AHNAK suppresses tumour proliferation and invasion by targeting multiple pathways in triple-negative breast cancer
- Bo Chen†1,
- Jin Wang†1,
- Danian Dai†1,
- Qingyu Zhou2,
- Xiaofang Guo2,
- Zhi Tian2,
- Xiaojia Huang1,
- Lu Yang1,
- Hailin Tang1Email author and
- Xiaoming Xie1Email author
© The Author(s). 2017
Received: 22 November 2016
Accepted: 31 March 2017
Published: 12 May 2017
AHNAK, also known as desmoyokin, is a giant protein with the molecular size of approximately 700 kDa and exerts diverse functions in different types of cancer.
In the present study, we demonstrated that AHNAK mRNA levels were down-regulated in 7 out of 8 human breast cancer cell lines, especially in triple - negative breast cancer (TNBC) cell lines. Moreover, in patients with TNBC, the expression of AHNAK gene was inversely correlated with the tumor status (P = 0.015), lymph node status (P < 0.001), lymph node (LN) infiltration (P < 0.001) and TNM stage (P < 0.001). Moreover, down-regulated AHNAK expression was considered an independent prognostic factor associated with the poor survival of patients with TNBC. Overexpression of AHNAK in two TNBC cell lines, MDA-MB-231 and BT549, suppressed the in vitro TNBC cell proliferation and colony formation, and inhibited the in vivo TNBC xenograft growth and lung metastasis. The tumor suppressing effect of AHNAK in TNBC was associated with the AKT/MAPK signaling pathway and Wnt/β-catenin pathway. Consistent results were observed when AHNAK was knockdown in BT20 and MDA-MB-435 cells.
Taken together, our results suggest that AHNAK acts as a tumor suppressor that negatively regulates TNBC cell proliferation, TNBC xenograft growth and metastasis via different signaling pathways.
KeywordsAHNAK Triple-negative breast cancer AKT MAPK Wnt/β-catenin pathway
It is well known that breast cancer is a devastating disease with extensive intra-tumour and inter-tumour heterogeneity [1, 2]. Triple-negative breast cancer (TNBC), which lacks the expression of ER, PR and HER2 , is a unique subtype of breast cancer with limited treatment options and poor prognosis, accounting for 15–20% of breast cancers . In China, although the overall incidence of breast cancer is lower than that in Western countries , the total number of patients with TNBC is relatively high. Despite advances in breast cancer treatment, the median overall survival (OS) for patients with TNBC is unfavourable compared with the mean OS for other breast cancer subtypes patients [6–8]. In this regard, it is necessary to investigate the molecular pathogenesis of TNBC and to explore novel therapeutic targets to improve the prognosis of TNBC patients.
AHNAK, also known as desmoyokin , is a large protein that was originally identified as a desmosomal plaque protein found at the periphery of the cytoplasmic plaque of desmosomes in the stratified squamous epithelia . AHNAK has been previously reported to be expressed in several intracellular locations, including the plasma membrane, cytoplasm and nucleus . Previous studies have indicated that AHNAK is involved in several important physiological activities, such as cardiac L-type Ca2+ channel function , neuronal cell differentiation and calcium signalling in T cells [13, 14]. In recent years, there has been increasing interest in understanding the function of AHNAK in various malignant tumours. So far, it has been demonstrated that the expression of AHNAK is variable in different types of cancer. For example, the expression of AHNAK is down-regulated in Burkitt lymphoma, small cell lung carcinoma and neuroblastoma [15, 16] but upregulated in glioma, mesothelioma, fibrosarcoma and prostate cancer . Due to its large size and protein structure, AHNAK can facilitate the binding of multiple proteins and can mediate signalling events [18, 19]. The results of a recent study using a transgenic mouse model of breast cancer and human breast cancer samples suggest that AHNAK can act as a tumour suppressor that mediates the negative regulation of cell growth via the modulation of the TGFβ/Smad signalling pathway . However, the expression profile of AHNAK in TNBC and its function have not been elucidated. In this study, we investigated the role of AHNAK in the pathogenesis of TNBC and assessed the effect of AHNAK on clinicopathological characteristics and prognosis by examining its expression in breast cancer cell lines and patient tissues and by characterizing its function in TNBC using both in vitro and in vivo models. We found that the expression of AHNAK is associated with the biological characteristics and prognosis of TNBC and the likelihood of lung metastasis. Moreover, the aggressive nature of TNBC with reduced expression of AHNAK was partly attributable to the activation of the AKT/MAPK and Wnt/β-catenin signalling pathways.
