Background
Glioblastoma (GBM) is one of the most lethal brain tumors. The average survival is 12–18 months and the 5-year overall survival is approximately 5% [
1]. Even with aggressive interventions including surgery, or combination of radiotherapy and chemotherapies, the prognosis for the patients remains poor. One of the major underlying contributing factors to GBM’s malignancy is the resistance to both traditional and targeted therapies. Due to the inherent heterogeneous nature of GBM, the signaling pathways involved in the acquired drug resistance represents a complicated task to decipher. The role of tumor microenvironment adds another layer of complexity. Glioma-associated microglial cells (GAMs) are functionally similar to that of tumor-associated macrophages in the peripheral system and interact with GBM cells intimately via intracellular communications [
2,
3]. GAMs have been found to secrete a spectrum of cytokines and signaling molecules to promote tumor proliferation, anti-apoptosis and angiogenesis/metastasis [
4,
5]. Thus, a better understanding of the intracellular molecular communications between GBM and GAMs will provide foundation for therapeutic development.
Emerging evidence indicates the importance of abundantly transcribed non-coding genes termed long non-coding RNAs (lncRNAs) in virtually every aspect of cell biology including glioma tumorigenesis. Recently, a lncRNA, termed SNHG15 has gained much attention. SNHG15 was shown to be elevated and associated with tumorigenic functions including proliferation and metastasis in a variety of cancer types including breast, lung, and liver [
6‐
8]. Notably, lncRNA SNHG15 level was positively linked to the histological grade, tumor node metastasis stage (TNM) and the poor overall survival in hepatocellular carcinoma [
8]. However, the potential role of SNHG15 in GBM tumorigenesis has not been fully investigated. Initially, we searched public databases and identified that an elevated level of lncSNHG15 in GBM cells as compared to normal brain tissues, and this elevation of lncSNHG15 was associated with a significantly higher risk of developing GBM and a shorter survival time in the patients.
In this study, we employed in vitro and in vivo assays to demonstrate the tumorigenic roles of lncSNHG15. First, we found that a significantly higher level of lncSNHG15 in TMZ-resistant clinical GBM samples and was associated with GBM malignant properties. Increased lncSNHG15 level was associated with an increased expression in markers of oncogenesis such as EGFR, CDK6 and stemness including Sox2 and β-catenin. More importantly, TMZ-resistant (TMZ-R) GBM cells were more capable of promoting M2-polarization of glioma associated microglia (M2-GAMs) than TMZ-sensitive (TMZ-S) counterparts. Down-regulation of lncSNHG15 resulted in reduced tumorigenesis, self-renewal and increased TMZ sensitivity and the reverse was true when increased. Interestingly, CDK6 inhibitor, palbociclib treatment suppressed GBM tumorigenesis as well as the generation of M2 GAMs. Palbociclib’s anti-GBM effects were associated with a reduced level of lncSNHG15 and increased level of tumor suppressor miR-627. Mechanistically, miR-627 could target not only CDK6 but also Sox2 and β-catenin. Finally, we used patient-derived xenograft model to demonstrate that palbociclib treatment alone significantly reduced GBM tumorigenesis and with a greater extent when combined with TMZ.
In summary, we provided preclinical insights into the functional roles of lncSNHG15/CDK6/miR-627 regulatory circuit in the development of GBM and polarization of GAMs. More importantly, the feasibility of employing palbociclib for treating TMZ-resistant GBM was examined and supported by both in vitro and in vivo models.
Materials and methods
Ethics approval and consent to participate
Clinical samples were collected from Harbin Medical University (Harbin, China). All enrolled patients gave written informed consent for their tissues to be used for scientific research. The study was approved by the Institutional Review Board (IRB) of the Harbin Medical University (Harbin, China), consistent with the recommendations of the declaration of Helsinki for biomedical research (Harbin Medical University, Harbin, China) and followed standard institutional protocol for human research. Moreover, the animal study protocol was approved by the Animal Care and User Committee at Harbin Medical University (Harbin, China) (Affidavit of Approval of Animal Use Protocol # Harbin Medical University).
Cell culture and clinical sample collection
Forty cancer tissues from the patients diagnosed with glioma (with different grades, please refer to Additional file
1: Table S1 for clinicalpathological features) were collected for this study. All tissue samples were pathologically confirmed and immediately snap-frozen in liquid nitrogen until RNA extraction. Written informed consent to the use of the tissue samples for research purposes was obtained from each patient. All procedures were conducted in accordance with the principles outlined in the Declaration of Helsinki, and all applicable international, national and/or institutional guidelines for the care and use of animals were followed. The study protocol was approved by the Ethics Committee of Harbin Medical University (Harbin, China). TMZ-resistant (termed TMZ-R) and TMZ-sensitive (termed TMZ-S) clinical samples were collected and cultured for further analyses in this study. The procedures used to isolate and culture TMZ-R and TMZ-S cells were according to a previously published study [
9]. The human microglial cell line, HMC3 used in our study was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). HMC3 cells were cultured according to the recommendations by ATCC, where EMEM (ATCC® 30–2003™) was used as the base medium and completed by adding 56 mL FBS (ATCC® 30–2020™) to a 500 mL of base EMEM.
