a novel mir-146a-pou3f2/smarca5 pathway regulates ......2020/09/24 · author manuscripts have been...

1

A novel miR-146a-POU3F2/SMARCA5 pathway regulates stemness and

therapeutic response in glioblastoma

Tiantian Cui1, Erica H. Bell1, Joseph McElroy2, Kevin Liu3, Ebin Sebastian1, Benjamin

Johnson1, Pooja Manchanda Gulati1, Aline Paixao Becker1, Ashley Gray3, Marjolein

Geurts4, Depika Subedi5, Linlin Yang1, Jessica Fleming1, Wei Meng1, Jill S.

Barnholtz-Sloan6, Monica Venere1, Qi-En Wang1, Pierre A. Robe7, S. Jaharul Haque1,

Arnab Chakravarti1

1Department of Radiation Oncology, Arthur G. James Hospital/Ohio State

Comprehensive Cancer Center, Columbus, OH, 43210, USA 2The Ohio State University Center for Biostatistics, Department of Biomedical

Informatics, Columbus, OH, 43210, USA 3The Ohio State University College of Medicine, Columbus, OH, 43210, USA 4Erasmus Medical Center Cancer Institute, Rotterdam, the Netherlands 5Berea College, Berea, KY, 40403, USA 6Department of Population and Quantitative Health Sciences and Case

Comprehensive Cancer Center, Case Western Reserve University School of

Medicine, Cleveland, OH, USA 7Department of Neurology and Neurosurgery, Brain Center Rudolf Magnus,

University Medical Center Utrecht, Utrecht, the Netherlands

Running Title: A novel miR-146a-POU3F2/SMARCA5 pathway in glioblastoma

*Corresponding Author:

Dr. Arnab Chakravarti

Ohio State University Comprehensive Cancer Center and Richard L. Solove

Research Institute

Arthur G. James Cancer Hospital

The Ohio State University Medical School

460 W. 10th Ave.

Room D252F

Columbus, Ohio 43210

Tel# 614-293-0672

Fax# 614- 293-1943

Email: [email protected]

Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were

disclosed.

on June 8, 2021. © 2020 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 24, 2020; DOI: 10.1158/1541-7786.MCR-20-0353

mailto:[email protected]://mcr.aacrjournals.org/

2

Abstract

Rapid tumor growth, widespread brain-invasion, and therapeutic resistance critically

contribute to glioblastoma (GBM) recurrence and dismal patient outcomes. Although

GBM stem cells (GSCs) are shown to play key roles in these processes, the

molecular pathways governing the GSC phenotype (GBM-stemness) remain poorly

defined. Here, we show that epigenetic silencing of miR-146a significantly correlated

with worse patient outcome and importantly, miR-146a level was significantly lower in

recurrent tumors compared with primary ones. Further, miR-146a overexpression

significantly inhibited the proliferation and invasion of GBM patient-derived primary

cells and increased their response to temozolomide (TMZ), both in vitro and in vivo.

Mechanistically, miR-146a directly silenced POU3F2 and SMARCA5, two

transcription factors that mutually regulated each other, significantly compromising

GBM-stemness and increasing TMZ response. Collectively, our data show that miR-

146a-POU3F2/SMARCA5 pathway plays a critical role in suppressing GBM-

stemness and increasing TMZ-response, suggesting that POU3F2 and SMARCA5

may serve as novel therapeutic targets in GBM.

Implications: MicroRNA-146a predicts favorable prognosis and the miR-146a-

POU3F2/SMARCA5 pathway is important for the suppression of stemness in GBM.

Key words: miR-146a, POU3F2/SMARCA5, primary and recurrent GBM, therapeutic

response, GSCs



http://mcr.aacrjournals.org/

3

Introduction

Glioblastomas (GBMs, World Health Organization grade IV gliomas) are the most

aggressive and lethal primary malignant tumors of the central nervous system in

adults (1). Recently the World Health Organization has classified GBM and lower

grade gliomas based on the neomorphic mutation status of isocitrate dehydrogenase

(IDH) 1 or 2, and irrespective of grades, gliomas with wild-type (wt) IDH1/2 have

worse clinical outcome compared with those with mutant IDH1/2 (1). More than 90%

of GBMs have wt-IDH1/2 and irrespective of IDH status patients with newly

diagnosed GBM receive the standard of care with maximal safe resection, followed

by adjuvant radiation and temozolomide (TMZ) chemotherapy (1-3). Despite the

aggressive multi-modality treatments, the median survival remains within the range of

12-15 months after diagnosis (4). This grim patient outcome is largely attributed to

rapid tumor growth, invasion of vital brain structures, and evolution of intrinsic and

acquired treatment resistance clones among other factors (4). However, we do not

have a clear understanding of the molecular signaling networks driving disease

progression and treatment resistance. Although a number of prognostic biomarkers,

including promoter methylation of O6-methylguanine-DNA methyltransferase (MGMT),

neomorphic mutation of IDH1/2, amplification and/or gain-of-function mutation of

epidermal growth factor receptor (EGFR), loss of function mutation/deletion of TP53

or PTEN, and CpG island methylation phenotype (CIMP) (5,6), have been identified

and characterized, the molecular mechanisms underlying the activation of signaling

pathways driving the treatment resistance and tumor recurrence have remained




4

poorly understood posing a great challenge in developing truly effective therapeutic

intervention. To this end, a number of studies demonstrate that a post-treatment

increase in GSC population markedly contributes to tumor recurrence and

therapeutic resistance mechanisms (7-9). GSCs possess potential of self-renewal

and multipotent differentiation, and are responsible for tumor initiation and

maintenance (7,10-13). These GSC functions are largely mediated by deregulation

of Wnt/β-catenin (14), Notch (15), NF-κB (16), and JAK-STAT (17) signaling

pathways among others resulting in an aberrant expression of downstream signature

molecules that drive the resistance and recurrence mechanisms in GBM. To this end,

we sought to identify molecular biomarkers/signatures of prognostic values and

significance that drive treatment resistance and recurrence in GBM, and to target

these drivers in preclinical GBM models for the development of novel therapeutic

interventions.

MicroRNAs (miRNAs) play regulatory roles through silencing the expression of target

genes by post-transcriptional degradation and translational repression. Evidence

shows that miRNAs are involved in regulating the progression and therapeutic

resistance mechanisms in a variety of malignancies including GBM by modulating

cancer stem cell functions (18). Previous studies have shown differential miRNA

expression in GBM tumor tissues; moreover, tumors harboring altered miRNA

expression had differential prognoses and treatment outcomes (19-22). Therefore,

the differentially expressed miRNAs may serve as biomarkers and/or drivers of

prognosis and treatment response in GBM. Very few studies have investigated the



https://www.sciencedirect.com/topics/medicine-and-dentistry/micrornahttp://mcr.aacrjournals.org/

5

expression of miRNA in primary and recurrent GBM (19,23). Hence, we undertook a

high-throughput molecular profiling approach to identify miRNAs that correlate with

clinical outcomes of patients with IDH1/2 wt GBM. We identified miR-146a as a tumor

suppressor miRNA in GBM. Subsequently, we have identified and characterized two

direct targets of miR-146a, POU domain, class 3, transcription factor 2 (POU3F2)

and SWI/SNF Related, Matrix Associated, Actin Dependent Regulator of Chromatin,

Subfamily A, Member 5 (SMARCA5), which are found to play key roles in tumor

growth and treatment response.

