GDF15 Repression Contributes to 5-Fluorouracil Resistance in Human Colon Cancer by Regulating Epithelial-Mesenchymal Transition and Apoptosis

Chemotherapy based on 5-fluorouracil (5-FU) is the standard approach for colon cancer treatment, and resistance to 5-FU is a significant obstacle in the clinical treatment of colon cancer. However, the mechanisms underlying 5-FU resistance in colon cancer cells remain largely unknown. This study aimed at determining whether 5-FU-resistant colon cancer cells undergo epithelial-mesenchymal transition (EMT) and apoptosis and the role of GDF15—a member of the transforming growth factor β/bone morphogenetic protein super family and a protein known to be involved in cancer progression—in the regulation of EMT and apoptosis of these cells, along with the underlying mechanisms. In vitro apoptosis detection assay, growth inhibition assay, transwell, and wound healing experiments revealed that 5-FU-resistant colon cancer cells possessed enhanced EMT and antiapoptotic ability. These cells also showed a stronger tendency to proliferate and metastasize in vivo. Quantitative reverse transcription-PCR and western blotting revealed that 5-FU-resistant colon cancer cells expressed lower levels of growth differentiation factor 15 (GDF15) than did 5-FU-sensitive colon cancer cells. Moreover, the transient GDF15 overexpression resensitized 5-FU-resistant colon cells to 5-FU. Collectively, these findings indicate the mechanism underlying the 5-FU resistance of colon cancer cells and provide new therapeutic targets for improving the prognosis of colon cancer patients.


Introduction
Resistance to 5-fluorouracil (5-FU) is the primary reason for the failure of traditional chemotherapy for colon cancer [1]. Despite significant advances in our understanding of 5-FU resistance, the underlying molecular mechanisms are not fully characterized. Evidence indicates that epithelialmesenchymal transition (EMT) and apoptosis are involved in tumor progression [2,3] and that drug-resistant cells often show accelerated proliferation and distant metastasis [4,5]. These findings suggest that both EMT and apoptosis are possibly involved in the development of chemotherapy resistance. Our previous results showed that growth differentiation factor 15 (GDF15) is downregulated in HCT-15/FU drugresistant colon cancer cells compared with drug-sensitive colon cells; these cells also showed accelerated proliferation and angiogenesis in vitro and in vivo. These results suggested that GDF15 may play a crucial role in the regulation of 5-FU sensitivity of colon cancer cells. However, the exact mechanism remains unknown.
GDF15, a member of transforming growth factor β (TGF-β)/bone morphogenetic protein (BMP) superfamily, is expressed in many types of tissues and has gained extensive attention with the increase in research on cancer progression [6,7]. However, the effects of GDF15 on tumor development are not well understood and are contradictory [6]. Like other TGF-β members, GDF15 can play a variety of biological roles via the regulation of the TGF-β/Smad signaling pathway. Previous studies have shown that GDF15 can promote EMT and metastasis in colorectal cancer [8] and contribute to radioresistance and cancer stemness of head and neck cancer [9]. However, the role of GDF15, especially in 5-FU resistance in colon cancer, is not yet fully understood.
In this study, we determined whether 5-FU-resistant colon cancer cells undergo EMT and apoptosis. Additionally, we investigated whether GDF15 is involved in the regulation of EMT and apoptosis and explored the underlying mechanisms. Our results show that the low expression of GDF15 contributes to 5-FU resistance in human colon cancer by regulating EMT and apoptosis via a Smad-associated signaling pathway. Thus, our findings will provide new insights into 5-FU resistance in colon cancer and shed light on a target for colon cancer gene therapy.

