Long noncoding RNA CCAT2 reduces chemosensitivity to 5‐fluorouracil in breast cancer cells by activating the mTOR axis

Abstract Breast cancer (BC) is the most prevalent cancer in women and the second leading cause for cancer‐related death in women. LncRNA CCAT2 is involved in BC cell drug sensitivity. Drug resistance of BC cells after chemotherapy is the main obstacle to therapeutic effects. This study explored whether BC cell drug sensitivity to 5‐Fu was related to lncRNA CCAT2‐regulated mTOR pathway. Normal breast tissues and BC tissues before/after neoadjuvant chemotherapy were collected, and CCAT2 expression was detected by RT‐qPCR. Correlation between CCATA2 expression and neoadjuvant chemotherapy efficacy was analysed using the Kendall's tau‐b correlation analysis. Normal breast epithelial cells and BC cell lines were cultured. BC cell lines were treated with 5‐Fu, and CCAT2 mRNA level in cells was detected. The 5‐Fu‐resistant MCF‐7/5‐Fu and MDA‐MB‐231/5‐Fu cells were treated with CCAT2 overexpression/knockdown or CCI‐779 (the mTOR pathway inhibitor). The mTOR pathway levels were detected. Expression of apoptosis‐related factors was identified. A subcutaneous xenograft model was carried out. High CCAT2 expression was detected in BC tissues and BC drug‐resistant cells after neoadjuvant chemotherapy, and a negative link was revealed between CCAT2 expression and efficacy of neoadjuvant chemotherapy. p‐mTOR/mTOR in 5‐Fu‐resistant BC cells with inhibited CCAT2 was decreased, while CCAT2 overexpression activated the mTOR pathway. IC50 value, proliferation, cells in S phase increased and apoptosis reduced after CCAT2 overexpression. After si‐CCAT2 or CCI‐779 treatment, the growth rate of transplanted tumours was inhibited, while promoted after CCAT2 overexpression. CCAT2 may reduce BC cell chemosensitivity to 5‐Fu by activating the mTOR pathway.


| INTRODUC TI ON
Breast cancer (BC) is the most common cancer in females with approximately 400,000 deaths per year worldwide. [1][2][3] As a severe public health problem in developed countries, it has also become a growingly important issue in developing countries, where the incidence of BC is growing at a rate of 5% per year. 4 Women of all ages are at risk of developing BC, and more than 90 percent of patients are possible to be cured through effective comprehensive treatment once diagnosed at early stage. 5 There are many established risk factors for BC, including aging, early menarche, family history, elder age at first live childbirth, late menopause, genetic causes, alcohol consumption, history of smoking, and obesity. 6 Radiotherapy plays a crucial part in BC treatment. 7 Besides, 5-fluorouracil (5-FU)-based combination chemotherapy is widely used for BC treatment, while recurrence and chemotherapy resistance are major obstacles leading to the high mortality in most patients. 8 Thereby, it is imperative to know the molecular mechanisms of chemotherapy resistance in BC for improving clinical efficacy.
Long noncoding RNAs (lncRNAs) are usually expressed in a specific manner, which exert specific functions in multiple kinds of diseases and human cancers. 9 More and more dysregulated lncRNAs are regarded as oncogenes that would promote the development and progression of cancers. 10 Additionally, the regulatory roles of ln-cRNAs in drug resistance are studied. For example, Leucci E recently pointed out that lncRNAs are applicable to sensitize cancer cells to multiple kinds of treatments. 11 Colon cancer-associated transcript 2 (CCAT2), mapping to 8q24, is upregulated in many tumour tissues and links with clinical characteristics and prognosis in multiple kinds of malignancies, like bladder cancer, gastric cancer, and cervical cancer. [12][13][14][15] In addition, CCAT2 is also proved to promote BC progression via the Wnt pathway. 16 The mammalian target of rapamycin (mTOR) is important in the extracellular and intracellular signals. 17,18 The mTOR pathway is downstream of PI3K/Akt, a well-known pathway mainly out of regulation in cancer, which is also involved in BC. 19 Now we tried to discuss the regulatory roles of CCAT2 in BC drug sensitivity to 5-Fu via the mTOR axis. 12 mg/kg d1-d5, 21 days as a cycle). Then, BC samples were harvested by core needle biopsy or excisional biopsy before chemotherapy, and by biopsy and chemotherapy after radical resection.

