Small GTPase Arl6 controls RH30 rhabdomyosarcoma cell growth through ciliogenesis and Hedgehog signaling
- Xiaotong Liu†1,
- Qiuhong Shen†1,
- Tingting Yu†1,
- Huijie Huang1,
- Ziyu Zhang1,
- Jie Ding1,
- Ying Tang1, 2,
- Ning Xu3 and
- Shen Yue1Email author
© The Author(s) 2016
Received: 8 June 2016
Accepted: 28 November 2016
Published: 12 December 2016
Rhabdomyosarcoma (RMS) originates from skeletal muscle precursors that fail to differentiate. Hedgehog (Hh) signaling and primary cilia contribute to the pathobiology of RMS.
Here we showed ADP ribosylation factor like GTPase 6 (ARL6) localizes at the base of primary cilium, controls ciliogenesis and Hh signaling. The transcription of Arl6 is dynamic during the differentiation of myoblasts, companying with the growth and elimination of primary cilia. Arl6 expression is significantly up regulated in cilia-dependent RMS cells and tissues. Knockdown of Arl6 inhibits proliferation and promotes apoptosis of RMS RH30 cells through defected ciliogenesis and reduced Hh activity.
Taken together, the functions of Arl6 in ciliogenesis and Hh signaling suggest it as a potential RMS drug target.
KeywordsPrimary cilia Hedgehog Rhabdomyosarcoma
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, which originates from skeletal muscle precursors that fail to differentiate . Current therapeutic approaches for RMS include multi-agent chemotherapy, radiation therapy and surgical resection, which have not resulted in significant improvement in outcome for patients with recurrence and/or metastasis . Recent investigations realized that dysregulation in RAS pathway , insulin-like growth factor , p53 , AKT/mTOR  and Hedgehog (Hh) signaling [6–8] is associated with the pathobiology of RMS, which suggested those signaling pathways as potential RMS drug targets.
Abnormal assembly of primary cilia is recently reported in RMS . Primary cilium is a microtubule-based cell protrusion present in every interphase cell, functions as a sensory organelle on the cell surface to detect a wide variety of stimuli [10, 11]. Many gene mutations cause defective ciliogenesis or ciliary transport, e.g. intraflagellar transport (IFT) genes, small GTPases and Bardet–Biedl syndrome (BBS) genes encoding proteins that form a complex termed the BBSome . The detailed mechanisms are still not very clear. Vertebrate Hh signaling cascade is initiated by the binding of three Hh ligands, Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh) to the 12-pass transmembrane receptor Patched-1 (Ptch1). Hh signaling is absolutely dependent on primary cilia, and requires the IFT machinery for the transport and processing of its signaling components in primary cilia, including transmembrane receptors, Ptch1, Smoothened (Smo), and downstream GLI-Kruppel family of transcription factors Gli1-3 as well .
ADP ribosylation factor like GTPase 6 (Arl6), also named as BBS3, is a member of the Ras superfamily of small GTPase, which is mutated in human Bardet–Biedl syndrome [13, 14]. Arl6 is required for the ciliary localization of the BBSome complex , which is necessary for ciliary membrane biogenesis and sorting membrane proteins to cilia [15, 16]. Studies from Caenorhabditis elegans showed that Arl6 undergoes IFT . Arl6 GTPase activity is required for Wnt signaling in cultured mammalian cells . Recent studies from Arl6 knockout mice also showed that loss of Arl6 affects retrograde transport of Smo inside cilia . However, the function of Arl6 in cilia-related RMS is still unknown.
Our data points to the role of Arl6 controlling RH30 RMS cell growth through ciliogenesis and Hedgehog signaling. During the in vitro differentiation of an established myoblast cell line, C2C12, we saw dynamic Arl6 expression and accompanying growth or elimination of primary cilia. Further more, Arl6 expression is significantly up-regulated in cilia-dependent RMS RH30 cells and tissues relative to normal skeletal muscles. RH30 cells with disrupted Arl6 expression show reduced ciliogenesis and crippled Hh activity, resulting in retarded cell growth as well as increased apoptosis.
