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LOXL1-AS1 contributes to metastasis in sonic-hedgehog medulloblastoma by promoting cancer stem-like phenotypes

Journal of Experimental & Clinical Cancer Research volume 43, Article number: 130 (2024) Cite this article

Abstract

Background

Medulloblastomas (MBs) are one of the most common malignant brain tumor types in children. MB prognosis, despite improvement in recent years, still depends on clinical and biological risk factors. Metastasis is the leading cause of MB-related deaths, which highlights an unmet need for risk stratification and targeted therapy to improve clinical outcomes. Among the four molecular subgroups, sonic-hedgehog (SHH)-MB harbors clinical and genetic heterogeneity with a subset of high-risk cases. Recently, long non-coding (lnc)RNAs were implied to contribute to cancer malignant progression, but their role in MB remains unclear. This study aimed to identify pro-malignant lncRNAs that have prognostic and therapeutic significance in SHH-MB.

Methods

The Daoy SHH-MB cell line was engineered for ectopic expression of MYCN, a genetic signature of SHH-MB. MYCN-associated lncRNA genes were identified using RNA-sequencing data and were validated in SHH-MB cell lines, MB tissue samples, and patient cohort datasets. SHH-MB cells with genetic manipulation of the candidate lncRNA were evaluated for metastatic phenotypes in vitro, including cell migration, invasion, sphere formation, and expressions of stemness markers. An orthotopic xenograft mouse model was used to evaluate metastasis occurrence and survival. Finally, bioinformatic screening and in vitro assays were performed to explore downstream mechanisms.

Results

Elevated lncRNA LOXL1-AS1 expression was identified in MYCN-expressing Daoy cells and MYCN-amplified SHH-MB tumors, and was significantly associated with lower survival in SHH-MB patients. Functionally, LOXL1-AS1 promoted SHH-MB cell migration and cancer stemness in vitro. In mice, MYCN-expressing Daoy cells exhibited a high metastatic rate and adverse effects on survival, both of which were suppressed under LOLX1-AS1 perturbation. Integrative bioinformatic analyses revealed associations of LOXL1-AS1 with processes of cancer stemness, cell differentiation, and the epithelial-mesenchymal transition. LOXL1-AS1 positively regulated the expression of transforming growth factor (TGF)-β2. Knockdown of TGF-β2 in SHH-MB cells significantly abrogated their LOXL1-AS1-mediated prometastatic functions.

Conclusions

This study proved the functional significance of LOXL1-AS1 in SHH-MB metastasis by its promotion of TGF-β2-mediated cancer stem-like phenotypes, providing both prognostic and therapeutic potentials for targeting SHH-MB metastasis.

Background

Medulloblastomas (MBs) are one of the most common malignant brain tumor types in children [1]. Although the 5-year overall survival from MB has been improved to 70% ~ 80% [2,3,4], approximately one-third of patients surviving the primary tumor still suffer from recurrence, mostly presented with metastasis [5]. Metastatic disease in MB comes with the highest fatality, is hard to predict, and lacks a standard regimen [6]. Risk-adapted treatment approaches are currently optimized to maintain the survival rate but also to spare or limit the irradiation dose to avoid long-term neurocognitive effects [3, 4, 7]. The need for molecular definition of high-risk cases and targeted therapy options remains unmet for MB, mainly due to our insufficient understanding of molecular factors that drive MB malignancy.

MBs are classified into four molecular subgroups, namely wingless (WNT)-activated, sonic-hedgehog (SHH)-activated, Group 3, and Group 4, which are considered different entities due to their distinct genetic and clinical characteristics [8]. Accounting for ∼30% of all MB cases, SHH-MB has an intermediate to good prognosis in children and a well-characterized molecular profile [3, 4, 8, 9]. However, this is also the most heterogeneous subgroup with significant biological variations and unfavorable outcomes in a subset of cases [7, 9,10,11]. This phenomenon can be linked to genetic alterations that support malignant phenotypes of tumor cells. Among such drivers, the MYCN proto-oncogene is a target of SHH signaling pathway and also a signature gene of SHH-MB [12, 13]. Both MYCN amplification and MYCN overexpression are frequently observed in SHH-MB compared to other subgroups [8, 12, 14, 15]. MYCN-amplified tumors are relatively rare, but have poor prognosis and high resistance to smoothened (SMO) inhibitors [11, 14, 16,17,18]. MYCN ectopic expression in neural/neuroepithelial stem cells can generate highly proliferative and metastatic brain tumors in vivo, whose transcriptomes resemble SHH-MB with enhanced stemness signatures [19,20,21]. Nevertheless, MYCN remains a very hard-to-drug target due to its pleiotropic roles in both cancer and fetal cerebellar development [15].

Tumor aggressiveness driven by MYCN can be acquired via dysregulation of MYCN-dependent genes, including long non-coding (lnc)RNAs, a type of non-coding RNAs. LncRNAs were recently characterized as specific regulators of diverse cancer behaviors, although our understanding about their roles in MB is just emerging [22, 23]. Interestingly, SHH-MB has a unique landscape of non-coding transcripts with potential relevance in tumor biology [24]. Identification of novel lncRNAs that contribute to SHH-MB metastasis can support practical strategies for risk stratification, regimen optimization, and targeted therapy development to further improve clinical outcomes.

In this study, we applied integrative screening approaches to identify lncRNA genes associated with MYCN expression in SHH-MB, among which lysyl oxidase-like 1-antisense 1 (LOXL1-AS1) appeared as a potential candidate. We sought to determine this lncRNA’s clinical relevance, its functions related to metastatic phenotypes, and its putative molecular effectors in SHH-MB.

