Anoctamin5 regulates cell migration and invasion in thyroid cancer

  • Authors:
    • Zhengyan Chang
    • Chunmiao Cai
    • Dongyan Han
    • Yaohui Gao
    • Qianyu Li
    • Lijin Feng
    • Wei Zhang
    • Jiayi Zheng
    • Jiaoying Jin
    • Huizhen Zhang
    • Qing Wei
  • View Affiliations

  • Published online on: September 1, 2017     https://doi.org/10.3892/ijo.2017.4113
  • Pages: 1311-1319
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Abstract

Anoctamin/TMEM16 family members have recently been identified as novel calcium-activated chloride channels, and dysregulation of many family members participates in tumorigenesis and progression. However, the exact role of anoctamin5 (ANO5), one member of this family, in thyroid cancer is still not clarified. In this study, we firstly found that the expression levels of ANO5 was significantly downregulated in thyroid cancer compared to adjacent normal tissue by mining the public GEO database. Subsequently, we further demonstrated that the expression levels of ANO5 was significantly downregulated in 69.5% (57/82) clinical thyroid cancer tissues using real-time PCR assay. Moreover, western blot assay also showed that ANO5 was downregulated in papillary thyroid cancer and follicular thyroid cancer compared to adjacent noncancerous tissues. Furthermore, some biological and functional in vitro experiments proved that ANO5 knockdown promotes thyroid cancer cell migration and invasion but overexpression of ANO5 inhibits these phenotypes. By analyzing gene set enrichment, we found that lower ANO5 expression was positively associated with JAK/STAT3 signaling pathway. Collectively downregulation of ANO5 promotes thyroid cancer cell migration and invasion by affecting JAK/STAT3 pathway.

Introduction

Papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC) are the most common types of thyroid cancer, accounting for 94% of all cases (1). Most of them are differentiated and have a good prognosis with a 10-year survival rate of >92% (2). But among clinical staging criteria, metastatic lymphadenopathy is one of the best predictors of a poor prognosis, recurrence and motility (35) as it likely reflects aggressive primary tumor biology (6,7) (seer.cancer.gov/statfacts/html/oralcav.html). So exploring the mechanism of lymph node metastasis will deepen our understanding of malignant characteristics of some differentiated thyroid cancer.

Anoctamin family (ANO, also known as TMEM16) contains 10 members which are identified as putative intracellular calcium activated chloride channels (8,9). It has been reported that some members of anoctamin family are overexpressed in cancer (10), moreover, overexpression of ANO1 and ANO6 can increase cancer cell migration (11,12). Anoctamin5 (ANO5), also referred to as TMEM16E, is one member of the anoctamin family. Somatic mutation or microdeletion of ANO5 usually results in muscular dystrophy (1318), but its exact role in tumorigenesis and cancer progression is still not clear.

We first evaluated the expression profile of this family in thyroid cancer by mining the public GEO database. We discovered and proved the downregulation of ANO5 expression in thyroid cancer. Thereafter, we revealed that downregulation of ANO5 is negatively associated with lymph node metastasis and inhibition of ANO5 promotes the migration and invasion of thyroid cancer cells. In addition, we also found that lower ANO5 expression was positively associated with JAK/STAT3 pathway which is well-known to be activated during cancer metastasis (19,20). The present results provided novel insight into ANO5's function in thyroid cancer metastasis.

Materials and methods

Tissue specimens and cell lines

Thyroid cancer tissue samples used in this study were harvested from Shanghai Tenth People's Hospital between November, 2013 and December, 2015. Written informed consent from all patients was obtained. Thyroid cancer cell lines TPC-1 and FTC-133 were obtained by Dr Lei Ye in Rui-Jin Hospital. TPC-1 cells were maintained in RPMI-1640 medium and FTC-133 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (both from HyClone, Logan, UT, USA) 100 U/ml penicillin, and 100 µg/ml streptomycin.

Antibodies and reagents

The target antibodies were purchased from Abcam.

