EpCAM is critical for tumor proliferation and oxaliplatin chemoresistance in EpCAM high / CD44 + colorectal cancer stem cells

Objectives: A small subpopulation of colorectal cancer stem cells (CSCs) possess the ability to self-renew and the capacity to initiate the original tumor. EpCAM high /CD44 + cells are regarded as CSCs in colorectal cancer. The present study was undertaken to investigate the signi ﬁ cance of EpCAM in the in vitro proliferation ability and oxaliplatin chemoresistance of EpCAM high /CD44 + colorectal CSCs. Methods: We applied fluorescence-activated cell sorting (FACS) to separate the EpCAM high /CD44 + subset from human colorectal cancer cell line HCT116. We also used siRNA targeting EpCAM to create EpCAM − /CD44 + subpopulation. Then we compared EpCAM high /CD44 + cells and EpCAM − /CD44 + cells for proliferation ability and the chemoresistance to oxaliplatin by CCK8 assay. Results: The EpCAM high /CD44 + subset comprises almost 6.25 ± 0.09% in cell line HCT116, and the EpCAM − /CD44 + cells displayed a signi ﬁ cantly lower proliferation ability and weaker oxaliplatin chemoresistance than the EpCAM high /CD44 + cells. Conclusions: EpCAM is critical for tumor proliferation and oxaliplatin chemoresistance in EpCAM high /CD44 + colorectal CSCs.


Introduction
The prevalence of colorectal cancer increases with age, and it is the 3rd most commonly diagnosed cancer in developed countries [1]. The significant risk factors of colorectal cancer are contributed by the increasingly ageing population, unfavorable modern dietary habits, low physical exercise, obesity, and smoking [2]. Surgery can be applied to remove tumors in almost 2/3 of patients with colorectal cancer that is localized. Radiation therapy is used to cure about 20% of patients with colorectal cancer, which has spread to other organs, most commonly to the liver [3]. Patients with cancer recurrence can be treated with surgery, and chemotherapy is still the primary therapeutic method for advanced colorectal cancer [4,5]. There has been substantial progress in the treatment of colorectal cancer recently [6].
Colorectal carcinomas consist of heterogeneous cells [7]. Colorectal cancer stem cells (CSC) have gained great attraction due to their high tumorigenicity and chemoresistance. Many studies have shown that three key pathways play a crucial role in stem cell proliferation and maintenance: the Notch pathway, the WNT pathway, and the bone morphogenetic protein (BMP) pathway [8][9][10]. Recent studies revealed molecular signatures of colorectal CSCs are associated with patient outcomes. Some CSC-directed therapies are being performed on patients to translate CSC discoveries into the clinics [11].
Several markers are used for colorectal CSC identification, such as epithelial cell adhesion molecule (EpCAM) [12], CD44 [13], CD166 [14], aldehyde dehydrogenase-1 (ALDH-1) [15] and CD26 [16]. EpCAM high CD44 + colorectal CSCs are considered putative colorectal CSCs rich in stem cell genes [17]. However, the significance of EpCAM in the in vitro proliferation ability and oxaliplatin chemoresistance of EpCAM high CD44 + colorectal CSCs is elusive. Therefore, this study is to elucidate the great value of EpCAM in EpCAM high CD44 + colorectal CSCs to provide the theoretical foundation for EpCAM-targeted therapies in the future.

Materials and methods
Chemical A 1 g/L stock solution of Oxaliplatin (Sigma, China) was prepared in a 5% glucose solution, which was diluted by RPMI-1640 medium (Gibco, USA) to make a working solution.

Cell culture
The human colorectal cancer cell lines (HCT116) were obtained from the Institute of Biophysics, Chinese Academy of Sciences. HCT116 cells were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, USA) and 1% streptomycin and penicillin (Sigma, China), which were maintained in an incubator supplied with 5% CO 2 at 37°C.

