LncRNA-MALAT1-mediated Axl promotes cell invasion and migration in human neuroblastoma

Abstract

Overexpression of Axl has been noted to correlate with several human cancers. However, the regulatory mechanisms and effects of Axl in human neuroblastoma development remain unclear. Here, we explore the expression of Axl in neurobalstoma and related upstream regulatory mechanisms of invasion and migration. We found that Axl was overexpressed in metastatic neuroblastoma tissues and positively associated with long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1. Meanwhile, our data suggested that metastasis-associated lung adenocarcinoma transcript 1 upregulated Axl expression in neuroblastoma cells, resulting in cell invasion and migration. Furthermore, we found that targeting Axl by inhibitor R428 significantly suppressed the abilities of tumor cell invasion and migration. In summary, these results suggested that Axl, which is regulated by long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1, may exert great influence on invasion and migration of neuroblastoma.

Keywords

Axl metastasis-associated lung adenocarcinoma transcript 1 neuroblastoma invasion migration

Introduction

Neuroblastoma, which arises from precursor neuroblast cells within the embryonic sympathetic nervous system, is one of the most common and aggressive malignancies in children.^1,2 As a very complicated disease which exhibits genetic and clinical heterogeneity, the patients suffering from neuroblastoma can be reclassified into low-, median- and high-risk stratifications.³ Currently, surgical resection, radiation, and chemotherapy are the main approaches for high-risk neuroblastoma treatment, but recurrence and metastasis are serious common events accounting for the poor prognostic outcomes.⁴ Therefore, it is vital to elucidate new mechanisms associated with neuroblastoma pathogenesis and identify useful new biomarkers to develop effective targeted treatment for clinical benefits.

Axl, together with Mer and Tyro3, are members of the Tyro-3–Axl–Mer (TAM) family of receptor tyrosine kinases (RTKs).⁵ The structure of Axl consists of two immunoglobulin (Ig)-like domains, pairs of fibronectin type III (FNIII) domains, a single transmembrane domain, and an intracellular kinase domain (17 tyrosine residues).⁶ Typically, binding of its ligand Gas6 to the extracellular domain leads to dimerization of Gas6–Axl complexes, which results in autophosphorylation of tyrosine residues on the intracellular tyrosine kinase domain and then increased activity of the downstream signaling pathways.⁷ Recently, Axl have been elucidated to mediate diverse cellular functions, including survival, differentiation, adhesion, proliferation, motility, and drug resistance in the development of many cancers, including hematologic and solid malignancies.^8,9 Our previous study has showed that Mer and Axl RTKs were co-expressed in human neuroblastoma, and inhibiting Axl leads to cell death and enhanced chemosensitivity in neuroblastoma.¹⁰ However, the expression patterns of Axl and related upstream regulatory mechanisms in neuroblastoma still remain largely unknown.

The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), also known as nuclear enriched abundant transcript 2 (NEAT2), is a type of long non-coding RNA (lncRNA) and demonstrated to be overexpressed in metastatic lung cancer tissues and several solid carcinomas.¹¹ Recent studies show that MALAT1 promotes migration, epithelial–mesenchymal transition (EMT), and metastasis in non-small-cell lung cancer,¹² bladder cancer,¹³ hepatocellular carcinoma,¹⁴ and colorectal cancer.¹⁵ Nevertheless, the function of MALAT1 in neuroblastoma remains unclear and the relationship among MALAT1 and migration also needs to be elucidated.

In this study, we explored the role of Axl in neuroblastoma. Our results showed that AXL was overexpressed in neuroblastoma and positively associated with an lncRNA MALAT1. Meanwhile, our data suggested that MALAT1-mediated Axl upregulation may play a vital role in invasion and migration. Furthermore, we found that Axl inhibitor R428 can suppress the ability of invasion and migration. Taken together, our results elucidated the roles of MALAT1-mediated Axl in cell invasion and migration, providing supports for these molecules as attractive novel targets for neuroblastoma therapy.