| Study subjects
Besides, the neoadjuvant chemotherapy efficacy in patients was assessed based on the unified standard formulated by WHO. In addition, before and after neoadjuvant chemotherapy, BC patients took the physical examination, coordinate mapping, breast B-mode ultrasound, and molybdenum target to evaluate the efficacy. Also, before chemotherapy, the size of tumours obtained from the excisional biopsy was quantified using B-mode ultrasonography. The changes of the tumours were followed up, and the efficacy was judged comprehensively by biopsy and pathology after radical resection. Samples were split into: complete remission (CR): no tumour detected using clinical means; partial remission (PR): reduced >50% in breast lumps; stable disease (SD): reduced <50% and increased <25% in breast lumps; progressive disease (PD): increased volume of breast lumps >25%. 20

| 5-ethynyl-2'-deoxyuridine (EdU) assay
Cells at the logarithmic growth phase were detached with trypsin and triturated into a single cell suspension. After counting, cells were seeded on 96-well plates at 1 × 10 4 cells per well. The culture liquid in each well was 200 μl. After 48 h, the cells entered an exponential growth phase. The cells were stained with EdU, and the 100μl complete culture medium containing EdU was put into each well for 2-h incubation. Afterwards, cells were rinsed in PBS twice and cultured in 100 μl 4% polyformaldehyde for 30 min. Next, cells were added with glycine (final concentration was 2 mg/ml) for oscillating on rockers for 5 min, added with 100 μl PBS for 5-min oscillating, and added with 0.5%Triton X-100 (100 μl) for 5 min oscillating. After PBS cleaning, 100 μl 1 × Apollo dyeing reaction solution was put into each well, and the dyeing solution was discarded after 30 min shaking. Later, cells were added with 0.5% Triton X-100 for 10 min shaking, followed by nucleus staining with 100 μl 1 × Hoechest 100 after discarding the 0.5% Triton X-100. After 30 min shaking, the dyeing reaction solution was removed, and cells were rinsed in PBS, observed under the microscope, and then analysed with Image-Pro Plus software.

| Flow cytometry
AnnexinV/propidiom iodide (PI) double staining method was used for cell apoptosis detection. After 48-h grouping, cells were harvested and adjusted to 1 × 10 6 /ml. Cell suspension (0.5 ml) was put into centrifugal tubes and added with 1.25 μl Annexin V-FITC (Nanjing Kaiji Biotechnology Co. Ltd.). After 15-min reaction in the dark, the cell suspension was centrifuged at 1000 rpm for 5 min with the supernatant removed. Next, cells were suspended with 0.5 ml pre-cooling combined buffer and added with 10 μl PI for immediate detection on a flow cytometer.
Cell cycle was detected using PI single staining. After grouping for 48 h, cells were fixed with −20°C pre-cooled 75% ice ethanol overnight at 4°C, then centrifuged, washed with cold PBS, and precipitated twice to remove the immobilization solution. Then cells were added with RNaseA and bathed in water for 30 min.
Afterwards, cells were dyed with PI, followed by even mixing. Finally, the red fluorescence was recorded via flow cytometry for cell cycle detection and counting the proportion of G0/G1, S, and G2/M cells.

| Subcutaneous xenograft nude mouse model
One hundred and forty BALB/C nude female mice (6 weeks, 16- week, and the tumour specimens were removed by surgical resection. The tumour specimens were subjected to pathological sections for immunohistochemistry and tissue homogenate for RT-PCR and Western blot to detect the corresponding indexes. After tumour removal, the nude mice were killed using the decapitation method.

| Immunohistochemistry
Tissue slices of transplanted tumours were taken and added with 30% H 2 O 2 to block the endogenous enzyme. The slices were heated to boil in antigen retrieval solution. After 5 min of cooling, the slices were reheated and cooled twice. After cooling, slices were added with 5% BSA solution with the excess liquid removed.
Then, slices were washed in PBS and developed in DAB (ZSGB-Bio). The staining results were observed under the microscope.
The cells with brown and yellow were regarded as positive expressions. Three slices were taken from each specimen. Five visual fields were selected at random from each slice to calculate the positive rate of Ki-67.

| Reverse transcription quantitative chain reaction (RT-qPCR)
The clinical sample tissues or cells or homogenate of transplanted tumour tissues were collected, and total RNA was extracted by  Table 1. The data were analysed using the 2 −ΔΔCt method. GAPDH was used as the internal parameter and normalized. The experiment was repeated three times.
After that, the membrane was washed with PBS +0.02% Tween 80 (PBST) three times, each time for 5 min, added with horseradish peroxidase labelled rabbit anti-mouse antibody (1:2000, Abcam), and incubated at room temperature for 2 h. Subsequently, the membrane was rinsed, added with ECL detection solution (Amersham Bioscience), and developed by conventional method X-ray film. The relative protein bands were analysed by Scion Image Analysis System (Scion Corporation). The relative content of protein was expressed as the ratio of the OD of the target protein to β-actin band.

| BC cell sensitivity to 5-Fu is negatively correlated with CCAT2 level
Before and after chemotherapy, CCAT2 expression was detected by RT-qPCR. It displayed that CCAT2 expression in BC tissues before neoadjuvant chemotherapy was higher than that in normal breast tissues. In addition, after chemotherapy, there were 30 cases of CR, 47 cases of PR, 17 of SD, and 6 of PD, with the total effective rate was 77.00%. Increased CCAT2 expression was found in residual tissues after chemotherapy ( Figure 1A   It showed that inhibition of the mTOR pathway attenuated the enhancement of CCAT2 on 5-Fu drug resistance in BC.

