A Novel Mir-146A-POU3F2/SMARCA5 Pathway Regulates Stemness and Therapeutic Response in Glioblastoma a C Tiantian Cui1, Erica H

Published OnlineFirst September 24, 2020; DOI: 10.1158/1541-7786.MCR-20-0353

MOLECULAR CANCER RESEARCH | CANCER GENES AND NETWORKS

A Novel miR-146a-POU3F2/SMARCA5 Pathway Regulates Stemness and Therapeutic Response in Glioblastoma A C 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 L. Fleming1, Wei Meng1, Jill S. Barnholtz-Sloan6, Monica Venere1, Qi-En Wang1, Pierre A. Robe7, S. Jaharul Haque1, and Arnab Chakravarti1

ABSTRACT ◥ Rapid tumor growth, widespread brain-invasion, and thera- Mechanistically, miR-146a directly silenced POU3F2 and peutic resistance critically contribute to glioblastoma (GBM) SMARCA5, two transcription factors that mutually regulated recurrence and dismal patient outcomes. Although GBM stem each other, significantly compromising GBM-stemness and cells (GSC) are shown to play key roles in these processes, the increasing TMZ response. Collectively, our data show that molecular pathways governing the GSC phenotype (GBM-stem- miR-146a–POU3F2/SMARCA5 pathway plays a critical role in ness) remain poorly defined. Here, we show that epigenetic suppressing GBM-stemness and increasing TMZ-response, sug- silencing of miR-146a significantly correlated with worse patient gesting that POU3F2 and SMARCA5 may serve as novel thera- outcome and importantly, miR-146a level was significantly peutic targets in GBM. lower in recurrent tumors compared with primary ones. Further, miR-146a overexpression significantly inhibited the proliferation Implications: miR-146a predicts favorable prognosis and the and invasion of GBM patient-derived primary cells and increased miR-146a–POU3F2/SMARCA5 pathway is important for the their response to temozolomide (TMZ), both in vitro and in vivo. suppression of stemness in GBM.

Introduction resection, followed by adjuvant radiation and temozolomide (TMZ) chemotherapy (1–3). Despite the aggressive multimodality treatments, Glioblastomas (GBM, WHO grade IV gliomas) are the most the median survival remains within the range of 12 to 15 months after aggressive and lethal primary malignant tumors of the central nervous diagnosis(4).Thisgrimpatientoutcomeislargelyattributedto system in adults (1). Recently, the WHO has classified GBM and rapid tumor growth, invasion of vital brain structures, and evolu- lower grade gliomas based on the neomorphic mutation status of tion of intrinsic and acquired treatment resistance clones among isocitrate dehydrogenase (IDH) 1 or 2, and irrespective of grades, other factors (4). However, we do not have a clear understanding gliomas with wild-type (wt) IDH1/2 have worse clinical outcome of the molecular signaling networks driving disease progression compared with those with mutant IDH1/2 (1). More than 90% of and treatment resistance. Although a number of prognostic bio- GBMs have wt-IDH1/2 and irrespective of IDH status patients with markers, including promoter methylation of O6-methylguanine- newly diagnosed GBM receive the standard of care with maximal safe DNA methyltransferase (MGMT), neomorphic mutation of IDH1/ 2,amplification, and/or gain-of-function mutation of EGFR,lossof function mutation/deletion of TP53 or PTEN,andCpGisland 1 Department of Radiation Oncology, Arthur G. James Hospital/Ohio State methylation phenotype (5, 6), have been identified and character- 2 Comprehensive Cancer Center, Columbus, Ohio. The Ohio State University ized, the molecular mechanisms underlying the activation of sig- Center for Biostatistics, Department of Biomedical Informatics, Columbus, Ohio. 3The Ohio State University College of Medicine, Columbus, Ohio. 4Erasmus naling pathways driving the treatment resistance and tumor recur- Medical Center Cancer Institute, Rotterdam, the Netherlands. 5Berea College, rence have remained poorly understood posing a great challenge in Berea, Kentucky. 6Department of Population and Quantitative Health Sciences developing truly effective therapeutic intervention. To this end, a and Case Comprehensive Cancer Center, Case Western Reserve University number of studies demonstrate that a posttreatment increase in 7 School of Medicine, Cleveland, Ohio. Department of Neurology and Neurosur- GSC population markedly contributes to tumor recurrence and gery, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, therapeutic resistance mechanisms (7–9). GSCs possess potential of the Netherlands. self-renewal and multipotent differentiation, and are responsible for Note: Supplementary data for this article are available at Molecular Cancer tumor initiation and maintenance (7, 10–13). These GSC functions Research Online (http://mcr.aacrjournals.org/). are largely mediated by deregulation of Wnt/b-catenin (14), Corresponding Author: Arnab Chakravarti, Ohio State University Comprehen- Notch (15), NF-kB (16), and JAK–STAT (17) signaling pathways sive Cancer Center and Richard L. Solove Research Institute, Arthur G. James among others resulting in an aberrant expression of downstream Cancer Hospital, The Ohio State University Medical School, 460 W. 10th Ave, signature molecules that drive the resistance and recurrence Room D252F, Columbus, Ohio 43210. Phone: 614-293-0672; Fax: 614- 293-1943; E-mail: [email protected] mechanisms in GBM. To this end, we sought to identify molecular biomarkers/signatures of prognostic values and significance that Mol Cancer Res 2020;XX:XX–XX drive treatment resistance and recurrence in GBM, and to target doi: 10.1158/1541-7786.MCR-20-0353 these drivers in preclinical GBM models for the development of 2020 American Association for Cancer Research. novel therapeutic interventions.

AACRJournals.org | OF1

Cui et al.

miRNAs play regulatory roles through silencing the expression of and U87MG/EGFRvIII cells were grown in DMEM medium (Gibco) target genes by posttranscriptional degradation and translational supplemented with 10% (v/v) FBS (Invitrogen), 100 U/mL penicillin/ repression. Evidence shows that miRNAs are involved in regulating streptomycin (Sigma), and maintained in a humidified atmosphere the progression and therapeutic resistance mechanisms in a variety with 5% CO2 at 37 C. GBM30, 08-387, 3359, GBM12, and GBM43 of malignancies including GBM by modulating cancer stem cell cells were cultured in neurobasal medium with EGF (20 ng/mL), FGF functions (18). Previous studies have shown differential miRNA (20 ng/mL), B27 (1), and GlutaMax (1), and sodium pyruvate (1) fi expression in GBM tumor tissues; moreover, tumors harboring in a humidi ed atmosphere with 5% CO2 at 37 C. All cells were altered miRNA expression had differential prognoses and treatment periodically tested for mycoplasma contamination using Universal outcomes (19–22). Therefore, the differentially expressed miRNAs Mycoplasma Detection Kit (ATCC30-1012K). may serve as biomarkers and/or drivers of prognosis and treatment response in GBM. Very few studies have investigated the expression qRT-PCR analysis of mRNA expression and miRNA expression of miRNA in primary and recurrent GBM (19, 23). Hence, For mRNA expression analysis, total RNA was extracted from GBM we undertook a high-throughput molecular profiling approach to cells using TRizol reagent as described previously (24). The primers are identify miRNAs that correlate with clinical outcomes of patients listed in Supplementary Table S1. For miRNA expression analysis, with IDH1/2 wt GBM. We identified miR-146a as a tumor sup- RNA (100 ng) was reverse transcribed using the TaqMan Advanced pressor miRNA in GBM. Subsequently, we have identified and MicroRNA Assay Kit (Applied Biosystems) with miRNA-specific characterized two direct targets of miR-146a, POU domain, class primers. Primers of Taqman MicroRNA Assays for hsa-miR-146a 3, transcription factor 2 (POU3F2) and SWI/SNF Related, Matrix (Assay ID: 000468) and RNU6B (Assay ID: 001093) were purchased Associated, Actin Dependent Regulator of Chromatin, Subfamily A, from Invitrogen. Relative expression level of miR-146a was calculated Member 5 (SMARCA5), which are found to play key roles in tumor by normalization with RNU6B expression in GBM cells. All the growth and treatment response. experiments were performed in triplicate.

