Palbociclib sensitizes ER-positive breast cancer cells to fulvestrant by promoting the ubiquitin-mediated degradation of ER-α via SNHG17/Hippo-YAP axis

The normal breast cancer cell lines T-47D and MCF-7 were purchased from ATCC and cultured in the medium of DMEM supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), and FBS (10%). The cells were cultured at 37 °C in a humidified atmosphere incubator with a 5% (v/v) CO₂. All reagents for cell culture were obtained from Gibco (Carlsbad, CA, USA). The construction of fulvestrant-resistant breast cancer cell lines (hereafter, T-47D-F and MCF-7-F) was constructed referring to the published descriptions [22].

Cell viabilities of T-47D-F and MCF-7-F were detected in vitro by using MTT assays. In brief, 5000 cells for both cell lines were seeded into each well of 96-well plates overnight to allow cell attachment. After that, the T-47D-F and MCF-7-F cells were treated with indicated concentrations of tamoxifen, exemestane, fulvestrant, abemacicilib, and palbociclib for 24 h. Then, 50 μl of 1 mg/ml MTT solution (Sigma-Aldrich, St. Louis, MO, USA, M2128) in PBS was added into each well and incubated at 37 °C for 3 h, and the purple crystal was dissolved by DMSO. Finally, the absorbance at 570 nm was read by a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Briefly, the 300 T-47D-F and MCF-7-F cells were placed in a 6-well plate individually, and treated with indicated drugs. After 10 days of culture, the cells were discarded culture medium. After washing with PBS buffer twice, the colonies were fixed with methanol for 10 min. After that, the colonies were stained with crystal violet solution (aladdin, Shanghai, China, C110703). Finally, the colonies (> 50 cells) were numbered under a microscope.

Human breast cancer cells MCF-7 were treated with Palbociclib or DMSO for 24 h, and three replications for each group were collected. Total RNA was extracted by Trizol reagent (Invitrogen, Carlsbad, CA, USA, 15596026) and subjected to LncRNA-seq by LC-Bio Technology CO. (Zhejiang, China). For transcriptome sequencing, MCF-7 cells were transfected with SNHG17 overexpression plasmid or empty vector for 24 h, and total RNA was extracted by Trizol reagent. Transcriptome sequencing and data analysis were also performed by LC-Bio Technology CO.

The total sequence of lncRNA SNHG17 was synthesized and then subcloned into the vector pCDH to construct the SNHG17 overexpression plasmid. DNA sequencing was used to confirm the integrity of the plasmid construction. The Lipofectamine 2000 (Invitrogen, Waltham, MA, USA, 11668-019) was applied to perform the transfection according to the manufacturer’s instructions. Briefly, the plasmid and lipofectamine 2000 (Invitrogen) were mixed and the complex was formed after incubation for 20 min at RT. The complex was then added to a cell culture medium drop by drop and incubated for 24 h.

The SNHG17 stably expressed MCF-7-F cell line was constructed using lentivirus. Shortly, we purchased SNHG17-overexpressed lentivirus (Lenti-SNHG17) and corresponding control lentivirus (Lenti-control) from GeneChem (Shanghai, China), and then MCF-7-F cells were infected with Lenti-SNHG17 and Lenti-control (MOI = 200) overnight, respectively. Finally, the SNHG17 stably expressed MCF-7-F cells were screened using puromycin.

