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Published on: September 8, 2021
Differential expression of ABCB5 in BRAF inhibitor-resistant melanoma cell lines
Jingjing Xiao1, Michael E Egger1, Kelly M McMasters1
1The Hiram C. Polk, Jr MD Department of Surgery, University of Louisville School of Medicine, Louisville, KY, 40292, USA.
Background:
More than 50% of metastatic melanoma patients have a specific mutation in the serine/threonine kinase BRAF. This results in constitutive activation of the RAS-RAF-MEK-ERK-MAP kinase pathway, which causes uncontrolled cell growth. Vemurafenib (PLX4032) is an oral chemotherapeutic agent that targets the specific mutation V600E in the BRAF protein. Initial response rates are high in patients with BRAF mutant melanoma treated with a BRAF inhibitor such as vemurafenib, but resistance nearly always develops and disease progression ensues. There are several different mechanisms by which melanoma develops BRAF inhibitor resistance. One potential component of resistance is increased drug efflux. Overexpressed ABCB5 (ATP-binding cassette transporter, subfamily B, member 5) has been shown to efflux anti-cancer drugs from cancer cells. The purpose of this study is to determine whether ABCB5 is highly expressed in BRAF inhibitor-resistant melanoma cells and to evaluate whether ABCB5 is involved in the development of resistance to BRAF inhibitors in cutaneous melanoma.
Methods:
We established three BRAF inhibitor-resistant melanoma cell lines with BRAF mutation. The expression level of ABCB5 in PLX-resistant cell lines was checked by real-time PCR and Western blot analysis. SK-MEL-2 melanoma cells with wild-type BRAF were used for comparison. The association of different levels of ABCB5 with the changes of ERK, p-ERK, Akt and p-Akt was also assessed by Western blotting. Re-sensitization of melanoma cells to PLX was tested by p-ERK inhibitor PD58059 and ABCB5 knockdown by ABCB5 siRNA, respectively.
Results:
We showed that ABCB5 was overexpressed in SK-MEL-28PLXr and A2058PLXr cells but not in A375PLXr cells. ABCB5 overexpression is associated with activation of p-ERK status but not Akt. Inhibition of p-ERK re-sensitized SK-MEL-28PLXr and A2058PLXr cells to PLX treatment, but knockdown of ABCB5 did not re-sensitize A2058 PLXr and SK-MEL-28 PLXr cells to PLX treatment.
Conclusion:
These results confirm that, even though ABCB5 was overexpressed in SK-MEL-28 and A2058 melanoma cells that develop resistance to BRAF inhibitors, ABCB5 may not be a major targetable contributor to BRAF resistance. p-ERK inhibition may play important roles in BRAF resistance in these two melanoma cell lines.
Insights
ABCB5 transporter overexpression in BRAF-mutant melanoma cells did not confer resistance to vemurafenib. While p-ERK inhibition resensitized cells, ABCB5 knockdown did not, suggesting p-ERK, not ABCB5, is key in BRAF inhibitor resistance.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Research
Background:
- BRAF mutations drive over 50% of metastatic melanomas, leading to uncontrolled cell growth via the MAPK pathway.
- Vemurafenib targets BRAF V600E mutations, initially effective but often overcome by resistance mechanisms.
- ATP-binding cassette transporter B5 (ABCB5) may contribute to drug efflux and cancer drug resistance.
Purpose of the Study:
- To investigate ABCB5 expression in BRAF inhibitor-resistant melanoma cells.
- To determine if ABCB5 is involved in the development of resistance to BRAF inhibitors.
Main Methods:
- Established BRAF inhibitor-resistant melanoma cell lines (SK-MEL-28PLXr, A2058PLXr, A375PLXr).
- Assessed ABCB5 expression using real-time PCR and Western blot.
- Analyzed signaling pathways (ERK, Akt) via Western blotting.
- Tested drug resensitization with p-ERK inhibitor (PD58059) and ABCB5 knockdown (siRNA).
Main Results:
- ABCB5 was overexpressed in SK-MEL-28PLXr and A2058PLXr cells, but not A375PLXr.
- ABCB5 overexpression correlated with increased p-ERK, not p-Akt.
- Inhibition of p-ERK resensitized resistant cells to vemurafenib.
- ABCB5 knockdown did not restore sensitivity to vemurafenib.
Conclusions:
- ABCB5 overexpression in some BRAF inhibitor-resistant melanoma cells does not appear to be a primary targetable mechanism for resistance.
- p-ERK pathway inhibition is implicated in vemurafenib resistance in these melanoma models.
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