BRAF-activated LncRNA functions as a tumor suppressor in papillary thyroid cancer

Long non-coding RNAs (lncRNAs) participate in cancer cell tumorigenesis, cell cycle control, migration, proliferation, apoptosis, metastasis and drug resistance. The BRAF-activated non-coding RNA (BANCR) functions as both an oncogene and a tumor suppressor. Here, we investigated BANCR's role in papillary thyroid carcinoma (PTC) by assessing BANCR levels in PTC and matched normal thyroid epithelial tissues from 92 patients using qRT-PCR. We also used lentiviral vectors to establish PTC cell lines to investigate the effects of BANCR overexpression on cancer cell proliferation, apoptosis, migration and invasion. Our results indicate BANCR levels are lower in PTC tumor tissues than control tissues. Decreased BANCR levels correlate with tumor size, the presence of multifocal lesions and advanced PTC stage. BANCR overexpression reduced PTC cell proliferation and promoted apoptosis, which inhibited metastasis. It also inactivated ERK1/2 and p38, and this effect was enhanced by treatment with the MEK inhibitor U0126. Finally, BANCR overexpression dramatically inhibited tumor growth from PTC cells in xenograft mouse models. These results suggest BANCR inhibits tumorigenesis in PTC and that BANCR levels may be used as a novel prognostic marker.


INTRODUCTION
The incidence of thyroid cancer, the most common endocrine malignancy, has increased by 3-fold over the last three decades. Approximately 80-85% of all thyroid cancers are papillary thyroid carcinoma (PTC) [1]. The 5-year survival rate of patients with PTC is over 95%; however, PTCs with more aggressive phenotypes can occasionally disseminate into distant tissues and lymph nodes or dedifferentiate into more lethal thyroid cancers [2]. Conventional management of PTC, including surgery, hormone therapy and radioactive iodine therapy are generally not curative in metastatic cases [1,3]. Therefore, novel therapeutic approaches and individual treatment strategies are needed to treat patients with metastatic thyroid cancer [2]. For example, the genetic and epigenetic alterations associated with PTC pathogenesis may serve as novel prognostic markers or therapeutic targets.
BRAF-activated lncRNA (BANCR) was first identified by Flockhart RJ et al. in 2012 via RNA sequencing. This molecule is a non-annotated, 693-bplong transcript on chromosome 9 that reduces cell migration in melanoma [8]. BANCR has been reported to function as a tumor suppressor in lung cancer [9] while acting as an oncogene in various types of cancer Research Paper www.impactjournals.com/oncotarget [9][10][11][12]. Here, we carried out in vitro and in vivo studies to elucidate BANCR's role in PTC.

BANCR expression is downregulated in PTC tissue and cell lines
To measure BANCR expression in PTC tissue, total RNA extracted from 92 paired human PTC samples and adjacent histologically normal tissues was assessed using SYBR Green qPCR analysis. As shown in Figure 1A-1B, BANCR expression was downregulated in 68.5% (63/92; P < 0.05) of the PTC tissue compared to the normal tissue.

BANCR overexpression inhibits PTC proliferation and induces apoptosis in vitro and in vivo
To elucidate the role of BANCR in PTC cell proliferation and apoptosis, we established BANCRoverexpressing PTC cell models using the TPC-1 and K1 cell lines and lentivirus vectors. Increased BANCR expression in these infected PTC cell lines was confirmed using qPCR ( Figure 2A). Proliferation of BANCRoverexpressing PTC cell lines TPC-1-BANCR and K1-BANCR was evaluated in vitro using a CCK-8 cell proliferation assay. Our results show that K1-BANCR proliferation is impaired compared to that of control cells (P < 0.05), while TPC-1-BANCR proliferation is unaffected ( Figure 2B). Apoptosis analysis by flow cytometry showed that BANCR overexpression promotes apoptosis in both the TPC-1-BANCR and K1-BANCR cell lines compared to TPC-1-NC (P < 0.05) and K1-NC (P < 0.05) control cells ( Figure 2C).
To investigate the effects of BANCR on PTC cell proliferation and apoptosis in vivo, we used subcutaneous xenograft mouse models engrafted with either K1-BANCR or control K1-NC cells. Average tumor weight and size were smaller in the K1-BANCR group than in the control group, indicating that tumor growth is inhibited in K1-BANCR mice ( Figure 3A-3B). Furthermore, Ki67 + tumor cell number as measured by IHC is reduced ( Figure 3C) and BANCR expression measured by qPCR is increased ( Figure 3D) in the K1-BANCR group compared with the control group.

