Cancer /testis antigen DDX53 is found in the sera of various cancers (Cho et al., 2002; Iwata et al., 2005; Liggins et al., 2010). Methylation has an important role for the expression regulation of DDX53 (Cho et al., 2003). DDX53 regulates the expression of cyclins and acts as an oncogene (Por et al., 2010). DDX53 confers resistance to various anti-cancer drugs (Kim et al., 2010). miR-200b-DDX53 negative feedback loop (Kim et al., 2013) and miR-217-DDX53 feedback loop (Kim et al., 2016) regulate responses to anti-cancer drugs.

miRNAs are known to regulate cancer stem-cell like properties. miR-200b enhances sensitivity to erlotinib in lung cancer cells and cancer stem cell markers (Ahmad et al., 2013). miR-1181 negatively regulates the cancer stem cell-like properties in pancreatic cancer (Jiang et al., 2015). The downregulation of miRNA-148a, inhibits the stem cell-like properties of glioblastoma (Lopez-Bertoni et al., 2015). miR-200c negatively regulates the expression of SOX-2 to suppress PI3K-AKT pathway (Lu et al., 2014). miR-638 negatively regulates mesenchymal-like transition by directly targeting SOX-2 (Ma et al., 2014). By targeting OCT4/SOX-2 expression, the Lin28B-Let7 pathway regulates stemness properties of oral squamous cell carcinoma cells (Chien et al., 2015).

Cancer/testis antigens are overexpressed in cancer stem-like cells (Yang et al., 2015). MAGEA-3 is expressed in side population (SP) and main population (MP) cells derived from human bladder cancer SW780 cells (Yin et al., 2014). Since DDX53 regulates anti-cancer drug-resistance, the regulatory role of DDX53 in cancer stem cell-like properties has been further investigated.

In this study, we found that anti-cancer drug-resistant cancer cells have cancer stem cell-like properties. DDX53 showed a co-expression with CD133, and binding to SOX-2, a marker of cancer stemness. DDX53 directly regulated the expression of SOX-2 and the cancer stem cell-like properties in Malme3M^R cells. We show a novel role of DDX53 in regulating cancer stem cell-like properties.

The melanoma cell lines (Malme3M, Malme3M^R) were grown in Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F-12 (DME/F12, Invitrogen). For all cell lines, media was supplemented with 10 % heat-inactivated fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin (GIBCO). CD133⁺ cells isolated from Malme3M^R, SNU387^R cells were maintained in serum-free DMEM/F12 (Gibco-BRL, USA) supplemented with 20 ng/ml epidermal growth factor (EGF) (Sigma, USA), 10 ng/ml basic fibroblast growth factor (bFGF) (Sigma, USA), and 20 ng/ml leukemia inhibitor factor (LIF) (Sigma, USA). Malme3M^R and SNU387^R cells were established from their parental Malme3M and SNU387 respectively by continuous exposure to celastrol (Kim et al., 2010). SNU387^R-AS-CAGE and Mame3M^R-AS-CAGE cells stably express anti-sense cDNA. Malme3M^R-miR-200b cells stably express miR-200b (Kim et al., 2013).

Western blot and immunoprecipitation were performed as describe (Kim et al., 2014).

The ChIP assay was performed using a ChIP assay kit (Upstate Biotechnology). Briefly, 2 × 10⁶ cells were crosslinked with 1% (v/v) formaldehyde (Sigma) at 37°C for 10 min and nuclear extracts were isolated. The nuclear lysates were immunoprecipitated with either anti-CAGE (1 μg/ml, Abcam), anti-SOX-2 (1 μg/ml, Santa Cruz) or rabbit IgG at 4°C overnight. Purified immunoprecipitated DNAs were subjected to PCR. human SOX-2 promoter-1 [5′-TAAGGCCTTTTG GCTA GGGC-3′ (sense) and 5′-AAACTAACTGTGGCTGGCGA-3′ (antisense)], SOX-2 promoter-2 [5′-TCTTGGTCTGCCCTTTGT GG-3′ (sense) and 5′-TCCAATCAACCTTCCTGCCC-3′ (antisense)], SOX-2 promoter-3 [5′-GGGCGGAGAGA GTGTTAC AG-3′ (sense) and 5′-GCACTGTATGGAGGTGGC TT-3′ (antisense)], SOX-2 promoter-4 [5′-GTGGGATGCCAGGAAGTT GA-3′ (sense) and 5′-TT GTTCTCCCGCTCATCCAC-3′ (antisense)] and SOX-2 promoter-5 [5′-AACTCCTGCACTG GCTG TTT-3′ (sense) and 5′-TTTGTATCCCCTCTCGCAGC-3′ (antisense)] were used to detect the binding of CAGE to the promoter sequences of SOX-2.