Cell culture and plasmids construction
Wild type (WT) and Arl6 knockout mouse embryonic fibroblasts (MEFs) were gifts from Dr. Val C. Sheffield (University of Iowa, Iowa City, IA, USA), and were immortalized following NIH3T3 protocol. A mouse myoblast cell line C2C12 and human RMS cell lines RD and RH30 are from ATCC. RD and RH30 are derived from tumors of the embryonal and alveolar origin, respectively. MEFs, RD and RH30 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin at 37 °C with 5% CO2. C2C12 cells were maintained with 15% FBS. Mouse Arl6 cDNA was obtained from MGC cDNA library (Open biosystems), and cloned into pRK5 vector with a red fluorescent protein (RFP) tag by PCR. The T31R and Q73L mutants of Arl6 were created using the Quickchange site-directed mutagenesis kit (Agilent Technologies).
The constructs expressing Arl6 short hairpin RNA (shArl6) (target sequence: GTCGAATTCCAATCTTGTT) and scramble control (shCon) were purchased from GeneChem (Shanghai, China). Briefly, the short hairpin RNAs were cloned into GV248, which has a hU6 promoter. The knock-down efficiency of shArl6 was validated by quantitative real-time polymerase chain reaction (Q-PCR). To generate stable cell lines, shArl6 or shCon plasmids were transfected in RD or RH30 cells using Fugene HD according to the manufacturer’s procedure (Promega, Madison, WI). Transfected cells were selected with 5 μg/ml puromycin for 2 weeks, and used for following experiments.
RNA isolation and quantitative PCR
Total RNA was isolated from cultured cells using the RNAiso reagent (TaKaRa, Shiga, Japan), and reverse transcription was carried out using the PrimeScript RT reagent Kit (TaKaRa). Standard reverse transcription polymerase chain reaction (RT-PCR) was carried out with the following primers: mouse Gli1 (5′-TCCAGCTTGGATGAAGGACCTTGT-3′ and 5′-AGCATATCTGGCACGGAGCATGTA-3′) and mouse Hypoxanthine-guanine phosophoribosyltransferase (HPRT) (5′-TATGGACAGGACTGAAAGAC-3′ and 5′-TAATCCAGCAGGTCAGCAAA-3′). Q-PCR was carried out using the FastStart SYBR Green Master mix (Roche, Germany) on a LightCycler 96 System (Roche) with primers for mouse Arl6 (5′-CACCGTCGAATTCCAATCTTG-3′ and 5′-ATGGCGTCACTAG- CACAAATATG-3′), mouse Gli1 (5′-GCTTGGATGAAGGACCTTGTG-3′ and 5′-GCTGATCCAGCCTAAGGTTCTC-3′), mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (5′-AGGTCGG- TGTGAACGGATTTG-3′ and 5′-GGGGTCGTTGATGGCAACA-3′), human Arl6 (5′-GTGTCTCAGTTGCTGTGTTTAG-3′ and 5′-AGCCAGTCTACACCTT- CTTG-3′), human Gli1 (5′-TCCTCTGAGACGCCATGTTC-3′ and 5′-CAGACAGTCCTTCTGTCCCCA-3′), and human GAPDH (5′-ATCATCCCTGCCTCTACTGG-3′ and 5′-GTCAGGTCCACCACTGACAC-3′). Experiments were repeated at least three times, and samples were analyzed in triplicate.
