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

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.

S ustaining proliferative signaling is one of the hallmarks of cancer 1 . CCND1 gene, which encodes the Cyclin D1 protein to drive cell proliferation, is the second most frequently amplified oncogene in human cancer across 26 histological types 2 . The interaction between Cyclin D1 and cyclin-dependent kinase 4/6 (CDK4/6), which leads to the phosphorylation and inactivation of retinoblastoma protein (Rb), is an essential regulatory step for G1-S transition and cell cycle progression 3-7 . Up to 20% of breast cancers have amplification of CCND1 gene [8][9][10] . Moreover, many dysregulated signaling pathways can lead to overexpressed Cyclin D1 protein in nearly 50% of breast cancers, most being estrogen receptor (ER)-positive luminal subtype. Endocrine therapy including tamoxifen is highly effective in reducing the recurrence of ER-positive early breast cancer by~40%, but de novo or acquired resistance still occurs in onethird of such patients, leading to tumor relapse and metastasis 11 . Several clinical studies demonstrated that amplification of CCND1 gene 12 or overexpression of Cyclin D1 protein 13,14 was associated with poor prognosis in ER-positive breast cancer patients received tamoxifen. Furthermore, Cyclin D1 is still necessary for the proliferation of tamoxifen-resistant breast cancer cells because small interfering RNAs (siRNAs) targeting Cyclin D1 blocked their cell growth 15 . Alternative proliferative signaling including phosphoinositide-3 kinase (PI3K) and fibroblast growth factor receptor pathway may be responsible for upregulated Cyclin D1 after tamoxifen resistance 16 . Therefore, alternative strategy is needed to block Cyclin D1 activity in cancer. It has not been reported yet whether Cyclin D1, a tightly regulated protein with a short half-life, is dysregulated at the posttranscriptional level after tamoxifen resistance.
Long non-coding RNA (lncRNA) is a large class of transcripts from non-protein coding regions of the genome that have >200 nucleotides in length. Recent studies showed that lncRNAs regulate many important pathological processes in cancer, such as tumorigenesis, proliferation, metastasis, and drug resistance 17,18 . LncRNAs function mainly through interacting with key regulatory proteins to regulate their function or expression 19 . Nevertheless, it remains unclear whether lncRNAs directly interact with Cyclin D1 to regulate its expression or function. In this study, we investigated lncRNAs that interact with Cyclin D1 and examined their functional significance in tamoxifen resistance of breast cancer.

Results
Upregulated Cyclin D1 protein is responsible for tamoxifen resistance in ER-positive breast cancer cells. Tamoxifenresistant MCF-7 and T47D breast cancer cell lines were established as reported before (Fig. S1a, b and Zhu et al. 20 ). To examine the status of Cyclin D1 expression in these tamoxifenresistant models, quantitative PCR (qPCR) and western blotting was done to compare the mRNA and protein levels of Cyclin D1 between parental (MCF7-Pa and T47D-Pa) and resistant (MCF7-Re and T47D-Re) breast cancer cells. Western blotting showed that the protein levels of Cyclin D1 in resistant cells were significantly higher than in parental cells (Fig. 1a). However, there was no difference in Cyclin D1 mRNA levels between parental and resistant cells (Fig. S1c), indicating post-transcriptional or posttranslational changes may be responsible for the upregulated Cyclin D1 protein in tamoxifen-resistant breast cancer cells.
To determine the functional significance of upregulated Cyclin D1 protein in tamoxifen resistance, Cyclin D1 was knocked down by siRNAs in tamoxifen-resistant MCF-7 and T47D cells ( Fig. S1d-f). It was found that siRNAs targeting Cyclin D1 not only restored tamoxifen sensitivity in MCF7-Re and T47D-Re cells ( Fig. 1b and S1g), but also resulted in cell cycle arrest at G1 phase (Fig. S1h, i), indicating that these tamoxifen-resistant breast cancer cells are still dependent on Cyclin D1 for cell cycle progression and upregulated Cyclin D1 is responsible for their tamoxifen resistance.
Identification of Cyclin D1-interacting long noncoding RNA 1 (DILA1). Recently, we and other investigators have shown that lncRNAs can bind to key signaling proteins and directly regulate their signaling pathways 19,21,22 . To determine whether lncRNAs bind to Cyclin D1 and regulate its function, MCF-7 cells with exogenous HA-tagged or untagged Cyclin D1 were established and subjected to RNA immunoprecipitation (RIP) using anti-HA antibody. RIP-sequencing (RIP-seq) was then performed to identify the lncRNAs that specifically binds to HA-tagged Cyclin D1 but not to untagged Cyclin D1 control. Hierarchical clustering analysis indicated that 51 lncRNAs were significantly enriched in the RNAs pulled down from cells with HA-tagged Cyclin D1 than the cells with untagged Cyclin D1 (greater than twofold and p < 0.05) ( Fig. 1c and Table S1). To identify lncRNAs that contribute to tamoxifen resistance, quantitative PCR with reverse transcription (RT-qPCR) showed that 6 of the 51 lncRNAs were increased both in tamoxifen-resistant MCF-7 ( Fig. S2a) and T47D (Fig. S2b) cells than in parental cells. To confirm whether these lncRNAs increased in tamoxifen-resistant cells indeed bind to Cyclin D1, RIP was performed using anti-HA antibody or IgG control in MCF7-Pa cells with ectopically expressed HAtagged Cyclin D1 and then subjected to RT-qPCR. LncD1-1, LncD1-5, and LncD1-8 were verified to specifically bind to Cyclin D1 (Fig. S2c). These three lncRNAs were then knocked down in MCF7-Re cells by respective siRNAs (Fig. S2d-f) and the sensitivity to tamoxifen was measured. It was found that only knockdown of lncD1-8 reversed tamoxifen resistance in MCF7-Re cells (Fig. S2g-i). Thus this Cyclin D1-interacting lncD1-8 that is upregulated and responsible for tamoxifen resistance is named as Cyclin D1-Interacting Long noncoding RNA 1 (DILA1).