RNA extraction and real-time PCR

Total RNA was extracted with TRIzol® (Invitrogen, Garlsbad, CA, USA), following the manufacturer's protocol. The OD260/OD280 ratio of RNA ranged from 1.8 to 2.0. Reverse transcription was performed in 20 µl reaction volume with 2 µg of RNA using M-MLv reverse transcriptase kit (Takara, Otsu, Japan). Quantitative real-time PCR was carried out using aBI 7900 Detection system with the SYBR Premix Ex Taq™ (Takara). Primer sequences specific to 38 genes and housekeeping gene GaDPH are listed in Table I.

Table I

Real-time PCR primers utilized in this study.

Table I

Real-time PCR primers utilized in this study.

GenesSequences
ADH1BF: CCCGGAGAGCAACTACTGC
R: AACCAGTCGAGAATCCACAGC
ANO5F: TTTTGGAAACAACGACAAGCCA
R: ACCATACTGGTGACGACAAGAG
BMP2F: ACCCGCTGTCTTCTAGCGT
R: TTTCAGGCCGAACATGCTGAG
CDH16F: GTCCCTAGAGCCTATCCACCT
R: TGCATTCACTTCAAAGGGTCC
CLCNKBF: GCCCTCCTTCTATGATGGCAC
R: CCTGCCCTTGGTGACAGTG
DLG2F: CCTCTACGTCAGAGCCATGTT
R: ATCGGGCACGTTCCTTTCTTT
DPP6F: CTACGCCGCCATCAATGATTC
R: GGGATAGTGGTAGGGCTTCAC
DPY19L2F: CTTCCAGTTCGTCCGTAATTCC
R: TCTCCCGTTCCAAAGATGAGAG
EDN3F: GGGACTGTGAAGAGACTGTGG
R: AGACACACTCCTTGTCCTTGTA
ERBB4F: GTCCAGCCCAGCGATTCTC
R: AGAGCCACTAACACGTAGCCT
ESRRGF: GCCCTCACTACACTGTGTGAC
R: CCTGCTAATTTGGACTGGTCTT
FABP4F: ACTGGGCCAGGAATTTGACG
R: CTCGTGGAAGTGACGCCTT
FHL1F: AAGAACCGCTTCTGGCATGAC
R: CCCCTTGTACTCCACGTTTTG
FOSF: CCGGGGATAGCCTCTCTTACT
R: CCAGGTCCGTGCAGAAGTC
GHRF: CCATTGCCCTCAACTGGACTT
R: AATATCTGCATTGCGTGGTGC
GNA14F: GAGCGATGGACACGCTAAGG
R: TCCTGTCGTAACACTCCTGGA
GPM6AF: ATTCCCTATGCCTCTCTGATTGC
R: GCCATCTCAAAGTAGGTTTGCAG
HGDF: ATTTACACCGAGTTTGGCAAGA
R: GGTCTCCTCAAAGACATCTATGC
ITPR1F: ATTGCTGGGGACCGTAATCC
R: TCCAATGTGACTCTCATGGCA
KIAA1324F: GGAGCTTCATGCCTGCAAAGA
R: CATCAAACCGAATGCCTGTGC
LIFRF: TGGAACGACAGGGGTTCAGT
R: GAGTTGTGTTGTGGGTCACTAA
LRP2F: GTTCAGATGACGCGGATGAAA
R: TCACAGTCTTGATCTTGGTCACA
PID1F: CGTGGAGTGCGAGAGCAAG
R: CTGGGAAACCTCTTCGGAGGA
PLA2R1F: TAAATCGGTTCTGACCCTGGA
R: GCCACCGTAAGGAAACGAG
PTHLHF: AAGGTGGAGACGTACAAAGAGC
R: CAGAGCGAGTTCGCCGTTT
RYR2F: CATCGAACACTCCTCTACGGA
R: GGACACGCTAACTAAGATGAGGT
SLC26A4F: TGGTGGGATCTGTTGTTCTGA
R: GGATCTGCCAAGTACCTCACT
SLC26A7F: GTGACCCAAGGATTGGCCTTT
R: GGCAACATGATGTCCCATTCC
SLC4A4F: GGGTGCCCTGACTGAAGTTC
R: GGTCGTGCCTGTCTTTTGCT
TFF3F: CCAAGCAAACAATCCAGAGCA
R: GCTCAGGACTCGCTTCATGG
TMEM171F: AACCGCTAAACGAGACAGACA
R: ACACAATCCCACAAGCACAATC
TMPRSS3F: TGGAAGGGTCACTACGCAAAT
R: AGTGGTGTAATGCAGTCACCT
TNFRSF11BF: GCGCTCGTGTTTCTGGACA
R: AGTATAGACACTCGTCACTGGTG
TPOF: GCCAACAAGCGGAGTGATTG
R: GGGCAGCATGTAAGGGAGAC
TPPPF: AGGGGTGACGAAAGCCATC
R: CGGACACATAGCCTGACTCG
WSCD2F: AAACCTGTGCGCTTCTTTACC
R: GTACCTGCGAGCAATGCTTGA
KLK7F: TAATGACCTCATGCTCGTGAAGC
R: CAGCCGGAGACAGTACAGG
GaPDHF: CTGGGCTACACTGAGCACC
R: AAGTGGTCGTTGAGGGCAATG

