Long non‑coding RNA UCA1 confers tamoxifen resistance in breast cancer endocrinotherapy through regulation of the EZH2/p21 axis and the PI3K/AKT signaling pathway10.3892/ijo.2024.5611

  • Authors:
    • Zhuo Li
    • Dehai Yu
    • Haijun Li
    • You Lv
    • Sijie Li
  • View Affiliations

  • Published online on: January 8, 2019     https://doi.org/10.3892/ijo.2019.4679
  • Pages: 1033-1042
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Abstract

Tamoxifen is the gold standard for breast cancer endocrinotherapy. However, drug resistance remains a major limiting factor of tamoxifen treatment. Long non‑coding (lnc) RNA serves an important role in drug resistance; however, the molecular mechanisms of tamoxifen resistance in breast cancer endocrinotherapy are largely unclear. lncRNA urothelial cancer associated 1 (lncRNA UCA1, UCA1) has been proven to be dysregulated in human breast cancer and promotes cancer progression. In the present study, it was demonstrated that UCA1 was significantly upregulated in breast cancer tissues compared with healthy tissues. Furthermore, the expression level of UCA1 was significantly greater in tamoxifen‑resistant breast cancer cells (LCC2 and LCC9) when compared with those in the tamoxifen‑sensitive breast cancer cells (MCF‑7 and T47D). UCA1 silencing in LLC2 and LLC9 cells increased tamoxifen drug sensitivity by promoting cell apoptosis and arresting the cell cycle at the G2/M phase. Notably, the induced overexpression of UCA1 in MCF‑7 and T47D cells decreased the drug sensitivity of tamoxifen. The molecular mechanism involved in UCA1‑induced tamoxifen‑resistance was also investigated. It was identified that UCA1 was physically associated with the enhancer of zeste homolog 2 (EZH2), which suppressed the expression of p21 through histone methylation (H3K27me3) on the p21 promoter. In addition, it was demonstrated that UCA1 expression was paralleled to the phosphorylation of CAMP responsive element binding protein (CREB) and AKT. When LCC2 cells were treated with the phosphoinositide 3‑kinase (PI3K)/protein kinase B (AKT) signaling pathway inhibitor LY294002, the phosphorylation levels of CREB and AKT were significantly downregulated. Taken together, it was concluded that UCA1 regulates the EZH2/p21 axis and the PI3K/AKT signaling pathway in breast cancer, and may be a potential therapeutic target for solving tamoxifen resistance.

Introduction

Breast cancer is the most common female malignancy and the second most common cause of cancer-associated fatality in the world (1). Approximately 70% of patients with breast cancer are estrogen receptor-positive (ER+) (2). Apart from surgery, endocrine therapy (including tamoxifen, fulvestrant and letrozole) has improved the overall survival and quality of life for patients with breast cancer (2-4). Among all endocrine therapies, tamoxifen is the most extensively used hormone therapy and functions as an estrogen antagonist in breast cancer (5,6). Although the majority of patients with ER+ breast cancer benefit from tamoxifen therapy, many tumors eventually recur because of tamoxifen resistance (7,8). Tamoxifen resistance can arise via several mechanisms, including loss of ERα, induction of abnormal estra-diol levels and alterations of coregulatory proteins, including amplified in breast cancer 1 and histone deacetylase (9-11).

An increasing number of long non-coding (lnc)RNAs in the human genome have been identified, and have provided new directions in cancer research (12). lncRNA, a class of non-protein coding transcripts with >200 nucleotides, regulates protein-coding genes during transcription and post-transcription in a sequence-specific manner (13-15). Importantly, lncRNAs in cancer cells are associated with the formation of tamoxifen resistance (16-18). However, only a few lncRNAs have been proposed to be clinically relevant biomarkers for tamoxifen resistance, such as H19 and homeobox antisense intergenic RNA (19-21). Searching for appropriate lncRNAs is valuable for the management of tamoxifen-resistance.

Out of the numerous cancer-associated lncRNAs, lncRNA urothelial carcinoma-associated 1 (UCA1) serves an important oncogenic role in several cancer types, including bladder cancer, colorectal cancer and gastric cancer (22). UCA1 has three exons that encode a 1.4-kb isoform and a 2.2-kb isoform (23). It was originally identified as a urine marker (the 1.4-kb isoform) in bladder cancer (24). Tuo et al (25) demonstrated that UCA1 can modulate breast cancer cell growth and apoptosis through downregulation of the tumor suppressor microRNA (miR)-143. Huang et al (23) reported that UCA1 can promote breast tumor growth by suppressing the level of p27. UCA1 is also associated with the poor prognosis of cancer. Bian et al (26) demonstrated that patients with colorectal cancer and higher UCA1 expression had a significantly poorer prognosis. Furthermore, it was reported that UCA1 expression was correlated with a reduction in recurrence-free survival in breast cancer (27). These findings highlight the important role of UCA1 in cancer development.

The polycomb group protein enhancer of zeste homolog 2 (EZH2) is a critical regulator of tumorigenesis (28,29). It has been demonstrated that the level of EZH2 is elevated in human bladder cancer, breast cancer, colon cancer and prostate cancer (30). Furthermore, the expression and mutation of EZH2 can regulate the level of H3K27me3 (31). In hepatocellular carcinoma, UCA1 repressed p27 expression through its association with EZH2, which suppresses p27Kip1 through H3K27me3 on the p27Kip1 promoter (32). However, the effects of UCA1 on EZH2 expression and the underlying molecular mechanisms in breast cancer are not fully understood.

The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is the most frequently altered pathway in human cancer, and previous studies have demonstrated that UCA1 regulates the cell cycle progression of bladder carcinoma cells via PI3K/AKT-dependent signaling (33,34). Notably, the knockdown of UCA1 inhibits AKT phosphorylation in breast cancer cells (35). Additionally, activation of the PI3K/AKT signaling pathway has been demonstrated to confer resistance to antiestrogens in tamoxifen-resistant breast cancer cells (36). Therefore, it would be useful to determine whether UCA1 is involved in the PI3K/AKT signaling pathway and if it induces tamoxifen resistance in breast cancer cells.