Demethylation tests, bisulfite modification, methylation- Materials and Methods specific PCR, and bisulfite sequencing Patient cohort, ethics statement, and RNA isolation GBM cells were plated and cultured on six-well plates. At 50% We used two patient cohorts in this study. Briefly, a total of 268 confluence, 10 mmol/L DAC (Sigma) was added to the medium on (n ¼ 268) FFPE tumor specimens without IDH1R132H mutation days 1 and day 3. Cells were then harvested for total RNA isolation and from patient cohort 1 with newly diagnosed GBM were used in the RT-qPCR analysis. Bisulfite modification, methylation-specific PCR study. All of the patients underwent tumor biopsy or resection at the (MSP), and bisulfate sequencing (BS) were carried out as described University of Utrecht (the Netherlands, cohort 1) from 2005 to previously (25). Primer pairs for MSP and BS are in Supplementary 2014. A total of 11 pairs (n ¼ 11) of fresh frozen tumor specimens Table S1. from patient cohort 2 with newly diagnosed GBM and matched recurrent GBM were used as a validation set in the study. All of the Western blot analysis patients underwent tumor resection at University Hospitals of Protein extraction and Western blot analysis were performed as Cleveland (cohort 2) from 2008 to 2015 and provided written described previously (26). The antibodies used in this study informed consent to participate in brain tumor research. The study are listed in Supplementary Table S2. Polyclonal goat anti-rabbit was approved by The Ohio State University, University Hospital of antibody (Cell Signaling Technology) and Western Blotting Detec- Cleveland, and University Medical Center Utrecht institutional tion System (Millipore) were used for exposure. review boards. Transfection of miR-146a mimics and inhibitors, siRNA miRNA expression analysis and MGMT promoter methylation treatment, and Dox-inducible stable cells analyses For functional studies, a specific miR-146 mimic/inhibitor Tumor samples were processed to generate miRNA expression (Invitrogen) and a respective negative control (Invitrogen) were data using the NanoString human v3a array, which contains 798 transfected into the GBM cells. For siRNA transfection, specific miRNA probes. DNA (250 ng) input was used per sample and the POU3F2 1/2 and SMARCA5 1/2 siRNAs (50 nmol/L; Dharmacon) MGMT-STP27 model was used to calculate MGMT promoter meth- and a negative control (Dharmacon) were used. For POU3F2 ylation status from Illumina EPIC data (24). overexpression, pcDNA3.1þ/C-(K)DYK-POU3F2 plasmid (Gen- Script) was used. Tet-On shmimic-inducible miR-146a construct Cell lines and cell culture was purchased from Dharmacon. All siRNAs were transfected into Normal human astrocytes (NHA) were purchased from Lonza. cells using Lipofectamine 2000 transfection reagent (Invitrogen). Human GBM cell lines U251, T98G, LN229, U87MG, LN18, were purchased from ATCC. No further authentication was performed Cell proliferation and invasion assays for NHAs and ATCC cell lines. U87MG/EGFRvIII cells were provided For cell proliferation assay, 24 hours posttransfection, cells by Dr. Deliang Guo (The Ohio State University). Primary GBM (1,000 cells/well) were seeded in 96-well plates and measured by patient-derived GBM30-luc cells were kindly provided by Dr. Balveen CellTiter-Glo (Promega) after indicated time points as described Kaur (The Ohio State University). Primary GBM patient-derived cells previously (24). Cell invasion assays were performed as described (08-387 and 3359) were kindly provided by Dr. Jeremy Rich (UC San previously (24). All the experiments were performed in triplicate. Diego) and originally isolated from tumor resections in accordance with approved institutional review board protocols. GBM12 and Colony formation and sphere formation assays GBM43 were kindly provided by Mayo Clinic. These cell lines were GBM cells (100 cells/well) were used for colony formation assay as authenticated by DNA profiling. U251, T98G, LN229, U87MG, LN18, described previously (24). All the experiments were performed in

OF2 Mol Cancer Res; 2020 MOLECULAR CANCER RESEARCH

A Novel miR-146a-POU3F2/SMARCA5 Pathway in Glioblastoma

triplicate. A total of 300 cells were mixed with neurobasal medium in a became moribund and the tumor tissues were harvested for miR- fi humidi ed atmosphere with 5% CO2 at 37 C. The number of spheres 146a expression detection. was counted 2 to 3 weeks after cell seeding. All the experiments were performed in triplicate. To evaluate the frequency of sphere- Statistical analysis forming cells (SFCf), cells were plated in 96-well plates in a limiting Analysis of Nanostring data was carried out in R (24). Cox regres- dilution manner (1, 5, 10, 20 cells/well) using FACS. The number of sion was used to identify the association between expression of wells containing spheres was counted after 2 weeks, and the SFCf miRNAs (continuous) and overall survival (OS) with age as a covari- was calculated using the ELDA software http://bioinf.wehi.edu.au/ able. miR-146a was then median dichotomized and the log-rank test software/elda/index.html (27). was employed to visualize the association between the expression and OS. Other statistical analyses were performed using the software Cell viability assay package SPSS 23.0 (SPSS). Descriptive statistics, that is, means SD, Twenty-four hours posttransfection, cells (2,000 cells/well) were are shown on the Figures. Two sample t tests or ANOVA were seeded in 96-well plates, then treated with different dose of TMZ, performed for data analysis for experiments with two groups or more 72 hours after treatment, cell viability was measured by CellTiter-Glo than two groups’ comparisons. Spearman correlation analyses was (Promega). applied to analyze the association between expression of miR-146a and POU3F2/SMARCA5. The publicly available CGGA datasets were Apoptosis assay directly analyzed from the CGGA Data Portal at http://www.cgga. An Annexin V-fluorescence isothiocyanate (FITC) Apoptosis org.cn/. The detailed information of the RNA-seq experiments and Detection Kit (Invitrogen) was used for apoptosis assay. At 72 hours software used can be found at the CGGA Data Portal at http://www. miR-146a overexpression, 106 cells were resuspended in 200 mL cgga.org.cn/. P values were calculated two-sided. P value less than 0.05 binding buffer with 10 mL Annexin-V-FITC and 5 mL propidium was defined as statistically significant. iodide, and incubated in the dark for another 30 minutes. Finally, cells were assessed using flow cytometric analysis. All the experiments were performed in triplicate. Results Decreased miR-146a expression is associated with shorter OS of Luciferase reporter assay patients with GBM The 30UTR of POU3F2 and SMARCA5 was synthesized, To investigate a potential association between miRNA expression annealed, and then inserted into the Xho l and Not I sites of the and GBM patient outcomes, miRNA expression profiling in GBM pCheck2-reporter luciferase vector downstream of the stop codon tumor specimens of a cohort of 268 patients with wt-IDH1/2 was of the gene for luciferase. To induce mutagenesis, the sequences conducted using Nanostring v3 technology (24). miR-146a was iden- complementary to the binding sites of miR-146a in the 30UTR was tified to be one of the top miRNAs, which upon univariable analysis replaced by the mutated sites (Supplementary Table S1). The with continuous expression values [hazard ratio (HR) ¼ 0.658; 95% constructs were confirmed by sanger sequencing. Cells were confidence interval (CI), 0.534–0.810; P < 0.001; Supplementary cotransfected with the wild type or mutated construct, pCheck2 Table S3] showed decreased miR-146a was associated with worse OS. plasmid, and equal amount of negative control or miR-146a mimic. Moreover, multivariable analysis (MVA) showed that independent of Luciferase assays were performed using the Dual Luciferase Report- clinical variables, which include age, sex, KPS, and treatment (HR ¼ er Assay System (Promega). 0.657; 95% CI, 0.527–0.712; P < 0.001; Supplementary Table S3), decreased miR-146a was significantly associated with worse prognosis. Xenograft tumor study Radiotherapy (RT) plus concomitant and adjuvant TMZ has been the Six- to eight-week-old athymic nude mice were obtained from standard of care for patients with GBM for over a decade, as opposed to the Target Validation Shared Resource at the Ohio State University. RT alone, as the addition of TMZ resulted in longer OS and progres- All animal procedures were approved by the Subcommittee on sion-free survival (PFS; ref. 28). Importantly, this benefit was observed Research Animal Care at The Ohio State University. To determine in patients harboring a methylated MGMT promoter (29). We also the frequency of tumor-initiating cells (TICf) using the limiting found that miR-146a continued to be a strong independent prognostic dilution assay, three cell doses (1 105,1 104,1 103)ofeach factor in a multimarker MVA with MGMT promoter methylation sample were injected subcutaneously into athymic nude mice. Mice status (HR ¼ 0.586; 95% CI, 0.450–0.764; P < 0.001; Supplementary were monitored for up to 4 weeks postinjection, and the tumor Table S4). The mediandichotomized miR-146a expression also cor- number per group within this period was used to calculate the TICf related with OS (Fig. 1A). These findings were further validated in two using aforementioned ELDA software (27). For the intracranial miRNA-array datasets (TCGA: https://www.cancer.gov/tcga and xenograft models, GBM cells (1 105 08-387/miR-146a/luc and CGGA: http://www.cgga.org.cn/) by MVA (Supplementary Tables GBM30/miR-146a/luc in 2 mL PBS), transduced with Tet-On S5 and S6). To further explore if miR-146a expression played a role inducible miR-146a and GFP-tagged luciferase plasmids, were in tumor recurrence, we compared miR-146a expression levels in implanted into the brain of the mouse. Doxycycline (Dox) grain- matched GBM primary and recurrent tumor pairs by Nanostring based diet (Thermo Fisher Scientific) was administered 1 day after technology. We found significantly decreased expression of miR-146a injection. For drug treatment studies, mice were treated with vehicle in recurrent tumors (Fig. 1B, cohort 1, n ¼ 30, 15 pairs), which was or 20 mg/kg of TMZ resuspended in vehicle by oral gavage once a then validated by RT-qPCR (Fig. 1C, cohort 1, n ¼ 28, 14 pairs). To day for 5 days, starting at day 5 postinjection. Luciferin (Perkin further validate this observation, 11 pairs of GBM primary and Elmer) solution (100 mg/kg) was used for imaging after tumor recurrent tumor tissues from another patient cohort (cohort 2) were formation. The IVIS Lumina II imaging platform from the Ohio analyzed by RT-qPCR. Herein, miR-146a expression was undetectable State University (OSU) Small Animal Imaging Core was used to in three pairs (data not shown), but six out of the remaining eight pairs detect and quantify the signal. Mice were sacrificed when they showed significantly lower expression of miR-146a in recurrent