Materials and Methods
2.1. Cell Culture. HCT-15 (human colon cancer cells) and HCT-15/FU cells were bought from the cell bank of Chinese Academy of Sciences (Shanghai, China) and were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS, Gibco, USA). HCT-15/FU cells were supplemented with 3.2 μg/ml 5-FU (the initial concentration of 5-FU was determined with the half maximal inhibitory concentration (IC 50 ) of HCT-15 cells, starting at 1/3 of IC 50 and increasing by 1/6-1/3 each time; the cells were cultured for 2-4 times at each concentration until stable HCT-15/FU cell line was obtained). All cells were incubated at 37°C with 5% CO 2 .
2.3. Cell Proliferation Assay. In total, 5 × 10 3 HCT-15 or HCT-15/FU cells were cultured in each well of a 96-well plate and treated with various concentrations of 5-FU (the concentrations of 5-FU were 0, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mM for HCT-15 cells and 0, 1, 2, 4, 8, 16, and 32 mM for HCT-15/5-FU cells). The viability of the cells was measured at the time point of 0 and 48 h using the MTS reagent (CellTiter 96® Aqueous One Solution Cell Proliferation assay, Promega). The optical density was measured at 490 nm after being incubated at 37°C for 2 h using an enzyme-labeled meter (Spectramax M3; Molecular Devices). Using GraphPad Prism 5.0 software to calculate the inhibition rate and draw a concentration effect curve, three independent tests were conducted for the cell proliferation assay.

Cell Migration
Assay. The migration ability of HCT-15 or HCT-15/FU cells was tested in a Transwell Boyden Chamber (8 mm pore size, 6.5 mm diameter) seeded in the upper chamber with 1 × 10 4 cells in 0.5 ml serum-free medium with 0.76 mM 5-FU, and the lower chamber was filled with 0.8 ml medium containing 10% FBS. After incubating for 48 h, cells were fixed with 100% methanol for 20 min and stained with 0.1% crystal violet for 15 min. The cells in the upper compartment were wiped off. Images were taken using a photomicroscope (Nikon, Japan) and quantified by counting at least three fields.

Wound
Healing Assay. HCT-15 or HCT-15/FU cells were cultured in 24-well plates. At 100% confluent, a 20 μL pipette tip was used to scratch a wound at the centre of the cell monolayer in the culture plates. The wounds between cells were washed twice with PBS and cultured with culture medium containing 0.76 mM 5-FU for another 48 h. Images of wounds were captured at 0, 12, 24, 36, and 48 h, and the wound healing area was calculated by ImageJ software.

Western Blotting Assay.
Whole proteins were extracted from cells using immunoprecipitation buffer and quantified using the BCA protein assay kit (Thermo scientific Pierce). After separation on a 10% SDS-PAGE gel, proteins were transferred to a PVDF membrane (Millipore) and blocked in 5% nonfat milk. Proteins were incubated with primary antibodies overnight at 4°C and secondary antibodies at room temperature for 2 h. TBST buffer was used to wash off the unbound antibodies. Protein bands were visualized by an ECL plus system (Beyotime). The specific primary antibodies used in western blot analysis were as follows: anti-     2.10. Statistical Analysis. Statistical analysis was conducted using GraphPad Prism 5.0 software. Data were expressed as the mean ± SEM. The differences between two groups were analyzed by Student's t-test, and three groups or more were analyzed by one-way ANOVA. P < 0:05 was considered statistically significant.

5-FU-Resistant Colon Cancer Cells Showed Increased
EMT and Decreased Apoptosis. We generated 5-FUresistant HCT-15/FU cells from the 5-FU-sensitive parental HCT-15 cells by continuously exposing them to gradually increasing concentrations of 5-FU. We treated the 5-FUsensitive and resistant cells with increasing doses of 5-FU for 48 h and examined the growth inhibition using MTS assays. As shown in Figure 1 (Figure 2(a)). These results further verified that 5-FU-resistant colon cancer cells had enhanced migration and antiapoptotic ability.
Our previous studies revealed that GDF15 was significantly higher in exosomes of HCT-15/FU cells as compared to exosomes of HCT-15 cells. We next investigated intracellular levels of GDF15. The mRNA and protein levels of GDF15 were decreased in the HCT-15/FU cells as compared with HCT-15 cells (Figure 2(b)). As a consequence of being a member of the BMP/TGF-β superfamily, as shown in Figure 2(b), the low expression of GDF15 in HCT-15/FU cells was accompanied by a reduction of P-Smad2/3. Therefore, we suspected that the inhibition of GDF15 and related Smad signaling pathways was associated with improved migration and antiapoptotic ability of 5-FU-resistant cells.  (Figure 3(g)). These results suggested that the low expression of GDF15 enhanced the migration and antiapoptosis ability of colon cancer cells and that the overexpression of GDF15 could partially reverse these effects by activating the Smad signaling pathway.

