Nuclear DLC1 exerts oncogenic function through association with FOXK1 for cooperative activation of MMP9 expression in melanoma

A Rho GTPase-activating protein (RhoGAP), deleted in liver cancer 1 (DLC1), is known to function as a tumor suppressor in various cancer types; however, whether DLC1 is a tumor-suppressor gene or an oncogene in melanoma remains to be clarified. Here we revealed that high DLC1 expression was detected in most of the melanoma tissues where it was localized in both the nuclei and the cytoplasm. Functional studies unveiled that DLC1 was both required and sufficient for melanoma growth and metastasis. These tumorigenic events were mediated by nuclear-localized DLC1 in a RhoGAP-independent manner. Mechanistically, mass spectrometry analysis identified a DLC1-associated protein, FOXK1 transcription factor, which mediated oncogenic events in melanoma by translocating and retaining DLC1 into the nucleus. RNA-sequencing profiling studies further revealed MMP9 as a direct target of FOXK1 through DLC1-regulated promoter occupancy for cooperative activation of MMP9 expression to promote melanoma invasion and metastasis. Concerted action of DLC1–FOXK1 in MMP9 gene regulation was further supported by their highly correlated expression in melanoma patients’ samples and cell lines. Together, our results not only unravel a mechanism by which nuclear DLC1 functions as an oncogene in melanoma but also suggest an unexpected role of RhoGAP protein in transcriptional regulation.


Quantitative polymerase chain reaction (qPCR)
Total RNA was extracted using MiniBEST Universal RNA Extraction Kit (Takara) and reverse transcribed for cDNA synthesis using PrimeScript RT Master Mix (Takara). All reactions including non-template controls were performed in triplicate on StepOnePlus Real-time PCR system (Applied Biosystem) using SYBR Premix Ex Taq II (Takara).
Human 36B4 was used for normalization. The list of primers for detection of gene expression are shown in Supplementary Table 2.

Chromatin immunoprecipitation (ChIP)
A total of 2 × 10 6 WM266-4 melanoma cells stably expressing Scramble control or DLC1 silencing shRNA were crosslinked by 1% formaldehyde and lysed, then digested using 5.0μL diluted micrococcal nuclease according to the manufacturer's protocol (Pierce Magnetic ChIP Kit, ThermoFisher). Supernatant was collected and sonicated for 6 × 30 s in a BioruptorPico Sonicator (Diagenode). The target size of chromatin fragments ranging from 100 bp to 600 bp was confirmed by 2% agarose gel electrophoresis. Chromatin fragments were immunoprecipitated by using normal rabbit IgG control (ThermoFisher) or 4μg anti-FOXK1 antibody (ChIP grade, ab18196, Abcam) at 4°C overnight. 20uL of ChIP grade magnetic beads (ThermoFisher) was then added into the chromatin-antibody mixture and incubated at 4 °C for 2 hours. DNA fragments were then purified and reserve-crosslinked based on manufacturer's instruction, followed by 40 cycles of quantitative PCR. The PCR products were further visualized by 2.5% agarose gel electrophoresis. The primers used for amplification of fragments spanning FOXK1 binding motif on MMP9 promoter are summarized in Supplementary Table 2. Data were analyzed and presented as the fold enrichment relative to IgG control.

Dual-luciferase reporter assay
Melanoma cells were transfected with pGL3-basic FireFly luciferase reporter vector (Promega) driven by human MMP9 proximal promoter (~ 1.5 kb) and Renilla luciferase reporter vector using PolyJet transfection reagent based on manufacturer's protocol.
Cells were harvested and lysed 48 hours post-transfection. The cell lysate was measured by PerkinElmer Victor 3 Multi-label Plate Reader using Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. The luminescence signal of the Renilla luciferase reporter activity was used for normalization of FireFly luciferase reporter activity.

Lentiviral production and transduction
7x10 6 293T cells in a 100mm culture dish with 90-100% confluence were cotransfected with a lentiviral expression vector, packaging plasmid psPAX.2 and envelope plasmid pMD2.G using PolyJet (SignaGen) according to the manufacturer's instruction. The cell culture medium containing the lentiviral particles was harvested 48-and 72-hour post transfection and filtered through a 0.22µm filter. 3x10 5 melanoma cells were infected with lentivirus particles expressing cDNA and/or shRNA and cultured in the presence of 8µg/ml Polybrene (Sigma) for 24 hours. After 48 hours transduction, infected melanoma cells were screened in presence of 1µg/ml puromycin (Life Technologies) for 96 hours to generate stable cell lines.