AACRJournals.org Mol Cancer Res; 2020 OF3

Cui et al.

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 patients with GBM with high (n ¼ 134) and low (n ¼ 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 ¼ 3; , P < 0.01; , P < 0.001. D, Expression of miR-146a in primary and recurrent GBM by RT-qPCR in cohort 2 (eight pairs; n ¼ 3; , P < 0.05; , P < 0.01). E, miR-146a expression in NHA and GBM cells by qRT-PCR analysis (n ¼ 3; , P < 0.001). F, qRT-PCR analysis of miR-146a expression in GBM cells after 10 mmol/L DAC treatment (n ¼ 3; , P < 0.01; , P < 0.001). G, Methylation status of miR-146a was detected by MSP using GBM cell lines. M, methylated product; U, unmethylated product. H, Methylation status of CpG sites in the promoter region of miR-146a by BS. Black square: methylated CpG site; grey square: partially methylated CpG site; white square: unmethylated CpG site. I, Methylation status of miR-146a was detected by MSP using GBM tissues. M, methylated product; U, unmethylated product.

OF4 Mol Cancer Res; 2020 MOLECULAR CANCER RESEARCH

A Novel miR-146a-POU3F2/SMARCA5 Pathway in Glioblastoma

tumors compared with the matched primary tumors (Fig. 1D, cohort (Fig. 2A–C), which was reproduced in additional patient-derived 2, n ¼ 16, eight pairs). Taken together, these data indicate that miR- xenografts (PDX) lines GBM12 and GBM43 with endogenous low 146a acts as a favorable prognostic biomarker in GBMs, and miR-146a expression of miR-146a (Supplementary Figs. S1A and S1B). In expression is significantly suppressed in recurrent patients with GBM consistence with this, inhibition of miR-146a increased the prolifer- further suggesting that miR-146a may play a role in the treatment ation of LN229 cells (Fig. 2D). In addition to rapid proliferation, response mechanisms in GBM. invasion is a defining hallmark of GBM cells (18). Accordingly, we determined the role of miR-146a on GBM cell invasion using a trans- miR-146a expression is partially regulated by promoter well Matrigel assay, which showed that overexpression of miR-146a methylation in GBM suppressed invasion of GBM cells in vitro (Fig. 2E; Supplementary To determine the endogenous expression of miR-146a in GBM cells, Fig. S2A), whereas inhibition of miR-146a increased invasion (Fig. 2F; we performed RT-qPCR in NHAs, six established GBM cell lines Supplementary Fig. S2B). Cell proliferation and invasion data were (U251, T98G, LN229, U87MG/EGFRvIII, U87MG, and LN18), and then substantiated by using additional GBM cell lines (U87 and three patient-derived primary GBM cell lines (08-387, 3359, and LN18; Supplementary Figs. S3A–S3C).Notably,weobservedthat GBM30). We found that the majority of GBM cell lines expressed overexpression of miR-146a in primary GBM cells significantly significantly lower levels of miR-146a, except LN229, compared with inhibited sphere formation, a characteristic feature of these cells the NHA cell line (Fig. 1E). This result suggests that miR-146a is (Fig. 2G and H). In consistence with previous studies, activation of significantly downregulated in GBM cells, which is consistent with a NF-BandERK1/2wasfoundtobeinhibited by overexpression of previous study demonstrating that miR-146a expression is lower in miR-146a in GBM cells (Supplementary Fig. S4). To study the role GBM tumors compared with adjacent normal tissues (30). Promoter of miR-146a on tumor growth in vivo, we generated two tetracycline hyper-methylation leading to transcriptional silencing of miRNAs has (doxycycline/Dox) inducible stable cell lines termed 08-387/miR- been found in a variety of cancer (31). Specifically, Wang and 146a and GBM30/miR-146a. Our data show that expression of miR- colleagues have reported that demethylation of miR-146a promoter 146a was induced after Dox treatment and proliferation was by 5-Aza-20-deoxycytidine (DAC) correlates with delayed progression inhibited in both cell lines (Supplementary Figs. S5A–S5D). Fur- of castration-resistant prostate cancer (32). Therefore, we explored thermore, an athymic nude xenograft mouse model was established, if miR-146a was silenced by promoter methylation in GBM and in which stable 08-387/miR-146a and GBM30/miR-146a cells were found that DAC treatment significantly increased the miR-146a implanted into the brain. Decreased tumor growth was observed by expression in eight out of nine cell lines examined, with exception of IVIS imaging in Dox-induced group of mice that had higher LN229 cell line that expressed a relatively higher endogenous level expression of miR-146a (Fig. 2I–2K) and an overall survival of of miR-146a (Fig. 1F). The methylation status of miR-146a was these mice was prolonged as revealed by the Kaplan–Meier plots further determined by methylation-specific PCR (MSP) in all nine (Fig. 2L and M). Representative H&E staining for the tumors GBM cell lines. As shown in Fig. 1G, seven of eight GBM cells are shown in Supplementary Fig. S5E. Dox-induced expression of showed partial methylation of the miR-146a promoter region miR-146a in vivo was further confirmed by qPCR (Fig. 2N and O). with downregulated miR-146a expression. Consistent with DAC Taken together, these data suggest that overexpression of miR-146a data, miR-146a promoter in LN229 cells was found to be unmethy- inhibits tumor growth and prolongs survival of the tumor cell lated. To confirm the MSP results and further evaluate the meth- implanted mice. ylation status of miR-146a in GBM cell lines, BS was performed for eight CpG sites (183, 160, 150, 142, 138, 136, 132, miR-146a enhances TMZ response in primary GBM models and 128) of the promoter region near the transcription start site. TMZ is the most widely used chemotherapy in patients with GBM. Consistent with the MSP results, a high level of methylation was To determine if miR-146a sensitizes GBM cells to TMZ, miR-146a was found in seven of eight cell lines with downregulated miR-146a overexpressed in U87MG/EGFRvIII and 08-387 cell lines and cell expression (Fig. 1H). The MSP result prompted us to analyze the viability was measured. We found that overexpression of miR-146a methylation status of miR-146a in GBM tumor tissues. We selected could significantly enhance TMZ-induced cell killing (Fig. 3A and B). tumor tissues with relatively low expression levels of miR-146a in In addition, fewer colonies were formed in the miR-146a overexpres- cohort 1 and, as expected, methylation of miR-146a was found in 9 sing group compared with the control group, after TMZ treatment, of 10 tumor tissues (Fig. 1I). These results suggest that hyper- which was consistent with