than to the different quantity of

In addition, a miRNA array analysis of Saos2-ΤΑp73α, Saos2-ΤΑp73β or Saos2-ΤΑp73γ clones versus their

EP

uninduced controls was performed in order to identify miRNAs in the miRNomes that are correlated with downregulation of EMT-factors (Fig. 1e). To identify putative direct miRNAs that are transactivated by

AC C

TAp73 isoforms and downregulate EMT-factors, we screened the top-50 list for miRNAs that fulfilled the following criteria: (a) are upregulated (> 2-fold increase) upon induction of either of the three TAp73 isoforms; (b) contain putative TAp73 responsive elements on their promoters; and (c) target EMT markers on the basis of in silico predictions (Table S2). The miRNA genes fulfilling all selection criteria were MIR3158 and MIR34A (grey-shaded in Table S2). For the confirmed p73 target MIR34A, a putative p53/p73 responsive element was detected -460bps upstream of the transcription start site (TSS) (p53/p73 RE: AAGCATGCAATTAATAAAAAGGGACCAGGTTT). For MIR3158, a putative p73 responsive element (p73 RE: TGGCGTGTGAAAAGTGCCCTGGGAGCCATGGGC) was detected at the position -71bps

8

ACCEPTED MANUSCRIPT upstream of the MIR3158 TSS. Of note, the three TAp73 isoforms revealed distinct miRNA signatures, with TAp73β and TAp73γ having more overlapping miRNAs compared to TAp73α, including miR-3158-5p and miR-34a-5p (Fig 1f). These isoform-specific miRNomes could account for the divergence of TAp73α from TAp73β and TAp73γ regarding their corresponding outcomes on EMT-factors expression levels and cell

RI PT

migration.

3.2. miR-3158-5p and miR-34a-5p are co-overexpressed within the TAp73β/TAp73γ-induced miRNomes

SC

The expression of miR-3158-5p and miR-34a-5p was validated in TAp73α-, TAp73β- and TAp73γ-expressing clones versus the corresponding uninduced controls using qPCR. The miR-3158-5p and miR-34a-5p levels

M AN U

were higher in TAp73β and TAp73γ but slightly lower in the TAp73α clone in comparison to their uninduced counterparts (Fig. 2a). MIR34A is a direct target of TAp73β [23] and a well-characterized tumor-suppressor miRNA gene [24]. MIR34A’s confirmed EMT-related targets ZEB1 and Snail are downregulated in the TAp73-expressing clones, in agreement with previous studies [13]. In contrast, MIR3158’s function either in

TE D

cell migration or in any other cellular function remains totally uncharacterized. Moreover, while miR-34a-5p expression is induced in the Saos2-p53 expressing clone versus its uninduced control, miR-3158-5p expression levels remain unaltered (Fig. 2a). This signifies that, unlike MIR34A the induction of which is

EP

p53/p73-dependent [24], MIR3158 is activated specifically by p73. Overall, our analysis indicated that MIR3158 and MIR34A are direct targets of p73, with the ability to downregulate EMT-factors. However, in

AC C

contrast to MIR34A, for which a p73/MIR34A/vimentin axis has been experimentally demonstrated previously, a similar p73/MIR3158/vimentin axis is only predicted for MIR3158 and needs to be validated. Toward this end, we have made an effort to experimentally validate this axis.

3.3. MIR3158 activation is triggered by TAp73β and TAp73γ, but blocked by TAp73α At this step, we evaluated whether the human MIR3158 is a direct target of TAp73 isoforms, as is the case of MIR34A. All three TAp73 isoforms bind to the predicted TAp73 binding site of MIR3158, as indicated by ChIP assays in the Saos2-ΤΑp73α, Saos2-ΤΑp73β and Saos2-ΤΑp73γ expressing clones versus their uninduced controls. Among the three TAp73 isoforms, TAp73α displayed the highest binding affinity to the 9

ACCEPTED MANUSCRIPT MIR3158 promoter (Fig. 2b). To further assess the ability of TAp73 isoforms to directly transactivate MIR3158, a luciferase plasmid with the TAp73 binding site-containing genomic fragment of the MIR3158 promoter located upstream to the luciferase gene was constructed and transiently co-transfected with each of the plasmids expressing TAp73α, ΤΑp73β and ΤΑp73γ in Saos2 cells. Both TAp73β and TAp73γ potently