Co-culture and GAM polarization
Co-culture assays of tumor cells with macrophages TMZ-R and TMZ-S GBM cells were co-cultured with HMC3 microglia (using Boyden Chamber) at a density of 1:10 or 1:5 in a 6-well plate. After 72 h, HMC3 were analyzed for their M1, M2 phenotypes using real-time PCR technique. This system was then used for testing palbociblic’s influence on GAM polarization, palbociblic (0.5 μM) was added into the TMZ-R and HMC3 co-culture system after the seeding and cultured for 72 h. The same experimental conditions were used for co-culturing lncSNHG15-silenced or overexpressed TMZ-R cells with HMC3 cells. Primer sequences for M1 M2 markers can be found in Additional file
2: Table S2. M1 M2 cytokines secreted into the culture medium were determined using M1/M2/MDSC Cytokines, ELISA Kit (Cat# MBS590066,
MyBiosources.com). The procedures were performed according to vendor’s protocols.
Total RNA isolation and qRT-PCR analyses
The isolation of total RNA and qRT-PCR were carried out as previously described [
10]. All experiments were repeated in triplicates. Please refer to Additional file
2: Table S2 for primer sequences used in this study.
Gene-silence and overexpression experiments
For gene silencing experiments, shRNA (Santa Cruz Biotechnology, USA) was constructed with sequences specifically against lncSNHG15. miR-627-5p mimic and inhibitor molecules and negative controls (NC) were purchased from GenePharma (Shanghai, China). For overexpression experiments, pcDNA3.1 (+) vector (GenePharma, Shanghai, China) was obtained to construct a pcDNA3.1-SNHG15 overexpressing plasmid. Please refer to Additional file
2: Table S2 for siRNA sequences
.
SDS-PAGE and Western blots
Expression analyses in this study were all carried out sing standard SDS-PAGE (10–12%). Cellular protein lysates were subsequently transferred to nitrocellulose membranes (Sigma), washed and incubated with primary antibodies in cold overnight, followed by washes and secondary antibodies incubation. Primary antibodies used were all purchased from Cell Signaling Technology unless otherwise specified. Please refer to Additional file
3: Figure S1. Full-size blots of Fig.
2e. Figure S2. Full-size blos of Fig.
4b. Figure S3. Full-size blots of Fig.
6a.
The colony formation assay was performed to determine the proliferation and evaluation the effects of gene manipulation and/or drug treatment of GBM cells. Briefly, GBM cells (500 cells per well) were incubated in 6-well plates. One week to 10 days, formed colonies were fixed with methanol and stained with 0.1% crystal violet. Colonies were quantified under a light microscope (Olympus Corp.). The experiments were done in triplicates.
MTT assay
Cell viability assay or (MTT assay) was used to determine the viability, drug and/or gene manipulation effects on the GBM cells. GBM cells (5000 cells per well) were seeded in 96-well plates. Control and cells with different treatment were incubated with 20 μL MTT (5 mg/mL) in each well at the indicated time points (cells were collected at 0, 24, and 48 h). DMSO (Sigma) was added (150 μL/well) to each well to dissolve the crystals. OD was read at 490 nm on a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The experiments were carried out in triplicates.
Preclinical mouse model for drug evaluation
Immune compromised NOD/SCID females (6 weeks old) were supplied by Animal Care facility of the Harbin Medical University (Harbin, China). The animal study protocol was approved by the Animal Care and User Committee at Harbin Medical University (Harbin, China) (Affidavit of Approval of Animal Use Protocol # Harbin Medical University). Patient-derived TMZ-R cells (500,000 cells per injection) were injected subcutaneously. Mice were monitored weekly for tumor development using a standard caliper. Mice were subdivided into 4 groups, vehicle control, TMZ only (4 mg/kg, p.o., 5 times a week), palbociclib only (75 mg/kg, i.p injection, 5 times a week) and combination. At the end of experiments, mice were humanely euthanized or when tumor burden became symptomatic. Tumor samples were harvested for further analyses. Tumor growth were calculated using the following formula where tumor volume = (a × b2)/2, where a is the long axis and b is the short axis of the tumor.
Statistical analysis
SPSS software (version 22.0, USA) was used to perform statistical analyses. The significant difference between different groups was analyzed using t-test or a chi-square test. The level of P value ≤0.05 is considered statistically significant.
Discussion
Glioblastoma multiforme (GBM) is one of the most malignant cancer types and currently there is no effective regiments for combating this deadly disease. Despite advancement in the development of chemotherapeutic agents including targeted therapeutic agents, the overall survival time often does not extend beyond 2 years post diagnosis. Recent experimental and clinical insights indicate the tumor microenvironment (TME) of GBM also play instrumental roles in promoting tumorigenesis and the development of drug resistance. Glioma-associated microglia (GAM) represents one of the key cell types that have been shown to contribute significantly towards GBM tumorigenesis and progression [
17,
18]. In this study, we examined the potential signaling networks involved in promoting TMZ resistance and the feasibility of using CDK6 inhibitor, palbociclib as a candidate agent for treatment.