the outcome of the viability experiments methylationofCpGislandsonthemiR-146apromotercontributes, (Fig. 3C and D). To further explore the effects of miR-146a on TMZ- in part, to the downregulation of miR-146a expression in GBM. induced apoptosis, we performed Annexin V apoptosis assay and measured caspase activation. As shown in Fig. 3E–G, overexpression Overexpression of miR-146a inhibits cell proliferation and of miR-146a markedly enhanced TMZ-induced apoptosis in 08-387 invasion in vitro and in vivo cells. Similar results were obtained in GBM30 cells (Supplementary Given that high expression of miR-146a was associated with better Figs. S6A and S6B). These findings demonstrate that miR-146a over- OS in patients with GBM and it was downregulated in GBM cells, we expression enhanced TMZ response and potentiated TMZ-induced hypothesized that miR-146a might play an important role in restrain- apoptosis in GBM cells. To further assess the function of miR-146a in ing GBM progression. To investigate the effects of miR-146a on cell TMZ response in vivo, an athymic nude xenograft mouse model proliferation and invasion in vitro, U87MG/EGFRvIII, GBM30, and was employed. Mice were randomized into four treatment groups, 08-387 cells, which expressed low endogenous levels of miR-146a, were 10 mice per group: (i) Dox()/TMZ(), (ii) Dox()/TMZ(þ), (iii) transfected with microRNA negative control (NC) or miR-146a Dox(þ)/TMZ(), and (iv) Dox(þ)/TMZ(þ). Mice were treated, as mimic. On the other hand, LN229 cells, which expressed a relatively indicated, with TMZ by oral gavage for 5 days, and euthanized when high level of miR-146a, were transfected with NC or a miR-146a displayed tumor-associated morbidity. Mice in Dox()/TMZ() inhibitor. We found that overexpression of miR-146a significantly group showed rapid tumor growth (23–25 days, median survival, inhibited proliferation of U87MG/EGFRvIII, GBM30, and 08-387 cells 25 days), Dox()/TMZ(þ) and Dox(þ)/TMZ() groups initially

AACRJournals.org Mol Cancer Res; 2020 OF5

Cui et al.

Figure 2 miR-146a inhibits cell proliferation and invasion in vitro and in vivo U87MG/EGFRvIII,GBM30,and08–387 cells were transiently transfected with miRNA NC or miR-146a mimics, respectively. LN229 cells were transiently transfected with NC or miR-146a inhibitors. Cell proliferation was measured at the different time points. n ¼ 4, , P < 0.001, compared with controls (A–D). The transwell invasion assay was conducted and numbers of invaded cells were quantiﬁed (E–F). n ¼ 3, , P < 0.01, , P < 0.001. Sphere formation assay was conducted and number of spheres was counted in 3 different areas and mean value was calculated (G and H). n ¼ 3, , P < 0.01. I, Luminescent imaging for mice with intracranial tumor formed from Dox()andDox(þ)groups on day 10, 17, and 25 after implanting. J–K, Quantiﬁcation of luminescence signal intensity. Student t-test was carried out for statistical analysis (mean SEM, n ¼ 10/group). , P < 0.05, , P < 0.01, , P < 0.001. L–M, Kaplan-Meier survival analysis shows that overall survival of mice injected with stable 08–387/miR-146a/ luc or GBM30/miR-146a/luc cells without or with Dox (n ¼ 10/group). N and O, Expression of miR-146a was detected after isolating RNAs from tumors harvested from mice brain when the mice became moribund. n ¼ 5, , P < 0.001.

OF6 Mol Cancer Res; 2020 MOLECULAR CANCER RESEARCH

A Novel miR-146a-POU3F2/SMARCA5 Pathway in Glioblastoma

Figure 3 miR-146a enhances TMZ sensitivity in vitro and in vivo in primary tumor model. A and B, Cell viability was determined in U87/EGFRvIII, and 08–387 cells after overexpression of miR-146a and treatment with different dose of TMZ. n ¼ 5, P < 0.01. 08–387 cells were transfected with NC or mIR-146a mimics, 24 h post transfection, cells were re-seeded and treated with DMSO or TMZ (100 mmol/L). Colony formation assay (C and D) was then carried out to determine the effect of miR-146a on the colony forming ability in 08–387 cells without or with TMZ treatment. n ¼ 3, , P < 0.01, , P < 0.001. Flow cytometry analysis (E and F) and western blot analysis (G) was conducted to confirm the effect of miR-146a on TMZ induced apoptosis in 08–387 cells. n ¼ 3, , P < 0.01. H, Luminescent imaging for mice in four groups with intracranial tumor formed 5 days after TMZ oral gavage. I, Kaplan-Meier survival analysis shows overall survival of the mice in these four groups without or with TMZ treatment (n ¼ 10/group). slowed tumor growth, but tumor still grew (33–52 and 33–53 days, multipotent progenitors. qPCR data revealed that GSCs expressed respectively, median survival, 38 days). Mice in Dox(þ)/TMZ(þ) miR-146a at markedly lower levels than the matched NGSCs, in group had significant smaller tumors (Fig. 3H) and had longer survival both 08-387 and 3359 cells (Fig. 4B). Interestingly, after a search of times (50–63 days, median survival, 55 days, Fig. 3I). Thus, miR-146a the GEO Profiles database (35), lower expression of miR-146a was not only inhibited tumor growth, but also enhanced TMZ sensitivity also found in GBM stem-like cells (Gene Expression database: both in vitro and in vivo in GBM. GSE23806, P ¼ 0.0304; Supplementary Fig. S7; ref. 36), which confirmed our findings. Further, overexpression of miR-146a resulted in Overexpression of miR-146a downregulates stemness of reduced proliferation of GSCs (Fig. 4C and D). Similarly, miR-146a GSCs overexpression reduced sphere formation frequency and sphere size GSCs are found to be primary drivers of tumor recurrence and by in vitro limiting dilution and sphere formation assays (Fig. 4E–H), therapeutic resistance (33). Because miR-146a was found to be down- as well as in vivo tumorigenicity by frequency of tumor-initiating regulated in recurrent GBM tumors (Fig. 1B–D), accordingly, we cells (TICf; Fig. 4I–J), indicating that overexpression of miR-146a hypothesized that overexpression of miR-146a would downregulate inhibited expansion of GSCs. Moreover, additional stemness markers stemness in GSCs. To test this hypothesis, we first determined the were assessed at mRNA levels, which revealed that overexpression of expression of miR-146a in GSCs and non-GSCs (NGSC), which were miR-146a significantly downregulated expression of SALL, SOX2, derived from human brain tumor specimens using CD133 selec- c-Myc, Nestin, and Oct4 in 08-387 GSCs (Supplementary Fig. S8). tion (34). Consistent with prior reports, the GSC-enriched fractions These data suggest that overexpression of miR-146a reduced the expressed high level of Olig2 and Oct4 (Fig. 4A), which are markers of stemness of GBM cells.