In the present study, the level of UCA1 expression was investigated in tamoxifen-resistant cells and compared with tamoxifen-sensitive cells. Induction of UCA1 overexpression in MCF-7 and T47D breast cancer cells and silencing of UCA1 in LLC2 and LLC9 breast cancer cells was performed to assess the drug sensitivity of the cells to tamoxifen. Furthermore, it was explored whether UCA1 was physically associated with EZH2. In addition, it was investigated whether UCA1 regulates tamoxifen resistance through a EZH2/p21 axis and the PI3K/AKT signaling pathway in breast cancer.

Materials and methods

Patients and specimens

A total of 10 hormone receptor-positive breast cancer specimens and 10 non-tumor specimens were randomly selected from the First Hospital of Jilin University (Changchun, China) between April 2015 and April 2017. All these participants were female. The breast cancer specimens were histologically diagnosed as breast carcinoma using ultrasound-guided core needle biopsy of the breast. In the 10 breast cancer specimens, 1 was at stage I, 5 were at stage II and 4 were at stage III. The age range of the 10 patients was from 37-68 years old, with a median age of 51. Evidence of bilateral disease and pregnancy concomitant with the diagnosis of breast cancer resulted in exclusion from the study. All samples were collected prior to tamoxifen therapy and stored in liquid nitrogen (−196°C) until use. Permission to use the clinical samples for research purposes was obtained and approved by the Ethics Committee of the First Hospital of Jilin University. Informed consents were obtained from all patients.

Cell culture

Human breast cancer cell lines MCF-7 (tamoxifen-sensitive), T47D (tamoxifen-sensitive), LCC2 (tamoxifen-resistant) and LCC9 (tamoxifen-resistant), were purchased from American Type Culture Collection (Manassas, VA, USA). All cancer cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA) 2 mM glutamine (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (HyClone). Cells were cultured at 37°C in an incubator with a humidified atmosphere containing 5% CO2.

Overexpression and knockdown of UCA1 in breast cancer cells

To induce the overexpression of UCA1 in breast cancer cells, the cDNA encoding UCA1 was polymerase chain reaction (PCR)-amplified. The primer sequences were as follows: UCA1, forward 5′-CGCGGATCCTTTATCAGGCATATTAG CTTTAA-3′ (BamHI) and reverse 5′-GCGAATTCTGACATTC TTCTGGACAATG-3′ (EcoRI). Following this, the PCR product was subcloned into the pGreen.puro lentivirus vector (SBI, Palo Alto, CA, USA) with BamHI and EcoRI restriction sites (Takara Biotechnology Co. Ltd., Dalian, China). Viral particles were harvested at 48 h post-cotransfection of the pGreen-UCA1-puro constructs with the packaging plasmid ps-PAX2 and the envelope plasmid pMD2G (SBI) into 293T cells using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). The empty vector was used as the control (lv-NC). MCF-7 and T47D cells were infected with the lentiviral particles (5×107 TU/ml; lv-UCA1 or lv-NC) plus 6 µg/ml polybrene (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The virus titers in the control and experimental groups were nearly the same as above (~5×107 TU/ml).

For the knockdown of UCA1, the small interfering (si)RNA targeting UCA1 (si-UCA1) and the scramble non-target control siRNA (si-NC) were synthesized by Shanghai GenePharma Co., Ltd., (Shanghai, China). si-UCA1 and si-NC sequences were as follows (37): si-UCA1, 5′-GTTAATCCAGGAGACAA AGA-3′; and si-NC, 5′-TTCTCCGAACGTGTCACGT-3′. LCC2 and LCC9 cells were transfected with equal amounts (100 nM) of si-UCA1 and si-NC using Lipofectamine 3000. All the following cellular or molecular experiments were carried out at 48 h post-transfection (38,39).

RNA extraction, reverse transcription-PCR (RT-PCR) and RT-quantitative PCR (RT-qPCR)

Total RNA was extracted from breast cancer tissues and cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. cDNA was reverse transcribed using 1 µg of total RNA and the SuperScript III First-Stand Synthesis Kit (Invitrogen; Thermo Fisher Scientific, Inc.). The expression level of UCA1 were determined on a PCR thermal cycler (T100, Bio-Rad Laboratories, Inc., Hercules, CA, USA) using 2X Taq PCR StarMix buffer (GeneStar, Beijing, China) or on a real-time PCR thermal cycler (ABI PRISM 7500, Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR Green Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). The endogenous control gene was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The RT-PCR amplification process was as follows: 1 cycle at 98°C for 2 min and 32 cycles at 95°C for 20 sec, 62°C for 15 sec, followed by 72°C for 15 sec; ending with an extension cycle at 72°C for 5 min. The qPCR amplification process consisted of 1 cycle at 95°C for 10 min, followed by 40 cycles at 95°C for 10 sec and 58°C for 30 sec. The results of RT-PCR were visual-ized using a 3% agarose gel and qPCR was performed using the 2−ΔΔCq method. All the oligonucleotide primers were synthesized by Takara. The primer sequences used were as follows (33): UCA1, forward 5′-CTTCTGCATAGGATCTG CAATCAG-3′ and reverse 5′-TTTTGTCCCCATTTTCCATCA TACG-3′; GAPDH, forward 5′-AGGTCGGAGTCAACGG ATTTG-3′ and reverse 5′-GTGATGGCATGGACTGTGGT-3′.

WST-1 assay to assess cell viability

Tamoxifen was purchased from Sigma-Aldrich; Merck KGaA. The stock solution of tamoxifen (500 µM) was prepared in 100% MeOH and maintained at 4°C. Working standard solutions at different concentrations were prepared by dilution in DMEM supplemented with 10% FBS. Solutions were added into the 96-well plate at a final concentration of 0, 0.01, 0.1, 1, 10 or 100 µM and incubated with cells for 24 h. Following this, 10 µl WST-1 (Roche Diagnostics, Shanghai, China) was added into each well and the cells were incubated at 37°C in the dark for 2 h. The absorbances of 450 and 630 nm were monitored. The relative cell viability percentage in each group was calculated by comparison to that of the control group.

