MKAD-21 Suppresses the Oncogenic Activity of the Mir-21/PPP2R2A/ERK Molecular Network in Bladder Cancer

Author Manuscript Published OnlineFirst on April 27, 2018; DOI: 10.1158/1535-7163.MCT-17-1049 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

MKAD-21 suppresses the oncogenic activity of the miR-21/PPP2R2A/ERK

molecular network in bladder cancer

Marina Koutsioumpa1, Hsiao-Wang Chen2, Neil O’Brien2, Filippos Koinis3, Swapna Mahurkar-

Joshi1, Christina Vorvis1, Artin Soroosh1, Tong Luo2, Shawnt Issakhanian2, Allan J Pantuck4,

Vassilis Georgoulias3, Dimitrios Iliopoulos1, Dennis J Slamon2 and Alexandra Drakaki2,4

1Center for Systems Biomedicine, Vatche & Tamar Manoukian Division of Digestive Diseases,

David Geffen School of Medicine, UCLA; 2Division of Hematology-Oncology, Department of

Medicine, Johnsson Comprehensive Cancer Center, David Geffen School of Medicine, UCLA;

3Laboratory of Tumor Cell Biology and Department of Medical Oncology, School of Medicine,

University of Crete, Heraklion, Greece; 4Institute of Urologic Oncology, Department of Urology,

David Geffen School of Medicine, UCLA.

Running title: PPP2R2A mediates miR-21-induced tumorigenic potential

Key words: MicroRNA-21, Protein Phosphatase 2 Regulatory Subunit Balpha,

Extracellular signal–regulated kinases, Bladder cancer

Grant support

This work was supported by funding provided to Dr. Drakaki by the Division of

Hematology/Oncology at David Geffen School of Medicine.

Disclosure of Potential Conflicts of Interest

Downloaded from mct.aacrjournals.org on September 26, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 27, 2018; DOI: 10.1158/1535-7163.MCT-17-1049 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

No potential conflicts of interest were disclosed.

Corresponding Author: Alexandra Drakaki, MD PhD, Assistant Professor of Medicine and

Urology, Director of GU Medical Oncology Program, co-Director of Research at GU Trials

Program, Division of Hematology/Oncology and Institute of Urologic Oncology, David Geffen

School of Medicine, UCLA, Wasserman Bldg, 300 Stein Plaza, Los Angeles, CA 90095 USA;

Email: [email protected]

Abstract

Bladder cancer (BC) represents a disease associated with significant morbidity and mortality.

MicroRNA-21 (MiR-21) has been found to have oncogenic activity in multiple cancers, including

BC, while inhibition of its expression suppresses tumor growth. Here, we examine the molecular network regulated by miR-21 in BC and evaluate the effects of intravenous (I.V.) and intraperitoneal (I.P.) administration of a novel miR-21 chemical inhibitor in vivo. LNA miR-21 reduced the oncogenic potential of BC cells, while the MKAD-21 chemically-modified antisense oligo against miR-21, dose-dependently blocked xenograft growth. I.V. administration of LNA miR-21 was more effective in suppressing tumor growth than was I.P. administration. Integration of computational and transcriptomic analyses in a panel of 28 BC lines revealed a 15-gene signature that correlates with miR-21 levels. Protein Phosphatase 2 Regulatory Subunit Balpha

(PPP2R2A) was one of these 15 genes and was experimentally validated as a novel miR-21 direct target gene. Gene network and molecular analyses showed that PPP2R2A is a potent negative regulator of the ERK pathway activation and BC cell proliferation. Importantly, we show that PPP2R2A acts as a mediator of miR-21-induced oncogenic effects in BC. Integrative analysis of human BC tumors and a large panel of human BC cell lines revealed a novel 15- gene signature that correlates with miR-21 levels. Importantly, we provide evidence that

PPP2R2A represents a new miR-21 direct target and regulator of the ERK pathway and BC cell growth. Furthermore, I.V. administration of the MKAD-21 inhibitor effectively suppressed tumor growth through regulation of the PPP2R2A-ERK network in mice.

Introduction

Bladder cancer (BC) is one of the most common genitourinary malignancies, accounting for 3.1 and 1.8% of the overall cancer mortality in males and females, respectively [1]. Urothelial carcinoma of the bladder, the most frequent histopathologic type of BC, has a variety of genetic and phenotypic characteristics. Many genetic factors, such as chromosomal abnormalities, genetic polymorphisms, mutations and epigenetic alterations contribute to tumorigenesis and the progression of this type of cancer [2]. More than half of all bladder malignancies of all grades and stages contain chromosome 9 alterations suggesting that chromosome 9 genes may be involved in early tumor development.

MiRNAs are single-stranded non-coding RNA molecules, 18-25 bases in length, that act as negative regulators of gene expression, through binding in the 3’ untranslated region (3'UTR) or coding sequence of genes [3]. Growing evidence supports, that dysregulation of miRNAs is associated with cancer initiation and progression in a variety of malignancies, suggesting that miRNAs may be used as molecular biomarkers for the diagnosis of cancer, prediction of prognosis or as therapeutic modalities. Pioneering data reported by several research groups have linked specific miRNA patterns associated with activation of different gene expression pathways in BC. For example, miRNA molecules aberrantly expressed in BCs have been proposed to target components of the RAS kinase signaling pathway or the p53 pathway, such as FGFR3, p53-binding protein homolog (mouse), Mdm2-p53-binding-protein homolog (mouse) and ataxia telangiectasia mutated gene products [4]. Thus, elucidation of the targets and molecular mechanisms triggered by aberrant miRNA expression is necessary for the potential clinical development of novel cancer therapeutics.

It has been reported that miR-21 is overexpressed in multiple human solid tumors, including BC

[5, 6]. A systematic review and meta-analysis showed that elevated miR-21 expression is

associated with poor overall survival and proposed that miR-21 may serve as follow-up marker for early detection of BC progression or recurrence [7]. Furthermore, miR-21:miR-205 ratio has been described as a tool for identifying the invasive capacity of bladder tumors with high sensitivity and specificity [6]. Several studies aiming to determine the molecular mechanism by which miR-21 functions in BC showed that the oncogenic role of miR-21 might be attributed to suppression of PTEN [8, 9]. MiR-21 has been shown to modulate cell proliferation and sensitivity to doxorubicin in transitional cell carcinomas of the urinary bladder. Particularly, BCL-

2 up-regulation upon miR-21 overexpression prevented T24 cells from apoptosis induced by doxorubicin [8]. In addition mechanistic studies, demonstrate that downregulation of miR-21 results in cell cycle arrest at the G1 phase and p53 phosphorylation at Ser46, accompanied by significant attenuation of proliferation and migration of BC cells [9].