RI PT

transactivated the MIR3158 promoter, whereas TAp73α failed to induce MIR3158 transactivation (Fig. 2c). Of particular note, although TAp73α binds strongly to the TAp73 responsive element (RE) of MIR3158, it fails to transactivate it and to increase miR-3158-5p levels (Fig. 2a, 2b and 2c). On the contrary, the isoforms TAp73β and TAp73γ bind to and transactivate MIR3158 at comparable levels. Such discrepancies between

SC

DNA binding activity and transcriptional efficiency on the same gene promoter and in the same cell context have been reported previously for other p73-regulated genes and provide the basis for the transactivation

M AN U

selectivity of TAp73 isoforms [14]. All TAp73 isoforms have the potential to transactivate the same p73 REbearing target genes, since they have identical transactivation and DNA binding domains. However, the unique C-terminus of each TAp73 isoform determines which target genes each isoform would selectively transactivate, thereby leading to divergent TAp73 isoform-specific outcomes [14]. MIR3158 is a

TE D

representative case, where TAp73α harbors TAp73 REs but does not potentiate MIR3158 transactivation, whereas TAp73β/TAp73γ isoforms, which have shorter C-terminal tails, antagonize this effect by enhancing

EP

MIR3158 transactivation even with lower promoter binding affinities (Fig. 2d).

3.4. miR-3158-5p downregulates vimentin and suppresses cell migration

AC C

Having characterized MIR3158 as a novel, TAp73-dependent target, the next step was to investigate whether it silences EMT-factors and impairs cell migration. To this end, we examined if miR-3158-5p suppresses its in silico predicted target vimentin, which has been found to be more potently downregulated in TAp73β- and TAp73γ-expressing clones. A TAp73/miR-3158-5p axis leading to vimentin inhibition could contribute in the retardation of cell migration which was observed in the TAp73-expressing clones. To confirm vimentin as a true target of miR-3158-5p, a luciferase construct carrying the 3’ UTR of human vimentin (containing the putative target sequence of miR-3158-5p along with its poly-A signal) downstream of the luciferase gene was constructed and transiently transfected in Saos2 cells. Cells were treated for 48h with either the miR-3158-5p mimic or the negative control (scrambled miRNA mimic). Treatment with the miR-3158-5p mimic reduced 10

ACCEPTED MANUSCRIPT the luciferase signal more than 55% in comparison to the negative control-treated cells (Fig. 2e). Vimentin protein levels in miR-3158-5p mimic-transfected Saos2 cells were also downregulated compared to the scrambled miR mimic negative control (Fig. 2g). Then, in order to examine the ability of miR-3158-5p to suppress cell migration, thereby “phenocopying” the effects of TAp73 induction, Saos2 cells were transiently

RI PT

transfected with either the miR-3158-5p mimic or its scrambled negative control. Wound healing assays 72h post-transfection revealed that miR-3158-5p reduced cell migration compared to the negative control-treated cells (Fig. 2f).

SC

3.5. The p73-regulated MIR3158 and MIR34A and their co-targets vimentin, β-catenin and lef1 form a network-module associated with cell migration

M AN U

Mammalian miRNAs exert their effects in an orchestrated, co-operating manner, by acting as member of functionally coherent groups or single-input modules of regulatory networks [25]. In this way, more than one miRNAs co-regulate a single mRNA target in order to eventually surpass a systemic effect threshold and produce a certain cell outcome [18]. Criteria of functional coherence between two miRNAs have been

TE D

recently determined and include close genomic proximity, common miRNA family, regulation by common transcription factors, common mRNA targets that are implicated in relevant biological processes [25, 26], coexpression in miRNomes [18]. The miR-3158-5p and miR-34α-5p, found co-expressed in the TAp73-induced

EP

miRNomes, fulfill several of these criteria, i.e. they (a) have common expression profiles in all Saos2-TAp73 expressing clones (upregulated in the TAp73β and TAp73γ clones, unaltered in the TAp73α clone), (b)

AC C

represent transcriptional targets of TAp73, (c) suppress cell migration, (d) target vimentin ([27] and this study). Based on the above, MIR34A emerges as a putative functional associate of MIR3158 in the p73induced miRNomes. To investigate this hypothesis thoroughly, we defined in silico the mRNA targets that hsa-miR-3158-5p and hsa-miR-34a-5p have in common. A total of 64 hsa-miR-3158-5p/hsa-miR-34a-5p coregulated predicted targets were defined (Table S3). Of those, 29 out of the 64 predicted co-targets are interconnected (Fig. 3), as revealed by STRING analysis (Supplementary Methods). This analysis also revealed two EMT-factors that are implicated in cell migration in addition to vimentin, that is β-catenin and lef1 [28, 29] (Fig. 3). Both proteins are downregulated upon doxocycline induction in the Saos-TAp73 clones (data not shown). Hence, a cell migration-associated network-module which consists of p73, MIR34A, MIR3158 and 11

ACCEPTED MANUSCRIPT the EMT-factors lef1, vimentin, beta-catenin was identified. No statistically significant enriched KEGG pathways were detected for MIR3158-specific targets. However, the MIR3158 targets are involved in cancerrelated KEGG pathways when examined in relation to MIR34A targets. Overall, 8 predicted MIR3158/MIR34A-coregulated targets, including the EMT-factors lef1 and β-catenin, participate in cancer-

RI PT

related pathways (Table S4).