Materials and methods

Tissue specimens, cell lines, and transfection

In all, 15 non-tumor normal tissues (NT; male, n = 8; female, n = 7), 19 primary neuroblastoma (pNB) tissues (male, n = 7; female, n = 12), and 28 metastatic neuroblastoma (mNB) tissues (male, n = 13; female, n = 15) were collected and stored at −80°C. The collection of tumor tissues was approved by our Institutional Review Board (IRB). A panel of neuroblastoma cell lines (NGP, SHSY5Y, NMB, SHEP21N, SKNAS, and SHEP2) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cells were cultured in RPMI 1640/DMEM (Hyclone, Logan, UT, USA) containing 10% inactivated fetal bovine serum (FBS; Hyclone), 100 Units/mL penicillin, and 100 mg/mL streptomycin (Hyclone). The small-molecule Axl inhibitor R428 was provided by Selleck Chemicals (Houston, TX, USA). Control siRNA (si-control), siRNA specific for Axl and MALAT1, pcDNA3.1 control, and pcDNA3.1-Axl were transfected into cells using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. MALAT1 was constructed into pCDH-CMV-MCS-EF1-Puro vector (LV-MALAT1), with empty vector (LV-control) as control. The verified recombined vectors were cotransfected with psAX2 and pMD.2G into 293T cells to produce the virus. Then, the culture supernatants were added into the neuroblastoma cells. Cells stably expressing MALAT1 were selected with 5 µg/mL puromycin.

RNA extraction and quantitative reverse transcription polymerase chain reaction

Total RNA was isolated from frozen tissues and cell lines using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Complementary DNA (cDNA) was obtained using Transcriptor First Strand cDNA Synthesis Kit (Roche, Brussel, Belgium). The relative level of MALAT1 and Axl to control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcripts was determined by quantitative polymerase chain reaction (qPCR) using GoTaq qPCR Master Mix with SYBR green (Promega, Madison, WI, USA). Primers for genes were as follows: Axl forward 5′-ATCAGCTTCGGCTAGGCAG-3′ and reverse 5′-TCCGCGTAGCACTAATGTTCT-3′; MALAT1 forward 5′-GACGGAGGTTGAGATGAAGC-3′ and reverse 5′-ATTCGGGGCTCTGTAGTCCT-3′.

Immunohistochemistry staining and western blot

Immunohistochemistry (IHC) and western blot were performed as previously described;¹⁰ 4-µm sections were mounted on a glass slide. After antigen retrieval, the sections were incubated with anti-Axl overnight, followed by a horseradish peroxidase (HRP)-labeled second antibody. The intensity of immunostaining was assessed under microscope. For western blot, the following primary antibodies were used: Axl, phospho-Axl (p-Axl), and GAPDH (Cell Signaling Technology, Beverly, MA, USA).

Transwell invasion assay

Cell invasion assay was performed using Transwell chamber (Corning, New York, NY, USA) coated with Matrigel (BD Biosciences, Bedford, MA, USA). Briefly, neuroblastoma cells were plated on the top chamber and suspended in serum-free medium. Medium containing 10% FBS was used as a chemoattractant in the bottom chamber. For some experiments, SKNAS and SHEP2 cells were treated with vehicle control or 100 nM R428. After being cultured 48 h, the non-invading cells were removed. The invaded cells on the lower membrane surface were fixed by methanol, stained, and counted.

Wound healing assay

Equal numbers of cells were seeded into plates, and then, an artificial wound was created onto the monolayer when cell confluence reached 90%. Then, the debris was removed by changing into serum-free medium. After 48 h, the migration of cells was observed and photographed under microscope.

Statistical analysis

For analysis of differences between the groups, one-way analysis of variance (ANOVA) or t-test was performed using GraphPad Prism. The results are expressed as the mean ± standard error of the mean (SEM), with p < 0.05 considered as statistically significant.

Results

AXL was overexpressed in mNB and positively associated with MALAT1

Axl expression was determined in histologically 19 primary neuroblastoma (pNB) tissues, 28 mNB tissues, and 15 NT by qRT-PCR and normalized to GAPDH. Axl expression was significantly upregulated in mNB compared with primary cancerous tissues and normal tissues (p < 0.01 and p < 0.001, respectively; Figure 1(a)). Examination of Axl expression by IHC also shows that Axl was overexpressed in primary and metastatic cancerous tissues (Figure 1(b)). Since lncRNAs exert biological effects by modulating gene expression¹⁶ and MALAT1 is well-known to be overexpressed in metastatic tumor tissues,^17,18 we next examined whether increased expression of Axl was associated to the MALAT1. As shown in Figure 1(c), MALAT1 expression was significantly higher in metastatic cancerous tissues compared with pNB and normal tissues (p < 0.001). We then examined whether increased expression of Axl was associated to the MALAT1 levels. Interestingly, we found a strong positive correlation between lncRNA MALAT1 and Axl expressions (r = 0.426, p < 0.01) in neuroblastoma tissues (Figure 1(d)). These results were consistent with the reanalysis of previously published dataset GSE16237 (Figure 1(e)), indicating that Axl was overexpressed in mNB and may be positively associated with MALAT1 expression.

