Metformin inhibits the development and metastasis of colorectal cancer

Metformin is a commonly used drug for the treatment of diabetes. Accumulating evidence suggests that it exerts anti-cancer effects in many cancers, including colorectal cancer. However, the underlying molecular mechanisms of colorectal cancer metastasis remain unclear. Colorectal cancer cell lines were treated with metformin, and cell proliferation, invasion, and migration were analyzed in vitro. The relationship between metformin and the AMPK–mTOR axis was assessed by Western blot analysis and transfection with small interfering RNA. A colorectal cancer xenograft mouse model was used to observe the effects of metformin on liver metastasis. Immunohistochemical analysis was performed on liver metastatic tumors. In in vitro experiments, metformin significantly inhibited the proliferation, migration, and invasion only in HCT116 and SW837 cells, but not in HCT8 and Lovo cells. Only in HCT116 and SW837, a change in AMPK–mTOR expression was observed in a dose-dependent manner. In colorectal cancer xenograft mice, the liver metastatic rate (10% vs. 50%, p = 0.05) and the number of liver metastatic nodules (0.1/body vs. 1.2/body, p = 0.04) were significantly lower in the metformin group. Tumor proliferation and EMT were decreased and apoptosis was promoted only in metastatic liver tumors of mice treated with metformin. The molecular mechanism of the anti-cancer effects of metformin involves repression of mTOR pathways via AMPK activation. Moreover, the differences in metformin sensitivity depend on the response of the AMPK–mTOR pathway to metformin. Our study provides a theoretical basis for the anti-metastatic treatment of colorectal cancer using metformin.


Introduction
Colorectal cancer (CRC) is one of the most common neoplasms in the world. Approximately 25% of patients initially present with metastatic CRC (synchronous metastases) [1]. Despite recent advances in the medical treatment of metastatic CRC, the 5-year survival rate of CRC patients with unresectable metastatic disease is reported to be less than 10% [2], 3. Therefore, it is important to develop novel approaches to prevent metastasis of CRC.
Metformin is a biguanide derivative widely used for the treatment of type 2 diabetes. Metformin exerts its effects by reducing hepatic glucose production and by increasing insulin sensitivity as well as glucose use by peripheral tissues. Recently, clinical studies of various cancer types have reported the anti-cancer effects of metformin [4], 5. Fransgaad et al. reported that metformin treatment was associated with 15% of all-cause mortality in CRC patients with diabetes compared with patients with insulin-treated diabetes [6]. Furthermore, a recent meta-analysis demonstrated that metformin was associated with increased overall survival and cancer-specific survival in CRC [7]. Therefore, the potential anti-cancer effects of metformin have gained great attention.
Previous experimental study has reported that the anticancer effects of metformin were induced by the activation of AMP-activated protein kinase (AMPK) [8]. AMPK activation leads to a reduction in mammalian target of rapamycin (mTOR) signaling, protein synthesis, and cell proliferation [9][10][11]. Furthermore, the administration of metformin significantly reduces the expression of epithelial-mesenchymal transition (EMT) markers in various types of cancer cells [12], 13. These findings suggest that metformin may be an optimal therapeutic agent for cancer treatment. However, there is a lack of experimental evidence of the anti-cancer effects of metformin, especially for metastatic disease.
The objective of this study was to clarify the inhibitory effect of metformin on liver metastasis of CRC. These data provide a rationale and experimental evidence for using metformin as possible option which contributes to the antimetastatic treatment for CRC.

Cell lines
The human CRC cell lines HCT116 (American Type Culture Collection, Human colon carcinoma, CCL-247), SW837 (JCRB cell bank, Human rectal adenocarcinoma, JCRB9115), Lovo (American Type Culture Collection, Human colon adenocarcinoma, CCL-229), and HCT8 (American Type Culture Collection, Human colon adenocarcinoma, CCL-244) were used in these studies and were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in standard culture conditions of humidified 5% CO 2 at 37 °C. All tissue culture reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Western blotting
Cells were lysed in radioimmunoprecipitation assay buffer with protease and phosphatase inhibitors. Proteins were quantified by protein assay (Nano Drop 2000c, Thermo Fisher Scientific, Waltham, MA), and 20 μg protein was separated by SDS-PAGE and transferred to 0.2 μm nitrocellulose membrane. Membranes were blocked in TBS, 0.1% Tween 20, and 5% BSA for 2 h before overnight incubation with primary antibodies diluted to 1:1000 in TBS, 0.1% Tween 20, and 5% BSA. Antibodies against phospho-mTOR, mTOR, phospho-AMPK, and AMPK were obtained from Cell Signaling Technology (Beverly, MA). The membranes were incubated for 1 h in HRP-conjugated secondary antibody diluted at 1:2000-1:10,000 in TBS and 0.1% Tween 20. Immunoreactive protein was detected using MultiImage ll (Alpha Innotech, San Leandro, CA) [14]. Western blot densitometry quantification was performed using ImageJ.