3.6. Exogenously overexpressed MIR3158 inhibits the downstream components of the network motif, suppresses cell migration and leads to anti-invasive outcomes of p53-mutant cancer cells

SC

The fact that MIR3158 (a) antagonizes cell migration and (b) is predicted to target genes that participate exclusively in cancer-related KEGG pathways, when examined in combination with MIR34A targets, indicate

M AN U

a putative anti-oncogenic role for MIR3158. Therefore, we examined if manipulation of the MIR3158 could modify this network-module and induce anti-oncogenic responses. In the p53-null Saos2-TAp73 experimental model, miR-3158-5p is forced to be expressed upon exogenous induction of its transcription factor TAp73. This system is ideal to unveil novel downstream targets of TAp73 in vitro, but it does not necessarily imply

TE D

that MIR3158 is associated with osteosarcomas in natura. To find a more suitable setting for simulation of its physiological role in the context of cancer, we sought for cancer types where miR-3158-5p is spontaneously and endogenously expressed in situ. To this end, we took into account that MIR3158 was originally detected

EP

and discovered in breast cancer tissue samples (Persson H personal communication; [30]). MIR3158, along with MIR34A expression, was also confirmed in 22 tumors representing benign and cancer stages of breast

AC C

cancer (Fig. S1). Inversely, vimentin, β-catenin and lef1 levels are frequently increased in breast cancer [28, 29]. Therefore, breast cancer emerged as an appropriate setting for further investigating the anti-oncogenic potential of this MIR3158-containing network. Based on the above evidence, we screened a panel of breast cancer cell lines for miR-3158-5p expression. We observed that cell lines with low invasive potential and proliferation rate expressed higher levels of miR-31585p, as opposed to the ones with high invasive potential and proliferation rate [31, 32] (Fig. 4a). Of the latter, both of the p53-mutant cell lines MDA-MB-468 and MDA-MB-231 have lower miR-3158-5p levels. MDAMB-468 has increased miR-34a [33], while MDA-MB-231 has reduced miR-34a [33] (Fig. 4b). In order to evaluate how modulation of MIR3158 expression affects the network-module underlying cell migration in 12

ACCEPTED MANUSCRIPT p53-mutant cancer cells with either high or low miR-34a expressing cell-context, MDA-MB-231 and MDAMB-468 cell lines were stably transfected with a human MIR3158-expressing plasmid. Two MDA-MB-231 clones stably expressing human MIR3158, namely MDA-MB-231-clone 3 and MDA-MB-231-clone 10, as well as two MDA-MB-468 clones stably expressing human MIR3158, namely MDA-MB-468-clone 3 and

RI PT

MDA-MB-468-clone 7, were used. Exogenous expression of MIR3158 downregulated vimentin, β-catenin and lef1 mRNA levels in all-four clones (Fig. 4c, 4d) and decreased cell migration (Fig. 4e, 4f), irrespective of the miR-34a expressing background. Taken together, the above findings indicate a consistent ability of MIR3158 to decrease cell migration by modulating the downstream components of the network-module in several

SC

cellular milieus. Aggressive and invasive phenotypes of these cancer cells were also ameliorated (Fig. 4g, 4h). Additionally, MIR3158 exogenous overexpression in all-four clones decreased proliferation rate (Fig. 5a, 5b)

M AN U

and impaired indefinite cell proliferation (Fig. 5c, 5d). It also led to formation of significantly smaller tumors upon subcutaneous injections in xenografts (Fig. 5e), significant prolongation of the latency period of tumor onset and decrease in the number of tumors grown in the injected sites compared to mock and parental

4. Discussion

TE D

controls (Fig. 5f and Table S5).