However, there is limited information regarding the molecular network that is regulated by miR-

21 and its functional and clinical significance in bladder oncogenesis. In this study we aimed to further define and characterize the miR-21 expression patterns associated with activation of certain gene expression pathways in BC.

Materials and Methods

Human Tissue Samples

RNA was extracted from 10 ‘normal’ (adjacent non-tumoral) and 74 BC tissues using RNAeasy

FPPE Kit (73504, Qiagen). All samples were classified according to the TNM Classification of

Malignant Tumors (UICC, 2014) staging and evaluated for histological type. The formalin-fixed paraffin-embedded (FFPE) human bladder tissues were obtained from the School of Medicine at the University of Crete in Greece and the UCLA Department of Pathology & Laboratory

Medicine. Patients provided written informed consent and this study was performed in accordance with the Declaration of Helsinki and was approved by the IRB committees of School

of Medicine at University of Crete and School of Medicine at University of California, Los

Angeles.

MKAD-21 structure

LNA miR-21 is an antisense oligonucleotide inhibitor against miR-21 seed sequence. LNA miR-

21 sequence is: 5’-TmCAGTCTGATAAGCT-3’, where red color corresponds to the nucleotides with locked nucleic acid modification, mC corresponds to 5’-methy-cytosine and the underlined sequence has phosphorothioate linkages between the nucleotides. All these chemical modifications in the LNA miR-21 inhibitor increase its resistance to nucleases, increasing its stability for in vivo studies.

Cell Cultures and Treatments

BC cell line 5637 was purchased from ATCC (2013) and RT-112 was purchased from Sigma-

Aldrich (2013) and were grown in RPMI 1640 (Gibco). BC cell lines KU19-19 and HBCLS2 were purchased from DSMZ (2015 and 2013, respectively) and were grown in RPMI-1640 (ATCC).

HBCLS1 cells were purchased from Cell Line Service (CLS) (2013) and were grown in RPMI-

1640 (ATCC). BC cell lines UMUC-1, UMUC-4, UMUC-5, UMUC-7, UMUC-9, UMUC-10,

UMUC-11, UMUC-13, UMUC-14, UMUC-15 and UMUC-17 were purchased from MD Anderson

(2013) and were grown in EMEM (ATCC). BC cell lines HT-1197, TCCSUP, HT-1376, UMUC-3,

SCABER and J82 were purchased from ATCC (2013) and were grown in EMEM (ATCC).

SW1710 cells were purchased from DSMZ (2015) and were grown in DMEM (ATCC). BC cell lines RT-4 and T24 were purchased from ATCC and were grown in MCCOYS (ATCC). BC cell lines BC-3C and CLS-439 were purchased from DSMZ (2015 and 2013, respectively) and were grown in MCCOYS (ATCC). SW780 cells were purchased from ATCC (2013) and were grown in

L-15 (ATCC). All the media used were supplemented with 10% Fetal Bovine Serum (FBS)

(Corning), 1% Antibiotic-Antimycotic (Gibco). All cell lines were used in the described experiments before reaching the 7th passage after thawing the cells out and were negative for mycoplasma prior performing these studies. Mycoplasma free verification was checked by PCR using a mixed primers based on 16S rRNA of most common species of mycoplasma [10].

Treatment of RT-112 and 5637 with 50nM or 100nM of LNA miR-21 or LNA scrambled negative control (LNA miR-scr) (199006-001, Exiqon) for 24h was followed by RNA extraction or functional assays. BC cells were transfected with siRNAs for Protein Phosphatase 2 Regulatory

Subunit Balpha (PPP2R2A), #1 (s5608) and #2 (s5609), control (4390846) using Lipofectamine

RNAiMax transfection reagent (13778150, Life Technologies) and incubated for 48h before

RNA or protein extraction. Treatment of cells with 100nM miR-21 mimic or mimic miR-scramble were performed using Lipofectamine RNAiMax transfection reagent and incubated for 48h before RNA or protein extraction. For PPP2R2A transient overexpression experiments, RT-112 and 5637 cells were treated with miR-21 mimic or mimic miR-scramble and transfected with pPM-C-His vector (PV032532) or Blank-Control Protein Vector (PV001, ABM) using Fugene 6

(E2691, Promega). After 24h, cells were subjected to functional assays or serum starved for

16h, followed by 10 min stimulation with FBS and analyzed by IB.

Animal studies

Xenograft models of RT-112 and 5637 BC cell lines were established in six-week-old CD-1 athymic nude mice (Charles River Laboratories). The following conditions were optimized for subcutaneous injection both cell lines: 1.0×107 cells with 50% matrigel/culture media (BD

Biosciences). When tumors reached an average size of 150-300mm3, mice were randomized into treatment groups.

For in vivo studies, we used MKAD-21 or LNA scramble negative control sequence (LNA miR- scr). Both were resuspended in RNAase-free water and diluted in sterile saline for intravenous

(I.V.) or intraperitoneal (I.P.) injection at doses of 5-15mg/kg. Tumor xenografts were measured with calipers 3 times/week, and tumor volume in mm3 was determined by multiplying height*width*length. Mouse weights were recorded daily. Upon termination of the efficacy studies xenograft tumor tissue was excised and snap-frozen in liquid nitrogen. Data were analyzed using StudyLog software from StudyDirector (San Francisco, CA). All statistics were calculated using Microsoft Excel. All animal work was carried out under a protocol approved by the UCLA Institutional Animal Care and Use Committee and the UCLA Animal Research

Committee.

Real-Time quantitative Polymerase Chain Reaction Analysis (RT-qPCR)

RNA purified from cells and tissues with TRIZOL (15596026, Life Technologies) was reverse- transcribed to form cDNA using the Universal cDNA synthesis kit (203300, Exiqon). Real-time

PCR was carried out using the SYBR Green master mix (203450, Exiqon) and primers for miR-

21 (204230, Exiqon) in a CFX384 Real Time PCR detection system (BioRad). MiR-21 expression levels were normalized to the levels of 5S rRNA (203906, Exiqon) and U6 snRNA

(203907, Exiqon).

Real-time PCR was employed to determine the expression levels of PPP2R2A. Reverse transcription was carried out using the iScript™ Reverse Transcription Supermix (1708841, Bio-

Rad). Real-time PCR was carried out using IQ SYBR Green supermix (1708882, BioRad). The primer sequences used for real-time PCR were acquired from previous studies [11] or designed using the NCBI Nucleotide Database (http://www.ncbi.nlm.nih.gov/nuccore), Primer3 v.0.4.0

(http://bioinfo.ut.ee/primer3-0.4.0) and UCSC In-Silico PCR (http://genome.ucsc.edu/cgi- bin/hgPcr) and are included in Supplementary Table S3. Gene expression levels were normalized to the levels of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin.

