Abstract
Background
Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of cancer in India with high incidence and rapid recurrence rates. Here, we aimed to investigate the role of β-catenin, a developmental pathway gene, in HNSCC therapy resistance, DNA damage response, recurrence and prognosis.
Methods
In total 80 HNSCC samples were included. Western blot, immunohistochemistry and qRT-PCR analyses were performed to assess β-catenin expression in the cut margin and tumor areas of each sample. Kaplan-Meier analyses were performed to correlate β-catenin expression with the survival and prognosis of HNSCC patients. In addition, chemo-resistance, DNA damage response and DNA repair capacities were evaluated in HNSCC-derived cell lines through LiCl-mediated up-regulation and siRNA-mediated silencing of β-catenin expression.
Results
We observed β-catenin up-regulation in cut margin areas of recurrent patients compared to their corresponding tumor regions, which subsequently could be associated with poor prognosis. In addition, we found that LiCl-mediated up-regulation of β-catenin in HNSCC-derived cells led to cisplatin resistance, evasion of apoptosis, enhanced DNA repair and enhanced migration. The effects of β-catenin silencing correlated with its putative role in chemo-resistance and DNA damage response.
Conclusion
From our results we conclude that β-catenin may contribute to HNSCC therapy resistance and disease relapse. As such, β-catenin may be explored as a therapeutic target along with conventional therapeutics.
Electronic supplementary material
The online version of this article (10.1007/s13402-017-0365-1) contains supplementary material, which is available to authorized users.
Keywords: Head and neck squamous cell carcinoma (HNSCC), β catenin, Cisplatin-resistance, Disease relapse, DNA damage repair and response, Prognosis
Introduction
Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent solid tumors within the oral and maxillofacial region [1]. The incidence of HNSCC in India is high due to the consumption of smokeless tobacco [2]. In addition, cigarette smoking and alcohol consumption synergistically contribute to disease progression [3]. The standard treatment of HNSCC includes induction chemotherapy by cisplatin, 5-flurouracil and docetaxel followed by radiotherapy and surgery [4]. Despite advances that have been made in these treatment modalities, the 5 year survival rate is still low due to chemotherapy resistance, invasion and metastasis of the cancer cells [5]. Different chemotherapeutic approaches have shown different responses and, as such, to play a crucial role in therapy resistance and disease relapse [6–9]. Enhanced DNA damage repair capacity, increased drug efflux capacity, overexpression of drug resistance genes and evasion from apoptosis are among the primary reasons associated with therapy resistance [10]. Hence, combinatorial therapeutic approaches, targeting factors responsible for DNA damage and DNA repair responses, along with conventional regimens may help to decrease therapy resistance and disease relapse, and to increase the survival of HNSCC patients.
The Wnt/β-catenin signaling pathway plays a major role in both normal embryonic developmental and in tumor development [11]. Aberrant Wnt/β-catenin signaling may promote the initiation and progression of several cancers, including colon cancer, hepatocellular carcinoma, gastric cancer and HNSCC [12]. Cisplatin is one of the most commonly used drugs to treat cancer, including lung, ovarian, cervical and bladder cancer, hepatocellular carcinoma and HNSCC [13]. Previous reports have suggested that the Wnt/β-catenin signaling pathway is a major contributor to cisplatin resistance in non-small cell lung cancer (NSCLC) [14] and, concordantly, β-catenin has been found to be highly expressed in cisplatin resistant lung cancer cells [15]. It has also been found that inhibition of β-catenin signaling may promote DNA damage in colorectal cancer cells elicited by benzopyrene [16]. Previously, we have reported that aberrant Wnt/β-catenin signaling in HNSCC may lead to genomic instability [1]. Hence, elucidation of the role of β-catenin in therapy resistance and DNA damage response in HNSCC may provide insight into its progression. Here, we have investigated the involvement of β-catenin in therapy resistance, DNA damage response and DNA repair with the objective to explore the putative usefulness of β-catenin as a prognostic/therapeutic marker in HNSCC.
Materials and methods
Patient sample collection and ethics statement
In total 80 samples from patients operated for HNSCC were collected at the time of surgical removal. The tumor tissues and their respective cut margin areas were stored appropriately. The study was approved by the institutional ethics committee of the School of Biotechnology and Kalinga Institute of Medical Sciences (KIMS), KIIT University, and conducted according to the Helsinki declaration. The human sample collection was carried out strictly as per institutional ethical board guidelines. Informed consent was obtained from all subjects or their nominees prior to participation in the study.
Cell culture
The colon cancer-derived cell line HCT-116 was procured from Imgenex Laboratories (Bhubaneshwar, Odisha, India). The HNSCC-derived cell lines UPCI-SCC-131 and CAL-27 were generously gifted by Dr. Susanta Roychoudhury, former Scientist of the Indian Institute of Chemical Biology (IICB), Kolkata, India and Dr. Amrita Suresh, Department of Head and Neck Oncology, Mazumdar Shaw Medical Center, Narayana Health, Bangalore, India, respectively. The cells were cultured as monolayers and maintained in DMEM (GIBCO, Life Technologies) supplemented with 1% antibiotics (100 U/ml penicillin and 10 mg/ml streptomycin (HIMEDIA), 10% FBS (GIBCO, Life Technologies) and 1% (w/v) L-glutamine (HIMEDIA) at 37 °C in a humidified incubator containing 5% CO2.
Reagents, antibodies and siRNAs
Cisplatin and lithium chloride (LiCl) were obtained from Cipla and Sigma-Aldrich, respectively. Anti-β-catenin and anti-mouse secondary antibodies were procured from Abcam (Cambridge, UK), whereas anti-GSK3β and anti-GAPDH antibodies were procured from Cell signaling and IMGENEX, respectively. DharmaFECT transcription reagent, β-catenin siRNA and scrambled siRNA were obtained from GE-Dharmacon.
Transient siRNA-mediated β-catenin silencing
UPCI-SCC-131 and HCT-116 cells were seeded in 6-well plates at a density of 1 × 105 cells/well and allowed to grow till 50% confluence. Subsequent scrambled siRNA and β-catenin siRNA transfections were carried out using DharmaFECT transcription reagent (Dharmacon) as per laboratory established protocol [17]. The silencing of β-catenin was confirmed by qRT-PCR and Western blotting, respectively.
β-catenin up-regulation by LiCl
UPCI-SCC-131 and CAL-27 cells were seeded in 6-well plates at a density of 2 × 105 cells/well and allowed to grow till 70% confluence. Next, the cells were treated with different concentrations of LiCl (2.5–10 mM) and incubated for 24 h at 37 °C in a humidified incubator. Up-regulation and nuclear translocation of β-catenin were confirmed by mRNA expression, protein expression and immunocytochemistry analyses, respectively.
RNA extraction and quantitative RT-PCR
Primary tissue samples and cells harvested after 48 h of transfection, 24 h of LiCl treatment and 24 h of cisplatin treatment were subjected to total RNA extraction using TRIZOL reagent (Invitrogen) as reported before [1]. Reverse transcription was performed using a TETRO cDNA synthesis kit (BIOLINE). Subsequent quantitative real-time PCR (qRT-PCR) analyses were performed for the β-catenin, ERCC1 and ABCG2 genes using KAPA SYBR® FAST qPCR Kit Master Mix (2×) Universal (Kapa Biosytems). β-actin was used as an endogenous control and mRNA fold changes were calculated using the 2-ΔΔCT method. The primer sequences are provided in Supplementary Table 1.
Western blot analysis
Western blot analyses were carried out on parental HCT-116, UPCI-SCC-131 and CAL-27 cells, UPCI-SCC-131 and HCT-116 β-catenin silenced cells, SCC-131 cells and CAL-27 β-catenin up-regulated cells and patient samples as per previously published protocol [1]. Cells and patient samples were harvested and re-suspended in RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 Mm NaCl, 1% NP-40, 0.25% sodium de-oxycholate, 1% Triton-X-100, 1 mM EDTA, Milli-Q water) to obtain protein lysates. 50 μg of the protein lysates were separated by 12% SDS-PAGE and transferred to PVDF membranes. Next, the membranes were blocked with 5% skimmed milk in PBST for 1 h at room temperature (RT) and probed with primary antibody (1:2000) at 4 °C overnight. After this, the membranes were washed with 0.25% PBST and probed with secondary antibody (1:4000) for 2 h at RT. Finally, the proteins were visualized by enhanced chemiluminescence using X-ray films (Kodak, India). All experiments were performed in triplicate and representative images of the blots are provided.
