Background
Colorectal cancer (CRC) is the most common malignant cancer in digestive tract worldwide [
1,
2]. Despite great progress in treatment of CRC, including 5-fluorouracil (5FU)-based chemotherapy, the combination strategies of 5-FU and oxaliplatin or irinotecan, the five-year survival rate is still dismal [
3,
4]. The existence of cancer stem cells (CSCs) that are the minority population of cells characterized by the capabilities of self-renewal, unlimited proliferation and differentiation into the multiple lineages of cancer cells has been regarded to be responsible for the failure of chemotherapy in CRC patients [
5,
6], which contributes to the poor prognosis of CRC patients [
7]. Indeed, multiple studies have shown that CSCs play crucial roles in the induction and maintenance of chemotherapeutic resistance in several human cancers [
8,
9]. Thus, improved understanding of the mechanisms that maintain CSCs properties will improve the efficacy of chemotherapy in patients with CRC via eradicating the CSC population.
Activator protein-2 (AP-2) factors are a conserved family of DNA-binding transcription factors in different species and five members have been identified in mammals, including AP-2α-ɛ [
10]. AP-2 factors play important roles in embryogenesis, and interestingly AP-2 factors expression is hardly detectable in most adult tissues [
11,
12]. However, overexpression of AP-2 factors has been observed in multiple human cancers. For example, TFAP2A expression was elevated in nasopharyngeal carcinoma, which promoted nasopharyngeal carcinoma proliferation and growth via inducing cyclooxygenase-2 expression [
13]; in addition, TFAP2B was reported to be overexpressed in non-small cell lung cancer (NSCLC) tissues and cell lines. TFAP2B knockdown by siRNA significantly attenuated cell growth and induced apoptosis in NSCLC cells in vitro and in a lung cancer subcutaneous xenograft model; conversely, upregulation of TFAP2B yielded an opposite effect [
14]. Furthermore, accumulating literatures have shown that AP-2 proteins play crucial roles in the development of therapeutic resistance in the treatment of cancers. TFAP2A was elevated in breast cancer tissue and cell lines and more highly expressed in tamoxifen resistant tumor tissues and cell lines [
15]. High expression of TFAP2C in breast cancer contributed to hormone resistance, which positively correlated with poor survival in breast cancer patients [
16]. Moreover, low expression of TFAP2E due to hypermethylation was significantly associated with nonresponse to chemotherapy in colorectal cancer [
17]. Therefore, these studies have indicated that different members of AP-2 proteins have stimulatory or inhibitory affect on chemotherapeutic response in cancer treatment, which may be environment and tumor type dependent.
The Hippo signaling pathway has been reported to be dysregulated in several biological processes and tumorigenesis of multiple human cancers [
18‐
20]. In the mammal, the Hippo pathway constitutes of four core kinase cassette components, including kinases MST1/2 and LATS1/2, as well as the adaptor proteins SAV1 and MOB1 [
20]. The Hippo signaling is active via tightly balancing the activity of YAP and TAZ, to low levels through phosphorylation– ubiquitination mechanisms [
21,
22]. While Hippo signaling is absent, unphosphorylated YAP1/TAZ enters the nucleus and induce the transcriptional activity of TEA domain (TEAD) family members (TEAD1–TEAD4) as the transcriptional co-activators, which further transcriptionally upregulate multiple downstream effectors to exert a pleiotropic role in tumor progression and metastasis [
23‐
25]. Moreover, accumulating studies has shown that the inactivation of Hippo signaling rendered resistance of cancer cells to chemotherapeutic drugs in various types of cancers. Chen et al. reported that simultaneous downregulation of MST1, LATS2, MOB1 and SAV1 by upregulation of miR-183c contributed to chemoresistance in pancreatic cancer [
26]; in addition, the study by Touil and colleagues showed that hyper-activation of YAP promoted resistace of colon cancer cells to 5-FU [
27]. Accordingly, the inactivation of Hippo pathway is considered as a crucial mediator in development of cancer chemoresistance, and better understanding of the mechanisms underlying the inactivation of Hippo pathway may provide new insights for the development of more effective cancer therapy.
In this study, we find that TFAP2C is significantly upregulated in CRC tissues and cells and high expression of TFAP2C correlates with advanced clinicopathological features, poor prognosis and disease progression in CRC patients. Furthermore, upregulation of TFAP2C enhances, while silencing TFAP2C inhibits CSCs characteristics and chemotherapeutic resistance in CRC cells in vitro and in vivo. Our results further reveal that TFAP2C promotes CSCs characteristics and chemoresistance via transcriptionally activating negative regulators of Hippo signaling, ROCK1 and ROCK2, resulting in inactivation of Hippo signaling in CRC cells. Therefore, our findings identify TFAP2C as a prognostic factor for CRC patients, as well as a therapeutic target to attenuate chemoresistance of CRC.