In this study, we identified MIR3158 as a novel target of p73. The TAp73β and TAp73γ isoforms transactivate

EP

MIR34A and MIR3158, which in turn downregulate the expression levels of EMT-factors lef1/βcatenin/vimentin and subsequently mitigate cancer cell migration and invasiveness. In particular, miR-3158-

AC C

5p was found to downregulate vimentin; moreover, induced expression of MIR3158 was demonstrated to exert anti-proliferative/anti-invasive effects on p53-mutant cancer cells. The newly identified p73-dependent target MIR3158 is an evolutionarily-young miRNA, classified (based on the conservation of its sequence) into the primate-specific group of miRNAs that represent the 55% of all human miRNA genes [34]. On the other hand, MIR34A is evolutionarily-old, as it appears for first time in Caenorhabditis elegans and is highly conserved across species [24]. The evolutionary age largely affects the overall behavior of miRNAs [34]. Compared to the evolutionarily-old miRNAs, evolutionarily-young miRNAs are lowly-expressed and tissue-specific, present milder phenotypes upon knockdown, are under tight

13

ACCEPTED MANUSCRIPT transcriptional regulation [34] and are associated with major body-plan innovations and phenotypic variations in mammals [35]. Consistently, MIR3158 exhibits low expression levels in all tissues, but is specifically enriched in brain (data mined from miRIAD database). Transactivation of MIR3158 is TAp73-dependent, but p53-independent, suggesting a tendency for selective participation in p73-regulated processes. In contrast,

RI PT

transactivation of MIR34A is TAp73- as well as p53-dependent, suggesting implication in common p53/p73pathways. It further suggests possible co-evolution of the ancient MIR34A with the ancient p53/p73 hybrid gene, before the p53/p73 gene split in cartilage fishes [14].

In line with the reported trend of miRNAs of different age to target the same or overlapping sets of genes [34],

SC

MIR34A and MIR3158 co-regulate targets that are implicated in cancer-related pathways. The tendency of MIR3158 to display tumor-suppressive profile in terms of predicted regulated targets when evaluated along

M AN U

with MIR34A, but not when examined individually, indicates that the function of an evolutionarily-young miRNA might be strongly influenced by evolutionarily-old associated miRNAs. To this end, it might act as an “assistant” to the tumor-suppressive MIR34A, by co-operatively enhancing downregulation of their common cancer-related targets.

TE D

The anti-proliferative and anti-invasive effect of exogenous MIR3158 overexpression in either high or low MIR34A-expressing backgrounds indicates that manipulation of MIR3158 could be considered in the context of anticancer targeting. The functional network in which MIR3158 participates is able to restore anti-

EP

oncogenic responses if re-activated in cells with defective p53-networks. Thus, its ability to produce antioncogenic phenotypes, and its effect on key EMT-related molecules could be proven to be a powerful ally in

AC C

the war against cancer progression. Unlike MIR34A, which is a “notorious” anti-oncogenic miRNA, primarily downregulated in several cancer tissues [24], the tissue-specific MIR3158 would not constitute one of the “common threats” which a tumor cell is accustomed to primarily disarm in order to gradually acquire its aggressive characteristics. A cancer cell might not be pre-conditioned to deal with the exogenous expression of a miRNA which was not supposed to be expressed in this tissue. Therefore, MIR3158’s overexpression might have the potential to substitute for ablated MIR34A-mediated processes, thus taking cancer cells by surprise and triggering “outsider”-like, anti-proliferative responses. Anti-oncogenic properties have also been reported for several primate-specific miRNAs, indicating a potential for this miRNA group in therapeutic

14

ACCEPTED MANUSCRIPT targeting [30, 36]. The value of primate-specific miRNAs as differential diagnostic markers in cancer has not been evaluated yet, but it could stimulate future research. In conclusion, we uncovered a p53-independent/p73-dependent functional network that consists of MIR34A/MIR3158/lef1/β-catenin/vimentin and restores anti-invasive responses in p53-mutant cancer cells.

RI PT

We suggest that re-activation of this network through overexpression of the p73-dependent target MIR3158 could have a potential translational value in the context of therapeutic targeting of p53-null tumors.

SC

Conflicts of interest

M AN U

The authors declare no conflicts of interest.

Funding

SG was supported by the General Secretariat of Research and Technology [ESPA Czech-Greek bilateral

TE D

cooperation (2012-2014); ESPA Greek-Turkey bilateral cooperation grant (2013-2015)]. SL was supported by “IKY fellowships of excellence for postgraduate studies in Greece-Siemens program” [SR 22893]. BV was supported by the Czech Science Foundation (project no. P206/12/G151) and by the National sustainability

AC C

EP

program of Ministry of Education Youth, Education and Sports of the Czech Republic (project no. LO1413).