Figure 1.

AXL was overexpressed in metastatic neuroblastoma and positively associated with MALAT1. (a) The expression levels of Axl in primary neuroblastoma (pNB) tissues, metastatic neuroblastoma (mNB) tissues, and normal tissues (NT) were evaluated by qRT-PCR. (b) Examination of the Axl expression by IHC. (c) qRT-PCR showed that the expression of MALAT1 was higher in metastatic cancerous tissues compared with primary and normal tissues. (d) The correlation between MALAT1 and Axl expression in neuroblastoma tissues. (e) Reanalysis of the correlation between MALAT1 and Axl from previously published dataset (GSE16237).

MALAT1 regulated Axl expression in neuroblastoma

Furthermore, we performed qRT-PCR to examine the messenger RNA (mRNA) expression levels of MALAT1 in various neuroblastoma cancer cell lines. As shown in Figure2(a), of the six neuroblastoma cancer cell lines investigated (NGP, SHSY5Y, NMB, SHEP21N, SKNAS, and SHEP2), SKNAS and SHEP2 neuroblastoma cancer cell lines expressed higher levels of MALAT1 than the others, while NGP and SHSY5Y expressed lower levels. These results were consistent with the follow-up western blot analysis for the Axl protein expression and phosphorylation levels. Since lncRNAs have been extensively reported for the essential mediators of gene transcription, we next examined whether alterative MALAT1 expression in neuroblastoma cells changes the levels of Axl and its phosphorylation, and MALAT1-specific siRNA or MALAT1-expressing vector was transfected into neuroblastoma cells. As expected in Figure 2(b), the qRT-PCR results confirmed that both si-MALAT1-1 and si-MALAT1-2 transfected neuroblastoma cancer cell lines SKNAS and SHEP2, which showed a lower MALAT1 expression level than their counterpart control cells. Interestingly, we found that the protein levels of both Axl and p-Axl were significantly reduced as MALAT1 levels decrease. Likewise, the expression of Axl and p-Axl was significantly increased in both NGP and SHSY5Y cells stably transfected with LV-MALAT1 (p < 0.01 and p < 0.05, respectively; Figure 2(c)). Taken together, these data suggested that MALAT1 could upregulate Axl expression in neuroblastoma cells.

Figure 2.

MALAT1 regulated Axl expression in neuroblastoma. (a) The expression levels of MALAT1, Axl, and phospho-Axl (p-Axl) were evaluated by qRT-PCR or western blot in various neuroblastoma cells. (b) si-MALAT1-1 or si-MALAT1-2 decreased the Axl expression and its phosphorylation in protein levels in SKNAS and SHEP2 cell lines. si-control acted as negative controls. (c) NGP and SHSY5Y cells were stably transfected with LV-MALAT1, and then, the Axl protein levels were examined by western blot.

MALAT1-mediated Axl promoted cell invasion and migration

To investigate the reciprocal effect of MALAT1 and Axl on invasion of neuroblastoma cells, the Axl ectopic expression vector (pcDNA3.1-Axl), si-MALAT1, LV-MALAT1, and si-Axl were used. As shown in Figure 3(a), overexpression of MALAT1 by stably transfecting LV-MALAT1 led to marked enhancement of the cellular invasion compared to the control in SKNAS cells (p < 0.01). Additionally, transfection with si-Axl led to downregulation of Axl and p-Axl levels and suppression of LV-MALAT1-mediated invasion. Measurement of the area of the wounds remaining showed that LV-MALAT1 decreased the area of the remaining wounds, compared with control in SKNAS cells (p < 0.05). However, cotransfection with si-Axl restored the area of the remaining wounds (p < 0.001; Figure 3(b)). While SKNAS cells expressed si-MALAT1 exhibited a decrease in invaded cells and an increase in area of the wounds remaining (Figure 3(c) and (d)), and re-expression of Axl by pcDNA3.1-Axl in neuroblastoma cells, as confirmed by Western blot analysis, restored the capacities of invasion and migration inhibited by si-MALAT1.

Figure 3.