Western blot analysis
After transfection or treatment as described, cells were lysed in radioimmunoprecipitation assay buffer (RIPA buffer) (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% vol/vol NP-40, 0.25% wt/vol sodium deoxycholate, 0.25% wt/vol NaF, 10 mmol/l β-glycerolphosphate, 1 mM Na3VO4, 1 mM DTT, and 1× Roche cOmplete Protease Inhibitor Cocktail) for 1 h at 4 °C. The lysate was clarified by centrifugation for 1 h at 20,000×g. The protein concentration was determined using a bicinchoninic acid assay and equal amounts of total protein from each of the samples was supplemented with 6× SDS loading buffer, heated at 95 °C for 5 min, subjected to SDS-PAGE, followed by western blotting with indicated antibodies. The source for antibodies are: anti-Arl6 antibodies (Sigma-Aldrich, St. Louis, MO); anti-myogenin and anti-β-actin antibodies (Santa Cruz Biotechnology, Dallas, TX).
Ptch1+/− mice (Jackson Laboratory, Bar Harbor, ME) were outcrossed onto the CD1 background for spontaneous RMS development. RMS and normal muscle tissues were dissected from gastrocnemius of hindlimbs of Ptch1+/− mice. Immunohistochemistry of tissue sections with antibodies to Gli1 (Cell Signaling Technologies, Danvers, MA) and Arl6 (Sigma-Aldrich) was performed using Polink-2 plus® Polymer HRP Detection System (ZSGB-BIO, China). Nuclei were counterstained with hematoxylin. Sections were visualized under 40× magnification using Leica DMI 3000B.
Immunofluorescent staining and confocal microscopy
Approximately 5 × 104 cells per well were seeded in Millicell EZ slides and cultured for 24 h. The cells were transfected, allowed to recover for 24–36 h. MEFs were treated with 0.5% FBS for 24 h to induce cilia growth. C2C12 or RMS cell lines were treated with 2% horse serum (HS) for 48 h to induce cilia growth. Cells were fixed with 4% paraformaldehyde for 10 min at 4 °C, and standard procedures for immunostaining were followed. Primary cilia were stained with mouse anti-acetylated tubulin (1:2000, Sigma-Aldrich) and corresponding Alexa-coupled secondary antibodies (1:200, Life Technologies). Tissue sections were immunostained with anti-acetylated tubulin. Confocal images were acquired on a Carl Zeiss LSM710 microscope system using Z-stack module of ZEN2011 program.
To measure cell growth, RD and RH30 cells stably expressing shArl6 or control shRNA were seeded at 3000 cells per well in 96-well plates. For 5 days, cells were incubated with 100 μl MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazoliumbromide, 5 mg/ml) for 4 h at 37 °C. Following MTT incubation, 100 μl DMSO was added to dissolve the crystals and the absorbance at 490 nm was measured using a microplate absorbance reader (Tecan, Austria). Each assay was performed in triplicates and repeated three times independently.
To measure cell proliferation, RD and RH30 cells stably expressed expressing shArl6 or control shRNA were seeded at 2 × 104 cells per well in 24-well plates and cultured for 48 h. Cells were incubated with 10 μM EdU for 2 h before the end of the culture period. Following culture, cells were, fixed with 3.7% formaldehyde, and then detected with Click-iT EdU Imaging Kit (Life Technologies, CA) according to the manufacturer’s procedure (Life Technologies, CA). The plates were visualized under 20× magnification using Leica DMI 3000B with Leica DFC490 Digital Camera. Quantification of percentage of EdU-positive cells was performed using the ImageJ program.
Cell cycle and apoptosis assays
Subconfluent RH30 stable cells were synchronized with 0.5% FBS for 24 h, then rescued with 10% FBS for 12 h before cell cycle analysis. To assess cell cycle properties, cells were collected and incubated with 100 μg/ml propidium iodide and 0.5 μg/ml RNase A for 30 min at the room temperature before subjecting to FACS analysis. To assess apoptosis, stable cells were cultured with 0.2% FBS for 72 h to induce apoptosis. The apoptotic cells were analyzed using the Annexin V: FITC apoptosis detection kit according to the manufacturer’s instruction (BD, USA).
