Schmidt et al. · Cell Reports · 2019

The lncRNA SLNCR Recruits the Androgen Receptor to EGR1-Bound Genes in Melanoma and Inhibits Expression of Tumor Suppressor p21

Schmidt Karyn, Carroll Johanna S., Yee Elaine, Thomas Dolly D., Wert-Lamas Leon, Neier Steven C., Sheynkman Gloria, Ritz Justin, Novina Carl D.

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The study

What was asked, and what was found

Schmidt et al., Cell Reports, 2019 asked how the melanoma long non-coding RNA SLNCR makes the androgen receptor oncogenic when no androgen is present, and whether that pairing explains why melanoma runs worse in men. The answer they reach is that SLNCR carries the androgen receptor to places on the genome where EGR1 is already sitting, and that the resulting complex flips EGR1 from switching the tumour suppressor p21 on to switching it off.

The AUM material sits in one experiment and it is a clean one. Short single stranded oligonucleotides were designed to sit on the stretch of SLNCR that the androgen receptor binds, two of them mimicking that stretch and two of them complementary to it, and all of them built so that they would not trigger RNase H cleavage of the transcript. They were added straight to the medium of the patient derived melanoma cultures WM1976 and WM858 with no transfection reagent, which the paper calls gymnotic delivery, and proliferation fell against the scramble arm. SLNCR itself did not fall. It sometimes rose 2 to 3 fold, which the authors read as a feedback response to losing SLNCR function. AUM BioTech is thanked in the acknowledgments, and Veenu Aishwarya is named there.

The rest of the study used transfected small interfering RNAs and expression vectors. Knocking down SLNCR moved 222 genes in WM1976 cells, chromatin immunoprecipitation found 9,974 androgen receptor binding regions in hormone deprived A375 cells, and knocking down either SLNCR or the androgen receptor raised p21 messenger RNA and protein, including in the p53 mutant line SK-MEL-28. Concentration and exposure time for the oligonucleotides are not stated in the paper; the sequences sit in a supplementary table not held here.

Key findings

Short single stranded oligonucleotides that sit on the androgen receptor binding site of SLNCR, delivered into melanoma cells with no transfection reagent, cut proliferation without lowering the long non-coding RNA itself.(Results, SLNCR and Ligand-Independent AR Increase Melanoma Cell Proliferation, Figures 2B and S3D)
Because proliferation fell while SLNCR levels rose, the effect follows from blocking the interaction rather than from removing the transcript.(Results, SLNCR and Ligand-Independent AR Increase Melanoma Cell Proliferation)
SLNCR carries the androgen receptor to sites already occupied by EGR1, and the complex turns EGR1 from an activator of the tumour suppressor p21 into a repressor of it.(Summary)
Removing either the long non-coding RNA or the androgen receptor raised p21 even in cells whose p53 is inactive.(Results, SLNCR and AR Cooperatively Inhibit Expression of the Cyclin-Dependent Kinase Inhibitor p21 in a p53-Independent Manner, Figures 4E and 4F)
The androgen receptor drove melanoma proliferation with no androgen present, which is why an RNA that recruits it matters.(Results, SLNCR and Ligand-Independent AR Increase Melanoma Cell Proliferation, Figure 2A)

Product: AUMblock sdASO
Targets: SLNCR · human · long non-coding RNA, bound and sterically blocked rather than degradedAR · human · protein, blocked at its interaction surface with SLNCR by mimic oligonucleotides
Models: WM1976 and WM858 melanoma short term cultures, which received the oligonucleotides · patient derived cellsA375 malignant melanoma, used for the small interfering RNA and chromatin work rather than for the oligonucleotides · cell lineSK-MEL-28, a p53 mutant melanoma line used for the p21 work · cell line
Delivery: added straight to the cell medium 24 hours after seeding, with no transfection reagent. The paper calls this gymnotic delivery.
Readouts: WST-1 proliferation assay, RT-qPCR, immunoblot, chromatin immunoprecipitation and sequencing, RNA sequencing, transcription factor activation array, RNA electrophoretic mobility shift assay, flow cytometry cell cycle analysis
Measured: effect of the steric blocking oligonucleotides on melanoma proliferation: significantly decreased. In Figure 2B all four oligonucleotides are marked ****p < 0.0001 against the scramble arm in WM1976, while in WM858 three of the four are marked ****p < 0.0001 and one, Mimic 2, is marked n.s. (gymnotic delivery to WM1976 and WM858, added 24 h after seeding, read to day 4, concentration not stated in the paper)change in SLNCR level caused by the steric blocking oligonucleotides: 2 to 3 fold increase on some occasions, with no decrease (gymnotic delivery, melanoma short term cultures)genes dysregulated by small interfering RNA knockdown of SLNCR: 222, of which 131 up and 91 down (WM1976, p < 0.01, versus a scramble small interfering RNA)androgen receptor binding regions found by chromatin immunoprecipitation and sequencing: 9,974 total, 5,717 with empty vector and 8,239 with the SLNCR1 vector (hormone deprived A375 melanoma cells)rise in p21 messenger RNA after knockdown of SLNCR: about 1.5 to 2.5 fold (WM1976 and A375 cells, small interfering RNA knockdown)
Figures: Figure 2 Proliferation of hormone deprived melanoma cells after androgen receptor knockdown in panel A, and after adding the two mimic and two antisense steric blocking oligonucleotides against a scramble arm to WM1976 and WM858 in panel B.Figure 4 p21 messenger RNA and protein after knockdown of SLNCR or the androgen receptor, in p53 wild type and p53 mutant melanoma cells.
Citation: Schmidt et al. The lncRNA SLNCR Recruits the Androgen Receptor to EGR1-Bound Genes in Melanoma and Inhibits Expression of Tumor Suppressor p21. Cell Reports 2019;27:2493-2507.e4. doi:10.1016/j.celrep.2019.04.101

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