Cell proliferation assays
Cell proliferation was assessed by MTT assay [15]. Cells were plated into 96-well plates at a density of 4 × 10 4 cells/ well. After 24 h incubation, 10 μl SP cell count reagent SF (Nacalai Tesque, Kyoto, Japan) was added to each well, and cells were further incubated for two hours. The viable cell number was directly proportional to the production of formazan following solubilization. Color intensity was measured at 450 nm using a Sunrise R microtiter plate reader (Tecan, Mannedorf, Switzerland). All experiments were performed in triplicate [16].

Cell migration assay
Cell monolayers were wounded with a plastic tip at 48 h after the initiation of metformin treatment. Cell migration was monitored for 24 h at 37 °C and photographed using an EVOS FL Cell Imaging System (Life Technologies, Carlsbad, CA) [14]. The original magnification was 10 × .

Cell invasion assay
Cells were trypsinized, and 50,000 cells resuspended in DME with 0.2% FBS were added to rehydrated Matrigelcoated Cell Culture Inserts (Corning, Corning, NY) and seeded in 24-well companion plates with DME and 10% FBS [17]. After 24 h, the non-invading cells and Matrigel in the upper chambers were removed using a cotton tip. The cells invading to the lower surface and the filters were fixed in methanol for 5 min at room temperature, and the nuclei were stained with hematoxylin and eosin (HE). The invading cells were counted at room temperature using an BX 51 System microscope (Olympus, Tokyo, Japan) and images were analyzed using ToupView software (ToupTek).

Plasmid transfection
Human AMPK siRNA and control siRNA were purchased from Shanghai GenePharma (Shanghai, China). For siRNA transfection, a total of 1.5 × 10 5 cells/well was seeded into six-well plates and transfected with 30 pmol AMPK siRNA or control siRNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA) in accordance with the manufacturer's instructions. Cell proliferation and wound healing were measured at 72 h post-transfection. Metformin treatments were initiated after completion of 48 h transfection.

In vivo studies
Liver metastasis was generated using a previously described splenic injection model [18]. Briefly, 6-weekold female severe combined immunodeficient (SCID) mice were anesthetized using ethyl ether, and the spleen was exteriorized via a 5 mm incision in the left upper abdomen. HCT116 cells (2 × 10 6 ) in 100 μL PBS were slowly injected into the spleen and allowed to flush to the liver for 1 min. Then, the spleen was removed, and homoeostasis was assured by ligation with a suture. In this study, a total of 20 female SCID mice were randomly divided into two groups (control and metformin groups). After splenic injection, metformin (Sumitomo Dainippon Pharma, Tokyo, Japan, 250 mg/kg body weight) was orally administrated every day for 42 days in the metformin group, whereas the control group received normal diet. The dosage selected in the present study was based on a previous report in which metformin suppressed ACF formation in a mouse model of azoxymethane-induced colon cancer [19]. At 42 days post-injection, all the mice were killed under ether anesthesia, and the livers were removed and weighed. Finally, surface liver metastases were counted under blinded conditions. All animal procedures were performed in the SCID mouse facility using protocols approved by the Keio Animal Care and Use Committee.

Immunohistochemistry
The liver metastases obtained from xenografts were immediately fixed in 20% formalin. Blocking of endogenous peroxidase with 3% hydrogen peroxide was performed on dewaxed and rehydrated slides for 30 min. The sections were washed several times with PBS, blocked with 10% normal swine serum (Vector Laboratories, Burlingame, CA) at room temperature, and then incubated with primary antibodies at 4 °C overnight. Antibodies against α-smooth muscle actin (α-SMA) and Ki-67 were purchased from Abcam (Cambridge, UK). After blocking with biotinylated anti-rabbit IgG blocking reagent (Vector Laboratories, Burlingame, CA), the sections were stained using the VECTASTAIN Elite ABC-HRP Kit (Vector Laboratories, Burlingame, CA) for 30 min at room temperature. After rinsing in PBS, all sections were visualized with 0.05% 3,3′-diaminobenzidine. The sections were then counterstained with HE at room temperature for 1 min. Then, for each section, field at 200 × magnification were analyzed. All positive cells in the field and ki-67 labeling index were photographed and counted. Results were expressed as the average of positive cells and presented with the means ± standard error (n = 3).