Immunohistochemistry
Immunohistochemical analyses of HNSCC FFPE tissue blocks were performed as per laboratory established protocol with minor modifications [1]. Briefly, tissues were cut into 3 μm sections and dried at 60 °C for 3 h, de-paraffinized and de-hydrated. Antigen retrieval was performed in 10 mM sodium citrate buffer pH 6.0 for 20 min in a microwave. Peroxide blocking was performed in the dark at RT for 30 min to quench endogenous peroxidase activity. Next, the sections were blocked with 5% BSA in PBS for 1 h at RT and then probed with a mouse monoclonal anti-β-catenin primary antibody (1:1000) at 4 °C overnight. On the next day, the sections were washed with PBS and incubated with a secondary antibody (1:1000) conjugated with HRP for 90 min at RT, followed by washing and treatment with DAB chromogen for 5 min at RT. Finally, the sections were washed and counterstained with hematoxylin, and images were captured using a Leica (DM 2000) bright-field microscope. Expression was scored by multiplying the percentage of positive cells (0–100%) with the intensity (weak: 1, moderate: 2 and strong: 3) to obtain a maximum score of 300. Scale bars were calculated using Image J software.
Cell viability assay
The viabilities of parental UPCI-SCC-131 and CAL-27 cells, LiCl-mediated β-catenin up-regulated UPCI-SCC-131 and CAL-27 cells, parental HCT-116 cells, scrambled siRNA transfected UPCISCC-131 and HCT-116 cells and β-catenin siRNA transfected UPCI-SCC-131 and HCT-116 cells, were determined by MTT assay. Cells were seeded in 96-well plates at a density of 1 × 104 cells per well and allowed to adhere overnight in a humidified incubator at 37 °C. On the next day, the cells were treated with different cisplatin concentrations (1–20 μM) for 24 h. After this treatment period MTT reagent [3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide)] was added to each well and incubated for 4 h at 37 °C to allow the formation of formazan crystals. Next, these crystals were dissolved in dissolution solution (11 mg SDS dissolved in 50 ml isopropanol and 50 ml 0.02 M HCl) and the absorbance was measured at 570 nm in an ELISA reader (Biotek, Germany). All experiments were performed in triplicate. The percentage cell viability was calculated relative to controls, and IC50 values were determined using GraphPad Prism 6 software.
Cell survival assay
The colony forming capacities of parental UPCI-SCC-131 and CAL-27 cells, LiCl-mediated β-catenin up-regulated UPCI-SCC-131 and CAL-27 cells, parental HCT-116 cells, scrambled siRNA transfected UPCI-SCC-131 and HCT-116 cells and β-catenin siRNA transfected UPCI-SCC-131 and HCT-116 cells were determined using a clonogenic cell survival assay. To this end, 500 cells/well were seeded in a 6-well cell culture plate and allowed to adhere overnight in a humidified incubator at 37 °C. Next, the cells were treated with increasing concentrations of cisplatin (1–15 μM) for 24 h. After that, the drug containing medium was replaced with fresh medium and the cells were allowed to form colonies for 7–8 days. Thereafter, the medium was removed and the cells were incubated with 0.2% crystal violet in methanol. Finally, the wells were washed with distilled water and colonies were counted using a gel documentation system (UVP, Germany). All experiments were performed in triplicate. The data were represented as number of colonies formed per 500 cells and as percentage cell survival relative to control.
Cell cycle and apoptosis assays
Cell cycle analyses were performed for parental UPCI-SCC-131 and CAL-27 cells, LiCl-mediated β-catenin up-regulated UPCI-SCC-131 and CAL-27 cells, parental HCT-116 cells, scrambled siRNA transfected UPCI-SCC-131 and HCT-116 cells and β-catenin siRNA transfected UPCI-SCC-131 and HCT-116 cells with or without cisplatin treatment (1–10 μM). The cells were seeded at a density of 1 × 105 cells/well in 6-well plates, collected by trypsinization, pelleted by centrifugation at 1200 rpm for 5 min and re-suspended in phosphate-buffered saline (PBS). Subsequently, the cells were fixed in 70% cold ethanol and stored overnight at −20 °C. Next, the cells were pelleted at 3000 rpm for 5 min and subsequently washed with PBS. Finally, the cells were re-suspended in PBS containing RNase A (100 μg/ml) and propidium iodide (50 μg/ml) and incubated for 45 min in the dark at room temperature, after which cell cycle distribution analyses were performed using FACS CANTO II (Becton & Dickinson, CA, USA) equipment with a count of 10,000 events per sample. Data analyses were performed using FACS diva software. Apoptotic cells were measured in the sub-G0 phase of the cell cycle.
Scratch wound healing assay
A scratch wound healing assay was used to study cellular migration and proliferation, and was performed as per protocol reported before [18]. Briefly, parental UPCI-SCC-131 and CAL-27 cells, LiCl-mediated β-catenin up-regulated UPCI-SCC-131 and CAL-27 cells, parental HCT-116 cells, scrambled siRNA transfected UPCI-SCC-131 and HCT-116 cells and β-catenin siRNA transfected UPCI-SCC-131 and HCT-116 cells were cultured in 6-well culture plates and allowed to grow till 90% confluence. Next, a ‘wound’ was created using a sterile micropipette tip by scratching the monolayer. The scratched cells were removed by rinsing with medium. Next, the cells were treated with different concentrations of cisplatin (1-10 μM) and allowed to grow for 24 h. Wound images were captured at 0 and 24 h using an Olympus inverted microscope at 10× magnification. The images were analyzed using Image J software and percentages of wound closure were calculated. All experiments were performed in triplicate and representative images were provided.
Immunocytochemistry analysis
Immunocytochemistry was performed as reported before [19]. Briefly, cells were grown on glass coverslips and subsequently fixed with 4% paraformaldehyde for 15 min at room temperature (RT), permeabilized with 0.25% PBST for 10 min at RT followed by blocking with 5% BSA (Bovine Serum Albumin) for 30 min. Next, the cells on the coverslips were probed with a primary antibody (1:2000) at 4 °C overnight. The next day, the cells were washed with PBS and probed with FITC/TRITC fluorophore tagged secondary antibodies (1:4000) for 1 h at RT. Finally, the cells on the coverslips were counterstained with 4, 6-diamidino-2-phenylindole (DAPI), mounted, sealed and evaluated using a fluorescence microscope (Olympus BX 61). Images were captured using Image Pro Express software.
γ-H2AX foci formation assay
To check the extent of DNA damage, γ-H2AX foci formation assays were performed as per protocol reported before [19]. Briefly, parental UPCI-SCC-131 cells, scrambled-siRNA transfected SCC-131 cells, β-catenin siRNA transfected SCC-131 cells and LiCl treated β-catenin up-regulated SCC-131 cells were seeded at a density of 1 × 105 on coverslips in 6-well plates, grown to 50–70% confluence and treated with increasing concentrations of cisplatin (1–10 μM). After incubation, the coverslips were washed and the cells were fixed in methanol:acetone (1:1), permeabilized with 0.25% PBST (1xPBS, 0.25% Triton-X100) at room temperature and blocked in 2.5% BSA in PBST for 1 h at room temperature. Next, an anti-γ-H2AX primary antibody (1:2000 dilution) was added to the coverslips and incubated overnight at 4 °C. After this, the coverslips were washed and incubated with FITC/TRITC fluorophore tagged secondary antibodies (1:4000 dilution) for 1 h at room temperature in the dark. Finally, the coverslips were counterstained with DAPI, mounted, sealed and evaluated using a fluorescence microscope (Olympus BX 61). Images were captured using Image Pro Express software.