AACRJournals.org Mol Cancer Res; 2020 OF7

Cui et al.

POU3F2 and SMARCA5 are direct targets of miR-146a in GBM POU3F2 gene), and SMARCA5 among others, were found as novel To identify putative target mRNAs of miR-146a, bioinformatics potential targets of miR-146a (Supplementary Fig. S9). Gene expres- analyses were performed employing different miRNA target predic- sion analysis using the TCGA datasets (http://gepia.cancer-pku.cn/) tion tools. The neural transcription factor BRN2 (encoded by the revealed that both POU3F2 and SMARCA5 were highly expressed

Figure 4 Overexpression of miR-146a inhibits stemness in GBM (A) OLIG2 and OCT4 were detected by western blot analysis in NGSCs and GSCs. B, Expression of miR-146awastestedinNGSCsandGSCs.n ¼ 3, , P < 0.05, , P < 0.01. C and D, Cell proliferation assay was performed in GSCs after transfection NC or miR-146a mimics. n ¼ 5, , P < 0.001. E–H, In vitro limiting dilution and sphere formation assays using 08–387 GSCs and 3359 GSCs after overexpression of NC or miR-146a mimics. For in 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 and G). Sphere formation assay was conducted and number of spheres was counted in 3 different areas and mean value was calculated (F and H). n ¼ 3, , P < 0.01. I, In vivo limiting dilution assay was used to estimate TICf in these xenografts. Tumor cells were isolated from xenografts and injected into nude mice subcutaneously in a limiting dilution manner. Images of tumors after 2 weeks of injection presented, and the number of xenografts counted to generate TICf using the online algorithm. J, Weight of tumors generated with 1 105 cells in NC and miR-146a-overexpressed mice bearing xenografts. n ¼ 4/group, , P < 0.05.

OF8 Mol Cancer Res; 2020 MOLECULAR CANCER RESEARCH

A Novel miR-146a-POU3F2/SMARCA5 Pathway in Glioblastoma

Figure 5 POU3F2 and SMARCA5 are direct targets of miR-146a. A and B, Expression of POU3F2 and SMARCA5 in TCGA datasets. Tumor tissue, n ¼ 163; Normal tissue, n ¼ 207 , P < 0.01. C and D, qPCR was performed to detect POU3F2 and SMARCA5 at mRNA level in U87MG, U87MG/EGFRvIII, T98G, GBM30, and 08–387 cells transfected with NC or miR-146a mimic. 72 hours post transfection, cells were harvested for RNA extraction. n ¼ 3, , P < 0.05, , P < 0.01, , P < 0.001. E and F, Expression of miR-146a, POU3F2 and SMARCA5 was detected by qPCR in Dox inducible stable cell line 08–387/miR-146a. n ¼ 3, , P < 0.05, , P < 0.01, , P < 0.001. G, Expression of POU3F2 and SMARCA5 at protein levels was detected in Dox inducible stable cell line 08–387/miR-146a by western blot analysis. H, Luciferase reporters containing putative miR-146a targeting sequences in the 30UTR of POU3F2 and SMARCA5 wild-type (wt) and mutated (mut) versions. I and J, U87MG/ EGFRvIII, GBM30, and 08–387 cells were co-transfected with the luciferase reporter constructs containing 30UTR (wt) or 30UTR (mut) together with NC or miR-146a mimics. Seventy-two hours post transfection, luciferase activity was measured and normalized. n ¼ 3, , P < 0.05, , P < 0.01.

AACRJournals.org Mol Cancer Res; 2020 OF9

Cui et al.