Materials and Methods

Patient cohort, ethics statement, and RNA isolation

We used two patient cohorts in this study. Briefly, a total of two hundred and sixty

eight (n=268) FFPE tumor specimens without IDH1R132H mutation from patient

cohort 1 with newly diagnosed GBM were used in the study. All of the patients

underwent tumor biopsy or resection at the University of Utrecht (The Netherlands,

cohort 1) from 2005 to 2014. A total of eleven pairs (n=11) of fresh frozen tumor

specimens from patient cohort 2 with newly diagnosed GBM and matched recurrent

GBM were used as a validation set in the study. All of the patients underwent tumor

resection at University Hospitals of Cleveland (cohort 2) from 2008 to 2015 and

provided written informed consent to participate in brain tumor research. The study

was approved by The Ohio State University, University Hospital of Cleveland, and

University Medical Center Utrecht institutional review boards.

miRNA expression analysis and MGMT promoter methylation analyses




6

Tumor samples were processed to generate miRNA expression data using the

NanoString human v3a array, which contains 798 miRNA probes. DNA (250 ng) input

was used per sample and the MGMT-STP27 model was used to calculate MGMT

promoter methylation status from Illumina EPIC data (24).

Cell lines and cell culture

Normal human Astrocytes (NHAs) were purchased from Lonza (Walkersville, MD).

Human GBM cell lines U251, T98G, LN229, U87MG, LN18, were purchased from

ATCC (Manassas, VA). No further authentication was performed for NHAs and ATCC

cell lines. U87MG/EGFRvIII cells were provided by Dr. Deliang Guo (The Ohio State

University). Primary GBM patient-derived GBM30-luc cells were kindly provided by Dr.

Balveen Kaur (The Ohio State University). Primary GBM patient-derived cells (08-387

and 3359) were kindly provided by Dr. Jeremy Rich (UC San Diego) and originally

isolated from tumor resections in accordance with approved institutional review board

protocols. GBM12 and GBM43 were kindly provided by Mayo Clinic. These cell lines

were authenticated by DNA profiling. U251, T98G, LN229, U87MG, LN18, and

U87MG/EGFRvIII cells were grown in DMEM medium (Gibco) supplemented with

10% (v/v) fetal bovine serum (FBS) (Invitrogen), 100U/ml penicillin/streptomycin

(Sigma), and maintained in a humidified atmosphere with 5% CO2 at 37°C. GBM30,

08-387, 3359, GBM12, and GBM43 cells were cultured in neurobasal medium with

EGF (20ng/ml), FGF (20ng/ml), B27 (1×), and GlutaMax (1×), and Sodium Pyruvate

(1×) in a humidified atmosphere with 5% CO2 at 37°C. All cells were periodically

tested for mycoplasma contamination using Universal Mycoplasma Detection Kit




7

(ATCC30-1012KTM).

qRT-PCR analysis of mRNA expression and miRNA expression

For mRNA expression analysis, total RNA was extracted from GBM cells using

TRIzol reagent as previously described (24). The primers are listed in STable 1. For

miRNA expression analysis, RNA (100 ng) was reverse transcribed using the

TaqMan Advanced MicroRNA Assay kit (Applied Biosystems) with miRNA-specific

primers. Primers of Taqman MicroRNA Assays for hsa-miR-146a (Assay ID: 000468)

and RNU6B (Assay ID: 001093) were purchased from Invitrogen. Relative

expression level of miR-146a was calculated by normalization with RNU6B

expression in GBM cells. All the experiments were performed in triplicate.

Demethylation tests, bisulfite modification, methylation-specific PCR and

bisulfite sequencing

GBM cells were plated and cultured on six-well plates. At 50% confluence, 10 µM

DAC (Sigma) was added to the medium on days 1 and day 3. Cells were then

harvested for total RNA isolation and RT-qPCR analysis. Bisulfite modification,

methylation-specific PCR (MSP) and bisulfate sequencing (BS) were carried out as

described previously (25). Primer pairs for MSP and BS are in STable 1.

Western blot analysis

Protein extraction and western blot analysis were performed as previously described

(26). The antibodies used in this study are listed in STable 2. Polyclonal goat anti-

rabbit antibody (Cell Signaling Technology) and Western Blotting Detection System

(Millipore) were used for exposure.




8

Transfection of miR-146a mimics and inhibitors, siRNA treatment, and Dox-

inducible stable cells

For functional studies, a specific miR-146 mimic/inhibitor (Invitrogen) and a

respective negative control (Invitrogen) were transfected into the GBM cells. For

siRNA transfection, specific POU3F2 1/2 and SMARCA5 1/2 siRNAs (50 nmol/L)

(Dharmacon) and a negative control (Dharmacon) were used. For POU3F2

overexpression, pcDNA3.1+/C-(K)DYK-POU3F2 plasmid (GenScript) was used. Tet-

On shmimic-inducible miR-146a construct was purchased from Dharmacon. All

siRNAs were transfected into cells using Lipofectamine 2000 transfection reagent

(Invitrogen).

Cell proliferation and invasion assays

For cell proliferation assay, twenty-four hours post-transfection, cells (1000 cells/well)

were seeded in 96-well plates and measured by CellTiter-Glo (Promega) after

indicated time points as previously described (24). Cell invasion assays were

performed as previously described (24). All the experiments were performed in

triplicate.

Colony formation and sphere formation assays

GBM cells (100 cells/well) were used for colony formation assay as described

previously (24). All the experiments were performed in triplicate. A total of 300 cells

were mixed with neurobasal medium in a humidified atmosphere with 5% CO2 at

37°C. The number of spheres was counted 2-3 weeks after cell seeding. All the

experiments were performed in triplicate. To evaluate the frequency of sphere-




9

forming cells (SFCf), cells were plated in 96-well plates in a limiting dilution manner

(1, 5, 10, 20 cells/well) using FACS. The number of wells containing spheres was

counted after 2 weeks, and the SFCf was calculated using the ELDA software

http://bioinf.wehi.edu.au/software/elda/index.html (27).

Cell viability assay

Twenty-four hours post-transfection, cells (2000 cells/well) were seeded in 96-well

plates, then treated with different dose of TMZ, 72 h after treatment, cell viability was

measured by CellTiter-Glo (Promega).

Apoptosis assay

An Annexin V-fluorescence isothiocyanate (FITC) Apoptosis Detection Kit (Invitrogen)

was used for apoptosis assay. At 72 h miR-146a overexpression, 106 cells were

resuspended in 200 µl binding buffer with 10 µl Annexin-V-FITC and 5 µl propidium

iodide, and incubated in the dark for another 30 min. Finally, cells were assessed

using flow cytometric analysis. All the experiments were performed in triplicate.

Luciferase reporter assay

The 3’UTR of POU3F2 and SMARCA5 was synthesized, annealed, and then inserted

into the Xho l and Not I sites of the pCheck2-reporter luciferase vector downstream of

the stop codon of the gene for luciferase. To induce mutagenesis, the sequences

complementary to the binding sites of miR-146a in the 3’UTR was replaced by the

mutated sites (STable 1). The constructs were confirmed by sanger sequencing.

Cells were co-transfected with the wild type or mutated construct, pCheck2 plasmid,

and equal amount of negative control or miR-146a mimic. Luciferase assays were



http://bioinf.wehi.edu.au/software/elda/index.htmlhttp://mcr.aacrjournals.org/

10

performed using the Dual Luciferase Reporter Assay System (Promega).

Xenograft tumor study

Six to eight week old athymic nude mice were obtained from the Target Validation

Shared Resource at the Ohio State University. All animal procedures were approved

by the Subcommittee on Research Animal Care at The Ohio State University. To

determine the frequency of tumor-initiating cells (TICf) using the limiting dilution

assay, three cell doses (1 × 105, 1 × 104, 1 × 103) of each sample were injected

subcutaneously into athymic nude mice. Mice were monitored for up to 4 weeks post-

injection, and the tumor number per group within this period was used to calculate

the TICf using aforementioned ELDA software(27). For the intracranial xenograft

models, GBM cells (1×105 08-387/miR-146a/luc and GBM30/miR-146a/luc in 2 μl

PBS), transduced with Tet-On inducible miR-146a and GFP-tagged luciferase

plasmids, were implanted into the brain of the mouse. Doxycycline (Dox) grain-based

diet (Thermo Fisher Scientific) was administered one day after injection. For drug

treatment studies, mice were treated with vehicle or 20 mg/kg of TMZ resuspended in

vehicle by oral gavage once a day for 5 days, starting at day 5 post-injection.