Methods

Clinical samples

This study included 70 MB cases from 1989 to 2019 at Taipei Veterans General Hospital (Taipei VGH) and Taipei Medical University Hospital (TMUH). All patients gave written informed consent to provide tissues and clinical data for research purposes. The collection and use of human specimens complied with the Declaration of Helsinki and followed a protocol approved by the Institutional Review Board of Taipei Medical University (IRB approval nos. 201701441A3 and N202202069). MB tissues obtained from the Taipei Medical University Joint Biobank (TMU-JBB) were separately processed to make a frozen tumor tissue repository and paraffin-embedded blocks for DNA/RNA profiling extraction and histological study, respectively.

Bioinformatic analysis

DNA/RNA profiling and clinical data of our MB cohort (n = 70) were described in a previous study [25]. For validation, an independent and non-overlapping MB cohort (n = 264) as part of a comprehensive dataset of pediatric tumors (OpenPedCan vers. 12) was retrieved from the Pediatric cBioPortal platform (https://pedcbioportal.org). Other MB datasets used in this study were accessed for analysis through the R2 public genomic archive platform (https://r2.amc.nl). Gene expression as normalized RNA-Seq read counts were retrieved for analyses. Differentially expressed gene (DEG) analysis and gene set enrichment analysis (GSEA) were performed using suitable packages in R (vers. 3.6.1).

Cell cultures

Two human SHH-MB cell lines, Daoy and ONS76, were originally obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Daoy cells were cultured in Eagle’s minimum essential medium (Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Cytiva) and 1% penicillin-streptomycin (PS) (Cytiva). ONS76 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% PS. HEK293FT cells obtained from Invitrogen (ThermoFisher Scientific, Waltham, MA, USA) were cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM) (Cytiva) supplemented with 10% FBS, 1% PS, and 1% non-essential amino acids (Cytiva). Cells were incubated at 37 °C in 5% CO2 and were routinely passaged upon 90% confluence.

Gene manipulation

For MYCN overexpression, complementary DNA (cDNA) of human MYCN (clone ID 3627330, Horizon Discovery, Cambridge, UK) was cloned into the pLenti4-Flag-CPO-v2 expression vector through the service of the National RNAi Core Facility (Academia Sinica, Taipei, Taiwan). For gene knockdown, small-hairpin (sh)RNAs targeting LOXL1-AS1 (shLOXL1-AS1) in the LV2N-U6/Puro vector were purchased from GenePharma (Shanghai, China), and shRNAs targeting TGFB2 (shTGFB2) in the pLKO.1-Puro vector were purchased from the National RNAi Core Facility (Academia Sinica). Detailed information about shRNA targeting sequences is given in Supplementary Table S1. The cDNA- or shRNA-containing lentiviruses were produced by transfection of HEK293FT cells using the Maestrofectin transfection reagent (Omics Bio, Taipei, Taiwan) according to the manufacturer’s protocol. Lentiviral transduction was performed on MB cells followed by selection with 250 μg/mL zeocin or 2.5 μg/mL puromycin to establish stable lines. For LOXL1-AS1 overexpression, the full-length cDNA of the LOXL1-AS1 transcript (2501 bp, NCBI accession no. NR_040066.1) in the pcDNA3.1 expression vector and the empty pcDNA3.1 vector as negative control were purchased from GenePharma, then subjected to direct transfection into SHH-MB cells using the Maestrofectin transfection reagent, followed by selection with 200 μg/mL G418 to establish a stable cell line.

Sphere-forming culture and sphere-formation assay

Monolayer cells were first detached with Accutase (BD Biosciences, Franklin Lakes, NJ, USA) until complete separation into single cells. Then, cells were washed three times with 1× phosphate-buffered saline (PBS) and seeded into ultra-low-attachment T175 flasks in a defined culture serum-free medium composed of DMEM/F12 (Cytiva) supplemented with 2% B-27 (ThermoFisher Scientific), 20 ng/mL of recombinant human basic fibroblast growth factor (bFGF; R&D Systems, Minneapolis, MN, USA), 20 ng/mL of recombinant human epidermal growth factor (hEGF; R&D Systems), and 1% PS [26]. A very low seeding density of 2000 cells/mL was applied to minimize cell aggregation. Fresh culture medium was added every 3 ~ 4 days for 2 weeks. Then, the entire culture of each sample was subjected to sphere counting, RNA extraction, or protein extraction. To evaluate sphere numbers and sizes, cells were gently re-distributed into a 96-well plate with a 3-mL plastic pipette for microscopic images, in which every spherical structure with a diameter of ≥100 μm was counted as a sphere using Fiji/ImageJ software. Sphere-formation efficiency was calculated as the number of spheres per 1000 cells seeded.

Cell migration and invasion assays

For the wound-healing assay, two- or three-well culture inserts (Ibidi, Gräfelfing, Germany) were placed in a culture dish, with 2.5 × 104 cells in 100 μL culture medium seeded into each well. After overnight incubation, the inserts were removed to create a gap. Cells were gently washed with 1× PBS and replenished with fresh culture medium for continued incubation. Microscopic images of the gap were captured at indicated time points. The gap area was quantified using Fiji/ImageJ software. For the transwell migration and invasion assays, hanging culture inserts with an 8-μm-pore membrane (Merck Millipore, Burlington, MA, USA) were placed in a 24-well plate. Additionally, the membrane was pre-coated with 50 μL of 1 mg/mL Matrigel (Life Sciences, Corning, NY, USA) in the invasion assay. In total, 5 × 104 cells in 200 μL serum-free medium were seeded into the insert (upper chamber) and 800 μL of culture medium with 1% FBS was added to the well (lower chamber). After incubation for 3 h for migration or 12 h for invasion, cells inside the insert were gently removed, and cells on the outside surface were fixed and stained with 0.5% crystal violet. Five random fields at 20× magnification were captured for automated cell counting using Fiji/ImageJ software. Alternatively, crystal violet from stained cells was dissolved in 100 μL of Sorenson’s solution and then quantified by measuring the absorbance at 570 nm.