Furthermore, RT-qPCR confirmed that DILA1 was expressed in MCF7-Pa and T47D-Pa cells and significantly upregulated in tamoxifen-resistant MCF7-Re and T47D-Re cells (Figs. 1d and S3a). Northern blotting also detected higher expression of DILA1 in MCF7-Re cells than in MCF7-Pa cells (Fig. S3b). To determine the abundance of DILA1 in breast epithelial cells, we examined the copy number of DILA1 in several breast cell lines using RT-qPCR and in vitro transcribed DILA-1 as standard. The results demonstrated that the copy numbers of DILA1 are higher in ER-positive breast cancer cells than in ER-negative breast cancer cells and immortalized breast epithelial cells (Fig. S3c, d). Moreover, the copy number of DILA1 was significantly increased in tamoxifen-resistant MCF-7 and T47D cells (385 ± 22 and 254 ± 22 copies per cell, respectively) than in the parental ones (130 ± 28 and 75 ± 5 copies per cell, respectively; Fig. S3d). Importantly, RIP-qPCR demonstrated that immunoprecipitation with anti-Cyclin D1 antibody specifically retrieved DILA1 in MCF7-Re cells, indicating that DILA1 binds to Cyclin D1 protein in an endogenous setting (Fig. 1f).
In UCSC (University of California Santa Cruz) Genome Browser, DILA1 is labeled as a long intergenic noncoding RNA named as ENST00000435697.1, which is an intergenic transcript of MIR99AHG. To determine the exact sequence of DILA1, we performed 5′ and 3′ RACE (rapid amplification of complementary DNA ends) based on the 716 nucleotides (nt) sequence in UCSC Genome Browser. The RACE results indicate that the full length of DILA1 is 1183 nt (Fig. 1f and Table S2). Furthermore, the lack of protein-coding potential of DILA1 was confirmed by ORF (open reading frame) Finder, validating DILA1 as an lncRNA. To determine the subcellular localization of DILA1, nuclear and cytoplasmic fractionation of MCF7-Re cells was performed and showed that the majority of DILA1 localized in the nucleus, similar to MALAT1 (Fig. 1g). Confocal microscopy of fluorescence in situ hybridization (FISH) (Fig. 1h) and RNAScope assay (Figs. 1i and S3e) also demonstrated that DILA1 located primarily in the nucleus and was upregulated in tamoxifen-resistant cells, suggesting that DILA1 may exert its biological function mainly in the nucleus.
To explore whether DILA1 directly interacts with Cyclin D1, we performed RNA pull-down assays with biotin-labeled DILA1 in MCF7-Re cells lysates and found that only DILA1, but not its antisense, pulled down Cyclin D1 protein, which was further confirmed with in vitro RNA pull-down of purified recombinant Cyclin D1 in a cell-free system (Fig. 1j), indicating the direct interaction between DILA1 and Cyclin D1 protein. Confocal microscopy also showed the co-localization of DILA1 with Cyclin D1 in the nucleus of MCF7-Re cells (Fig. 1k).

DILA1 promotes cell proliferation and tamoxifen resistance.
Since DILA1 mainly localizes in the nucleus and siRNAs were reported to be less efficient than antisense oligonucleotides (ASOs) for modulating nuclear-localized lncRNAs 23 , DILA1 was knocked down by ASOs in tamoxifen-resistant MCF7 and T47D cells to examine its function. RT-qPCR showed that both ASOs efficiently reduced DILA1 expression in MCF7-Re and T47D-Re cells (Fig. S4a, b). Knocking down of DILA1 by ASOs not only slowed down the cell growth but also restored their sensitivity to tamoxifen in MCF7-Re and T47D-Re cells, demonstrated by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay (Figs. 2a and S4c), colony-formation assay (Figs. 2b and S4d) and 5ethynyl-2′-deoxyuridine (EdU) incorporation by fluorescence microscopy (Fig. 2c). Moreover, cell cycle analysis by flow cytometry showed that DILA1-ASOs caused G1/S cell cycle arrest in MCF7-Re and T47D-Re cells, which was further increased by tamoxifen (Figs. 2d and S4e). These results suggest that DILA1 is necessary for cell proliferation and tamoxifen resistance.
To determine whether DILA1 is sufficient to drive cell proliferation and cause tamoxifen resistance, DILA1 was ectopically expressed in parental MCF7 and T47D cells by transfecting with PCDH-puro expression vector carrying the DILA1 sequence (Fig. S4f, g). Consistent with the results of DILA1-ASOs, overexpression of DILA1 in MCF7-Pa and T47D-Pa cells promoted cell proliferation and tamoxifen resistance (Figs. 2e-g and S4h, i). DILA1 accelerated cell cycle progression by decreasing the percentage of G1 cells, which was not affected by tamoxifen (Figs. 2h and S4j). Together, these results indicate that DILA1 is not only necessary but also sufficient to promote cell proliferation and cause tamoxifen resistance.