BANCR overexpression suppresses PTC cell migration and invasion
To investigate BANCR's role in PTC cell migration and invasion, we performed wound-healing and transwell invasion assays using TPC-1-BANCR and K1-BANCR cells. The wound-healing assay results revealed that BANCR overexpression slows down the closing rate of scratch wounds compared with that of control cells (P < 0.05) ( Figure 4A-4B). The transwell invasion assay results showed that increased BANCR expression inhibits TPC-1-BANCR and K1-BANCR (P < 0.05) cell invasion compared with controls ( Figure 4C).

ERK1/2 and P38, but not AKT or JNK, are inactivated by BANCR upregulation
To investigate whether the ERK/MAPK signaling pathway is involved in the upregulation of BANCR observed in PTC cells, the expression levels of total and phosphorylated ERK, AKT, MEK, JNK and P38 were measured by western blot. As shown in Figure 5A, phosphorylated ERK and phosphorylated P38 were downregulated in K1-BANCR cells, but not in TPC-1-BANCR cells, while the total levels of these molecules (i.e., both phosphorylated and non-phosphorylated) did not change in either cell line. However, neither total nor phosphorylated AKT and JNK levels were affected in either cell line. In addition, treatment with the MEK inhibitor U0126 inhibited proliferation (P < 0.05) ( Figure 5B) and impaired invasion (P < 0.05) ( Figure 5C) in K1-BANCR cells relative to control K1-NC cells. Furthermore, levels of phosphorylated ERK were reduced in K1-BANCR cells following treatment with U0126 ( Figure 5D).

BANCR downregulation correlates with poor prognosis in PTC patients
To test whether BANCR correlates with prognosis in PTC patients, we measured the correlation between BANCR expression and several pathological parameters in PTC patients. Our results show that downregulation of BANCR expression correlates with tumor size (P < 0.05), the presence of multifocal lesions (P < 0.05) and advanced pathological stage (P < 0.05) in PTC tissues. However, BANCR expression did not correlate with other clinicopathologic parameters, such as age (P = 0.214), gender (P = 0.711), extrathyroidal extension (P = 0.406), or lymph node metastasis (P = 0.992) in PTC tissue compared with normal thyroid tissues (Table 1). These results suggest that low BANCR levels are associated with poor outcomes in PTC patients.

DISCUSSION
Ectopic expression of BANCR shown in multiple cancer types may due to tumor heterogenicity; these findings motivated us to explore the effects of BANCR in PTC and to delineate the associated mechanisms. In the current study, we found that BANCR levels were www.impactjournals.com/oncotarget  decreased in 92 human PTC tissues compared to adjacent normal thyroid tissues. We also found that lower BANCR expression correlates with advanced tumor stage and size. We observed elevated BANCR levels in 86.2% earlystage PTC and 13.8% late-stage PTC tumors. Our results suggest that PTC tumors with high BANCR levels are more differentiated than those with low BANCR levels and that BANCR may function as a tumor suppressor in PTC.
We also investigated the role of BANCR in PTC cells utilizing BANCR-overexpressing TPC-1 and K1 cells as in vitro models. Our results show that BANCR overexpression inhibits cell proliferation, migration and invasion while inducing tumor cell apoptosis. In xenograft mouse models, K1 cells overexpressing BANCR exhibited lower tumor proliferation. Our in vitro and in vivo data show that BANCR acts as a tumor suppressor that inhibits tumor growth and metastasis, in line with the clinicopathological data. In a previous study [12] Activation of the MAPK signaling pathway promotes human cancer cell proliferation and survival, particularly in PTC [3,13]. BRAF and RAS mutations activate the MAPK signaling pathway in thyroid cancer, inducing the expression of phosphorylated ERK1/2, JNK, and P38 kinase, amplifying oncogenicity [3]. BANCR is normally activated by BRAF; however, 30-60% of PTCs contain the BRAF V600E mutation, which has been associated with higher mortality [14] and recurrence [15,16] rates. BRAF-activated BANCR has been shown to promote cell migration and proliferation in melanoma [10] and lung carcinoma [17] via the MAPK signaling pathway. However, whether BANCR exerts MAPK-dependent effects in PTC remains unclear. Here we demonstrate that BANCR overexpression in the BRAF V600E -mutated PTC cell line K1 inactivates ERK1/2 and P38. Furthermore, this inactivation was blocked by the MAPK pathway selective inhibitor U0126, without altering BRAF's effects in wildtype TPC-1 cells. These results suggest interplay between the ERK/MAPK signaling pathway and BANCR in PTC, highlighting BANCR as a potential therapeutic target for the treatment of BRAF V600E -positive thyroid cancer.
In this study, we report that BRAF-activated BANCR may function as a tumor suppressor via the ERK/MAPK signaling pathway in PTC. However, our results might be biased towards early-stage PTC given the disparity between the number of early-stage (n = 66) vs. late-stage (n = 26) PTC patients in our sample. This difference might result from new diagnostic methods that allow detecting PTC earlier, yielding more patients   with micro-papillary cancer (early stage; tumor size < 0.5 cm) than with papillary cancer (late stage; tumor size > 0.5 cm). Interestingly, the results from our cell proliferation and apoptosis assays varied for TPC-1 and K1 cells overexpressing BANCR. This might be attributed to the different BRAF status of these cells, warranting further investigations.
In conclusion, we demonstrated that BANCR induces proliferation, migration and invasiveness in BRAF-mutated PTC cells partially via the ERK/MAPK signaling pathway and that BANCR expression is downregulated in human PTC tissues. Therefore, our results suggest that BANCR may serve as a potential therapeutic target and that decreased BANCR levels could be used as a novel prognostic marker for PTC.  The following clinicopathological characteristics were documented: age, gender, and PTC features, including tumor size, location, multifocal lesions, bilateral lesions, extrathyroidal extension, lymph node metastasis and TNM classification. The resected tissue samples were immediately snap-frozen in liquid nitrogen and stored at -80°C for further use.