Flow cytometry for cell cycle and apoptosis analysis

LCC2 cells transfected with si-UCA1 (si-UCA1 LCC2) were treated with 10 µM tamoxifen for 24 h, trypsinized, collected and washed with PBS. For cell cycle analysis, cells were fixed in pre-cold 70% ethanol for 20 min and stored at -20°C. Following this, cells were washed with PBS and stained with a solution containing 3.5 µM Tris-HCl (pH 7.6), 10 mM NaCl, 50 µg/ml propidium iodide (PI) (Sigma-Aldrich; Merck KGaA), 20 µg/ml RNase and 0.1% igepal CA-630 (Sigma-Aldrich; Merck KGaA) for 20 min on ice to label DNA. Subsequently, LCC2 cells were analyzed using a FACSCaliber flow cytometer with a FlowJo software (version 10.0, BD Biosciences, Franklin Lakes, NJ, USA). The percentages of cells at different phases were calculated from three independent experiments.

For cell apoptosis analysis, cells were stained using an Annexin V-FITC/PI double staining apoptosis detection kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions, and analyzed with a FACSCaliber flow cytometer. The cells in the different portions represented the different cell states as follows: The late-apoptotic cells were present in the upper right portion, the viable cells were present in the lower left portion and the early apoptotic cells were present in the lower right portion.

RNA immunoprecipitation (RIP) assay

The RIP experiment was performed in LCC2 cells using the Magna RIP R NA-Binding Protein Immunoprecipitation Kit (EMD Millipore, Billerica, MA, USA) according to the manufacturer's instructions. EZH2 antibody for the RIP assay was purchased from Abcam (1:500; #ab186006; Shanghai, China). Samples were treated with proteinase K (Thermo Fisher Scientific, Inc.) to digest the protein for 1 h at 37°C and the immunoprecipitated RNA was isolated. Final analysis of co-precipitated RNA was performed using qPCR and demonstrated as fold enrichment of UCA1.

Chromatin immunoprecipitation (ChIP) assay

The ChIP experiment was performed in LCC2 cells using the EZ ChIP Chromatin Immunoprecipitation Kit (#17-371, EMD Millipore) according to the manufacturer's instructions. Briefly, LCC2 cells were incubated with formaldehyde for 10 min to generate DNA-protein cross-links; the crosslinked chromatin DNAs were sonicated into 200 to 1,000-bp-sized fragments. Subsequently, immunoprecipitation was performed using anti-EZH2 antibody (1:1,000; #07-689, EMD Millipore) and anti-H3K27me3 antibody (1:1,000; #17-622, EMD Millipore), or normal IgG (1:200, EZ ChIP Chromatin Immunoprecipitation Kit) as control. Precipitated chromatin DNA was recovered and analyzed by qPCR. The primer sequences of p21 promoter were as follows: Forward (40), 5′-AGACCATGTGGACCTGTCACTG-3′ and reverse 5′-GTTTGGAGTGGTAGAAATCTGTC-3′.

Western blot analysis

Cell samples were lysed using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing protease inhibitor. The total protein concentration was determined using a BCA protein assay kit (Beyotime Institute of Biotechnology). A total of 20 µg of total protein was loaded per lane and separated by SDS-PAGE (10 or 12% gels) and transferred to polyvinylidene fluoride membrane (Roche). The membranes were blocked in 5% skimmed milk diluted with Tris-buffered saline/Tween-20 (Tris-HCl 20 mmol/l, NaCl 150 mmol/l, 0.1% Tween-20, pH 7.5) at room temperature for 1 h and subsequently incubated overnight at 4°C with primary antibodies: Anti-AKT (1:1,000, #ab8805, Abcam), anti-phospho(p)-AKT (1:2,000, #ab8933, Abcam), CAMP responsive element binding protein (CREB, 1:2,000, #ab178322, Abcam), anti-p-CREB (1:1,000, #ab10564, Abcam), anti-GAPDH (1:2,000, #ab181603, AbMart Bio-tech Co. Ltd., Shanghai, China), anti-B cell lymphoma/leukemia-2 (Bcl-2, 1:2,000, #ab196495, Abcam), anti-cleaved caspase-3 (1:1,000, #9661, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-cleaved caspase-9 (1:1,000, #52873, Cell Signaling Technology, Inc.), anti-cyclin D1 (1:1,000, #2978, Cell Signaling Technology, Inc.) and anti-p21 (1:1,000, #2947, Cell Signaling Technology, Inc.). Subsequently, the membranes were incubated with anti-mouse (1:5,000, SAB3701214, Sigma-Aldrich; Merck KGaA) or rabbit (1:5,000, SAB3700852, Sigma-Aldrich; Merck KGaA) horseradish peroxidase-conjugated secondary antibodies at 37°C for 1 h. The immunoreactive bands were visualized using the ECL western blot substrate (Promega Corporation, Madison, WI, USA) and the relative band density was analyzed by Quantity-one software (version 4.6, Bio-Rad Laboratories, Inc.).

Suppression of the PI3K signaling pathway

The PI3K signaling pathway was suppressed by the PI3K inhibitor LY294002 (Cell Signaling Technology, Inc.). LCC2 cells were treated with 50 µM LY294002 for 24 h in DMEM supplemented with 10% FBS. Subsequent qPCR and western blot analysis were conducted at 24 h post-inhibition.

Statistical analysis

Data were presented as the mean ± standard error of the mean of at least three independent experiments. Statistical significance between two groups was determined using one-way analysis of variance followed by an LSD or Dunnett's post hoc test or the Student's t-test. P<0.05 was considered to indicate a statistically significant difference. All analyses were performed using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA).

Results

UCA1 expression is upregulated in tamoxifen-resistant breast cancer cells

Firstly, 1 normal breast tissue and 3 breast cancer tissues were randomly selected from the 20 samples, and the level of UCA1 expression was detected using RT-PCR. The PCR results revealed that the level of UCA1 expression was significantly increased in breast cancer tissues compared with normal tissues (P<0.001 and P<0.01; Fig. 1A). Following this, the UCA1 expression levels in all the 10 normal breast tissues and 10 breast cancer tissues were assessed by qPCR. As indicated in Fig. 1B, the mean expression level of UCA1 in the breast cancer group was 4.68-fold greater when compared with that in the normal control group (P<0.0001). These data indicated a positive association between breast cancer and the expression of UCA1.

