5‐FU inhibits migration and invasion of CRC cells through PI3K/AKT pathway regulated by MARCH1

Abstract Colorectal cancer is a major health problem with a significant impact on the patients' quality of life. 5‐Fluorouracil is the most common chemotherapy drug used for this type of cancer. While its molecular mechanism is the inhibition of DNA synthesis via the inhibition of thymine nucleotide synthetase, its complete anticancer mechanism is not clear. Membrane‐associated RING‐CH‐1 (MARCH1) is an E3 ubiquitin ligase that plays an important role in antigen presentation. However, MARCH1 has not been studied in the context of colorectal cancer. In this study, we demonstrated that MARCH1 is highly expressed in colorectal cancer tissues and cell lines. Furthermore, migration and invasion of colorectal tumor cells were inhibited via transfection with small interfering RNAs to suppress the expression of MARCH1. The western blot analysis showed that MARCH1 regulates epithelial–mesenchymal transition and the PI3K/AKT pathway. Moreover, 5‐fluorouracil inhibited the proliferation, migration, and invasion of tumor cells, via the targeting of MARCH1 and the consequent downregulation of the PI3K/AKT pathway, impacting the progression of epithelial–mesenchymal transition. In conclusion, our study shows that MARCH1 may play a role as an oncogene in colorectal cancer and may represent a new target molecule of 5‐fluorouracil.


| INTRODUCTION
Colorectal cancer (CRC) is one of the most common tumors in the digestive tract. The pathogenesis of CRC is unclear; it may be related to the malignant change of colorectal polyps, chronic inflammation of the colorectal mucosa, dietary habits, or heredity factors (Mármol et al., 2017;Thanikachalam & Khan, 2019). Despite considerable improvements in the treatment of CRC, its incidence is increasing due to metastasized tumors (Piawah & Venook, 2019). Of note, metastasis and invasion are also the leading causes of poor tumor prognosis. Epithelial-mesenchymal transition (EMT) plays an important role in tumor migration and invasion. It refers to the process of the conversion of epithelial cell morphology into an interstitial phenotype under the stimulation of external signals (Cao et al., 2015). Therefore, studying EMT and its role in invasion and proliferation in the context of CRC cells may provide new therapeutic targets to prevent metastasis and recurrence. 5-Fluorouracil (5-FU) is one of the most used and effective chemotherapy drugs for CRC (Grem, 2001). Most patients receive 5-FU-based treatment regimens; both intravenous and oral 5-FU for CRC have become the main systemic treatment since the 1990s. 5-FU is an antimetabolite drug; the hydrogen at position C-5 of uracil is replaced by fluorine. Many studies have focused on the biomodulation of 5-FU to improve its therapeutic anticancer effectiveness and selective cytotoxicity (Goirand et al., 2018). Owing to genetic and epigenetic differences of CRC patients, however, the incidence of 5-FU resistance is gradually increasing. Consequently, exploring specific targeted molecules or pathways of 5-FU resistance may help improve patients' outcomes in the future.
The membrane-associated RING-CH (MARCH) family comprises E3 ubiquitin ligases located on the plasma and organelle membranes, consisting of an N-terminal ring finger and zero, two, or more C-terminal transmembrane domains (Bauer et al., 2017). Of the 11 members of the MARCH family, some play important roles in immune regulation, such as endoplasmic reticulum degradation, protein-energy control, and membrane transport Nakamura, 2011). MARCH1, expressed by antigen-presenting cells (APCs), dendritic cells (DCs), and B cells in lymphoid tissues (e.g., the spleen), participates in the regulation of antigen presentation via the ubiquitination of major histocompatibility complex (MHC)-II and CD86 (Corcoran et al., 2011;Wilson et al., 2018). Many studies have reported the role of MARCH1 in the immune system. However, MARCH1 also takes part in the development of cancers. A study found that MARCH1 is overexpressed in ovarian cancer tissues and that MARCH1 silencing inhibited the proliferation, invasion, and migration of SKOV3 cells via the mediation of the nuclear factor-κB (NF-κB) and the Wnt/β-catenin pathways (Meng et al., 2016).
However, there is no research on the role of MARCH1 in CRC.
In this study, we aimed to investigate the role and expression of MARCH1 in CRC tissues and cell lines. Furthermore, we also investigated its role in metastasis and tumor invasion, as well as its response to 5-FU treatment. We demonstrate that MARCH1 is highly expressed in CRC tumor tissues and cell lines. Moreover, the results show that MARCH1 downregulation inhibits EMT and regulates the migration and invasion of CRC cells via the PI3K/AKT pathway. Furthermore, we show that the levels of MARCH1 are decreased in CRC cells after treatment with 5-FU. Notably, we identified the underlying mechanism: 5-FU not only downregulates the expression of MARCH1, but also inhibits the expression of the PI3K/AKT pathway to repress the proliferation, migration, and invasion of CRC in vitro. This article mainly proposes that 5-FU plays a role in the inhibition of CRC malignant biological behaviors via the downregulating of MARCH1 through the PI3K/AKT pathway.