COMET assay
Single cell gel electrophoresis (COMET) assays were performed to analyze the extent of DNA damage in parental UPCI-SCC131 cells, scrambled-siRNA transfected SCC-131 cells, β-catenin siRNA transfected SCC-131 cells and LiCl treated β-catenin up-regulated SCC-131 cells as per protocol reported before, with minor modifications [17, 20]. Cells were seeded in 6-well plates at a density of 1 × 105 cells per well and allowed to grow till 60–70% confluence. Next, the cells were treated with increasing concentrations of cisplatin (1–10 μM) and incubated for 24 h in a humidified CO2 incubator at 37 °C. Subsequently, the slides were coated with 0.5% agarose and allowed to dry overnight at 37 °C. Cells were suspended in 0.5% low melting point agarose and added to the dried slides, after which the agarose was allowed to solidify at 4 °C and kept in a pre-chilled lysis solution for 4 h. Next, the slides were transferred to an electrophoresis tank and run at 300 mA for 30 min at room temperature in the dark. After this step, the slides were immersed in neutralization buffer overnight at 4 °C. The next day, the slides were stained with PI (1 μg/ml) after which imaging was carried out using a fluorescent microscope (Olympus BX 61). Images were captured using ImagePro Express software and analyzed using the Image J plugin OpenComet [21]. The extent of DNA damage is represented by Olive tail moment and presented in a graph.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 6 software. A χ2 test was used to evaluate associations between β-catenin expression and clinico-pathological characteristics. Kaplan-Meier analyses and log-rank tests were used to determine disease-free and overall survival rates. A p-value < 0.05 was considered as statistically significant. Two-way Anova and t tests were performed to assess statistical significance.
Results
High β-catenin expression in cut margins is associated with recurrence in HNSCC patients
In order to assess whether β-catenin expression is associated with HNSCC, we conducted a meta-analysis using publicly available Oncomine datasets [22]. Specifically, we used three HNSCC datasets reported by Peng et al., Ginos et al. and Cromer et al., respectively [23–25]. Through this meta-analysis, a higher β-catenin expression was revealed in HNSCC samples compared to normal mucosa samples (Fig. 1a). Previously, we also reported a clinico-pathological correlation between β-catenin expression and telomere dysfunction in HNSCC [1]. Hence, we conclude that β-catenin may play a role in HNSCC development and/or progression.
Fig. 1.
Differential β-catenin expression in cut-margin and tumor tissues of recurrent and non-recurrent HNSCC patients. a β-catenin mRNA expression data extracted from the Peng, Gino and Cromer datasets, respectively, presented as log2 median-centered HNSCC versus normal mucosa ratios. [0 = Normal mucosa; 1 = Tumor of HNSCC patient]. b Representative blots showing differential expression of β-catenin in cut margin (CM) and Tumor (T) areas of recurrent and non-recurrent patients. c-d Quantitative representation of β-catenin protein expression in CM and T areas of recurrent and non-recurrent patients. e-f Comparative immunohistochemical analysis and graphical representation of β-catenin expression in CM and T areas of recurrent and non-recurrent patients. g Graphical representation of quantitative β-catenin mRNA expression in CM and T areas of recurrent and non-recurrent patients
In the current study 80 HNSCC patients were included. The detailed clinico-pathological characteristics of these patients are listed in Supplementary Table 2. To assess the expression of β-catenin protein in surgically removed cut margin (CM) tissue samples and tumor tissue samples, Western blot analyses were carried out. We found an increased expression of β-catenin in the CM areas of recurrent HNSCC cases compared to their tumor counterparts (Fig. 1b, c). No significant differences in β-catenin expression were observed between CM samples and tumor samples of non-recurrent cases (Fig. 1b, d). Subsequently, immunohistochemical (IHC) analyses were performed to assess the localization of β-catenin in CM tissue and tumor tissue samples of recurrent and non-recurrent HNSCC cases. We observed a strong expression of β-catenin in the cellular membranes, cytoplasm and nuclei of the CM tissues of recurrent HNSCC cases, whereas weak membranous signals were observed in the tumor tissues of these cases (Fig. 1e, f). In non-recurrent HNSCC cases, the expression of β-catenin was found to be minimal in the CM areas, while a strong β-catenin expression was observed both in the cellular nuclei and cytoplasm of the tumor tissues of these cases (Fig. 1e, f). These findings were subsequently confirmed by qRT-PCR, i.e., an increased β-catenin mRNA expression was observed in the CM areas of recurrent HNSCC cases compared to their tumor tissue counterparts. In the non-recurrent HNSCC cases no significant differences in β-catenin mRNA expression were observed between the respective CM and tumor tissues (Fig. 1g).
Next, univariate analyses were performed to evaluate putative associations between high β-catenin expression in the CM or tumor tissues and different prognostic factors such as age, gender, histological tumor grade, tumor site, recurrence, lymph node metastasis, bone metastasis, skeletal muscle involvement, lympho-vascular and perineural invasion and other co-morbid factors using a χ2 test (Table 1). By doing so, we found that a high β-catenin expression in the CM margins was significantly associated with recurrence in HNSCC patients.
Table 1.
Association of β-catenin expression with clinicopathological characteristics of 80 HNSCC patients
| Clinicopathological characteristics | No. of patients | High β-catenin expression | P value | |
|---|---|---|---|---|
| Cut margin | Tumor | |||
| Age | ||||
|
> 60 ≤ 60 |
31 49 |
17 30 |
14 19 |
0.5719 |
| Gender | ||||
|
Male Female |
68 12 |
40 8 |
28 4 |
0.6091 |
| Histological Grade | ||||
|
Well Moderate Poor |
36 39 5 |
21 23 2 |
15 16 3 |
0.7390 |
| Site of tumor | ||||
|
Tongue Buccal mucosa Cheek |
24 50 6 |
17 26 4 |
7 24 2 |
0.2807 |
| Recurrence | 0.0055** | |||
| Recurrent | 26 | 21 | 5 | |
| Non-recurrent | 54 | 26 | 28 | |
| Lymph node metastasis | 0.4479 | |||
| Yes | 23 | 12 | 11 | |
| No | 57 | 35 | 22 | |
| Lymphovascular Invasion (LVI) | 0.1885 | |||
| Yes | 6 | 2 | 4 | |
| No | 74 | 45 | 29 | |
| Perineural Invasion (PNI) | 0.6549 | |||
| Yes | 19 | 12 | 7 | |
| No | 61 | 35 | 26 | |
| Bone metastasis | 0.6086 | |||
| Yes | 9 | 5 | 4 | |
| No | 71 | 42 | 29 | |
| Skeletal muscle involvement | 0.3620 | |||
| Yes | 3 | 1 | 2 | |
| No | 77 | 46 | 31 | |
| Co-morbidities | 0.2940 | |||
| No comorbidities | 50 | 29 | 21 | |
| Cardiac diseases/Hypertension | 16 | 11 | 5 | |
| Diabetes | 21 | 9 | 12 | |
| Others | 5 | 4 | 1 | |
**P<0.01 – Statistically significant
High β-catenin expression in cut margin areas is associated with poor prognosis in recurrent HNSCC patients
In order to evaluate the prognostic relevance of β-catenin expression, we assessed its impact on disease-free and overall HNSCC patient survival using Kaplan-Meier analyses and log rank tests. After analysis of the survival rates in the 80 HNSCC patients in relation to β-catenin expression, no significant differences in disease-free and overall survival were observed in patients exhibiting high or low β-catenin expression levels in the tumor samples (Fig. 2a, b). Next, the HNSCC patients were segregated into two cohorts, i.e., recurrent and non-recurrent, for which the disease-free and overall survival rates were analyzed. By doing so, we observed a significantly reduced disease-free and overall survival in recurrent patients compared to the non-recurrent patients (Fig. 2c, d). We also investigated the impact of high β-catenin expression in the CM and tumor samples of recurrent HNSCC patients on survival. We found that both the disease-free and overall survival rates were lower in recurrent patients exhibiting a high β-catenin expression in the CM areas compared to those with a high expression in the tumor samples (Fig. 2e, f).
Fig. 2.