Normalized expression levels were quantified to the respective control. Bars represent means ±

Standard Error (SE); experiments were performed in quadruplicates for each condition.

Dynamic Monitoring of Cell Proliferation

Real-time cell proliferation analysis based on the application of electrical cell substrate impedancechanges(https://lifescience.roche.com/wcsstore/RASCatalogAssetStore/Articles/BIO

CHEMICA_4_08_p14-16.pdf) was performed using the xCELLigence RTCA instrument (ACEA

Biosciences). The presence of cells affects the local ionic environment at the electrode solution interface. Cell status is represented by a dimensionless parameter termed Cell Index which is derived as the relative change in measured electrical impedance, after subtraction of the background measurements from media alone. Local ionic environment varies according to cell size, cell morphology and strength of adhesion of the cells to the surface of the electrode, resulting in changes of the electrode impedance. 5*103 RT-112 or 5637 cells were seeded in quadruplicates of an E-Plate 96 with interdigitated microelectrode arrays integrated in the bottom of each well. Subsequently, the E-Plate 96 was mounted on the SP Station of the xCELLigence RTCA system which is placed in a standard temperature-controlled CO2 incubator under humidity saturation. The RTCA Software preinstalled on the RTCA control unit allows automatic selection of wells for measurement and real-time data acquisition within preprogrammed 15 min time intervals. Bars represent means ± Standard Deviation (SD); experiments were performed in quadruplicates for each condition.

Anchorage-Independent Cell Growth Assay

Triplicate samples of 35*104 RT-112 cells from each treatment were assayed in 48-well plates for colony formation using the CytoSelect Cell Transformation kit (CBA-135, Cell Biolabs, Inc).

The number of colonies was quantified after 7 days by counting the entire area of each well

divided in 5 fields, using a grid and an Evos microscope at a 20X magnification. Data are expressed as the mean number of colonies per field ± SE.

Invasion assay

Invasion in matrigel has been conducted by using standardized conditions with BD BioCoat

Matrigel invasion chambers (354480, BD Biosciences) according to manufacturer’s protocol.

Assays for RT-112 and 5637 cells were conducted using 10% FBS-containing media as chemoattractant for 24h and 48h, respectively. Non-invading cells on the top side of the membrane were removed while invading cells were fixed and stained with 0.1% crystal violet,

22h post seeding. The cells that migrated through the filter were quantified by counting the entire area of each filter divided in four fields, using a grid and an Evos microscope at a 20X magnification. Data are expressed as the mean number of invading cells per field ± SE.

Luciferase Assay

5637 cancer cells were transfected with the PPP2R2A_pLightSwitch_ 3_UTR Reporter vector carrying human PPP2R2A 3’UTR (S812284, SwitchGear Genomics) and the pLenti CMV Puro

LUC (w168-1) (17477, Addgene) containing luciferase reporter gene. At 24 h, the cells were transfected with 100 nM mimic miR-scr or miR-21 mimic and 48h later luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega). Data were expressed

± SE of the mean of three independent experiments.

Immunoblot Analysis

Total cell extracts and protein lysates were separated by Sodium Dodecyl Sulfate

Polyacrylamide Gel Electrophoresis and transferred to PVDF membranes following standard procedures. Protein extraction from xenograft tissues was performed by grinding frozen

specimens in liquid nitrogen into a powder with a mortar and a pestle, followed by the addition of

RIPA buffer (R0278, Sigma). The samples were incubated with agitation for 1h at 40C and clarified by centrifugation at 13,000 RPM for 20 minutes. The following antibodies were used for immunoblot analysis: PPP2R2A (5689), CREB (9104), phospho-p44/p42 mitogen-activated protein kinase (MAPK) (Thr202/Tyr204) (4370), total-p44/p42 MAPK (4695) (Cell Signaling

Technology).

Statistical Analysis

Quantitative data are expressed as means ± SD or SE of the mean, as indicated, or as boxes and whiskers representing the 25th and 75th percentile (the lower and upper quartiles, respectively), using Origin 9.1 Software. Statistical analyses were performed using one-way

ANOVA and Student’s t-test (or both) or Pearson correlation. For animal studies, statistical differences between treatment arms at specific time points were performed using a two-tailed paired Student t-test. P values of <0.05 were considered statistically significant.

Accession Numbers

The datasets generated during the current study are available in the GEO repository

(http://www.ncbi.nlm.nih.gov/geo/) under the following accession numbers: GSE97782 and

GSE97943.

Results

1) LNA miR-21 blocks the oncogenic properties of bladder cancer cells

To investigate the therapeutic potential of targeting miR-21 in BC in vitro, we efficiently suppressed its expression by treatment with LNA miR-21. LNA miR-21 compound was highly efficient in blocking miR-21 expression levels as determined by RT-qPCR analysis, in RT-112

and 5637 BC cell lines (Fig. 1A). The impact of miR-21 inhibition of expression on BC anchorage-independent growth was evaluated (Fig. 1B). Specifically, LNA miR-21 significantly reduced the number of colonies formed by RT-112 cells. Consistent with these findings, LNA miR-21 significantly decreased the invasive capacity of RT-112 and 5637 BC cells (Fig. 1C and

D). 50nΜ of LNA miR-21 was determined as the dose to maximally block these miR-21- mediated phenotypic biologic activities in BC cells, under the current experimental conditions.

2) Therapeutic potential of LNA miR-21 to block BC tumor growth in vivo

To substantiate our findings in an in vivo experimental setting, we designed an experimental approach to study the effects of miR-21 blockade in mice bearing human BC xenografts. Nude mice were inoculated with either 5637 or RT-112 cells and developed palpable tumors in 5 days post cell injection. Mice were then treated with sterile saline or LNA miR-scr or increasing concentrations of MKAD-21 which is a chemically-modified (phosphorothioate backbone and locked-nuclei-acid) antisense oligo against miR-21, by I.V. tail vein or I.P. injections every 5 days. In both 5637 and RT-112 xenografts, administration of MKAD-21 (15 mg/kg) resulted in decreased tumor size at the endpoint of the experiment relative to the respective controls, while minor alterations in the body weight of the mice were observed. Importantly, I.V. administration of MKAD-21 was more effective than I.P. administration. Specifically, 15 mg/kg of MKAD-21 given I.V. every 5 days for a total of 3 doses/cycles reduced the growth rate of the tumors, while mice body weight remained essentially stable (Fig. 2A and B). Furthermore, growth curves of the tumors originating from mice bearing 5637 xenografts, which were treated with increasing dosages of MKAD-21, show the threshold effect of the latter in BC progression (Fig. 2C). The efficiency of miR-21 inhibition of expression caused by the LNA miR-21 was validated by RT- qPCR in the excised tumors (Supplementary Fig. S1).

