LncRNA DILA1 inhibits Cyclin D1 degradation and contributes to tamoxifen resistance in breast cancer

Abstract

Cyclin D1 is one of the most important oncoproteins that drives cancer cell proliferation and associates with tamoxifen resistance in breast cancer. Here, we identify a lncRNA, DILA1, which interacts with Cyclin D1 and is overexpressed in tamoxifen-resistant breast cancer cells. Mechanistically, DILA1 inhibits the phosphorylation of Cyclin D1 at Thr286 by directly interacting with Thr286 and blocking its subsequent degradation, leading to overexpressed Cyclin D1 protein in breast cancer. Knocking down DILA1 decreases Cyclin D1 protein expression, inhibits cancer cell growth and restores tamoxifen sensitivity both in vitro and in vivo. High expression of DILA1 is associated with overexpressed Cyclin D1 protein and poor prognosis in breast cancer patients who received tamoxifen treatment. This study shows the previously unappreciated importance of post-translational dysregulation of Cyclin D1 contributing to tamoxifen resistance in breast cancer. Moreover, it reveals the novel mechanism of DILA1 in regulating Cyclin D1 protein stability and suggests DILA1 is a specific therapeutic target to downregulate Cyclin D1 protein and reverse tamoxifen resistance in treating breast cancer.

Fig. 1. A Cyclin D1-interacting lncRNA DILA1 is overexpressed in tamoxifen-resistant breast cancer cells with upregulated Cyclin D1 protein.

a Western blotting showing Cyclin D1 protein in parental and tamoxifen-resistant MCF-7 (MCF7-Pa, MCF-Re) and T47D (T47D-Pa, T47D-Re) cells. GAPDH was used as a loading control. b MCF-Re cells, transfected with negative control siRNA (NC) or one of the two siRNAs targeting Cyclin D1 (siD1-1 and siD1-2), were treated with tamoxifen for 48 h. Relative cell numbers were determined by a cell counter. c Heatmap of the enriched long noncoding RNAs by RIP–sequencing in MCF-7 cells with ectopically expressed HA-Cyclin D1 and untagged Cyclin D1 control. (fold change >2, p < 0.05). p values were determined by negative binomial generalized linear models. No adjustments were made for multiple comparisons. d RT-qPCR showing the expression of DILA1 in MCF7-Pa and MCF-Re cells. e Binding of DILA1 to CyclinD1 protein in MCF7-Re cells, assayed by RIP, followed by RT-qPCR. IgG and GAPDH were used as negative controls. f The full length of DILA1 (ENST00000435697.1) in UCSC Genome Browser (upper) and determined by 5′ and 3′ RACE (lower). g RT-qPCR showing the nuclear and cytoplasmic fraction of DILA1 in MCF-Re cells, with GAPDH and MALAT1 as cytoplasmic and nuclear control, respectively. h Confocal FISH images showing nuclear localization of DILA1 (green) in MCF7-Pa and MCF-Re cells. i RNAScope showing subcellular localization and relative expression of DILA1 (red) in MCF7-Pa and MCF7-Re cells. j RNA pull-down showing the interaction between Cyclin D1 and DILA in vitro (MCF7-Re cell lysates or recombinant GST-Cyclin D1 protein). Biotin-labeled DILA1 detection by anti-biotin antibody as a control. k Confocal FISH images showing the co-localization of Cyclin D1 (red) and DILA1 (green) in MCF7-Re cells. For a, h–k, representative images of three biologically independent experiments are shown. For b, d, e, g, n = 3 biologically independent experiments, means ± s.d. are shown, and p values were determined by two-tailed Student’s test. For h, i, k, scale bars represented 10 μm.

Fig. 2. DILA1 promotes cell proliferation and tamoxifen resistance.

a–d MCF-Re cells were transfected with NC or ASOs targeting DILA1 and then treated with 3 μM tamoxifen (Tam). a MTT assay showing relative cell growth at 0, 24, 48, and 72 h. Representative images of colony formation (b) or EdU incorporation by fluorescence microscopy (c). Flow cytometry showing the cell cycle distribution of cells (d). e–h MCF7-Pa cells were transfected with control vector or vector expressing DILA1 and then treated with 3 μM tamoxifen (Tam). MTT assay showing relative cell growth at 0, 24, 48, and 72 h (e). Representative images of colony formation (f) or EdU incorporation by fluorescence microscopy (g). Flow cytometry showing the cell cycle distribution of cells (h). For c, g, scale bars represented 100 μm. For all experiments, n = 3 biologically independent experiments. For a–h, means ± s.d. were shown, and p values were determined by two-tailed Student’s test.

Fig. 3. DILA1 inhibits Cyclin D1 degradation via the ubiquitin–proteasome pathway.

a, b Western blotting showing Cyclin D1 protein in MCF7-Pa (a) and MCF7-Re (b) cells treated with tamoxifen. c Western blotting showing Cyclin D1 protein in MCF7-Pa and MCF7-Re cells treated with CHX for the indicated time (left). The quantification of Cyclin D1 degradation rate by gray scale analysis (right). d Western blotting showing Cyclin D1 protein in MCF7-Pa and MCF7-Re cells treated with MG132 for the indicated time. e Western blotting showing Cyclin D1 protein in MCF7-Re cells transfected with NC or DILA-ASOs and then treated with MG132 for 24 h. f Western blotting showing Cyclin D1 protein in MCF7-Re cells transfected with NC or DILA-ASOs and then treated with CHX for the indicated time (left). The quantification of Cyclin D1 degradation rate by gray scale analysis (right). g Western blotting showing Cyclin D1 protein in MCF7-Pa cells transfected with control vector or vector expressing DILA1 and then treated with CHX (left). The quantification of Cyclin D1 degradation rate by gray scale analysis (right). h Ubiquitinated Cyclin D1 detected by immunoprecipitation with anti-Cyclin D1 antibody or IgG control and immunoblotting with anti-ubiquitin antibody in MCF7-Pa and MCF-Re cells. i Ubiquitinated Cyclin D1 detected by immunoprecipitation with anti-Cyclin D1 antibody or IgG control and immunoblotting with anti-ubiquitin antibody in MCF-Re cells transfected with NC or DILA-ASOs. For a–i, GAPDH was used as a loading control. Representative images of three biologically independent experiments are shown. For c, f, g, means ± s.d. are shown, and p values were determined by two-tailed Student’s test.