Flow cytometry sorting
After washing with Phosphate buffer solutions (PBS), HCT116 cells were stained with FITC-conjugated anti-CD44 and APC-conjugated anti-EpCAM antibodies for 30 min on ice. The BD FACS Aria III cell sorter was applied to analyze and isolate the EPCAM high /CD44 + cells.

Gene silencing of EpCAM
The mismatched control and the siRNA targeting human EpCAM were designed and synthesized from RiboBio (Guangzhou, China). Mismatched control siRNA sequences were as follows: sense, UUCUCCGAACGUGUCACGUdTdT; antisense, ACGUGACACGUUCGG AGAAdTdT. The siRNA sequences for EPCAM were as follows: sense, CAATGCCAGTGTACTTCAGTT; antisense, AACTGAAGTACACTGGCA TTG. After HCT116 was transfected with 16.7 nM annealed negative mismatched control siRNA or siRNA targeting human EPCAM in OPTI media, a siRNA-mate reagent (Genepharma, Shanghai, China) was used as the transfection reagent based on the manufacturer's instruction.

RT-PCR
An RNA purification kit (New England Biolabs, USA) was used to isolate total RNA based on the manufacturer's instructions. A Pri-meScript RT Reagent Kit (TaKaRa, Japan) was chosen to perform reverse transcription (RT). ChamQ Universal SYBR qPCR Master Mix (Vazyme, China) was applied to perform a real-time polymerase chain reaction (PCR). LightCycler 96 System (Roche, Switzerland) was selected to carry out PCR amplification and fluorescence detection. β-Actin was chosen as the internal control. The C T method (2 −ΔΔCT ) was applied to calculate comparative expression ratios of the target sample to the control group for each sample. The primers for RT-PCR are in Table 1.

Western blotting
Whole proteins were extracted from HCT116 cells. Thirty mg of protein from each group was separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis gel, then transferred to a polyvinylidene difluoride membrane. Firstly, the membrane was blocked with 5% nonfat dry milk diluted in Tris-buffered saline, then incubated with mouse anti-human EpCAM monoclonal antibody (ABCAM, USA) or mouse anti-human beta Actin monoclonal antibody (ZSGB-BIO, Beijing, CN). Subsequently, a horseradish peroxidase-linked antimouse IgG antibody (ZSGB-BIO, Beijing, CN) was incubating the membranes. Protein bands were visualized by a chemiluminescence horseradish peroxidase (HRP) substrate (Millipore, USA) and analyzed by ImageJ software.
The assay to test the proliferation ability A CCK8 kit (Biyuntian Biological Technology Co., LTD, China) was used to compare the proliferation ability of EpCAM high /CD44 + and EPCAM − /CD44 + HCT116 cells. 1 × 10 3 cells were cultured into each well of a 96-welate for 24 h, and the old medium was removed. Then 10 μL CCK8 in a 90 μL new medium was added to each well, which was cultured for another 4. A multimode plate reader (EnSpire, USA) was selected to calculate the cell viability based on the absorbance at 450nm. The detection time was set at 0day, 1 day, 2 day, 3 day, 4 day, and 5 day.
The assay to test the chemoresistance A CCK8 kit (Biyuntian Biological Technology Co., LTD, China) was used to compare the chemoresistance of EpCAM high /CD44 + and EpCAM − /CD44 + HCT116 cells. The cells were seeded into the 96 well plates, and the cells were treated with 2, 4, 8, 16, 32, or 64 mg/L Oxaliplatin for 24 h. Then the old medium was removed, and 10 μL CCK8 in 90 μL medium was added to each well, which was cultured for another 4. A multimode plate reader (EnSpire, USA) was used to calculate the cell viability based on the absorbance at 450nm.