3) Transcriptomic analysis and miR-21 levels in a panel of 28 bladder cancer cell lines

Using comparative qPCR analysis, we screened the levels of miR-21 transcript in a panel of 28

BC cell lines (Fig. 3A), that were further stratified into 3 groups (high, intermediate, low) according to their baseline miR-21 expression. Transcriptomic analysis revealed a gene signature consisting of 15 candidates that negatively correlate with miR-21 expression (Fig. 3B).

As a next step, in silico complementarity analysis between miR-21 sequence and the 3’UTR sequences for all 15 genes found to be inversely correlated with miR-21 expression was employed, using the RNA22 v2 microRNA target detection [12]. Interestingly, the coding region of PPP2R2A was predicted to have a potent binding site for miR-21 (Fig. 3C).

4) PPP2R2A is targeted directly by miR-21 through interaction with its 3’UTR

Based on the findings above, we hypothesized that PPP2R2A is a downstream effector of miR-

21 activity. Efficient miR-21 enforced expression by a miR-21 mimic in vitro, as assessed by RT- qPCR analysis (Fig. 4A), reduced PPP2R2A mRNA expression levels in both RT-112 and 5637 cells (Fig. 4B). A 3’UTR luciferase assay in 5637 BC cells transfected with a miR‐21 mimic revealed a significant decrease in the luciferase activity of PPP2R2A‐3′UTR vector, relatively to mimic miR-scr, validating, at the molecular level, the direct interaction between miR-21 and the

3’UTR of PPP2R2A (Fig. 4C). To explore whether miR‐21 regulated PPP2R2A expression in vivo, RT-qPCR and IB were performed to detect the PPP2R2A levels in mice bearing 5637 BC xenografts treated with MKAD-21 or LNA miR-scr (Fig. 4D). The excised tumors demonstrated elevated PPP2R2A levels after treatment with LNA miR-21, suggesting their direct and inverse correlation.

5) PPP2R2A acts as a tumor suppressor in bladder cancer

The product of PPP2R2A gene belongs to the phosphatase 2 regulatory subunit B family, the major determinant of substrate specificity of the heterotrimeric serine/threonine Protein

Phosphatase 2. PPP2R2A represents one of the four major Ser/Thr phosphatases implicated in the negative control of cell growth and division [13]. Despite substantial evidence, pointing to its role as a tumor suppressor in bladder carcinoma [14, 15], PPP2R2A expression status and genomic alterations remain undefined. Given that PPP2R2A levels were found significantly decreased in BC patients (Fig. 5A), we sought to determine the functional role of PPP2R2A in

BC cells. We first silenced its expression by using 2 different siRNAs (Supplementary Fig. S2) and conducted gene profiling analysis using the Agilent Human 44K expression array platform

(Fig. 5B). In total, 498 and 914 genes were found to be differentially regulated by PPP2R2A expression in RT-112 cells by using siPPP2R2A#1 and siPPP2R2A#2, respectively. Notably, an overlap of 237 differentially expressed genes (DEGs) was consistently found with PPP2R2A suppression in RT-112 cells (Fig. 5C). Network analysis of the common deregulated genes, using the IPA Software, highlighted Extracellular signal–regulated kinases (ERK) as the central hub (Fig. 5D) and predicted cancer/cellular growth and proliferation and as major functionally relevant networks downstream of PPP2R2A (Supplementary Table S1). In the same line, by mapping the differentially up- and down-regulated genes to well established ("canonical") pathways, cell cycle-related events were distinguished as the top rated canonical pathways

(Supplementary Table S2). The concept that PPP2R2A expression possibly regulates ERK pathway activation, as well as the proliferative capacity of BC cells, was further confirmed by IB analysis for the phosphorylated and thus activated p44/p42 Mitogen-activated protein kinase

(Fig. 5E) and real-time cell growth monitoring showing elevated proliferation of BC cells transiently depleted of PPP2R2A by an siRNA. Specifically, the characteristic kinetic trace of BC cells is reminiscent of an exponentially growing state (Fig. 5F). Differences in Cell Index measurements are significant after 24 or 3.3 hours of monitoring RT-112 and 5637 cells,

respectively (Supplementary Table S3). Taken together, these data revealed that reduction of

PPP2R2A levels results in activation of the ERK signaling pathway and elevation of BC cell proliferative capacity.

6) PPP2R2A is a mediator of miR-21-induced tumorigenic potential of bladder cancer cells

To explore whether the oncogenic activity of miR-21 in BC is linked to the effects of its downstream target, PPP2R2A, we examined the association of miR-21 levels with ERK signaling pathway activation. Significantly higher levels of p44/p42 MAPK are observed upon

PPP2R2A suppression both in BC cells and mouse xenografts treated with miR-21 innhibitor relatively to the respective controls Fig. 6A and 6B. Importantly, simultaneous miR-21 and

PPP2R2A overexpression in BC cells (Supplementary Fig. S3), showed reversal of the miR-21- induced ERK signaling activation (Fig. 6C), cell proliferation (Fig. 6D) and anchorage- independent growth (Fig. 6E) of RT-112 and 5637 cell lines. Statistically significant differences in Cell Index measurements of PPP2R2A-overexpressing versus control cells, upon miR-21 treatment, are shown in Supplementary Table S4. Taken together, these data reveal that miR-

21 function and oncogenic activity in BC is mediated, at least in part, through the suppression of

PPP2R2A expression and activation of the ERK signaling pathway.

Discussion

Through integration of both molecular and clinical data from bladder tumors and interrogation of a large panel of human bladder cancer (BC) cell lines, we found a novel 15-gene signature that negatively correlates with miR-21 levels. Interestingly, one of these 15 genes, named Protein

Phosphatase 2 Regulatory Subunit B Isoform A (PPP2R2A), was proven to be a direct miR-21 target and a negative regulator of the extracellular signal–regulated kinases (ERK) signaling

pathway and BC cell growth. Moreover, we determined the minimum dose and route of administration for MKAD-21 to suppress effectively BC tumor growth in vivo.