Trizol (Invitrogen) was used to extract the total RNA from cell samples. The HiScript 1st Strand cDNA Synthesis Kit (Vazyme Biotech, Nanjing, China) was used to synthesize cDNA. Realtime PCR was performed on ABI-7500 using SYBR Green master mixture reagent (Thermofisher, Waltham, MA, USA, A46012). The PCR protocol was as follows: initial denaturation at 95 °C for 10 min, denaturation at 95 °C for 15 s for 40 cycles, and annealing and extension at 60 °C for 1 min. The 2^−ΔΔCt method was used to determine the relative expression of the target. β-actin is used as an internal reference gene. The primers were listed in the Table.1

The T-47D-F and MCF-7-F cells were lysed in RIPA lysis buffer containing a 1% protease inhibitor cocktail (Sigma, St. Louis, MO, USA, P8215) for protein extraction. After electrophoresis on 10%, 12%, or 15% SDS–PAGE gels, proteins were electroblotted onto PVDF membranes (Millipore, Darmstadt, Germany, IPFL00010). After blocking with 5% non-fat milk for 2 h at room temperature, the membranes were then incubated with indicated primary antibodies at 4 °C overnight and secondary antibodies for 2 h at room temperature. After reacting with the HRP substrate, the chemiluminescence signals were visualized. The primary antibodies used in this study were as follows: YAP (CST, 14,074; dilution rate:1:1000), p-YAP (CST, 13,619; dilution rate:1:500), LATS1 (CST, 14,074; dilution rate:1:1000), p-LATS1 (CST, 9157; dilution rate:1:500), ER-α (CST, 8644; dilution rate: 1:1000), Ubiquitin (P4D1) (CST, 70,990, dilution rate:1:1000), and β-actin (Proteintech, 20,536–1-ap; dilution rate:1:5000).

The SNHG17-overexpressed MCF-7-F cells for immunoprecipitation assays were firstly lysed by IP lysis buffer (Thermo Scientific, Massachusetts, USA, 87,787). Cell lysates (with 250–500 μg protein contained) were incubated at 4 °C with 1 μg anti-ER-α (CST, 8644; dilution rate: 1:50) and 4 μl flag-beads (Sigma, St. Louis, MO, USA, M8823) overnight. Then the protein-antibody-beats complexes was detected by WB experiments.

Eighteen female Balb/c nude mice (4 weeks old, 20 g) were obtained from Vital River Laboratories (Beijing, China) to generate a xenograft mice model. Animal experiments were performed in compliance with the regulations and guidelines of institutional animal care of Zhejiang Cancer Hospital, and conducted according to the AAALAC and the IACUC guidelines. Xenografts were established by subcutaneously injecting 5 × 10⁶ SNHG17 stably expressed MCF-7-F cells into the right flank of nude mice. Tumor size was determined using a micrometer caliper. Tumor volume (mm³) was calculated using the following formula: V = (a × b × c)/2. After the largest tumors reached a volume of about 50–80 mm³, mice were randomly separated into three groups: (i) pCDH + control, (ii) pCDH + fulvestrant and palbociclib, and (iii) SNHG17 + fulvestrant and Palbociclib. 5 mg/kg fulvestrant was administered subcutaneously once per week, 25 mg/kg palbociclib was administered intraperitoneally daily, and all drugs treatment continued for 4 weeks. In this process, tumor size was measured every two days. After treatment, mice were euthanized and tumors were excised and weighed. The tumors were subjected to IHC experiments.

For IHC experiments, 6 μM thick sections obtained from xenografts tissues were de-paraffinized and rehydration in graded ethanol. For antigen retrieval, slides were immersed in 0.01 M citrate buffer, pH 6.0, using a steamer at 95 °C. Subsequently, sections were incubated with primary antibodies (ki67, LATS1, and ER-α, diluted at 1:500 or 250 respectively in PBS) at 4 °C overnight. After washing with PBS, horseradish peroxidase-conjugated secondary antibodies were incubated. Slides were incubated with diaminobenzidene substrate for color development and counterstained with hematoxylin.

Fulvestrant, a selective ER down-regulator, is the typical endocrine therapy agent prescribed for patients with ER-positive breast cancer [23]. Here, we selected normal breast cancer cell lines (T-47D and MCF-7) and constructed fulvestrant-resistant cell sublines (hereafter, T-47D-F and MCF-7-F) to investigate whether the fulvestrant could inhibit human breast cancer cells or not. As shown in Fig. 1a, fulvestrant exhibited an apparent inhibitory effect on the cell survival of T-47D and MCF-7, while fulvestrant mildly suppressed cell survival even at a very high dose such as 1000 nM in MCF-7-F cells (Fig. 1b). Therefore, T-47D-F and MCF-7-F were used for the following exploration.