Generation of Arl6-knockout RH30 cells
To make CRISPR/Cas9 constructs, a pair of single guide RNAs (sgRNAs) targeted human Arl6 were designed and cloned into pX330 vector (from Addgene) . Arl6 sgRNAs target sequences are 5′-TGCCACTATTATCTAGCCCAAGG-3′ and 5′-GACAGACTTTCAGTCTTGCTTGG-3′. To generate Arl6 knockout cells, RH30 cells were co-transfected with CRISPR/Cas9 plasmids and the ones encoding puromycin selection marker. 48 h after transfection, transfection-positive cells were selected using puromycin. After 5 days of selection, survived RH30 cells were seeded at 30 cells per well in 96-well plates and screened for Arl6 levels by western blotting.
Statistical analyses were performed in the GraphPad Prism 5.0 environment. Comparisons between indicated groups were performed using independent-samples t test. P values less than 0.05 were considered statistically significant. *p < 0.05, **p < 0.01, and ***p < 0.001.
Arl6 controls ciliogenesis and Hh signaling from the basal body to the primary cilium
It was reported that Arl6 functions at or near the ciliary gate to modulate mammalian ciliary assembly or disassembly and ciliary signaling, for example Wnt Signaling . To understand the function of Arl6 at the base, we immortalized Arl6−/− MEFs derived from E13.5 Arl6 knockout mice . Our immortalized Arl6−/− MEFs showed reduced numbers and length of primary cilia (Fig. 1c, d), although ciliary staining was normal in tissue sections from kidney, eye, and pancreas of Arl6−/− mice . Vertebrate Hh signaling absolutely requires IFT . To assess the role of Arl6 in Hh signaling, we quantified the transcriptional responses of endogenous Gli1 by RT-PCR and Q-PCR in Arl6−/− MEFs, and found that lack of Arl6 led to marked curtailment of Gli1 activation (Fig. 1e, f; Additional file 1: Figure S1). Knockdown of Arl6 leads to a 42.5% decrease of Hh pathway activation. This result is consistent with the disruption of ciliogenesis in Arl6−/− MEFs. Taken together, the above results show that Arl6 is required for ciliogenesis and vertebrate Hh signaling.
Arl6 is expressed dynamically during muscle differentiation
Although primary cilia are missing in mature muscle, they grow in myoblasts. Recent study reported that assembly and disassembly of primary cilia are controlled during the differentiation of myoblasts . To understand whether Arl6 contributes to ciliogenesis during muscle differentiation, we induced the differentiation of C2C12 myoblasts with 2% HS for 3 days, and analyzed ciliary formation and Arl6 expression at different time points. The increased expression of differentiation marker Myogenin over time indicates C2C12 cells differentiated well as we expected (Additional file 2: Figure S2B). We found that primary cilia were assembled during the early stage of myogenic differentiation (0–24 h after HS treatment), subsequently disappeared during the later period (24–72 h after HS treatment) through immunofluorescent staining of acetylated tubulin (Additional file 2: Figure S2A). The numbers and length of cilia reached the peak at 24 h time point (Fig. 2b, c). This result is consistent with that of primary mouse myoblasts . Interestingly, the transcription of Arl6 was also dynamically regulated during the differentiation of C2C12 cells. The mRNA level of Arl6 was also at the peak at 24 h time point according to Q-PCR analysis (Fig. 2d). So the regulation of Arl6 expression synchronized with that of ciliogenesis. These results suggested that Arl6 participates ciliogenesis during muscle differentiation.
Up-regulation of Arl6 is related to RMS
Arl6 promotes RH30 cell growth and inhibits ciliogenesis in RH30 cells
To further characterize the tumor suppression properties of Arl6 knockdown, we examined the ability of RH30 cells to undergo apoptosis when stably expressing shArl6. Apoptosis was induced by maintaining the cells in DMEM supplemented with 0.2% FBS for 24 h and quantified by using Annexin V and propidium iodide double staining. Flow cytometry analyses indicated that only around 1.5% of RH30 cells expressing shCon became Annexin V positive, but this percentage increased to 11.6% in the cells expressing shArl6 after serum depletion (Fig. 5c, d). This result indicated that knockdown of Arl6 promotes the apoptosis of RH30 cells, and suggested that cell cycle arrest at G2/M phase is involved in apoptosis induction.