| Combination effects of CCAT2 and mTOR pathway on 5-Fu drug-resistant cell apoptosis in BC
The results showed that ( Figure 4)

| CCAT2 mediates the BC occurrence and its sensitivity to 5-Fu
After the successful modelling, the volume of transplanted tumours gradually expanded. As is shown in Figure 5A, the growth rate of transplanted F I G U R E . 3 Combination effect of CCAT2 and mTOR signaling pathway on 5-Fu drug resistance in BC. Note: A, cell growth curve; B, IC50 value in each group; C, EdU assay was performed to detect cell proliferation. The measurement data were expressed as mean ±standard deviation. Paired t-test was used for comparisons between two groups and one-way ANOVA was used for comparisons. Tukey's multiple comparisons test was used for the post hoc test. Compared with the empty plasmid group, * p < 0.05, compared with the CCI-779 group, # p < 0.05 tumours was faster in the CCAT2 group than in the NC group; the growth rate of transplanted tumours in the si-CCAT2 and CCI-779 groups was slower. The volume of transplanted tumours in the CCAT2 + CCI-779 group was less than that in the CCAT2 group (all p < 0.05). After removal of tumour in the nude mice, the mRNA level of CCAT2 was detected by RT-qPCR. Compared with the NC group, the level of CCAT2 was clearly increased in the CCAT2 group and CCAT2 + CCI-779 group and was clearly decreased in the si-CCAT2 group ( Figure 5B). The number of brownish yellow positive expressing tumour cells of proliferation marker protein Ki67 was detected by immunohistochemistry. The results

F I G U R E . 4
Combination effect of CCAT2 and mTOR pathway on 5-Fu drug-resistant cell apoptosis in BC. Note: A, flow cytometry was conducted to detect cell apoptosis; B, flow cytometry was applied to detect cell cycle; C, Western blot analysis was used to test apoptosisrelated factors. The measurement data were expressed as mean ±standard deviation. Paired t-test was used for comparisons between two groups and one-way ANOVA was used for comparisons. Tukey's multiple comparisons test was used for the post hoc test. Compared with the empty plasmid group, * p < 0.05, compared with the CCI-779 group, # p < 0.05 revealed that ( Figure 5C

| DISCUSS ION
Although many efforts have been made in early diagnosis and treatment methods, BC survival rate keeps low due to poor prognosis. 23 In addition, the process of BC treatment becomes more complex due to different therapeutic responses and outcomes. 24 32 Besides, abnormal proliferation and anti-apoptotic signals transmitted by the mTOR axis are involved in malignancy. 33,34 Further, activation of Akt, mTOR, and p70S6K has proved to be related to a more severe prognosis in BC patients. 35 Cells with highly expressed Akt were more sensitive to mTOR inhibitors, while inhibiting mTOR might recover their sensitivity to chemotherapies. 35,36 Inhibitors targeting mTOR may suppress drug resistance. 37 Previous evidence showed that CCAT2 knockout repressed endometrial cancer cell growth and invasion by binding to miR-216b, and miR-216b could negatively regulate Bcl-2 that could active the mTOR pathway. 38 Additionally, CCAT2 genetic polymorphisms were closely related to the susceptibility of lung cancer cells to platinum-based chemotherapy response; thus, it may be an underlying biomarker for the disease prediction and chemotherapy response. 39 Furthermore, inhibition of the mTOR pathway could reverse the anti-apoptosis effect of CCAT2 on 5-Fu drug-resistant BC cells.
Close interactions were found between apoptosis and autophagy via a common axis, among which the PI3K/AKT/mTOR is important in tumourigenesis. 40 Functionally, CCAT2 is considered to be a therapeutic target because its depletion can block cancer cell proliferation and invasiveness. 41 Besides, it is reported that higher CCAT2 expression is strongly linked with the more malignant molecular process in patients with gastric cancer (GC), and silencing CCAT2 prevented GC progression. 42 Silencing CCAT2 triggered enhancement of GC cell apoptosis by suppression of PI3K and mTOR. 43 Moreover, CCAT2 enhanced BC cell migration and decreased chemosensitivity to 5-FU. 30 In line with our study, silencing CCAT2 suppressed endometrial cancer cell growth by inactivation of the mTOR pathway. 38 Finally, in vivo experiments found that overexpression of CCAT2 in vivo can increase the drug resistance to 5-Fu in BC-resistant transplanted tumour tissue, but inhibition of mTOR pathway can reverse the effect of CCAT2 on 5-Fu resistance in BC-resistant transplanted tumour tissue, which was consistent with our cell experiment results.
To summarize, CCAT2 was at a high level in BC tissues and cells, and CCAT2 may reduce chemosensitivity to 5-Fu in BC cells by activating the mTOR pathway, manifesting CCAT2 may be a potential biomarker and therapeutic target for BC patients.

ACK N OWLED G EM ENTS
This work was partially supported by grants from the Natural

CO N FLI C T O F I NTE R E S T
The authors declare no potential conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.