High β-catenin expression in cut margins leads to a poor prognosis in recurrent HNSCC patients. a-b Kaplan-Meier curves of disease-free and overall survival in high versus low β-catenin expressing HNSCC patients. c-d Kaplan-Meier curves of disease-free and overall survival of recurrent and non-recurrent HNSCC patients (e-f) Kaplan-Meier curves for disease-free and overall survival of recurrent patients exhibiting higher β-catenin expression in cut margin (CM) compared to tumor (T) areas. P < 0.05 is considered statistically significant
Differential β-catenin expression in colon cancer and HNSCC-derived cells
In order to validate the above findings from HNSCC patient samples, we decided to perform a comparative β-catenin expression analysis in the colon cancer-derived cell line HCT-116 and the HNSCC-derived cell lines UPCI-SCC-131 and CAL-27. The HCT-116 cell line was selected since it has previously been reported [26, 27] that these cells are heterozygous for β-catenin (i.e., one wild-type allele and one mutant allele), which results in stabilization of β-catenin and, hence, activation of the Wnt signaling cascade. We found that the HNSCC-derived cells UPCI-SCC-131 and CAL-27 exhibited lower β-catenin expression levels than the HCT-116 cells (Fig. 3a-c). Next, we set out to explore whether the high β-catenin expression may play any role in chemo-resistance. We found that the parental HCT-116 cells showed an increase in IC50 value, as well as in colony forming, survival and migration capacities, compared to β-catenin silenced HCT-116 cells when treated with different concentrations of cisplatin (1–20 μM) (Supplementary Fig. 2). We also found that the percentage of early apoptotic cells decreased in the parental HCT-116 cells compared to that in the β-catenin silenced HCT-116 cells when treated with 1 μM cisplatin (Supplementary Fig. 2). These findings led us to speculate that an increased β-catenin expression may play a role in chemo-resistance.
Fig. 3.
Differential β-catenin expression in colon cancer and HNSCC-derived cells. a Western blot analysis of β-catenin expression in HCT-116 cells, UPCI-SCC-131 cells and CAL-27 cells. b qRT-PCR expression analysis of β-catenin in HCT-116 cells, UPCI-SCC-131 cells and CAL-27 cells. (c) Representative immunofluorescence images showing increased nuclear localization of β-catenin in HCT-116 cells compared to HNSCC cells (UPCI-SCC-131, CAL-27)
LiCl treatment up-regulates the expression and nuclear localization of β-catenin in HNSCC cells
Previously, it has been shown that LiCl treatment leads to activation of the Wnt/β-catenin pathway in different types of cancer, including ovarian cancer, lung cancer and hepatocellular cancer [15, 28]. Hence, we decided to assess the effect of LiCl treatment on activation of the Wnt/β-catenin pathway in HNSCC-derived cells. We found that after treatment with increasing concentrations of LiCl (2.5, 5, 10 mM), 5 mM LiCl treatment led to an increase in β-catenin expression and a decrease in GSK-3β expression in both the UPCI-SCC-131 and CAL-27 cell lines (Fig. 4a-f). In addition, we observed a nuclear localization of β-catenin upon treatment with 5 mM LiCl in both HNSCC cell lines (Fig. 4g, h).
Fig. 4.
LiCl treatment up-regulates β-catenin expression and promotes nuclear localization in HNSCC cells. a and d Western blots showing β-catenin expression after treatment with different concentrations of LiCl (2.5-10 mM) in UPCI-SCC-131 and CAL-27 cells. b and e Relative protein expression of β-catenin after treatment with increasing concentrations of LiCl in UPCI-SCC-131 and CAL-27 cells. c and f mRNA expression levels of β-catenin post LiCl treatment in UPCI-SCC-131 and CAL-27 cells. g-h Representative immunofluorescence images showing increased nuclear localization of β-catenin at 5 mM LiCl treatment in UPCI-SCC-131 cells and CAL-27 cells
β-catenin up-regulation leads to increases in the viability, growth and migration of cisplatin treated HNSCC cells
Next, we set out to determine whether LiCl-mediated up-regulation of β-catenin expression has any effect on malignancy-associated characteristics of cisplatin treated HNSCC cells. First, the effect of cisplatin on the viability of parental cells and HNSCC cells in which β-catenin was up-regulated by LiCl was tested using a MTT assay. After treatment of the cells with increasing concentrations (1–20 μM) of cisplatin for 24 h, we found that this treatment resulted in decreases in cell viability in a dose-dependent manner in both the UPCI-SCC-131 and CAL-27 cells. However, a ~2-fold increase in cisplatin IC50 value was observed in LiCl-mediated β-catenin up-regulated UPCI-SCC-131 cells compared to its non-upregulated parental cells, and a 1.55-fold increase in cisplatin IC50 value in LiCl-mediated β-catenin up-regulated CAL-27 cells compared to its non-upregulated parental cells, respectively (Fig. 5a). Next, the effect of cisplatin on the growth capacity of HNSCC cells was assessed using a clonogenic assay. We found that β-catenin up-regulated UPCI-SCC-131 cells showed an increase in colony forming ability compared to non-upregulated parental UPCI-SCC-131 cells when treated with increasing doses of cisplatin (1–15 μM). Similar results were obtained with β-catenin up-regulated CAL-27 cells compared to non-upregulated parental CAL-27 cells (Fig. 5b, c). These preliminary results suggested that β-catenin may play a role in cisplatin resistance in HNSCC cells. Additionally, we assessed the role of β-catenin in cisplatin resistance of HNSCC cells by measuring the percentage early apoptotic cells in the Sub-G0 phase of the cell cycle. We found that a 4 μM cisplatin treatment led to a 2.26-fold decrease in the apoptotic cell population in LiCl-mediated β-catenin up-regulated UPCI-SCC-131 cells compared to non-upregulated parental cells. Similarly, a 1.53-fold decrease in the apoptotic cell population was observed in LiCl-mediated β-catenin up-regulated CAL-27 cells after 4 μM cisplatin treatment compared to non-upregulated parental cells (Fig. 5d). We also evaluated the effect of LiCl-mediated up-regulation of β-catenin on the migration of HNSCC cells using a scratch wound healing assay as reported before [18]. We found that β-catenin up-regulation in UPCI-SCC-131 cells resulted in a 2.19-fold increase in cellular migration compared to non-upregulated parental cells after 1 μM cisplatin treatment. This pattern was similar after treatment of the cells with 4 μM and 10 μM cisplatin, respectively. Likewise, we observed a 1.45-fold increase in migration in β-catenin up-regulated CAL-27 cells compared to its non-upregulated parental counterpart. Again, increased cellular migration was observed after treatment of the cells with different concentrations of cisplatin (1–10 μM) (Fig. 5e, f). Together, these results suggest that β-catenin up-regulation leads to cisplatin resistance in HNSCC cells.
Fig. 5.