Luciferin (Perkin Elmer) solution (100mg/kg) was used for imaging after tumor

formation. The IVIS Lumina II imaging platform from the Ohio State University (OSU)

Small Animal Imaging Core was used to detect and quantify the signal. Mice were

sacrificed when they became moribund and the tumor tissues were harvested for

miR-146a expression detection.

Statistical analysis




11

Analysis of Nanostring data was carried out in R (24). Cox regression was used to

identify the association between expression of miRNAs (continuous) and overall

survival with age as a covariable. miR-146a was then median dichotomized and the

log rank test was employed to visualize the association between the expression and

overall survival. Other statistical analyses were performed using the software

package SPSS 23.0 (SPSS, Chicago, USA). Descriptive statistics, i.e., means ± SD,

are shown on the Figures. Two sample t-tests or ANOVA were performed for data

analysis for experiments with two groups or more than two groups’ comparisons.

Spearman correlation analyses was applied to analyze the association between

expression of miR-146a and POU3F2/SMARCA5. The publically available CGGA

datasets were directly analyzed from the CGGA Data Portal at

http://www.cgga.org.cn/. The detailed information of the RNA-Seq experiments and

software used can be found at the CGGA Data Portal at http://www.cgga.org.cn/. P

values were calculated two-sided. P value less than 0.05 was defined as statistically

significant.

Results

Decreased miR-146a expression is associated with shorter overall survival (OS)

of GBM patients

To investigate a potential association between miRNA expression and GBM patient

outcomes, miRNA expression profiling in GBM tumor specimens of a cohort of 268

patients with wt-IDH1/2 was conducted using Nanostring v3 technology (24). miR-

146a was identified to be one of the top miRNAs, which upon univariable analysis



http://www.cgga.org.cn/http://www.cgga.org.cn/http://mcr.aacrjournals.org/

12

(UVA) with continuous expression values (HR=0.658, 95%CI: 0.534-0.810, p

13

(data not shown), but 6 out of the remaining 8 pairs showed significantly lower

expression of miR-146a in recurrent tumors compared with the matched primary

tumors (Figure 1d, cohort 2, n=16, 8 pairs). Taken together, these data indicate that

miR-146a acts as a favorable prognostic biomarker in GBMs, and miR-146a

expression is significantly suppressed in recurrent GBM patients further suggesting

that miR-146a may play a role in the treatment response mechanisms in GBM.

MicroRNA-146a expression is partially regulated by promoter methylation in

GBM

To determine the endogenous expression of miR-146a in GBM cells, we performed

RT-qPCR in normal human astrocytes (NHAs), 6 established GBM cell lines (U251,

T98G, LN229, U87MG/EGFRvIII, U87MG, and LN18), and 3 patient-derived primary

GBM cell lines (08-387, 3359, and GBM30). We found that the majority of GBM cell

lines expressed significantly lower levels of miR-146a, except LN229, compared with

the NHA cell line (Figure 1e). This result suggests that miR-146a is significantly

downregulated in GBM cells, which is consistent with a previous study demonstrating

that miR-146a expression is lower in GBM tumors compared with adjacent normal

tissues (30). Promoter hyper-methylation leading to transcriptional silencing of

miRNAs has been found in a variety of cancer (31). Specifically, Wang et al. have

reported that demethylation of miR-146a promoter by 5-Aza-2’-deoxycytidine (DAC)

correlates with delayed progression of castration resistant prostate cancer(32).

Therefore, we explored if miR-146a was silenced by promoter methylation in GBM

and found that DAC treatment significantly increased the miR-146a expression in 8




14

out of 9 (8/9) cell lines examined, with exception of LN229 cell line that expressed a

relatively higher endogenous level of miR-146a (Figure 1f). The methylation status of

miR-146a was further determined by methylation specific PCR (MSP) in all 9 GBM

cell lines. As shown in Figure 1g, 7/8 GBM cells showed partial methylation of the

miR-146a promoter region with downregulated miR-146a expression. Consistent with

DAC data, miR-146a promoter in LN229 cells was found to be unmethylated. To

confirm the MSP results and further evaluate the methylation status of miR-146a in

GBM cell lines, bisulfite sequencing (BS) was performed for 8 CpG sites (−183, −160,

−150, −142, −138, −136, −132, and −128) of the promoter region near the

transcription start site. Consistent with the MSP results, a high level of methylation

was found in 7/8 cell lines with downregulated miR-146a expression (Figure 1h). The

MSP result prompted us to analyze the methylation status of miR-146a in GBM tumor

tissues. We selected tumor tissues with relatively low expression levels of miR-146a

in cohort 1 and, as expected, methylation of miR-146a was found in 9/10 tumor

tissues (Figure 1i). These results suggest that hyper-methylation of CpG islands on

the miR-146a promoter contributes, in part, to the downregulation of miR-146a

expression in GBM.

Overexpression of miR-146a inhibits cell proliferation and invasion in vitro and

in vivo

Given that high expression of miR-146a was associated with better OS in GBM

patients and it was downregulated in GBM cells, we hypothesized that miR-146a

might play an important role in restraining GBM progression. To investigate the




15

effects of miR-146a on cell proliferation and invasion in vitro, U87MG/EGFRvIII,

GBM30, and 08-387 cells, which expressed low endogenous levels of miR-146a,

were transfected with microRNA negative control (NC) or miR-146a mimic. On the

other hand, LN229 cells, which expressed a relatively high level of miR-146a, were

transfected with NC or a miR-146a inhibitor. We found that overexpression of miR-

146a significantly inhibited proliferation of U87MG/EGFRvIII, GBM30, and 08-387

cells (Figure 2a-c), which was reproduced in additional patient-derived xenografts

(PDX) lines GBM12 and GBM43 with endogenous low expression of miR-146a

(Figure S1a-b). In consistence with this, inhibition of miR-146a increased the

proliferation of LN229 cells (Figure 2d). In addition to rapid proliferation, invasion is a

defining hallmark of GBM cells (18). Accordingly, we determined the role of miR-146a

on GBM cell invasion using a trans-well matrigel assay, which showed that

overexpression of miR-146a suppressed invasion of GBM cells in vitro (Figure 2e,

Figure S2a), whereas inhibition of miR-146a increased invasion (Figure 2f, Figure

S2b). Cell proliferation and invasion data were then substantiated by using additional

GBM cell lines (U87 and LN18, Figure S3a-c). Notably, we observed that

overexpression of miR-146a in primary GBM cells significantly inhibited sphere

formation, a characteristic feature of these cells (Figure 2g-2h). In consistence with

previous studies, activation of NF-ĸB and ERK1/2 was found to be inhibited by

overexpression of miR-146a in GBM cells (Figure S4). In order to study the role of

miR-146a on tumor growth in vivo, we generated two tetracycline (doxycycline/Dox)

inducible stable cell lines termed 08-387/miR-146a and GBM30/miR-146a. Our data




16

show that expression of miR-146a was induced after Dox treatment and proliferation

was inhibited in both cell lines (Figure S5a-d). Furthermore, an athymic nude

xenograft mouse model was established, in which stable 08-387/miR-146a and

GBM30/miR-146a cells were implanted into the brain. Decreased tumor growth was

observed by IVIS imaging in Dox-induced group of mice that had higher expression

of miR-146a (Figure 2i-2k) and an overall survival of these mice was prolonged as

revealed by the Kaplan Meier plots (Figure 2l-2m). Representative H&E staining for

the tumors are shown in Figure S5E. Dox-induced expression of miR-146a in vivo

was further confirmed by qPCR (Figure 2n-2o). Taken together, these data suggest

that overexpression of miR-146a inhibits tumor growth and prolongs survival of the

tumor cell implanted mice.