Decreased degradation is responsible for upregulated Cyclin D1 protein in tamoxifen-resistant cells. To study at which level Cyclin D1 protein was dysregulated in tamoxifen-resistant cells, western blotting was done to measure the protein expression of Cyclin D1 in parental and tamoxifen-resistant MCF-7 and T47D cells before and after tamoxifen treatment at different time points. It was found that Cyclin D1 protein remained steady without tamoxifen treatment and significantly decreased after tamoxifen treatment in MCF7-Pa and T47D-Pa cells (Figs. 3a and S5a), whereas Cyclin D1 protein in MCF7-Re and T47D-Re cells accumulated over time, regardless of tamoxifen treatment (Figs. 3b and S5b). However, there was no significant change at the levels of Cyclin D1 mRNA in resistant cells over time (Fig. S5c, d). The upregulated Cyclin D1 protein with similar mRNA level in tamoxifen-resistant cells compared to parental cells indicate that either translational or posttranslational dysregulation may be responsible for the increased Cyclin D1 protein in tamoxifen-resistant cells.
To further elucidate the mechanism of Cyclin D1 upregulation, the levels of Cyclin D1 protein in parental and resistant breast cancer cells were examined when cycloheximide (CHX) or MG132 was used to inhibit de novo protein synthesis or proteasome degradation respectively. When protein synthesis was inhibited by CHX, Cyclin D1 protein decreased rapidly and its half-life was~1 h in MCF7-Pa and T47D-Pa cells, but Cyclin D1 protein remained high and its half-life significantly increased to >2 h in MCF7-Re and T47D-Re cells. (Figs. 3c and S5e), indicating that the protein stability of Cyclin D1 in tamoxifen-resistant cells is increased. However, when proteasome degradation was inhibited by MG132 for~2 h to see whether there is a difference of protein synthesis, Cyclin D1 levels remained steady in MCF7-Re and MCF7-Pa cells (Fig. 3d), indicating that the protein synthesis of Cyclin D1 was not different between MCF7-Re and MCF7-Pa cells. Together with similar Cyclin D1 mRNA levels in MCF7-Re and MCF7-Pa cells, these results suggest that protein degradation but not protein synthesis was responsible for the upregulated Cyclin D1 protein in tamoxifen-resistant breast cancer cells.

DILA1
inhibits Cyclin D1 degradation via ubiquitin-proteasome pathway. To study the mechanism that Cyclin D1-interacting DILA1 leads to tamoxifen resistance, the expression of DILA1 was knocked down with ASOs in MCF7-Re cells in the absence or presence of CHX or MG132. It was found that Cyclin D1 protein was significantly downregulated by DILA1 ASOs but remained high in the presence of MG132 (Fig. 3e). Nevertheless, DILA1-ASOs markedly decreased Cyclin D1 protein expression in the presence of CHX treatment, with the halflife of Cyclin D1 decreased from >2 h to <1 h in MCF7-Re cells (Fig. 3f), suggesting that DILA1-ASOs accelerated the degradation of Cyclin D1 protein in MCF7-Re cells that overexpressed DILA1. Consistent with the results of DILA1-ASOs in MCF7-Re cells, overexpression of DILA1 in MCF7-Pa cells significantly increased the Cyclin D1 protein expression and extended the half-life of Cyclin D1 protein from <1 h to~2 h under CHX treatment (Fig. 3g). These results indicate that DILA1 increases the protein expression of Cyclin D1 by inhibiting its degradation.
Cyclin D1 and CDK4/6 play a crucial role in G1/S cell cycle progression by phosphorylating and inactivating the Rb, a tumor suppressor that restrains G1/S cell cycle transition 32 . We found a higher level of Ser780-phosphorylated Rb (p-Rb(Ser780)) in tamoxifen-resistant MCF-7 (Fig. S6b) and T47D (Fig. S6c) cells than in the parental ones, while total Rb protein levels were similar. Moreover, DILA1-ASOs in MCF7-Re cells or overexpressed DILA1 in MCF7-Pa cells decreased or increased the p-Rb(Ser780) levels, respectively, without changing the total Rb levels (Fig. S6d, e). Together, these results indicate that DILA1 enhances the classical Ser780 phosphorylation of Rb and accelerates G1/S cell cycle progression.
To identify the exact sequence of DILA1 that binds Cyclin D1, a series of DILA1 deletion mutants were constructed (Fig. S6f) and examined for their binding to Cyclin D1 by RNA pull-down assay with MCF7-Re cell lysates or purified recombinant Cyclin D1.
It was found that the DILA1 mutants missing the 1000-1183 nt lost the binding to Cyclin D1 and the 1000-1183 nt of DILA1 showed the affinity to bind Cyclin D1 comparable to full-length DILA1, suggesting that the sequence of 1000-1183 nt in DILA1 is essential and sufficient for the interaction between DILA1 and Cyclin D1 (Fig. 4h). Moreover, enhanced crosslinking IP and qPCR (eCLIP-qPCR) was performed to identify the lncRNA fragment that bound and was protected by Cyclin D1 from RNAase digestion. Among the 11 pairs of primers, only the primer pair 11 designed for the 1000-1183 nt region of DILA1 successfully amplified DILA1 segment in eCLIP-qPCR, confirming that the 1000-1183 nt of DILA1 was the main region responsible for binding with Cyclin D1 (Fig. 4i). To determine the functional role of the 1000-1183 nt of DILA1, the last part of DILA1 (1000-1183 nt, DILA1-S6) was ectopically expressed in MCF7-Pa cells. It was found that DILA1-S6 inhibited the phosphorylation of Cyclin D1 at Thr286, decreased the nuclearto-cytoplasm translocation and ubiquitination of Cyclin D1, and caused tamoxifen resistance (Fig. S7).