Current knowledge and concepts concerning the involvement of miRNAs in cancer have emerged from the study of cell culture and animal model systems featuring miRNA overexpression and/or ablation. These studies have demonstrated causal links between miRNAs and cancer development emphasizing their potential value in diagnostic, prognostic and possibly therapeutic strategies [16, 17]. A growing body of evidence now suggests that miRNAs may contribute to bladder oncogenesis, progression and metastasis. Examination of the differential expression of miRNAs between clinical BC and normal bladder tissue has led to the elucidation of an 11-miRNA expression signature. miRNAs downregulated in BC, such as miR-145, miR-143 and miR125b, are known to be tumor suppressors, whereas miRNAs found to be upregulated, such as miR-21, miR-183, miR-96, miR17-5p and miR-20a, are oncogenic

[17]. Our analysis of the molecular and clinical data from bladder tumors as well as investigation of BC cell lines reveal a novel correlation of differentially expressed genes with elevated miR-21 levels and have a similar expression pattern in actual BC tumor specimens [18-20]. By associating the levels of miR-21 with the expression status of its candidate downstream effectors, we provide molecular insights into the functional role of miR-21 as a mediator of key molecular drivers in BC.

MiRNA target prediction software identified a miR-21 binding site in the 3′ UTR of PPP2R2A and dual-luciferase reporter assays confirmed that the latter is a direct target of miR-21.

Confirmation of this functional interaction was demonstrated with overexpression of miR-21, which reduced both PPP2R2A mRNA and protein expression in BC cell lines and human BC xenografts. Our mechanistic studies show that blockage of miR-21 expression results in the deactivation of the ERK signaling pathway, while overexpression of miR-21 triggers the same effects as PPP2R2A silencing in BC cells. Perhaps most importantly, concomitant enforced

miR-21 and PPP2R2A expression, rescued the miR-21-induced phenotype in BC cells lines, supporting the notion that PPP2R2A acts as a functional mediator of miR-21. Several findings lead to the conclusion that PP2A, as well as some of its regulatory subunits, such as PPP2R2A, have definitive roles as tumor suppressors in a large variety of cancers [21-23]. Specifically,

PPP2R2A is located on chromosome 8p21.2 and encodes B55α regulatory subunit of PP2A.

Loss of PPP2R2A is a common event in non-small cell lung cancer, and it results in the impairment of the homologous recombination repair pathway. Additionally, shRNA-mediated silencing of PPP2R2A has been shown to be a potent oncogenic signal in experimental models of colon carcinogenesis [14]. Furthermore, in patients with acute myeloid leukemia PPP2R2A has been suggested to act as an adverse prognostic factor given that low levels of its expression in blast cells have been associated with shorter complete remission duration [24].

The kinases that have been found to interact with PP2A include calcium-calmodulin-dependent protein kinase I.V.[25], p70 S6 kinase [26], p21-activated kinases [27], p38 kinase [28], casein kinase II [29], IκB kinases [30], MAPK [31, 32] and Akt [33, 34]. In support of our current data, several reports using overexpression and RNA interference approaches, as well as in vitro dephosphorylation assays, have demonstrated the roles of the B55 regulatory subunits in the

MAPK signaling pathway in multiple systems [35-37]. Specifically, Van Kanegan and colleagues elucidated the role of PPP2R2A in targeting the PP2A heterotrimer to dephosphorylate and inactivate ERKs and indicated that PPP2R2A suppression leads to hyperactivation of ERK stimulated by constitutively active Mitogen-Activated Protein Kinase 1. Along with the modulation of the Akt and ERK signaling pathways by PP2A regulatory subunits, it was further demonstrated that PPP2R2Aα plays a role in regulating cell proliferation and survival, consistent with our results showing the tumor suppressive actions of PPP2R2A in BC [38].

Previous studies have described the degradation of unmodified miRNAs [39]. A modified oligonucleotide that has a locked 2'-O 4'-C methylene bridge in its structure, it is highly

resistance to enzymatic degradation, resulting to higher structural rigidity and increased selective affinity to the RNA counter strand [40]. The biological effects, as well as the safety issues derived from using LNA miR inhibitors for degradation of miRNAs, have been reported in several studies suggesting them as suitable candidates for therapeutic applications [39, 41]. By derivatization with phosphorothioate linkages, enzymatic degradation is delayed and pharmacokinetics is enhanced. Phosphorothioate backbone modifications adequately stabilize the oligonucleotides against degradation and result in a high degree of binding to plasma proteins reducing rapid elimination [42]. In a recent study, the pharmacokinetic and pharmacodynamic properties of LNA anti-miR-221 in NODSCID mice and Cynomolgus monkeys were reported. These studies show that LNA anti-miR-221 has a short half-life, optimal tissue bioavailability, minimal urine excretion in both mice and monkeys and is detectable in mice vital organs and in xenografted tumors for up to 3 weeks after treatment [43]. These studies highlight the suitability of LNA miRs for clinical use. MiR-21 which has been shown to be strongly overexpressed in glioblastomas, was silenced in vitro using LNA modified antisense oligos and resulted in a significant reduction of tumor cell viability and elevated intracellular levels of caspases [44]. Importantly, miR-21 inhibitors were found to be promising multiple myeloma therapeutic agents when delivered in vitro and in vivo [45]. With this background, we tested LNA and phosphorothioate modified antisense oligo against miR-21 for its antitumor efficacy in human BC xenografts as a novel therapeutic approach. We show that I.V. administration of a low dose of MKAD-21 drastically reduced human BC xenograft tumor growth when compared to intraperitoneal administration. Further studies are warranted to investigate the molecular mechanisms underlying this novel inhibitor and to further evaluate its safety, in order to establish its potential value for treatment of BC patients with high miR-21 expression.

Collectively, the current study contributes to the understanding of the molecular mechanisms underlying the malignant features of the BC phenotype. We define a novel 15-gene signature

that correlates with miR-21 levels in BC cells and demonstrate using several methodologic approaches, that there is a direct targeting of PPP2R2A by miR-21. The tumorigenic potential of miR-21 was shown to depend on its downstream effector PPP2R2A, ultimately regulating ERK activation and BC cell growth. Finally, in view of our in vivo data showing the efficacy of a chemically modified antisense oligo against miR-21 (MKAD-21), the potential therapeutic applications of this compound should now be considered more closely for BC treatment.

