DEPDC1B Promotes Melanoma Angiogenesis and Metastasis through Sequestration of Ubiquitin Ligase CDC16 to Stabilize Secreted SCUBE3

Abstract The ability of melanoma to acquire metastasis through the induction of angiogenesis is one of the major causes of skin cancer death. Here, it is found that high transcript levels of DEP domain containing 1B (DEPDC1B) in cutaneous melanomas are significantly associated with a poor prognosis. Tissue microarray analysis indicates that DEPDC1B expression is positively correlated with SOX10 in the different stages of melanoma. Consistently, DEPDC1B is both required and sufficient for melanoma growth, metastasis, angiogenesis, and functions as a direct downstream target of SOX10 to partly mediate its oncogenic activity. In contrast to other tumor types, the DEPDC1B‐mediated enhancement of melanoma metastatic potential is not dependent on the activities of RHO GTPase signaling and canonical Wnt signaling, but is acquired through secretion of signal peptide, CUB domain and EGF like domain containing 3 (SCUBE3), which is crucial for promoting angiogenesis in vitro and in vivo. Mechanistically, DEPDC1B regulates SCUBE3 protein stability through the competitive association with ubiquitin ligase cell division cycle 16 (CDC16) to prevent SCUBE3 from undergoing degradation via the ubiquitin‐proteasome pathway. Importantly, expression of SOX10, DEPDC1B, and SCUBE3 are positively correlated with microvessel density in the advanced stage of melanomas. In conclusion, it is revealed that a SOX10‐DEPDC1B‐SCUBE3 regulatory axis promotes melanoma angiogenesis and metastasis, which suggests that targeting secreted SCUBE3 can be a therapeutic strategy against metastatic melanoma.

7.4) depending on the assay and the formulars are shown as below table. Protease and phosphatase inhibitors cocktail (100x, ThermoFisher, #78430) was supplemented to the lysis buffer before lysing cells. For thorough lysis, the cell lysate was incubated on ice for 40 minutes with intermittent vortex every 10 minutes. After protein concentration measurement, 40 -60 µg of proteins lysates were mixed with 4x Laemmli loading buffer supplemented with 10% βmercaptoethanol (β-ME) (Bio-Rad, #1610747) and heated at 95°C for 5 minutes to denature the proteins. Proteins were stacked and separated through SDS-PAGE and subjected to antibody incubation. ECL HRP substrate (advansta, #K-12045-D50) was to give out chemiluminescence signals. Film-image the signal using darkroom development techniques. The signal intensity was controlled by exposure time. After proper fixation, the films were washed in running water, oven-dried and scanned to keep the image. The protein bands were quantified using Image J.

RHOA/RAC1 activation assay
The RHOA activation assay was done using Rhotekin RBD beads (Cytoskeleton, #RT02). The RAC1 activation assay was done using PAK-PBD beads (Cytoskeleton, #PAK02). Melanoma cells with desired treatment were cultured no more than 70% confluency. Cells were washed using ice-cold PBS and collected in IP lysis buffer supplemented with 1% proteinase inhibitors.
Followed by 5-minute lysis on ice, the cell debris was separated using centrifugation at 16,000rcf for 3 minutes. Supernatant containing the active RHOA/RAC1 was transferred to a new 1.5 mL tube and 50 µL were saved for protein concentration measurement. 300-500 µg total protein was incubated with either Rhotekin beads (50 µg per reaction) or PAK-PBD beads (10 µg per reaction) at 4°C for 2 hours. Beads were then collected by centrifugation at 4°C at a speed of 1,000 rcf for 3 minutes and washed for 4 times. Proteins bound to the beads were then eluted using 2x Laemmli buffer and denatured under 95°C for 10 minutes. All volume obtained was loaded into 12% SDS-PAGE for further western blot analysis.

b-catenin TOPFlash luciferase reporter assays
To test the activity of β-catenin, 2 µg DNA in total containing M50 8x TOPFlash and Renilla in a ratio of 10:1 was transfected into 106 melanoma cells using polyjet. The negative control was transfected with only Renilla without the presence of any reporter. Cells were collected 48 hours after transfection and lysed in passive lysis buffer provided in the Dual-luciferase Reporter Assay System kit (Promega, #E1910) on a shaker at room temperature for 15 minutes. 20 µL cell lysate was added each well in an untransparent 96-well plate followed by addition of 100 µL LAR II solution and subjected to firefly luciferase measurement. 100 µL stop solution was added afterwards for the Renilla luciferase measurement. One empty well containing no cell lysate was also measured for background signal. Luciferase activity relative to Renilla was calculated and normalized with control group.

Calculation method using Image J
The calculation of "blood vessel total area" on xenograft tissue sections in Figure 2g was performed based on the immunofluorescent staining against CD31. Two xenograft tumors from each group were sectioned and three adjacent sections were picked for H&E staining, fluorescent-IHC staining and DAB-IHC staining respectively. Triplicates were obtained every 100 µm apart and five random images were taken in each section subjected to calculation by Image J software. n = 2 x 3 x 5 = 30. The calculation of "blood vessel total area" and "tumor total area" on lung metastasis tissue sections in Figure 2h were performed based on the H&E staining. 7 lungs dissected from intravenous injection were fixed and sectioned for H&E staining. High magnification images were taken and stitched into a full section image before subjected to analysis using Image J. Blood vessel area containing red blood cell and tumor nodule area darkly stained were identified by color threshold identification function and the area was measured.     Tables   Table S1-