RNA-sequencing (RNA-Seq) and real-time reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analysis

Total cellular RNA was extracted with NC RNA extraction reagent (EBL Biotechnology, Taipei, Taiwan) following the manufacturer’s instructions. Cellular RNA samples were sent to the Taipei Medical University Core Facility Center for library preparation and sequencing. A PrimeScript RT reagent kit (Takara Bio, Shiga, Japan) was used for RNA reverse-transcription into cDNA. Then, a real-time RT-qPCR was performed using gene-specific primer pairs (listed in Supplementary Table S2), a QuantiNova SYBR Green PCR kit (Qiagen, Hilden, Germany), and LightCycler 96 (Roche, Basel, Switzerland), all according to the manufacturers’ instructions. The 2-ΔΔCt method was applied to calculate gene expressions, with HSPCB being the housekeeping gene for normalization.

Protein extraction and Western blot analysis

Whole-cell lysates were collected, determined of protein concentration, and blotted following a previously described protocol [27] using specific antibodies: anti-N-Myc (cat. no. D4B2Y #51705, Cell Signaling Technology, Danvers, MA, USA), anti-Sox2 (cat. no. GTX101507, GeneTex, Irvine, CA, USA), anti-Oct4 (cat. no. GTX101507, GeneTex), anti-Nanog (D73G4, cat. no. #4903, Cell Signaling Technology), anti-Bmi1 (D20B7, cat. no. #6964, Cell Signaling Technology), anti-TGF-β2 (cat. no. MAB612, R&D Systems), and anti-β-actin (cat. no. GTX109639, GeneTex). All first antibodies were probed at a 1:1000 dilution overnight at 4 °C, while second antibodies, including anti-mouse (cat. no. 111-035-146, Jackson ImmunoResearch, West Grove, PA, USA) and anti-rabbit (cat. no. 111-035-144, Jackson ImmunoResearch), were probed at a 1:104 dilution for 1 h at room temperature. Blots were visualized using a chemiluminescent detection kit (T-Pro Biotechnology, New Taipei City, Taiwan) and Amersham Imager 600 (Cytiva Life Sciences). Fiji/ImageJ software was used to quantify bands with all values normalized to the β-actin signal.

In situ hybridization and immunohistochemical (IHC) staining

In situ hybridization was performed using an RNAScope 2.5 HD Assay-RED kit (Advanced Cell Diagnostics, Newark, CA, USA) following the manufacturer’s protocol. Gene-specific probes for human LOXL1-AS1 with human peptidyl-prolyl cis-trans isomerase B (PPIB) as a positive control and bacterial dapB as a negative control were designed by the manufacturer. RNA expression was determined by automated detection of chromogenic dots using the Color Deconvolution and Weka Classifiers plug-ins and was normalized to the positive control RNA signal. IHC staining was performed with an anti-Mycn antibody (cat. no. #51705, Cell Signaling Technology) and Novolink Polymer Detection System kit (Leica Biosystems, Richmond, IL, USA) according to the manufacturer’s instructions. Protein expression was determined by quantification of the chromogenic signal intensity. All images were analyzed with Fiji/ImageJ software.

Animal studies

This study was approved by the Taipei Medical University (TMU) Ethics Committee for the use of a xenograft brain tumor model (LAC-2021-0475). Luciferase-tagged Daoy or Daoy-MYCN cells were prior checked for stable luciferase activity and the absence of mycoplasma infection, and then washed and suspended in 1× PBS and Matrigel (Life Sciences) at a ratio of 2:1. In total, 4 × 105 cells in 10 μL were injected into the left cerebral hemisphere (2.5 mm lateral, 0.14 mm posterior to the bregma, and 3 mm in depth) of 6 ~ 8-week-old NOD/SCID mice (National Laboratory Animal Center, Taipei, Taiwan). Bioluminescence imaging (BLI) was performed at 3 and 7 days post-inoculation (dpi) to confirm the presence of xenograft cells and then weekly until the endpoint at day 63 (for Daoy vs. Daoy-MYCN) or day 56 (for Daoy-MYCN shNC vs. shLOXL1-AS1). The brain and spinal cord were obtained post-mortem for a histological study. All animal care, monitoring, and experiments followed guidelines of the TMU Laboratory Animal Center where the animal study was conducted.

Statistical analysis

Data are presented as the mean ± standard deviation (SD) with the difference between two groups determined by a two-tailed Student’s t-test unless otherwise specified in the figure legends. Correlations between two variables were assessed using a simple linear regression and Pearson’s correlation coefficients. Survival analyses were carried out using a Kaplan-Meier curve and the Gehan-Breslow-Wilcoxon test. The cut-off values of gene expression for samples grouping was determined using the maxstat and survminer packages (surv_cutpoint function, minprop = 0.25) in R software. All statistical analyses were performed with GraphPad Prism vers. 9 (GraphPad, San Diego, CA, USA) and Microsoft Office Excel vers. 16 (Microsoft, Redmond, WA, USA). p < 0.05 (*) was considered statistically significant.