Fig. 4. Hairpin A of DILA1 interacts with Thr286 of Cyclin D1 and inhibits its phosphorylation and nuclear-to-cytoplasmic redistribution.

a Western blotting showing the levels of Cyclin D1, phosphorylated Cyclin D1 (p-D1) (Thr286), p-D1 (Ser-90), and GSK3β in MCF7-Pa and MCF-Re cells. b–d Western blotting showing the levels of Cyclin D1, p-D1(Thr286), and GSK3β in DILA1-silenced MCF-Re cells (b), in DILA1-overexpressed MCF7-Pa cells (c), and in DILA1 and constitutively activated GSK3β (GSK3β-CA) both overexpressing MCF7-Pa cells (d). GAPDH was used as a loading control. e–g Western blotting showing the levels of nuclear and cytoplasmic Cyclin D1 in MCF7-Pa and MCF-Re cells (e), in DILA1-silenced MCF-Re cells (f), and in DILA1-overexpressed MCF7-Pa cells (g). GAPDH and Lamin A/C was used as cytoplasmic and nuclear controls, respectively. h RNA pull-down showing the interaction between Cyclin D1 and full-length or serial truncation mutants of DILA1. i eCLIP-qPCR assay indicating the exact DILA1 region responsible for Cyclin D1 binding in MCF7-Re cells (bottom). A schematic diagram of DILA1 primers (P1–P11) designed for eCLIP-qPCR, covering the full length of DILA1 (top). j RNA pull-down showing the interaction between Cyclin D1 and DILA1 or DILA mutant with deletion of hairpin A (DILA1-mA). k RNA pull-down showing the interaction between DILA1 and HA-tagged full length or truncation mutants (FL (1–295), T1 (20–295), T2 (91–295), and T3 (1–256)) of Cyclin D1 proteins in MCF-7 cells. AS (antisense) was used as a negative control. l In vitro kinase assay showing the phosphorylation of Cyclin D1 at Thr286 by active GSK3β-CA or inactive GSK3β-KD in the absence or presence of DILA1 or DILA1-mA. m RIP-qPCR of DILA1 immunoprecipitated with anti-HA antibody or IgG in MCF7-Re cells with ectopically expressed HA-Cyclin D1 or HA-Cyclin D1(T286A). n eCLIP-qPCR assay in MCF7-Re cells with HA-Cyclin D1 or HA-Cyclin D1(T286A) overexpressed. For i, m, n, n = 3 biologically independent experiments, means ± s.d. are shown, and p values were determined by two-tailed Student’s test. o Western blotting showing the levels of Cyclin D1 and p-D1(Thr286) in MCF7-Re cells transfected with control vector or vector expressing Cyclin D1(T286A) and then transfected with NC or DILA1-ASOs. For a–h, j–l, o, representative images of three biologically independent experiments are shown.

Fig. 5. DILA1 promotes tamoxifen resistance in vivo.

MCF7-Re cells were inoculated into the mammary fat pads of NOD/SCID mice. When tumors became palpable, MCF7-Re tumors were injected with NC or DILA1-ASOs intratumorally. The tumor picture (a) and the tumor growth curves (b) are shown and compared among the groups. c Immunohistochemistry (IHC) staining of Ki67, Cyclin D1, p-D1(Thr286), and p-Rb(Ser780) and in situ hybridization (ISH) staining of DILA1 in the tumors. Representative images of six xenografts from each group are shown. d–h The quantification of IHC scores of Ki67(d), Cyclin D1(e), p-D1(Thr286) (f), and p-Rb(Ser780) (g) and ISH scores of DILA1 (h). For b, d–h, n = 6 mice per group, means ± s.d. are shown, and p values were determined by two-tailed Student’s test. Scale bars represents 50 µm.

Fig. 6. High DILA1 expression is associated with higher Cyclin D1 protein expression, tamoxifen resistance, and poor prognosis in ER-positive breast cancer patients.

a ISH staining for DILA1 and IHC staining for Cyclin D1, p-D1(Thr286), p-Rb(Ser780), and Ki67 in breast cancer specimen from ER-positive patients with or without relapse. Representative images of tumor specimens from ER-positive patients with (n = 36) or without relapse (n = 154) are shown. Scale bars, 50 μm. b–e The distribution of Cyclin D1, p-D1(Thr286), p-Rb(Ser780), and Ki67 determined by the staining index in the high (n = 109) or low (n = 81) DILA1-expressing groups. n = 190, p values were determined by two-tailed Chi-square test. f, g The distribution of DILA1 (f) and Cyclin D1 (g) by the staining index in the samples of ER-positive patients with (n = 36) or without relapse (n = 154). n = 190, means ± s.d. are shown, p values were determined by two-tailed Student’s test. h, i Relapse-free survival of ER-positive breast cancer patients with low and high DILA1 (h) or Cyclin D1 (i) was analyzed by Kaplan–Meier plots. n = 190, p values were determined by two-tailed log-rank test.