[i] F, forward; R, reverse.

Protein extraction and western blot analysis

Total cellular proteins were extracted using cell lysis buffer containing 50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 10% 2-mercaptoethanol, and protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Then protein concentration was determined using the BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein (30 µg) was subjected to electrophoresis by SDS-PAGE on the 10% gel and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% bovine serum albumin (BSA) and 0.1% Tween-20 in PBS for 2 h at room temperature. After incubation with the appropriate primary antibody overnight at 4°C with anti-ANO5 (1:500), anti-STAT3 (1:1,000), anti-p727-STAT3 (1:1,000) (all from Abcam), and anti-GAPDH (1:3,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) antibodies, membranes were washed and incubated with the IRDye 800CW secondary antibodies for 1 h at room temperature. The labeled protein bands were detected using the Odyssey Infrared Imaging system (Li-COR Biosciences, Lincoln, NE, USA). GADPH was used as a loading control.

Construction of ANO5 expression vectors

Human full-length ANO5 cDNA (GenBank accession no. NM_001142649.1) was acquired from normal thyroid tissues. Primers for PCR amplification were designed as follows: forward, 5′-ATA TCT AGA ATG GGC GAC CCG GAT CTC CTG GAA G-3′ and reverse, 5′-ACG CGG CCG CTT AGA GTG TTG ATT TAG CCA GCT G-3′. The PCR product was subcloned into the pCDH vector (Systembioscience, Inc.) and verified by restriction digestion and DNA sequencing.

Lentivirus production

All recombinant lentiviruses were produced by transfecting HEK293T cells according to standard protocols. In brief, sub-confluent HEK293T cells were co-transfected with 4 µg pCDH-ANO5 plasmid vector, 3 µg PLP1, 3 µg PLP2 and 2 µg PLP-VSVG in 50 µl Lipofectamine 2000. The medium was changed after 16 h and supernatant was harvested 72 h later.

siRNA synthesis

One siRNA against ANO5 was chemically synthesized and the sequences were listed as follows: siRNA-1269, 5′-GCU GUA GUU GGC UUA GCU UTT-3′; siRNA-2487, 5′-GCU CAU AGC AUA GGU GUU UTT-3′. The non-targeting nucleotides were used as a negative control siRNA-NC, 5′-UUC UCC GAA CGU GUC ACG UTT-3′.

In vitro migration and invasion assays

Cell migration/invasion assays were performed using 24-well Transwells (6.5 mm pore size, Costar), coated without (migration) or with (invasion) Matrigel. TPC-1 and FTC-133 cells were starved in serum-free media for 12 h, trypsinized and washed three times in RPMI-1640/DMEM containing 0.1% BSA. Cells (1×104) were seeded into the upper chamber, and 600 µl medium containing 10% FBS was placed in the lower chamber at 37°C in 5% CO2. After 48 h incubation, Matrigel and cells remaining in the upper chamber were removed. Cells on the lower surface of the membrane were fixed in 4% paraformaldehyde and stained with Coomassie Brilliant Blue, photographed and counted under a dissecting microscope. Every experiment was repeated three times.