References

1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D: Global cancer statistics. CA Cancer J Clin 2011, 61:69-90. 2. Knowles MA: Molecular pathogenesis of bladder cancer. Int J Clin Oncol 2008, 13:287-297. 3. Carthew RW, Sontheimer EJ: Origins and Mechanisms of miRNAs and siRNAs. Cell 2009, 136:642-655. 4. Zabolotneva AA, Zhavoronkov A, Garazha AV, Roumiantsev SA, Buzdin AA: Characteristic patterns of microRNA expression in human bladder cancer. Front Genet 2012, 3:310. 5. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone R, Iorio M, Roldo C, Ferracin M, et al: A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A 2006, 103:2257-2261. 6. Wszolek MF, Rieger-Christ KM, Kenney PA, Gould JJ, Silva Neto B, Lavoie AK, Logvinenko T, Libertino JA, Summerhayes IC: A MicroRNA expression profile defining the invasive bladder tumor phenotype. Urol Oncol 2011, 29:794-801.e791. 7. Xie Y, Ma X, Chen L, Li H, Gu L, Gao Y, Zhang Y, Li X, Fan Y, Chen J, Zhang X: MicroRNAs with prognostic significance in bladder cancer: a systematic review and meta-analysis. Sci Rep 2017, 7:5619. 8. Tao J, Lu Q, Wu D, Li P, Xu B, Qing W, Wang M, Zhang Z, Zhang W: microRNA-21 modulates cell proliferation and sensitivity to doxorubicin in bladder cancer cells. Oncol Rep 2011, 25:1721- 1729. 9. Lei M, Xie W, Sun E, Sun Y, Tian D, Liu C, Han R, Li N, Liu M, Han R, Liu L: microRNA-21 Regulates Cell Proliferation and Migration and Cross Talk with PTEN and p53 in Bladder Cancer. DNA Cell Biol 2015, 34:626-632. 10. Uphoff CC, Drexler HG: Elimination of mycoplasmas from infected cell lines using antibiotics. Methods Mol Biol 2011, 731:105-114. 11. Vorvis C, Hatziapostolou M, Mahurkar-Joshi S, Koutsioumpa M, Williams J, Donahue TR, Poultsides GA, Eibl G, Iliopoulos D: Transcriptomic and CRISPR/Cas9 technologies reveal FOXA2 as a tumor suppressor gene in pancreatic cancer. Am J Physiol Gastrointest Liver Physiol 2016, 310:G1124-1137. 12. Miranda KC, Huynh T, Tay Y, Ang YS, Tam WL, Thomson AM, Lim B, Rigoutsos I: A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes. Cell 2006, 126:1203-1217.

13. Shi Y: Serine/threonine phosphatases: mechanism through structure. Cell 2009, 139:468-484. 14. Kalev P, Simicek M, Vazquez I, Munck S, Chen L, Soin T, Danda N, Chen W, Sablina A: Loss of PPP2R2A inhibits homologous recombination DNA repair and predicts tumor sensitivity to PARP inhibition. Cancer Res 2012, 72:6414-6424. 15. Cristobal I, Manso R, Rincon R, Carames C, Senin C, Borrero A, Martinez-Useros J, Rodriguez M, Zazo S, Aguilera O, et al: PP2A inhibition is a common event in colorectal cancer and its restoration using FTY720 shows promising therapeutic potential. Mol Cancer Ther 2014, 13:938-947. 16. Jansson MD, Lund AH: MicroRNA and cancer. Mol Oncol 2012, 6:590-610. 17. Iorio MV, Croce CM: MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics. A comprehensive review. EMBO Mol Med 2012, 4:143-159. 18. Armstrong DA, Green BB, Seigne JD, Schned AR, Marsit CJ: MicroRNA molecular profiling from matched tumor and bio-fluids in bladder cancer. Mol Cancer 2015, 14:194. 19. Catto JW, Miah S, Owen HC, Bryant H, Myers K, Dudziec E, Larre S, Milo M, Rehman I, Rosario DJ, et al: Distinct microRNA alterations characterize high- and low-grade bladder cancer. Cancer Res 2009, 69:8472-8481. 20. Dyrskjot L, Ostenfeld MS, Bramsen JB, Silahtaroglu AN, Lamy P, Ramanathan R, Fristrup N, Jensen JL, Andersen CL, Zieger K, et al: Genomic profiling of microRNAs in bladder cancer: miR- 129 is associated with poor outcome and promotes cell death in vitro. Cancer Res 2009, 69:4851-4860. 21. Pallas DC, Shahrik LK, Martin BL, Jaspers S, Miller TB, Brautigan DL, Roberts TM: Polyoma small and middle T antigens and SV40 small t antigen form stable complexes with protein phosphatase 2A. Cell 1990, 60:167-176. 22. Liu X, Sempere LF, Ouyang H, Memoli VA, Andrew AS, Luo Y, Demidenko E, Korc M, Shi W, Preis M, et al: MicroRNA-31 functions as an oncogenic microRNA in mouse and human lung cancer cells by repressing specific tumor suppressors. J Clin Invest 2010, 120:1298-1309. 23. Calin GA, di Iasio MG, Caprini E, Vorechovsky I, Natali PG, Sozzi G, Croce CM, Barbanti-Brodano G, Russo G, Negrini M: Low frequency of alterations of the alpha (PPP2R1A) and beta (PPP2R1B) isoforms of the subunit A of the serine-threonine phosphatase 2A in human neoplasms. Oncogene 2000, 19:1191-1195. 24. Ruvolo PP, Qui YH, Coombes KR, Zhang N, Ruvolo VR, Borthakur G, Konopleva M, Andreeff M, Kornblau SM: Low expression of PP2A regulatory subunit B55alpha is associated with T308 phosphorylation of AKT and shorter complete remission duration in acute myeloid leukemia patients. Leukemia 2011, 25:1711-1717. 25. Westphal RS, Anderson KA, Means AR, Wadzinski BE: A signaling complex of Ca2+-calmodulin- dependent protein kinase IV and protein phosphatase 2A. Science 1998, 280:1258-1261. 26. Peterson RT, Desai BN, Hardwick JS, Schreiber SL: Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycinassociated protein. Proc Natl Acad Sci U S A 1999, 96:4438-4442. 27. Westphal RS, Coffee RL, Jr., Marotta A, Pelech SL, Wadzinski BE: Identification of kinase- phosphatase signaling modules composed of p70 S6 kinase-protein phosphatase 2A (PP2A) and p21-activated kinase-PP2A. J Biol Chem 1999, 274:687-692. 28. Lee T, Kim SJ, Sumpio BE: Role of PP2A in the regulation of p38 MAPK activation in bovine aortic endothelial cells exposed to cyclic strain. J Cell Physiol 2003, 194:349-355. 29. Heriche JK, Lebrin F, Rabilloud T, Leroy D, Chambaz EM, Goldberg Y: Regulation of protein phosphatase 2A by direct interaction with casein kinase 2alpha. Science 1997, 276:952-955.