LiCl-mediated β-catenin up-regulation promotes cisplatin resistance, increases cellular proliferation and inhibits apoptosis in HNSCC cells. a Viability of parental and LiCl-treated β-catenin up-regulated UPCI-SCC-131 cells after treatment with different concentrations of cisplatin (1–20 μM). b-c Colony forming capacity and survival of parental and LiCl-treated β-catenin up-regulated UPCI-SCC-131 cells after cisplatin treatment (1–15 μM). d Graphical representation of percentage apoptotic cells (Sub-G0) in parental and LiCl-treated β-catenin up-regulated UPCI-SCC-131 cells after cisplatin treatment (1–10 μM). e and g Representative images of scratch wounds in parental and LiCl-treated β-catenin up-regulated UPCI-SCC-131and CAL-27 cells at 0 and 24 h after cisplatin treatment (1–10 μM). f and h Graphical representations of percentages wound closure after 24 h cisplatin treatment in parental and LiCl-treated β-catenin up-regulated UPCI-SCC-131 and CAL-27 cells
β-catenin silencing leads to increased cisplatin sensitivity in HNSCC cells
In order to explore the role of β-catenin in cisplatin resistance of HNSCC cells in further detail, we set out to transiently silence β-catenin expression by siRNA in UPCI-SCC-131 cells. After having confirmed efficient β-catenin silencing at both the mRNA and protein level (Supplementary Fig. 3 a-c), we investigated its effect on cell cycle progression. We found that β-catenin silencing led to a substantial S phase cell cycle arrest compared to that of scrambled siRNA transfected and non-silenced parental cells (Supplementary Fig. 3 d). We also found that β-catenin silencing led to a decrease in cell viability post cisplatin treatment in silenced cells compared to that in scrambled siRNA transfected cells. The IC50 value in β-catenin silenced cells was 2.13 μM compared to 3.7 μM in its scrambled siRNA transfected counterpart (Fig. 6a). In support of these observations, we found by clonogenic assay that β-catenin silencing led to a dose-dependent decrease in the colony forming ability of the cells post cisplatin treatment (1–15 μM) compared to its scrambled siRNA transfected counterpart (Fig. 6b, c). Cell cycle analysis further revealed an increase in cisplatin-induced apoptosis in β-catenin silenced UPCI-SCC-131 cells compared to scrambled siRNA transfected cells. After 4 μM of cisplatin treatment a 1.66-fold increase in the early apoptotic population was observed in β-catenin silenced cells compared to its scrambled siRNA transfected counterpart (Fig. 6d). Based on these results, we conclude that β-catenin silencing leads to an increase in cisplatin induced apoptosis in UPCI-SCC-131 cells compared to its scrambled siRNA transfected counterpart. In addition, we observed a 3.92-fold decrease in cellular migration in β-catenin silenced UPCI-SCC-131 cells compared to scrambled siRNA transfected cells after 1 μM cisplatin treatment. A significantly reduced migratory ability of β-catenin silenced UPCI-SCC-131 cells was also observed at cisplatin concentrations of 4 μM and 10 μM (Fig. 6e, f). Hence, loss of β-catenin expression may have resulted in an impaired migratory capacity of HNSCC cells after a chemotherapeutic (cisplatin) insult.
Fig. 6.
Silencing of β-catenin sensitizes HNSCC cells to cisplatin and promotes apoptosis. a Viability of scrambled siRNA and siRNA-β-catenin transfected UPCI-SCC-131 cells after cisplatin treatment (1–20 μM). b-c Colony forming capacity and survival of scrambled siRNA and β-catenin siRNA transfected UPCI-SCC-131 cells after cisplatin treatment (1–15 μM). d Graphical representation of percentage apoptotic cells (Sub-G0) in scrambled siRNA and β-catenin siRNA transfected UPCI-SCC-131 cells after cisplatin treatment (1–10 μM). e Representative images of scratch wounds in scrambled siRNA and β-catenin siRNA transfected UPCI-SCC-131 cells at 0 and 24 h after cisplatin treatment (1–10 μM). (f) Graphical representation of percentages wound closure after 24 h cisplatin treatment in scrambled siRNA and β-catenin siRNA transfected UPCI-SCC-131 cells
β-catenin plays a role in DNA damage repair
Next, we set out to investigate a possible involvement of β-catenin in DNA damage repair after cisplatin treatment. To this end, the expression of β-catenin was evaluated in parental UPCI-SCC-131 cells, LiCl-mediated β-catenin up-regulated UPCI-SCC-131 cells, β-catenin silenced and scrambled siRNA transfected UPCI-SCC-131 cells post cisplatin treatment (1-10 μM). A dose-dependent increase in β-catenin expression was observed in LiCl-mediated β-catenin up-regulated cells compared to its non-upregulated parental counterpart after treatment with 1 μM and 4 μM cisplatin. But, after 10 μM cisplatin treatment, we found that the expression of β-catenin was lower in β-catenin up-regulated cells compared to its parental non-upregulated cells. In contrast, we found that β-catenin silencing led to a dose-dependent decrease in the expression of β-catenin in silenced cells compared to scrambled siRNA transfected cells when treated with different concentrations of cisplatin (1-10 μM) (Fig. 7a, b).
Fig. 7.
Role of β-catenin in DNA damage response, repair and drug resistance in HNSCC cells. a Western blot results and graphical representation of relative β-catenin protein expression after treatment with different concentrations of cisplatin (1–10 μM) in parental UPCI-SCC-131 cells, LiCl-mediated β-catenin up-regulated UPCI-SCC-131 cells, scrambled siRNA and β-catenin siRNA transfected UPCI-SCC-131 cells. b β-catenin mRNA expression in parental UPCI-SCC-131 cells, LiCl-treated β-catenin up-regulated cells, scrambled siRNA and β-catenin siRNA transfected UPCI-SCC-131 cells after cisplatin treatment (1–10 μM). c Representative images of γ-H2AX foci in parental UPCI-SCC-131 cells, LiCl-treated β-catenin up-regulated cells, scrambled siRNA and β-catenin siRNA transfected cells after treatment with cisplatin (1–10 μM). d Graphical representation of the number of γ-H2AX foci/nuclei in parental UPCI-SCC-131 cells, LiCl-treated β-catenin up-regulated cells, scrambled siRNA and β-catenin siRNA transfected cells after treatment with different concentrations of cisplatin (1–10 μM). e Representative images of comet tail formation in parental UPCI-SCC-131 cells, LiCl-treated β-catenin up-regulated cells, scrambled siRNA and β-catenin siRNA transfected cells post cisplatin treatment (1–10 μM). f Graphical representation of percentages of olive tail moment in parental UPCI-SCC-131 cells, LiCl-treated β-catenin up-regulated cells, scrambled siRNA and β-catenin siRNA transfected cells after cisplatin treatment (1–10 μM). g-h ERCC1 and ABCG2 mRNA expression in parental UPCI-SCC-131 cells, LiCl-treated β-catenin up-regulated cells, scrambled siRNA and β-catenin siRNA transfected cells post cisplatin treatment (1–10 μM)
Subsequently, we investigated the DNA damage repair capacity of UPCI-SCC-131 cells under LiCl-mediated β-catenin up-regulated and β-catenin silenced conditions after cisplatin treatment using a γ-H2AX assay. We found a 4.77-fold decrease in γ-H2AX foci/nuclei in β-catenin up-regulated cells compared to its non-upregulated parental cells after 1 μM cisplatin treatment. A similar result was observed in 4 μM and 10 μM treated β-catenin up-regulated cells (Fig. 7c, d). In contrast, we found a 1.5-fold increase in γ-H2AX foci/nuclei in β-catenin silenced cells compared to scrambled siRNA transfected cells after 1 μM cisplatin treatment (Fig. 7c, d). Treatment of β-catenin silenced cells with 4 μM and 10 μM cisplatin also resulted in decreased γ-H2AX foci/nuclei.
These results were further confirmed in UPCI-SCC-131 parental cells, LiCl-mediated β-catenin up-regulated and β-catenin silenced UPCI-SCC-131 cells after cisplatin treatment using a COMET assay as reported before [17, 20]. This assay revealed a 2.21-fold decrease in percentage olive tail moment in β-catenin up-regulated cells compared to its parental non-upregulated cells after treatment with 4 μM cisplatin (Fig. 7e, f). In contrast, we found that 4 μM cisplatin treatment led to a ~2-fold increase in percentage olive tail moment in β-catenin silenced cells compared to its scrambled siRNA transfected counterpart (Fig. 7e, f). Hence, we conclude that LiCl-mediated β-catenin up-regulated cells exhibit less DNA damage or enhanced DNA damage repair compared to its parental cells when treated with increasing concentrations of cisplatin, whereas silencing of β-catenin leads to increased DNA damage or reduced DNA damage repair compared to its scrambled siRNA transfected counterparts.
We also assessed the expression of the drug resistance-associated genes ERCC1 and ABCG2 in parental and LiCl-mediated β-catenin up-regulated UPCI-SCC-131 cells, and in β-catenin silenced and scrambled siRNA transfected UPCI-SCC-131 cells after cisplatin treatment (1-10 μM). We found that LiCl-mediated up-regulation of β-catenin led to increases in ERCC1 and ABCG2 expression compared to its non-upregulated parental cells after treatment with 1 μM and 4 μM cisplatin (Fig. 7g, h). A reduced level of ABCG2 expression was, however, observed in β-catenin up-regulated cells compared to its non-upregulated parental counterparts when treated with 10 μM cisplatin. In addition, we found that β-catenin silencing led to a dose-dependent reduction in ERCC1 and ABCG2 expression (Fig. 7g, h). These results indicate that in cisplatin treated HNSCC cells the DNA damage response and repair capacity was decreased in the absence of β-catenin. Hence, we conclude that β-catenin may play an important role in modulating DNA damage repair responses and drug (cisplatin) resistance in HNSCC cells.