across different cancer types compared with normal tissues (Supple- positively correlated (Supplementary Figs. S14A and S14B and S15A mentary Figs. S10A and S11A), particularly in GBM (Fig. 5A and B). and S15B). To confirm the finding in vitro, we knocked down either Notably, high expression of both of POU3F2 and SMARCA5 correlated POU3F2 or SMARCA5 in 08-387 cells (Supplementary Figs. S15C and with worse OS of glioma patients (Supplementary Figs. S10B and S15D), and found that expression of SMARCA5 and POU3F2 was S11B), and these were negatively correlated with miR-146a expression decreased at both protein levels (Supplementary Figs. S15C and S15D). (Supplementary Figs. S10C and S11C), as expected. To determine Collectively, these data confirmed that the tumor suppressive potential whether POU3F2 and SMARCA5 are putative targets, different GBM of miR-146a is largely mediated by the miR-146a/POU3F2/SMARCA5 cells were transfected with miR-146a mimic, which resulted in signif- axis (Fig. 6I) in GBM. icant reduction of both POU3F2 and SMARCA5 expression in U87MG, U87MG/EGFRvIII, T98G, GBM30, and 08-387 cell lines at the mRNA level (Fig. 5C and D). To confirm changes in expression of Discussion POU3F2 and SMARCA5 in the Dox-inducible stable 08-387/miR-146a Rapid growth, invasion, and resistance to RT and TMZ are largely cell model (Fig. 5E), we treated 08-387/miR-146a cells with different attributed to the GSC population in GBM tumors. GSCs are capable of dose of Dox, reduction of POU3F2 and SMARCA5 at both mRNA and executing these pathologic functions through the activation of a protein levels was observed (Fig. 5F and G). To exclude the off-target number of signal transduction pathways, including the PI3Kinase, effect, we detected expression of TRAF6, which is a known miR-146a Wnt/b-catenin, Notch, NF-kB, and Jak-Stat among others. Activation target (37), and as expected, reduction of TRAF6 was observed when of these pathways results in aberrant expression of a variety of neural miR-146a was overexpressed by either miR-146a mimics (Supple- stem cell- and GSC-related genes, which contribute to the maintenance mentary Fig. S12A) or Dox treatment (Supplementary Fig. S12B). To of proliferation, invasion, RT/TMZ-resistance functions in GSCs. further investigate whether POU3F2 and SMARCA5 are direct and Many of these regulatory genes happen to be oncogenes and tumor specific targets of miR-146a, luciferase reporter containing wild-type/ suppressor genes, which are posttranscriptionally suppressed by mutated 30UTR of POU3F2 and SMARCA5 were constructed tumor suppressive miRNAs and onco-miRNAs, respectively (38). By (Fig. 5H). Wild-type or mutated 30UTR reporters of POU3F2 and NanoString analysis of miRNA expression in human GBM specimens, SMARCA5 were cotransfected with NC/miR-146a mimics into we have identified and validated that miR-146a expression was U87MG/EGFRvIII, GBM30, and 08-387 cells, respectively. Consistent correlated with favorable patient overall survival. Its expression was reduction in luciferase activity by miR-146a was observed only with partly suppressed by its promoter methylation in primary GBM, and wild-type 30UTR construct, but not with the mutant construct (Fig. 5I further it was noted that expression of this miRNA was significantly and J). Collectively, these data suggest that miR-146a suppressed lower in recurrent GBM indicating that miR-146a may have further POU3F2 and SMARCA5 by directly targeting their 30-UTRs in GBM significance in tumor progression or recurrence. These findings led to cells. the hypothesis that miR-146 could be a tumor suppressor miRNA in GBM and that it might negatively control therapeutic resistance Tumor suppression by miR-146a is mediated by POU3F2 and mechanisms likely by suppressing the GSC population and/or their SMARCA5 and both proteins are positively correlated in GBM functions. It is worth noting that miR-146a functions as a tumor Once we found that tumor suppressive and TMZ-sensitizing func- suppressor miRNA in a number of cancers including NSCLC (39) and tions of miR-146a were mediated by knocking down the expression of prostate cancer (40), and as an onco-miRNA in others that include its direct targets POU3F2 and SMARCA5, we sought to determine the cervical, head and neck, and hepatocellular cancers (41–43). Kim and individual relative contributions of these stemness-related transcrip- colleagues reported that miR-146a was one of the miRNAs that tion factors to miR-146a functions. Among the primary GBM cell involved in long survival GBM subclass, but did not provided lines, we determined that 08-387 expressed relatively high levels of definitive evidence (44). To test our first hypothesis, we took two both POU3F2 and SMARCA5, which was consistent with a relatively reciprocal approaches: first, overexpression in primary PDX lines low expression of miR-146a. We observed that 08-387 cells’ prolifer- that expressed relatively lower levels of endogenous miR-146; and ation, invasion, and TMZ-response were significantly reduced by second, inhibition of miR-146a in cells, namely LN229 that siRNA-mediated knockdown of either POU3F2 or SMARCA5 expres- expressed significantly higher level of miR-146. In these reciprocally sion (Fig. 6A–F). We noted that knockdown of either target was engineered cell models, we measured cell proliferation, invasion, sufficient to produce effects on cell proliferation, invasion, and TMZ- and stemness-markers in vitro, and tumor growth and overall response comparable with the effects produced by overexpression of mouse survival in orthotopically implanted xenograft tumors. Our miR-146a. To substantiate this notion, miR-146a was inhibited in results clearly suggest that miR-146a functions as a tumor suppres- LN229 cells that expressed higher level of miR146a (Fig. 1E), which sive miR in GBM. This observation is in good agreement with were then cotransfected with POU3F2 siRNA and/or SMARCA5 another recent report that shows that ectopic expression of miR- siRNA (Fig. 6G). Subsequent functional assays demonstrated that 146a inhibits glioma development (45). miR-146a inhibition significantly induced cell proliferation and inva- Next, we tested if miR-146a sensitizes GBM to TMZ and RT. To this sion, which were significantly reversed by knocking down either end, our in vitro data clearly indicate that miR-146 overexpression POU3F2 or SMARCA5 expression (Fig. 6H and I). In addition, sensitized PDX lines to TMZ in vitro, and in vivo which, in turn, miR-146a did not demonstrate a tumor suppressive phenotype when significantly prolonged the mouse survival. The PDX cell line models POU3F2/SMARCA5 was overexpressed (Supplementary Figs. S13A– employed here, were sensitive to low dose (2Gy) of ionizing radiation S13C). Thus, either knocking down POU3F2 or SMARCA5 can mimic limiting the evaluation of RT-sensitizing potential of miR-146a. the effect of miR-146a in GBM, which provoked us to investigate the Once we found that tumor suppressive and TMZ-sensitizing func- correlation between POU3F2 and SMARCA5 in patients with GBM. tions of miR-146a were mediated by knocking down the expression of First, we analyzed mRNA expression of POU3F2 and SMARCA5 from its direct targets POU3F2 and SMARCA5, we sought to determine the both TCGA (https://www.cbioportal.org/) and CGGA (http://www. relative contributions of these stemness-related transcription factors to cgga.org.cn/) datasets, and found that POU3F2 and SMARCA5 were miR-146a functions in GBM. POU3F2 (BRN2), a master regulator of

OF10 Mol Cancer Res; 2020 MOLECULAR CANCER RESEARCH

A Novel miR-146a-POU3F2/SMARCA5 Pathway in Glioblastoma

Figure 6 Tumor suppression by miR-146a is mediated by POU3F2 and SMARCA5. A–C, 08–387 cells were transiently transfected with either POU3F2 or SMARCA5 siRNA-1/2. Western blot analysis was performed to detect POU3F2/SMARCA5 protein expression in 08–387 cells transfected with siRNA control or POU3F2/SMARCA5 siRNAs (A). B, Cell proliferation was tested by CellTiter Glo. n ¼ 5, , P < 0.01, , P < 0.001. C, Cell invasion assay was performed using matrigel transwell membranes. n ¼ 3, , P < 0.01. D and E, TMZ induced cell apoptosis was detected after 72 h treatment with TMZ by western blot analysis. F–H, LN229 cells were transiently transfected with NC or miR-146a inhibitor alone, or simultaneously with either POU3F2 and/or SMARCA5 siRNA. The protein expression of POU3F2 and SMARCA5 was determined using western blotting (F). Cell proliferation at 48 h and 96 h was determined using methylene blue (G). n ¼ ,5,P < 0.01. The matrigel transwell invasion assay was carried out to quantify the invaded cells (H). n ¼ 3, , P < 0.001. I, Model of the mechanism by which miR-146a inhibits tumor progression and induce TMZ sensitivity in GBM.

AACRJournals.org Mol Cancer Res; 2020 OF11

Cui et al.