Results

Establishment of a MYCN-driven SHH-MB model with a high rate of metastasis in vivo

To target SHH-MB metastasis, we first aimed to develop a suitable model in xenograft mice. Previous studies demonstrated de novo SHH-MB formation in mice which had been transplanted with neural stem cells aberrantly expressing MYCN [19,20,21]. MYCN withdrawal, on the other hand, abated cancer stemness properties of tumor-propagating cells in a MYCN-dependent MB mouse model [28]. We hypothesized that in MB cells with a low MYCN level, MYCN overexpression could promote their malignant features to drive metastasis in vivo. Compared to other human MB cell lines, Daoy cells have a gene expression profile closely resembling SHH-MB and can recapitulate the cancer stemness of human brain tumor-initiating cells [29]. Therefore, we ectopically expressed MYCN in Daoy cells (Daoy-MYCN) and then inoculated these cells into mouse brains to observe tumor formation and metastasis (Fig. 1A-B). Compared to mice injected with Daoy, those injected with Daoy-MYCN exhibited early-onset metastasis, a striking increase in the tumor burden, and a lower survival rate (Fig. 1C-D). BLI signals were detected in the head region indicating intracranial tumors and in the body region indicating spinal cord metastases (Fig. 1E). Histological studies confirmed the presence of xenograft tumors at inoculation site in both groups, and particularly at other sites of the central nervous system, including the cerebellum, brainstem, and spinal cord, in Daoy-MYCN group (Fig. 1F). Therefore, MYCN overexpression produced a highly aggressive phenotype of Daoy cells observed in xenografted mice.

Fig. 1
figure 1

Identification of dysregulated long non-coding (lnc)RNA targets in highly malignant MYCN-expressing sonic-hedgehog medulloblastoma (SHH-MB) cells and clinical samples. A Schematic outline of the experiment design. B Western blot analysis of MYCN expression in ONS76, Daoy, and Daoy-MYCN cells. C-E NOD/SCID mice were randomly divided into two groups for orthotopic inoculation with Daoy or Daoy-MYCN cells. Data are presented as (C) total photon flux measurements, (D) overall survival analysis, and (E) BLI of representative mice in each group from 3 to 56 dpi. F H&E staining images of the brain and spinal cord of a representative mouse from each group. G Scatterplot (left) showing all non-coding genes found in SHH-MB clinical samples (MYCN amplification positive versus negative, x-axis) and in cell line samples (Daoy-MYCN vs. Daoy, y-axis). Genes differentially expressed in both MYCN-amplified tumors and Daoy-MYCN cells are labeled with gene names and marked as red dots. Heatmap (right) of the top differentially expressed genes in MYCN-amplified SHH-MB tumors with their expression levels in Daoy and Daoy-MYCN cells. H RT-qPCR validation of five annotated lncRNA genes in ONS76, Daoy, and Daoy-MYCN cells. I Immunohistochemical staining of MYCN protein and in situ hybridization of LOXL1-AS1 RNA in Daoy and Daoy-MYCN cells (left images) and quantitative signals (right bar charts). Quantitative data are presented as mean ± SD of replicates from representative of three independent experiments. BLI, bioluminescence imaging; dpi, days post-inoculation. * p < 0.05, ** p < 0.01, *** p < 0.001

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Screening for dysregulated lncRNAs in SHH-MB cells identifies LOXL1-AS1 as a potential MYCN-associated gene

To explore potential lncRNAs in malignant SHH-MB, we performed RNA-Seq to achieve the first record of MYCN-dependent lncRNAs in Daoy-MYCN cells. To ensure clinical relevance, we used DNA/RNA profiling data from 24 SHH-MB patients and grouped those harboring MYCN amplification (n = 4) to compare against the remaining cases, resulting in differentially expressed lncRNAs in these cases. Then, Daoy cell RNA-Seq data were integrated to further screen out the top nine genes with the highest expression levels in both MYCN-amplified tumors and MYCN-expressing cells (Fig. 1G). Five of these nine candidates have been fully annotated and were subjected to validation. Expression of the gene LOXL1-AS1 was significantly elevated in Daoy-MYCN compared to Daoy in RT-qPCR (Fig. 1H), which was further confirmed by in situ hybridization (Fig. 1I). Explorative in silico genomic screening for predicted transcription factor-binding sites detected enrichment of a MYCN-binding motif upstream of the LOXL1-AS1 transcription start site (Supplementary Fig. S1), suggesting a possible interaction via promoter region binding. Altogether, our integrative screening identified LOXL1-AS1 as a potential MYCN-associated lncRNA in SHH-MB.

LOXL1-AS1 is highly expressed in MYCN-positive tissues and was correlated with poor outcomes in the SHH-MB subgroup

Next, we validated the relevance of LOXL1-AS1 in SHH-MB clinical samples and cohorts. LOXL1-AS1 RNA signals were found to be higher in MYCN-positive SHH-MB tissues than in MYCN-negative ones (Fig. 2A-B). A correlation between MYCN and LOXL1-AS1 expression was found in primary cell lines of SHH-MB, despite statistical insignificance likely due to small sample size (Fig. 2C). Notably, two primary cell lines with MYCN amplification were also highly expressed with both MYCN and LOXL1-AS1. LOXL1-AS1 expression was also higher in MB compared to the normal brain or other brain tumors (Figs. 2D, S2A). Survival analyses uncovered that high LOXL1-AS1 expression was correlated with poor outcomes in MB regardless of the subgroup (Fig. 2E) and more significantly in the SHH subgroup (Figs. 2F, S2B). SHH-MB cases were further sub-categorized into three subtypes of SHH-α, -β, and -γ in children [9], where LOXL1-AS1 expression was significantly associated with worse survival in SHH-α and SHH-γ patients (Fig. 2G-I). Collectively, LOXL1-AS1 is a novel marker predictive of poor outcomes in SHH-MB.

