MiR-205-5p inhibition by locked nucleic acids impairs metastatic potential of breast cancer cells

Mir-205 plays an important role in epithelial biogenesis and in mammary gland development but its role in cancer still remains controversial depending on the specific cellular context and target genes. We have previously reported that miR-205-5p is upregulated in breast cancer stem cells targeting ERBB pathway and leading to targeted therapy resistance. Here we show that miR-205-5p regulates tumorigenic properties of breast cancer cells, as well as epithelial to mesenchymal transition. Silencing this miRNA in breast cancer results in reduced tumor growth and metastatic spreading in mouse models. Moreover, we show that miR-205-5p knock-down can be obtained with the use of specific locked nucleic acids oligonucleotides in vivo suggesting a future potential use of this approach in therapy.


Introduction
Despite advances in breast cancer treatment, metastases still remain the major cause of patients death due to therapeutic failure and disease recurrence. It is currently believed that a subpopulation of cells within the tumor displaying stem-like properties, such as self renewal and ability to differentiate, drives tumor progression and metastasis, and is in general more resistant to therapies, leading to a worst clinical outcome [1][2][3][4] . These cells have been indicated as: cancer stem cells (CSCs) or tumorinitiating cells 5 .
Breast cancer was the first solid tumor in which CSCs were identified and characterized by the expression of specific surface markers including CD44+, CD24-and EpCam+ 6 . Breast CSCs (BCSCs) are also characterized by epithelial-mesenchymal plasticity 7 and undergo epithelial to mesenchymal transition (EMT), as well as mesenchymal to epithelial transition (MET) during the different stages of metastatic spreading. EMT is a program involved in tissue morphogenesis induced during embryogenesis by which epithelial cells take on a mesenchymal phenotype, loosing their polarity and acquiring the ability of motility and invasion. Thanks to EMT and MET cancer cells can switch from epithelial to mesenchymal states and vice versa, increasing their ability to invade stromal and vessel tissues, leading to the formation of secondary tumors 8 . In cancer progression, EMT is reactivated and is promoted by the tumor microenvironment leading to the induction of several transcription factors like Zeb1 and Snail and to the activation of intracellular signaling pathways 9 enabling cells to move to new sites, allowing the formation of metastatic lesions.
Accumulating evidences in the last years support the involvement of microRNAs in EMT modulation by targeting genes related to EMT machinery 10,11 . MiRNAs are small regulatory non-coding RNAs, controlling gene transcription through base pairing with the 3′-untranslated regions (3′-UTR) of specific target mRNAs, leading to the downregulation of gene expression 12 . However, miRNAs activity may change depending on the cellular context and stimuli, indeed the same gene can be regulated by different miRNAs, thus explaining the dualistic and often controversial role of miRNAs.
MiR-205, is one of the most studied miRNAs in breast cancer for its involvement in cellular proliferation, migration and differentiation and it was defined "angel or devil" in cancer, acting either as an onco-suppressor or as an oncogene 13 . Indeed, miR-205 has been shown to inhibit EMT in several tumors, targeting Zeb1 and Zeb2 14 but has also been reported to promote proliferation and metastasis in other models 15,16 . We have previously reported that patient derived BCSCs express high levels of miR-205-5p compared with more differentiated tumor cells 17 . We found that miR-205-5p upregulation controls CSCs phenotype targeting Her2, p63 and Epidermal growth factor receptor (EGFR) contributing to targeted therapy resistance 17 . Here we further investigate the role of this miRNA in breast cancer progression and show that miR205-5p silencing impairs the metastatic potential of BCSCs in vitro and in vivo modulating EMT program. Moreover, we show that miR205-5p silencing can be obtained with the use of specific locked nucleic acids (LNA) oligonucleotides in vivo suggesting this approach as a future potential therapeutic tool.