To identify the role of cilia assembly in RMS cell proliferation, we tried to knockdown IFT88 in RH30 cells (Additional file 3: Figure S3A). IFT88 is a well-known central component of the IFT complex B, essential for cilia assembly. Knockdown of IFT88 blocked cilia assembly, retarded Hh signaling, and suppressed cell proliferation (Additional file 3: Figure S3). The siIFT88 data identified that blocking cilia assembly suppresses Hh signaling and cell proliferation in RH30 cells.
To further investigate the functions of Arl6 in ciliogenesis and cell proliferation of RH30 cells, we used the CRISPR/Cas9 genome editing technology to knockout Arl6. Western blotting showed that the expression of Arl6 was significantly decreased in RH30 cells edited by Arl6 CRISPR, indicated Arl6 was knockout in most cells of the selected individual clone (Additional file 4: Figure S4A). The ciliogenesis and cell proliferation was also suppressed in edited RH30 cells when compared with that in untransfected cells (Additional file 4: Figure S4B–D). The data again indicated that Arl6 promotes cell growth and ciliogenesis in RH30 cells.
Hh signal induces the transcription of Arl6
RMS is the most common pediatric soft-tissue sarcoma that displays defective myogenic differentiation. Understanding the mechanisms in RMS development is important to invent innovative treatment strategies. In this study, we investigated the role of a small GTPase Arl6 in regulating RMS cell growth. Our research indicates that Arl6 is an oncogene in RMS cells. Arl6 expression is significantly up regulated in cilia-dependent human RMS cells RH30 and mouse RMS tissues. Knockdown of Arl6 obstructs the proliferation and promotes apoptosis of RH30 through defected ciliogenesis and reduced Hh activity.
Ciliogenesis is required for the differentiation of diverse cell types, for example, cardiomyocyte , adipocyte , myocyte [9, 26], osteocyte , and mesenchymal stem cells as well . Assembly and disassembly of primary cilia are dynamical during the myogenic development. Primary cilia were assembled during the early stage of myogenic differentiation, subsequently disappeared during the later period. Interruption of cilia assembly with knockdown of IFT genes enhances the cell proliferation of myoblasts and abolishes the differentiation . However, primary cilia assembly and disassembly are deregulated in RMS that originates from poorly differentiated myoblasts. Some kinds of RMS cells (A204, RH30, and Rh36) are ciliated . We found that interruption of cilia assembly with knockdown of Arl6 obstructed cell proliferation and promoted apoptosis in ciliated RH30 cells. It suggests that the ciliary signaling transductions regulating cell cycle are different between myoblasts and RMS cells.
Arl6 is a ciliary protein, which accumulates at the base of primary cilia in our in vitro cultured MEFs and ciliated murine inner medullary collecting duct (IMCD3) cells , as well as in C. elegans . This localization of Arl6 suggests its function in ciliogenesis and ciliary trafficking. However, recent study in Arl6 knockout mice indicated that Arl6 is dispensable in ciliogenesis, since the cilia morphology was completely normal in tissue sections from kidney, eye, and pancreas of Arl6−/− mice . There are numerous mice studies show that the phenotypic effects of single gene knockout are influenced by the genetic background . It is possible that genetic redundancy masks some essential functions of Arl6 in those knockout mice. It is also possible that some spontaneous mutations during immortalization and in vitro short-term serum starvation potentiate cilia defect in Arl6−/− MEFs. In our study, primary cilia numbers were significantly decreased in in vitro cultured Arl6−/− MEFs compared with WT cells. As a small GTPase, the localization of Arl6 in primary cilia depends on the binding of GTP. T31R, a mutant of Arl6 reported in BBS3 pathology, appears strictly bound to GDP, although WT Arl6 is primarily bound to GTP. T31R mutant was on longer accumulated in basal body of primary cilia in our experiment. Interestingly, it was reported that expression of T31R leaded to stunted cilia in IMCD3 cells . This result is similar to the loss of ciliogenesis in our MEFs and RH30 cells which lost Arl6. Those findings suggest that Arl6 regulates ciliogenesis in some situations.