30. Fu DX, Kuo YL, Liu BY, Jeang KT, Giam CZ: Human T-lymphotropic virus type I tax activates I- kappa B kinase by inhibiting I-kappa B kinase-associated serine/threonine protein phosphatase 2A. J Biol Chem 2003, 278:1487-1493. 31. Ory S, Zhou M, Conrads TP, Veenstra TD, Morrison DK: Protein phosphatase 2A positively regulates Ras signaling by dephosphorylating KSR1 and Raf-1 on critical 14-3-3 binding sites. Curr Biol 2003, 13:1356-1364. 32. Liu Q, Hofmann PA: Protein phosphatase 2A-mediated cross-talk between p38 MAPK and ERK in apoptosis of cardiac myocytes. Am J Physiol Heart Circ Physiol 2004, 286:H2204-2212. 33. Borgatti P, Martelli AM, Tabellini G, Bellacosa A, Capitani S, Neri LM: Threonine 308 phosphorylated form of Akt translocates to the nucleus of PC12 cells under nerve growth factor stimulation and associates with the nuclear matrix protein nucleolin. J Cell Physiol 2003, 196:79-88. 34. Trotman LC, Alimonti A, Scaglioni PP, Koutcher JA, Cordon-Cardo C, Pandolfi PP: Identification of a tumour suppressor network opposing nuclear Akt function. Nature 2006, 441:523-527. 35. Silverstein AM, Barrow CA, Davis AJ, Mumby MC: Actions of PP2A on the MAP kinase pathway and apoptosis are mediated by distinct regulatory subunits. Proc Natl Acad Sci U S A 2002, 99:4221-4226. 36. Van Kanegan MJ, Adams DG, Wadzinski BE, Strack S: Distinct protein phosphatase 2A heterotrimers modulate growth factor signaling to extracellular signal-regulated kinases and Akt. J Biol Chem 2005, 280:36029-36036. 37. Adams DG, Coffee RL, Jr., Zhang H, Pelech S, Strack S, Wadzinski BE: Positive regulation of Raf1- MEK1/2-ERK1/2 signaling by protein serine/threonine phosphatase 2A holoenzymes. J Biol Chem 2005, 280:42644-42654. 38. Kuo YC, Huang KY, Yang CH, Yang YS, Lee WY, Chiang CW: Regulation of phosphorylation of Thr- 308 of Akt, cell proliferation, and survival by the B55alpha regulatory subunit targeting of the protein phosphatase 2A holoenzyme to Akt. J Biol Chem 2008, 283:1882-1892. 39. Trang P, Weidhaas JB, Slack FJ: MicroRNAs as potential cancer therapeutics. Oncogene 2008, 27 Suppl 2:S52-57. 40. Braasch DA, Corey DR: Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA. Chem Biol 2001, 8:1-7. 41. Di Martino MT, Gulla A, Gallo Cantafio ME, Altomare E, Amodio N, Leone E, Morelli E, Lio SG, Caracciolo D, Rossi M, et al: In vitro and in vivo activity of a novel locked nucleic acid (LNA)- inhibitor-miR-221 against multiple myeloma cells. PLoS One 2014, 9:e89659. 42. Eckstein F: Phosphorothioate oligodeoxynucleotides: what is their origin and what is unique about them? Antisense Nucleic Acid Drug Dev 2000, 10:117-121. 43. Gallo Cantafio ME, Nielsen BS, Mignogna C, Arbitrio M, Botta C, Frandsen NM, Rolfo C, Tagliaferri P, Tassone P, Di Martino MT: Pharmacokinetics and Pharmacodynamics of a 13-mer LNA-inhibitor-miR-221 in Mice and Non-human Primates. Mol Ther Nucleic Acids 2016, 5:e326. 44. Griveau A, Bejaud J, Anthiya S, Avril S, Autret D, Garcion E: Silencing of miR-21 by locked nucleic acid-lipid nanocapsule complexes sensitize human glioblastoma cells to radiation-induced cell death. Int J Pharm 2013, 454:765-774. 45. Leone E, Morelli E, Di Martino MT, Amodio N, Foresta U, Gulla A, Rossi M, Neri A, Giordano A, Munshi NC, et al: Targeting miR-21 inhibits in vitro and in vivo multiple myeloma cell growth. Clin Cancer Res 2013, 19:2096-2106.

Figure Legends

Figure 1.

Effects of miR-21 blockage on anchorage-independent growth and invasiveness in vitro. A,

Effect of 50nM LNA miR-scr or LNA miR-21 on the expression of miR-21 in RT-112 and 5637 cells, as assessed by RT-qPCR. B, Colonies formed by RT-112 cells upon miR-21 inhibition. C and D, Effect of miR-21 suppression on BC cells’ invasive capacity. For each cell line, representative images of colonies or invading cells are shown in the upper panel and the respective quantification in the lower panel. Cells were treated with 50 or 100nM LNA miR-scr or

LNA miR-21. LNA miR-scr, cells treated with scrambled sequence control; LNA miR-21, cells treated with miRCURY locked nucleic acid miR-21 inhibitor (for in vitro experiments). Data are expressed as the mean number of colonies or invading cells per field ± SE. Asterisks denote statistical significant differences, * P<0.05, *** P<0.001

Figure 2.

Activity of a chemically modified miR-21 inhibitor in human bladder cancer xenografts. A, Mice inoculated with 5637 human BC cells (n=8 mice per group) were treated with 15 mg/kg MKAD-

21 by I.V. tail vein injection every 5 days for the time indicated. Tumors from two mice in each group were collected on day 14 of the study. Statistically significant difference between the vehicle and the MKAD-21 (15mg/kg) treatment in 5637 mouse xenografts is denoted. B, Mice inoculated with RT-112 human BC cells (n=5 mice per group) were treated with 15 mg/kg

MKAD-21 by I.V. tail vein or by l.P. injection every 5 days for the time indicated. C, Mice inoculated with 5637 human BC cells (n=5 mice per group) were treated with increasing concentrations of MKAD-21 by I.V. tail vein injection every 5 days for the time indicated.

Statistically significant difference between the LNA miR-scr and the MKAD-21 (15mg/kg)

treatment in 5637 mouse xenografts is denoted. Vehicle control, mice treated with sterile saline;

LNA miR-scr, mice treated with 15mg/kg scrambled sequence control; MKAD-21, mice treated with miRCURY phosphorothioate backbone and locked nucleic acid miR-21 inhibitor at the indicated dosage. Data represent mean tumor volume ± SEM (left panel) and percentage values of tumor volume change (middle panel) and mean relative (to day 0 of treatment) body weights within each treatment arm during the dosing phase (right panel). Asterisks denote statistical significant differences compared to vehicle and scrambled RNA controls or lower dose treatment arms, * P<0.05

Figure 3.