According to these results, the expression levels of UCA1 in tamoxifen-sensitive cells, MCF-7 and T47D, and in the tamoxifen-resistant cells, LCC2 and LCC9, were assessed using qPCR (Fig. 1C). It was revealed that the level of UCA1 expression in LCC2 and LCC9 cells was >20-fold greater when compared with that in MCF-7 and T47D cells (P<0.001), suggesting a positive association between tamoxifen resistance and UCA1 expression in breast cancer cells.

UCA1 affects the cell viability of breast cancer cells treated with tamoxifen

In order to further confirm the contribution of UCA1 to tamoxifen resistance, the WST-1 assay was performed to detect the cell survival rate following UCA1 knockdown or overexpression in breast cancer cells.

The delivery efficiencies of the lentivirus carrying UCA1 DNA and the siRNA were assessed. As indicated in Fig. 2A, UCA1 expression was significantly elevated by 21.67- and 22.97-fold in lentivirus-transduced MCF-7 and T47D cells compared with the lv-NC group, respectively (P<0.001). Furthermore, UCA1 expression was significantly downregulated to 0.2-fold and 0.23-fold in the UCA1-siRNA transfected LCC2 and LCC9 cells when compared with the si-NC group, respectively (Fig. 2B; P<0.001).

Following treatment with increasing concentrations of tamoxifen (0, 0.01, 0.1, 1, 10 and 100 µM), it was observed that the cell survival rates of UCA1-overexpressed cells were significantly increased compared with the lv-NC group in the presence of 1 or 10 µM tamoxifen in MCF-7 cells and in the presence of 1, 10 or 100 µM tamoxifen in T47D cells (Fig. 2C and D; P<0.05 and P<0.01). Conversely, UCA1 silencing significantly decreased the cell survival rate compared with the si-NC group in the presence of 10 or 100 µM tamoxifen in LCC2 cells or in the presence of 1, 10 or 100 µM tamoxifen in LCC9 cells (Fig. 2E and F; P<0.05 and P<0.01). Specifically, the cell survival rates significantly changed in the 10 µM tamoxifen treatment group compared with the 1 µM tamoxifen treatment group (Fig. 2C-F; P<0.05 or P<0.01). However, in the 100 µM tamoxifen treatment group, the cell survival rates of the control and the experimental group were significantly decreased compared with the 10 µM tamoxifen treatment group, indicating that a high concentration of tamoxifen promoted non-specific cytotoxicity (Fig. 2C-F; P<0.01).

Flow cytometry results indicated that the cell apoptosis rate of si-UCA1 LCC2 cells (35%) and si-UCA1 LCC9 cells 41.8%) was significantly increased following 10 µM tamoxifen treatment when compared with the negative control (si-NC, 5.39% and 4.18%; Fig. 3A-D; P<0.001). Several apoptosis-associated factors were also measured by western blot analysis. Results indicated that the expression levels of Bcl-2, cleaved caspase-3 and cleaved caspase-9 were significantly increased in si-UCA1 LCC2 cells (Fig. 3E and F; P<0.05 and P<0.001). These data suggest that UCA1 contributed to the tamoxifen resistance in breast cancer cells.

UCA1 silencing promotes G2/M phase cell cycle arrest following tamoxifen treatment

A previous study have demonstrated that UCA1 could promote bladder cancer progression (33). Therefore, the cell cycle distribution in LCC2 cells post-UCA1 knockdown was assessed. si-UCA1 LCC2 cells treated with 10 µM tamoxifen for 24 h exhibited significant G2/M phase arrest (Fig. 4A-C; P<0.01), and the expression level of cell cycle-associated factor p21 was significantly upregulated and the expression level of cyclin D1 was significantly downregulated (Fig. 4D; P<0.001 and P<0.01).

UCA1 recruits EZH2 to the p21 promoter and represses p21 expression

It was reported that EZH2 could inhibit the expression of p21 and that p21 is a target of UCA1 (41). It was speculated in the present cell model that p21 may also be suppressed by UCA1 through the recruitment of EZH2 on the p21 promoter. Therefore, RIP analysis was performed. The results indicated that, compared with the IgG control antibody, UCA1 was significantly enriched by EZH2 antibody (Fig. 5A; P<0.01).

ChIP analysis was further performed to demonstrate whether UCA1 inhibited p21 expression by interacting with EZH2. As indicated in Fig. 5B, EZH2 and H3K27me3 could bind to the p21 promoter region directly. However, in the si-UCA1 LCC2 cells, the binding of EZH2 and H3K27me3 to the p21 promoter region was significantly weakened (P<0.01). This finding suggested that UCA1 repressed the expression of p21 via the recruitment of EZH2 and H3K27me3.

UCA1 contributes to tamoxifen resistance in breast cancer cells through the PI3K/AKT signaling pathway

CREB-binding protein, a key nuclear transcription factor in the PI3K/AKT signaling pathway, serves an important role in cell cycle progression (42). A previous study demonstrated that cell cycle progression was greatly arrested in UCA1 knockdown cells, and CREB expression levels were significantly downregulated simultaneously (33). In the present study, it was investigated whether UCA1 could influence the expression of CREB. As indicated in Fig. 6A, CREB and p-CREB expression levels were reduced in si-UCA1 LCC2 cells. Band density analysis revealed that the level of CREB and the p-CREB expression significantly decreased 3.06-fold and 2.1-fold when compared with the control group (Fig. 6B; P<0.001 and P<0.01).

Considering that the PI3K/AKT signaling pathway is pivotal for the maintenance of normal cell cycle progression and is associated with CREB expression (43,44), it was further assessed whether the PI3K/AKT signal pathway could regulate the expression of CREB in si-UCA1 LCC2 cells in the present study. As indicated in Fig. 7, the expression levels of AKT and p-AKT were significantly reduced in si-UCA1 LCC2 cells (P<0.0001 and P<0.01), suggesting that UCA1 was involved in the activation of AKT.