As CDK4/6 inhibitor is used as a combination agent for endocrine therapy, we chose abemacicilib and palbociclib to further confirm whether they could sensitize fulvestrant in ER-positive breast cancer cells. Firstly, we treated T-47D and MCF7 cells with various doses of CDK inhibitors abemacicilib and Palbociclib for 24 h, and MTT assay was used to check the cytotoxicity of two drugs. As shown in Fig. 1c and d, both abemacicilib and palbociclib caused a dose-dependent reduction in the cell viability in T-47D-F and MCF-7-F cells. Next, we also further confirm whether they could sensitize T-47D-F and MCF-7-F cells to fulvestrant. The data of the MTT assay suggested that co-treated with indicated concentrations of palbociclib can obviously strengthen fulvestrant cytotoxicity in T-47D-F and MCF-7-F cells (Fig. 1e), while no similar effect was observed in abemacicilib (data were not shown). Concurrently, as shown in Fig. 1f, co-treatment of palbociclib and fulvestrant apparently decreased efficiency to form colonies compared with control, palbociclib alone and fulvestrant alone groups in both T-47D and MCF-7 cells. The clonogenic survival fraction was numbered 206 ± 49 and 128 ± 22 for the combined group, 527 ± 97 and 364 ± 72 for the control, 401 ± 91, 276 ± 67 and 375 ± 88, 292 ± 58 for the fulvestrant alone, and palbociclib alone groups on T-47D and MCF-7 cells in the right histogram, respectively. Taken together, these results certified that palbociclib strengthened the sensitivity of ER-positive human breast cancer cells to fulvestrant.

To identify the major mechanism involved in the sensitization function of Palbociclib on fulvestrant, high-throughput sequencing of LncRNA was performed in MCF-7-F cells treated with Palbociclib. At first, the RNA of MCF-7-F cells with palbociclib treated or untreated was isolated. Depending on the differentially expressed genes (DEGs) obtained from the sequencing, Volcano plots, and heatmap were generated and shown in Fig. 2a and b respectively. Furthermore, the enrichment analysis of Gene Ontology (GO, Fig. 2c) and Kyoto Encyclopedia of Genes and Genomes (KEGG, Fig. 2d) were performed in DEGs. We also chose the top 7 significantly differently expressed targeted lncRNAs for further identification in the palbociclib-treated MCF-7-F cells by RT-qPCR assay. As shown in Fig. 2e, palbociclib treatment remarkably reduced the expressions of lncRNA TYMSOS, SNHG17, TMPO-AS1, and TDRKH-AS1 in MCF-7-F cells. Although SNHG17 is not the most significantly suppressed lncRNA, it is reported that this lncRNA acts as an oncogene in multiple cancer types including breast cancer [24, 25]. We constructed the overexpression plasmid for SNHG17, and its transfection efficiency in both T-47D-F and MCF-7-F cells was verified by RT-PCR (Fig. 3a). The ectopic expression of SNHG17 alone obviously increased the colony formation in T-47D-F and MCF7-F cells in contrast to their control cells (Fig. 3b). More interestingly, SNHG17 overexpression also attenuated the synergistic effect of Palbociclib and fulvestrant in both two cell lines (Fig. 3b). The clonogenic survival fraction was numbered that 238 ± 25 and 255 ± 13 for the control, 291 ± 22 and 322 ± 14 for the SNHG17 alone groups, 80 ± 15 and 108 ± 8 for the fulvestrant and palbociclib combined groups, and 206 ± 42 and 175 ± 20 for the three combined groups in T-47D-F and MCF7-F cells in the right histogram, respectively (Fig. 3c). Taken together, these results suggested that Palbociclib enhanced the sensitivity of ER-positive breast cancer cells to fulvestrant by targeting SNHG17, at least partially.