Gene set enrichment analysis (GSEA)

GSEA was carried out using GSEA software according to literature (21,22). Firstly we classified thyroid cancer samples (GSE3678) into ANO5 high expression and ANO5 low expression group according to ANO5 expression, subsequently three gene sets including KEGG, Hallmark and BioCarta were chosen to conduct GSEA.

Statistical analysis

The statistical difference of quantitative variables was evaluated with Student's t-test using GraphPad Prism 5 software and R ×64 3.2.2 software, p<0.05 was considered statistically significant.

Results

ANO5 is downregulated in thyroid cancer

In order to find differentially expressed genes in thyroid cancer and adjacent normal tissue, a GEO dataset (GSE3678) which contains seven cancerous and seven normal tissues was chosen. A total of 38 genes were discovered to display at least 3-fold alterations (Fig. 1A). Furthermore, we evaluated expression of these 38 genes in clinical thyroid cancer tissues and adjacent noncancerous tissues by real-time PCR (Fig. 1B). We found that the expression of some genes including TPO (23), ANO5, ERBB4 (24) and SLC4A4 (25) genes were in accordance with the results of GEO gene expression atlas (Fig. 1A), then we focused on ANO5 and its family through biological information analysis and previous literature studies. In order to investigate the role of anoctamin family in thyroid cancer progression, we first measured the expression profile of this family in thyroid cancer tissue samples by mining a public database (GSE3678). Interestingly, only ANO5 was significantly downregulated in thyroid cancer compared to adjacent noncancerous tissues (Fig. 1C). Subsequently we confirmed that 69.5% (57/82) thyroid cancer showed up to 2-fold downregulation of ANO5 by real-time PCR assay (Fig. 1D). Similarly, western blot assay also proved that ANO5 is downregulated in PTC and follicular thyroid cancer compared to adjacent noncancerous tissues (Fig. 1E). Collectively, these data revealed that ANO5 expression is significantly downregulated in thyroid cancer.

Downregulation of ANO5 is positively associated with lymph node metastasis of thyroid cancer

In order to explore the correlationship between downregulation of ANO5 and clinical characteristics, we analyzed RNA-seq data of thyroid cancer from TCGA database. We found that ANO5 expression is significantly associated with lymph node stage (N0 or N1, p=0.01) and neoplasm histologic type (follicular or classical/papillary, p=6.97E-05) (Table II), ANO5 expression levels in thyroid cancer with lymph node metastasis is lower than that without lymph node metastasis (Fig. 2A). Real-time PCR also confirmed the downregulation of ANO5 in thyroid cancer with lymph node metastasis compared to that without lymph node metastasis (Fig. 2B). In addition, we proved, as shown in Table III, ANO5 expression was significantly associated with lymph node metastasis (lymph node negative vs. lymph node positive, p=0.0038, χ2=8.376) in our in-house samples. There are no significant association between ANO5 expression and other tumor characteristics, such as age, gender, size, Hashimoto background or multifocal disease (Table III). In order to carry out cellular and functional experiments, we next evaluated ANO5 expression in thyroid cancer cell lines, including FTC-133 and TPC-1, by real-time PCR, data show that ANO5 is undetectable in PTC cell line (Fig. 3A). In total, these findings indicate that downregulation of ANO5 is positively associated with lymph node metastasis of thyroid cancer.

Table II

The correlation of ANO5 expression with clinical characteristics of thyroid cancer from TCGA.

Table II

The correlation of ANO5 expression with clinical characteristics of thyroid cancer from TCGA.

Clinical characteristicNo. of patientsANO5
p-value
HighLow
Age (years)
 ≥601176552
 <603791831960.169
Gender
 Male1346272
 Female3621861760.312
Recurrence
 Yes462224
 No4362152210.848
Overall survival (month)
 ≥60974156
 <603992071920.090
Neoplasm histologic type
 Classical/usual354161193
 Follicular10068326.97E-05
 Tall cell3516190.990
Tumor stage
 T11417170
 T216486780.717
 T316779880.594
 T42210120.669
Metastasis stage
 M0276136140
 M19540.711
Lymph node stage
 N0224122102
 N1222941280.010

Table III

The correlation of ANO5 expression with clinical characteristics of PTC.