Cell culture and treatment
Four PTC cell lines (TPC-1, K1, BCPAP and CGTH-W3) and a normal human thyroid epithelial cell line (Nthy-ori 3-1) were used. Nthy-ori-3 and TPC-1 cell lines were provided by Dr. Haixia Guan. K1 was gifted by Dr. Zebing Liu. BCPAP cell lines were purchased from the Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China). The CGTH-W3 cell line was obtained from Dr. Jun Xiang. All cell lines were cultured in RPMI 1640 (GIBCO) supplemented with 10% fetal bovine serum (FBS; GIBCO), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C in 5% CO 2 . For inhibitor treatment experiments, the cell lines were treated with either DMSO or 10 μM U0126 for 48 h.

Total RNA extraction, reverse transcription and quantitative real-time PCR
Total RNA was extracted from tissues and cultured cells using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. A total of 1 μg of RNA was reverse-transcribed using a PrimeScript RT reagent kit (Takara, Dalian, China). For quantitative real-time PCR (qPCR), cDNA was amplified using SYBR Green Premix Ex Taq (Takara, Dalian, China) following the manufacturer's instructions. BANCR expression was normalized against GAPDH mRNA expression in three independent experiments. The following primers were used for qPCR: BANCR forward sequence, 5′-ACAGGACTCCATGGCAAACG-3′; reverse sequence, 5′-ATGAAGAAAGCCTGGTGCAGT-3′. The results were analyzed and calculated relative to cycle threshold values and then converted into fold changes.

Generation of BANCR-overexpressing cell lines
Recombinant lentiviruses packing genomes encoding human full-length BANCR (LV-BANCR) and a negative control sequence (LV-NC) were purchased from GENECHEM (Shanghai, China). TPC-1 and K1 cells were infected with either LV-BANCR or LV-NC plus 5 μg/ml polybrene (GENECHEM, Shanghai, China). Infection efficiency was examined by qPCR 48 h after infection, and the cells were selected with 2 μg/ml puromycin to create stable overexpression cell lines. The infected TPC-1 and K1 cells were denoted as TPC-1-BANCR, K1-BANCR, TPC-1-NC and K1-NC in accordance with the lentiviral construct used in each. BANCR expression was confirmed by qPCR.

CCK8 cell proliferation assay
Cell proliferation was determined using a cell counting kit-8 (CCK-8) assay according to the manufacturer's protocol. Briefly, cells were seeded into 96-well plates at 4 × 10 3 cells/well. An aliquot of 10 μl CCK-8 solution was added to each well, and the plate was incubated for 4 h at 37°C. At the indicated time points, the absorbance at 450 nm was measured using a spectrophotometer. For each group, data from five wells were pooled.