MicroRNA-146a enhances TMZ response in primary GBM models

TMZ is the most widely used chemotherapy in GBM patients. To determine if miR-

146a sensitizes GBM cells to TMZ, miR-146a was overexpressed in

U87MG/EGFRvIII and 08-387 cell lines and cell viability was measured. We found

that overexpression of miR-146a could significantly enhance TMZ-induced cell killing

(Figure 3a-b). In addition, fewer colonies were formed in the miR-146a

overexpressing group compared with the control group, after TMZ treatment, which

was consistent with the outcome of the viability experiments (Figure 3c-3d). To

further explore the effects of miR-146a on TMZ-induced apoptosis, we performed

Annexin V apoptosis assay and measured caspase activation. As shown in Figure

3e-3g, overexpression of miR-146a markedly enhanced TMZ-induced apoptosis in




17

08-387 cells. Similar results were obtained in GBM30 cells (Figure S6a-b). These

findings demonstrate that miR-146a overexpression enhanced TMZ response and

potentiated TMZ-induced apoptosis in GBM cells. To further assess the function of

miR-146a in TMZ response in vivo, an athymic nude xenograft mouse model was

employed. Mice were randomized into 4 treatment groups, 10 mice per group: (i)

Dox(-)/TMZ(-), (ii) Dox(-)/TMZ(+), (iii) Dox(+)/TMZ(-), and (iv) Dox(+)/TMZ(+). Mice

were treated, as indicated, with TMZ by oral gavage for 5 days, and euthanized when

displayed tumor-associated morbidity. Mice in Dox(-)/TMZ(-) group showed rapid

tumor growth (23-25 days, median survival, 25 days), Dox(-)/TMZ(+) and

Dox(+)/TMZ(-) groups initially slowed tumor growth, but tumor still grew (33-52 days

and 33-53 days, respectively, median survival, 38 days). Mice in Dox(+)/TMZ(+)

group had significant smaller tumors (Figure 3h) and had longer survival times (50-63

days, median survival, 55 days, Figure 3i). Thus, miR-146a not only inhibited tumor

growth, but also enhanced TMZ sensitivity both in vitro and in vivo in GBM.

Overexpression of miR-146a downregulates stemness of GSCs

GSCs are found to be primary drivers of tumor recurrence and therapeutic resistance

(33). Because miR-146a was found to be downregulated in recurrent GBM tumors

(Figure 1b-1d), accordingly, we hypothesized that overexpression of miR-146a would

downregulate stemness in GSCs. To test this hypothesis, we first determined the

expression of miR-146a in GSCs and non-GSCs (NGSCs), which were derived from

human brain tumor specimens using CD133 selection (34). Consistent with prior

reports, the GSC-enriched fractions expressed high level of Olig2 and Oct4 (Figure




18

4a), which are markers of multipotent progenitors. qPCR data revealed that GSCs

expressed miR-146a at markedly lower levels than the matched NGSCs, in both 08-

387 and 3359 cells (Figure 4b). Interestingly, after a search of the GEO Profiles

database (35), lower expression of miR-146a was also found in GBM stem-like cells

(Gene Expression database: GSE23806, p=0.0304, Figure S7)(36), which confirmed

our findings. Further, overexpression of miR-146a resulted in reduced proliferation of

GSCs (Figure 4c-4d). Similarly, miR-146a overexpression reduced sphere formation

frequency and sphere size by in vitro limiting dilution and sphere formation assays

(Figure 4e-4h), as well as in vivo tumorigenicity by frequency of tumor-initiating cells

(TICf) (Figureure 4i-4j), indicating that overexpression of miR-146a inhibited

expansion of GSCs. Moreover, additional stemness markers were assessed at

mRNA levels, which revealed that overexpression of miR-146a significantly

downregulated expression of SALL, SOX2, c-Myc, Nestin, and Oct4 in 08-387 GSCs

(Figure S8). These data suggest that overexpression of miR-146a reduced the

stemness of GBM cells.

POU3F2 and SMARCA5 are direct targets of miR-146a in GBM

To identify putative target mRNAs of miR-146a, bioinformatics analyses were

performed employing different miRNA target prediction tools. The neural transcription

factor BRN2 (encoded by the POU3F2 gene), and SMARCA5 among others, were

found as novel potential targets of miR-146a (Figure S9). Gene expression analysis

using the TCGA datasets (http://gepia.cancer-pku.cn/) revealed that both POU3F2

and SMARCA5 were highly expressed across different cancer types compared with



http://gepia.cancer-pku.cn/http://mcr.aacrjournals.org/

19

normal tissues (Figure S10a and Figure S11a), particularly in GBM (Figure 5a-b).

Notably, highexpression of both of POU3F2 and SMARCA5 correlated with worse

overall survival of glioma patients (Figure S10b and Figure S11b), and these were

negatively correlated with miR-146a expression (Figure S10c and Figure S11c), as

expected. To determine whether POU3F2 and SMARCA5 are putative targets,

different GBM cells were transfected with miR-146a mimic, which resulted in

significant reduction of both POU3F2 and SMARCA5 expression in U87MG,

U87MG/EGFRvIII, T98G, GBM30, and 08-387 cell lines at the mRNA level (Figure

5c-d). To confirm changes in expression of POU3F2 and SMARCA5 in the Dox-

inducible stable 08-387/miR-146a cell model (Figure 5e), we treated 08-387/miR-

146a cells with different dose of Dox, reduction of POU3F2 and SMARCA5 at both

mRNA and protein levels was observed (Figure 5f-5g). To exclude the off-target effect,

we detected expression of TRAF6, which is a known miR-146a target (37), and as

expected, reduction of TRAF6 was observed when miR-146a was overexpressed by

either miR-146a mimics (Figure S12a) or Dox treatment (Figure S12b). To further

investigate whether POU3F2 and SMARCA5 are direct and specific targets of miR-

146a, luciferase reporter containing wild-type/mutated 3’UTR of POU3F2 and

SMARCA5 were constructed (Figure 5h). Wild-type or mutated 3’UTR reporters of

POU3F2 and SMARCA5 were co-transfected with NC/miR-146a mimics into

U87MG/EGFRvIII, GBM30, and 08-387 cells, respectively. Consistent reduction in

luciferase activity by miR-146a was observed only with wild-type 3’UTR construct,

but not with the mutant construct (Figure 5i-5j). Collectively, these data suggest that




20

miR-146a suppressed POU3F2 and SMARCA5 by directly targeting their 3’-UTRs in

GBM cells.

Tumor suppression by miR-146a is mediated by POU3F2 and SMARCA5 and

both proteins are positively correlated in GBM

Once we found that tumor suppressive and TMZ-sensitizing functions of miR-146a

were mediated by knocking down the expression of its direct targets POU3F2 and

SMARCA5, we sought to determine the individual relative contributions of these

stemness-related transcription factors to miR-146a functions. Among the primary

GBM cell lines, we determined that 08-387 expressed relatively high levels of both

POU3F2 and SMARCA5, which was consistent with a relatively low expression of

miR-146a. We observed that 08-387 cells’ proliferation, invasion, and TMZ-response

were significantly reduced by siRNA-mediated knockdown of either POU3F2 or

SMARCA5 expression (Figure 6a-f). We noted that knockdown of either target was

sufficient to produce effects on cell proliferation, invasion, and TMZ-response

comparable to the effects produced by overexpression of miR-146a. To substantiate

this notion, miR-146a was inhibited in LN229 cells that expressed higher level of

miR146a (Figure 1e), which were then co-transfected with POU3F2 siRNA and/or

SMARCA5 siRNA (Figure 6g). Subsequent functional assays demonstrated that miR-

146a inhibition significantly induced cell proliferation and invasion, which were

significantly reversed by knocking down either POU3F2 or SMARCA5 expression

(Figure 6h-6i). In addition, miR-146a did not demonstrate a tumor suppressive

phenotype when POU3F2/SMARCA5 was overexpressed (Figure S13a-c). Thus,




21

either knocking down POU3F2 or SMARCA5 can mimic the effect of miR-146a in

GBM, which provoked us to investigate the correlation between POU3F2 and

SMARCA5 in GBM patients. Firstly, we analyzed mRNA expression of POU3F2 and

SMARCA5 from both TCGA (https://www.cbioportal.org/) and CGGA

(http://www.cgga.org.cn/) datasets, and found that POU3F2 and SMARCA5 were

positively correlated (Figure S14a-b and Figure S15a-b). To confirm the finding in

vitro, we knocked down either POU3F2 or SMARCA5 in 08-387 cells (Figure S15c-d),

and found that expression of SMARCA5 and POU3F2 was decreased at both protein

levels (Figure S15c-d). Collectively, these data confirmed that the tumor suppressive

potential of miR-146a is largely mediated by the miR-146a/POU3F2/SMARCA5 axis

(Figure 6i) in GBM.