Table III

The correlation of ANO5 expression with clinical characteristics of PTC.

Clinical characteristicNo. of patientsANO5 expression (ct)
p-valueχ2
>30≤30
Age (years)
 <453217150.32440.971
 ≥45502129
Gender
 Male2514110.24561.349
 Female572633
Microcarcinoma
 Yes3316170.68460.165
 No492623
Hashimoto's thyroiditis
 Yes4320230.84150.04
 No391920
Multifocal
 Yes2913160.23561.407
 No533122
LN metastasis
 Yes3222100.00388.376
 No501832

[i] T indicates papillary thyroid cancer tissues. Ct=30 is the average of cancer tissues.

Knockdown of ANO5 promotes FTC-133 cell migration and invasion

To understand whether ANO5 knockdown affects thyroid cancer cells migration and invasion, we synthesized siRNA against ANO5 (siANO5) and transfected FTC-133 cells which have higher ANO5 expression. Real-time PCR and western blot results indicate that siRNA significantly decreased ANO5 expression (Fig. 3A and B). Cell migration assay showed that knockdown of ANO5 increased the migrated cell number (Fig. 3C). Meanwhile, inhibition of ANO5 also promoted FTC-133 cell invasion (Fig. 3D). Our results prove that knockdown of ANO5 promotes FTC-133 cell migration and invasion.

Overexpression of ANO5 inhibits FTC-133 and TPC-1 cell migration and invasion

Next we detect the effect of ANO5 overexpression on thyroid cancer cell migration and invasion. Lentivirus expressing ANO5 was constructed and TPC-1 and FTC-133 cells infected. Real-time PCR and western blotting proved that ANO5 successfully expressed in TPC-1 and FTC-133 cells (Fig. 4A and B). Cell migration and invasion assays showed that ectopic expression of ANO5 decreased the invasive and migrated cell number of TPC-1 and FTC-133 cells (Fig. 4C and D). Collectively these data demonstrate that overexpression of ANO5 inhibits FTC-133 and TPC-1 cell migration and invasion.

ANO5 activates the JAK/STAT3 pathway in thyroid cancer

To explore the mechanism by which ANO5 regulates thyroid cancer cells migration and invasion, we carried out GSEA using public datasets (GSE3678). We found that lower ANO5 expression was negatively associated with some important signaling pathways such as TPO, KRAS, p53 and VEGF (Fig. 5A). In addition, the results also showed that lower ANO5 expression was positively associated with JAK/STAT3 pathway which is well-known to be activated during cancer metastasis (19,20) (Fig. 5B). Western blot results indicated that overexpression of ANO5 suppressed phosphorylation of STAT3 but silencing of ANO5 increased the phosphorylation of STAT3 (Fig. 5C). Collectively these data demonstrated that ANO5 can regulate JAK/STAT3 signaling pathway in thyroid cancer.

Discussion

Most of thyroid cancers are well differentiated and have good prognosis, but lymph node metastasis usually increase the risk of recurrence and mortality (35). In this study, for the first time, we identified that ANO5 gene was documented to be expressed in 7 papillary thyroid carcinoma samples. Moreover, TCGa databases showed that expression of ANO5 in PTC with LN metastases (n=166) is lower than those without LN metastases (n=185) (Fig. 2B). We found ANO5 is downregulated in thyroid cancer tissues including PTC and FTC (Fig. 1E), downregulation of ANO5 promotes thyroid cancer cell migration and invasion (Fig. 3C and D), while overexpression of ANO5 has the opposite effect (Fig. 4C and D). Identifying the molecular events that regulate thyroid cancer metastasis holds promise for developing more effective prevention for human thyroid cancer. One of the major signaling pathways that is aberrantly activated and is critical for thyroid tumor metastasis is the JAK/STAT3 pathway by GSEA (Fig. 5B). The phosphorylated STAT3 protein can translocate into the nucleus, where it activates the transcription of various genes that regulate vital cellular functions, including cell proliferation and metastasis (26). These data suggested a relationship between ANO5 and JAK/STAT3 pathway activation, but it remained to be determined if the JAK/STAT3 pathway is required for thyroid cancer metastasis. These data indicated that ANO5 is a potential tumor suppressor gene in thyroid cancer, and downregulation of ANO5 participates in lymph node metastasis. Thus, the functional effect of ANO5 in PTC metastasis and has potential clinical value for developing gene therapy to treat PTC and subsequent lymph node or distant metastases and improving prognosis.