Discussion
Several lines of evidence indicate that the Wnt/β-catenin pathway plays a role in HNSCC [29]. Previously, we found that β-catenin may play an important role in regulating two major pathways, i.e., the telomere maintenance and the hTERT regulatory pathways [1]. We also found that HNSCC cases with high β-catenin levels in their cut margins suffered from rapid recurrences, poor prognosis and poor overall survival [30]. These findings prompted us to look in more detail into the role of β-catenin in disease relapse and therapy resistance in primary HNSCC patient samples as well as in HNSCC-derived cell lines. A meta-analysis of three publicly available HNSCC datasets again revealed an association between β-catenin expression and HNSCC progression. Additional mRNA and protein expression analyses of surgically removed tumor tissues and its cut margin (CM) counterparts revealed an up-regulated β-catenin expression in the CM regions of recurrent HNSCC samples compared to that in non-recurrent HNSCC samples. Subsequent immunohistochemistry analyses revealed the presence of a transcriptionally active form of β-catenin in the nucleus, along with cytoplasmic expression, in the CM regions of the recurrent HNSCC cases. We speculate that this nuclear expression may have led to minimal residual disease and a rapid proliferation of cancer cells post chemotherapy/radiotherapy and surgical excision, thereby resulting in recurrence.
Univariate analysis of prognostic factors such as age, gender, histological tumor grade, tumor site, lymph node metastasis, bone metastasis, skeletal muscle involvement, lympho-vascular invasion, perineural invasion and other co-morbid factors yielded no significant association with β-catenin expression in the CM and tumor regions in HNSCC patients. We found, however, that a high β-catenin expression in the peri-tumor (CM) areas was markedly associated with recurrence. The univariate analyses also revealed that comorbid factors exhibited a significant association with recurrence (Supplementary Table 3). Additional Kaplan-Meier analyses revealed no significant differences in the disease-free and overall survival rates of HNSCC patients based on β-catenin expression. Survival analysis in recurrent patients revealed that patients having higher β-catenin expression levels in the CM areas exhibited reduced disease-free and overall survival rates compared to patients having higher β-catenin expression levels in the tumor tissues. These observations suggest a correlation between β-catenin up-regulation in CM areas of HNSCC cases with disease recurrence and survival.
The primary cause of HNSCC relapse is drug resistance [31]. Cancer cells may mediate drug resistance through the generation of enhanced DNA repair and drug efflux capacities, and the evasion of apoptosis [10]. Previous reports indicate that the Wnt/β-catenin signaling pathway is one of the primary pathways associated with cisplatin resistance [15, 32, 33]. Here, we found that constitutive overexpression/stabilization of β-catenin in HCT-116 cells contributes to cisplatin resistance. We also found that a high β-catenin expression was associated with a poor prognosis and the occurrence of recurrences in our cohort of HNSCC patients. Since we found that β-catenin expression was relatively low in the HNSCC-derived cell lines UPCI-SCC-131 and CAL-27, we up-regulated β-catenin in these cell lines through LiCl treatment to mirror the conditions observed in HNSCC patients and, subsequently, evaluated their resistance to cisplatin. Others have reported that LiCl treatment results in GSK-3β inhibition, leading to an increased nuclear localization of β-catenin [34]. Accordingly, we found that 5 mM LiCl treatment led to an increased nuclear accumulation, as well as increased β-catenin mRNA and protein expression in the HNSCC-derived cell lines. We also found that β-catenin up-regulated UPCI-SCC-131 and CAL-27 cells exhibited increased cisplatin resistance compared to their parental counterparts, as assessed by MTT and scratch wound healing assays. In order to further confirm the role of β-catenin in cisplatin resistance we transiently silenced β-catenin expression using siRNA. By doing so, we indeed observed an increased sensitivity of these cells to cisplatin, resulting in a reduced wound healing capacity and a significant increase in S phase cell cycle arrest. These results led us to conclude that β-catenin may play a role in modulating the chemo-sensitivity of HNSCC cells.
We also found that increased β-catenin expression led to enhanced DNA damage repair induced by cisplatin, whereas β-catenin silencing led to increased DNA damage in HNSCC cells. It is well established that stabilized β-catenin, when translocated to the nucleus, acts as a transcriptional activator in several downstream pathways, one of which is the DNA damage repair pathway. We found that β-catenin up-regulated HNSCC cells, when treated with increased concentrations (1-10 μM) of cisplatin, showed decreased γ-H2Ax foci/nuclei, indicating less double strand break (DSB) sites, which corresponds with enhanced DNA repair. Conversely, we found that β-catenin silenced cells showed increased γ-H2Ax foci/nuclei upon treatment with increasing cisplatin concentrations, which corresponds to decreased DNA repair. These results were further confirmed using an alkaline COMET assay and led us to conclude that stabilization and nuclear translocation of β-catenin is involved in promoting cisplatin resistance through increasing DNA damage repair efficiency in HNSCC cells.
Previously, it has been reported that cisplatin interacts with DNA through intra-strand crosslinks and exerts its cytotoxic effects through the formation of cisplatin-DNA adducts [35]. The primary repair option for this type of adducts is nucleotide excision repair (NER), and ERCC1 has been found to play an important role in the repair of cisplatin induced adducts [36]. Others have previously assessed the expression of ABCG2, which is primarily associated with drug resistance in cancer cells [37]. Here, we found an increased expression of ERCC1 along with ABCG2 in β-catenin up-regulated cells after cisplatin treatment. Conversely, ERCC1 and ABCG2 downregulation was observed in β-catenin silenced cells, indicating that β-catenin up-regulation may enhance DNA repair by increasing its resistance to cisplatin in HNSCC cells.
In conclusion, we found that the expression of β-catenin correlates with the occurrence of relapse in HNSCC patients. Our data also suggest that β-catenin may play a role in cisplatin resistance through modulating DNA damage response which, in turn, may underlie HNSCC relapse. This work may form a basis for future studies aimed at developing novel strategies to combat therapy resistance and disease relapse in HNSCC.
Electronic supplementary material
Silencing of β-catenin in HCT-116 cells. (a) mRNA level expression of β-catenin in HCT-116 cells after transfected with scrambled siRNA and siRNA- β-catenin. (b) Representative western blot results and graphical representation of β-catenin expression in scrambled siRNA and siRNA-β-catenin transfected HCT-116 cells. (GIF 64 kb)
β-catenin is plays an important role in chemoresistance in HCT-116 cells. (a) Cell viability assay of parental, scrambled-siRNA transfected and siRNA-β-catenin transfected HCT-116 cells after treatment with different concentrations of cisplatin. (b,c) Determination of colony forming capacity and cell survival of parental, scrambled-siRNA and siRNA-β-catenin transfected HCT-116 cells after treatment with different concentrations of cisplatin by clonogenic assay. (d) Graphical representation of percentage apoptotic cells (Sub G0) in parental, scrambled-siRNA and siRNA-β-catenin transfected HCT-116 cells after cisplatin treatment. (e) Representative image of wound in parental, scrambled-siRNA and siRNA-β-catenin transfected cells at 0 h and 24 h after cisplatin treatment. (f) Graphical representation of percentage wound closure after 24 h of cisplatin treatment in parental, scrambled-siRNA and siRNA-β-catenin transfected HCT-116 cells. (GIF 674 kb)
Silencing of β-catenin in UPCI-SCC-131 HNSCC cell line and its effect on cell cycle progression. (a) UPCI-SCC-131 cells were transfected with scrambled siRNA and siRNA- β-catenin for 48 h. mRNA expression of β-catenin in scrambled siRNA and siRNA- β-catenin transfected UPCI-SCC-131 cells . (b) Representative western blot results of β-catenin expression in scrambled siRNA and siRNA-β-catenin transfected UPCI-SCC-131 cells. (c) Graphical representation of relative protein expression of β-catenin in scrambled-siRNA and siRNA-β-catenin transfected UPCI-SCC-131 cells. (d) Representative histograms of cell cycle analysis in scrambled-siRNA transfected cells and siRNA-β-catenin transfected cells. (e) Graphical representation of percentage distribution of cells in scrambled-siRNA transfected cells and siRNA-β-catenin transfected UPCI-SCC-131 cells. (GIF 120 kb)
Sequences of the primers used in this study. (PDF 166 kb)
Clinico-pathological characteristics of 80 HNSCC patients. (PDF 15 kb)
Association of co-morbidity factors with recurrence in HNSCC patients. (PDF 8 kb)
Acknowledgements
This work was supported by a grant from the Department of Atomic Energy (DAE), Board of Research for Nuclear Sciences (BRNS), Government of India, Grant Number 2013/35/45/BRNS and by the MSSB (Molecular Stress and Stem Cell Biology) group.