miR-205-5p regulates the tumorigenic potential of BCSCs
Recently we reported that miR-205-5p is overexpressed in BCSCs compared with more differentiated cancer cells and by regulating the expression of different molecules including p63, participates to sustain a stem-like phenotype of breast cancer cells, desensitizing these cells to targeted therapy 17 .
To further delineate the contribution of miR-205-5p in the regulation of BCSCs properties, we investigated if this miRNA also influenced their tumorigenic potential. To this end, we stably knocked down miR-205-5p with short hairpin RNA (shRNA) lentiviral vectors in two patientderived BCSC lines and we examined the effect of miR-205 silencing on proliferation in vitro. As shown in Fig. 1a, miR-205 suppression reduced the proliferation of both BCSCs tested.
Then, in order to efficiently silence miR-205 in a way with potential in vivo applications, we took advantage of the LNA-phosphorothioate (PS) oligonucleotides technology. Recently, LNA have been used to inhibit endogenous small non-coding RNAs in vitro and in vivo [18][19][20] and therapeutic silencing of disease-associated miRNAs by LNA antisense oligonucleotide is under investigations in clinical trials for human cancers, autoimmune and cardiac disorders [21][22][23] .
Thus, we tested the effect of miRCURY LNA anti-miR-205-5p (LNA-i-miR-205-5p) oligonucleotides in vitro, treating BCSCs cells with two different molar concentrations (40 and 120 pmol). As shown in Fig. 1b (and Supplementary 1A), LNA-i-miR205-5p reduced miR-205-5p expression levels at both concentrations used and as early as 24 h after treatment reaching the maximum effect of inhibition at 120 pmol of LNA anti-miR-205 after 72 h of treatment.
To confirm the anti-proliferative effect of miR-205 knock-down, we treated BCSCs with 40 pmol of LNA-i-miR-205-5p or a scrambled control every 72 h. Figure 1c (and Supplementary 1B) shows that cell proliferation was significantly reduced after 7 days of treatment. Figure 1d (and Supplementary 1B) confirms that miR-205 levels are downregulated after 7 days of culture in treated samples.
The effect on proliferation was paralleled by altered expression of typical BCSCs surface markers such as CD44 and its splice variant CD44v6 24 , CD24 and alpha-6-Integrin (CD49f) evaluated both by quantitative reverse transcriptase-PCR (qRT-PCR) and fluorescence-activated cell sorting (FACS) silencing mir-205-5p by shRNA or LNA. Silencing miR-205-5p by shRNA results in significant increase in CD24 levels and decrease of CD49f, full-length CD44 levels and CD44v6 splice variant, the last known as a bona fide marker of BCSCs (Figs. 2a, b and Supplementary 2A-B). Similar results were obtained, treating BCSCs with LNA-i-mir-205-5p or a scramble control. FACS analysis in Fig. 3a and qRT-PCR in Fig. 3b show that LNA-i-miR-205-5p treatment reduced significantly the expression of CD44v6 and CD49f both at mRNA and protein level, whereas CD24 and CD44 expression levels remained unchanged. Moreover, treatment with LNA-i-miR-205-5 results in a 20% reduction of aldehyde dehydrogenase (ALDH1) activity confirming reduced stemness (Fig. 3c). Altogether these data, suggest that reducing mir-205-5p expression, switch CSCs into a less "aggressive" phenotype.
We then tested the effect of miR-205-5p silencing in vivo, by injecting these cells subcutaneously into NOD-SCID-GAMMA (NSG) immune-compromised mice. As shown in Fig. 4a, miR-205-5p silencing resulted in a modest but significant tumor growth rate reduction, as well as an increased survival evaluated by sacrificing the mice when tumors reached a cut off of 1 cm 3 . Figure 4b confirms downregulation of miR-205-5p in tumors while staining of sections for Ki67 shows a lower proliferation activity in tumors from silenced cells compared with controls ( Fig. 4c).
We then tested the in vivo effect of systemic delivery of miR-205-5p LNA inhibitor. We subcutaneously injected NSG mice with BCSCs and the day after we intraperitoneally treated mice with six doses (20 mg/kg) of LNA anti-miR-205 or with a scrambled control. Treatment was performed immediately after cells injection as we envisage the potential use of this LNA in adjuvant setting. Significant inhibition of tumor growth was observed after the last LNA injection (on the 18th experimental day) and tumor volumes differed significantly between control and treated groups up to 25 days (Fig. 4d), when mice were