Acknowledgements

The authors would like to thank Dr. Helena Persson, Dr. Panagiotis Politis and Dr. Jerome Zoidakis for their helpful and constructive comments.

15

ACCEPTED MANUSCRIPT References

AC C

EP

TE D

M AN U

SC

RI PT

[1] B. Vogelstein, D. Lane, A.J. Levine, Surfing the p53 network, Nature, 408 (2000) 307-310. [2] S.M. Joruiz, J.C. Bourdon, p53 Isoforms: Key Regulators of the Cell Fate Decision, Cold Spring Harbor perspectives in medicine, (2016). [3] J. Chakraborty, S. Banerjee, P. Ray, D.M. Hossain, S. Bhattacharyya, A. Adhikary, S. Chattopadhyay, T. Das, G. Sa, Gain of cellular adaptation due to prolonged p53 impairment leads to functional switchover from p53 to p73 during DNA damage in acute myeloid leukemia cells, The Journal of biological chemistry, 285 (2010) 33104-33112. [4] K. John, V. Alla, C. Meier, B.M. Putzer, GRAMD4 mimics p53 and mediates the apoptotic function of p73 at mitochondria, Cell death and differentiation, 18 (2011) 874-886. [5] B. Ory, M.R. Ramsey, C. Wilson, D.D. Vadysirisack, N. Forster, J.W. Rocco, S.M. Rothenberg, L.W. Ellisen, A microRNA-dependent program controls p53-independent survival and chemosensitivity in human and murine squamous cell carcinoma, The Journal of clinical investigation, 121 (2011) 809-820. [6] S. Zhang, L. Zhou, B. Hong, A.P. van den Heuvel, V.V. Prabhu, N.A. Warfel, C.L. Kline, D.T. Dicker, L. Kopelovich, W.S. El-Deiry, Small-Molecule NSC59984 Restores p53 Pathway Signaling and Antitumor Effects against Colorectal Cancer via p73 Activation and Degradation of Mutant p53, Cancer research, 75 (2015) 3842-3852. [7] A. Puisieux, T. Brabletz, J. Caramel, Oncogenic roles of EMT-inducing transcription factors, Nature cell biology, 16 (2014) 488-494. [8] R. Kalluri, R.A. Weinberg, The basics of epithelial-mesenchymal transition, The Journal of clinical investigation, 119 (2009) 1420-1428. [9] J. Winter, S. Jung, S. Keller, R.I. Gregory, S. Diederichs, Many roads to maturity: microRNA biogenesis pathways and their regulation, Nature cell biology, 11 (2009) 228-234. [10] Z. Fang, R. Du, A. Edwards, E.K. Flemington, K. Zhang, The sequence structures of human microRNA molecules and their implications, PloS one, 8 (2013) e54215. [11] P.V. Nazarov, S.E. Reinsbach, A. Muller, N. Nicot, D. Philippidou, L. Vallar, S. Kreis, Interplay of microRNAs, transcription factors and target genes: linking dynamic expression changes to function, Nucleic acids research, 41 (2013) 2817-2831. [12] D. Sengupta, S. Bandyopadhyay, Topological patterns in microRNA-gene regulatory network: studies in colorectal and breast cancer, Molecular bioSystems, 9 (2013) 1360-1371. [13] H. Hermeking, MicroRNAs in the p53 network: micromanagement of tumour suppression, Nature reviews. Cancer, 12 (2012) 613-626. [14] S. Logotheti, A. Pavlopoulou, S. Galtsidis, B. Vojtesek, V. Zoumpourlis, Functions, divergence and clinical value of TAp73 isoforms in cancer, Cancer metastasis reviews, 32 (2013) 511-534. [15] M. Beitzinger, L. Hofmann, C. Oswald, R. Beinoraviciute-Kellner, M. Sauer, H. Griesmann, A.C. Bretz, C. Burek, A. Rosenwald, T. Stiewe, p73 poses a barrier to malignant transformation by limiting anchorageindependent growth, The EMBO journal, 27 (2008) 792-803. [16] Y. Zhang, W. Yan, Y.S. Jung, X. Chen, Mammary epithelial cell polarity is regulated differentially by p73 isoforms via epithelial-to-mesenchymal transition, The Journal of biological chemistry, 287 (2012) 1774617753. [17] A.K. Thakur, J. Nigri, S. Lac, J. Leca, C. Bressy, P. Berthezene, L. Bartholin, P. Chan, E. Calvo, J.L. Iovanna, S. Vasseur, F. Guillaumond, R. Tomasini, TAp73 loss favors Smad-independent TGF-beta signaling that drives EMT in pancreatic ductal adenocarcinoma, Cell death and differentiation, 23 (2016) 1358-1370. [18] E. Skourti, S. Logotheti, C.K. Kontos, A. Pavlopoulou, P.T. Dimoragka, I.P. Trougakos, V. Gorgoulis, A. Scorilas, I. Michalopoulos, V. Zoumpourlis, Progression of mouse skin carcinogenesis is associated with the orchestrated deregulation of mir-200 family members, mir-205 and their common targets, Molecular carcinogenesis, 55 (2016) 1229-1242. [19] S. Logotheti, D. Papaevangeliou, I. Michalopoulos, M. Sideridou, K. Tsimaratou, I. Christodoulou, K. Pyrillou, V. Gorgoulis, S. Vlahopoulos, V. Zoumpourlis, Progression of mouse skin carcinogenesis is associated