In order to further verify whether UCA1 could regulate CREB through the PI3K/AKT signaling pathway, LCC2 cells were treated with the PI3K inhibitor LY294002 for 24 h. qPCR analysis revealed that LCC2 cells treated with LY294002 exhibited significantly decreased UCA1 expression levels (Fig. 8A; P<0.01). Furthermore, the phosphorylation of CREB and AKT was also significantly repressed in LCC2 cells were treated with LY294002 (Fig. 8B and C; P<0.001 and P<0.05). Taken together, these results further indicated that UCA1 regulated the activation of CREB and impacted cell cycle progression through PI3K/AKT-dependent signaling.

Discussion

Breast cancer currently remains the most common female malignancy in the world (45). Tamoxifen is the most frequently used endocrinotherapy for ER+ breast cancer (46). Despite great treatment advances in improving the survival rate of patients with breast cancer, almost 30% of patients treated with tamoxifen may develop resistance to the drug (47). Numerous studies have focused on the function of lncRNA, and emerging evidence has demonstrated that lncRNAs significantly contribute to various aspects of cancer biology and have been identified as critical players of drug resistance in cancer therapy (44). However, the underlying mechanisms for tamoxifen resistance are largely unknown. In the present study, it was indicated that UCA1 expression was significantly increased in tamoxifen-resistant breast cancer compared with tamoxifen-sensitive breast cancer. Following the knockdown of UCA1, breast cancer cells exhibited a significant increase in G2/M phase cell cycle arrest.

UCA1 has been reported to be upregulated and to exert its oncogenic activity and enhance chemoresistance in several cancer types (23,26,35,48). It has been reported that UCA1 can increase chemosensitivity through a CREB-miR-196a-5p paradigm in bladder cancer (49). Various studies have demonstrated that UCA1 expression is elevated in breast cancer. For example, Liu et al (50) revealed that UCA1 regulates tamoxifen resistance through the Wnt/β-catenin signaling pathway in breast cancer. Consistent with these reports, in the present study it was demonstrated that UCA1 was significantly increased in tamoxifen-resistant breast cancer. Following treatment with tamoxifen, the expression levels of Bcl-2 and cleaved caspase-3 and -9 were increased in si-UCA1 LCC2 and si-UCA1 LCC9 cells, which demonstrated that UCA1 contributed to tamoxifen drug resistance in breast cancer cells. Bcl-2 protein is a critical component in cell apoptotic signaling. It blocks the increased permeability of the mitochondrial membrane and prevents the release of cytochrome c (51). Several studies have reported lncRNA-mediated sequestering of miR expression, whereas some miRs can directly target Bcl-2 and affect the function of Bcl-2 (52-54). It was presumed that UCA1 regulated Bcl-2 through a similar manner. However, the exact reason for this change remains to be further studied.

The PI3K/AKT signaling pathway serves an important role in cell growth, cell cycle distribution, apoptosis and survival of human cancer (55). AKT and CREB are two key molecules in this pathway. lncRNA may regulate the activation of the PI3K/AKT signaling pathway and affect tumorigenesis and drug sensitivity. For example, miR-21 can modulate tamoxifen sensitivity of breast cancer cells through the PI3K/AKT/mTOR signaling pathway (56). In the present study, it was demonstrated that knockdown of UCA1 in LCC2 cells induced an apparent G2/M phase arrest and altered the expression of p21 and cyclin D1.

A previous study reported that p21 transcription could be repressed through recruitment of EZH2, which was mediated by UCA1 in renal cell carcinoma cells (40). EZH2 is a histone methyltransferase that catalyzes the trimethylation of H3K27me3 of target genes. The levels of EZH2 are frequently elevated in breast cancer (30). The present study indicated that p21 transcription was repressed by EZH2 through H3K27me3, which was mediated by UCA1 in breast cancer cells. These data demonstrated that UCA1 could modulate the cell cycle through EZH2 and H3K27me3 in breast cancer cells.

CREB, a proto-oncogenic transcription factor, is crucial in cell cycle regulation of breast cancer cells (57). In the present study, the association between the expression of UCA1 with the expression of CREB was assessed by western blot analysis. Results demonstrated that CREB and p-CREB expression levels were significantly decreased when UCA1 was suppressed. CREB is mediated by various protein kinases, including AKT and PI3K (58). Likewise, it was indicated in the present study that AKT expression was positively associated with UCA1 expression. A previous study reported that CREB could be positively regulated by AKT kinase activity (33). Furthermore, the present results confirmed that the expression levels of p-AKT and p-CREB were inhibited by the PI3K inhibitor, LY294002, and this was consistent with a previous report (33). These data demonstrated that UCA1 could regulate CREB through AKT via PI3K/AKT signaling.

In conclusion, to the best of our knowledge the present study demonstrated for the first time that UCA1 regulates tamoxifen resistance through the EZH2/p21 axis and the PI3K/AKT signaling pathway in breast cancer (Fig. 9). Based on the present results, UCA1 may be considered a novel biomarker of poor response to tamoxifen and a potential therapeutic intervention target of breast cancer endocrinotherapy.

Funding

The present study was supported in part by grants from the Natural Science Foundation of Jilin University (Bethune plan B) (grant no. 2015311 was awarded to ZL).

Availability of data and materials

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

SL contributed to the design of the study and wrote the manuscript. ZL and DY performed the experiments and analyzed the data. HL and YL helped perform the analysis with constructive discussions. All authors have read and approved this manuscript.

Ethics approval and consent to participate

Permission to use the clinical samples for research purposes was obtained and approved by the Ethics Committee of the First Hospital of Jilin University.

Patient consent for publication

Informed consents were obtained from all patients.

Competing interests

The authors declare no conflict of interest.

Acknowledgments

Not applicable.