First, we found that an emerging tumorigenic lncRNA, SNHG15 is elevated in the GBM clinical samples and associated with a significantly shorter survival time (Fig.
1). We also provide clinical validation using our own clinical samples demonstrating a significantly higher lncSNH15 level in the TMZ-R cells than TMZ-S cells, implicating lncSNHG15’s potential involvement in drug resistance (Fig.
2). We then showed that TMZ-R cells with a higher level lncSNHG15 possessed a significantly higher self-renewal capacity, coincident with the higher expression of Sox2, β-catenin and EGFR, all of which have been attributed to GBM’s ability to fend off therapeutics [
19‐
21]. In addition, our observation was supported by a recent study where lncSNHG15 level was found elevated in the endothelial cells and involved in promoting angiogenesis [
22]. In addition, an increased level of lncSNHG15 was linked to the increased metastatic potential of non-small cell lung cancer [
23] and liver metastasis in colon cancer [
24]. Results from this study and others support the tumorigenic role of lncSNHG15.
Notably, we added another layer of complexity in terms of lncSNHG15’s role in GBM tumorigenesis where the increased lncSNH15 level was associated to the propensity to promote M2 polarization in the GAM. This association was supported by the observations where lncSNHG15 silencing significantly reduced M2 polarization while towards M1 polarization (Fig.
3). Moreover, we showed a positive correlation between the level of lncSNHG15 and oncogenic/stemness markers such as EGFR, CDK6, SOX-2 and β-catenin (Fig.
4). This observation added another possible target for drug development for treating GBM since these markers not only have been documented to be elevated in GBM but also contribute towards malignancy. For instance, β-catenin signaling was linked to the generation of CD133+ glioma stem cells [
25] as well as linked to the increased oncogenic activity of EGFR [
26]; more importantly, CDK6 has been shown to be an emerging target for drug development for GBM where CDK6 expression/activity is elevated in GBM cells and inhibitor of CDK6 could significantly suppressed GBM in vitro and in vivo [
27]. Based on these studies and our observations, we have further supported the tumorigenic role of lncSNHG15 in GBM.
To further explore the connection between CDK6 and lncSNHG15, we showed that treating TMZ-R cells with CDK6 inhibitor, palbociclib also led to the decreased level of lncSNHG15; palbociclib-treated TMZ-R cells showed a significantly decreased ability to generate M2 GAM (Fig.
5). Consistently, palbociblic treatment (suppression of CDK6) led to decreased expression of EGFR, Sox2 and β-catenin, similarly seen in the lncSNHG15-silenced TMZ-R cells. More importantly, lncSNHG15 knockdown and palbociclib treatment both led to an increased sensitivity towards TMZ. Mechanistically, we identified that a tumor suppressor, miR-627-5p targeted CDK6 at its 3’UTR and palbociclib treatment led to an increased level of this tumor suppressor (Fig.
6). Interestingly, in a previous report, miR-627 was found to target a histone demethylase (JMJD1A) and show anti-cancer function in colon cancer [
28]. Since JMJD1A is an epigenetic regulator, it was logical that palbociclib-induced miR-627 level could suppress tumorigenesis via a myriad of targets. Finally, our PDX study showed that the addition of palbociblic could overcome TMZ-resistance in vivo (Fig.
7).
Palbociclib (trade name: Ibrancea, approved by FDA in February 2015 to treat HR+/HER2- breast cancer), amebaciclib and ribociclib (trade name: Kisqali, approved by FDA in March 2017 to treat HR+/HER2- breast cancer) are recently developed CDK4/6 inhibitors currently undergoing clinical testing as potential chemotherapeutics for the treatment of primary or secondary brain tumors [
29]. Palbociclib was shown to promote survival in a genetic mouse model of brain stem glioma, but its unbound brain-to-plasma partition coefficient was only 5 min after intravenous administration of 1 mg/kg [
30]. There is preliminary evidence and preclinical data that ribociclib, abemaciclib and palbociclib cross the brain–blood barrier with data suggesting that albemaciclib may be more efficient in crossing the blood–brain barrier compared to palbociclb. Several clinical trials are ongoing to evaluate CDK4/6 inhibitors in patients with brain metastases.
In a Phase I dose escalation safety study, palbociclib was examined in 3 weeks on 1 week off schedule in 41 patients with advanced malignancies. Therecommended phase II dose, at which neutropenia was the sole significant toxicity was determined to be 125 mg once daily. As dose limiting toxicities, a reversible neutropenia was identified. Grade 3 hematological toxicities included neutropenia (12%) and anemia (7%). Non-hematological toxicity was mild, including fatigue, nausea and diarrhea. There was a clear signal for clinical activity of the drug: Thirty-seven patients were evaluable for tumor response; 10 (27%) had stable disease for ≥4 cycles of whom six derived prolonged benefit (≥10 cycles) [
31]. Beyond breast cancer, new indications for palbociclib are currently being evaluated in other malignancies such as sarcoma, pancreatic cancer, head & neck cancer, NSCLC, brain tumors, or even hematological malignancies.
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