The siRNA or control siRNA was purchased from Bioneer (Korea). To test the effect of miR-200b in Malme3M or Malme3M^R-miR-200b cell lines, miR-200b inhibitor (Bioneer, Korea) was used to knockdown the expression of miR-200b. The sequences used were 5′-UCAUCAUUACCCA GUAUUA-3′ (miR-200b inhibitor) and 5′-GCAUAUAUCUA UUCCACUA-3′ (control inhibitor).

Total miRNA was isolated using the mirVana miRNA isolation kit (Ambion). miRNA was extended by a poly (A) tailing reaction using the A-Plus Poly (A) Polymerase Tailing Kit (Cell Script). CDNA was synthesized from miRNA with poly (A) tail using a poly (T) adaptor primer and qScript™ reverse transcriptase (Quanta Biogenesis). The expression level of miRNA gene was quantified with SYBR Green qRT-PCR kit (Ambion) using a miRNA-specific forward primer and a universal poly (T) adaptor reverse primer. For quantitative PCR, SYBR PCR Master Mix (Applied Biosystems) was used in a CFX96 Real-Time System thermocycler (BioRad).

Cell viability was determined by using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; Sigma).

Caspase-3 activity was measured according to the manufacturer’s instructions (BioVision, USA).

Colonies were stained with 0.01% crystal violet and counted.

Briefly, 1 × 10⁶ cells were incubated with 100 μl of 3 % BSA in PBS for 1 h on ice, then labeled with PE-conjugated anti-CD133 (Miltenyi Biotec, Germany), anti-CAGE (Abcam, Cambridge, UK) antibody bound to an Alexa-488 secondary antibody for 1 h. After washing three times with PBS, labeled cells were analyzed using flow cytometry using a FACSCalibur (BD Biosciences, USA)

For tumor spheroid forming assay, CD133⁺ and CD133⁻ cells were plated (5 × 10⁴ cells/well) in ultralow attachment plates (Corning Inc.) in DMEM/F12 stem cell medium. Cells were maintained at 37°C in a humidified 5% CO₂ incubator and fed with 0.2 ml of fresh stem cell medium on days 2, 4 and 6. The total number of spheres was counted after 7 days by inverted microscopy (Olympus). To examine the self-renewal ability of CD133⁺ and CD133⁻ cells, primary spheres were dissociated with trypsin and 0.05% EDTA. Single cell suspension obtained from primary spheres were counted and replated in 6-well ultralow attachment plates. Secondary spheroids derived from single cells of primary spheres were examined by inverted microscopy (Olympus).

The expression of CAGE in spheroids formed from CD133⁺ cells was analyzed by immunofluorescence staining. CD133⁺ cells cultured in 24-well plates were fixed with 4% paraformaldehyde in PBS containing 0.2% Triton X-100 for 30 min at 4°C. After washing with PBS, incubated with 5% BSA in PBS for 1 h and then stained with primary anti-CAGE an tibody at 4°C overnight. Spheroids were washed three times with PBS and incubated with secondary antibody Alexa-488-labeled goat anti-rabbit IgG for 1 h. Following the incubation, spheroids were washed three times with PBS and nuclei were stained with DAPI. Spheroids were observed under the fluorescence microscopy (Nikon TS 100, Nikon).

Paraffin-embedded tissue sections were immunostained using the Vecta stain ABC Elite Kit (Vector Laboratories). Tissue sections were deparaffinized with xylene and washed in ethanol. Endogenous peroxidase activity is blocked with 3% hydrogen peroxide and H₂O for 10 min. Slides were then blocked with 5% normal goat serum in TBS containing 0.1% Tween-20 (TBS-T) for 1 h. For immunohistochemistry staining, a primary antibody to DDX53 (1:100, Santa Cruz), MDR1 (1:100, Santa Cruz), SOX-2 (1:100, Santa Cruz) or IgG (1:100, Santa Cruz) was added and incubation continued at 4°C for 24 h. After washing with TBS-T, incubation with biotinylated secondary antibody was continued for 30 min. After washing, slides were incubated in the ABC complex for 30 min, and then stained with diaminobenzidine (DAB, Sigma).

Animal experiments were performed under an approved protocol by the Institutional Animal Care and Use Committee of Kangwon National University (KW-160329-2). All animals were housed in a laminar air-flow cabinet under aseptic conditions. To examine the tumorigenic potential of CD133⁺ cells, athymic nude mice (BALB/c nu/nu, 5–6-week-old females) were used. Cells (1 × 10⁶) were injected subcutaneously into the dorsal flank area of the mice. To test the effect of CAGE on in vivo tumorigenic potential, control siRNA (5 μM/kg) or CAGE siRNA (5 μM/kg) was injected intravenously after establishment of sizable tumor every three days for 20 days. At the end of experiments, all animals were sacrificed and tumors were removed. Tissues were fixed in 10% neutral-buffered formalin (Sigma, USA) and embedded in paraffin for histological studies or snap-frozen for protein analysis by Western blot. Also tumor fragments were subjected to isolate CD133⁺ and CD133⁻ cells using MACs system as described previously.

