Ⅰ. INTRODUCTION
Biological characteristics such as rapid growth rate, invasive capability, and metastatic potential of cancer cells affect the aggressiveness of cancer and are major factors in determining the prognosis of cancer patients9). These aggressive biological characteristics are known to be promoted (strengthened) during the process of epithelial- mesenchymal transition (EMT), which is characterized by a decreased expression of epithelial markers and/or an increased expression of mesenchymal markers. Cells that have acquired a mesenchymal phenotype through EMT display resistance to chemotherapeutic and apoptotic reagents, as well as increased motility and invasiveness13). Thus, EMT may be one of the most important events that contribute to aggravating the prognosis of cancer. EMT is not only intrinsically acquired through multiple genetic and epigenetic changes, but is also induced by non-genetic extrinsic factors derived from the tumor microenvironment, such as hypoxia18).
Hypoxia is commonly observed in cancer tissues because the formation of blood vessels cannot keep up with the growth rate of anaplastic cancer cells, especially in highly malignant tumors. Though cancer malignancy is promoted by selective genetic mutations, growing data corroborate the role of hypoxia in the transformation of cancer cells into a more aggressive phenotype, and these changes may be mediated by EMT6,7).
There has been no significant progression in the prognosis of oral cancer, mostly oral squamous cell carcinoma (OSCC), in the past decades, when compared with the other types of cancers such as leukemia, lung, and breast cancers1,11,19). Prognosis of these cancers has been significantly improved by the development of a variety of chemotherapeutic reagents, as well as the enhanced efficacy of radiotherapy. No effective chemotherapeutic reagent has been found for oral cancer, whereas numerous medications have been found and effectively utilized for other types of cancers14,15). Oral cancer cells exhibit resistance to most chemotherapeutic reagents that are effective in other types of cancers, even though the factors involved in the acquisition of aggressive behavior of OSCC cells have been continuously studied.
Taken together, identifying genes involved in the acquisition of EMT characteristics in oral cancer cells under hypoxic conditions may be critical for developing effective cancer therapies. However, few studies on the functional link between hypoxia and EMT-related genes have been conducted, and the information on the link remains limited, especially in oral cancer cells6). Through studies identifying the contributing factors that exacerbate the prognosis of OSCC, we can get insights into potential therapeutic strategies for oral cancer. In this study, we investigated hypoxia- induced changes in mRNA expression profiles that can influence EMT in OSCC cells. In addition, the present study described and discussed the functional significance of a particular gene in the pathogenesis of OSCC.
Ⅱ. MATERIALS & METHODS
1. Cell culture and hypoxic treatment
The human oral squamous cell carcinoma (OSCC) cell line, YD10B, gifted from Professor JI Yook (College of Dentistry, Yonsei University, South Korea), was used. They were grown in a 3:1 mixture of Dulbecco’s Modified Eagle’s Medium and Ham’s nutrient mixture F12 (Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (FBS; Atlas Biologicals, Fort Collins, CO) and 1% penicillin-streptomycin (GIBCO-BRL, Rockville, MD). The cells were maintained at 37°C in a humidified 5% CO2-95% air incubator. For hypoxic treatment, YD10B OSCC cells were incubated in a multi-gas incubator (MCO-5M, Sanyo, Japan) that was equilibrated at 1% O2, 5% CO2, and balanced N2.
2. Cell viability assay
Cells were seeded in 96-well plates and then incubated overnight. On the next day, the plates were further incubated for various periods in normoxic or hypoxic chambers. After incubation, the medium was removed, and 100 μL of MTT (5 mg/mL) was added to each well. The formazan crystals that formed were solubilized in 200 μL of DMSO. The colored solution was quantified at 570 nm absorbance using a spectrophotometer. Cell viability was determined as percent of the control. Each condition was performed in triplicate, and data were obtained from at least 3 separate experiments.