Apoptosis detection by TUNEL assay
Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining was conducted with the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Chemicon, Billerica, MA) in accordance with the manufacturer's instructions. The sections were then counterstained with 4,6-diamidino-2-phenylindole (DAPI). Images of TUNEL staining were taken with the EVOS FL Cell Imaging System (Life Technologies, Carlsbad, CA). All TUNEL-positive cells in the field were analyzed similarly as in the case of immunohistochemistry.

Statistical analysis
All data are expressed as means ± standard error (SE). Data analyses were performed by one-way and two-way ANOVA using Stata/SE 12.1 for Mac (Stata Corporation, College Station, TX). Statistically significant differences were considered at p < 0.05 and markedly significant differences were considered at p < 0.01.

Inhibitory effect and chemosensitivity of metformin on cell proliferation
To examine the effects of in vitro metformin treatment AMPK-mTOR signaling, Western blot analysis was performed. As a result, HCT116 and SW837 cells exhibited a decrease in p-mTOR levels that was inversely proportional to the increase in p-AMPK levels with metformin treatment (Fig. 1a, b), while HCT8 and Lovo cells showed no decrease in p-mTOR levels with metformin treatment (Fig. 1c, d). As shown in Fig. 2a and b, significant dose-dependent inhibition of proliferation was observed in HCT116 and SW837 cells treated with metformin. In contrast, no significant effect was observed on HCT8 or Lovo cell proliferation, regardless of the increasing doses of metformin, as shown in Fig. 2c and d. Metformin can phosphorylate AMPK and subsequently inhibit mTOR activity via the AMPK-mTOR pathway. This finding suggested that metformin sensitivity varies among CRC cell lines, and the anti-proliferative effect of metformin is mediated by the suppression of mTOR caused by AMPK phosphorylation. We hypothesized that the AMPK-mTOR pathway plays a critical role in the differences in reactivity between the sensitivity of CRC to metformin and its insensitivity to metformin.

Differential inhibitory effect of metformin on cell migration and invasion
We also tested the effect of metformin on the invasive behavior of CRC cells. In the cell invasion assay, metformin significantly reduced the invasion of metformin-sensitive HCT116 and SW837 (Fig. 3a). However, metformin-insensitive Lovo also showed a significant decrease in the number of invaded cells (Fig. 3b). Overall, metformin elicited greater inhibition of cell motility in metformin-sensitive CRC cells than in metformin-insensitive CRC cells.

Transient AMPK knockdown inhibits proliferation and migration of metformin-sensitive CRC cells
To assess whether the anti-cancer effects of metformin treatment resulted from activation of AMPK and inhibition of mTOR, we transfected metformin-sensitive CRC cells with AMPK siRNA or non-specific control siRNA and then treated them with metformin. Specific knockdown of AMPK by the corresponding siRNA was confirmed by western blot analysis and densitometric analysis. The levels of p-mTOR were elevated in HCT116 cell lines (Fig. 4a). In the proliferation and migration assay, metformin treatment significantly reduced the cell variability and wound healing in HCT116 control cells but not in HCT116 cells transfected with AMPK siRNA (Fig. 4b). Additionally, although the elevation of p-mTOR expression was not observed in SW837, transfection with AMPK siRNA decreased the anti-proliferative activity of metformin compared with nonspecific control siRNA (Fig. 4c). These findings suggest that inhibition of AMPK could abolish the anti-cancer effects of metformin in metformin-sensitive CRC cell lines. Therefore, it is suggested that activation of the AMPK-mTOR pathway in response to metformin plays an essential role in the anticancer effects of metformin against CRC.

Oral administration of metformin inhibits the liver metastasis of CRC cells in vivo
To examine whether metformin treatment of metformin-sensitive CRC cells affects growth and metastasis in vivo, we injected highly tumorigenic, metformin-sensitive HCT116 cells into SCID mice and monitored liver metastasis. As  Fig. 5, the number of mice with liver metastases was greater in the control group than in the metformintreated group. Liver metastases were less frequently observed in the metformin-treated group (metformin-treated group 10% vs. control group 50%, p = 0.05). Furthermore, the number of metastatic nodules was significantly smaller in the metformin-treated group (metformin-treated group 1.2/body vs. control group 0.1/body, p = 0.04). These findings suggested that metformin inhibits the development of liver metastasis in metformin-sensitive CRC xenografts in SCID mice.