Analysis of apoptosis by flow cytometry
Cells were harvested, washed in phosphatebuffered saline (PBS), and stained with Annexin V-PE and 7-AAD in Annexin-binding buffer (BD Biosciences, Mississauga, ON) for 15 min at room temperature in the dark. As controls, cells were either not stained, stained with Annexin V-PE only or stained with 7-AAD only. Following the addition of 400 μl binding buffer, the cells were immediately analyzed by flow cytometry. Apoptotic cells were marked based on Annexin V expression (Annexin V+/7-AAD-and Annexin V+/7-AAD+), quantified and compared to the controls from each group. The experiment was performed in triplicate.

Wound-healing assay
To evaluate cell migration, a wound-healing assay was performed. To accomplish this, 2 × 10 5 cells/well were seeded into 6-well plates and cultured until the cells reached 90% confluence. Artificial wounds were created by scraping using a sterile 200 μl pipette tip. The streaked cells were then washed with PBS 3 times and cultured in medium containing 1% FBS for 48 h. Cell migration was observed at 0 h and 48 h by analyzing phase-contrast images that were captured using an inverted microscope. The distance between the two edges of a wound was calculated using Image J software.

Transwell cell invasion assay
To perform a transwell invasion assay, 2 × 10 4 cells were suspended in 100 μl serum-free medium and placed on the non-coated membranes of the upper chambers of a transwell plate insert (24-well insert, 8 μm pore size; BD Biosciences). Following this, 600 μl medium with 10% FBS was added into each lower chamber. After incubation at 37°C for 24 h, the cells remaining on the upper membranes were removed with a cotton swab. The migrated or invaded cells were fixed with 4% paraformaldehyde in PBS buffer, stained with 0.1% crystal violet, counted, and photographed under an inverted microscope over 5 different fields per filter. The experiments were independently repeated in triplicate.

Western blotting
Cells were lysed in RIPA protein extraction reagent supplemented with a protease inhibitor cocktail (Roche, CA, USA) and phosphatase inhibitor cocktail (Roche). Protein concentration was measured using a bicinchoninic acid assay (BCA). Equal amounts of total protein lysate were separated by 10% SDS-PAGE and transferred onto PVDF membranes. After blocking in 5% non-fat milk, the membranes were probed with targeted primary antibodies overnight at 4°C and detected by incubating with specific secondary antibodies for 2 h at room temperature. ECL chromogenic substrate with HRP was used to visualize the protein bands, and the intensities of the bands were quantified using Image J software. GAPDH served as an internal loading control. Primary anti-human antibodies against total Akt, Erk1/2, P38, and JNK and phosphorylated-Akt, Erk1/2, P38, and JNK were purchased from Cell Signaling Technology (MA, USA).

Tumour xenograft animal model
All animal experiments were performed based on a protocol that was approved by the Shanghai Experimental Animal Center (Chinese Academy of Sciences). Specific pathogen-free (SPF) female athymic nude mice between six and eight weeks of age were purchased from the Shanghai Laboratory Animal Center. After one week of being housed and fed in a designated animal room, each mouse was subcutaneously inoculated into the right scapula with either 1 × 10 7 K1-BANCR cells or control K1-NC cells in 50% Matrigel (BD Biosciences). Tumor size and weight were monitored twice a week. At 25 days after inoculation, the mice were sacrificed to assess tumor size. Tumors from each mouse (n = 4) were randomly selected for immunohistochemical (IHC) analysis. Fresh frozen tumor sections were used for mRNA analysis.

Immunohistochemistry
Formalin-fixed, paraffin-embedded xenograft tissue was used for IHC analysis. Briefly, 5 μm-thick paraffin sections were deparaffinized in xylene and rehydrated in a 100%, 95%, 75% ethanol gradient. Then, the tissue sections were incubated in 30% H 2 O 2 for 30 min to quench endogenous peroxidase activity. After antigen retrieval in heated 10 mM citrate buffer for 10 min, the tissue sections were immunostained with mouse anti-human Ki-67 primary antibody overnight at 4°C. Horseradish peroxidaseconjugated mouse secondary antibody was added for 1 h at room temperature. A DAB substrate development system was used to detect protein expression. Images were obtained using an Olympus IX71 inverted microscope. Ki67+ cells were counted using Image J software.

Statistical analysis
All statistical analyses were performed using GraphPad Prism 5.1. The results are presented as the mean ± the standard error of the mean. The statistical significance of inter-group differences was determined using the Student's t-test. Correlation analyses were assessed using the Pearson correlation and linear regression analyses.