miR-205-5p controls BCSCs metastatic potential promoting EMT
We then investigated if miR-205 also regulated the invasive potential of BCSCs. To this end, we initially performed a transwell invasion assay showing that miR-205 silencing results in a decrease (almost 50%) of the invasive ability of BCSCs (Fig. 5a). As shown in Fig. 5b this is also accompanied by reduced expression of EMT related genes, such as Twist and Vimentin, both at protein and mRNA level, whereas the expression level of Slug and Snail remain unchanged (data not shown) suggesting that silencing this miR mainly affects the EMT pathway driven by Twist family and not by the Snail/Slug one.
To further investigate a role of miR-205 in invasion, we used an experimental metastatic model by injecting 2 × 10 5 BCSCs infected with shmiR-205 construct or with an empty vector in the tail vein of NSG immunecompromised mice. Injection of cancer cells in this model has been shown to result in the formation of metastatic lesions in the lung of animals 25 .
Indeed, after 32 days mice were sacrificed and lung metastasis were counted. Consistently with the reduction of invasive potential shown in vitro, we observed a significant reduction of the number and the size (Fig. 5c) of lung metastasis. In addition, immunohistochemistry analysis of lung lesions (Fig. 5d) shows that metastatic lesions of the miR-205 silenced group have a significant reduced proliferation rate (less Ki67-positive cells).
Treatment with LNA anti-miR-205-5p also reduced the invasive potential of BCSCs in vitro in both cell lines tested ( Fig. 6a and Supplementary 1C). Moreover, we checked the expression level of EMT regulator genes in vitro ( Fig. 6b and Supplementary 1D) and ex vivo from tumor xenograft (Figure Supplementary 1E). LNA-i-miR-205-5p treatment leads to a reduction trend of Twist and Vimentin mRNA levels and a significant reduction in their protein levels in line with the results obtained by shmiR-205-5p. Conversely Zeb1, a direct target of miR-205-5p, as previously reported, is upregulated upon miR-205-5p silencing (Supplementary 1F), suggesting as recently described that EMT transcription factors have nonredundant functions and are not all activated at the same time [26][27][28] .
Finally, we evaluated the effect of LNA-i-miR-205-5p on the metastatic potential of BCSCs in vivo using the same model described above and treating animals with four doses of LNA-i-miR-205 or a scramble control. At the end of the experiment (30 days after cells injection), we collected lungs and counted and analyzed the metastasis. As shown in Fig. 6c, treatment with LNA anti-miR-205-5p significantly reduced the number and the size of lung metastasis. In addition, metastases of LNA-i-miR-205-5p treated group showed lower percentage of Ki67-positive cells (Fig. 6d). All these data confirmed that miR-205 inhibition impairs cell proliferation, motility and invasion ability.

Discussion
Currently, targeting oncogenic miRNAs represents a new strategy to overcome human cancer and is widely recognized that a subpopulation of cancer cells within the tumor, with stem-like features, plays a pivotal role in cancer progression and recurrence. We have previously shown that miR-205-5p is upregulated in BCSCs compared with more differentiated tumor cells, contributing to sustain a stem-like phenotype, also driving targeted therapy resistance 17 . These findings led us to hypothesize that miR-205-5p could be involved in tumorigenic and metastatic potential of BCSCs.
In this study, we silenced mir-205-5p in vitro and in vivo with either shRNA or LNA PS antisense oligonucleotides, chemically modified to ensure stability, specificity and high binding affinity for the target miRNA. Our in vitro data show that miR-205-5p knock-down results in reduced proliferative ability of these cells paralleled by a reduced expression of CSC markers such as CD44v6, CD44 and alpha-6-Integrin. These results provide additional insights into the role of miR-205 in promoting the expansion of stem cells, as previously (see figure on previous page) Fig. 3 miR-205-5p LNA inhibition reduces the stem-like phenotype of BCSCs. a FACS analysis of CD24, CD44 and CD44v6 expression of BCSCs#3 after 24 h of treatment with 120 pmol of miRCURY LNA miR-205-5p inhibitor (LNA-i-miR-205-5p: blue line) or the scramble control (scr: red line) (upper panel). Data are presented as mean ± SD with T-test analysis of the percentage fold over control of receptors expression in three different experiments (lower panel). b Real-time PCR of CD24, CD44, CD44v6 and CD49f receptors of BCSCs#3 treated for 24 h as described above. Data are presented as mean ± SD with T-test analysis. c Representative FACS analysis of ALDH1 activity of BCSCs after 24 h of treatment with 120 pmol of LNA miR-205-5p inhibitor or the scramble control using Aldefluor Assay. Histograms (lower panel) show the mean ± SD with T-test analysis of the percentage fold over control of ALDH activity in two different experiments described for the progenitors and stem cells populations in skin during early development 29 . Indeed, knocking-out this miRNA in mice causes neonatal lethality with compromised epidermal and hair follicle growth, and impaired proliferation of basal cells in stratified epithelium, by modulating the phosphatidylinositol 3 kinase pathway 29 . Thus, our findings support the hypothesis that in CSCs, genes involved in normal early development like miR-205 are switched on and upregulated.
Along with others, we have previously shown that miR-205 regulates EMT targeting Zeb1 14,17 , here we show that miR-205-5p silencing reduces the invasive ability of BCSCs, modulating the expression of EMT-associated genes like Vimentin and Twist. In line with in vitro results, we show that reduced expression of miR-205 in vivo significantly impairs tumor growth and metastasis formation. Indeed the acute treatment of mice with LNA anti-miR-based therapy, significantly reduces the growth of the primary tumor, as well as the number and the size of lung metastasis derived from BCSCs injection. Furthermore, the systemic delivery of LNA anti-miR205 resulted in an effective mir-205 silencing with no major adverse effect, suggesting the potential safety of this approach.
Several studies 18,30 have reported the efficiency of LNA anti-miR-based therapies and miRNA-based therapeutics are currently entering clinical trials; 31 for instance "Miravirsen", LNA anti-miR122, is the first miRNA-targeted drug to enter phase II clinical trial for its use against HCV infection 21,32 . Thus, the discovery and the development of new miRNAs therapeutics, used alone or in combination with other drugs, against cancer and other disorders, avoiding off-target effects, still remains the great challenge of medicine. Our work supports the idea that LNA anti-miR205 can be possibly used as an adjuvant therapy, to overcome breast cancer progression and metastatic spreading. However, caution should be used in suggesting this approach as miR-205-5p has been shown to act also as a tumor suppressor in other models and further work is required to clearly understand this pathway in order to select patients that might benefit from this approach.
For the authentication of BCSCs was used the short tandem repeat (STR) system (GlobalFiler STR Kit; Applied Biosystems) and the ABIPRISM 3130 genetic analyzer (Applied Biosystems) as previously reported 5 .
Custom miRCURY LNA microRNA inhibitor in vivo Ready were designed and purchased from Exiqon (Denmark) with the following sequences: i-hsa-miR-205-5p 5′-CCGGTGGAATGAAGG-3′ and Scrambled control 5′-ACGTCTATACGCCCA-3′. The oligonucleotides were High Performance Liquid Chromatography (HPLC) and Na salt exchanged purity level and had full PS backbones. They were custom designed to work in human cells in vitro and in mice xenograft of human breast cancer.