Discussion

Rapid growth, invasion, and resistance to RT and TMZ are largely attributed to the

GSC population in GBM tumors. GSCs are capable of executing these pathological

functions through the activation of a number of signal transduction pathways,

including the PI3Kinase, Wnt/β-catenin, Notch, NF-κB and Jak-Stat among others.

Activation of these pathways results in aberrant expression of a variety of neural

stem cell- and GSC-related genes, which contribute to the maintenance of

proliferation, invasion, RT/TMZ-resistance functions in GSCs. Many of these

regulatory genes happen to be oncogenes and tumor suppressor genes, which are

post-transcriptionally suppressed by tumor suppressive miRNAs and onco-miRNAs,

respectively (38). By NanoString analysis of miRNA expression in human GBM



https://www.cbioportal.org/http://www.cgga.org.cn/http://mcr.aacrjournals.org/

22

specimens, we have identified and validated that miR-146a expression was

correlated with favorable patient overall survival. Its expression was partly

suppressed by its promoter methylation in primary GBM, and further it was noted that

expression of this miRNA was significantly lower in recurrent GBM indicating that

miR-146a may have further significance in tumor progression or recurrence. These

findings led to the hypothesis that miR-146 could be a tumor suppressor miRNA in

GBM and that it might negatively control therapeutic resistance mechanisms likely by

suppressing the GSC population and/or their functions. It is worth noting that miR-

146a functions as a tumor suppressor miRNA in a number of cancers including

NSCLC(39) and prostate cancer (40), and as an onco-miRNA in others that include

cervical, head and neck, and hepatocellular cancers (41-43). Kim et al. reported that

miR-146a was one of the miRNAs that involved in long survival GBM subclass, but

did not provided definitive evidence (44). To test our first hypothesis, we took two

reciprocal approaches: first, overexpression in primary PDX lines that expressed

relatively lower levels of endogenous miR-146, and second, inhibition of miR-146a in

cells, namely LN229 that expressed significantly higher level of miR-146. In these

reciprocally engineered cell models, we measured cell proliferation, invasion, and

stemness-markers in vitro, and tumor growth and overall mouse survival in

orthotopically implanted xenograft tumors. Our results clearly suggest that miR-146a

functions as a tumor suppressive miR in GBM. This observation is in good

agreement with another recent report that shows that ectopic expression of miR-146a

inhibits glioma development (45).




23

Next, we tested if miR-146a sensitizes GBM to TMZ and RT. To this end, our in vitro

data clearly indicate that miR-146 overexpression sensitized PDX lines to TMZ in

vitro, and in vivo which, in turn, significantly prolonged the mouse survival. The PDX

cell line models employed here, were sensitive to low dose (2Gy) of ionizing radiation

limiting the evaluation of RT-sensitizing potential of miR-146a.

Once we found that tumor suppressive and TMZ-sensitizing functions of miR-146a

were mediated by knocking down the expression of its direct targets POU3F2 and

SMARCA5, we sought to determine the relative contributions of these stemness-

related transcription factors to miR-146a functions in GBM. POU3F2 (BRN2), a

master regulator of neuronal differentiation, is a POU-domain transcription factor well

described in developmental biology (46). It has been reported that inhibiting BRN2

expression led to significantly reduced proliferation, migration, and invasion in SCLC,

prostate cancer, as well as melanoma (47-50). Importantly, targeting BRN2 is a

strategy to treat or prevent neuroendocrine differentiation in prostate cancer (50).

SMARCA5 (hSNF2H) is a member of SWI/SNF family, and contains helicase and

ATPase activities. hSNF2H promotes tumor growth in ovarian cancer and glioma

(51,52). It has also been shown that miR-100 affects stem cell self-renewal and cell

proliferation in part by targeting SMARCA5(53). Interestingly, Zhou et al. reported a

reverse correlation between expression of miR-146a and SMARCA5 in a bladder

tumor cell model (54), however, they did not provide evidence about the regulation

between miR-146a and SMARCA5. Among the primary GBM cell lines we examined,

08-387 expressed relatively high level of both POU3F2 and SMARCA5, which was




24

consistent with a relatively low expression of miR-146a. We observed that 08-387

cells’ proliferation, invasion, and TMZ-response were significantly reduced by siRNA-

mediated knockdown of either POU3F2 or SMARCA5 expression. We noted, with

surprise, that knockdown of either target was sufficient to produce effects on cell

proliferation, invasion, and TMZ-response comparable to the effects produced by

overexpression of miR-146a. To gain an insight of these unexpected observations,

we conducted a series of experiments which revealed that POU3F2 and SMARCA5

positively regulate each other in the absence of miR-146a involvement/modulation.

The study has potential limitations. First, miR-146a expression was tested in a small

sample size for primary and recurrent GBM, a large patient cohort is needed. Second,

the PDX cell line models employed here, were sensitive to low dose (2Gy) of ionizing

radiation limiting the evaluation of RT-sensitizing potential of miR-146a. Third, given

the fact that POU3F2 is a transcriptional regulator, it might regulate expression of

SMARCA5 through direct binding or indirectly through other targeted genes. Further

studies are needed to investigate the mechanism of the regulation between POU3F2

and SMARCA5 to identify the novel therapeutic strategies for GBM.

In summary, focusing on the tumor suppressive functions of miR-146a in GBM, this

study revealed the following novel findings: 1) Decreased miR-146a expression is

associated with shorter OS independent of MGMT methylation status in GBM; 2)

Promoter methylation-induced silencing of miR-146a drives tumor progression and

therapeutic resistance in glioblastoma; 3) miR-146a inhibits the stemness of GBM

cells via directly targeting POU3F2 and SMARCA5 whose expression were positively




25

correlated in GBM. Data from our human cohorts and in vitro/vivo mechanistic

analysis strongly implicate that miR-146a plays a significant role in the progression

and therapeutic sensitivity in GBM, making it or POU3F2 and SMARCA5 to be

potentially attractive and novel therapeutic targets.

Author Contributions

T.C. designed and performed experiments, analyzed results and wrote the

manuscript; E.H.B. and S.J.H. designed experiments and edited the manuscript. J.M.

analyzed Nanostring data, patient survival data and edited the manuscript. K.L., E.S.,

A.G., M.G., D.S., L.Y. performed experiments and edited the manuscript. E.S., B.J.,

and P.M.G performed the animal work. A.P.B. analyzed results and edited the

manuscript. J.F., W.M., M.V. edited the manuscript. Q.E.W. analyzed results and

edited the manuscript. P.A.R. provided patient samples and clinical annotations for

cohort 1 and edited the manuscript. J.S.B-S. provided patient samples and clinical

annotations for cohort 2 and edited the manuscript. A.C. coordinated the project and

edited the manuscript.