Compliance with ethical standard
Ethical approval
This study was approved by the institutional ethics committee of the School of Biotechnology and Kalinga Institute of Medical Sciences (KIMS), KIIT University, and was conducted according to the Helsinki declaration. The human sample collection was carried out strictly according to the institutional ethical board guidelines.
Conflict of interest
The authors declare no conflict of interest.
Informed consent
Informed consent was obtained from all subjects or their nominees prior to participation in the study.
References
- 1.S. Padhi, A. Saha, M. Kar, C. Ghosh, A. Adhya, M. Baisakh, N. Mohapatra, S. Venkatesan, M.P. Hande, B. Banerjee, Clinico-pathological correlation of β-catenin and telomere dysfunction in head and neck squamous cell carcinoma patients. J Cancer 6, 192–202 (2015). 10.7150/jca.9558 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.P. Joshi, S. Dutta, P. Chaturvedi, S. Nair, Head and neck cancers in developing countries. Rambam Maimonides Med J 5, e0009 (2014). 10.5041/rmmj.10143 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.E.M. Smith, L.M. Rubenstein, T.H. Haugen, E. Hamsikova, L.P. Turek, Tobacco and alcohol use increases the risk of both HPV-associated and HPV-independent head and neck cancers. Cancer Causes Control: CCC 21, 1369–1378 (2010). 10.1007/s10552-010-9564-z [DOI] [PubMed] [Google Scholar]
- 4.S. Vishak, B. Rangarajan, V.D. Kekatpure, Neoadjuvant chemotherapy in oral cancers: Selecting the right patients. Indian J Med Paediatr 36, 148–153 (2015). 10.4103/0971-5851.166716 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.S. Ghuwalewala, D. Ghatak, P. Das, S. Dey, S. Sarkar, N. Alam, C.K. Panda, S. Roychoudhury, CD44(high)CD24(low) molecular signature determines the Cancer Stem Cell and EMT phenotype in Oral Squamous Cell Carcinoma. Stem Cell Res 16, 405–417 (2016). 10.1016/j.scr.2016.02.028 [DOI] [PubMed] [Google Scholar]
- 6.M. Bruggemann, C. Pott, M. Ritgen, M. Kneba, Significance of minimal residual disease in lymphoid malignancies. Acta Haematol 112, 111–119 (2004). 10.1159/000077566 [DOI] [PubMed] [Google Scholar]
- 7.S. Bhattacharyya, V. Sekar, B. Majumder, D.G. Mehrotra, S. Banerjee, A.K. Bhowmick, N. Alam, G.K. Mandal, J. Biswas, P.K. Majumder, N. Murmu, CDKN2A-p53 mediated antitumor effect of Lupeol in head and neck cancer. Cell Oncol 40, 145–155 (2017). 10.1007/s13402-016-0311-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.I.P. Ribeiro, F. Caramelo, F. Marques, A. Domingues, M. Mesquita, L. Barroso, H. Prazeres, M.J. Juliao, I.P. Baptista, A. Ferreira, J.B. Melo, I.M. Carreira, WT1, MSH6, GATA5 and PAX5 as epigenetic oral squamous cell carcinoma biomarkers - a short report. Cell Oncol 39, 573–582 (2016). 10.1007/s13402-016-0293-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.L. Kadletz, G. Heiduschka, R. Wiebringhaus, E. Gurnhofer, U. Kotowski, G. Haymerle, M. Brunner, C. Barry, L. Kenner, ELMO3 expression indicates a poor prognosis in head and neck squamous cell carcinoma - a short report. Cell Oncol 40, 193–198 (2017). 10.1007/s13402-016-0310-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.H. Zahreddine, K.L. Borden, Mechanisms and insights into drug resistance in cancer. Front Pharmacol 4, 28 (2013). 10.3389/fphar.2013.00028 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.K.Q. Liu, Z.P. Liu, J.K. Hao, L. Chen, X.M. Zhao, Identifying dysregulated pathways in cancers from pathway interaction networks. BMC Bioinf 13, 126 (2012). 10.1186/1471-2105-13-126 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.E.M.F. de Sousa, L. Vermeulen, Wnt signaling in cancer stem cell biology. Cancers 8 (2016). doi: 10.3390/cancers8070060 [DOI] [PMC free article] [PubMed]
- 13.S. Dasari, P.B. Tchounwou, Cisplatin in cancer therapy: molecular mechanisms of action. Eur J Pharmacol 740, 364–378 (2014). 10.1016/j.ejphar.2014.07.025 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.D.J. Stewart, Wnt signaling pathway in non-small cell lung cancer. J Natl Cancer Inst 106, djt356 (2014). 10.1093/jnci/djt356 [DOI] [PubMed] [Google Scholar]
- 15.J. Zhang, J. Liu, H. Li, J. Wang, β-catenin signaling pathway regulates cisplatin resistance in lung adenocarcinoma cells by upregulating Bcl-xl. Mol Med Rep 13, 2543–2551 (2016). 10.3892/mmr.2016.4882 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.M. Kabatkova, O. Zapletal, Z. Tylichova, J. Neca, M. Machala, A. Milcova, J. Topinka, A. Kozubik, J. Vondracek, Inhibition of beta-catenin signalling promotes DNA damage elicited by benzo[a]pyrene in a model of human colon cancer cells via CYP1 deregulation. Mutagenesis 30, 565–576 (2015). 10.1093/mutage/gev019 [DOI] [PubMed] [Google Scholar]
- 17. A. Saha, S. Padhi, M. Kar, S. Roy, P. Maiti, B. Banerjee, Role of TRF2 in efficient DNA repair, spheroid formation and cancer stem cell maintenance. Oncomedicine 2, 71–79 (2017). 10.7150/oncm.18373
- 18.A. Saha, S. Shree Padhi, S. Roy, B. Banerjee, Method of detecting new cancer stem cell-like enrichment in development front assay (DFA). J Stem Cells 9, 235–242 (2014) [PubMed] [Google Scholar]
- 19.A. Saha, S. Shree Padhi, S. Roy, B. Banerjee, HCT116 colonospheres shows elevated expression of hTERT and beta-catenin protein - a short report. J Stem Cells 9, 243–251 (2014) [PubMed] [Google Scholar]
- 20.J.P. Newman, B. Banerjee, W. Fang, A. Poonepalli, L. Balakrishnan, G.K. Low, R.N. Bhattacharjee, S. Akira, M. Jayapal, A.J. Melendez, R. Baskar, H.W. Lee, M.P. Hande, Short dysfunctional telomeres impair the repair of arsenite-induced oxidative damage in mouse cells. J Cell Physiol 214, 796–809 (2008). 10.1002/jcp.21276 [DOI] [PubMed] [Google Scholar]
- 21.B.M. Gyori, G. Venkatachalam, P.S. Thiagarajan, D. Hsu, M.V. Clement, OpenComet: an automated tool for comet assay image analysis. Redox Biol 2, 457–465 (2014). 10.1016/j.redox.2013.12.020 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.D.R. Rhodes, S. Kalyana-Sundaram, V. Mahavisno, R. Varambally, J. Yu, B.B. Briggs, T.R. Barrette, M.J. Anstet, C. Kincead-Beal, P. Kulkarni, S. Varambally, D. Ghosh, A.M. Chinnaiyan, Oncomine 3.0: genes, pathways, and networks in a collection of 18,000 cancer gene expression profiles. Neoplasia 9, 166–180 (2007). 10.1593/neo.07112 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.M.A. Ginos, G.P. Page, B.S. Michalowicz, K.J. Patel, S.E. Volker, S.E. Pambuccian, F.G. Ondrey, G.L. Adams, P.M. Gaffney, Identification of a gene expression signature associated with recurrent disease in squamous cell carcinoma of the head and neck. Cancer Res 64, 55–63 (2004). 10.1158/0008-5472.CAN-03-2144 [DOI] [PubMed] [Google Scholar]