3. Gene expression by the EMT PCR array
RNA was isolated from oral cancer cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and converted to first strand cDNA using the RT2 First Strand Kit. cDNA was added to RT2 qPCR Master Mix, which contains SYBR Green and a reference dye. RT2 Profiler PCR array panel was used to profile the changes in the gene expression of 84 EMTrelated key markers. Real-time PCR thermal cycling was performed using the ABI 7500 thermal cycler. Fold changes in mRNA expression were determined with the ΔΔCt method. Differences between normoxic and hypoxic OSCC cells were analyzed by the Array Data Analysis Web Portal (http://www.sabiosciences.com/pcrarraydataanalysis).
4. Western blot analysis
Cells were harvested, pelleted by centrifugation, washed with ice-cold PBS, and lysed on ice for 20 min with RIPA buffer. The cell lysates were analyzed using antibodies against HIF-1 α, β-catenin, fibronectin, TCF-4 (Cell Signaling, Beverly, MA), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA).
5. Transient Transfections and luciferase assay.
All transient transfections were performed using the Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA). The Tcf/Lef reporter plasmid, TOP-flash, was used as a reporter construct. In brief, cells were co-transfected with 2 μg of the reporter construct pTOP-flash and/or a mutant β-catenin vector, a constitutive active form of β-catenin. To serve as an internal control for transfection, cells were co-transfected with a Renilla luciferase vector. Luciferase activities were measured 48 h after transfection.
6. Statistical analysis
Data were obtained from at least 3 independent experiments, and the results were presented as the mean values ± SD. Comparisons between two groups was performed using Student’s t-tests (GraphPad Prism 5.03, GraphPad Software, Inc., La Jolla, CA). P values less than 0.05 were considered statistically significant.
Ⅲ. RESULTS
1. Hypoxia induces changes in EMT-related mRNA expression profiles of OSCC cells
To determine whether hypoxia affects the cellular proliferation of OSCC, cells were grown in a hypoxic chamber. As shown in Fig. 1, hypoxia significantly suppressed the growth of YD10B OSCC cells in a time-dependent manner.
Normoxic and hypoxic oral cancer cells were compared using mRNA expression profile arrays that profiled the expression of 84 EMT-related genes. Twenty-four EMT-related genes had expressions that were at least twofold higher in the hypoxic OSCC cells than that in the normoxic cells (Fig. 2). Hypoxic OSCC cells were found to overexpress Il1rn by 25.9-fold and have a 2.79-fold downregulation of TCF-4 relative to the normoxic cells. In addition to the overexpression of Fn1 (6.1 fold), Snai1 (4.7 fold), Bmp1 (3.9 fold), Itga5 (3.1 fold), and Snai2 (2.0 fold), Tmeff1 (-2.08 fold) and Krt14 (-2.43 fold) were downregulated (Table 1 and 2).
2. Hypoxia downregulated transcription and translation of TCF-4 in OSCC cells
Both hypoxia and EMT are known to be related to the aggressiveness of cancer cells, including invasive capability and chemoresistance6). We, thus, hypothesized that hypoxia promotes the expression of EMT-related genes in oral cancer cells. However, the PCR array study revealed TCF-4, a major transcription factor involved in EMT-related changes in cancer cells, to be the most significantly downregulated gene. The suppressed mRNA expression of TCF-4 in hypoxic oral cancer cells was further validated by real-time PCR. The hypoxic status of the cells was confirmed by observing the increase in the mRNA level of VEGF, which is known to be dramatically induced by hypoxia. In addition, other hypoxiainducible genes, such as PAI-1 and Wnt-5a, were also upregulated in hypoxic oral cancer cells. To determine whether the suppressed expression of TCF-4 was reflected at the translational level, the protein expression of TCF-4 was examined using western blot analysis. TCF-4 protein was significantly downregulated by hypoxia in a time-dependent manner, whereas HIF-1α, a representative protein that accumulates under hypoxic conditions, as well as fibronectin and β-catenin, was upregulated.