The RNAfold software predicts that a stable hairpin (1000-1046 nt, hairpin A) structure exists within 1000-1183 nt of DILA1 (Fig. S6g). Indeed, RNA pull-down assay with MCF7-Re cell lysates or purified recombinant Cyclin D1 showed that mutation of hairpin A in DILA1 (DILA1-mA) completely abolished the binding between DILA1 and Cyclin D1 (Fig. 4j), suggesting that hairpin A was the site of DILA1 that directly interacts with Cyclin D1.
To determine whether DILA1 can directly inhibit the phosphorylation of Cyclin D1 by GSK3β, purified inactive (GSK3β-KD) and constitutively active form (GSK3β-CA) of GSK3β 30 was added to purified recombinant Cyclin D1 protein in the absence or presence of DILA1 or DILA1-mA. In vitro phosphorylation assay showed that GSK3β-CA, but not GSK3β-KD, efficiently phosphorylated Cyclin D1 at Thr286. Furthermore, DILA1, but not DILA1-mA, significantly inhibited the phosphorylation of Cyclin D1 by GSK3β (Fig. 4l). More interestingly, RIP assay showed that immunoprecipitation of HA-tagged Cyclin D1, but not HA-tagged Cyclin D1 mutant at threonine 286 (T286A), enriched DILA1 (Fig. 4m), suggesting NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19349-w ARTICLE that the threonine at 286 is the site of Cyclin D1 that directly interacts with DILA1. eCLIP-qPCR assays were performed using an anti-HA antibody in MCF7-Re cells transfected with vectors expressing HA-Cyclin D1 or HA-Cyclin D1(T286A). As expected, only the primer pair 11 amplified DILA1 segment from the precipitates in cells with HA-tagged Cyclin D1 but not in cells with HA-tagged Cyclin D1 mutant at threonine 286 (T286A) (Fig. 4n).
Furthermore, it was found that DILA1-ASOs had no effect on the expression of Cyclin D1 T286A that was ectopically expressed in MCF7-Re cells (Fig. 4o). In total, these results indicate that the hairpin A of DILA1 directly interacts with the Thr286 at Cyclin

DILA1 promotes tamoxifen resistance in vivo.
To investigate the role of DILA1 in regulating tamoxifen resistance of ERpositive breast cancer in vivo, MCF7-Re cells were inoculated into the mammary fat pads of NOD/SCID mice. Mice were treated with tamoxifen or control when tumors became palpable. DILA1-ASOs or control was then injected into tumors every 2 days. Consistent with the results in vitro, DILA1-ASOs significantly decreased tumor volumes than control oligos, and tamoxifen treatment further shrunk the tumors, suggesting that DILA1-ASOs inhibits the tumor growth and restores the sensitivity to tamoxifen in tamoxifen-resistant tumors (Fig. 5a, b). Immunohistochemistry (IHC) staining showed that the expression of Ki67, Cyclin D1, and p-Rb(Ser780) were markedly lower in tumors with DILA1-ASO treatment and were further decreased by tamoxifen treatment, while the expression of Thr286phosphorylated Cyclin D1 (p-D1(Thr286)) was increased with DILA1-ASO treatment, and the efficiency of DILA1 knockdown was confirmed by ISH ( Fig. 5c-h). These results indicate that antagonizing DILA1 significantly decreases cancer cell proliferation, inhibits Cyclin D1 protein expression and its downstream Rb protein phosphorylation, and reverses tamoxifen resistance in vivo.
MCF7-Pa cells with stably overexpressed DILA1 or vector control by lentiviral infection were inoculated into the mammary fat pads of NOD/SCID mice. Tumors with overexpressed DILA1 were significantly larger than vector control and resistant to tamoxifen treatment (Fig. S8a, b). IHC staining and ISH staining showed that overexpression of DILA1 led to a markedly higher expression of Ki67, Cyclin D1, and p-Rb(Ser780) but lower p-D1 (Thr286) levels in the tumor that were not affected by tamoxifen (Fig. S8c-h).
High DILA1 expression is associated with higher Cyclin D1 protein expression, tamoxifen resistance, and poor prognosis in ER-positive breast cancer patients. To determine whether the findings above are clinically relevant, the expression of DILA1 and Cyclin D1, p-D1(Thr286), p-Rb(Ser780), and Ki67 protein was evaluated in the primary tumor samples from 190 ERpositive breast cancer patients who received adjuvant tamoxifen treatment. The cut-off value of staining intensity (staining index (SI)) score to determine high or low DILA1 expression was calculated by receiver operating characteristic curve and it indicated that SI = 3 was the optimal score to separate the DILA1-high and DILA1-low groups (Fig. S9a). The DILA1-high group exhibited higher Cyclin D1, p-Rb(Ser780), and Ki67 expression but lower p-D1(Thr286) expression, whereas the DILA1-low group showed lower expression of Cyclin D1, p-Rb(Ser780), and Ki67 but higher expression of p-D1(Thr286) (Fig. 6a-e and Table S4). Collectively, these results indicate that DILA1 regulates the expression of Cyclin D1 and subsequent Rb phosphorylation in vivo.
