Downregulation of lncRNA TSLNC8 promotes melanoma resistance to BRAF inhibitor PLX4720 through binding with PP1α to re-activate MAPK signaling

Abstract

Purpose: Approximately 60% of patients with melanoma harbor BRAF mutation and targeting BRAF offers enormous advance in the treatment of those patients. Unfortunately, the efficacy of the BRAF inhibitors is usually restricted by the onset of drug resistance. Therefore, better understanding of the adaptive drug resistance mechanisms is essential for the development of alternative therapeutic strategies, and offers more promising measures to promote the short duration of response to BRAF inhibitors.

Methods: The levels of tumor suppressive long noncoding RNA on chromosome 8p12 (TSLNC8) were evaluated by qPCR. The MTT assay, colony formation assay, apoptosis assay, and in vivo xenograft tumor model were performed to assess the functions of TSLNC8 on drug resistance. Western blotting, RNA pull-down, and RNA immunoprecipitation (RIP) assays were applied to investigate the mechanisms of TSLNC8 in melanoma.

Results: Herein, our findings demonstrate that TSLNC8 is significantly downregulated in BRAF inhibitor-resistant melanoma tissues and cells. Moreover, downregulation of TSLNC8 in BRAF inhibitor sensitive cells reduces the toxicity response to BRAF inhibitor PLX4720, and inhibits apoptosis of melanoma cells-treated with PLX4720. Further assay elucidates that TSLNC8 can bind with the catalytic subunit of protein phosphatase 1α (PP1α) to regulate its distribution, and Downregulation of TSLNC8 results in PP1α cytoplasmic accumulation, thus re-activating the MAPK signaling. Eventually, the overexpression of TSLNC8 in BRAF inhibitor PLX4720-resistant melanoma cells restores the sensitive to BRAF inhibitor.

Conclusion: Collectively, our research provides a compelling rationale for resistance to BRAF inhibitor in melanoma, and the patient might benefit from the combinatorial therapy of BRAF inhibitors and lncRNA TSLNC8.

Keywords: BRAF mutation; MAPK signaling; PP1α; TSLNC8.

Fig. 1

LncRNA TSLNC8 is significantly downregulated in melanoma tissues and cells that are resistant to BRAF inhibitor. a TSLNC8 expression is significantly down-regulated in melanoma as compared to normal skin tissues using data from availably public dataset GEPIA. b TSLNC8 expression in paired tissue samples from acquired resistance to BRAF inhibitor patients that were acquired resistant to BRAF inhibitors (treated samples) is lower than that in matched tissues prior to treatment (pretreated samples). c TSLNC8 expression is significantly downregulated in BRAF inhibitor-resistant cells as compared to corresponding parental sensitive cells. *P < 0.05

Fig. 2

Downregulation of TSLNC8 in BRAF inhibitor-sensitive cells reduces the sensitivity to BRAF inhibitor. a Relative TSLNC8 expression in indicated melanoma stable cell line. b MTT assay showed that overexpresssion of TSLNC8 significantly increase the sensitivity of melanoma cells to different concentration of BRAF inhibitor PLX4720, while downregulation of TSLNC8 showed the opposite results. c The IC50 value for BRAF inhibitor PLX4720 (72 h) is significantly declined in TSLNC8-overexpressing cells, while increased in TSLNC8-silenced cells. *P < 0.01; **P < 0.01; ***P < 0.001

Fig. 3

Downregulation of TSLNC8 inhibits the apoptosis of melanoma cells-treated with-PLX4720 in vitro. a The protein levels of Bim and cleaved caspase 3 in indicated cell that were treated with 20 nM PLX4720 for 72 h by western blotting assay. b Representative images (left panel) and quantification (right panel) of the indicated cells that were treated with 20 nM PLX4720 for 72 h by colony formation assay. c Representative images of the indicated cells that were treated with 20 nM PLX4720 for 72 h by flow cytometry assay. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 4

Overexpression of TSLNC8 significantly decreases in vivo tumorigenesis of melanoma cells. a The overexpression of TSLNC8 markedly decreases tumor growth rate. b Luciferase signal in the indicated tissues through ex vivo imaging assay. c Tumor weight formed by indicated cells. e TSLNC8 levels in mice tumors. **P < 0.01; ***P < 0.001

Fig. 5

TSLNC8 can bind with PP1α to regulate MAPK signaling. a Relative distribution of TSLNC8 in melanoma cells using qPCR assay. U2 is served as the nuclear marker, and β-actin as the cytoplasmic marker. b RNA pull-down assay showed that the protein PP1α presents in the complexes pulled down by biotinylated TSLNC8. c RIP assay showed that TSLNC8 was enriched in the lysate pulled down by the antibody to PP1α, but not in the lysate by IgG. d Western blotting assay showed that TSLNC8 can change the distribution of PP1α and regulate the MAPK signaling. e Western blotting assay showed that p-MEK and p-ERK levels are also significantly reduced in mice tumor injected with TSLNC8-silenced cells when compared with control. **P < 0.01; ***P < 0.001

Fig. 6

The colony formation assay a and apoptosis assay b showed that upregulation of TSLNC8 restores the toxicity response to BRAF inhibitor PLX4720 in BRAF inhibitor-resistant cells. **P < 0.01; ***P < 0.001