Correlation of miR-21 expression levels with differentially expressed genes in 28 bladder cancer cell lines. A, MiR-21 expression levels in 28 BC cell lines, as assessed by RT-qPCR. qPCR levels (High, Medium, Low) of miR21 are annotated along with cell line name. B, 28 BC cell lines were arrayed against their pooled reference mix and the name of each cell line is listed on

Y axis. The 15 DEGs are listed on X axis and attached in hierarchical tree to indicate the distance of relation between each gene. The logarithmic color scheme depicts the expression levels of DEGs expression from higher to lower in red > black > green order. C, In silico complementarity analysis between miR-21 sequence and the coding and 3’UTR sequences for all the 15 genes found to be inversely correlated with miR-21 expression was performed, using the RNA22 v2 microRNA target detection. Criteria that were applied, include a size seed of 7 and 11 as the minimum number of paired up bases in heteroduplex and maximum folding energy in heteroduplex, respectively. H: High; M: Medium; L: Low. Data are shown as the mean

± SE.

Figure 4.

PPP2R2A expression depends on miR-21 levels in vitro and in vivo. A, Effect of 100nM mimic miR-scr or miR-21 mimic on the expression of miR-21 in RT-112 and 5637 cells, as assessed by RT-qPCR. B, Transient transfection of miR-21 mimic mitigated the PPP2R2A mRNA levels, as assessed by RT-qPCR. C, Luciferase activity mediated by PPP2R2A 3’UTR was evaluated

28 h after transfection of the PPP2R2A_pLightSwitch_ 3_UTR Reporter and pLenti CMV Puro

LUC vectors in 5637 cells. Dual luciferase reporter assay showed that the 3’UTR of PPP2R2A luciferase activity was significantly inhibited by miR-21 enforced expression. D, PPP2R2A protein levels in the excised tumors from 5637 bearing mouse xenografts treated with MKAD-21

(20 mg/kg), as assessed by IB analysis. Tumors were collected on day 27 of the study. CREB was used as a loading control. Mimic miR-scr, cells treated with 100nM scrambled sequence control; miR-21 mimic, cells treated with 100nM miR-21 mimic. Data are shown as the mean ±

SE. Asterisks denote statistical significant differences, *** P<0.001

Figure 5.

Effects of PPP2R2A suppression on the gene signature and proliferative capacity of bladder cancer cells. A, Differential expression of PPP2R2A in urinary bladder carcinoma versus normal tissues, as assessed by RT-qPCR. B, Heat maps showing the DEGs in RT-112 cells upon

PPP2R2A suppression using siPPP2R2A#1 (left panel) and siPPP2R2A#2 (right panel). Data was filtered using a P value cutoff of 0.05 and a fold change cutoff of 1.5. Clustering dendrograms show the relative expression values according to the following coloring scheme: red = high, black = moderate, green = low. C, Venn diagrams demonstrating the overlap of commonly DEGs upon PPP2R2A suppression by using two different siRNAs. D, Top scoring network (score of 30 focus molecules) generated by IPA with ERK as central regulator under the function ‘Cell Cycle, Cell-To-Cell Signaling and Interaction, Cellular Growth and

Proliferation’. Nodes represent genes and the relationship between the nodes are indicated by

edges. Upregulated proteins are indicated in red and downregulated proteins in green.

Molecules found by the data mining tools of IPA (build tools) are shown in gray. E, IB analysis for the phosphorylated p44/p42 MAPK (Thr202/Tyr204) and total-p44/p42 MAPK in RT-112 and

5637 cells after PPP2R2A suppression with 2 different siRNAs. F, Effect of transient PPP2R2A silencing on BC cell proliferation, as assessed by the xCELLigence system. siC#1, cells transfected with a negative control scramble siRNA; siPPP2R2A#1, cells transfected with siRNA#1 for PPP2R2A; siPPP2R2A#2, cells transfected with siRNA#2 for PPP2R2A. Data are represented in box-and-whisker plots showing median and 25th and 75th percentiles for each category of sample.

Figure 6.

PPP2R2A overexpression rescues the miR-21-induced biological effects in bladder cancer cells.

IB for phosphorylated p44/p42 MAPK (Thr202/Tyr204) and total-p44/p42 MAPK in A, BC cells and B, excised tumors originating from 5637 bearing mouse xenografts, after treatment with

50nM LNA miR-21 or MKAD-21 (20 mg/kg) and the respective scramble controls. C, The effect of PPP2R2A enforced expression on ERK pathway activation in miR-21 mimic- or mimic miR- scr-treated, serum starved- and FBS-stimulated BC cells, as assessed by IB analysis. Cells treatment with 100nM mimic miR-scr or miR-21 mimic was followed by transient transfection of pPM-C-His vector or Blank-Control Protein Vector. D, Dynamic monitoring of BC cellular proliferation upon miR-21 and PPP2R2A parallel overexpression, using the xCELLigence RTCA

SP system. E, PPP2R2A overexpression causes reversal of miR-21-mediated induction of the colony formation ability of RT-112 cells. Representative images of colonies are shown in the upper panel and the respective quantification in the lower panel. Mimic miR-scr+empty vector, cells treated with 100nM mimic miR-scr for 24h followed by transfection with Blank-Control

Protein Vector; Mimic miR-scr+PPP2R2A vector, cells treated with 100nM mimic miR-scr for

24h followed by transfection with pPM-C-His vector; miR-21 mimic+empty vector, cells treated with 100nM mimic miR-21 for 24h followed by transfection with Blank-Control Protein Vector; miR-21 mimic+PPP2R2A vector, cells treated with 100nM mimic miR-21 for 24h followed by transfection with pPM-C-His vector. Data are shown as the mean ± SE. Asterisks denote statistical significant differences, * P<0.05, *** P<0.001

MKAD-21 suppresses the oncogenic activity of the miR-21/PPP2R2A/ERK molecular network in bladder cancer

Marina Koutsioumpa, Hsiao-Wang Chen, Neil O'Brien, et al.

Mol Cancer Ther Published OnlineFirst April 27, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-17-1049

Supplementary Access the most recent supplemental material at: Material http://mct.aacrjournals.org/content/suppl/2018/04/27/1535-7163.MCT-17-1049.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mct.aacrjournals.org/content/early/2018/04/27/1535-7163.MCT-17-1049. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.