Then the correlation between the expression of DILA1 or Cyclin D1 and the clinicopathological parameters was analyzed. Higher DILA1 expression was significantly associated with clinical stage, lymph node metastasis, and Ki67 staining (p < 0.05) but not age, tumor size, or Her-2 status (Table S5). Higher Cyclin D1 expression was significantly associated with the level of Ki67 expression but not with other factors analyzed in this patient cohort (Table S6). ISH and IHC staining showed that the expression of DILA1, Cyclin D1, and p-Rb(Ser780) protein was significantly higher in the samples from relapsed patients than from non-relapsed patients (Figs. 6f, g and S9b). Importantly, Spearman rank correlation analysis showed a positive correlation between DILA1 and Cyclin D1 protein in the patients' samples (r = 0.57, p < 0.0001; Fig. S9c).
Kaplan-Meier survival curve analysis (K-M analysis) indicated that higher DILA1, Cyclin D1, and p-Rb(Ser780) protein expression in these ER-positive breast cancer patients was associated with shorter relapse-free survival (Figs. 6h, i and S9d). Then we performed K-M analysis to examine the association between the overall survival and the expression of DILA1, Cyclin D1, and p-Rb. The results showed that Cyclin D1, DILA1, or p-Rb, were not significantly associated with the overall survival, although CyclinD1 (p = 0.064) and DILA1 (p = 0.073) showed a statistically insignificant trend (Fig. S9e-g). This may be caused by the relatively small patient number in this cohort, different treatments after relapse, etc. Moreover, univariate and multivariate Cox proportional hazards regression analysis showed that only DILA1 expression was an independent prognosis predictor in ER-positive breast cancer (Table S7). Collectively, increased expression of DILA1, Cyclin D1, and p-Rb(Ser780) protein are associated with tamoxifen resistance and poor prognosis of ER-positive breast cancer patients who received adjuvant tamoxifen treatment, consistent with the findings in tamoxifen-resistant breast cancer cell lines.

Discussion
In this study, we identified an lncRNA named DILA1 that specifically binds to the Thr286 of Cyclin D1 protein and inhibited NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19349-w ARTICLE its phosphorylation, leading to decreased ubiquitination and degradation of Cyclin D1 (Fig. S10). The subsequent upregulated Cyclin D1 protein accelerated cell proliferation and resulted in tamoxifen resistance in breast cancer cells. Knocking down DILA1 decreased the expression of Cyclin D1 protein and reversed tamoxifen resistance of breast cancer cells both in vitro and in vivo. More importantly, high expression of DILA1 was associated with overexpressed Cyclin D1 protein and poor prognosis in ER+ breast cancer patients who received tamoxifen.
Excessive cell proliferation is one of the hallmarks of cancer. Cyclin D1 protein, one of the most important oncoproteins in human cancer, accelerates cell proliferation by engaging CDK4/6 to phosphorylate Rb and drive G1-S transition. It has been well established that overexpressed Cyclin D1 protein is caused by the   protein with the half-life of only~24 min 31 and the dysregulated degradation also frequently leads to its overexpression 36 . Although a number of agents were shown to induce Cyclin D1 degradation in vitro 37 , the mechanism was not clear and specific. Thus no effective way can be used to target Cyclin D1 degradation clinically yet. Our study showed that DILA1 specifically interacted with Cyclin D1 and regulated its degradation, indicating that DILA1 could be a specific target to control the posttranslational regulation of Cyclin D1 protein.
The majority of breast cancer is ER positive and suitable for endocrine therapy, including tamoxifen. Tamoxifen can inhibit the ER signaling and the ensuing transcription of CCND1 gene, decreasing the relapse rate by 40% in ER-positive breast cancer patients. Nevertheless, the overexpression of Cyclin D1 was found to be associated with poor prognosis and tamoxifen resistance in these patients. Indeed, two tamoxifen-resistant breast cancer cell line models used in this study showed that Cyclin D1 protein was further increased in resistant cells. Interestingly, this upregulation in resistant cells was not caused by enhanced transcription by alternative upstream pathways but by suppressed degradation of Cyclin D1, indicating previously unappreciated importance of Cyclin D1 degradation in tamoxifen resistance.
We 19 and others have shown that lncRNAs can directly interact with key signaling proteins to regulate their function. It was reported that lncRNA ABHD11-AS1 38 interacted with Cyclin D1 and promoted the growth of endometrial cancer cells, but the mechanism was unclear. LncRNA was also shown to regulate the transcription of CCND1 39 . Our results indicate that DILA1 is a lncRNA that directly interacts with Cyclin D1 and regulates its degradation. More importantly, DILA1 was upregulated in tamoxifen-resistant cells and the breast cancer patients who relapsed after tamoxifen treatment, suggesting that DILA1 is a good biomarker to predict poor prognosis and tamoxifen resistance in ER-positive breast cancer patients. Knocking down DILA1 increased Cyclin D1 degradation and reversed tamoxifen resistance both in vitro and in vivo, indicating that DILA1 is a therapeutic target in regulating Cyclin D1 degradation and improve the efficacy of tamoxifen. It was suggested that combination therapies that simultaneously target CCND1 transcription of Cyclin D1 protein turnover may be helpful in treating cancer 40 .