Acknowledgments

We thank Dr. Jeremy Rich (UC San Diego, USA) for providing primary GBM patient-

derived cells. We also thank The Ohio State University (OSU) Comprehensive

Cancer Center Small Animal Imaging Core, The Ohio State University (OSU)

Genomics Shared Resource (GSR), The Ohio State University (OSU) Target

Validation Shared Resource (TVSR), and The Ohio State University (OSU)

Comprehensive Cancer Center Pathology Core Facility supported in part by grant




26

P30 CA016058, National Cancer Institute, Bethesda, MD. This work was also

supported by National Cancer Institute [R01CA169368 (to A.C), R01CA11522358 (to

A.C), R01CA1145128 (to A.C), R01CA108633 (to A.C), R01CA188228 (to A.C., R.B.,

K.L., and J.S.B-S.), 1RC2CA148190 (to A.C), and U10CA180850-01 (to A.C.)]; A

Brain Tumor Funders Collaborative Grant (to A.C.); Ohio State University

Comprehensive Cancer Center Award (to A.C.), and the T&P Bohnenn Fund for

Neuro-Oncology Research (grant to P.A.R.).

References

1. Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D,

Cavenee WK, et al. The 2016 World Health Organization Classification of

Tumors of the Central Nervous System: a summary. Acta Neuropathol

2016;131(6):803-20 doi 10.1007/s00401-016-1545-1.

2. Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, et

al. Effects of radiotherapy with concomitant and adjuvant temozolomide

versus radiotherapy alone on survival in glioblastoma in a randomised phase

III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol

2009;10(5):459-66 doi 10.1016/S1470-2045(09)70025-7.

3. Ryken TC, Kalkanis SN, Buatti JM, Olson JJ. The role of cytoreductive

surgery in the management of progressive glioblastoma. J Neuro-Oncol

2014;118(3):479-88 doi 10.1007/s11060-013-1336-7.

4. Ostrom QT, Cioffi G, Gittleman H, Patil N, Waite K, Kruchko C, et al. CBTRUS

Statistical Report: Primary Brain and Other Central Nervous System Tumors

Diagnosed in the United States in 2012-2016. Neuro Oncol

2019;21(Supplement_5):v1-v100 doi 10.1093/neuonc/noz150.

5. Weller M, Pfister SM, Wick W, Hegi ME, Reifenberger G, Stupp R. Molecular

neuro-oncology in clinical practice: a new horizon. Lancet Oncol

2013;14(9):e370-9 doi 10.1016/S1470-2045(13)70168-2.

6. Thakkar JP, Dolecek TA, Horbinski C, Ostrom QT, Lightner DD, Barnholtz-

Sloan JS, et al. Epidemiologic and molecular prognostic review of

glioblastoma. Cancer Epidemiol Biomarkers Prev 2014;23(10):1985-96 doi

10.1158/1055-9965.EPI-14-0275.

7. Chen J, Li YJ, Yu TS, McKay RM, Burns DK, Kernie SG, et al. A restricted cell

population propagates glioblastoma growth after chemotherapy. Nature

2012;488(7412):522-+ doi 10.1038/nature11287.

8. Auffinger B, Tobias AL, Han Y, Lee G, Guo D, Dey M, et al. Conversion of

differentiated cancer cells into cancer stem-like cells in a glioblastoma model




27

after primary chemotherapy. Cell Death Differ 2014;21(7):1119-31 doi

10.1038/cdd.2014.31.

9. Tamura K, Aoyagi M, Wakimoto H, Ando N, Nariai T, Yamamoto M, et al.

Accumulation of CD133-positive glioma cells after high-dose irradiation by

Gamma Knife surgery plus external beam radiation. J Neurosurg

2010;113(2):310-8 doi 10.3171/2010.2.JNS091607.

10. De Bacco F, D'Ambrosio A, Casanova E, Orzan F, Neggia R, Albano R, et al.

MET inhibition overcomes radiation resistance of glioblastoma stem-like cells.

EMBO Mol Med 2016;8(5):550-68 doi 10.15252/emmm.201505890.

11. Lathia JD, Mack SC, Mulkearns-Hubert EE, Valentim CLL, Rich JN. Cancer

stem cells in glioblastoma. Gene Dev 2015;29(12):1203-17 doi

10.1101/gad.261982.115.

12. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, et al.

Identification of human brain tumour initiating cells. Nature

2004;432(7015):396-401 doi 10.1038/nature03128.

13. Liu G, Yuan X, Zeng Z, Tunici P, Ng H, Abdulkadir IR, et al. Analysis of gene

expression and chemoresistance of CD133+ cancer stem cells in

glioblastoma. Mol Cancer 2006;5:67 doi 10.1186/1476-4598-5-67.

14. Zhang N, Wei P, Gong A, Chiu WT, Lee HT, Colman H, et al. FoxM1 promotes

beta-catenin nuclear localization and controls Wnt target-gene expression

and glioma tumorigenesis. Cancer Cell 2011;20(4):427-42 doi

10.1016/j.ccr.2011.08.016.

15. Wang JL, Wakeman TP, Lathia JD, Hjelmeland AB, Wang XF, White RR, et al.

Notch Promotes Radioresistance of Glioma Stem Cells. Stem Cells

2010;28(1):17-28 doi 10.1002/stem.261.

16. Kim SH, Ezhilarasan R, Phillips E, Gallego-Perez D, Sparks A, Taylor D, et al.

Serine/Threonine Kinase MLK4 Determines Mesenchymal Identity in Glioma

Stem Cells in an NF-kappaB-dependent Manner. Cancer Cell 2016;29(2):201-

13 doi 10.1016/j.ccell.2016.01.005.

17. Cao Y, Lathia JD, Eyler CE, Wu Q, Li Z, Wang H, et al. Erythropoietin

Receptor Signaling Through STAT3 Is Required For Glioma Stem Cell

Maintenance. Genes Cancer 2010;1(1):50-61 doi

10.1177/1947601909356352.

18. Beyer S, Fleming J, Meng W, Singh R, Haque SJ, Chakravarti A. The Role of

miRNAs in Angiogenesis, Invasion and Metabolism and Their Therapeutic

Implications in Gliomas. Cancers (Basel) 2017;9(7) doi

10.3390/cancers9070085.

19. Matos B, Bostjancic E, Matjasic A, Popovic M, Glavac D. Dynamic expression

of 11 miRNAs in 83 consecutive primary and corresponding recurrent

glioblastoma: correlation to treatment, time to recurrence, overall survival and

MGMT methylation status. Radiol Oncol 2018;52(4):422-32 doi 10.2478/raon-

2018-0043.

20. Huang SW, Ali ND, Zhong L, Shi J. MicroRNAs as biomarkers for human

glioblastoma: progress and potential. Acta Pharmacol Sin 2018;39(9):1405-13




28

doi 10.1038/aps.2017.173.

21. Henriksen M, Johnsen KB, Andersen HH, Pilgaard L, Duroux M. MicroRNA

Expression Signatures Determine Prognosis and Survival in Glioblastoma

Multiforme-a Systematic Overview. Mol Neurobiol 2014;50(3):896-913 doi

10.1007/s12035-014-8668-y.

22. Low SYY, Ho YK, Too HP, Yap CT, Ng WH. MicroRNA as potential modulators

in chemoresistant high-grade gliomas. J Clin Neurosci 2014;21(3):395-400

doi 10.1016/j.jocn.2013.07.033.

23. Bo LJ, Wei B, Li ZH, Wang ZF, Gao Z, Miao Z. Bioinformatics analysis of

miRNA expression profile between primary and recurrent glioblastoma. Eur

Rev Med Pharmaco 2015;19(19):3579-86.

24. Cui T, Bell EH, McElroy J, Becker AP, Gulati PM, Geurts M, et al. miR-4516

predicts poor prognosis and functions as a novel oncogene via targeting

PTPN14 in human glioblastoma. Oncogene 2019;38(16):2923-36 doi

10.1038/s41388-018-0601-9.

25. Cui T, Chen Y, Yang L, Knosel T, Zoller K, Huber O, et al. DSC3 expression is

regulated by p53, and methylation of DSC3 DNA is a prognostic marker in

human colorectal cancer. Brit J Cancer 2011;104(6):1013-9 doi

10.1038/bjc.2011.28.