References

1 

Beiki O, Hall P, Ekbom A and Moradi T: Breast cancer incidence and case fatality among 4.7 million women in relation to social and ethnic background: A population-based cohort study. Breast Cancer Res. 14:R52012. View Article : Google Scholar : PubMed/NCBI

2 

Lim E, Metzger-Filho O and Winer EP: The natural history of hormone receptor-positive breast cancer. Oncology (Williston Park). 26:pp. 688–694. pp. 6962012

3 

Regan MM, Neven P, Giobbie-Hurder A, Goldhirsch A, Ejlertsen B, Mauriac L, Forbes JF, Smith I, Láng I, Wardley A, et al: BIG 1-98 Collaborative Group; International Breast Cancer Study Group (IBCSG): Assessment of letrozole and tamoxifen alone and in sequence for postmenopausal women with steroid hormone receptor-positive breast cancer: The BIG 1-98 randomised clinical trial at 8·1 years median follow-up. Lancet Oncol. 12:1101–1108. 2011. View Article : Google Scholar : PubMed/NCBI

4 

Colditz GA: Relationship between estrogen levels, use of hormone replacement therapy, and breast cancer. J Natl Cancer Inst. 90:814–823. 1998. View Article : Google Scholar : PubMed/NCBI

5 

Lumachi F, Brunello A, Maruzzo M, Basso U and Basso SM: Treatment of estrogen receptor-positive breast cancer. Curr Med Chem. 20:596–604. 2013. View Article : Google Scholar : PubMed/NCBI

6 

Higgins MJ and Stearns V: CYP2D6 polymorphisms and tamoxifen metabolism: Clinical relevance. Curr Oncol Rep. 12:7–15. 2010. View Article : Google Scholar : PubMed/NCBI

7 

Raha P, Thomas S and Munster PN: Epigenetic modulation: A novel therapeutic target for overcoming hormonal therapy resistance. Epigenomics. 3:451–470. 2011. View Article : Google Scholar : PubMed/NCBI

8 

Hurvitz SA and Pietras RJ: Rational management of endocrine resistance in breast cancer: A comprehensive review of estrogen receptor biology, treatment options, and future directions. Cancer. 113:2385–2397. 2008. View Article : Google Scholar : PubMed/NCBI

9 

Anzick SL, Kononen J, Walker RL, Azorsa DO, Tanner MM, Guan XY, Sauter G, Kallioniemi OP, Trent JM and Meltzer PS: AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. Science. 277:965–968. 1997. View Article : Google Scholar : PubMed/NCBI

10 

Ravdin PM, Fritz NF, Tormey DC and Jordan VC: Endocrine status of premenopausal node-positive breast cancer patients following adjuvant chemotherapy and long-term tamoxifen. Cancer Res. 48:1026–1029. 1988.PubMed/NCBI

11 

Paridaens R, Sylvester RJ, Ferrazzi E, Legros N, Leclercq G and Heuson JC: Clinical significance of the quantitative assessment of estrogen receptors in advanced breast cancer. Cancer. 46(Suppl): 2889–2895. 1980. View Article : Google Scholar : PubMed/NCBI

12 

Di Gesualdo F, Capaccioli S and Lulli M: A pathophysiological view of the long non-coding RNA world. Oncotarget. 5:10976–10996. 2014. View Article : Google Scholar : PubMed/NCBI

13 

Li Z, Shen J, Chan MT and Wu WK: TUG1: A pivotal oncogenic long non-coding RNA of human cancers. Cell Prolif. 49:471–475. 2016. View Article : Google Scholar : PubMed/NCBI

14 

Han P and Chang CP: Long non-coding RNA and chromatin remodeling. RNA Biol. 12:1094–1098. 2015. View Article : Google Scholar : PubMed/NCBI

15 

Kornienko AE, Guenzl PM, Barlow DP and Pauler FM: Gene regulation by the act of long non-coding RNA transcription. BMC Biol. 11:592013. View Article : Google Scholar : PubMed/NCBI

16 

Zhang HY, Liang F, Zhang JW, Wang F, Wang L and Kang XG: Effects of long noncoding RNA-ROR on tamoxifen resistance of breast cancer cells by regulating microRNA-205. Cancer Chemother Pharmacol. 79:327–337. 2017. View Article : Google Scholar : PubMed/NCBI

17 

Li X, Wu Y, Liu A and Tang X: Long non-coding RNA UCA1 enhances tamoxifena resistance in breast cancer cells through a miR-18a-HIF1α feedback regulatory loop. Tumor Biol. 37:14733–14743. 2016. View Article : Google Scholar

18 

Cai Y, He J and Zhang D: Suppression of long non-coding RNA CCAT2 improves tamoxifen-resistant breast cancer cells' response to tamoxifen. Mol Biol. 50:821–827. 2016.In Russian.

19 

Chen G, Wang Z, Wang D, Qiu C, Liu M, Chen X, Zhang Q, Yan G and Cui Q: lncRNADisease: A database for long-non-coding RNA-associated diseases. Nucleic Acids Res. 41D:D983–D986. 2013.

20 

Lottin S, Adriaenssens E, Dupressoir T, Berteaux N, Montpellier C, Coll J, Dugimont T and Curgy JJ: Overexpression of an ectopic H19 gene enhances the tumorigenic properties of breast cancer cells. Carcinogenesis. 23:1885–1895. 2002. View Article : Google Scholar : PubMed/NCBI

21 

Gupta RA, Shah N, Wang KC, Kim J, Horlings HM, Wong DJ, Tsai MC, Hung T, Argani P, Rinn JL, et al: Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature. 464:1071–1076. 2010. View Article : Google Scholar : PubMed/NCBI

22 

Xue M, Chen W and Li X: Urothelial cancer associated 1: A long noncoding RNA with a crucial role in cancer. J Cancer Res Clin Oncol. 142:1407–1419. 2016. View Article : Google Scholar

23 

Huang J, Zhou N, Watabe K, Lu Z, Wu F, Xu M and Mo YY: Long non-coding RNA UCA1 promotes breast tumor growth by suppression of p27 (Kip1). Cell Death Dis. 5:e10082014. View Article : Google Scholar : PubMed/NCBI

24 

Wang XS, Zhang Z, Wang HC, Cai JL, Xu QW, Li MQ, Chen YC, Qian XP, Lu TJ, Yu LZ, et al: Rapid identification of UCA1 as a very sensitive and specific unique marker for human bladder carcinoma. Clin Cancer Res. 12:4851–4858. 2006. View Article : Google Scholar : PubMed/NCBI