LNA transfection
miRCURY LNA miR-205-5p inhibitor and the scramble control were used at concentration of 40 pmol (or 120 pmol where indicated) and the mixture was added directly to cells seeded into six-well ultra-low attachment plated. Successful miR205-5p inhibition was evaluated by qRT-PCR.

Virus generation and infections
Lentiviruses were produced by transient co-transfection of a three-plasmid expression system in the packaging 293T cells, using the calcium phosphate transfection kit (Invitrogen, Life Technologies) as previously described 17 . Then, BCSCs were infected with viral supernatant as previously described 17 and infection efficiency was assessed by
Real-time PCR was performed by using SsoAdvanced Universal Probe Supermix (Bio-Rad) and Universal Probe Library, Probe #21 (Roche) using the following primers: miR-205-5p Fw 5′-GCGGCGGTGTAGTGTTTCCTA-3′ and universal Reverse primer: 5′-GTGCAGGGTCCG AGGT-3′. miR-205-5p expression was calculated relative to U44 snoRNA with the following primer: 5′-GCGGCGGCCTGGATGATGATAG-3′. All qRT-PCR were performed with an amplification protocol as follows: one cycle of 95°C for 30 s and 40 cycles of 95°C for 15 s and 60°C for 30 s on CFX96 Touch detection system (Bio-Rad) and relative quantification of miRNA expression was calculated according to the comparative method of 2-ΔΔCT .
Detection was performed with SuperSignal West Dura extended duration substrate kit (Thermo Scientific, USA).

FACS analysis
For flow cytometric analysis of CD44, CD24 and CD44v6 surface markers, 2 × 10 5 cells per sample, for all two BCSCs lines tested (BCSC#1 and BCSC#3), were washed in PBS, resuspended in 100 μl of specific antibody diluted in PBS, and incubated for 30  ALDH activity was measured following Aldefluor kit manufacturer's instructions (Stemcell Technologies) upon 24 h of treatment with 120 pmol LNA miR-205 inhibitor or a scramble control.
A FACSCantoII flow cytometer, running with FACS-DiVa software (BD Biosciences), was used for sample acquisition and analysis.
All antibodies were titrated under assay conditions, in order to obtain optimal dilutions. Instrument performances and data reproducibility were sustained and checked by using the Cytometer Setup & Tracking Module (BD Biosciences). In order to assess nonspecific fluorescence, fluorescence minus one controls were used. Compensation was calculated using CompBeads (BD Biosciences) and single stained samples.
After 24 h, non-invaded cells were removed and crystal violet stain solution was added for 10 min. Percentage of invasion was calculated counting and averaging the number of stained cells in five random fields within each Transwell insert, dividing this number by the area of the microscope viewing field and then multiplying this number by the entire area of the Transwell insert. Then, the total number of cells per insert was normalized by the number of cells seeded.

