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ADAM9 functions as a transcriptional regulator to drive angiogenesis in esophageal squamous cell carcinoma

Hypoxia and angiogenesis play key roles in the pathogenesis of esophageal squamous cell carcinoma (ESCC), but regulators linking these two pathways to drive tumor progression remain elusive. Here we provide evidence of ADAM9's novel function in ESCC progression. Increasing expression of ADAM9 was correlated with poor clinical outcomes in ESCC patients. Suppression of ADAM9 function diminished ESCC cell migration and in vivo metastasis in ESCC xenograft mouse models. Using cellular fractionation and imaging, we found a fraction of ADAM9 was present in the nucleus and was uniquely associated with gene loci known to be linked to the angiogenesis pathway demonstrated by genome-wide ChIP-seq. Mechanistically, nuclear ADAM9, triggered by hypoxia-induced translocation, functions as a transcriptional repressor by binding to promoters of genes involved in the negative regulation of angiogenesis, and thereby promotes tumor angiogenesis in plasminogen/plasmin pathway. Moreover, ADAM9 suppresses plasminogen activator inhibitor-1 gene transcription by interacting with its transcription factors at the promoter. Our findings uncover a novel regulatory mechanism of ADAM9 as a transcriptional regulator in angiogenesis and highlight ADAM9 as a promising therapeutic target for ESCC treatment.

IHC analysis was performed to study the expression of ADAM9 in human ESCC samples as previously described [2]. Briefly, goat anti-mouse ADAM9 antibody (AF949, R&D systems, Wiesbaden, Germany) was used to perform IHC staining using horseradish peroxidase-conjugated avidin-biotin complex (ABC) in a Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA). Tissue sections were counterstained with hematoxylin and mounted. The staining was evaluated by pathologists who were unaware of the clinicopathological parameters and clinical outcomes of the patients.

RNA-sequencing and analysis
The RNA-sequencing of control and ADAM9 KO KYSE170 samples were performed by Welgene Biotech Co. Ltd. (Taipei, Taiwan). Briefly, total RNA was extracted by Trizol Reagent (Invitrogen, USA) and the RNA quality was measured using a Bioanalyzer 2100 with a RNA 6000 LabChip kit (Agilent Technologies, USA) with an RNA Integrity Number ≥ 7.
The library was constructed using Agilent's SureSelect Strand-Specific RNA Library Preparation Kit for 2X150bp (Paired-End) and AMPure XP beads (Beckman Coulter, USA) size selection. The sequence was determined using Illumina's sequencing-by-synthesis (SBS) technology (Illumina, USA) NovaSeq. The sequencing results (FASTQ reads) were generated using Welgene Biotech's pipeline based on Illumina's base calling program bcl2fastq v2.20.
We further performed functional enrichment analysis of the differential expression genes as previously described [3].

Gene knockdown and knockout in ESCC cells
Lentiviral shRNA targeting ADAM9, SERPINE1, and PLAT was used as previous described [4] and reagent were obtained from the National RNAi Core Facility, Institute of Molecular Biology, Genomic Research Center, Academia Sinica. The human library is referred to as TRC-Hs 1.0. ESCC cells were infected with lentiviral suspensions and selected with 2 μg/ml puromycin to knock down ADAM9 expression. The efficiency of the gene knockdown was validated by Western blot analysis. A CRISPR RNA-guided Cas9 nuclease gene targeting system was used to knock out the ADAM9 gene in ESCC cells with a ToolGen kit (ToolGen Inc.) as previously described [5].

Plasmids, transfection, and generation of stable cell lines
The plasmids of HA-ADAM9 WT and catalytic mutant E348A were constructed as previously described [5]. The different fragments of SERPINE1 promoter were constructed in the luciferase reporter plasmid pGL3-basic with MluI and XhoI sites, and then they were cotransfected with Renilla luciferase reporter plasmids into the indicated cells for detecting promoter activity using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).

tPA protein and activity assay
Cells were seeded on 6cm dishes at 80-90% confluency. After 24h incubation, the

Time-lapse migration assay
This assay was conducted as previously described [4]. Briefly, cells were cultured on collagen-coated dishes (10 μg/mL, 3 mL) in serum-free media. The migration of cells was captured with CCD video cameras (AxioCam MRm, Zeiss) at 20 min intervals for a total 16 hours by inverted microscopes (Axio Observer Z1, Zeiss). Accumulated migration distance was determined by using the Track Point function of Image J.

Endothelial tube formation
The detailed procedures have been previously described [5]. Briefly, Matrigel was added to each well of a μ-slide (ibidi GmbH, Munich, Germany) and allowed to polymerize for 30 min at 37 °C. Cell suspensions (50 μL) of HUVECs treated with conditioned media from the indicated cells were plated in the Matrigel-coated wells at 7500 cells/well in serumfree M199 medium for 4 h at 37 °C in a 5% CO2 humidified atmosphere incubator. After 4-h incubation, capillary-like structures formed, and the images were captured using a Cytation™ 5 plate reader (BioTek, Winooski, VT, USA). The images were analyzed by measuring the total length of the tubules per well with ImageJ software (National Institutes of Health, Bethesda, MD, USA).