This study was approved by the Ethics Committee of Sun Yat-Sen University Cancer Centre Health Authority. A total of 221 matched human triple-negative breast cancer (TNBC) tissues and 51 their adjacent normal mammary tissues (Normal 1), 20 non-triple-negative breast cancer (NTNBC) tissues and the corresponding paired normal adjacent tissues (Normal 2) were collected between October 2001 and September 2009 at Sun Yat-Sen University Cancer Centre. The details of the NTNBC samples are given in the supplementary information (Additional file 1: Table S1). The resected tissues were immediately cut and stored in RNAlater (Ambion). The collection and use of tissues followed procedures that are in accordance with the ethical standards formulated in the Declaration of Helsinki.
Cell cultures and transfection
All the cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA), including normal mammary epithelial cell lines (184A1, MCF-10A), human breast cancer cell lines (MDA-MB-231, BT549, BT-20, MCF-7, T47D, BT474, MDA-MB-435 and BT-483) and human embryonic kidney 293 T cells. All of the cell lines were passaged in our laboratory for less than six months and maintained according to the supplier’s instructions. The cell lines were found to be free of mycoplasma infection and their authenticity was verified by DNA fingerprinting before use.
Lentivirus-mediated AHNAK-expressing vector (EX-V0190-Lv122) and control plasmids were purchased from GeneCopoeia (Rockville, MD, USA). According to the manufacturer’s instructions, the AHNAK cDNA-containing plasmids were transfected into 293 T cells (1 × 106) for 48 h to generate lentiviral particles. Lipofectamine® 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) was used. The control groups included the vector-transfected group (EX-NEG-Lv122). The viral supernatant was subsequently collected and used to infect MDA-MB-231 and BT549 cells. Seventy-two hours post-transfection, western blotting was performed to determine the transfection efficiency (Additional file 2: Figure S1A). BT-20 and MDA-MB-435 cells were used for transfection with AHNAK siRNA. Five microlitres of control or targeted siRNAs were transfected with Lipofectamine 2000 (Invitrogen) according to the protocol provided by Santa Cruz (sc-97060). The cells were grown for 48 h before assessing gene and protein knockdown efficiencies by western blotting (Additional file 2: Figure S1B).
Quantitative RT-PCR analysis (qRT-PCR)
Total RNA was extracted from the cells using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). A NanoDrop ND-1000 instrument was used to determine the concentrations of the RNA samples. The integrity of RNA was assessed by electrophoresis on a denaturing agarose gel. Reverse transcription and qRT-PCR reactions were performed using a QuantiFast SYBR® Green RT-PCR Kit (QIAGEN, Germantown, MD, USA), which is a one-step RT-PCR kit. Each reaction was performed in triplicate. The primer sequences are given as follows: AHNAK: 5′-ATGCTCCAGGGCTCAACCT-3' (forward) and 5'-CGTGCCCCAACGTTAAGCTT-3' (reverse); β-actin: 5'-CGCGAGAAGATGACCCAGAT-3' (forward) and 5′-GGGCATACCCCTCGTAGATG-3' (reverse); Wnt1: 5'-ATGGGGCTCTGGGCGCTGTTG-3' (forward) and 5'-TCACAGACACTCGTGCAGTAC-3' (reverse); c-Myc: 5’-AGAAATGTCCTGAGCAATCACC-3' (forward) and 5’-AAGGTTGTGAGGTTGCATTTGA-3' (reverse); β-catenin: 5′- CCGCATGGAAGAAATAGTTGAAG-3′ (forward) and 5′- CAATTCGGTTGTGAACATCCC-3′ (reverse). The real-time PCR assays were performed with the Bio-Rad IQTM5 Multicolour Real-Time PCR Detection System (USA). The specificity of the amplification products was confirmed by melting curve analysis. The values were normalized to internal controls and fold changes were calculated through relative quantification (2-ΔΔCt).
Cell proliferation assay
Cells were seeded on 6-well plates at the desired cell concentrations. The numbers of cells were counted after 1, 2, 3 and 4 days of incubation using a Coulter Counter (Beckman Coulter, Fullerton, USA) in triplicate.
Colony growth assays
Six-well plates were covered with a layer of 0.6% agar in medium supplemented with 20% foetal bovine serum. Cells were prepared in 0.3% agar and seeded in triplicate. Then the plates were incubated in a CO2 incubator at 37 °C for two weeks. Crystal violet was used to stain the colonies, and the colonies were counted.
Immunohistochemistry (IHC) staining was performed as described previously . The concentration used for AHNAK (Santa Cruz Biotechnology) was 1:50.