To further elucidate the mechanism by which SNHG17 influences the sensitivity of fulvestrant in ER-positive breast cancer cells, mRNA sequencing was performed in MCF-7-F cells with SNHG17 overexpressed. According to the differentially expressed genes, the heatmap and Volcano plots were shown in Fig. 4a and b respectively. GO and KEGG pathway enrichment analyses were also performed, and the data from KEGG revealed that cytokine-cytokine receptor interaction, Hippo signaling pathway, and transcriptional misregulation in cancer were significantly enriched (Fig. 4c and d). It has been reported that the dysregulation of the Hippo-YAP signaling pathway has remarkable implications for the development of tumors, the activated large tumor suppressor 1 (LATS1) promoted the phosphorylation of YAP and then resulted in the degradation of YAP [26]. Thus, we next investigated whether the Hippo signaling pathway was activated under SNHG17 overexpression in ER-positive breast cancer cells. As shown in Fig. 4e, the inhibitory phosphorylation of YAP was inhibited and the YAP expression was up-regulated. Moreover, the expression of LATS1 and p-LATS1 were both suppressed, and the estrogen receptor-α (ER-α) expression level was significantly up-regulated.

LATS1/LATS2 was reported to be related to the degradation of ER-α, and this effect is independent on their kinase activity or their downstream effectors YAP and TAZ. LATS induces the ubiquitin-mediated degradation of ER-α in a Ddb1–cullin4-associated-factor 1 (DCAF1)-dependent manner [27]. Therefore, MCF-7-F cells were transfected with LncRNA SNHG17 or not, and immunoprecipitation (IP) and western blotting (WB) assays were performed to explore the ubiquitination of ER-α. As shown in Fig. 5a, palbociclib treatment promoted the ubiquitination of ER-α, while overexpressing SNHG17 attenuated that result, indicating that the effect of palbociclib was closely related to the ubiquitin-mediated degradation of ER-α. For further validation, the MCF-7-F cells with LncRNA SNHG17 transfected or not were co-treated with or without palbociclib and fulvestrant. As shown in Fig. 5b, overexpressing SNHG17 not only notably down-regulated the protein level of LATS1 and up-regulated the protein level of ER-α in MCF-7-F cells but also recovered the promotional effect of the co-treatment of palbociclib and fulvestrant on LATS1 level and the inhibitory effect on ER-α level. Therefore, we could come to the conclusion that palbociclib might sensitize the cytotoxicity of fulvestrant to ER-positive breast cancer cells by inhibiting the expression of SNHG17, then increasing the expression of LATS1, and eventually, promoting the ubiquitin-mediated degradation of ER-α.

To further determine the role of SNHG17 in fulvestrant resistance in ER-positive breast cancer, SNHG17 stably overexpressed MCF-7-F cell line was constructed using lentivirus. After subcutaneously injected in nude mice, MCF-7-F cells-derived xenografts were obtained and followed by co-treatment of palbociclib and fulvestrant. Our data revealed that the combined administration of palbociclib and fulvestrant effectively suppressed tumor growth. However, overexpression of SNHG17 significantly attenuated the role of the two drugs (Fig. 6a and b). ki67 is a classic biomarker for cell proliferation, and we next used IHC assay to examine its expression in tumors. As shown in Fig. 6c, co-treatment with palbociclib and fulvestrant obviously inhibited the ki67 expression compared with these in the control, while SNHG17 effectively reversed this function. We also detected the expression of LATS1 and ER-α. As shown in Fig. 6d and e, palbociclib, and fulvestrant treatment markedly upregulated the LATS1 expression and downregulated the ER-α expression, while overexpression of SNHG17 reversed the above results.