16

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

with increased ERalpha levels and is repressed by a dominant negative form of ERalpha, PloS one, 7 (2012) e41957. [20] S. Logotheti, I. Michalopoulos, M. Sideridou, A. Daskalos, S. Kossida, D.A. Spandidos, J.K. Field, B. Vojtesek, T. Liloglou, V. Gorgoulis, V. Zoumpourlis, Sp1 binds to the external promoter of the p73 gene and induces the expression of TAp73gamma in lung cancer, The FEBS journal, 277 (2010) 3014-3027. [21] A. Mukhopadhyay, B. Deplancke, A.J. Walhout, H.A. Tissenbaum, Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans, Nature protocols, 3 (2008) 698-709. [22] Y. Jin, Z. Chen, X. Liu, X. Zhou, Evaluating the microRNA targeting sites by luciferase reporter gene assay, Methods in molecular biology, 936 (2013) 117-127. [23] M. Agostini, P. Tucci, R. Killick, E. Candi, B.S. Sayan, P. Rivetti di Val Cervo, P. Nicotera, F. McKeon, R.A. Knight, T.W. Mak, G. Melino, Neuronal differentiation by TAp73 is mediated by microRNA-34a regulation of synaptic protein targets, Proceedings of the National Academy of Sciences of the United States of America, 108 (2011) 21093-21098. [24] M. Agostini, R.A. Knight, miR-34: from bench to bedside, Oncotarget, 5 (2014) 872-881. [25] Y. Xiao, C. Xu, J. Guan, Y. Ping, H. Fan, Y. Li, H. Zhao, X. Li, Discovering dysfunction of multiple microRNAs cooperation in disease by a conserved microRNA co-expression network, PloS one, 7 (2012) e32201. [26] J. Xu, C.X. Li, Y.S. Li, J.Y. Lv, Y. Ma, T.T. Shao, L.D. Xu, Y.Y. Wang, L. Du, Y.P. Zhang, W. Jiang, C.Q. Li, Y. Xiao, X. Li, MiRNA-miRNA synergistic network: construction via co-regulating functional modules and disease miRNA topological features, Nucleic acids research, 39 (2011) 825-836. [27] R. Du, W. Sun, L. Xia, A. Zhao, Y. Yu, L. Zhao, H. Wang, C. Huang, S. Sun, Hypoxia-induced downregulation of microRNA-34a promotes EMT by targeting the Notch signaling pathway in tubular epithelial cells, PloS one, 7 (2012) e30771. [28] C. Gilles, M. Polette, M. Mestdagt, B. Nawrocki-Raby, P. Ruggeri, P. Birembaut, J.M. Foidart, Transactivation of vimentin by beta-catenin in human breast cancer cells, Cancer research, 63 (2003) 26582664. [29] G.B. Jang, J.Y. Kim, S.D. Cho, K.S. Park, J.Y. Jung, H.Y. Lee, I.S. Hong, J.S. Nam, Blockade of Wnt/betacatenin signaling suppresses breast cancer metastasis by inhibiting CSC-like phenotype, Scientific reports, 5 (2015) 12465. [30] H. Persson, A. Kvist, N. Rego, J. Staaf, J. Vallon-Christersson, L. Luts, N. Loman, G. Jonsson, H. Naya, M. Hoglund, A. Borg, C. Rovira, Identification of new microRNAs in paired normal and tumor breast tissue suggests a dual role for the ERBB2/Her2 gene, Cancer research, 71 (2011) 78-86. [31] O.C. Kousidou, A.E. Roussidis, A.D. Theocharis, N.K. Karamanos, Expression of MMPs and TIMPs genes in human breast cancer epithelial cells depends on cell culture conditions and is associated with their invasive potential, Anticancer research, 24 (2004) 4025-4030. [32] L.A. Gordon, K.T. Mulligan, H. Maxwell-Jones, M. Adams, R.A. Walker, J.L. Jones, Breast cell invasive potential relates to the myoepithelial phenotype, International journal of cancer. Journal international du cancer, 106 (2003) 8-16. [33] S. Yang, Y. Li, J. Gao, T. Zhang, S. Li, A. Luo, H. Chen, F. Ding, X. Wang, Z. Liu, MicroRNA-34 suppresses breast cancer invasion and metastasis by directly targeting Fra-1, Oncogene, 32 (2013) 4294-4303. [34] Y. Zhu, G. Skogerbo, Q. Ning, Z. Wang, B. Li, S. Yang, H. Sun, Y. Li, Evolutionary relationships between miRNA genes and their activity, BMC genomics, 13 (2012) 718. [35] R. Niwa, F.J. Slack, The evolution of animal microRNA function, Current opinion in genetics & development, 17 (2007) 145-150. [36] C. Koufaris, Human and primate-specific microRNAs in cancer: Evolution, and significance in comparison with more distantly-related research models: The great potential of evolutionary young microRNA in cancer research, BioEssays : news and reviews in molecular, cellular and developmental biology, 38 (2016) 286-294.