25 

Tuo YL, Li XM and Luo J: Long noncoding RNA UCA1 modulates breast cancer cell growth and apoptosis through decreasing tumor suppressive miR-143. Eur Rev Med Pharmacol Sci. 19:3403–3411. 2015.PubMed/NCBI

26 

Bian Z, Jin L, Zhang J, Yin Y, Quan C, Hu Y, Feng Y, Liu H, Fei B, Mao Y, et al: lncRNA-UCA1 enhances cell proliferation and 5-fluorouracil resistance in colorectal cancer by inhibiting miR-204-5p. Sci Rep. 6:238922016. View Article : Google Scholar : PubMed/NCBI

27 

Li GY, Wang W, Sun JY, Xin B, Zhang X, Wang T, Zhang QF, Yao LB, Han H, Fan DM, et al: Long non-coding RNAs AC026904.1 and UCA1: A 'one-two punch' for TGF-β-induced SNAI2 activation and epithelial-mesenchymal transition in breast cancer. Theranostics. 8:2846–2861. 2018. View Article : Google Scholar :

28 

Ringrose L and Paro R: Epigenetic regulation of cellular memory by the Polycomb and Trithorax group proteins. Annu Rev Genet. 38:413–443. 2004. View Article : Google Scholar : PubMed/NCBI

29 

Haupt Y, Alexander WS, Barri G, Klinken SP and Adams JM: Novel zinc finger gene implicated as myc collaborator by retro-virally accelerated lymphomagenesis in E mu-myc transgenic mice. Cell. 65:753–763. 1991. View Article : Google Scholar : PubMed/NCBI

30 

Sauvageau M and Sauvageau G: Polycomb group proteins: Multi-faceted regulators of somatic stem cells and cancer. Cell Stem Cell. 7:299–313. 2010. View Article : Google Scholar : PubMed/NCBI

31 

Yap DB, Chu J, Berg T, Schapira M, Cheng SW, Moradian A, Morin RD, Mungall AJ, Meissner B, Boyle M, et al: Somatic mutations at EZH2 Y641 act dominantly through a mechanism of selectively altered PRC2 catalytic activity, to increase H3K27 trimethylation. Blood. 117:2451–2459. 2011. View Article : Google Scholar :

32 

Hu JJ, Song W, Zhang SD, Shen XH, Qiu XM, Wu HZ, Gong PH, Lu S, Zhao ZJ, He ML, et al: HBx-upregulated lncRNA UCA1 promotes cell growth and tumorigenesis by recruiting EZH2 and repressing p27Kip1/CDK2 signaling. Sci Rep. 6:235212016. View Article : Google Scholar : PubMed/NCBI

33 

Yang C, Li X, Wang Y, Zhao L and Chen W: Long non-coding RNA UCA1 regulated cell cycle distribution via CREB through PI3-K dependent pathway in bladder carcinoma cells. Gene. 496:8–16. 2012. View Article : Google Scholar : PubMed/NCBI

34 

Xie X, Pan J, Wei L, Wu S, Hou H, Li X and Chen W: Gene expression profiling of microRNAs associated with UCA1 in bladder cancer cells. Int J Oncol. 48:1617–1627. 2016. View Article : Google Scholar : PubMed/NCBI

35 

Chen S, Shao C, Xu M, Ji J, Xie Y, Lei Y and Wang X: Macrophage infiltration promotes invasiveness of breast cancer cells via activating long non-coding RNA UCA1. Int J Clin Exp Pathol. 8:9052–9061. 2015.PubMed/NCBI

36 

You D, Jung SP, Jeong Y, Bae SY, Lee JE and Kim S: Fibronectin expression is upregulated by PI-3K/Akt activation in tamoxifen-resistant breast cancer cells. BMB Rep. 50:615–620. 2017. View Article : Google Scholar : PubMed/NCBI

37 

Xue M, Li X, Li Z and Chen W: Urothelial carcinoma associated 1 is a hypoxia-inducible factor-1α-targeted long noncoding RNA that enhances hypoxic bladder cancer cell proliferation, migration, and invasion. Tumour Biol. 35:6901–6912. 2014. View Article : Google Scholar : PubMed/NCBI

38 

Motawi TK, Abdelazim SA, Darwish HA, Elbaz EM and Shouman SA: Modulation of tamoxifen cytotoxicity by caffeic acid phenethyl ester in MCF-7 breast cancer cells. Oxid Med Cell Longev. 2016:30171082016. View Article : Google Scholar

39 

Grigsby JG, Parvathaneni K, Almanza MA, Botello AM, Mondragon AA, Allen DM and Tsin AT: Effects of tamoxifen versus raloxifene on retinal capillary endothelial cell proliferation. J Ocul Pharmacol Ther. 27:225–233. 2011. View Article : Google Scholar : PubMed/NCBI

40 

Lu Y, Liu WG, Lu JH, Liu ZJ, Li HB, Liu GJ, She HY, Li GY and Shi XH: lncRNA UCA1 promotes renal cell carcinoma proliferation through epigenetically repressing p21 expression and negatively regulating miR-495. Tumour Biol. 39:10104283177016322017. View Article : Google Scholar : PubMed/NCBI

41 

Chen WM, Huang MD, Sun DP, Kong R, Xu TP, Xia R, Zhang EB and Shu YQ: Long intergenic non-coding RNA 00152 promotes tumor cell cycle progression by binding to EZH2 and repressing p15 and p21 in gastric cancer. Oncotarget. 7:9773–9787. 2016.PubMed/NCBI

42 

Giordano A and Avantaggiati ML: p300 and CBP: Partners for life and death. J Cell Physiol. 181:218–230. 1999. View Article : Google Scholar : PubMed/NCBI

43 

Du J, Tong A, Wang F, Cui Y, Li C, Zhang Y and Yan Z: The Roles of PI3K/AKT/mTOR and MAPK/ERK Signaling Pathways in Human Pheochromocytomas. Int J Endocrinol. 2016:52869722016. View Article : Google Scholar : PubMed/NCBI