- 24.C.H. Peng, C.T. Liao, S.C. Peng, Y.J. Chen, A.J. Cheng, J.L. Juang, C.Y. Tsai, T.C. Chen, Y.J. Chuang, C.Y. Tang, W.P. Hsieh, T.C. Yen, A novel molecular signature identified by systems genetics approach predicts prognosis in oral squamous cell carcinoma. PloS one 6, e23452 (2011). 10.1371/journal.pone.0023452 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.A. Cromer, A. Carles, R. Millon, G. Ganguli, F. Chalmel, F. Lemaire, J. Young, D. Dembele, C. Thibault, D. Muller, O. Poch, J. Abecassis, B. Wasylyk, Dentification of genes associated with tumorigenesis and metastatic potential of hypopharyngeal cancer by microarray analysis. Oncogene 23, 2484–2498 (2004). 10.1038/sj.onc.1207345 [DOI] [PubMed] [Google Scholar]
- 26.S. Sekine, T. Shibata, M. Sakamoto, S. Hirohashi, Target disruption of the mutant β-catenin gene in colon cancer cell line HCT116: preservation of its malignant phenotype. Oncogene 21, 5906–5911 (2002). 10.1038/sj.onc.1205756 [DOI] [PubMed] [Google Scholar]
- 27.A.B. Sparks, P.J. Morin, B. Vogelstein, K.W. Kinzler, Mutational analysis of the APC/beta-catenin/Tcf pathway in colorectal cancer. Cancer Res 58, 1130–1134 (1998) [PubMed] [Google Scholar]
- 28.E. Beurel, M. Kornprobst, M.J. Blivet-Van Eggelpoel, C. Ruiz-Ruiz, A. Cadoret, J. Capeau, C. Desbois-Mouthon, GSK-3beta inhibition by lithium confers resistance to chemotherapy-induced apoptosis through the repression of CD95 (Fas/APO-1) expression. Exp Cell Res 300, 354–364 (2004). 10.1016/j.yexcr.2004.08.001 [DOI] [PubMed] [Google Scholar]
- 29.S.G. Shiah, Y.S. Shieh, J.Y. Chang, The Role of Wnt Signaling in Squamous Cell Carcinoma. J Dent Res 95, 129–134 (2016). 10.1177/0022034515613507 [DOI] [PubMed] [Google Scholar]
- 30.Padhi SS, Kar M, Saha A, Baisakh MR, Mohapatra N, Banerjee BN. Rapid recurrence and poor prognosis with altered levels of β-catenin in 3 cases of head and neck squamous cell carcinoma patients. Head Neck Oncol 6, 39 (2014)
- 31.Y. Yamano, K. Uzawa, K. Saito, D. Nakashima, A. Kasamatsu, H. Koike, Y. Kouzu, K. Shinozuka, K. Nakatani, K. Negoro, S. Fujita, H. Tanzawa, Identification of cisplatin-resistance related genes in head and neck squamous cell carcinoma. Int J Cancer 126, 437–449 (2010). 10.1002/ijc.24704 [DOI] [PubMed] [Google Scholar]
- 32.D.W. Shen, L.M. Pouliot, M.D. Hall, M.M. Gottesman, Cisplatin resistance: a cellular self-defense mechanism resulting from multiple epigenetic and genetic changes. Pharmacol Rev 64, 706–721 (2012). 10.1124/pr.111.005637 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.M.K. Mohammed, C. Shao, J. Wang, Q. Wei, X. Wang, Z. Collier, S. Tang, H. Liu, F. Zhang, J. Huang, D. Guo, M. Lu, F. Liu, J. Liu, C. Ma, L.L. Shi, A. Athiviraham, T.C. He, M.J. Lee, Wnt/beta-catenin signaling plays an ever-expanding role in stem cell self-renewal, tumorigenesis and cancer chemoresistance. Genes Dis 3, 11–40 (2016). 10.1016/j.gendis.2015.12.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.N. Kneidinger, A.O. Yildirim, J. Callegari, S. Takenaka, M.M. Stein, R. Dumitrascu, A. Bohla, K.R. Bracke, R.E. Morty, G.G. Brusselle, R.T. Schermuly, O. Eickelberg, M. Konigshoff, Activation of the WNT/beta-catenin pathway attenuates experimental emphysema. Am. J. Respir. Crit. Care Med. 183, 723–733 (2011). 10.1164/rccm.200910-1560OC [DOI] [PubMed] [Google Scholar]
- 35.A.M. Florea, D. Busselberg, Cisplatin as an anti-tumor drug: cellular mechanisms of activity, drug resistance and induced side effects. Cancers 3, 1351–1371 (2011). 10.3390/cancers3011351 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.A. Basu, S. Krishnamurthy, Cellular responses to Cisplatin-induced DNA damage. J Nucleic Acids 2010, 1–16 (2010). 10.4061/2010/201367 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.R.W. Robey, O. Polgar, J. Deeken, K.W. To, S.E. Bates, BCG2: determining its relevance in clinical drug resistance. Cancer Metastasis Rev 26, 39–57 (2007). 10.1007/s10555-007-9042-6 [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Silencing of β-catenin in HCT-116 cells. (a) mRNA level expression of β-catenin in HCT-116 cells after transfected with scrambled siRNA and siRNA- β-catenin. (b) Representative western blot results and graphical representation of β-catenin expression in scrambled siRNA and siRNA-β-catenin transfected HCT-116 cells. (GIF 64 kb)
β-catenin is plays an important role in chemoresistance in HCT-116 cells. (a) Cell viability assay of parental, scrambled-siRNA transfected and siRNA-β-catenin transfected HCT-116 cells after treatment with different concentrations of cisplatin. (b,c) Determination of colony forming capacity and cell survival of parental, scrambled-siRNA and siRNA-β-catenin transfected HCT-116 cells after treatment with different concentrations of cisplatin by clonogenic assay. (d) Graphical representation of percentage apoptotic cells (Sub G0) in parental, scrambled-siRNA and siRNA-β-catenin transfected HCT-116 cells after cisplatin treatment. (e) Representative image of wound in parental, scrambled-siRNA and siRNA-β-catenin transfected cells at 0 h and 24 h after cisplatin treatment. (f) Graphical representation of percentage wound closure after 24 h of cisplatin treatment in parental, scrambled-siRNA and siRNA-β-catenin transfected HCT-116 cells. (GIF 674 kb)
Silencing of β-catenin in UPCI-SCC-131 HNSCC cell line and its effect on cell cycle progression. (a) UPCI-SCC-131 cells were transfected with scrambled siRNA and siRNA- β-catenin for 48 h. mRNA expression of β-catenin in scrambled siRNA and siRNA- β-catenin transfected UPCI-SCC-131 cells . (b) Representative western blot results of β-catenin expression in scrambled siRNA and siRNA-β-catenin transfected UPCI-SCC-131 cells. (c) Graphical representation of relative protein expression of β-catenin in scrambled-siRNA and siRNA-β-catenin transfected UPCI-SCC-131 cells. (d) Representative histograms of cell cycle analysis in scrambled-siRNA transfected cells and siRNA-β-catenin transfected cells. (e) Graphical representation of percentage distribution of cells in scrambled-siRNA transfected cells and siRNA-β-catenin transfected UPCI-SCC-131 cells. (GIF 120 kb)
Sequences of the primers used in this study. (PDF 166 kb)
Clinico-pathological characteristics of 80 HNSCC patients. (PDF 15 kb)
Association of co-morbidity factors with recurrence in HNSCC patients. (PDF 8 kb)