We have not explored the mechanism how DILA1 is regulated and increased in tamoxifen-resistant cells and this warrants further investigation. The strategy to efficiently target DILA1 in breast cancer remains to be optimized. Whether DILA1 plays such an important role in other types of cancer will be explored in future studies. Moreover, this study examined the role of DILA1 only in tamoxifen-resistant breast cancer cells and patients. Whether DILA1 plays a similar role in breast cancer that received the treatment of aromatase inhibitors or CDK4/6 inhibitors remains unknown and needs further study.
In summary, this study demonstrates the novel role of lncRNA DILA1 in regulating Cyclin D1 protein at the posttranslational level and leading to tamoxifen resistance of breast cancer. Different from targeting CCND1 transcription or CDK4/6, antagonizing DILA1 may be an alternative or complementary way to downregulate Cyclin D1 protein in cancer treatment.

Methods
Cell culture and transfection. MCF-7 and T47D breast cancer cell lines and 293T cell lines were obtained from the ATCC (American Type Culture Collection). Both of them were authenticated using short tandem repeat multi-amplification and tested to be mycoplasma negative. MCF-7 cells and 293T cells were cultured in DMEM (Gibco, USA) medium, supplemented with 10% fetal bovine serum (FBS; Hyclone, USA). T47D cells were cultured in RPMI-1640 medium (Gibco, USA), supplemented with 10% FBS (Hyclone, USA).
For transfection of siRNA or ASOs, cells were plated at 2 × 10 4 per well in 24well plate and transfected with specific siRNAs (100 nM; GenePhrama, China) or ASOs (50 nM; RiboBio, China) mixed with RNAiMax (13778150, Invitrogen) according to the manufacturer's instructions. The sequences of siRNAs and ASOs are listed in Table S3.
For transduction of MCF-7 and T47D cells, cells were plated at 0.5 × 10 5 cells per well in 24-well plates and transduced with lentiviral particles with 5 µg/ml Polybrene. After 2 days, puromycin was added into the medium at a concentration of 3 µg/ml to select stably transduced cells.
RIP and RIP-seq. RIP was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (17-700, Millipore) according to the manufacturer's instructions. Briefly, the cultured cells were lysed by RIP lysis buffer containing protease inhibitor cocktail and RNase inhibitor. Antibodies were added to the lysate and incubated with rotation at room temperature for 60 min. The magnetic beads were added to the mixture and incubated with rotation at 4°C for 2 h. The beads were washed three times and the RNAs pulled down were extracted by TRIzol™ LS (10296028, Invitrogen) and subjected to RIP-seq or RIP-qPCR analysis.
For RIP-seq, anti-HA antibody (H9658, Sigma, dilution 1:100) was used to perform RIP reaction in MCF-7 cells with exogenous 5′-HA-tagged (MCF7-HA-D1) or untagged Cyclin D1 (MCF7-D1). The ribosomal RNAs were removed by a Low Input RiboMinus™ Eukaryote System (A15027, Invitrogen). Then the harvested RNAs were reverse transcribed into cDNA sequencing library using the KAPA Stranded RNA-Seq Library Preparation Kit (KK8400, Illumina). The quality of cDNAs was analyzed by the Agilent Bioanalyzer 2100 and then sequenced using the Illumina NextSeq 500 platform. The reads were mapped to human genome using TopHat2 and visualized on the ensemble (http://asia.ensembl.org/index. html) and UCSC browser (http://genome.ucsc.edu). The lncRNAs interacted with HA-tagged Cyclin D1 were selected according to the screening criteria: fold change of fragments per kilobase of transcript per million mapped reads was >2 and the p value was <0.05. Untagged Cyclin D1 was used as a negative control to rule out any RNAs non-specifically bound to anti-HA antibody.
Quantitative PCR with reverse transcription. TRIzol® Reagent (Life Technologies, USA) was used to extract total RNA from breast cancer cells. RNA was reverse-transcribed into cDNA using PrimeScript RT Master Mix (RR036A, Takara). Real-time quantitative PCR was performed using TB Green Premix Ex Taq II (RR820A, Takara) according to the manufacturer's recommendations. The reactions were carried out in the LightCycler480 system with gene-specific primers. The primers were designed on the website of Primer Bank (https://pga.mgh. harvard.edu/primerbank/) and PrimerBlast (https://www.ncbi.nlm.nih.gov/tools/ primerblast/index.cgi?LINK_LOC=BlastHome). All the primer sequences used in this research are listed in Table S3.
Cell number counting, MTT, colony formation, and EdU assays. For cell number counting, the cultured cells were digested by 0.4% trypsin and counted using a cell counter (IC 1000, Countstar). For MTT analysis, MTT powder (3580MG250, Biofrox) was dissolved in sterile phosphate-buffered saline (PBS) at a concentration of 5 mg/ml, added into the cells cultured in 96-well plates with a 1:10 ratio. After incubation at 37°C for 4 h, the absorbance was measured at a wavelength of 490 nm by a microplate spectrophotometer. For colony-formation assay, cells (1000/well) were planted in 6-well plates and cultured in medium with tamoxifen (3 μM) for 2 weeks, then were fixed by 4% paraformaldehyde and stained with crystal violet for colony number count. EdU proliferation was performed according to the protocol of Cell-Light EdU Apollo567 In Vitro Kit (C10310-1, RIOBIO). In brief, cells were planted in 96-well plates (3000/well) and treated with tamoxifen (3 μM) for 48 h. After the addition of EdU, the cells were cultured for 4 h, fixed by 4% paraformaldehyde, and stained by Apollo®567 and 4,6-diamidino-2-phenylindole (DAPI). Images were taken by fluorescence microscopy.