Methods
Cell lines and cell culture
The normal colon epithelial cell CMEC was purchased from PriCells, and all colorectal cancer cell lines, including RKO, CW-2, SW948, HCT116, SW480 COLO 210DM and COLO 205, were obtained from Shanghai Chinese Academy of Sciences cell bank (China). All were cultured in RPMI-1640 medium (Life Technologies, Carlsbad, CA, US) supplemented with penicillin G (100 U/ml), streptomycin (100 mg/ml) and 10%fetal bovine serum (FBS, Life Technologies) and cultured at 37 °C in a humidified atmosphere with 5% CO2.
Patients and tumor tissues
A total of eight paired fresh colorectal cancer tissues with matched adjacent normal tissues and individual 378 paraffin-embedded, archived CRC tissues were obtained during surgery at the The First Hospital of Jilin University (Changchun, China) between January 2008 and December 2011 (Additional file
1: Table S1 and Additional file
2: Table S2). Patients were diagnosed based on clinical and pathological evidence, and the specimens were immediately snap-frozen and stored in liquid nitrogen tanks. For the use of these clinical materials for research purposes, prior patients’ consents and approval from the Institutional Research Ethics Committee were obtained.
Total RNA from tissues or cells was extracted using TRIzol (Life Technologies) according to the manufacturer’s instructions. Messenger RNA (mRNA) were polyadenylated using a poly-A polymerase-based First-Strand Synthesis kit (TaKaRa, DaLian, China) and reverse transcription (RT) of total mRNA was performed using a PrimeScript RT Reagent kit (TaKaRa) according to the manufacturer’s protocol. Complementary DNA (cDNA) was amplified and quantified on ABI 7500HT system (Applied Biosystems, Foster City, CA, USA) using SYBR Green I (Applied Biosystems). Additional file
3: Table S3 lists the primers used in the reactions. Real-time PCR was performed according to a standard method, as described previously [
28]. Primers for TFAP2C were synthesized and purified by RiboBio (Guangzhou, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous controls for miRNA or mRNA respective. Relative fold expressions were calculated with the comparative threshold cycle (2
-ΔΔCt) method according to the previous study [
29].
Plasmid, small interfering RNA and transfection
Human TFAP2C cDNA was purchased form (Vigene Biosciences, Shandong, China) and cloned into the pSin-EF2 plasmid (addgene #16578, Cambridge, MA, USA). Knockdown of endogenous TFAP2C was performed by cloning two short hairpin RNA (shRNA) oligonucleotides into the pSUPER-puro-retro vector (OligoEngine, Seattle, WA, USA). The full sequence and two separate shRNA fragments of TFAP2C are listed in Additional file
4: Table S4. The luciferase reporter system of pGL6-TA promoter-luc was used to examine the transcriptional activity of ROCK1 and ROCK2, and the sequences of ROCK1 and ROCK2 promoter was presented in Additional file
4: Table S4. Small interfering RNA (siRNA) for YAP and TAZ knockdown was obtained from Ribobio (Guangzhou, China). Transfection of siRNAs and plasmids was performed using Lipofectamine 3000 (Life Technologies) according to the manufacturer’s instructions.
Western blotting analysis
Nuclear/cytoplasmic fractionation was separated by using Cell Fractionation Kit (Cell Signaling Technology, USA) according to the manufacturer’s instructions, and the whole cell lysates were extracted using RIPA Buffer (Cell Signaling Technology). Western blot was performed according to a standard method, as described previously [
30]. Antibodies against TFAP2C, BCL2 and BCL2L1, p-MST1/2, p-LATS1, LATS1, p-YAP, YAP, ROCK1 and ROCK2 were purchased from Cell Signaling Technology (Cambridge, USA), and MST1 and TAZ from Abcam. The membranes were stripped and reprobed with an anti–α-tubulin antibody (Cell Signaling Technology) as the loading control.