44 

Liang MH, Wendland JR and Chuang DM: Lithium inhibits Smad3/4 transactivation via increased CREB activity induced by enhanced PKA and AKT signaling. Mol Cell Neurosci. 37:440–453. 2008. View Article : Google Scholar

45 

Plantamura I, Cosentino G and Cataldo A: MicroRNAs and DNA-damaging drugs in breast cancer: Strength in numbers. Front Oncol. 8:3522018. View Article : Google Scholar : PubMed/NCBI

46 

Rocca A, Maltoni R, Bravaccini S, Donati C and Andreis D: Clinical utility of fulvestrant in the treatment of breast cancer: A report on the emerging clinical evidence. Cancer Manag Res. 10:3083–3099. 2018. View Article : Google Scholar : PubMed/NCBI

47 

Xia H and Hui KM: Mechanism of cancer drug resistance and the involvement of noncoding RNAs. Curr Med Chem. 21:3029–3041. 2014. View Article : Google Scholar : PubMed/NCBI

48 

Cheng N, Cai W, Ren S, Li X, Wang Q, Pan H, Zhao M, Li J, Zhang Y, Zhao C, et al: Long non-coding RNA UCA1 induces non-T790M acquired resistance to EGFR-TKIs by activating the AKT/mTOR pathway in EGFR-mutant non-small cell lung cancer. Oncotarget. 6:23582–23593. 2015. View Article : Google Scholar : PubMed/NCBI

49 

Pan J, Li X, Wu W, Xue M, Hou H, Zhai W and Chen W: Long non-coding RNA UCA1 promotes cisplatin/gemcitabine resistance through CREB modulating miR-196a-5p in bladder cancer cells. Cancer Lett. 382:64–76. 2016. View Article : Google Scholar : PubMed/NCBI

50 

Liu H, Wang G, Yang L, Qu J, Yang Z and Zhou X: Knockdown of long non-coding RNA UCA1 Increases the Tamoxifen Sensitivity of Breast cancer cells through inhibition of Wnt/β-catenin pathway. PLoS One. 11:e01684062016. View Article : Google Scholar

51 

Adams JM and Cory S: The Bcl-2 protein family: Arbiters of cell survival. Science. 281:1322–1326. 1998. View Article : Google Scholar : PubMed/NCBI

52 

Deng J, Deng H, Liu C, Liang Y and Wang S: Long non-coding RNA OIP5-AS1 functions as an oncogene in lung adenocar-cinoma through targeting miR-448/Bcl-2. Biomed Pharmacother. 98:102–110. 2018. View Article : Google Scholar

53 

Taylor MA, Sossey-Alaoui K, Thompson CL, Danielpour D and Schiemann WP: TGF-β upregulates miR-181a expression to promote breast cancer metastasis. J Clin Invest. 123:150–163. 2013. View Article : Google Scholar

54 

Srivastava N, Manvati S, Srivastava A, Pal R, Kalaiarasan P, Chattopadhyay S, Gochhait S, Dua R and Bamezai RN: miR-24-2 controls H2AFX expression regardless of gene copy number alteration and induces apoptosis by targeting antiapoptotic gene BCL-2: A potential for therapeutic intervention. Breast Cancer Res. 13:R392011. View Article : Google Scholar : PubMed/NCBI

55 

Vanhaesebroeck B, Leevers SJ, Ahmadi K, Timms J, Katso R, Driscoll PC, Woscholski R, Parker PJ and Waterfield MD: Synthesis and function of 3-phosphorylated inositol lipids. Annu Rev Biochem. 70:535–602. 2001. View Article : Google Scholar : PubMed/NCBI

56 

Yu X, Li R, Shi W, Jiang T, Wang Y, Li C and Qu X: Silencing of microRNA-21 confers the sensitivity to tamoxifen and fulvestrant by enhancing autophagic cell death through inhibition of the PI3K-AKT-mTOR pathway in breast cancer cells. Biomed Pharmacother. 77:37–44. 2016. View Article : Google Scholar : PubMed/NCBI

57 

de Groot RP, Ballou LM and Sassone-Corsi P: Positive regulation of the cAMP-responsive activator CREM by the p70 S6 kinase: An alternative route to mitogen-induced gene expression. Cell. 79:81–91. 1994. View Article : Google Scholar : PubMed/NCBI

58 

Linnerth NM, Greenaway JB, Petrik JJ and Moorehead RA: cAMP response element-binding protein is expressed at high levels in human ovarian adenocarcinoma and regulates ovarian tumor cell proliferation. Int J Gynecol Cancer. 18:1248–1257. 2008. View Article : Google Scholar : PubMed/NCBI

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March-2019
Volume 54 Issue 3

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Spandidos Publications style
Li Z, Yu D, Li H, Lv Y and Li S: Long non‑coding RNA UCA1 confers tamoxifen resistance in breast cancer endocrinotherapy through regulation of the EZH2/p21 axis and the PI3K/AKT signaling pathway10.3892/ijo.2024.5611. Int J Oncol 54: 1033-1042, 2019.
APA
Li, Z., Yu, D., Li, H., Lv, Y., & Li, S. (2019). Long non‑coding RNA UCA1 confers tamoxifen resistance in breast cancer endocrinotherapy through regulation of the EZH2/p21 axis and the PI3K/AKT signaling pathway10.3892/ijo.2024.5611. International Journal of Oncology, 54, 1033-1042. https://doi.org/10.3892/ijo.2019.4679
MLA
Li, Z., Yu, D., Li, H., Lv, Y., Li, S."Long non‑coding RNA UCA1 confers tamoxifen resistance in breast cancer endocrinotherapy through regulation of the EZH2/p21 axis and the PI3K/AKT signaling pathway10.3892/ijo.2024.5611". International Journal of Oncology 54.3 (2019): 1033-1042.
Chicago
Li, Z., Yu, D., Li, H., Lv, Y., Li, S."Long non‑coding RNA UCA1 confers tamoxifen resistance in breast cancer endocrinotherapy through regulation of the EZH2/p21 axis and the PI3K/AKT signaling pathway10.3892/ijo.2024.5611". International Journal of Oncology 54, no. 3 (2019): 1033-1042. https://doi.org/10.3892/ijo.2019.4679