26. Cui TT, Chen Y, Yang LL, Knosel T, Huber O, Pacyna-Gengelbach M, et al.

The p53 target gene desmocollin 3 acts as a novel tumor suppressor through

inhibiting EGFR/ERK pathway in human lung cancer. Carcinogenesis

2012;33(12):2326-33 doi 10.1093/carcin/bgs273.

27. Hu YF, Smyth GK. ELDA: Extreme limiting dilution analysis for comparing

depleted and enriched populations in stem cell and other assays. J Immunol

Methods 2009;347(1-2):70-8 doi 10.1016/j.jim.2009.06.008.

28. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, et al.

Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.

N Engl J Med 2005;352(10):987-96 doi 10.1056/NEJMoa043330.

29. Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, et al.

MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl

J Med 2005;352(10):997-1003 doi 10.1056/NEJMoa043331.

30. Hu HQ, Sun LG, Guo WJ. Decreased miRNA-146a in glioblastoma multiforme

and regulation of cell proliferation and apoptosis by target Notch1. Int J Biol

Marker 2016;31(3):E270-E5 doi 10.5301/jbm.5000194.

31. Ha M, Kim VN. Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol

2014;15(8):509-24 doi 10.1038/nrm3838.

32. Wang XL, Gao HT, Ren LX, Gu JF, Zhang YP, Zhang Y. Demethylation of the

miR-146a promoter by 5-Aza-2 '-deoxycytidine correlates with delayed

progression of castration-resistant prostate cancer. Bmc Cancer 2014;14 doi

Artn 30810.1186/1471-2407-14-308.

33. Osuka S, Van Meir EG. Overcoming therapeutic resistance in glioblastoma:

the way forward. J Clin Invest 2017;127(2):415-26 doi 10.1172/Jci89587.

34. Dermawan JKT, Hitomi M, Silver DJ, Wu QL, Sandlesh P, Sloan AE, et al.




29

Pharmacological Targeting of the Histone Chaperone Complex FACT

Preferentially Eliminates Glioblastoma Stem Cells and Prolongs Survival in

Preclinical Models. Cancer Res 2016;76(8):2432-42 doi 10.1158/0008-

5472.Can-15-2162.

35. Barrett T, Troup DB, Wilhite SE, Ledoux P, Rudnev D, Evangelista C, et al.

NCBI GEO: mining tens of millions of expression profiles--database and tools

update. Nucleic Acids Res 2007;35(Database issue):D760-5 doi

10.1093/nar/gkl887.

36. Schulte A, Gunther HS, Phillips HS, Kemming D, Martens T, Kharbanda S, et

al. A distinct subset of glioma cell lines with stem cell-like properties reflects

the transcriptional phenotype of glioblastomas and overexpresses CXCR4 as

therapeutic target. Glia 2011;59(4):590-602 doi 10.1002/glia.21127.

37. Stickel N, Prinz G, Pfeifer D, Hasselblatt P, Schmitt-Graeff A, Follo M, et al.

MiR-146a regulates the TRAF6/TNF-axis in donor T cells during GVHD.

Blood 2014;124(16):2586-95 doi 10.1182/blood-2014-04-569046.

38. Esquela-Kerscher A, Slack FJ. Oncomirs - microRNAs with a role in cancer.

Nat Rev Cancer 2006;6(4):259-69 doi 10.1038/nrc1840.

39. Li YL, Wang J, Zhang CY, Shen YQ, Wang HM, Ding L, et al. MiR-146a-5p

inhibits cell proliferation and cell cycle progression in NSCLC cell lines by

targeting CCND1 and CCND2. Oncotarget 2016;7(37):59287-98 doi

10.18632/oncotarget.11040.

40. Huang Y, Tao T, Liu C, Guan H, Zhang G, Ling Z, et al. Upregulation of miR-

146a by YY1 depletion correlates with delayed progression of prostate cancer.

Int J Oncol 2017;50(2):421-31 doi 10.3892/ijo.2017.3840.

41. Wang X, Tang S, Le SY, Lu R, Rader JS, Meyers C, et al. Aberrant expression

of oncogenic and tumor-suppressive microRNAs in cervical cancer is required

for cancer cell growth. PLoS One 2008;3(7):e2557 doi

10.1371/journal.pone.0002557.

42. Hung PS, Chang KW, Kao SY, Chu TH, Liu CJ, Lin SC. Association between

the rs2910164 polymorphism in pre-mir-146a and oral carcinoma progression.

Oral Oncol 2012;48(5):404-8 doi 10.1016/j.oraloncology.2011.11.019.

43. Xu T, Zhu Y, Wei QK, Yuan Y, Zhou F, Ge YY, et al. A functional polymorphism

in the miR-146a gene is associated with the risk for hepatocellular carcinoma.

Carcinogenesis 2008;29(11):2126-31 doi 10.1093/carcin/bgn195.

44. Kim TM, Huang W, Park R, Park PJ, Johnson MD. A Developmental

Taxonomy of Glioblastoma Defined and Maintained by MicroRNAs. Cancer

Res 2011;71(9):3387-99 doi 10.1158/0008-5472.Can-10-4117.

45. Mei J, Bachoo R, Zhang CL. MicroRNA-146a inhibits glioma development by

targeting Notch1. Mol Cell Biol 2011;31(17):3584-92 doi 10.1128/MCB.05821-

11.

46. Dominguez MH, Ayoub AE, Rakic P. POU-III transcription factors (Brn1, Brn2,

and Oct6) influence neurogenesis, molecular identity, and migratory

destination of upper-layer cells of the cerebral cortex. Cereb Cortex

2013;23(11):2632-43 doi 10.1093/cercor/bhs252.




30

47. Ishii J, Sato H, Sakaeda M, Shishido-Hara Y, Hiramatsu C, Kamma H, et al.

POU domain transcription factor BRN2 is crucial for expression of ASCL1,

ND1 and neuroendocrine marker molecules and cell growth in small cell lung

cancer. Pathol Int 2013;63(3):158-68 doi 10.1111/pin.12042.

48. Boyle GM, Woods SL, Bonazzi VF, Stark MS, Hacker E, Aoude LG, et al.

Melanoma cell invasiveness is regulated by miR-211 suppression of the

BRN2 transcription factor. Pigm Cell Melanoma R 2011;24(3):525-37 doi

10.1111/j.1755-148X.2011.00849.x.

49. Pinner S, Jordan P, Sharrock K, Bazley L, Collinson L, Marais R, et al.

Intravital Imaging Reveals Transient Changes in Pigment Production and

Brn2 Expression during Metastatic Melanoma Dissemination. Cancer Res

2009;69(20):7969-77 doi 10.1158/0008-5472.Can-09-0781.

50. Bishop JL, Thaper D, Vahid S, Davies A, Ketola K, Kuruma H, et al. The

Master Neural Transcription Factor BRN2 Is an Androgen Receptor-

Suppressed Driver of Neuroendocrine Differentiation in Prostate Cancer.

Cancer Discov 2017;7(1):54-71 doi 10.1158/2159-8290.Cd-15-1263.

51. Sheu JJ, Choi JH, Yildiz I, Tsai FJ, Shaul Y, Wang TL, et al. The roles of

human sucrose nonfermenting protein 2 homologue in the tumor-promoting

functions of Rsf-1. Cancer Res 2008;68(11):4050-7 doi 10.1158/0008-

5472.CAN-07-3240.

52. Zhao XC, An P, Wu XY, Zhang LM, Long B, Tian Y, et al. Overexpression of

hSNF2H in glioma promotes cell proliferation, invasion, and chemoresistance

through its interaction with Rsf-1. Tumor Biol 2016;37(6):7203-12 doi

10.1007/s13277-015-4579-4.