Mouse xenograft
NSG mice were purchased from Jackson Laboratory and bred in the animal facility of CeSI-MeT, G. D'Annunzio University of Chieti. Animal care and experimental procedures were approved by the Ethics Committee for Animal Experimentation of the institute according to the Italian law.
Seven weeks old female NSG mice were randomly divided into two groups (six mice per group) and were injected unilaterally with 1 × 10 5 BCSC#3 control and BCSC#3 sh-mir-205-5p into the fourth mammary fat pad. Cells were suspended in 200 μl of PBS 1:6 Matrigel (BD Biosciences).
Tumor growth was monitored twice a week using calipers and animals were sacrificed when tumors reached 1 cm 3 of volume. Tumor volume was calculated as 0.5 × d1 2 x d2, where d1 and d2 are the smaller and larger diameters, respectively. Tumor xenografts and organs were collected and fixed in 10% neutral-buffered formalin, paraffin embedded, sectioned and stained with hematoxylin and eosin (H&E; BioOptica).

In vivo experimental metastasis assay
For experimental lung metastasis assay, BCSC#3 control and BCSC#3 sh-mir-205-5p cells were resuspended in PBS and then 2 × 10 5 cells (in 0.2 mL) were injected via the lateral tail vein of 7 weeks old female NSG mice (six mice per group). Mice were killed 35 days after injection and lungs were harvested and fixed in 10% neutralbuffered formalin.
To optimize the detection of microscopic metastases and ensure systematic uniform and random sampling, lungs were cut transversally, to the trachea, into 2.0 mm thick parallel slabs with a random position of the first cut in first 2 mm of the lung, resulting in 5-8 slabs for lung. The slabs were then embedded cut surface down and sections were stained with hematoxylin and eosin. Slides were independently evaluated by two pathologists.

LNA in vivo treatment
Seven weeks old female NSG mice were randomly divided into two groups (five mice per group) and were injected unilaterally with 1 × 10 5 BCSC#3 into the fourth mammary fat pad. The day after cells injection, we treated mice with 20 mg/kg LNA-i-miR-205-5p or with LNA scrambled control (Exiqon) intraperitoneally, twice a week, for 3 weeks. The experiment was conducted for 25 days when mice were sacrificed. Tumors and organs were collected and one part of each organ was preserved for RNA isolation and the other part was fixed in 10% neutral-buffered formalin, paraffin embedded, sectioned and stained with H&E for histology analysis.
For experimental metastasis assay, 7 weeks old female NSG mice were divided into two groups (four mice per group) and were injected with 2 × 10 5 BCSC#3 via the lateral tail vein. The day after injection, mice were intraperitoneally treated with 20 mg/kg LNA-i-miR-205-5p or with LNA scrambled control (Exiqon) twice a week for 2 weeks. Mice were sacrificed after 30 days and lungs were collected and sectioned as described above (in vivo experimental metastasis assay paragraph).

Immunohistochemistry
For immunohistochemistry, slides were deparaffinized, serially rehydrated and, after the appropriate antigen retrieval procedure, stained with monoclonal mouse antihuman Ki67 (M7240, Dako), followed by the appropriate secondary antibody. Immunoreactive antigens were detected using streptavidin peroxidase (Thermo Scientific) and the DAB Chromogen System (Dako). After chromogen incubation, slides were counterstained in hematoxylin (BioOptica) and images were acquired by Leica DMRD optical microscope (Leica). The percentage of KI67-positive cells was evaluated on digital images of 4-6 tumors or lungs per group (4-6 × 200 microscopic fields per sample); clear brown nuclei were regarded as positive cells and the percentage of labeling index (number of positive cells/total cells × 100) was calculated for each field, by two pathologists, independently.

Statistical analysis
For statistical analysis, GraphPad Prism 6 software was used, using T-test or ANOVA test with p < 0.05.
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