Briefly, total protein was extracted and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes. The protein bands were probed with antibodies against AHNAK (Santa Cruz Biotechnology), Akt, phospho-Akt (Ser473), ERK1/2, phospho-ERK1/2 (Tyr202/Y204), phospho-c-Raf (Ser296), phospho-MEK1/2 (Ser221) (Cell Signaling Technology, Beverly, MA), c-myc, Wnt-1 and β-actin (Abcam, Cambridge, UK) overnight at 4 °C followed by incubation with HRP-conjugated second antibodies (Santa Cruz, CA, USA) (1:3500) and detected by enhanced chemiluminescence. The dilutions used for the anti-AHNAK and anti-β-actin antibodies were 1:200 and 1:5000, respectively. The dilution used for the other antibodies was 1:1000. β-actin was used as the protein-loading control.
Mouse xenograft model
All the animal procedures were performed in accordance with institutional guidelines. Ethical approval was obtained from the Institute Research Ethics Committee of Sun Yat-sen University Cancer Center. MDA-MB-231 or BT549 cells were stably transfected with AHNAK or vector and collected and suspended in PBS at a concentration of 1 × 107 cells/ml. Then, 200 μl of cancer cell suspension was subcutaneously inoculated into the dorsal flanks of nude mice ((female, 4–6 w; five in each group) using 1-ml syringes. The tumour size was measured every four days. After 28 days, the mice were euthanized, and the tumours were weighed. The tumour volumes were determined according to the following formula: A × B2/2, where A is the largest diameter and B is the diameter perpendicular to A . To assay the effect of AHNAK on tumour metastasis, 1 × 105 MDA-MB-231 or BT549 cells infected with AHNAK or vector were injected into the tail veins of nude mice (eight in each group). The cells were suspended in PBS at a concentration of 2 × 106 cells/ml, and 50 μl of cancer cell suspension was injected into each mouse using a microsyringe. Necropsies were performed after 28 days. The numbers of microscopic metastases in the lungs per H&E-stained section from individual mice were determined.
The expression levels of the AHNAK transcript in breast cancers and normal breast tissues were determined from the Oncomine database (www.oncomine.org). The threshold was set at a two-fold difference in expression between cancers and normal tissues with a P-value < 0.01. The Cancer Genome Atlas (TCGA)  and METABRIC  datasets were analysed and the figures were generated using the cBio Cancer Genomics Portal (http://cbioportal.org) [25, 26]. All TCGA data included in this manuscript are in compliance with the TCGA publication guidelines.
Statistical analyses were performed using the SPSS 16.0 software. Comparisons between groups were conducted with t-tests and χ2 tests. The Kaplan-Meier method was used to plot overall survival curves and relapse-free survival curves, and the log-rank test was used for comparison. Survival was calculated from the time of pathological diagnosis. Univariate and multivariate analyses (Cox proportional hazards regression model) were performed to identify the independent factors relevant to patient survival. The differences were considered statistically significant at P < 0.05.
Bioinformatics analysis of AHNAK expression in human breast cancer
AHNAK expression was significantly down-regulated in triple-negative breast cancer and correlated with the clinicopathological characteristics and prognosis of TNBC patients
Association between AHNAK and clinicopathological characteristics in triple-negative breast cancer
(n = 221)
Tumor status (T)
Lymph node status (N)
G1 + G2
Prognostic value of AHNAK for overall survival in triple-negative breast cancer patients by Univariate and Multivariate analyses
Age (<50 vs. ≥50 years)
Menopause (Yes vs. No)
BMI (<25 vs. ≥ 25)
Histological grade(G1 + G2 vs. G3)
TNM Staging(I + II vs. III + IV)
Ahnak(low vs. high)
AHNAK effected TNBC cell line proliferation and colony formation invasion
Overexpression of AHNAK in TNBC cell lines inhibited in vivo tumour growth and lung metastasis
AHNAK targets AKT/MAPK signalling and the Wnt/β-catenin pathway
TNBC is a subtype of breast cancer that has some of the worst patient prognoses of all breast cancer subtypes and is not sensitive to normal endocrine therapy or targeted therapy against breast cancer . Studies of molecular mechanisms in TNBC are extremely necessary. By mining the literature, we found that AHNAK is a very large protein that is involved in many cellular processes and pathways . Down-regulation of AHNAK prevents cortical actin cytoskeleton reorganization. AHNAK forms a multimeric complex with actin and the Annexin 2/S100A10 complex at the plasma membrane, which suggests that AHNAK interacts with the cortical actin cytoskeleton as part of a submembranous complex . Previous research also showed that AHNAK could potentiate TGFβ-induced transcriptional activity of R-Smad, which leads to the negative regulation of cell growth by stimulating the localization of Smad3 into the nucleus . Meanwhile, cell- or tissue-specific processes or pathways regulated by AHNAK are quite distinct and are related to the cell or tissue type . Although several proteins have been identified to interact with AHNAK, the function of AHNAK in breast cancer remains undefined.