53. Deng L, Shang L, Bai S, Chen J, He X, Martin-Trevino R, et al. MicroRNA100

inhibits self-renewal of breast cancer stem-like cells and breast tumor

development. Cancer Res 2014;74(22):6648-60 doi 10.1158/0008-5472.CAN-

13-3710.

54. Zhou W, Thiery JP. Loss of Git2 induces epithelial-mesenchymal transition by

miR146a-Cnot6L-controlled expression of Zeb1. J Cell Sci 2013;126(Pt

12):2740-6 doi 10.1242/jcs.126367.

Figure Legends

Figure 1 Expression of miR-146a in GBM patient samples and cell lines. (a)

Correlation between miR-146a expression and OS by Kaplan-Meier analysis of GBM

patients with high (n=134) and low (n=134) expression of miR-146a. (b) Expression

of miR-146a in primary and recurrent GBM by Nanostring analysis. (c) Expression of

miR-146a in primary and recurrent GBM by qRT-PCR in cohort 1(14 pairs). n=3,

**P

31

RT-qPCR in cohort 2 (8 pairs). n=3, *P

32

or GBM30/miR-146a/luc cells without or with Dox (n = 10/group). (n-o) Expression of

miR-146a was detected after isolating RNAs from tumors harvested from mice brain

when the mice became moribund. n=5, ***P

33

vitro limiting dilution, cells were plated in a limiting dilution manner in 96-well plates,

and number of wells containing spheres calculated after 2-3 weeks to calculate the

SFCf (e,g). Sphere formation assay was conducted and number of spheres was

counted in 3 different areas and mean value was calculated (f,h). n=3, **P

34

GBM30, and 08-387 cells were co-transfected with the luciferase reporter

constructs containing 3′UTR (wt) or 3′UTR (mut) together with NC or miR-146a

mimics. Seventy-two hours post transfection, luciferase activity was measured and

normalized. n = 3, *P

Primary Recurrent

7.5

7.0

6.5

6.0

5.5

5.0 p=0.0047 No

rmali

zed

miR

-146

a L

ev

el

(Nan

os

trin

g)

a b

e f

U251

M U

T98G

M U

U87MG/EGFRvIII

M U

U87MG

M U

LN18

M U

08-387

M U

3359

M U

GBM30

M U

LN229

M U

g

Case 1

M U

Case 2

M U

Case 3

M U

Case 4

M U

Case 5

M U

Case 6

M U

Case 7

M U M U M U M U

Case 8 Case 9 Case 10

h

i

c

Cohort 1

**

***

***

**

*** ***

*** ***

***

** **

d

Cohort 2

** ** ** **

* *

*** ***

**

***

***

***

***

**

** **

CpG sites -183 -160 -150 -142 -138 -136 -132 -128

U87MG

GBM30

LN18

U87MG/EGFRvIII

U251

08-387

T98G

3359

LN229

Promoter Region

Figure 1




GBM30

08-387

NC miR-146a

GBM30/miR-146a/luc

a b c d

e f g h

i

j k

Dox (+) Dox (-)

10

17

25

Dox (+) Dox (-)

Radiance

(p/s/cm2/sr)

2.0

1.5

1.0

0.5

0

1×107

08-387/miR-146a/luc GBM30/miR-146a/luc

m

08-387/miR-146a/luc

LN229

***

***

*** ***

*** *** ***

**

** **

**

* **

***

** p

miR-NC miR-146a

DMSO

TMZ

(100µM)

Annexin V

PI

DMSO TMZ 100µM

NC

miR-146a

Dox (-) TMZ (-)

Dox (-) TMZ(+)

Dox (+) TMZ(-)

Dox (+) TMZ(+)

3.0

2.0

1.0

1×106

4.0

5.0

6.0

Radiance

(p/s/cm2/sr)

a b c d

e f

h

**

***

**g

c-caspase 3

08-387

TMZ(µM)

c-parp

parp

caspase 3

U87MG/EGFRvIII

NC miR-146a

0 100 500 0 100 500

NC miR-146a

0 50 100 0 50 100

i

08-387/miR-146a/luc

** p

Cell number

No. of Tumor

(injection sites)

NC miR-146a

2× 105 4(4) 4(4)

2× 104 4(4) 3(4)

2× 103 3(4) 2(4)

TICf 1/1443 1/9158

TICf

(TIC/104) 6.93 1.09

p 0.0296 08-387GSCs

e

i j

g

TICf

Fra

cti

on

of

wells w

ith

ou

t

tu

mo

r s

ph

ere

s (

log

2)

Number of Cells

miR-146a

08-387GSCs

NC

Fra

cti

on

of

wells w

ith

ou

t

tu

mo

r s

ph

ere

s (

log

2)

Number of Cells

miR-146a

3359GSCs

NC

Group NC miR-146a

Estimate

(Range)

1 in 12.7

(9.44-17.0)

1 in 21.8

(15.51-30.7)

p 0.0145

Group NC miR-146a

Estimate

(Range)

1 in 15.6

(11.4-21.3)

1 in 31.4

(21.3-46.4)

p 0.00457

SFCf

SFCf

*

f

h

**

**

OCT4

OLIG2

08-387 3359

GAPDH

a b c d

*

**

***

***

(Days)

Figure 4




Dox 1.0µM - +

SMARCA5

08-387/miR146a

GAPDH

POU3F2

GBM

6

5

4

3

2

1

0

PO

U3F

2 E

xp

res

sio

n

(TC

GA

, lo

g)

T N GBM

6

5

4

3

2

1

0

SM

AR

CA

5 E

xp

res

sio

n

(TC

GA

, lo

g)

T N

a b

e

c

d

f g h

i j

** ** **

*

**

***

***

**

**

** **

* * * *

**

*

**

***

**

** *

*

** **

*

Figure 5




0 50 100 0 50 100

siCtrl siSMARCA5

TMZ(µM)

GAPDH

parp

c-parp

SMARCA5

0 50 100 0 50 100

siCtrl siPOU3F2

TMZ(µM)

GAPDH

parp

c-parp

POU3F2

a

c d e

POU3F2

GAPDH

SMARCA5

GAPDH

POU3F2

SMARCA5

GAPDH

miR-146a inhibitor

siPOU3F2

siSMARCA5

+ - + + +

- - + - +

- - - + +

f g

h

** ** **

**

miR-146a inhibitor

siPOU3F2

siSMARCA5

+ - + + +

- - + - + - - - + +

+ - + + +

- - + - + - - - + +

**

**

+ - + + +

- - + - + - - - + +

miR-146a inhibitor

siPOU3F2

siSMARCA5

***

b

*** *

*

**

**

LN229

08-387 08-387

i

Figure 6




Published OnlineFirst September 24, 2020.Mol Cancer Res Tiantian Cui, Erica Hlavin Bell, Joseph McElroy, et al. stemness and therapeutic response in glioblastomaA novel miR-146a-POU3F2/SMARCA5 pathway regulates

Updated version

10.1158/1541-7786.MCR-20-0353doi:

Access the most recent version of this article at:

Material

Supplementary

http://mcr.aacrjournals.org/content/suppl/2020/09/24/1541-7786.MCR-20-0353.DC1

Access the most recent supplemental material at:

Manuscript

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

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

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mcr.aacrjournals.org/content/early/2020/09/24/1541-7786.MCR-20-0353To request permission to re-use all or part of this article, use this link



http://mcr.aacrjournals.org/lookup/doi/10.1158/1541-7786.MCR-20-0353http://mcr.aacrjournals.org/content/suppl/2020/09/24/1541-7786.MCR-20-0353.DC1http://mcr.aacrjournals.org/cgi/alertsmailto:[email protected]://mcr.aacrjournals.org/content/early/2020/09/24/1541-7786.MCR-20-0353http://mcr.aacrjournals.org/

Article FileFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6

a novel mir-146a-pou3f2/smarca5 pathway regulates ......2020/09/24 · author manuscripts have been...

Documents