17

ACCEPTED MANUSCRIPT

Figure Legends Figure 1. TAp73-induced miRNomes inhibit EMT-factors and cell migration in an isoform-specific manner. (A) p53 directly transactivates miRNA gene targets which, in turn, silence EMT-factors and reduce

RI PT

cell migration. (B) Wound healing assays in days 0, 2 and 6 of doxocycline-induction of ΤΑp73α, ΤΑp73β or ΤΑp73γ isoform in the Saos-TAp73 study system. (C) Western blots for representative EMT markers Zeb-1, vimentin and Snail at the corresponding time points. (D) The levels of ΤΑp73α, ΤΑp73β or ΤΑp73γ generated by doxocycline induction are comparable, as indicated by Western blot analysis using anti-HA antibody. (E)

SC

Heat-map of the miRNA array analysis of Saos2 doxocycline-inducible clones stably transfected with

M AN U

ΤΑp73α, ΤΑp73β or ΤΑp73γ isoform versus their uninduced controls, containing the 50 top-listed miRNAs. The heat map diagram shows the result of a two-way hierarchical clustering of microRNAs and samples. Each row represents a miRNA and each column represents a sample. The miRNA clustering tree is shown on the left. MiRNA arrays for each of the three isoforms were normalized to their respective uninduced controls. (F) Venn diagrams of the 25 miRNAs of the 50 top-listed miRNAs that are found upregulated upon TAp73

TE D

isoforms induction.

Figure 2. miR-3158-5p is a miR-34a-coexpressed, p53-independent and TAp73-dependent target, that

EP

downregulates vimentin and decreases cell migration. (A) qPCR validations of miR-34a-5p and miR-31585p expression upon TAp73 isoforms induction (+dox) in Saos-TAp73 and Saos-p53 clones versus the

AC C

corresponding uninduced (-dox) controls. (B) In vivo binding of ΤΑp73α, ΤΑp73β and ΤΑp73γ isoforms to the predicted TAp73 binding site of the MIR3158 promoter using ChIP assays with anti-HA tag antibodies. The reaction product was enhanced by qPCR. The fold enrichment represents the increase of the expression levels of the qPCR product in TAp73α, TAp73β and TAp73γ expressing clones relative to their corresponding uninduced controls. TAp73 binding to MIR3158 promoter region was compared versus the region downstream the MIR3158 gene (negative control). (C) A luciferase plasmid containing the TAp73 binding site containing region of miR-3158-5p promoter was constructed and transiently co-transfected with expression plasmids of TAp73α, ΤΑp73β and ΤΑp73γ in Saos2 cells. Cells were then subjected to luciferase assays. (D) The

18

ACCEPTED MANUSCRIPT differential effect of TAp73 isoforms on MIR3158 promoter. (E) A pGL3 luciferase plasmid, containing a 3’UTR target region of vimentin, was co-transfected into Saos2 cells with either a negative control miRNA or miR-3158-5p mimic miRNA. Lusiferace assay shows that mir-3158-5p downregulates the expression of vimentin at about 55%. (F) Wound healing assays on Saos2 cells in presence of miR-3158-5p mimic for 72h

RI PT

compared to the untreated or the negative control-treated Saos2 cells. (G) Vimentin protein levels in Saos2 cells treated with miR-3158-5p mimic compared to the untreated or the negative control-treated Saos2 cells.