3. Hypoxia suppressed β-catenin signaling despite the upregulation of β-catenin under hypoxic conditions in OSCC cells
Though TCF-4 is a key downstream effector of the Wnt/β -catenin signaling pathway and a transcription factor that modulates the expression of a diverse set of genes by forming a large complex with β-catenin, target gene regulation is mainly controlled by the level of Wnt and its binding to the cell surface, which results in the stabilization and translocation of β-catenin4). The activity of the Wnt/β-catenin signaling pathway was monitored using TOP- flash luciferase assay; transcriptional activity was found to be significantly suppressed in hypoxic oral cancer cells (Fig. 4). Interestingly, expression of the transcriptional target genes of Wnt/β-catenin signaling, such as CD44, showed no change, even in the presence of the constitutively active form of β-catenin and decreased transcriptional activity (Fig. 3).
Ⅳ. DISCUSSION
Hypoxia, one of the major environmental factors encountered by malignant tumor cells, primarily inhibits the growth of cells by restricting numerous molecules and organelles that are essential for cell proliferation. However, some cells can survive and adapt to this harsh condition by modulating numerous factors that are involved in promoting cellular malignancy20). These cells display higher durability and resistance to therapeutic modalities compared to normoxic cells. Studies reveal that hypoxia can further perturb the expression levels of many mRNAs that are already dysregulated in cancer, especially genes that promote the aggressiveness of cancer cells10,12). These hypoxia-responsive mRNAs are critical not only for tumor invasion and metastasis, but also for tumor progression and development of chemoresistance, based on EMT-related changes5). In this study, we found significant differences in the EMT-related mRNA expression profiles of normoxic and hypoxic oral cancer cells, including the genes Snai1 and Snai2, which are major transcription factors involved in EMT. Expression levels of various Wnt genes were also increased in hypoxic cells compared to that in normoxic cells.
Wnt signaling is one of the key pathways that are involved in the development of cancer malignancy, including EMT. Wnt binding to receptors on the cell surface initiates the signaling cascade and triggers the stabilization and nuclear translocation of β-catenin4). Then, the translocated β-catenin forms a complex with nuclear TCF-4 and activates the expression of EMT-specific transcription factors, as well as the transcription of numerous target genes that are related to malignant transformation21). The increased expression of various Wnt ligands in hypoxic OSCC cells in the present study suggests that hypoxia may contribute to tumor progression by activating Wnt signaling, as well as HIF-1 signaling, which is known as a classical signal transduction pathway in hypoxic tumor cells2,17). However, hypoxia suppressed the transcriptional activity of β-catenin in oral cancer cells by downregulating TCF-4 expression transcriptionally and translationally, even though Wnt ligands and β-catenin were significantly increased. It reported that hypoxia suppresses the transcriptional regulation of the β-catenin/ TCF-4 complex via the competitive binding of HIF-1α with TCF-4, emphasizing the importance of the HIF-1 pathway8).
On the contrary, it is interesting that the expression of the downstream target genes of the β-catenin/TCF-4 complex was not decreased, even with the significant decrease in TCF-4 expression and the suppression of β-catenin/TCF-4 signaling in hypoxic oral cancer cells. This may be partly explained by the findings that TCF-4 acts as a repressor of transcription of those target genes by binding to corepressors such as Groucho in the absence of β-catenin3). It. supported the repressive role of TCF-4 by observing the significant downregulation of TCF-4 in breast cancer tissue relative to that in normal breast tissue16). In this study, hypoxia-induced reduction of TCF-4 expression did not exacerbate the transcription of growth- controlling genes in oral cancer. In addition, a reduction in the amount of TCF-4 may not reduce the overall response to Wnt-dependent gene transcription by its association with β-catenin; however, it is not clear whether the reduction of TCF-4 is enough to contribute to EMT changes.
In conclusion, hypoxia can modulate the biological behaviors of oral cancer cells and enhance their malignant characteristics by changing EMT-related gene expression profiles through various pathways. It is plausible that the hypoxia-induced modulation of EMT-related genes may underlie the acquisition of aggressiveness such as chemoresistance, as well as invasion and metastasis. Thus, further advancement of our knowledge on hypoxia-induced regulation of mRNAs and cancer-susceptible genes would facilitate the development of a new therapeutic strategy aimed to prevent hypoxia- induced progression of malignant tumors, including cellular adaptation and resistance to therapeutic remedies.