MALAT1-mediated Axl promoted cell invasion and migration. (a and c) Transwell assays revealed that LV-MALAT1/si-MALAT1 significantly altered the invasion capacities of SKNAS cells, and the effects were reversed by si-Axl/pcDNA3.1-Axl. (b and d) Wound healing assays demonstrated that cell migration abilities would be altered by LV-MALAT1/si-MALAT1 in SKNAS cells, and the effects were restored by si-Axl/pcDNA3.1-Axl.

The Axl inhibitor R428 suppressed neuroblastoma cell invasion and migration

To manipulate the Axl function in neuroblastoma cancer cells, an Axl inhibitor R428 was used. As expected, Axl phosphorylation was effectively inhibited by 100 nM R428 (Figure 4(a)). We also detected the Axl-dependent signaling pathways and found marked inhibition of Akt and extracellular signal–regulated protein kinases 1 and 2 (ERK1/2) phosphorylation. Furthermore, there was a significant dose-dependent increase in the percentage of apoptotic cells upon R428 treatment (Figure 4(b)). Quantification of invaded cells showed that R428 significantly reduced neuroblastoma cell invasion abilities (p < 0.05; Figure 4(c) and (d)). For cell migration analysis, wound healing assays showed that treatment with 100 nM R428 increased the area of remaining wounds in both SKNAS and SHEP2 cells (p < 0.001 and p < 0.05, respectively; Figure 4(e) and (f)). These data suggested that suppression of Axl activity with the small-molecule inhibitor R428 may be used to inhibit neuroblastoma metastasis in the future.

Figure 4.

The Axl inhibitor R428 suppressed neuroblastoma cell invasion and migration. (a) SKNAS and SHEP2 cells were treated with vehicle control or recommended 100 nM R428 for 24 h, and then, the protein expression of phospho-Axl (p-Axl), Axl, phospho-Akt (p-Akt), Akt, phospho-ERK1/2 (p-ERK1/2) and ERK1/2 was analyzed by western blot. GAPDH was used as the internal control. (b) SKNAS cells were treated with different concentrations of R428 (0, 0.1, 1, 10, and 100 µM) for 48 h, and the percentage of apoptosis cells was detected by flow cytometry. (c) and (d) Transwell assays showed the effect of the inhibitor on cell invasion in SKNAS and SHEP2 cells. (e) and (f) Wound healing assays showed the effect of R428 on cell migration.

Discussion

Axl is a transmembrane RTK which belongs to the TAM (Tyro3, Axl, and Mer) family. Previous studies have shown that Axl is upregulated and acts as an oncogene in several carcinomas, making it as a novel target for cancer therapy.^19,20 Here, we explored the Axl expression in neuroblastoma tissues and found that metastatic cancerous tissues had a significantly higher Axl expression compared with the primary ones, suggesting that Axl may be associated with neuroblastoma metastasis.

Researchers have shown that Axl activates fibroblast growth factor receptor pathway to potentiate survival signals in B-cell chronic lymphocytic leukemia cells.²¹ The Gas6/Axl/PI3K/Akt pathway also protects cells from apoptosis via various mechanisms.²² The downstream pathways of Axl-dependent signaling, which is responsible for cell-specific pathophysiological process, has been to be well documented and elucidated. However, the direct upstream regulation and mechanisms of Axl at the protein, translational, and post-transcriptional levels leave a large gap in the field.

In this study, we identified a significant positive correlation between the levels of lncRNA MALAT1 and Axl in neuroblastoma. It has been reported that lncRNA MALAT1 advances the development of several cancers by regulating oncogenes.²³ To gain a better understanding of the roles of MALAT1 in modulating Axl expression and function in neuroblastoma, we changed MALAT1 levels and found coordinated alteration in Axl expression and activation. Furthermore, Transwell assays and wound healing demonstrated that MALAT1-mediated Axl upregulation promoted invasion and migration of neuroblastoma cells. Several Axl inhibitors have exhibited effective anti-tumor activity in preclinical studies.²⁴ Our results also revealed that the small-molecule Axl inhibitor R428 significantly induced cell apoptosis and reduced neuroblastoma cell invasion and migration. However, it is unclear how MALAT1 regulates Axl. More potential molecular mechanism pathways should be explored in the future.

In conclusion, this study revealed a novel pathway, involving MALAT1-mediated Axl, which plays a vital role in neuroblastoma cell invasion and migration. Moreover, targeting Axl with its inhibitor suppressed neuroblastoma cell metastasis. These findings therefore provide important evidence for further development of more potent MALAT1/Axl inhibitors for the treatment of neuroblastoma.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

References

Brodeur

. Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer 2003; 3: 203–216.