Figure 3. A p73-controled network consisting of miR-3158-5p/miR-34a-5p and their predicted inter-

SC

associated mRNA co-targets. p53 transactivates MIR34A, while TAp73 isoforms transactivate both MIR34A and MIR3158 genes. The 29 of the 64 predicted co-regulated targets of MIR34A and MIR3158 form a protein

M AN U

interaction network. The nodes represent the genes/gene products and the connecting lines (edges) represent the interactions between the nodes. The thickness of the lines shows the degree of confidence for the interactions between the nodes (thicker lines represent highly confident interactions). The crosses indicate the mRNA targets that are functionally related and build-up a functional network module underlying cell

TE D

migration.

Figure 4. Anti-invasive effect of exogenous human MIR3158 expression in p53-mutant cancer cells. (A)

EP

Q-PCR for miR-3158-5p in a panel of breast cancer cell lines with low or high invasive potential. (B) The status of miR-3158-5p in association with miR-34a in the highly invasive and proliferative, p53-mutant MDA-

AC C

MB-468 and MDA-MB-231 cell lines. (C) qPCR of vimentin, β-catenin and lef1 levels in MDA-MB-231 cells expressing MIR3158 in comparison to their mock counterparts. (D) Same as (c) for MIR3158-expressing MDA-MB-468 clones. (E) Wound healing assays on MDA-MB-231 clones stably expressing MIR3158 as compared to their parental and mock-transfected counterparts. (F) Wound healing assays on MDA-MB-468 clones stably expressing MIR3158. (G) Invasion assay analysis (X10 and X40 magnitude) for the MDA-MB231 clones stably expressing MIR3158 compared to the mock and parental cells. (H) Invasion assay analysis (X10 and X40 magnitude) for the MDA-MB-468 clones stably expressing MIR3158. The asterisks (*) denote statistically significant (p<0.05) changes.

19

ACCEPTED MANUSCRIPT Figure 5: Anti-proliferative effect of exogenous human MIR3158 expression in p53-mutant cancer cells. (A) Proliferation assays on MDA-MB-231 clones stably expressing MIR3158 revealed a time-dependent reduction in the proliferation rate as compared to their parental and mock-transfected counterparts. (B) Same results are observed for the MDA-MB-468 clones stably expressing MIR3158. (C) Colony formation assays

RI PT

on MDA-MB-231 and MDA-MB-468 clones stably expressing MIR3158 compared to the corresponding mock controls. (D) Graph representation of the quantitative colony assay results of (C). (E) Subcutaneous injection of MDA-MB-231 and MDA-MB-468 clones stably expressing MIR3158 in BalB/c SCID mice resulted to a reduction of the tumor size compared to their mock-transfected counterparts. (F) Graph

SC

representation of the mean tumor volume with time in an approximately 11-week period.

M AN U

Supplementary Material

Figure S1: qPCR for MIR34A and MIR3158 expression in tumor samples excised from 22 breast cancer

AC C

EP

TE D

patients (P1-P22). Values are expressed relative to the pooled sample RQ value.

20

ACCEPTED MANUSCRIPT

A.

C.

M AN U

SC

B.

RI PT

Fig.1

EP

F.

AC C

D.

TE D

E.

ACCEPTED MANUSCRIPT

Fig. 2 B.

M AN U

SC

RI PT

A.

C.

TE D

E.

EP AC C

F.

D.

G.

ACCEPTED MANUSCRIPT

Fig. 3 TAp73

RI PT

p53

MIR34A

M AN U

SC

MIR3158

AC C

EP

TE D

+

+

+

ACCEPTED MANUSCRIPT

B.

Fig. 4 MDA-MB-231

MDA-MB-468

miR-3158

low

low

miR-34a

low

high

vimentin

high

low

invasion

high

high

proliferation

D.

high

M AN U

SC

C.

RI PT

A.

E.

G.

AC C

EP

TE D

F.

H.

high

ACCEPTED MANUSCRIPT

Fig. 5

C.

F.

AC C

EP

E.

TE D

M AN U

D.

RI PT

B.

SC

A.

ACCEPTED MANUSCRIPT

•

MIR3158 was identified as a p73-dependent target.

•

Functional characterization of the unstudied novel miRNA, MIR3158.

•

miR-3158-5p downregulates vimentin and suppresses cell migration

•

Elucidation of a p73/MIR3158-regulated network mediating cancer cell migration and

AC C

EP

TE D

M AN U

SC

RI PT

invasiveness