Anti-EGFR antibody sensitizes colorectal cancer stem-like cells to Fluorouracil-induced apoptosis by affecting autophagy

Recent reports suggest that colorectal carcinoma (CRC) may be sustained by a small subpopulation of cells, termed cancer stem cells (CSCs), which have drug resistance properties as a key reason for chemotherapy failure. The epidermal growth factor receptor (EGFR) controls CRC initiation and progression. Monoclonal antibody against EGFR (cetuximab) has been used in treatment of several cancers. However, the effects of cetuximab on CSCs in the CRC chemotherapy remain unclear. Here, we studied the effects of cetuximab on the CSC-like cells in Fluorouracil (5-FU)-treated CRC cells. CSC-like cells were independently isolated from CRC cells using CD133, CD44 or EphB2-high as markers and confirmed by tumor sphere formation assay. We found that 5-FU increased the apoptotic death of CSC-like CRC cells. Co-application of cetuximab augmented the apoptotic death of CSC-like CRC cells by 5-FU, seemingly through inhibition of 5-FU-induced increases in cell autophagy in CSC-like CRC cells. Together, our data suggest that EGFR monoclonal antibody may sensitize CSC-like CRC cells to 5-FU-induced apoptosis by affecting autophagy.


INTRODUCTION
Colorectal carcinoma (CRC) is the third common cancer in humans [1][2][3]. Although the primary CRC are highly curable, some CRC may migrate to distal tissues, resulting in poor prognosis [4]. Recent reports suggest that CRC may be sustained by specific cells called cancer stem cells (CSCs). CRCs have potential innate drug resistance properties, leading to chemotherapy failure [4]. Moreover, CSCs are supposed to be responsible for the majority of the cancer invasiveness and metastases, which highlights the significance of treating CSCs rather than the complete cancer mass during cancer therapy [4].
The epidermal growth factor receptor (EGFR) signaling pathway is involved in the initiation and progression of CRC [29][30][31][32]. Cetuximab is a FDAapproved EGFR chemeric human-murine monoclonal antibody against EGFR (cetuximab). However, the effects of cetuximab on CSCs in the chemotherapy of CRC remain unclear.
Here, we studied the effects of cetuximab on the CSC-like cells in Fluorouracil (5-FU)-treated CRC cells. CSC-like cells were independently isolated from CRC

Research Paper
Oncotarget 81403 www.impactjournals.com/oncotarget cells using CD133, CD44 or EphB2-high as markers and the features of these cells as CSC-like cells were proven by tumor sphere formation assay. We found that 5-FU increased the apoptotic death of CSC-like CRC cells, in an CCK-8 assay and an apoptotic assay. Co-application of cetuximab augmented the apoptotic death of CSC-like CRC cells by 5-FU, seemingly through inhibition of 5-FUinduced increases in cell autophagy in CSC-like CRC cells. Together, our data suggest that EGFR monoclonal antibody may sensitize CSC-like CRC cells to 5-FUinduced apoptosis by affecting autophagy.

CD133-positive cells are enriched with CSCs in CRC
In order to examine the effects of cetuximab on the CSC population of CRC cells treated with chemotherapeutic drugs (e.g. 5-FU), we isolated CSC-like cells from CRC cell lines using different CSC markers, CD133, CD44 and EphB2, independently. We chose two CRC cell lines, HT-29 and SW480, in our study. HT-29 cells express low levels of EGFR, and has wildtype KRAS. On the other hand, SW480 express high levels of EGFR, and has mutated KRAS [37]. Hence, these two lines are very good representatives in analyzing the effects of cetuximab on the CSC population of CRC cells treated with 5-FU.
First, we isolated CD133+ cells vs CD133− cells from either HT-29 cells ( Figure 1A), or SW480 cells ( Figure 1B). To confirm that CD133+ cells may be enriched for CSCs, we performed tumor sphere formation assay. We found that CD133+ cells formed significantly more spheres than CD133− cells, in either HT-29 cells ( Figure 1C), or SW480 cells ( Figure 1D). Quantification was shown in Figure 1E. Hence, CD133-positive cells are enriched with CSCs in CRC.

CD44-positive cells are enriched with CSCs in CRC
Then, we isolated CD44+ cells vs CD44− cells from either HT-29 cells (Figure 2A), or SW480 cells ( Figure 2B). To confirm that CD44+ cells may be enriched for CSCs, we performed tumor sphere formation assay. We found that CD44+ cells formed significantly more spheres than CD44− cells, in either HT-29 cells ( Figure 2C), or SW480 cells ( Figure 2D). Quantification was shown in Figure 2E. Hence, CD44-positive cells are enriched with CSCs in CRC.

EphB2-high cells are enriched with CSCs in CRC
Finally, we isolated EphB2-high cells vs EphB2low cells from either HT-29 cells ( Figure 3A), or SW480 cells ( Figure 3B). To confirm that EphB2-high cells may be enriched for CSCs, we performed tumor sphere formation assay. We found that EphB2-high cells formed significantly more spheres than EphB2-low cells, in either HT-29 cells ( Figure 3C), or SW480 cells ( Figure 3D). Quantification was shown in Figure 3E. Hence, EphB2-high cells are enriched with CSCs in CRC. Thus, these enriched CSCpopulations (CD133+; CD44+; EphB2-high) were independently used for analyzing the effects of cetuximab on the CSC population of CRC cells treated with 5-FU.