Other than ANO5, some ANO family members such as ANO1-4 and ANO6-10 have been reported to be related to tumors. Previous studies reported that ANO1, another member of anoctamin family, is upregulated in gastrointestinal stromal tumors (27) and head and neck squamous cell carcinomas (28), inhibition of ANO1 can suppresses tumor invasion (2932). We measured ANO5 expression in PTCs using a public database, and found that in contrast to ANO1 expression, ANO5 is downregulated in cancer tissues (Fig. 1C). Additional study of ANO5 revealed that it negatively regulate lymph node metastases of PTC, a role opposing that of ANO1 in other tumors (33,34). It has been reported that overexpression of ANO6 also increase cancer cell migration (12). ANO7 has been reported to participate in the development of breast (35) and prostate (36) cancers, the monoantiboby targeting the extracellular regions of ANO7 has a potential application for immunotherapy (37). ANO5 itself functions as a Cl channel, but its activation requires higher Ca2+ concentration than other aNO members (38). Thus, some antagonism may exist among different aNO family members with respect to tumors, and this warrants further study.

In conclusion, we identified that ANO5 is downregulated in thyroid cancer and downregulation of ANO5 promotes thyroid cancer cell migration and invasion. In addition, we found that the expression level of ANO5 was correlated with activation of JAK/STAT3 pathway in thyroid cancer, suggesting a potential application of ANO5 as a biomarker. Altogether, our results demonstrate that targeting JAK/STAT3 pathway, using siRNA knockdown of ANO5, effectively promote lymph node metastasis of thyroid cancer, therefore, could be a potential novel therapeutic approach for treating lymph thyroid cancer. Thus, we confirmed that ANO5 is a novel potential biomarker of thyroid cancer and its expression correlates with lymph node metastasis. To further uncover the effect of ANO5 on proliferation and cell cycle and the detailed molecular mechanism of lymph node metastasis of thyroid cancer is necessary for clinical gene therapy in the future.

Acknowledgments

We would like to thank Lei Ye (Rui Jin Hospital) for providing us with TPC-1 and FTC-133 cells. This study was supported by the grants from the National Natural Science Foundation of China (grant nos. 81472501 and 81502197).

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October-2017
Volume 51 Issue 4

Print ISSN: 1019-6439
Online ISSN:1791-2423

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Copy and paste a formatted citation
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Spandidos Publications style
Chang Z, Cai C, Han D, Gao Y, Li Q, Feng L, Zhang W, Zheng J, Jin J, Zhang H, Zhang H, et al: Anoctamin5 regulates cell migration and invasion in thyroid cancer. Int J Oncol 51: 1311-1319, 2017
APA
Chang, Z., Cai, C., Han, D., Gao, Y., Li, Q., Feng, L. ... Wei, Q. (2017). Anoctamin5 regulates cell migration and invasion in thyroid cancer. International Journal of Oncology, 51, 1311-1319. https://doi.org/10.3892/ijo.2017.4113
MLA
Chang, Z., Cai, C., Han, D., Gao, Y., Li, Q., Feng, L., Zhang, W., Zheng, J., Jin, J., Zhang, H., Wei, Q."Anoctamin5 regulates cell migration and invasion in thyroid cancer". International Journal of Oncology 51.4 (2017): 1311-1319.
Chicago
Chang, Z., Cai, C., Han, D., Gao, Y., Li, Q., Feng, L., Zhang, W., Zheng, J., Jin, J., Zhang, H., Wei, Q."Anoctamin5 regulates cell migration and invasion in thyroid cancer". International Journal of Oncology 51, no. 4 (2017): 1311-1319. https://doi.org/10.3892/ijo.2017.4113