neuronal differentiation, is a POU-domain transcription factor well inhibits the stemness of GBM cells via directly targeting POU3F2 described in developmental biology (46). It has been reported that and SMARCA5 whose expression were positively correlated in inhibiting BRN2 expression led to significantly reduced proliferation, GBM. Data from our human cohorts and in vitro/vivo mechanistic migration, and invasion in SCLC, prostate cancer, as well as analysis strongly implicate that miR-146a plays a significant role in melanoma (47–50). Importantly, targeting BRN2 is a strategy to treat the progression and therapeutic sensitivity in GBM, making it or or prevent neuroendocrine differentiation in prostate cancer (50). POU3F2 and SMARCA5 to be potentially attractive and novel SMARCA5 (hSNF2H) is a member of SWI/SNF family, and contains therapeutic targets. helicase and ATPase activities. hSNF2H promotes tumor growth in ovarian cancer and glioma (51, 52). It has also been shown that miR- Disclosure of Potential Conflicts of Interest 100 affects stem cell self-renewal and cell proliferation in part by J.S. Barnholtz-Sloan reports grants from NIH/NCI during the conduct of the targeting SMARCA5 (53). Interestingly, Zhou and colleagues reported study. No potential conflicts of interest were disclosed by the other authors. a reverse correlation between expression of miR-146a and SMARCA5 in a bladder tumor cell model (54), however, they did not provide Authors’ Contributions evidence about the regulation between miR-146a and SMARCA5. T. Cui: Formal analysis, validation, investigation, visualization, methodology, Among the primary GBM cell lines we examined, 08-387 expressed writing-original draft, writing-review and editing. E.H. Bell: Investigation, writing- review and editing. J. McElroy: Investigation. K. Liu: Investigation. E. Sebastian: relatively high level of both POU3F2 and SMARCA5, which was Investigation. B. Johnson: Investigation. P.M. Gulati: Investigation. A.P. Becker: consistent with a relatively low expression of miR-146a. We observed Investigation. A. Gray: Investigation. M. Geurts: Investigation. D. Subedi: that 08-387 cells’ proliferation, invasion, and TMZ-response were Investigation. L. Yang: Investigation. J.L. Fleming: Investigation. W. Meng: significantly reduced by siRNA-mediated knockdown of either Investigation. J.S. Barnholtz-Sloan: Investigation. M. Venere: Investigation. POU3F2 or SMARCA5 expression. We noted, with surprise, that Q.-E. Wang: Investigation. P.A. Robe: Investigation. S.J. Haque: Investigation, knockdown of either target was sufficient to produce effects on cell writing-review and editing. A. Chakravarti: Supervision, project administration. proliferation, invasion, and TMZ-response comparable to the effects produced by overexpression of miR-146a. To gain an insight of these Acknowledgments unexpected observations, we conducted a series of experiments which We thank Dr. Jeremy Rich (UC San Diego, USA) for providing primary GBM patient-derived cells. We also thank The Ohio State University (OSU) Com- revealed that POU3F2 and SMARCA5 positively regulate each other in prehensive Cancer Center Small Animal Imaging Core, The Ohio State University the absence of miR-146a involvement/modulation. The study has (OSU) Genomics Shared Resource (GSR), The Ohio State University (OSU) Target potential limitations. First, miR-146a expression was tested in a small Validation Shared Resource (TVSR), and The Ohio State University (OSU) Com- sample size for primary and recurrent GBM, a large patient cohort is prehensive Cancer Center Pathology Core Facility supported in part by grant no. needed. Second, the PDX cell line models employed here, were P30 CA016058, NCI. This work was also supported by NCI [R01CA169368 sensitive to low dose (2 Gy) of ionizing radiation limiting the eval- (to A. Chakravarti), R01CA11522358 (to A. Chakravarti), R01CA1145128 (to A. Chakravarti), R01CA108633 (to A. Chakravarti), R01CA188228 (to A. Chakravarti, uation of RT-sensitizing potential of miR-146a. Third, given the fact K. Liu, and J.S. Barnholtz-Sloan), 1RC2CA148190 (to A. Chakravarti), and that POU3F2 is a transcriptional regulator, it might regulate expression U10CA180850-01 (to A. Chakravarti); A Brain Tumor Funders Collaborative Grant of SMARCA5 through direct binding or indirectly through other (to A. Chakravarti); Ohio State University Comprehensive Cancer Center Award (to targeted genes. Further studies are needed to investigate the mecha- A. Chakravarti), and the T&P Bohnenn Fund for Neuro-Oncology Research (grant to nism of the regulation between POU3F2 and SMARCA5 to identify the P.A. Robe). novel therapeutic strategies for GBM. In summary, focusing on the tumor suppressive functions of The costs of publication of this article were defrayed in part by the fi payment of page charges. This article must therefore be hereby marked miR-146a in GBM, this study revealed the following novel ndings: advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate (i) Decreased miR-146a expression is associated with shorter OS this fact. independent of MGMT methylation status in GBM; (ii) Promoter methylation-induced silencing of miR-146a drives tumor progres- Received April 20, 2020; revised July 24, 2020; accepted September 21, 2020; sion and therapeutic resistance in glioblastoma; (iii) miR-146a published first September 24, 2020.

References 1. Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, 6. Thakkar JP, Dolecek TA, Horbinski C, Ostrom QT, Lightner DD, Cavenee WK, et al. The 2016 World Health Organization classification of Barnholtz-Sloan JS, et al. Epidemiologic and molecular prognostic review tumors of the central nervous system: a summary. Acta Neuropathol 2016; of glioblastoma. Cancer Epidemiol Biomarkers Prev 2014;23:1985–96. 131:803–20. 7. Chen J, Li YJ, Yu TS, McKay RM, Burns DK, Kernie SG, et al. A restricted cell 2. Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, population propagates glioblastoma growth after chemotherapy. Nature 2012; et al. Effects of radiotherapy with concomitant and adjuvant temozolomide 488:522. versus radiotherapy alone on survival in glioblastoma in a randomised phase 8. Auffinger B, Tobias AL, Han Y, Lee G, Guo D, Dey M, et al. Conversion of III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 2009;10: differentiated cancer cells into cancer stem-like cells in a glioblastoma model 459–66. after primary chemotherapy. Cell Death Differ 2014;21:1119–31. 3. Ryken TC, Kalkanis SN, Buatti JM, Olson JJ. The role of cytoreductive 9. Tamura K, Aoyagi M, Wakimoto H, Ando N, Nariai T, Yamamoto M, et al. surgery in the management of progressive glioblastoma. J Neuro Oncol 2014; Accumulation of CD133-positive glioma cells after high-dose irradiation by 118:479–88. Gamma Knife surgery plus external beam radiation. J Neurosurg 2010;113: 4. Ostrom QT, Cioffi G, Gittleman H, Patil N, Waite K, Kruchko C, et al. CBTRUS 310–8. statistical report: primary brain and other central nervous system tumors 10. De Bacco F, D’Ambrosio A, Casanova E, Orzan F, Neggia R, Albano R, et al. MET diagnosed in the United States in 2012–2016. Neuro Oncol 2019;21:v1–100. inhibition overcomes radiation resistance of glioblastoma stem-like cells. 5. Weller M, Pfister SM, Wick W, Hegi ME, Reifenberger G, Stupp R. Molecular EMBO Mol Med 2016;8:550–68. neuro-oncology in clinical practice: a new horizon. Lancet Oncol 2013;14: 11. Lathia JD, Mack SC, Mulkearns-Hubert EE, Valentim CLL, Rich JN. Cancer stem e370–9. cells in glioblastoma. Gene Dev 2015;29:1203–17.