Arl6 is critical for recruiting the polymerized BBSome to lipid vesicles as a coat and for targeting the BBSome to cilia but not the assembly of it . Normal assembly and ciliary trafficking of BBSome is required for ciliary Hh signaling. Loss of BBS7, a subunit of BBSome, results in accumulation of Smoothened and Patched1 in cilia and decreases Hh response . Loss of Arl6 affects the retrograde transport of Smoothened in cilia and leads to decreases in ciliary Hh signaling . Our quantitative PCR for Gli1 from immortalized Arl6−/− MEFs showed a slight increase without Hh and a significant decrease when stimulated with Hh compared with WT MEFs. The deregulation of Hh pathway activation is due to the deregulated transport at ciliary base lack of Arl6. Taken together with the function of Arl6 in ciliogenesis, Arl6 is critical to ciliary Hh signaling. In ciliated RMS cells and tissues, the Hh signaling was aberrantly activated accompanying with the high Arl6 expression. Aberrant activation of Hh signaling leads to oncogenic consequences involving deregulation of cell cycle and cell proliferation not only in RMS but also in other tumors as well [32, 33]. Hence, knockdown of Arl6 could inhibit the proliferation and promote of apoptosis of RMS cells through blockage of ciliary hedgehog signaling. Our study also showed that Hh signal could induce the transcription of Arl6. So there may be a positive feedback between Hh signaling and Arl6 to accelerate aberrant development of RMS.
In conclusion, our data provides evidence that the small GTPase Arl6 contributes to RMS cell growth due to its function in ciliogenesis and ciliary Hh signaling. While Hh signal induces the transcription of Arl6 to form a positive feedback loop to maintain the pathway activation. The function of Arl6 may be dependent on its binding of GTP, since of the GTP-bound Arl6 localized at the basal body of primary cilia. The data supports that targeting Arl6 may be successful in the treatment of ciliated RMS. More research is needed to develop the compounds inhibiting the expression or GTP-binding of Arl6 for tumor therapy.
ADP ribosylation factor like GTPase 6
mouse embryonic fibroblast
fetal bovine serum
red fluorescent protein
Arl6 short hairpin RNA
shRNA scramble control
quantitative real-time polymerase chain reaction
reverse transcription polymerase chain reaction
glyceraldehyde 3-phosphate dehydrogenase
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
single guide RNAs
Arl6 tagged with RFP
murine inner medullary collecting duct
standard error of the mean
XTL and QHS participated in the acquisition and analysis of data, and drafted the manuscript. YTT participated in the data acquisition and manuscript revision. HJH, ZYZ, JD and YT participated in the acquisition and analysis of data. NX participated in the design of the study. SY conceived of the study, participated in its design and data analysis, and drafted the manuscript. All authors read and approved the final manuscript.
We wish to thank Dr. Val C. Sheffield for Arl6 knockout MEFs, and Dr. Steven Y Cheng and Dr. Yaoyu Chen for scientific suggestions, and Dr. Zhaoxia Sun and Dr. Stephanie Jerman for help of CRISPR/Cas9 technology.
The authors declare that they have no competing interests.
Availability of data and materials
The data supporting the conclusions of this article is available to all interested researchers upon request.
Ethics approval and consent to participate
This study was approved by the Research Ethics Committee of Nanjing Medical University.
This work was supported by Grants from National Natural Science foundation of China (81101497 and 81572720 to SY), and by grants from Natural Science Foundation of Jiangsu Province (BK20141438 to SY and BK20130895 to NX). The funders had no role in the design of the study, data collection and interpretation, or writing the manuscript.
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