Flow cytometry for cell cycle. Flow cytometry was performed on a flow cytometer (Becton Dickinson) to analyze cell cycle distribution. The cells (~10 6 ) were fixed by 75% cold ethanol for >24 h and stained with 200 μl propidium iodide containing 5 μl RNAase and analyzed by flow cytometer. The gating strategy was used to exclude cell debris and aggregates (Fig. S10).
Subcellular RNA fractionation. Nuclear and cytoplasmic RNA was purified according to the manufacturer's recommendations of the Nuclear and Cytoplasmic RNA Purification Kit (Invitrogen, AM1921), and then the expression of DILA1 in different subcellular fractionations was analyzed by RT-qPCR.
Determination of DILA1 copy numbers. Increasing numbers of in vitro transcribed DILA1 were used as standard samples for RT-qPCR, and the standard curve was generated according to DILA1 copy numbers and corresponding threshold cycle (CT) value. DILA1 in 5 × 10 5 cells from multiple cell lines was subjected for RT-qPCR and the DILA1 copy number per cell was determined according to their CT values compared to the standard curve and then divided by the cell number.
Ubiquitination assay. Cultured cells were treated with 20 μM MG132(C2211, Sigma) for 6 h and then lysed by IP lysis buffer containing protease and phosphatase inhibitors on ice for 30 min. Anti-Cyclin D1 antibody (RB-010-P, Invitrogen, dilution 1:100) or IgG was added into the lysate and incubated with rotation overnight at 4°C. Dynabeads Protein G (10003D, Invitrogen) was added into the mixture and incubated at 4°C for 2 h, boiled in SDS loading buffer, and used for western blotting analysis. Antibody against ubiquitin (3936, CST, dilution 1:1000) was used to detect the ubiquitination of Cyclin D1.
5′ and 3′ rapid amplification of cDNA ends (RACE). 5′ and 3′ RACE was conducted using the SMARTer RACE 5′/3′ Kit (634859, Takara) according to the manufacturer's instructions. Briefly, total RNA was extracted from MCF7-Re cells, then the 5′-and 3′-RACE-Ready first-strand cDNA was synthesized with 5′-and 3′-CDS primer. The cDNAs were subjected to PCR reaction using universal primer mix with 5′ or 3′ gene-specific primers. Gene-specific primers are listed in Table S3. The amplified cDNA was purified and cloned into the p-EASY-T5-zero cloning vector (CT501-02, TransGen Biotech). The vectors were further transfected into Trans-T1 Phage Resistant chemically competent cells (CT501-03, TransGen Biotech) and amplified, then the plasmids were extracted and sequenced.
RNA FISH and in situ hybridization. Digoxin (Dig)-conjugated LNA oligonucleotide probes (5′DIG-TACAGCAATGTCAAGGCACGAT-3′DIG) were custommade and synthesized by Exiqon (267053814, QIAGEN). Briefly, cells plated in confocal dishes (2 × 10 4 /dish) were fixed by 4% formaldehyde in PBS for 20 min at room temperature, digested with 0.4% trypsin for 5 min at room temperature, and permeabilized in PBS containing 0.05% Triton X-100 for 3 min on ice. For the paraffin-embedded sections, dewaxed and rehydrated tissues were digested in 10% trypsin for 40 min at room temperature. Hybridization was carried out at 54°C overnight in hybridization solution with a probe concentration at 25 nM.
RNAScope. RNAScope assay was performed to detect the single-molecule RNA using the RNAScope® Assay Kit (Advanced Cell Diagnostics, CA, USA). Fourteen paired double-Z oligonucleotide probes targeting 2-1152 nt of DILA1 were designed using the custom software (Hs-MIR99AHG-O1, NPR-0007680). The experiment was performed according to the manufacturer's instructions. In brief, cultured cells were fixed by 10% neutral formalin at room temperature for 30 min, incubated in a hydrogen peroxide solution for 15 min, and digested in protease III solution for 20 min. The cells were then hybridized with target probes at 40°C for 2 h in a hybridization oven. After signal amplification, the cells were conjugated with TSA Plus Cy3 fluorescence at 40°C for 30 min and blocked by the HRP blocker. Then the cells were counterstained with DAPI and observed by confocal microscopy.
Enhanced crosslinking IP and qPCR. eCLIP-qPCR was performed as reported before [41][42][43] . Briefly, cells were cultured in medium with 4-thiouridine (100 μM; T4509, Sigma) for 16 h. Then cells were washed twice by cold PBS and crosslinked with ultraviolet (365 nm,150 mJ/cm 2 ) and then lysed with NP-40 lysis buffer (FNN0021, Invitrogen) containing protease inhibitors and 1 mM dithiothreitol (P2325, Invitrogen). RNase T1 (AM2283, Invitrogen) was added to the supernatant at a final concentration of 1 U/μl and incubated at 22°C for 15 min. Then Cyclin D1 antibody (RB-010-P, Invitrogen, dilution 1:100) or HA-tag antibody (H9658, Sigma, dilution 1:100) was added and incubated at 4°C with rotation overnight. Forty microliters of dynabeads Protein G (10003D, Invitrogen) was added and incubated at 4°C for 3 h. The pellets were incubated in NP-40 lysis buffer with DNase I (18047019, Invitrogen) at a concentration of 1 U/5 μl for 15 min at 37°C. The immunoprecipitated protein-RNA complex was eluted from the beads by heat denaturing. After SDS-PAGE and nitrocellulose membrane transfer, the 35-110 KD region (a region of 75 kDa (~220 nt of RNA) above the Cyclin D1 protein size) is excised and treated with proteinase K (25530049, Invitrogen) to isolate RNA for next qPCR analysis. Primers were designed every 200 nt with 100-nt overlapping intervals to cover the full length of DILA1, listed in Table S3.