Chromatin immunoprecipitation (ChIP)
Cells were cultured as described above. Cross-linking was performed with formaldehyde (Merck) at a final concentration of 1% and terminated after 5 min by the addition of glycine at a final concentration of 0.125 M. Cells were harvested with SDS buffer, pelleted by centrifugation, and resuspended in IP buffer. Chromatin was sheered by sonication (HTU SONI 130; Heinemann) to generate DNA fragments with an average size of 500 bp. Preclearing and incubation with anti-Flag (F1804, Sigma) antibodies or IgG control (M-7023, Sigma) for 16 h was performed as previously described (Menssen et al. 2007). Washing steps and the reversal of cross-linking were performed as described previously (Frank et al. 2001). Immunoprecipitated DNA was analyzed by qPCR and the primers of ROCK1 and ROCK2 promoter was presented in Additional file
3: Table S3. Enrichment was expressed as the percentage of the input for each condition.
Anchorage-independent growth assay
A total of 500 cells were trypsinized and suspended in 2 ml of complete medium containing 0.3% agar (Sigma). This experiment was performed as previously described [
31] and carried out three times independently for each cell line.
Cell counting Kit-8 analysis
For cell counting kit-8 analysis, cells (2 × 10
3) were seeded into 96 well plates and the specific staining process and methods were performed according to the previous study [
32].
The cells were trypsinized as single cell and suspended in the media with 10% FBS. The indicated cells (300 cells per well) were seeded into of 6-well plate for ~ 10–14 days. Colonies were stained with 1% crystal violet for 10 min after fixation with 10% formaldehyde for 5 min. Plating efficiency was calculated as previously described [
33]. Different colony morphologies were captured under a light microscope (Olympus).
Flow cytometric analysis
Flow cytometric analyzed of apoptosis were using the FITC Annexin V Apoptosis Detection Kit I (BD, USA), and was performed previously described [
34]. The cell’s inner mitochondrial membrane potential (Δψm) was detected by flow cytometric using MitoScreen JC-1 staining kit (BD), and was presented as protocol described. Briefly, cells were dissociated with trypsin and resuspended at 1 × 10
6 cells/ml in Assay Buffer, and then incubated at 37 °C for 15 min with 10 μl/ml JC-1. Before analyzed by flow cytometer, cells were washed twice by Assay Buffer. Flow cytometry data were analyzed using FlowJo 7.6 software (TreeStar Inc., USA).
Caspase-9 or Caspase-3 activity assays
Activity of caspase-9 or caspase-3 was analysis by spectrophotometry using Caspase-9 Colorimetric Assay Kit or Caspase-3 Colorimetric Assay Kit (Keygen, China), and was presented as protocol described. Briefly, 5 × 106 cells or 100 mg fresh tumor tissues were washed with cold PBS and resuspended in Lysis Buffer and incubated on ice for 30 min. Mixed the 50 μl cell suspension, 50 μl Reaction Buffer, and 5 μl Caspase-3/− 9 substrate, and then incubated at 37 °C for 4 h. The absorbance was measured at 405 nm, and BCA protein quantitative analysis was used as the reference to normal each experiment groups.
Side population analysis
The cell suspensions were labeled with Hoechst 33,342 (Molecular probes – #H-.
3570) dye for side population analysis as per standard protocol [
35]. Briefly, cells were resuspended at 1× pre-warmed OptiMEM (Gibco, USA) containing 2% FBS (Gibco, USA) at a density of 10
6/mL. Hoechst 33,342 dye was added at a final concentration of 5 lg/mL in the presence or absence of verapamil (50 lmol/L; Sigma) and the cells were incubated at 37 °C for 90 min with intermittent shaking. At the end of the incubation, the cells were washed with OptiMem containing 2% FBS and centrifuged down at 4 °C, and resuspended in ice-cold OptiMem containing 2% FBS and 10 mmol/L HEPES. Propidium iodide (Sigma, USA) at a final concentration of 2 lg/mL was added to the cells to gate viable cells. The cells were filtered through a 40-lm cell strainer to obtain single cell suspension before sorting. Analysis and sorting was done on a FACS AriaI (Becton Dickinson). The Hoechst 33,342 dye was excited at 355 nm and its dual-wavelength emission at blue and red region was plotted to get the SP scatter.
Cells (500 cells/well) were seeded into 6-well Ultra Low Cluster plates (Corning) and cultured as previously described [
36]. After 10–12 days, the number of cell spheroids (tight, spherical, non-adherent masses > 50 μm in diameter) were counted, and images of the spheroids were scored under an inverse microscope (spheroids formation efficiency = colonies/input cells× 100%).