| CRC clinical samples
Twenty clinical CRC samples and adjacent peritumoral tissues were collected from patients who had experienced radical resection of CRC at Binzhou Medical University Hospital. Tumor tissues and adjacent tissues were collected within 30 min from the body. The adjacent tissues were taken from noncancerous tissues about 5 cm away from the tumor tissue.
After taking the tissues, they were divided into about 0.5 cm tissue pieces and stored in a liquid nitrogen tank. These patients did not undergo radiotherapy and chemotherapy and signed informed consent before surgery. This study was approved by the Ethics Committee of Binzhou penicillin-streptomycin solution (Hyclone) at 37℃ in 5% CO 2 atmosphere, and 0.25% trypsin (Biological Industries) was used to passage the cell lines till they reached 90% confluence. The cells which were planted (3 × 10 5 per well) on six-well culture dishes or planted (5 × 10 3 per well) on 96-well culture dishes were transfected with 50 nM siRNA-MARCH1

| Cell proliferation assay
Cell proliferation assays were performed using a cell counting kit-8 (CCK-8; Dojindo). According to the manufacturer's instructions, 2000 cells were seeded in 96-well plates overnight before treatment.
CCK-8 reagent was added to each well, and after incubation with the reagent for 2 h at 37°C. The absorption and reference wavelength was measured at 450 nm. After normalizing the 0 h optical density average value, Graphpad Prism 7.0 software was used to draw the proliferation curve of each group of cells over time.

| Colony formation assay
One thousand cells were seeded in six-well plates per well, treated with 5-FU after 24 h, and grown for 14 days. Then the colonies were stained with 0.1% crystal violet solution (Sangon Biotech), washed with PBS, and imaged with a photomicroscope. Colonies containing over 50 cells were counted.

| Cell apoptosis assay
The apoptosis assays were performed using an Annexin V, Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit (Dojindo) according to the manufacturer's instructions. We collected at least 10,000 cells after transfection, washed them twice with cold PBS, and used 100 µl annexin V binding solution to make cell suspension.
The cell suspension was incubated with 5 μl of annexin V-FITC and 5 μl of propidium iodide (PI) for 15 min, followed by apoptosis analysis by flow cytometry (BD Biosciences).

| Transwell assay
Cell migration and invasion assay were performed using 6.5 mm transwell insert chambers with an 8.0 µm pore polycarbonate membrane. The cells (3000) were cultured in serum-free medium, placed in the upper chambers, and coated with Matrigel basement membrane matrix (Corning) for 2 h at 37°C before the cells were added.
The medium with 20% FBS was added to the down chamber. The cells were incubated for 24-48 h, and cells that did not migrate through the pores were removed with a cotton swab. Then the upper chambers were fixed in 4% paraformaldehyde, stained with 0.5% crystal violet (Sangon Biotech), and counted under a photomicroscope. However, there was no need for the Matrigel coating for the cell migration assay.

| Immunohistochemistry
All tissue microarray slide containing tumor and adjacent tissues were provided by Binzhou Medical University Hospital. All tissues were fixed in 4% paraformaldehyde overnight and subsequently embedded in paraffin wax. The embedded-tissues were cut into 4-μm sections which were stained for analysis. The sections were deparaffinized using dimethylbenzene followed by antigen retrieval by heating for 20 min in ethylenediaminetetraacetic acid buffer (pH = 9.0) in a microwave oven. The staining intensity score of tumor tissues and adjacent nontumor tissues was as follows: 0 (negatively stained), 1 (weakly stained), 2 (moderately stained), 3 (strongly stained).

| Statistic analysis
SPSS 23.0 was used to analyze the data, and GraphPad Prism 7.0 software was used for statistical drawing. The measurement data is expressed as mean ± standard deviation (mean ± SD). The t test

5-FU treatment inhibits malignant behaviors of CRC cells, partially due
to the regulation of the MARCH1 and PI3K/AKT pathways.

| DISCUSSION
CRC is the leading cause of the sharp increase in cancer morbidity and mortality in the world. The early detection and appropriate treatments have improved patients' survival in recent decades (Meester et al., 2019). Of note, metastasis is one of the most important events that lead to CRC progression and poor prognosis.
Therefore, the investigation of the mechanisms behind metastasis, looking for biomarkers of CRC, and the development of better therapeutics targeting specific molecules are strategies of great significance for the prevention and treatment of CRC.

MARCH1 functions as an E3 ubiquitin ligase and plays an
important role in the immune system. According to the literature,

Foxp3 + T regulatory cells (Tregs) suppress dendritic cells (DCs)
and antigen presentation via the production of interleukin 10 (IL-10), promoting the elevation of MARCH1 mRNA levels (Chattopadhyay & Shevach, 2013). Further, another study con-  which revealed that 5-FU may inhibit migration and invasion by inhibiting EMT. The classic anticancer mechanism of 5-FU is to block the catalytic process of thymidylate synthase and further interfere with DNA synthesis (Diasio & Harris, 1989). Importantly, in this study, we found that MARCH1 was downregulated after 5-FU treatment in a dose-dependent manner, which may indicate a potentially new mechanism of action of 5-FU treatment in the context of CRC. In addition, we show that 5-FU can suppress malignant behaviors of CRC cell lines via the downregulation p-PI3K and p-AKT. Our results suggest that 5-FU also exerts an anticancer effect through the inhibition of the MARCH1-PI3K/AKT-EMT pathway.

| CONCLUSION
In conclusion, our experiments further solidify the carcinogenic role of MARCH1 in CRC. Importantly, MARCH1 is associated with the malignant biological behavior of migration and invasion in CRC cell lines. MARCH1 could regulate PI3K/AKT pathway and further influence the progression of EMT, which is important in tumor distant metastasis. Furthermore, we also found that 5-FU was able to suppress the progression of CRC in vitro by downregulating the expression of MARCH1, partially through the inhibition of PI3K/AKT pathway and EMT.
It is worth noting that our study has some limitations. For example, we did not collect enough clinical samples and follow-up information to confirm the relationship between MARCH1 expression and patients' prognosis. Our future research will focus on this mechanism. Finally, this study suggests a new target molecule for 5-FU treatment and lays the foundation for the future development of therapeutic drugs targeting MARCH1.