Northern blotting. Northern blotting was performed using the NorthernMax™ Kit (AM1940, Invitrogen) according to the manufacturer's instructions. Briefly, total RNA was extracted using GeneJET RNA purification kits (K0731, Thermo Scientific), separated using agarose gel electrophoresis and then transferred to a positively charged nylon membrane, and crosslinked using an ultraviolet light. The Dig-labeled LNA probes complementary to DILA1 were hybridized to the membrane at 54°C overnight, then incubated with anti-Dig AP-conjugate antibody (11093274910, Roche, dilution 1: 5000) for 30 min, added with CSPD substrate. The signal was detected by enhanced chemiluminescence assay.
In vitro phosphorylation assay. Glutathione S-transferase (GST) fusion protein GST-cyclin D1 was purchased from Abcam (ab85247). Biotin-labeled RNA was prepared as described in the RNA pull-down procedure. 293T cells were transfected with plasmids carrying activated V5-GSK3-β or kinase-deficient V5-GSK3-β, respectively. After 48 h of transfection, cells were washed with cold PBS and lysed by IP lysis buffer, then immunoprecipitated using anti-V5 tag antibody (R960-25, Invitrogen, dilution 1:100). V5-GSK3-β immune complexes were added into kinase buffer (9802S, CST) containing 100 mM ATP (9804S, CST) and 1 μg of recombinant GST-Cyclin D1. DILA1 or the hairpin A deletion mutant was added into the reaction mixture as indicated. The reaction mixture was incubated at 30°C for 1 h, then boiled in SDS loading buffer at 98°C for 10 min. Thr286-phosphorylated Cyclin D1 was detected by western blotting.
RNA pull-down. RNA pull-down was performed using the MAGNETIC RNA PULL-DOWN Kit (20164, Pierce) according to the manufacturer's instructions. Briefly, RNAs for in vitro experiments were transcribed using the Transcript Aid T7 High Yield RNA Synthesis Kit (0441, Thermo Scientific) according to the manufacturer's instructions and then biotinylated using the components from the PULL-DOWN Kit. For proper secondary structure formation, 1 µg of biotinylated RNA in RNA structure buffer was heated to 95°C for 2 min, put on ice for 3 min, and then left at room temperature for 30 min. Folded biotin-labeled RNA was then added into streptavidin magnetic beads rotated at room temperature for 60 min. The beads were then added to the cell lysates or recombinant GST-Cyclin D1 protein and then incubated with rotation for 2 h at 4°C. Then the beads were washed five times and boiled in SDS loading buffer for 10 min at 98°C. Finally, the retrieved proteins were analyzed by western blotting.
Animal experiment. The animal experiments were approved by Sun Yat-Sen University laboratory animal care and use committee. Four-week-old female NOD/ SCID mice were purchased from Vital River Laboratory. Mice were housed under a specific pathogen-free condition of 12 h light/12 h dark cycle in a temperature-and humidity-controlled cage and were fed ad libitum. 17β-Estrogen pellets (0.72 mg, 60-day release, innovative research of America) were implanted subcutaneously 1 week before inoculating cells. In all, 1 × 10 7 MCF7-Re, MCF7-Vector, or MCF7-DILA1 cells were suspended in 0.1 ml sterile PBS and orthotopically injected directly into the mammary fat pads of mice. After tumors were palpable, MCF7-Re xenograft mice were randomly divided into six groups: (1) negative control group, (2) ASO-1 group, (3) ASO-2 group, (4) tamoxifen group, (5) tamoxifen+ASO-1 group, and (6) tamoxifen+ASO-2 group. MCF7-vector xenograft mice or MCF7-DILA1 xenograft mice were each divided into two groups, with or without tamoxifen, respectively. In the tamoxifen treatment group, 1 week after inoculation, tamoxifen time-released pellet (5 g, 60-day release, innovative research of America) was implanted subcutaneously per mouse. From the next day, 5 nM ASOs per tumor were injected intratumorally every 2 days in ASO treatment group. Four weeks later, all mice were euthanized, and tumor volumes were measured. All tumors were collected for ISH and IHC staining. Statistical analysis. Statistical analyses were performed using GraphPad Prism 8.0. and IBM SPSS Statistics 25. All in vitro and animal experiment results were expressed as means ± s.d. and two-tailed Student's test were used to calculate the p value. Survival curves were constructed using the K-M analysis and compared using two-tailed log-rank test. Two-tailed Spearman rank correlation analysis was done to analyze the association between DILA1 and Cyclin D1 expression. Correlation between DILA1 expression and clinical parameters was determined by two-tailed Chi-square test. Univariate and multivariate Cox proportional hazards regression model was performed by IBM SPSS Statistics 25. p < 0.05 was considered statistically significant.