Colony formation assay
Single cells (5x10 2 ) stably expressing cDNA and/or shRNA in complete medium were seeded in each well of a 6-well plate. Plates were incubated at 37 °C for 1 week for A375 and 2 weeks for WM266-4, during which culture medium was refreshed every 3 days for A375 and 1 day for WM266-4, respectively. Following methanol (Merck) fixation and 0.1% crystal violet (Sigma) staining, the number of colonies formed in each well was calculated by Quantity One Software (Bio-Rad).

AlamarBlue (Proliferation) assay
Single cell suspension containing 1x10 3 A375 or WM266-4 melanoma cells was seeded in each well of a 96-well plate and incubated at 37°C. After 24 hours, each well was replaced with 100µL of complete medium containing 10% AlamarBlue (Life Technologies) and incubated at 37°C for 2 hours with light protection. The measurement of the absorbance reading at 570nm and 600nm in each well was recorded for quantification followed by replacement of fresh complete medium. The growth curve of the cells with different treatments was monitored for 4 to 11 days with 6 replicates.

EDU incorporation staining
The Click-iT EdU Alexa Fluor 488 Imaging Kit (ThermoFisher) was used. Briefly, before adding the EdU treatment, plated 3×10 4 melanoma cells on sterile coverslips in 24well plate and allowed them to recover overnight. Replace half of the media with fresh media containing 20µM of EdU to the final concentration of 10µM and then incubate 2 hours for A375 and 4 hours for WM266-4 at 37°C, respectively. Afterwards, cells were fixed, permeabilized and incubated with Click-iT reaction cocktail based on the manufacturer's protocol. The EdU-labeled cells and Hoechst were captured by inverted fluorescent microscope (Nikon). The percentage of positive EdU-stained cells in all Hoechst-labeled cells was presented.

Transwell invasion assays
Melanoma cell lines with different treatments were resuspended as single cell (5x10 4 ) in plain medium, then seeded on the transparent PET membrane of cell culture insert (8µm, Falcon) coated with 150uL of Matrigel (2.5mg/mL, Corning). Cells were allowed to invade through the Matrigel and membrane driven by FBS in the lower chamber for 12 hours (A375) or 48 hours (WM266-4). Cells failed to invade were removed by the cotton swap. After 100% methanol fixation and DAPI (1µg/mL, Sigma) staining, the number of positive DAPI signal that representing invaded cells was counted.

RHOA pull-down activation assay
The detection of RHOA activity was performed by using RHOA Pull-down Activation Assay Biochem Kits (Cytoskeleton) based on manufacturer's protocol. Melanoma cells at 70% confluence were washed twice with cold PBS and lysed with IP Buffer supplemented with 1% protease and phosphatase inhibitors (ThermoFisher) for 3 minutes on ice. Cell lysates were then centrifuged at 16,000g for 3 min. The supernatant (400µg) was then immediately incubated with Rhotekin-RBD (50µg) beads at 4°C for 2 hours to pull down RHOA-GTP. The beads were then washed three times by wash buffer and subjected to western blotting using mouse monoclonal antibodies against RHOA (Santa Cruz). Total RHOA in whole cell lysates was served as loading control.

GST pull-down assay
The fragments of DLC1 (636-1091 aa) and FOXK1 (1-391 aa) were cloned into pGEX-6P-2 and pET-28a vector, respectively, which were kindly provided by Q Hao from the School of Biomedical Sciences in the University of Hong Kong. GSTtagged DLC1 and His-tagged FOXK1 truncations were transformed into BL21 E. coli strain with chloramphenicol resistance. Competent cells were cultured in 200mL LB medium at 37°C until the OD600 reached 0.6. For protein induction, 0.3mM IPTG was added into the medium and maintained at 16°C for 20 hours. Cell pellets were then resuspended in lysis buffer (20mM Tris-HCl, 150mM NaCl, 1mM DTT) and sonicated to obtain optimal protein concentration, followed by incubation with Glutathione beads (ThermoFisher) at 4°C for 1 hour. GST-bound beads were subsequently incubated with His-tagged FOXK1 fragment for 2 hours and subjected to SDS-PAGE. pGEX-6P-2 empty vector was served as a negative control.