Fig. 2
figure 2

Tissue expression of LOXL1-AS1 and its association with survival in sonic-hedgehog medulloblastoma (SHH-MB) cohorts. A-B Immunohistochemical staining of MYCN protein and in situ hybridization of LOXL1-AS1 and positive control RNA in 13 SHH-MB samples divided into two groups based on the MYCN signal (positive or negative), including (A) representative staining images and (B) quantitative LOXL1-AS1 signals. Scale bars, 20 μm. C Correlation between MYCN and LOXL1-AS1 in MB primary cell lines (Brain Tumor Resource Lab). Two SHH cell lines with MYCN amplifications are specifically indicated. Simple linear regression analysis: SHH, n = 7, R = 0.687, p = 0.0883; Group 3, n = 14, R = -0.531, p = 0.0509; Group 4, n = 4, R = -0.144, p = 0.8557. D LOXL1-AS1 expression in the normal brain and in different types of brain tumors in a public dataset (Donson, R2 platform). PA, pilocytic astrocytoma; GBM, glioblastoma; EPN, ependymoma; MB, medulloblastoma. E-I Survival analyses of LOXL1-AS1 expression in our cohort (TMU), including (E) all MB cases, (F) the SHH subgroup of MB, and (G-I) three subtypes of SHH-MB. * p < 0.05, ** p < 0.01, *** p < 0.001

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LOXL1-AS1 enhances the stemness features and promotes the migratory and invasive abilities of SHH-MB cells in vitro

To investigate the effects of LOXL1-AS1 in vitro, we silenced LOXL1-AS1 expression in Daoy-MYCN cells and overexpressed LOXL1-AS1 in Daoy and ONS76 cells (Fig. 3A). To evaluate stemness-related characteristics, we maintained SHH-MB cells in a defined sphere-forming condition widely used to enrich cancer stemness in vitro [26]. Results showed that the known stemness regulators, including Nanog, Oct4, Sox2, and Bmi1, decreased under LOXL1-AS1 depletion and increased under ectopic LOXL1-AS1 expression (Fig. 3B-C, E, G). The sphere-formation capacity, reflected by the sphere number and size, was significantly impaired in LOXL1-AS1-knockdown cells but was augmented in LOXL1-AS1-overexpressing cells (Fig. 3D, F, H). Furthermore, LOXL1-AS1 silencing in Daoy-MYCN cells significantly reduced cell migratory phenotypes observed via wound-healing and transwell migration-invasion assays (Fig. 4A, D), while LOXL1-AS1 overexpression in Daoy and ONS76 cells produced corresponding increases (Fig. 4B-C, E-F). Taken together, LOXL1-AS1 facilitated metastatic features of SHH-MB cells, including expression of stemness factors, formation of tumor spheres, and the capabilities of cell migration and invasion in vitro.

Fig. 3
figure 3

Effects of LOXL1-AS1 on stemness phenotypes of sonic-hedgehog medulloblastoma cells. A RT-qPCR analysis of LOXL1-AS1 expression in the Daoy-MYCN, Daoy, and ONS76 cell lines relative to the parental group. B Western blot analysis of four stemness markers in the Daoy-MYCN, Daoy, and ONS76 cell lines. Quantification values below each band are relative to the parental group. C-H RT-qPCR analysis of four stemness markers in the (C) Daoy-MYCN, (E) Daoy, and (G) ONS76 cell lines relative to the parental group. Sphere-formation assay of the (D) Daoy-MYCN, (F) Daoy, and (H) ONS76 cell lines, including microscopic images at different magnifications (left images) and the number and sizes of spheres of ≥150 μm in diameter (right bar chart). Scale bars, 500 μm in 4× and 100 μm in 20× magnification. Quantitative data are presented as mean ± SD of replicates from representative of three independent experiments. NC, negative control; ns, non-significant. * p < 0.05, ** p < 0.01, *** p < 0.001

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Fig. 4
figure 4

Effects of LOXL1-AS1 on the migration and invasion capabilities of sonic-hedgehog medulloblastoma cells. A-C Wound-healing assay of the (A) Daoy-MYCN, (B) Daoy, and (C) ONS76 cell lines. D-F Transwell migration and invasion assays of the (D) Daoy-MYCN, (E) Daoy, and (F) ONS76 cell lines, including quantification of cell migration (left bar chart) and invasion (right bar chart). Quantitative data are presented as mean ± SD of replicates from representative of three independent experiments. NC, negative control; ns, non-significant. * p < 0.05, ** p < 0.01, *** p < 0.001

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Perturbation of LOXL1-AS1 in MYCN-expressing SHH-MB cells significantly delays early-onset metastasis and improves survival in vivo

To evaluate the effect of LOXL1-AS1 depletion in vivo, the highly metastatic Daoy-MYCN cells with LOXL1-AS1 knockdown were chosen for transplantation into mouse brains (Fig. 5A). LOXL1-AS1-knockdown resulted in significantly improved overall survival and metastasis-free survival (Fig. 5B-C). Although all mice eventually developed tumors, those injected with LOXL1-AS1-knockdown cells exhibited a notable delay in the occurrence of metastasis compared to those injected with knockdown-negative control cells (Fig. 5D). Seven of 10 mice in the control group experienced metastasis and five of them died rapidly within the first 3 weeks after inoculation (Fig. 5E). In contrast, mice in the LOXL1-AS1-knockdown groups had low metastasis rates and none died prior to 5 weeks of cell transplantation (Fig. 5F). These knockdown groups, however, experienced high primary tumor burdens later, most likely due to their prolonged survival time. Histological study of tissues collected at the end-point confirmed the presence of xenograft tumors at the site of inoculation and at other common sites of leptomeningeal spread (Supplementary Fig. S3). Interestingly, higher LOXL1-AS1 signals were detected in metastatic lesions regardless of the mouse group (Fig. 5G). Our in vivo results suggested that LOXL1-AS1 is a crucial factor not for primary tumor growth but for the development of leptomeningeal metastasis, which severely affects survival.

