Materials
Recombinant human ADAMTS1 (2197-AD) containing a zinc-dependent metalloprotease (ZnMc) domain was purchased from R&D Systems (Minneapolis, MN). Recombinant human ADAMTS1 (RPB973Hu02) containing thrombospondin (TSP) type I domains was purchased from Cloud-Clone (Houston, TX). Pifithrin-α (PFT-α, P4359), dimethyl sulfoxide (DMSO, D2650), and crystal violet (C0775) were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies employed in the western blot, dot blot, and coimmunoprecipitation (Co-IP) analyses were as follows: Bid (no. 2002), Bad (no. 9239), Bak (no. 12,105), Bim (no. 2933), poly(ADP ribose polymerase (PARP; no. 9532), cleaved PARP (no. 5625), phosphorylated (p)-EGFR (no. 3777), p-Src (no. 2101), p-Akt (no. 9271), p-extracellular signal-regulated kinase (ERK; no. 4370), p-signal transduction and activator of transcription 3 (Stat3; no. 9145), Akt (no. 9272), ERK (no. 4695), Stat3 (no. 9139; Cell Signaling Technology, Danvers, MA); EGFR (sc-03), Wilm’s tumor 1 (WT1; sc-7385), Lamin A/C (sc-7292), p-ErbB2 (SC-81507), ErbB (SC-33684), p53 (sc-126; Santa Cruz Biotechnology, Santa Cruz, CA); versican V1 (DPEAAE) (ab19345), Bcl-2 (ab32124; Abcam, Cambridge, MA); GAPDH (60,004–1-Ig), α-tubulin (66,031–1-Ig; Proteintech, Chicago, IL); ADAMTS1 (AF5867; R&D Systems); and c-Jun (GTX101135; GeneTex, Irvine, CA).
Data collection from bioinformatics analyses
Messenger RNA (mRNA) expression levels and corresponding survival data of patients from the Cancer Genome Atlas Kidney Renal Clear Cell Carcinoma (TCGA-KIRC) were obtained from UCSC Xena (https://xena.ucsc.edu/). The cDNA microarray data, GSE53757, containing 72 paired RCC tumors and normal renal tissues, and the single cell (SC) RNA scequecing data of GSE159115, comprising eight renal tumor specimens and six benign human kidney specimens from RCC were acquired from Gene Expression Omnibus (GEO) datasets. The probe used for analyzing ADAMTS1 was 222162_s_at. Violin plots for detecting ADAMTS1, EGFR, and tissue inhibitor of metalloproteinase 3 (TIMP3) expressions in kidney cancer tissues were retrieved from available online data at TNMplot (https://tnmplot.com/analysis/)(23). For TCGA RNA sequencing data, gene expressions were normalized by fragments per kilobase per million (FPKM) and log2-transformed. To analyze the correlation between anoikis resistance and the expression of ADAMTS10, EGFR, and VCAN, we utilized the “negative regulation of anoikis” pathway derived from the gene ontology database. We performed a single sample gene set enrichment analysis (ssGSEA) to calculate the score of this pathway for each patient in the TCGA-KIRC dataset. A Spearman correlation analysis was then conducted to evaluate the relationship between the “negative regulation of anoikis” pathway and the expression levels of ADAMTS10, EGFR, and VCAN. For the single-cell RNA sequencing (scRNA-seq) analysis, a differentially expressed gene analysis was performed to compared the expression of ADAMTS1, EGFR, and VCAN between tumor and benign epthelial cells. We utilized a uniform manifold approximation and projection (UMAP) plot to demonstrate the distribution of tumor and benign cells based on their transcription profiles from the scRNA-seq data.
Cell lines and cell culture
The Caki-1, 786-O, and Achn RCC cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in minimum essential medium (MEM) (Caki-1 and Achn) or RPMI-1640 medium (786-O) with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in 5% CO2.
Construction of the E402Q mutation of ADAMTS1
The pLex-MCS-ADAMTS1 expression construct was kindly provided by Dr. Tsang-Chih Kuo (National Taiwan University, Taipei, Taiwan). Metalloproteinase activity in ADAMTS1 was inactivated by generating a mutation of glutamate (Glu, E) into glutamine (Gln, Q) on amino acid 402 using a QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). The following primers were used to generate E402Q: ADAMTS1-E402Q-F: GTT AAA CAC GTG GCC TAA CTG ATG GGC TGT GGT GAA GGC and ADAMTS1-E402Q-R: GCC TTC ACC ACA GCC CAT CAG TTA GGC CAC GTG TTT AAC.