Here, we demonstrated the functional significance of AHNAK in TNBC. Using public datasets from Oncomine (www.oncomine.org), the AHNAK mRNA level was found to be reduced in breast cancer, although there was a huge variation among different types of cancers. Meanwhile, both the TCGA and METABRIC datasets showed that the level of AHNAK mRNA was significantly decreased in the samples classified as basal-like (most TNBCs have basal-like characteristics). Consistent with previous findings , we found that the expression of AHNAK is low in several TNBC cell lines and that AHNAK might play a tumour suppressive role. In addition, we also found that AHNAK expression was markedly decreased in TNBC patient samples, and the expression of AHNAK is negatively correlated with some vital clinicopathological characteristics, such as tumour status (P = 0.015), lymph node status (P < 0.001), lymph node (LN) infiltration (P < 0.001) and TNM stage (P < 0.001) of TNBC, as well as prognosis of TNBC patients. From in vitro studies, we found that overexpression of AHNAK could inhibit proliferation and colony formation of TNBC cell lines. Conversely, knocking down AHNAK expression could promote the proliferation and colony formation of TNBC cell lines. As indicated, transfection with AHNAK-overexpressing vectors could decrease the growth and metastasis breast cancer xenografts. Thus, we confirmed the function of AHNAK in suppressing tumour progression.
In previous studies, AHNAK has been suggested to be involved in signalling pathways, such as the reorganization of the actin cytoskeleton network, the PI3K–PKB pathway to engage effector proteins, the formation of pseudopodial protrusions, and adaptation events to reprogram tumour cell biology [17, 34]. The Wnt/β-catenin signalling pathway plays critical roles in development and tissue homeostasis [35, 36]. As many previous studies have indicated, the Wnt/β-catenin signalling pathway also plays a vital role in breast cancer [37–39]. The role of Wnt signalling in primary TNBC and as a predictor of lung and brain metastasis has been described [40, 41]. A meta-analysis indicated that the Wnt pathway is activated in TNBC and that increased Wnt/β-catenin signalling is associated with metastatic disease and poor prognosis . Notable Wnt transcriptional targets upregulated in TNBC include MYC , matrix metallopeptidase 7 (MMP7) , VEGF , MET , CD44 , snail (SNAI1)  and survivin (BIRC5) . In this study, we explored the association between AHNAK and the Wnt/β-catenin signalling pathway and demonstrated that, in TNBC, AHNAK indeed regulated the expression of several important genes belonging to the Wnt/β-catenin signalling pathway, such as Wnt-1, β-catenin and c-myc, both at the mRNA level and at the protein level. Previous studies have confirmed that there is a link between AHNAK and c-myc. Overexpression of AHNAK could down-regulate c-myc and cyclin D1/D2, resulting in cell cycle arrest and growth retardation . A recent study also showed that, in the absence of the ectopic expression of c-myc, the down-regulation of AHNAK could generate safer induced pluripotent stem cells (iPSCs) . Moreover, we identified PI3K/AKT and MAPK/ERK as key signalling pathways involved in the inhibition of tumour cell proliferation mediated by AHNAK. The constitutive activation of PI3K/AKT and MAPK/ERK signalling pathways is an important event in breast cancer, as they regulate multiple cellular processes to promote cancer growth, survival, and metastasis [50, 51]. However, there are several limitations in our study that should be addressed. First, although we found that AHNAK affected some pathways to some extent, the specific details of these mechanisms are still unknown. In addition, it remains unclear whether the proposed role for AHNAK is limited to only the triple-negative subtype of breast cancer. Therefore, further studies will be needed to determine the exact function of AHNAK.
In conclusion, our study demonstrates the potential tumour suppressive role and lung metastasis-inhibiting effect of AHNAK in TNBC. Moreover, AHNAK could, at least partially, affect the AKT/MAPK and the Wnt/β-catenin signalling pathways, which are important tumour-related signalling pathways in TNBC. Taken together, although more in-depth mechanisms and prognostic roles for AHNAK in TNBC need to be confirmed in the future, our findings provide a preliminary basis to explore AHNAK as a potential therapeutic candidate in TNBC.
This work was supported by funds from the National Natural Science Foundation of China (81472575, 81672598, Xiaoming Xie; 81472469, Hailin Tang) and the Science and Technology Planning Project of Guangzhou and Guangdong (2015B090901050, 2015B020211002, Xiaoming Xie; 2016A020214009, Hailin Tang).
XX and HT designed the experiment and critically revised the paper; BC, QZ, XG and ZT performed most of the experiments; JW, DD, XH and LY contributed to collected the patients data; BC, JW and DD performed the statistical and bioinformatic analysis; HT cultured cells and built animal models; BC wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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