Fig. 5
figure 5

Effects of LOXL1-AS1 perturbation on metastasis occurrence and survival rates in orthotopic xenograft mice. A Schematic outline of the animal study. NOD/SCID mice were randomly divided into three groups for inoculation with Daoy-MYCN -shNC, -shLOXL1-AS1 #1196, and -shLOXL1-AS1 #2278 cells. B Overall survival analysis of the three groups. C Metastasis-free survival analysis of the three groups. D BLI of all mice in each group from 3 to 56 dpi. E-F Data analysis at 21 dpi, including (E) the number of mice with only intracranial signals versus with both intracranial and metastatic signals and (F) the number of dead mice in each group. The presence and location of tumors were confirmed by a histological study of brains and spinal cords at the end-point. G LOXL1-AS1 signal detected from in situ hybridization of the brain and spinal cord tissue of representative mice from each group. See also Supplementary Fig. S3. BLI, bioluminescence imaging; dpi, days post-inoculation. * p < 0.05, ** p < 0.01

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RNA-Seq data analyses reveal LOXL1-AS1 potentially regulating pathways and genes involved in cell migration and stemness-differentiation

LncRNAs can act via a wide range of mechanisms, many of which are linked to modulating gene expressions. To identify downstream targets responsible for the significant changes observed in Daoy-MYCN cells, we performed thorough RNA-Seq profiling of two knockdown samples (shLOXL1-AS1 #1196 and #2278) compared to the negative control (shNC). These RNA-Seq data were first subjected to unbiased GSEA screening (Supplementary Fig. S4A). The top up- and down-regulated gene sets with both significance and agreement between the two knockdown samples were identified and showed that LOXL1-AS1 could be involved in the PI3K/AKT/mTOR pathway, epithelial-mesenchymal transition (EMT), and cell adhesion, differentiation, and stemness (Figs. 6A, S4B). Analysis of DEGs were explored to produce a curated list of important genes involved in each process (Fig. 6B). Next, we adopted a second approach in which totals of 401 DEGs in both knockdown samples were used as input for an investigative GSEA (Supplementary Fig. S4C). Results of the top enriched gene sets with the top overlapping genes revealed the involvement of neurogenesis, neuron generation, cell adhesion, and cell proliferation processes (Fig. 6C). These data suggested that the phenotypical changes observed upon LOXL1-AS1 alteration in SHH-MB cells could result from dysregulation of pro-migratory and pro-stemness signaling pathways.

Fig. 6
figure 6

RNA-sequencing analyses of dysregulated pathways and putative effector genes of LOXL1-AS1. A Gene set enrichment analysis (GSEA) plots of Daoy-MYCN-shLOXL1-AS1 #1196 versus -shNC samples in six gene sets of interest. See also Supplementary Fig. S4A-B. B Heatmap of differentially expressed genes (DEGs) closely associated with pathways or processes of interest. C GSEA of the top DEGs found in both knockdown samples. Bar chart (top) shows the false discovery rate (FDR) and p value of the top nine enriched pathways. Matrix table (below) includes the top 20 enriched genes and their presence in each pathway (red, present; gray, absent). See also Supplementary Fig. S4C. D Venn diagrams of DEGs found in each knockdown sample overlapping with an additional set of genes that have strong correlations with LOXL1-AS1 found in a public SHH-MB dataset (Pfister-223, R2 platform). Genes at the center overlap are specifically indicated. See also Supplementary Fig. S5A. E Bar chart of six candidate genes summarizing their differential expressions in SHH-MB cells (left y-axis) and correlations with LOXL1-AS1 in SHH-MB patient dataset (right y-axis). F RT-qPCR validation of TGFB2 in the Daoy-MYCN, Daoy, and ONS76 cell lines. Quantitative data are presented as mean ± SD of replicates from representative of three independent experiments. NC, negative control. ** p < 0.01, *** p < 0.001. G-I TGFB2 expression in the OpenPedCan cohort including (G) expression levels across four molecular MB subgroups, (H) correlation with LOXL1-AS1 in SHH-MB, and (I) survival analysis

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TGF-β2 is a putative effector of LOXL1-AS1 with prognostic value and prometastatic functions in SHH-MB

To identify the functional effector of LOXL1-AS1, we stringently narrowed down candidate genes which exhibited consistent associations with LOXL1-AS1 in SHH-MB. A collection of DEGs found in Daoy-MYCN-shLOXL1-AS1 cells was intersected with another collection of genes correlated with LOXL1-AS1 in SHH-MB tumors (Figs. 6D, S5A), resulting in six dysregulated candidate genes (Figs. 6E, S5B). RT-qPCR validation in SHH-MB cells further confirmed that TGFB2, encoding TGF-β2, had the most significant changes under LOXL1-AS1 alterations (Figs. 6F, S5C). Importantly, TGFB2 was among the genes participating in pathways discovered from our GSEA results (Fig. 6B, C). Among MB subgroups, TGFB2 had the highest mRNA basal level and the strongest correlation with LOXL1-AS1 in SHH-MB (Figs. 6G-H, S5D-E), as well as an association with unfavorable outcomes (Figs. 6I, S5F).

Next, we defined the functional role of TGF-β2 by investigating whether it was sufficient to abrogate metastatic phenotypes promoted by LOXL1-AS1 in SHH-MB cells. To this end, we used shRNA to silence endogenous TGF-β2 in Daoy-MYCN and Daoy-LOXL1-AS1 cells (Fig. 7A). The metastatic phenotypes of Daoy-LOXL1-AS1 cells were significantly impaired by TGF-β2 knockdown, reflected via decreases in expression of stemness markers (Fig. 7B-C), sphere formation (Fig. 7D), and cell migration and invasion (Fig. 7E-F). TGF-β2 knockdown in Daoy-MYCN cells also resulted in similar effects (Fig. 7B-C, G-I), which were previously observed under LOXL1-AS1 silencing (Figs. 3-4). Taken together, TGF-β2 is regulated by LOXL1-AS1 and mediates the prometastatic effects of LOXL1-AS1 in SHH-MB cells.