Metformin therapy inhibited tumor proliferation and EMT and promoted apoptosis in vivo
At cellular level, metformin can inhibit cell proliferation and metastatic behavior. In order to further investigate the mechanism underlying the metformin-induced inhibitory effect of CRC metastasis in vivo, we evaluated cell proliferation, apoptosis and invasion, using Ki-67 labeling index, TUNEL assay and α-SMA immunohistochemistry, respectively, in Fig. 6a. The Ki-67 labeling index was significantly reduced in the metformin-treated group. Additionally, TUNELpositive cells were significantly increased, and α-SMAexpressing CRC cells were reduced in the metformin-treated group (Fig. 6b). These results suggested that metformin is able to inhibit cell proliferation and metastatic behavior in vivo.

Discussion
The findings of this study demonstrated that metformin induced the inhibition of liver metastasis, depending on the expression of phosphorylated mTOR. We found that the anti-cancer effects of metformin differed among different CRC cell lines. In this study, metformin inhibited the phosphorylation of mTOR via AMPK activation only in metformin-sensitive CRC cells. Additionally, metformin reduced cell motility by decreasing cell migration and invasion, thus inhibiting CRC metastasis. We also observed a significant decrease in liver metastasis in an animal xenograft model with metformin-sensitive HCT116 cells. These findings suggested that phosphorylation of mTOR via AMPK activation has an important role in the CRC metastasis inhibitory effect of metformin. The results of the current study provide fundamental evidence for a new therapeutic strategy with metformin in the treatment of CRC patients with metastatic liver disease.
Several studies investigating the clinical efficacy of metformin reported its preventive effect against CRC. Recently, a meta-analysis showed that metformin use significantly reduces colorectal adenoma and cancer incidence [20]. A phase 3 randomized control trial conducted in non-diabetic patients also demonstrated that metformin reduced the prevalence and number of metachronous colorectal adenomas or polyps [21]. Additionally, the synergistic effects of metformin with chemotherapeutic agents for CRC patients were also investigated. In a phase 2 trial, both metformin plus 5-FU and metformin plus irinotecan showed feasible anti-cancer effects in patients with refractory CRC [22,23]. However, conflicting results were also reported. Fransgard et al. reported that there was no association between metformin treatment and recurrence-free or disease-free survival after surgery for colorectal cancer in their registry-based study of 25,785 patients [24]. Another population-based study conducted in England also did not support a protective association between metformin and cancer-specific survival in colorectal cancer patients [25]. The synergistic effect of metformin was also questioned. A subgroup analysis of RCTs reported that no relationship was found between metformin use and postoperative survival of resected stage III colon cancer patients receiving adjuvant oxaliplatin-based chemotherapy (FOLFOX/XELOX) [26,27]. This study indicated that varying sensitivity for metformin treatment among CRC cell lines might be the cause of these conflicting results. However, the clinical efficacy of metformin in CRC treatment remains controversial. Hence, elucidation of the optimal indication and usage of metformin in CRC patients is urgently required.
To our knowledge, this is the first study demonstrating that metformin mediated inhibition of liver metastasis of CRC cells both in vitro and in vivo. On the basis of the results of this study, it is suggested that the anti-proliferative and anti-metastatic effects of metformin are associated with the inhibition of liver metastasis of CRC. Previously, the anti-proliferative effects of metformin combined with chemotherapeutics have been explored [28,29]. However, few studies have focused on the anti-metastatic effects of metformin monotherapy [30]. The results of this study, which demonstrated the anti-metastatic effects of metformin in a xenograft model of orthotopic liver metastasis, support the use of metformin in clinical practice. Further studies investigating the therapeutic potential of metformin for CRC liver metastasis are required.