Castel

Villamon

Canete

. Neuroblastoma in adolescents: genetic and clinical characterisation. Clin Transl Oncol 2010; 12: 49–54.

Maris

Hogarty

Bagatell

. Neuroblastoma. Lancet 2007; 369: 2106–2120.

Maris

Matthay

. Molecular biology of neuroblastoma. J Clin Oncol 1999; 17: 2264–2279.

Linger

Keating

Earp

. TAM receptor tyrosine kinases: biologic functions, signaling, and potential therapeutic targeting in human cancer. Adv Cancer Res 2008; 100: 35–83.

Brandao

Migdall-Wilson

Eisenman

. TAM receptors in leukemia: expression, signaling, and therapeutic implications. Crit Rev Oncog 2011; 16: 47–63.

Stitt

Conn

Gore

. The anticoagulation factor protein S and its relative, Gas6, are ligands for the Tyro 3/Axl family of receptor tyrosine kinases. Cell 1995; 80: 661–670.

Linger

Keating

Earp

. Taking aim at Mer and Axl receptor tyrosine kinases as novel therapeutic targets in solid tumors. Expert Opin Ther Targets 2010; 14: 1073–1090.

Neubauer

Fiebeler

Graham

. Expression of Axl, a transforming receptor tyrosine kinase, in normal and malignant hematopoiesis. Blood 1994; 84: 1931–1941.

10.

Wang

. Inhibition of Mer and Axl receptor tyrosine kinases leads to increased apoptosis and improved chemosensitivity in human neuroblastoma. Biochem Biophys Res Commun 2015; 457: 461–466.

11.

Batista

Chang

. Long noncoding RNAs: cellular address codes in development and disease. Cell 2013; 152: 1298–1307.

12.

Diederichs

Wang

. MALAT-1, a novel noncoding RNA, and thymosin beta4 predict metastasis and survival in early-stage non-small cell lung cancer. Oncogene 2003; 22: 8031–8041.

13.

Ying

Chen

Wang

. Upregulated MALAT-1 contributes to bladder cancer cell migration by inducing epithelial-to-mesenchymal transition. Mol Biosyst 2012; 8: 2289–2294.

14.

Zhang

Wan

. Long noncoding RNA plays a key role in metastasis and prognosis of hepatocellular carcinoma. Biomed Res Int 2014; 2014: 780521.

15.

Liu

. Resveratrol inhibits invasion and metastasis of colorectal cancer cells via MALAT1 mediated Wnt/β-catenin signal pathway. PLoS One 2013; 8: e78700.

16.

Moran

Perera

Khalil

. Emerging functional and mechanistic paradigms of mammalian long non-coding RNAs. Nucleic Acids Res 2012; 40: 6391–6400.

17.

Gutschner

Hammerle

Eissmann

. The noncoding RNA MALAT1 is a critical regulator of the metastasis phenotype of lung cancer cells. Cancer Res 2013; 73: 1180–1189.

18.

Tano

Mizuno

Okada

. MALAT-1 enhances cell motility of lung adenocarcinoma cells by influencing the expression of motility-related genes. FEBS Lett 2010; 584: 4575–4580.

19.

Liu

Koul

. Axl kinase as a novel target for cancer therapy. Oncotarget 2014; 5: 9546–9563.

20.

Vajkoczy

Knyazev

Kunkel

. Dominant-negative inhibition of the Axl receptor tyrosine kinase suppresses brain tumor cell growth and invasion and prolongs survival. Proc Natl Acad Sci USA 2006; 103: 5799–5804.

21.

Sinha

Boysen

Nelson

. Axl activates fibroblast growth factor receptor pathway to potentiate survival signals in B-cell chronic lymphocytic leukemia cells. Leukemia 2016; 30: 1431–1436.

22.

Sawabu

Seno

Kawashima

. Growth arrest-specific gene 6 and Axl signaling enhances gastric cancer cell survival via Akt pathway. Mol Carcinog 2007; 46: 155–164.

23.

Zhang

Liu

. Long non-coding RNA MALAT1 promotes tumour growth and metastasis in colorectal cancer through binding to SFPQ and releasing oncogene PTBP2 from SFPQ/PTBP2 complex. Br J Cancer 2014; 111: 736–748.

24.

Holland

Pan

Franci

. R428, a selective small molecule inhibitor of Axl kinase, blocks tumor spread and prolongs survival in models of metastatic breast cancer. Cancer Res 2010; 70: 1544–1554.