OF12 Mol Cancer Res; 2020 MOLECULAR CANCER RESEARCH

A Novel miR-146a-POU3F2/SMARCA5 Pathway in Glioblastoma

12. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, et al. Identification 34. Dermawan JKT, Hitomi M, Silver DJ, Wu QL, Sandlesh P, Sloan AE, et al. of human brain tumour initiating cells. Nature 2004;432:396–401. Pharmacological targeting of the histone chaperone complex FACT preferen- 13. Liu G, Yuan X, Zeng Z, Tunici P, Ng H, Abdulkadir IR, et al. Analysis of gene tially eliminates glioblastoma stem cells and prolongs survival in preclinical expression and chemoresistance of CD133þ cancer stem cells in glioblastoma. models. Cancer Res 2016;76:2432–42. Mol Cancer 2006;5:67. 35. Barrett T, Troup DB, Wilhite SE, Ledoux P, Rudnev D, Evangelista C, et al. NCBI 14. Zhang N, Wei P, Gong A, Chiu WT, Lee HT, Colman H, et al. FoxM1 promotes GEO: mining tens of millions of expression profiles—database and tools update. beta-catenin nuclear localization and controls Wnt target-gene expression and Nucleic Acids Res 2007;35:D760–5. glioma tumorigenesis. Cancer Cell 2011;20:427–42. 36. Schulte A, Gunther HS, Phillips HS, Kemming D, Martens T, Kharbanda S, et al. 15. Wang JL, Wakeman TP, Lathia JD, Hjelmeland AB, Wang XF, White RR, et al. A distinct subset of glioma cell lines with stem cell-like properties reflects the Notch promotes radioresistance of glioma stem cells. Stem Cells 2010;28:17–28. transcriptional phenotype of glioblastomas and overexpresses CXCR4 as ther- 16. Kim SH, Ezhilarasan R, Phillips E, Gallego-Perez D, Sparks A, Taylor D, et al. apeutic target. Glia 2011;59:590–602. Serine/threonine kinase MLK4 determines mesenchymal identity in glioma stem 37. Stickel N, Prinz G, Pfeifer D, Hasselblatt P, Schmitt-Graeff A, Follo M, et al. cells in an NF-kappaB-dependent manner. Cancer Cell 2016;29:201–13. MiR-146a regulates the TRAF6/TNF-axis in donor T cells during GVHD. 17. Cao Y, Lathia JD, Eyler CE, Wu Q, Li Z, Wang H, et al. Erythropoietin receptor Blood 2014;124:2586–95. signaling through STAT3 is required for glioma stem cell maintenance. 38. Esquela-Kerscher A, Slack FJ. Oncomirs - microRNAs with a role in cancer. Genes Cancer 2010;1:50–61. Nat Rev Cancer 2006;6:259–69. 18. Beyer S, Fleming J, Meng W, Singh R, Haque SJ, Chakravarti A. The role of 39. Li YL, Wang J, Zhang CY, Shen YQ, Wang HM, Ding L, et al. MiR-146a-5p miRNAs in angiogenesis, invasion and metabolism and their therapeutic inhibits cell proliferation and cell cycle progression in NSCLC cell lines by implications in gliomas. Cancers 2017;9:85. targeting CCND1 and CCND2. Oncotarget 2016;7:59287–98. 19. Matos B, Bostjancic E, Matjasic A, Popovic M, Glavac D. Dynamic expression of 40. Huang Y, Tao T, Liu C, Guan H, Zhang G, Ling Z, et al. Upregulation of miR- 11 miRNAs in 83 consecutive primary and corresponding recurrent glioblas- 146a by YY1 depletion correlates with delayed progression of prostate cancer. toma: correlation to treatment, time to recurrence, overall survival and MGMT Int J Oncol 2017;50:421–31. methylation status. Radiol Oncol 2018;52:422–32. 41. Wang X, Tang S, Le SY, Lu R, Rader JS, Meyers C, et al. Aberrant expression of 20. Huang SW, Ali ND, Zhong L, Shi J. MicroRNAs as biomarkers for human oncogenic and tumor-suppressive microRNAs in cervical cancer is required for glioblastoma: progress and potential. Acta Pharmacol Sin 2018;39:1405–13. cancer cell growth. PLoS One 2008;3:e2557. 21. Henriksen M, Johnsen KB, Andersen HH, Pilgaard L, Duroux M. MicroRNA 42. Hung PS, Chang KW, Kao SY, Chu TH, Liu CJ, Lin SC. Association between the expression signatures determine prognosis and survival in glioblastoma multi- rs2910164 polymorphism in pre-mir-146a and oral carcinoma progression. forme-a systematic overview. Mol Neurobiol 2014;50:896–913. Oral Oncol 2012;48:404–8. 22.LowSYY,HoYK,TooHP,YapCT,NgWH.MicroRNAaspotential 43. Xu T, Zhu Y, Wei QK, Yuan Y, Zhou F, Ge YY, et al. A functional polymorphism modulators in chemoresistant high-grade gliomas. J Clin Neurosci 2014; in the miR-146a gene is associated with the risk for hepatocellular carcinoma. 21:395–400. Carcinogenesis 2008;29:2126–31. 23. Bo LJ, Wei B, Li ZH, Wang ZF, Gao Z, Miao Z. Bioinformatics analysis of miRNA 44. Kim TM, Huang W, Park R, Park PJ, Johnson MD. A developmental taxonomy expression profile between primary and recurrent glioblastoma. Eur Rev Med of glioblastoma defined and maintained by microRNAs. Cancer Res 2011;71: Pharmaco 2015;19:3579–86. 3387–99. 24. Cui T, Bell EH, McElroy J, Becker AP, Gulati PM, Geurts M, et al. miR-4516 45. Mei J, Bachoo R, Zhang CL. MicroRNA-146a inhibits glioma development by predicts poor prognosis and functions as a novel oncogene via targeting PTPN14 targeting Notch1. Mol Cell Biol 2011;31:3584–92. in human glioblastoma. Oncogene 2019;38:2923–36. 46. Dominguez MH, Ayoub AE, Rakic P. POU-III transcription factors (Brn1, Brn2, 25. Cui T, Chen Y, Yang L, Knosel T, Zoller K, Huber O, et al. DSC3 expression is and Oct6) influence neurogenesis, molecular identity, and migratory destination regulated by p53, and methylation of DSC3 DNA is a prognostic marker in of upper-layer cells of the cerebral cortex. Cereb Cortex 2013;23:2632–43. human colorectal cancer. Brit J Cancer 2011;104:1013–9. 47. Ishii J, Sato H, Sakaeda M, Shishido-Hara Y, Hiramatsu C, Kamma H, et al. POU 26. Cui TT, Chen Y, Yang LL, Knosel T, Huber O, Pacyna-Gengelbach M, et al. The domain transcription factor BRN2 is crucial for expression of ASCL1, ND1 and p53 target gene desmocollin 3 acts as a novel tumor suppressor through neuroendocrine marker molecules and cell growth in small cell lung cancer. inhibiting EGFR/ERK pathway in human lung cancer. Carcinogenesis 2012; Pathol Int 2013;63:158–68. 33:2326–33. 48. Boyle GM, Woods SL, Bonazzi VF, Stark MS, Hacker E, Aoude LG, et al. 27. Hu YF, Smyth GK. ELDA: Extreme limiting dilution analysis for comparing Melanoma cell invasiveness is regulated by miR-211 suppression of the BRN2 depleted and enriched populations in stem cell and other assays. J Immunol transcription factor. Pigm Cell Melanoma R 2011;24:525–37. Methods 2009;347:70–8. 49. Pinner S, Jordan P, Sharrock K, Bazley L, Collinson L, Marais R, et al. Intravital 28. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, et al. imaging reveals transient changes in pigment production and Brn2 expression Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. during metastatic melanoma dissemination. Cancer Res 2009;69:7969–77. N Engl J Med 2005;352:987–96. 50. Bishop JL, Thaper D, Vahid S, Davies A, Ketola K, Kuruma H, et al. The master 29. Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, et al. neural transcription factor BRN2 is an androgen receptor-suppressed driver of MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J neuroendocrine differentiation in prostate cancer. Cancer Discov 2017;7:54–71. Med 2005;352:997–1003. 51. Sheu JJ, Choi JH, Yildiz I, Tsai FJ, Shaul Y, Wang TL, et al. The roles of human 30. Hu HQ, Sun LG, Guo WJ. Decreased miRNA-146a in glioblastoma multiforme sucrose nonfermenting protein 2 homologue in the tumor-promoting functions and regulation of cell proliferation and apoptosis by target Notch1. Int J Biol of Rsf-1. Cancer Res 2008;68:4050–7. Marker 2016;31:E270–5. 52. Zhao XC, An P, Wu XY, Zhang LM, Long B, Tian Y, et al. Overexpression of 31. Ha M, Kim VN. Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol 2014; hSNF2H in glioma promotes cell proliferation, invasion, and chemoresistance 15:509–24. through its interaction with Rsf-1. Tumor Biol 2016;37:7203–12. 32. Wang XL, Gao HT, Ren LX, Gu JF, Zhang YP, Zhang Y. Demethylation of the 53. Deng L, Shang L, Bai S, Chen J, He X, Martin-Trevino R, et al. MicroRNA100 miR-146a promoter by 5-Aza-20-deoxycytidine correlates with delayed progres- inhibits self-renewal of breast cancer stem-like cells and breast tumor develop- sion of castration-resistant prostate cancer. BMC Cancer 2014;14:308. ment. Cancer Res 2014;74:6648–60. 33. Osuka S, Van Meir EG. Overcoming therapeutic resistance in glioblastoma: the 54. Zhou W, Thiery JP. Loss of Git2 induces epithelial-mesenchymal transition by way forward. J Clin Invest 2017;127:415–26. miR146a-Cnot6L-controlled expression of Zeb1. J Cell Sci 2013;126:2740–6.

AACRJournals.org Mol Cancer Res; 2020 OF13

A Novel miR-146a-POU3F2/SMARCA5 Pathway Regulates Stemness and Therapeutic Response in Glioblastoma

Tiantian Cui, Erica H. Bell, Joseph McElroy, et al.

Mol Cancer Res Published OnlineFirst September 24, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-20-0353

Supplementary Access the most recent supplemental material at: Material http://mcr.aacrjournals.org/content/suppl/2020/09/24/1541-7786.MCR-20-0353.DC1

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://mcr.aacrjournals.org/content/early/2020/10/28/1541-7786.MCR-20-0353. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.