Statistical analysis
Statistical difference was analyzed by GraphPad Prism version 7.0 (GraphPad Software Inc., California, USA). The statistical significance of the data from three independent experiments was

Results
The proportion of the EpCAM high /CD44 + subset in the human colorectal cancer cell line HCT116 The EpCAM high /CD44 + cells are regarded as colorectal CSCs. Anti-CD44 and anti-EPCAM antibodies were used to separate EpCAM high /CD44 + subpopulation from the human colorectal cancer cell line HCT116 (Figure 1), which comprises 6.25 ± 0.09% of the whole cell number.
The EpCAM silencing efficacy in the EpCAM high /CD44 + cells The EpCAM high /CD44 + cells were treated with siRNA targeting EpCAM, RT-PCR was used to detect the mRNA levels of EpCAM, and Western Blotting was applied to evaluate the protein levels of EpCAM. As shown in Figure 2A,B, the mRNA and protein levels of EpCAMdecreased significantly after the treatment of EpCAM high /CD44 + cells with siRNA targeting EpCAM.

The comparison between EpCAM high /CD44 + cells and EpCAM − /CD44 + cells in the chemoresistance
To determine the significance of EpCAM in chemoresistance, CCK8 assay was applied to compare the chemoresistance of EpCAM − /CD44 + cells with EpCAM high /CD44 + cells after treatment with different concentrations of oxaliplatin (2,4,8,16 or 64 mg/L) for 24, 48 or 72 h. The results in Figure 4A-C revealed that EpCAM − /CD44 + cells showed a significantly lower oxaliplatin chemoresistance than EpCAM high /CD44 + cells control.

Discussion
About 945,000 patients are diagnosed with colorectal cancer, and almost 492,000 patients die from this disease globally each year [18]. The new molecular basis of colorectal cancer has resulted in the emergence of targeted therapeutic methods tested in clinics. Recent research has highlighted that colorectal CSCs play a crucial role in cancer chemoresistance and relapse. Targeting colorectal CSCs through monoclonal antibodies or small molecule inhibitors in vivo studies has demonstrated the effectiveness of these treatments [11]. CD44 is a multifunctional glycoprotein present on the cell surface, and EpCAM is an epithelial cell adhesion molecule [19,20]. Some investigations suggested that EpCAM high /CD44 + cells are considered as CSCs in colorectal cancer [21]. This study applied fluorescence-activated cell sorting (FACS) to separate the EPCAM high /CD44 + subset from human colorectal cancer cell line HCT116. After quantifying the average proportion of the EPCAM high /CD44 + cells in the cell line HCT116, we used siRNA targeting EpCAM to create EpCAM − /CD44 + subpopulation. Subsequently, we compared EPCAM high /CD44 + cells and EpCAM − /CD44 + cells for proliferation ability and the chemoresistance to oxaliplatin. Our results suggested that the EpCAM high / CD44 + subset comprises almost 6.25 ± 0.09% cell line HCT116. It also turned out that the EpCAM − /CD44 + cells displayed a significantly lower proliferation ability and weaker oxaliplatin chemoresistance than the EPCAM high / CD44 + cells.
The preliminary result of this research will emphasize the significance of EpCAM in the EPCAM high /CD44 + cells to act as a potential target in curing colorectal cancer in the coming years. Antibodies (catumaxomab or edrecolomab) binding to EpCAM combined with oxaliplatin might be a novel method to eliminate EPCAM high /CD44 + cells [22], which will increase the overall survival rates for patients with colorectal cancer. This is sure to reduce the dose of oxaliplatin and improve the patients' quality of life due to the limited toxicity. This study has one limitation: we didn't explore the detailed mechanism at the cellular level. We didn't compare EPCAM high /CD44 + cells and EpCAM − /CD44 + cells for the tumorigenicity and oxaliplatin chemoresistance in tumor xenograft models. Thus, we need to perform further cell experiments to investigate the molecular mechanism and validate its effect on animal models.
In summary, EpCAM is critical for tumor proliferation, and oxaliplatin chemoresistance in EpCAM high /CD44 + colorectal CSCs, which indicates targeting EpCAM may be a method to eliminate EPCAM high /CD44 + cells to cure patients with colorectal cancer.