Establishment of gene knockdown (KD) and overexpression of RCC cells
Short hairpin (sh) RNAs for ADAMTS1, VCAN, and EGFR were obtained from the the RNA Technology Platform and Gene Manipulation Core Facility at Academic Sinica (Taipei, Taiwan), including targeting sequences for shADAMTS1 (CCA CAG GAA CTG GAA GCA TAA), shVCAN (ATG GAT GTG TTC AAC CTT AAT), and shEGFR (GCC AAG CCA AAT GGC ATC TTT). To produce the lentivirus, 293 T packaging cells were cotransfected with shRNA constructs or pLex-MCS-ADAMTS1 together with pCMVDR8.91 and pMD.G. according to protocols from our previous study [24]. After overnight incubation, the transfection medium was removed and replaced with fresh culture medium. Viral supernatants were collected at 24 and 48 h. Then, RCC cells were infected with fresh lentivirus-containing medium (supplemented with 8 μg/mL polybrene) for 24 h and subjected to the following assays.
The pcDNA3-TIMP3 plasmid was acquired from Dr. Shun-Fa Yang (Chung Shan Medical University, Taichung, Taiwan). RCC cells were transfected with either 2 μg of an empty vector or pcDNA3-TIMP3 using the Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA) for 6 h, following the manufacturer’s guidelines.
Western blot analysis
Cell lysates of the experimental groups were extracted using PRO-PREPTM protein extraction buffer (iNtRON Biotechnology, Seongnam, Korea) supplemented with protease inhibitor cocktails. Proteins were quantified using bovine serum albumin (BSA) and Coomassie Brilliant Blue G-250 dye (Bio-Rad protein assay). Approximately ~15–30 µg of protein was then loaded and separated via sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS–PAGE), followed by transfer onto polyvinylidene difluoride (PVDF) membranes. Specific primary antibodies were used to detect targeted proteins. After three washes of blots in Tris Buffered Saline with Tween 20 (TBST), incubation with a secondary antibody at room temperature was performed. Finally, chemiluminescence was utilized for detection.
Anoikis resistance assay
Caki-1 and 786-O cells, modified for ADAMTS1, EGFR, or VCAN expression, or exposed to ADAMTS1-indicative recombinant proteins, were initially plated at a density of 5 × 104 cells/well in six-well ultralow-attachment plates for either 24 or 48 h. Afterwards, suspended cells were harvested and reseeded into three separate wells of 96-well plates for an additional ~3– 4 h. Subsequently, cell viability was evaluated using the CCK-8 viability assay (Abcam).
Zebrafish xenotransplantation model
Transgenic Tg (fli1: EGFP) zebrafish were utilized to establish a zebrafish xenograft model to assess the RCC distant metastatic potential. A total of 400 Caki-1 or 786-O RCC cells expressing a vector control, ADAMTS1-overexpression, ADAMTS1 + shVCAN, or ADAMTS1 + shEGFR, labeled with CM-DiI dye (ThermoFisher Scientific, Waltham, MA), were injected into a zebrafish yolk sac under anesthesia with 0.1 mg/mL tricaine. Zebrafish injected with tumor cells were maintained at 34 °C for a specified duration. Fluorescence signals present in the trunk and end-tail of the fish, indicating RCC cancer cell metastatic activity, were monitored daily using a Zeiss Axiophot fluorescence microscope (Carl Zeiss Microimaging, Gottingen, Germany). Distant metastasis signals were quantified using ImageJ software (National Institutes of Health, Bethesda, MD).
Human phosphoreceptor tyrosine kinase (RTK) array
The phosphorylation status of RTKs was analyzed by collecting 300 μg of total protein lysates from ADAMTS1-manipulated Caki-1 cells. Subsequently, proteins were harvested and subjected to incubation with a membrane-based RTK antibody array (ARY001B; R&D) according to the manufacturer’s instructions. Following the reaction with specific antibodies and target proteins, signals were detected using a chemiluminescent horseradish peroxidase (HRP) substrate. Spot densities were then normalized against respective reference array spots and further against the controls.