Fig. 7
figure 7

Effects of transforming growth factor (TGF)-β2 silencing on metastatic phenotypes of sonic-hedgehog medulloblastoma cells with high LOXL1-AS1 expression. A RT-qPCR analysis of TGF-β2 expression in Daoy-LOXL1-AS1 and Daoy-MYCN cells. B RT-qPCR analysis of four stemness markers in Daoy-LOXL1-AS1 and Daoy-MYCN cells relative to the parental group. C Western blot analysis of four stemness markers in Daoy-LOXL1-AS1 and Daoy-MYCN cells. Quantification values below each band are relative to the parental group. D, G Sphere-formation assay of (D) Daoy-LOXL1-AS1 and (G) Daoy-MYCN cells, including microscopic images (left images), and the number and sizes of spheres of ≥150 μm in diameter (right bar chart). Scale bars, 500 μm in 4× and 100 μm in 20× magnification. E, H Wound-healing assay of (E) Daoy-LOXL1-AS1 and (H) Daoy-MYCN cells. F, I Transwell migration and invasion assays of (F) Daoy-LOXL1-AS1 and (I) Daoy-MYCN cells including quantification of cell migration (left bar chart) and invasion (right bar chart). Quantitative data are presented as mean ± SD of replicates from representative of three independent experiments. NC, negative control; ns, non-significant. * p < 0.05, ** p < 0.01, *** p < 0.001

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Discussion

Metastasis, either at diagnosis or at recurrence, is the adverse feature that contributes the most to mortality in MB [5]. In this study, we recapitulated metastasis in vivo using SHH-MB cells that expressed the MYCN malignant driver. An integrative screening approach was applied to identify the LOXL1-AS1 lncRNA as a potential prometastatic factor. Recently, LOXL1-AS1 has emerged as an oncogenic lncRNA in several cancer types [30, 31]. A study by Gao et al., to our knowledge the first and only report dedicated to LOXL1-AS1 in MB, suggested its involvement in the aggressiveness of Group 3 MB [30]. Here, we focused on the SHH subgroup of MB in which LOXL1-AS1 expression was highly prognostic. We further described in detail the contribution of LOXL1-AS1 to critical aspects of the metastasis hallmark, including cancer stemness, and discovered TGF-β2 to be a mediator of these prometastatic functions in SHH-MB.

Apart from the high prognostic value of MYCN amplification status in SHH-MB, MYCN mRNA expression was elevated in WNT-MB and SHH-MB but its prognostic value remains inconclusive across all subgroups [12, 32, 33]. Our RNA-Seq validation confirmed the same findings, with the MYCN mRNA level being the highest but a suboptimal indicator of clinical outcomes in SHH-MB (Supplementary Fig. S2C-F). LOXL1-AS1 expression, on the other hand, was not high in the SHH subgroup or any of its subtypes (Supplementary Figs. S2G-I), but was superior in correlating poor outcomes compared to MYCN. This highlights the pathological context-specific characters of lncRNAs like LOXL1-AS1, in contrast to MYCN which is a universal transcription factor with pleiotropic roles in both normal and disease processes [15].

In MB, metastasis occurs in a distinct way of leptomeningeal dissemination where cancer cells are seeded via cerebral spinal fluid to form multiple lesions across the central nervous system [34]. Stemness features of spreading MB cells, i.e., the cells’ ability to initiate and propagate to form a secondary population, is particularly relevant to this mode of metastasis. As suggested from our animal data, high LOXL1-AS1 expression seemed to be an advantage for SHH-MB cells to establish metastatic tumors during leptomeningeal spread. This aligns with recent studies showing that primary and metastatic MB tissues have distinct genetic profiles [5] and clonal selection can give rise to divergent cell populations [35]. To this end, we further pointed out the previously unknown role of LOXL1-AS1 in SHH-MB cell’s stemness. Among stemness markers mediated by LOXL1-AS1, Sox2 was highly expressed and was correlated with poor prognosis in the SHH subgroup [36, 37]. Sox2 expression defines both tumor-initiating and tumor-propagating MB cells capable of self-renewal and resistance to SMO inhibitors [38, 39]. Oct4 and Bmi1 are predictive of shorter survival times [40] and can maintain a stem-like cellular state to drive MB formation and metastasis [21, 41]. Bmi1 also regulates multiple genes associated with tumor-stromal interactions and cell adhesion in MB [42], whereas little is known about the role of Nanog in this tumor.

The TGF-β family is a well-established mediator of cancer stem cell potential and metastatic niches via either canonical Smad signaling or non-canonical pathways like PI3K/AKT/mTOR [43]. In brain malignancies, TGF-β1 and TGF-β2 are known as tumor growth factors [44], while a recent study revealed that TGF-β1 signaling from T cells can promote SHH-MB progression [45]. However, no previous study investigated the role of TGF-β2 in the SHH subgroup. Herein, we discovered TGF-β2 to be a LOXL1-AS1-dependent gene and confirmed its pro-metastatic functions in SHH-MB. While TGF-β2 and Sox2 were both transcriptionally mediated by LOXL1-AS1, these two genes were also strongly correlated in SHH-MB (Supplementary Fig. S5G). This indicates a crucial role of LOXL1-AS1 in regulating a group of pro-stemness drivers to support SHH-MB metastasis.