Tumor xenografts
Four-week-old BALB/c-nu female mice weighing 15–20 g were maintained in a standard pathogen-free environment where the animals were housed in sterile cages under laminar flow hoods in a 20–26 °C temperature controlled room with a 12-h light/12-h dark cycle and fed autoclaved chow and water. The 6-week-old BALB/c-nu mice were randomly divided into four groups (n = 6 per group). Cells (1 × 106, 1 × 105 1 × 104 and 1 × 103) were inoculated subcutaneously together with Matrigel (final concentration of 25%) into the inguinal folds of the nude mice respectively. To study the effect of TFAP2C on chemoresistance of CRC cells, The 4–6 week-old BALB/c-nu mice were randomly divided into four groups (n = 6 per group) and the indicated cells (2 × 106) were inoculated subcutaneously into the inguinal folds of the nude mice. After seven days for cells inoculation, the mice were injected intraperitoneally 50 mg/kg.d 5-FU for 4 weeks. Tumor volume was determined using an external caliper and calculated using the eq. (L × W2)/2. On day 38, animals were euthanized, tumors were excised, weighed and stored in liquid nitrogen tanks.
Immunohistochemistry
The immunohistochemistry procedure and scoring of TFAP2C expression levels were performed as previously described [
37]. Scores given by two independent investigators were averaged for further comparative evaluation of TFAP2C expression. The proportion of tumor cells was scored as follows: 0 (no positive tumor cells); 1 (< 10% positive tumor cells); 2 (10–35% positive tumor cells); 3 (35–70% positive tumor cells) and 4 (> 70% positive tumor cells). The staining intensity was graded according to the following criteria: 0 (no staining); 1 (weak staining, light yellow); 2 (moderate staining, yellow brown) and 3 (strong staining, brown). The staining index (SI) was calculated as the product of the staining intensity score and the proportion of positive tumor cells. Using this method of assessment, we evaluated TFAP2C expression in CRC samples by determining SI, with scores of 0, 1, 2, 3, 4, 6, 8, 9 or 12. An SI score 4 was the median SI of all sample tissues. High and low TFAP2C expression was stratified by the follow criteria: SI ≤ 4 was used to define tumors with low TFAP2C expression; SI ≥6 was used to define tumors with high TFAP2C expression.
Luciferase assay
Cells (4 × 10
4) were seeded in triplicate in 24-well plates and cultured for 24 h, and the luciferase reporter assay was performed as previously described [
38]. Cells were transfected with 100 ng pROCK1 or ROCK2 promoter reporter luciferase plasmid, plus 5 ng pRL-TK Renilla plasmid (Promega) using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s recommendation. Luciferase and Renilla signals were measured 36 h after transfection using a Dual Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s protocol.
Immunofluorescence
Cells were seeded on glass culture slides (BD Biosciences) and fixed with 4% cold methanol at − 20 °C for 10 min. Subsequently, cells were blocked with 10% goat serum for 1 h and incubated with primary antibodies against YAP (Cell Signaling Technology, Cat. 14,074), TAZ (Abcam, Cat. ab224239) or Phalloidin-iFluor 488 Reagent - CytoPainter (Abcam, Cat. ab176753) at room temperature for 2 h and then incubated with secondary antibody FITC for 1 h at room temperature. After counterstained with DAPI (Invitrogen), the slide was observed under a confocal microscope (Zeiss).
Statistical analysis
All values are presented as means ± standard deviation (SD). Significant differences were determined using GraphPad 5.0 software (USA). Student’s t-test was used to determine statistical differences between two groups. One-way ANOVA was used to determine statistical differences between multiple testing. The chi-square test was used to analyze the relationship between TFAP2C expression and clinicopathological characteristics. Survival curves were plotted using the Kaplan Meier method and compared by log-rank test. P < 0.05 was considered significant. All the experiments were repeated three times.
Discussion
In the current study, we found that TFAP2C was upregulated in CRC tissues and cells, which correlated with advanced clinicopathological features, poor prognosis and disease progression in CRC patients. Furthermore, upregulation of TFAP2C enhanced, while silencing TFAP2C attenuated stemness and chemotherapeutic resistance in CRC cells in vitro and in vivo. Our results further reveal that TFAP2C promoted CSCs characteristics and chemoresistance via transcriptionally upregulating ROKC1 and ROCK2 expression, leading to inactivation of Hippo signaling. Therefore, our findings uncover a novel mechanism by which TFAP2C promotes CSCs characteristics and chemoresistance in CRC.