Real-time reverse-transcription quantitative polymerase chain reaction (RT–qPCR)
mRNA was isolated from specified experimental groups of RCC cells using Trizol (Invitrogen, Carlsbad, CA). Approximately 1 μg of total RNA was then employed for reverse transcription to complementary DNA (cDNA) using an iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA). The resulting cDNA was subsequently utilized in RT–qPCR assays with a TOOLS 2 × SYBR qPCR Mix kit (BIOTOOLS, New Taipei City, Taiwan), as detailed in our prior study[25]. Primers used in the RT–qPCR are listed as follows: ADAMTS1_F: TTT TGT TCA CAC ACT TGC CGT T and ADAMTS1_R: CAG TGC CAG TTT ACA TTT GGG G; VCAN_F: AAC GGC TTT GAC CAG TGC GA and VCAN_R: ATC AGG GGG AGG GAA GCC TG; TIMP3_F: ACC GAG GCT TCA CCA AGA TG and TIMP3_R: CCC CGT GTA CAT CTT GCC AT; EGFR_F: AGG CAC GAG TAA CAA GCT CAC and EGFR_R: ATG AGG ACA TAA CCA GCC ACC; and GAPDH_F: 5′-CTGGAGAAACCTGCCAAGTATGAT-3′ and GAPDH_R: 5′-TTCTTACTCCTTGGAGGCCATGTA-3′. We also designed the following primer set to detect expression levels of VCAN isoforms, V0_F: CAG CCC CCA GCA AGC ACA AAA TT and V0_R: TCA GCC ATT AGA TCA TGC ACT GG; V1_F: GAA CCC TGT ATC GTT TTG AGA ACC and V1_R: TCA GCC ATT AGA TCA TGC ACT GG; V2_F: CAG CCC CCA GCA AGC ACA AAA TT and V2_R: GCA TAC GTA GGA AGT TTC AGT AGG; and V3_F: GAA CCC TGT ATC GTT TTG AGA ACC and V3_R: GCA TAC GTA GGA AGT TTC AGT AGG.
Transwell invasion assay
An invasion assay was conducted using 24-well Transwell assays (with an 8-μm pore size; Corning Costar, Corning, NY). Approximately 5 × 104 RCC cells were seeded into the upper chamber coated with Matrigel (BD Biosciences, Bedford, MA) and incubated for 48 h. After incubation, cells were fixed with methanol for 20 min and stained with 0.5% crystal violet for an additional ~3–4 h. Cells in the upper chamber were carefully removed using cotton buds. Quantification was performed by counting invaded cells in five individual microscopic fields (×100 magnification) of stained cells.
Dot blot assay
Conditioned medium (CM) from specified RCC cells was collected and centrifuged at a minimum speed of 13,000 rpm to remove cell debris. Around 300 μL of the supernatant was carefully loaded onto nitrocellulose membranes using a 96-well GFE960 Dot Blotter (GenePure, Taichung, Taiwan) and drawn slowly through the membrane by a suction pump. Antibody hybridization and signal detection were carried out following the western blot protocol.
Luciferase reporter assay
An EGFR promoter construct was purchased from GeneCopoeia (clone HPRM45993) (Rockville, MD) and transfected into RCC cells with 1 μg plasmid DNA for the final 48 h to examine promoter activity, which stably manipulated ADAMTS1 expression. For CM treatment, RCC cells were first transfected with reporter constructs for 24 h, and the cells were then further treated with the indicated CM for another 24 h. Medium from RCC cells was collected, and Gaussia luciferase activity was determined using a Secrete-Pair™ Gaussia Luciferase Assay kit (GeneCopoeia). In the meantime, cell lysates were harvested to determine Renilla luciferase activity as the internal control by a luciferase assay kit (Promega, Madison, WI).
Subcellular fractionation
Isolation of cytoplasmic fractionation was achieved using a cytosolic lysis buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, and 1% NP-40), followed by centrifugation at 13,000 rpm for 5 min. The resulting supernatant, containing cytosolic proteins, was collected. The pellet was then lysed using PRO-PREPTM protein extraction buffer and subjected to an additional centrifugation step at 13,000 rpm for 15 min. Each fraction obtained was separated by SDS–PAGE and probed for ADAMTS1. Fraction purity was evaluated by detecting GAPDH for the cytoplasmic fraction and lamin A/C for the nuclear fraction.
Immunoprecipitation (IP) assay
Total protein from RCC cells was extracted using NETN buffer (20 mM Tris at pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40). Approximately 1.5 mg of protein was incubated with an anti-ADAMTS1 antibody (AF5867) at 4 °C for 24 h, followed by incubation with Protein A Sepharose beads for ~1–2 h. After washing the beads five times with NETN buffer, bound proteins were eluted and boiled with 6× sample buffer. The resulting bound proteins were then analyzed by western blotting.
Statistical analysis
Values from both in vitro and in vivo studies are presented as the mean ± standard deviation (SD). A Student’s t-test was employed for data analysis when comparing two groups. In the analysis of clinical data, Spearman correlations were used to assess the correlation between ADAMTS1 and EGFR or VCAN expression. Associations of gene expressions with overall survival (OS) and disease-specific survival (DSS) were evaluated using a log-rank test. For comparisons involving three groups, a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was conducted to compare each group. The expression cutoff to define high or low expression groups was determined based on the minimum log-rank test p value. Gene expressions from paired tumor/normal tissues were compared using a paired t-test.
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