Cav‐1 Upregulation Slows Migration Velocity in BLM Melanoma
Authors: Kinsey S Skyler , Baer Robert w 
Published: November 2023

Caveolin‐1 (Cav‐1) is a membrane‐associated and cytosolic protein that influences a number of cellular signaling cascades. Changes in Cav‐1 expression levels are thought to be responsible for changes in metastatic behaviour of a number of cancers, including melanoma. However, the effect of changes in Cav‐1 expression level seem dependent on cell‐type and direction of expression change. In highly migratory, metastatic melanoma, Cav‐1 expression levels tend to be low. The purpose of this study was to determine whether Cav‐1 upregulation alters EMT markers, cell division or basal and CXCL‐8‐stimulated migration velocity in human BLM melanoma. Wild‐type BLM cells were stably transfected (Lipofectamine 2000, Invitrogen) with a Myc‐DDK‐tagged human Cav‐1 gene cloned into a pCMV6‐Entry vector (Origene, TrueOrf). Permanent selection was achieved with G418 (1 mg/ml) antibiotic. Cell extracts from wild‐type and transfected BLM cells were run on 10%‐PAGE gels and transferred to PVDF membranes. Appropriate 1° antibodies were used to confirm Cav‐1 upregulation and to test for the presence of the EMT marker N‐cadherin using infrared 2° antibody detection (LiCor). Proliferation of wild‐type and Cav‐1 transfected cells were measured using BrdU assay. Cells were plated in 96‐well plates and incubated for 24 hours in RPMI 1640, 10% FBS. BrdU was added to the cells and allowed to incubate for 12 hours. Cells were fixed, the DNA denatured, and mouse anti‐BrdU antibody added to detect the incorporated BrdU. BrdU uptake was measured using HRP‐linked 2° antibody and [TMB] detected at A450 nm using a spectrophotometer (Epoch Microplate Spectrophotometer). Migration velocity was measured in wild‐type and transfected cells using a wound‐healing assay. Cells were seeded at 250,000 cells per well in 24‐well plates and incubated overnight in serum free RPMI 1640 media. Wells were scratched with pipette tip and plated in RPMI 1640, 10% FBS. Wells received a range of unbound CXCL8 doses (0–100 ng/ml). Wound images were collected at 15 min intervals and quantified at 4 hour intervals using ImageJ. Immunoblot analysis confirmed increased expression of Myc‐DDK‐tagged Cav‐1 in the transfected BLM cells. Immunohistochemical staining also confirmed increased expression of Cav‐1 in the transfected cells and showed that subcellular distribution of Cav‐ 1 is similar in the wild‐type and transfected cells. The EMT marker N‐cadherin, which was largely absent in the wild‐type cells, showed increased expression in the Cav‐1 transfected BLMs. BrdU assay shows that there is no significant difference in cell division over 12 hours between wild‐type and Cav‐1 transfected cells by Student's t‐test (p = 0.54). Migration velocity by wound‐closure assay decreased with Cav‐1 overexpression. Wild‐type cells migrated at 14.0 ± 4.9 μm / h (mean ± sd). Transfected cells migrated at 7.0 ± 2.2 μm / h. This was statistically significant by Student's t‐test (p < 0.001). In both wildtype and Cav‐1 transfected BLM melanoma, we were unable to demonstrate altered cell migration rates when unbound CXCL8 was administered over a 24 hour period in RPMI media containing 10% FBS. In summary, Cav‐1 expression in BLM melanoma is associated with an increased N‐cadherin expression, no change in cell division by BrdU assay, and slowed migration by wound‐healing assay. More work will be necessary to elucidate the underlying mechanisms.

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Supported by the Graduate Program Committee, KCOM