Study of the subcellular localization of the human ATP-binding cassette transporter isoform,ABCB5β, expressed in melanoma

Abstract

Human ATP-binding cassette (ABC) transporter superfamily comprises 44 membrane
transporters classified into five families (A, B, C, D, and G). There are also 4 non-transporter ABC proteins that belong to family E and F. ABC transporters are responsible for translocating an array of endogenous and xenobiotic substances across cellular membranes. Many of these transporters are involved in cancer multidrug resistance. Among these transporters, ABCB5 has been described as a marker of skin progenitor cells, melanoma stem cells, and as a marker of limbal stem cells. ABCB5 encodes a full transporter (ABCB5FL) and a half transporter (ABCB5β). ABCB5FL was cloned from testis and ABCB5β from melanocytes. There are also many additional transcript variants, including ABCB5α, but they are too short to form functional transporters. Recently, ABCB5β has attracted substantial attention due to its roles in promoting proliferation, metastasis, and invasive capacities in melanoma cells. Given the high expression of ABCB5β in melanoma, we directed our research toward this isoform. In this study, we explored the subcellular localization of ABCB5β using a combination of subcellular fractionation and immunofluorescence analyses. To conduct this investigation, we tested commercial anti-ABCB5 antibodies and engineered several tagged ABCB5β cDNA constructs.Our study focuses on two cell lines, HeLa (cervical cancer) and MelJuSo (melanoma) and
revealed that GFP- and HA-tagged ABCB5β is primarily located in the endoplasmic reticulum. Transferring HA-ABCB5β to a low-expression plasmid yielded similar results, as well as treating the cells with a known inducer of chaperone activity (SAHA). In summary, our findings support that ABCB5β predominantly resides in the ER under normal cellular conditions. Additionally,we observed that the expression of the close homolog ABCB1 increases after ABCB5 knockdown in MelJuSo cells, raising the possibility that the former could compensate for the latter. However, it appears unlikely that this compensation would cover all ABCB5-dependent functions in these cells, considering that MelJuSo cells with a knocked-down ABCB5 gene exhibited decreased proliferation. In summary, we demonstrated that ABCB5β is
predominantly localized to the ER even after the treatment with SAHA, which restored the folding and increased the total level of ABCB5β expression to some extent. However, remains unclear the role of ABCB5β in the ER of melanoma cells and whether it can function either as a homodi-mer or as heterodimer.

Date of Award	27 Oct 2023
Original language	English
Awarding Institution	University of Namur
Sponsors	UNamur-CERUNA
Supervisor	Jean-Pierre Gillet (Supervisor), Marielle Boonen (Co-Supervisor), Henri François Renard (President), Olivier De Backer (Jury), Abele Rupert (Jury) & Gergely Szakacs (Jury)