Discussion
Resistance to 5-FU is a major obstacle in the treatment of colon cancer. Therefore, it is critical to understand the mechanisms of 5-FU resistance. Drug-resistant cells exhibit accelerated proliferation and distant metastasis [11][12][13]. Consistent with previous studies, the present findings demonstrate that 5-FU resistance in colon cancer cells is mechanistically associated with their enhanced migration and antiapoptotic ability by inhibiting the expression of GDF15 and related signaling pathways. EMT is a dynamic process that is essential for the development of multicellular organisms. Its influence on tumor progression is clearly established [14]. Increasing evidence suggests that EMT can enable tumor cells to acquire metastatic features and develop therapeutic resistance and immune escape ability [15]. Our results indicated that HCT-15/FU    BioMed Research International cells underwent EMT (through the modulation of the expression of N-cadherin, E-cadherin, and MMP14), which is related to the inhibition of the Smad signaling pathway. In vivo experiments also confirmed the absence of the epithelial marker (E-cadherin) in the HCT-15/FU group. Whether drug-resistant colon cancer cells are more prone to distant metastasis, such as to the liver, lungs, and peritoneum, via EMT needs a further study. 5-FU plays an antitumor role via inhibition of thymine nucleotide synthase and then by interfering with DNA synthesis. Under the effect of chemotherapy drugs, tumor cells are subjected to stress damage and appear apoptotic [16]. In this study, we found that upon 5-FU treatment, the apoptosis of HCT-15/FU cells was significantly reduced, compared to HCT-15 cells; this was further confirmed by changes in apoptotic executive proteins (Bcl-2, BAX, and cleaved caspase-3). These results suggested that apoptosis is associated with 5-FU resistance in colon cancer cells. However, the regulatory network of apoptosis is complex and involves multiple processes and a cascade of reactions. Identifying the critical regulatory points of apoptosis involved in drug resistance will have profound significance in reversing drug resistance in colon cancer and enhancing treatment. Future studies should pay more attention to this aspect.
Evidence suggests that GDF15 plays different roles in different stages of tumor progression [17]. It inhibits early tumor promotion but accelerates tumor progression in advanced cancers [17]. It can also promote drug or radiation resistance in tumor cells by acting on the Smad signaling pathway [9]. Previous studies have confirmed that GDF15 is closely associated with tumor stem cells and can be used as a marker to reflect the prognosis of colon cancer [7,18,19]. In our study, we found that 5-FU resistance was caused due to the inhibition of GDF15 and the downstream Smad signaling pathway in colon cancer. The overexpression of GDF15 can resensitize 5-FU-resistant HCT-15/FU cells to 5-FU. Our in vivo experiment also validated that HCT-15/FU with low GDF15 expression had more robust proliferation capacity and metastasis tendency. These results demonstrate that GDF15 might act as a tumor suppressor gene in colon cancer. Many mechanisms result in the inactivation of tumor suppressor genes, such as loss of heterozygosity [20][21][22], DNA methylation [23,24], or acetylation [25,26]. Future studies should focus on the mechanism through which GDF15 is

Conclusion
Overall, our findings suggest that 5-FU resistance in colon cancer cells is associated with the repression of GDF15 and the subsequent enhancement of the EMT and antiapoptotic ability of cancer cells. Although we did not show the mechanism of GDF15 repression, we have provided evidence for its significance in 5-FU resistance in colon cancer cells and identified a gene therapy target for reversing drug resistance and improving the prognosis of colon cancer patients. Bcl-2-associated X protein

Data Availability
All data generated or analyzed during our study are available from the corresponding author on reasonable request.

Ethical Approval
All animal experiments were performed in accordance with a protocol approved by the Experimental Animal Welfare Ethics Review Committee of National Institutes for Food and Drug Control, China.

Conflicts of Interest
The authors declare that they have no competing interests.