(A) Malme3M^R-CD133 (+) and Malme3M^R-CD133 (−) cells were subjected to flow cytometry. (B) Western blot was performed. (C) Spheroids derived from Malme3M^R-CD133 (+) cells or Malme3M^R-CD133 (−) cells were subjected to immunofluorescence staining for expression analysis of DDX53. Scale bars represent 50 μm. (D) Malme3M^R cells were transfected with the indicated siRNA (each at 10 nM). Western blot was performed after 48 h.

(A) Malme3M^R-CD133 (+) and Malme3M^R-CD133 (−) cells were subjected to tumor spheroid forming assays. Malme3M^R cells were transfected with the indicated siRNA (each at 10 nM) for 48 h, followed by Western blot (right panel). (B) Chemoinvasion assays were performed. **p < 0.005. (C) Colony formation assays were performed. (D) At 48 h after transfection, Malme3M^R cells were subjected to tumor spheroid formation assays and cell lysates were subjected to Western blot (right panel). **p < 0.005. Scale bars represent 50 μm. (E) At 48 h after transfection with the indicated construct (each at 1 μg), Malme3M cells were subjected to tumor spheroid formation assays and cell lysates were subjected to Western blot (right panel). **p < 0.005. Scale bars represent 50 μm.

(A, B) Cell lysates from the indicated cancer cells were subjected to Western blot. (C) Cell lysates isolated from the indicated cancer cells were subjected to ChIP assays. (D) Cell lysates from the indicated cancer cells were subjected to immunoprecipitation, followed by Western blot (upper panel). At 48 h after transfection with the indicated siRNA, cell lysates were subjected to Western blot (right panel).

(A) Malme3M cells were treated with taxol (1 μM). Cell lysates prepared at each time point were subjected to Western blot. (B) At 24 h after transfection with the indicated siRNA (each at 10 nM), Malme3M^R cells were treated with taxol (1 μM) for 24 h, followed by Western blot. (C) At 24 h after transfection with the indicated siRNA (each at 10 nM), Malme3M^R cells were treated with various concentrations of taxol for 24 h, followed by MTT assays. *p < 0.05. (D) At 24 h after transfection with the indicated siRNA (each at 10 nM), Malme3M^R cells were treated with taxol (1 μM) for 24 h, followed by caspase-3 activity assays. **p < 0.005. (E) After transfection with indicated siRNA (each at 10 nM) along with the indicted construct (each at 1 μg), Malme3M^R cells were subjected to chemoinvasion and migration assays. *p < 0.05.

(A) The level of the indicated miRNAs from Malme3M^R-CD133 (+) and Malme3M^R -CD133 (−) cells was determined by qRT-PCR. *p < 0.05. (B) At 48 h after transfection with the indicated inhibitor (each at 10 nM), Western blot was performed. (C) At 24 h after transfection with the indicated inhibitor (each at 10 nM), Malme3M cells were treated with celastrol (1 μM) or taxol (1 μM). Tumor spheroid formation assays were performed. (D) At 48 h after transfection with the indicated inhibitor (each at 10 nM), ChIP assays employing the indicated antibody (2 μg/ml) were performed. (E) The indicated cancer cells were transfected with the indicated inhibitor (each at 10 nM), followed by tumor spheroid formation assays (upper panel) and Western blot (lower panel).

(A) Malme3M^R-CD133(+) cells (10⁶ cells) were subcutaneously injected into athymic nude mice. The indicated siRNA (50 μM/kg) was intravenously injected 7 times over a period of 32 days. The extent of tumorigenic potential was determined. *p < 0.05. (B) Immunohistochemistry staining employing tumor tissue was performed, as described. (C) Immunoprecipitation and Western blot employing tumor tissue lysates were performed.

(A) 1^o xhCD133 (+) and 1^o xhCD133 (−) cells were from tumor tissues formed from Malme3M^R cells. 2^o xhCD133 (+) and 2^o xhCD133 (−) cells were from tumor tissues formed from 1^o xhCD133 (+) cells injected into nude mice. (B) 1^o xhCD133 (+) and 1^o xhCD133 (−) cells were subjected to tumor spheroid formation assays in the absence or presence of celastrol (1 μM) or taxol (1 μM). Primary spheroids obtained from 1^o xhCD133 (+) or 1^o xhCD133 (−) cells were dissociated to yield single cell suspension. Thus obtained single suspension was subjected to tumor spheroid formation to obtain secondary tumor spheroids. *p < 0.05. (C) 2^o xhCD133 (+) and 2^o xhCD133 (−) cells were subjected to tumor spheroid formation assays. Primary spheroids obtained from 2^o xhCD133 (+) or 2^o xhCD133 (−) cells were dissociated to yield single cell suspension. Thus obtained single suspension was subjected to tumor spheroid formation to obtain secondary tumor spheroids. *p < 0.05; **p < 0.005; ***p < 0.0005. (D) Western blot was performed.