Accumulating studies have shown that aberrant expression of AP-2 proteins was implicated in the development, progression and metastasis in several human cancers. For example, TFAP2C expression was elevated in lung carcinoma and high expression of TFAP2C promoted cell cycle activation and lung carcinoma cell tumorigenesis via by upregulating the oncogenic miR-183 and downregulating tumor-suppressive miRNA-33a [
40]. Moreover, low TFAP2B expression was reported in primary neuroblastomas, which correlated with poor prognosis via promoting proliferation and cell cycle progression [
41]. Furthermore, different members of AP-2 proteins play opposite roles in the same tumor type. In breast cancer, TFAP2A functioned as a tumor suppressor; conversely, TFAP2C played an oncogenic role in the development and progression of breast cancer [
42]. These studies indicated that although belonged to the AP-2 family, different members of AP-2 proteins function as either oncogene or tumor suppressor in cancers. In the current study, we found that TFAP2C was remarkably elevated in CRC tissues and high expression of TFAP2C correlated with advanced clinicopathological features, poor prognosis and disease progression in CRC patients. Furthermore, upregulation of TFAP2C enhanced the chemotherapeutic resistance in CRC cells; conversely silencing TFAP2C yielded an opposite effect. Therefore, our findings indicate that TFAP2C plays an oncogenic role in CRC via promoting chemoresistance and stemness of CRC cells.
TFAP2C has been reported to be upregulated in various types of cancer, including lung carcinoma, breast cancer, and high expression of TFAP2C significantly correlated with poor prognosis via promoting the growth and proliferation [
40,
43,
44]. However, other studies have shown that TFAP2C played a tumor suppressive role in several human cancers, such as melanoma, endometrial cancer [
45,
46]. Interestingly, Bogachek and colleagues reported that TFAP2C regulated multiple breast cancer-related genes, and loss of TFAP2C induced epithelial-mesenchymal transition in breast cancer cells [
47]. These studies suggest that the pro- and anti-tumor roles of TFAP2C are function and tumor type dependent. However, the clinical significance and biological role of TFAP2C in colorectal cancer remain largely unknown. In this study, high expression of TFAP2C was observed in CRC tissues, which correlated with advanced clinicopathological features, poor prognosis and disease progression in CRC patients. Moreover, our results demonstrated that TFAP2C promoted the chemoresistance and stemness of CRC cells in vitro and in vivo, further determining the tumor-stimulatory role of TFAP2C in CRC.
Numerous studies have reported that several regulatory mechanisms were responsible for the inactivation of Hippo signaling, which played an important role in the chemotherapeutic resistance of cancer. Studies have consistently shown that inactivation of Hippo signaling by downregulation of the Hippo pathway components mammalian MST1/2 andLATS1/2 contributed to resistance of cancer cells to chemotherapeutic drugs [
48‐
50]. Furthermore, upregulation of YAP or TAZ conferred chemotherapeutic resistance in multiple cancer types [
51‐
53]. Recently, it was reported that Rho-associated protein kinase (ROCK) repressed activity of Hippo signaling through inhibition of LATS activity and cell polarity in the outside cells [
39]. Importantly, Cao and colleagues reported that TFAP2C inhibited Hippo signaling dependent on ROCK activity [
54]. It remains unclear, however, how ROCK mediates the inhibitory effect of TFAP2C on Hippo signaling activity and whether TFAP2C/ROCK/Hippo signaling promotes chemotherapeutic resistance in CRC. In this study, our results revealed that TFAP2C transcriptionally activated ROCK1 and ROCK2 via binding to the promoter region of ROCK1 and ROCK2 in CRC cells. Importantly, the stimulatory effects of TFAP2C on chemoresistance and stemness in CRC cells were effectively attenuated by the specific inhibitor of ROCK1 and ROCK2, Y-27632. Thus, our results uncover a novel mechanism by which TFAP2C promotes chemotherapeutic resistance and stemness in CRC cells.
It has been widely documented that AP-2 members may be used as a prognostic marker in numerous human tumor types. A study by Ikram et al. showed that low TFAP2B expression caused by CpG methylation of the TFAP2B locus in primary neuroblastomas significantly promoted proliferation and cell cycle progression and low expression of TFAP2B significantly associated with unfavorable prognostic markers as well as adverse patient outcome [
41]. Conversely, TFAP2A and TFAP2B correlated with good overall and disease-free survival in breast cancer patients [
55]. These studies indicated that different AP-2 proteins predict different prognosis in different tumor types. Furthermore, TFAP2C has been extensively reported to be a poor prognostic marker in several human cancers, including breast cancer, lung carcinoma, [
16,
40,
44]. However, the correlation of TFAP2C with prognosis of CRC patients remains unknown. In this study, our results found that TFAP2C expression level was increased in CRC tissues and high expression of TFAP2C correlated with advanced clinicopathological features. More importantly, Kaplan-Meier survival analysis revealed that CRC patients with high TFAP2C expression exhibited shorter overall survivals and progression-free survivals. Thus, these findings identify TFAP2C as a potential prognostic marker in CRC patients.