EGFR inhibition increases 5-FU-induced apoptotic death in CSC-like CRC cells
Cultured CD133+ HT-29 cells, or SW480 cells were treated with/without 5-FU. Moreover, the 5-FU-treated cells were also treated with cetuximab, or control IgG. After 24 hours, the cells were analyzed. We found that 5-FU significantly reduced the cell viability of CD133+ CRC cells, in an CCK-8 assay ( Figure 4A), seemingly by increasing the apoptotic cell death ( Figure 4B-4C). Co-application of cetuximab augmented the apoptotic death of CD133+ CRC cells by 5-FU ( Figure 1A-1C). We got similar results, using CD44+ or EphB2 cells in this study (not shown). Thus, EGFR inhibition increases 5-FUinduced apoptotic death in CSC-like CRC cells.

EGFR inhibition reduces 5-FU-induced cell autophagy in CSC-like CRC cells
Since autophagy and apoptosis are closely related and may affect each other at molecular level, we thus examined whether EGFR inhibition may alter cell autophagy in 5-FU-treated CSC-like CRC cells. LCII vs LC I levels are a golden standard for evaluating cellular autophagy activity. We found that 5-FU induced cell autophagy in CD133+ CRC cells, which was significantly attenuated by cetuximab ( Figure 5A-5B). Similar results were obtained when we used either CD44+ cells, or EphB2-high cells ( Figure 5C-5F). Thus, 5-FU may not only induce apoptotic cell death of CSC-likes in CRC, but also induce cell autophagy to contradict apoptotic cell death to allow some cells to survive the treatment. However, cetuximab may inhibit the autophagy to increase the sensitivity of CSC-like CRC cells to chemotherapy ( Figure 6).

DISCUSSION
CSCs are characterized by their drug resistance properties. Treatments targeting CSCs thus could improve the therapeutic outcome of rapidly growing cancers and highly metastatic cancers [5][6][7][8].
In the current study, we showed that 5-FU treated CRC cells underwent apoptotic cell death. However, the effects of 5-FU on CRC cells could be augmented by cowww.impactjournals.com/oncotarget treatment of the CRC cells with cetuximab, which has been widely used in treating malignant cancer in clinic. Here, we examined two CRC cell lines, among which SW480 has mutated KRAS while HT29 has wild-type KRAS. Those cell lines thus represent CRC cells with or without mutated KRAS. Since mutated KRAS is resistant      to EGFR-targeted mAbs [38], our data that show coapplication of cetuximab with 5-FU is effective for both lines may suggest that the effects of cetuximab on cell death are indirectly and may result from the changes of sensitivity of CRC cells to 5-FU. This conclusion is also supported by the data that a control group of cetuximab alone of same dosage did not have significant effects on cell death.
Interestingly, we found that the effects of cetuximab on CRC cells appeared to be on CSC-like cells, which were independently isolated from CRC cells using CD133, CD44 or EphB2-high as markers. Of note, these markers have been used to enrich CSC cells, but not purify them. Using these markers independently increased the reliability of the conclusion on CSC cells, which were further proved by tumor sphere formation assay, which is a gold method for validating CSCs. We found that 5-FU increased the apoptotic death of CSC-like CRC cells in an apoptotic assay. Moreover, the changes in cell number were confirmed in a CCK-8 assay.
Then, we studied the mechanisms underlying the cetuximab augmented CRC cell death. We found that 5-FU treatment decreased CRC cell viability in a dosedependent manner. However, this 5-FU-induced CRC cell death appeared to be attenuated by augmentation in autophagy-associated cell survival. Thus, 5-FU may induce both apoptotic cell death and autophagic cell survival in CRC cells, while autophagy could be a negative feedback from CRC cells to resist 5-FU. In another word, 5-FU damaged CRC cells, the CSC-like cells from which upregulated autophagy associated proteins to enhance autophagic cell survival against the effects of 5-FU. The cetuximab treatment inhibited the autophagy of CSC-like cells from CRC, resulting in improved elimination of the CRC. A limitation of the current study is that the effect of cetuximab/5-FU adjunctive treatment in vivo was not investigated, which should be addressed in future studies. Moreover, further dissection of the details of the involved signaling pathway is highly needed.
Together, our data suggest that EGFR monoclonal antibody may sensitize CSC-like CRC cells to 5-FUinduced apoptosis by affecting autophagy. Cetuximab treatment may be a promising treatment targeting chemoresistance of CSC-likes from CRC.

Protocol approval
All the experimental methods in the current study has been approved by the research committee at Jilin University. All the experiments have been carried out in accordance with the guidelines from the research committee at Jilin University.

Cell line culture and treatment
From all published CRC cell lines, we selected HT-29 and SW480 in our study. HT-29 is a colorectal adenocarcinoma from a 44 year-old female, and has been describe before [33]. SW480 is a colorectal adenocarcinoma from a 50 year-old male, and has been describe before [34]. Both lines were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA), and maintained in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 15% fetal bovine serum (FBS; Sigma-Aldrich, St Louis, MO, USA) in a humidified chamber with 5% CO 2 at 37°C. 5-FU (Sigma-Aldrich) was prepared in a stock of 1 mmol/l and applied to the cultured CRC cells at 2 µmol/l [35,36]. Cetuximab is an EGFR chemeric human-murine monoclonal antibody, and was applied to the cultured CRC cells at 0.5 mg/ml. Isotype-match IgG of same concentration was used as a control.

Cell viability assay
The CCK-8 detection kit (Sigma-Aldrich) was used to measure cell viability according to the manufacturer's instructions. Briefly, cells were seeded in a 96-well microplate at a density of 5 × 10 4 /ml. After 24 h, cells were treated with resveratrol. Subsequently, CCK-8 solution (20 ml/well) was added and the plate was incubated at 37 o C for 2 h. The viable cells were counted by absorbance