The present study has several limitations. The exact mechanism of how MYCN regulates LOXL1-AS1 remain to be determined, which can be via different mechanisms like transcriptional activation via direct promoter region binding [46] or indirectly via intermediate dependent factors [47]. The way LOXL1-AS1, TGF-β2, and other stemness regulators interact at the molecular level also warrant deeper investigation. The low number of cases in our MB cohort should also be noted when interpreting results from patient data, which was partially resolved by validation with a larger and non-overlapping MB cohort. Finally, our animal model performed in mice intracranially engrafted with SHH-MB cell lines can be further improved by using patient-derived primary cells collected at recurrence. Once overcoming the low establishment rate of only around 35% [48, 49], this type of model can more accurately recapitulate MB recurrence and metastasis in response to conventional therapies.

Findings about LOXL1-AS1’s specific expression, prometastatic function, and prognostic value in SHH-MB make it a promising candidate for translational studies. Since the analysis of disseminating tumor cells in the cerebrospinal fluid is emerging [50], biomarkers like LOXL1-AS1 could be further evaluated for molecular stratifications to assist risk-adapted therapy. Furthermore, with recent advances in precise gene delivery and lncRNA breakdown in vivo, therapeutic models targeting LOXL1-AS1 can be developed to reduce metastatic events in such high-risk cases of SHH-MB.

Conclusions

In conclusion, the MYCN-associated lncRNA LOXL1-AS1 contributes to SHH-MB metastasis by promoting TGF-β2-mediated cancer stem-like phenotypes. This study discovered a novel molecular target with both prognostic and therapeutic implications to minimize tumor recurrence, potentiating the de-escalation of cytotoxic regimens to improve clinical outcomes in children bearing this dismal type of brain tumor.

Availability of data and materials

Data generated and/or analyzed in the current study are available from the corresponding author upon reasonable request.

Abbreviations

BLI:

Bioluminescence imaging

DEG:

Differentially expressed gene

EMT:

Epithelial mesenchymal transformation

GSEA:

Gene set enrichment analysis

lncRNA:

Long non-coding RNA

MB:

Medulloblastoma

RNA-Seq:

RNA sequencing

SHH:

Sonic-hedgehog

WNT:

Wingless

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Acknowledgements

The authors would like to express appreciation for Ms. Chia-Yen Hsueh and Dr. Le Thi Huynh Trang for their helpful guidance with the experiments. We thank the Taipei Medical University Core Facility Center and Laboratory Animal Center for providing technical support in RNA-sequencing and animal experiments, the RNA Technology Platform and Gene Manipulation Core Facility (RNAi Core) of the National Core Facility for Biopharmaceuticals (Academia Sinica, Taipei, Taiwan) for providing shRNA reagents, and Enago (www.enago.com) for the English language review.

Funding

This work was supported by fundings from the Ministry of Science and Technology (NSTC 112-2314-B-169-001, MOST 112-2314-B-001-007, MOST 111-2314-B-038-078-MY3 and MOST 110-2314-B-038-137-MY3) and the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE).

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Authors and Affiliations

  1. International Ph.D. Program for Translational Science, College of Medical Science and Technology, Taipei Medical University, Taipei, 11031, Taiwan

    Anh Duy Do, Le Hien Giang, Che-Chang Chang & Shian-Ying Sung

  2. Department of Physiology, Pathophysiology and Immunology, Pham Ngoc Thach University of Medicine, Ho Chi Minh City, 700000, Vietnam

    Anh Duy Do

  3. Graduate Institute of Clinical Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, 11031, Taiwan

    Kuo-Sheng Wu, Shing-Shung Chu, Tai-Tong Wong & Shian-Ying Sung

  4. Department of Biology and Genetics, Hai Phong University of Medicine and Pharmacy, Hai Phong, 180000, Vietnam

    Le Hien Giang

  5. Agricultural Biotechnology Research Center, Academia Sinica, Taipei, 11529, Taiwan

    Yu-Ling Lin

  6. The Ph.D. Program for Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, 11031, Taiwan

    Che-Chang Chang, Chia-Ling Hsieh & Shian-Ying Sung

  7. Neuroscience Research Center, Taipei Medical University Hospital, Taipei, 11031, Taiwan

    Che-Chang Chang, Tai-Tong Wong, Chia-Ling Hsieh & Shian-Ying Sung

  8. Pediatric Brain Tumor Program, Taipei Cancer Center, Taipei Medical University, Taipei, 11031, Taiwan

    Tai-Tong Wong

  9. Division of Pediatric Neurosurgery, Department of Neurosurgery, Taipei Neuroscience Institute, Taipei Medical University Hospital, Taipei Medical University, Taipei, 11031, Taiwan

    Tai-Tong Wong

  10. Institute for Drug Evaluation Platform, Development Center for Biotechnology, Taipei, 11571, Taiwan

    Chia-Ling Hsieh

  11. TMU Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei, 11031, Taiwan

    Shian-Ying Sung

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Contributions

Conceptualization, A.D.D. C.-L.H. and S.-Y.S; validation, K.-S.W., S.-S.C., L.H.G. and A.D.D.; formal analysis, K.-S.W. and A.D.D.; investigation, T.-T.W., C.-C.C., Y.-L.L., S.-Y.S. and C.-L.H; writing—original draft preparation, A.D.D.; writing—review and editing, C.-L.H. and S.-Y.S.; funding acquisition, S.-Y.S., T.-T.W., Y.-L.L., C.-C.C. and C.-L.H. All authors read and approved the final manuscript.

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Correspondence to Chia-Ling Hsieh or Shian-Ying Sung.

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Do, A.D., Wu, KS., Chu, SS. et al. LOXL1-AS1 contributes to metastasis in sonic-hedgehog medulloblastoma by promoting cancer stem-like phenotypes. J Exp Clin Cancer Res 43, 130 (2024). https://doi.org/10.1186/s13046-024-03057-0

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Journal of Experimental & Clinical Cancer Research

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