The mTOR has gained attention as a potential therapeutic target for various types of cancers. At present, as a semisynthetic rapamycin analog, the mTOR inhibitor everolimus is widely accepted as the treatment option for patients with clear-cell renal cell cancer, pancreatic neuroendocrine tumor, and breast cancer. However, clinical trials have failed to demonstrate the clinical efficacy of mTOR inhibitors in   [31,32]. Perhaps it is due to multiple resistance mechanisms, including feedback of PIK3CA-AKT signaling which is another upstream signaling of mTOR [33,34]. For the mTOR targeting therapy for CRCs, the new approach to bypass the resistance mechanisms should be considered. Previously, it is well known that phosphorylated mTOR in turn regulates phosphorylation of numerous protein targets associates cell metabolism, including 4EBP1 and p70-S6K. [35,36]. In this study, we found that the anti-proliferative effect was a dose-dependent decrease in p-mTOR levels in inverse proportion to the increase in p-AMPK levels. It is suggested that the AMPK-initiated mTOR inhibition is important for the anti-proliferative effect of metformin on CRC cells. Importantly, we also found that the anti-proliferative effects of metformin varied among different CRC cell types. Evidence for sensitivity of metformin in CRC cell lines is still limited. Further investigation is needed for optimal patient selection in clinical application of metformin.
On the other hand, the distinct effect of metformin between metformin-sensitive and -insensitive CRC cell lines were not observed in invasion and migration assays. We speculate that there are anti-metastatic effects of metformin independent of the AMPK-mTOR pathway. This evidence also supported our hypothesis. Metformin can activate an AMPK-independent signaling pathway, which inhibits EMT through different mechanisms [37,38]. Additionally, it was also reported that metformin inhibits EMT in rectal cancer cells by suppressing the TGF-β pathway [39]. These AMPKindependent pathways may be the reason of contraindicated results in wound healing assay and invasion assay for Lovo cell line in current study. Our results indicated both AMPK-mTOR-dependent and -independent mechanisms have critical roles in the inhibitory effect of metformin in CRC cell The numbers of positive cells in each immunohistochemistry (microscopic quantitative analysis and ki-67 labeling index). Data are presented with the means ± standard error (n = 3). *p < 0.05 vs. control group [35,36] lines. Further investigation focused on the mechanisms of the anti-cancer effects of metformin is needed to identify CRC patients who can benefit from metformin treatment.
In this study, we first demonstrated that metformin inhibited CRC liver metastasis in vivo. Additionally, we found that decreased proliferation, increased apoptosis, and inhibition of EMT in liver metastasis are associated with the inhibitory effects of metformin. Previously, metformin showed inhibitory effects on the metastasis of CRC cells in several studies. Kang et al. reported that metformin reduced IL-6-induced EMT in colon cancer cell lines [40]. Another group showed that metformin decreases EMT in CRC cells by regulating the SNAIL-miR-34 and ZEB-miR-200 system [41]. However, the exact underlying mechanism of metformin in anti-metastatic regulation, especially in CRC, remains elusive. There are no published findings showing that metformin induces apoptosis in CRC, and the absence of cell mortality after treatment with the drug has previously been observed in prostate and breast cancer cells [42,43]. Nevertheless, we could observe a cytocidal effect of metformin in vivo that could be due to the microenvironment of liver metastasis. It is reported that CRC metastatic colonization of the liver occurs in the hypoxic microenvironment, and CRC cells have inadequate levels of ATP [44,45]. Additionally, metformin may induce apoptosis only in nutrient-poor conditions [46]. Taken together, it is plausible that metformin has cytocidal effects on CRC cells that are induced by nutrient-poor conditions at liver metastasis sites. However, further detailed experiments are required.
This study had several limitations. First, SCID mice lack an immune system, which is thought to play a critical role in both the initiation and progression of cancer metastasis and may also modify the response to metformin therapy. Thus, further investigation using mouse models with an intact immune system may be needed. Second, comparisons of metformin and other m-TOR inhibitors were not conducted in this study. Rapamycin, an original inhibitor of mTOR, and its rapalogs mainly inhibit mTORC1 activity and are classified as first-generation mTOR inhibitors. However, recent studies have shown that, unlike rapamycin, metformin not only prevents phosphorylation of mTORC1 complex components, but also inhibits mTORC2 complex components [47]. Dual inhibition of both mTORC1 and mTORC2 may lead to more effective inhibition of cancer cell proliferation than blocking mTORC1 alone [48]. Further studies exploring the anti-cancer efficacy of metformin in detail are needed. Finally, in this study, metformin was supplied by oral administration. Therefore, it is suspected that the concentration of metformin in vivo differed from the concentration in vivo.
In conclusion, our data indicate that metformin inhibits the metastasis of CRC by upregulating AMPK and inhibiting mTOR expression in vivo. This finding is extremely important because the main cause of mortality in patients with CRC is the development of metastasis and the scarcity of therapeutic options. Further experiments will focus on the underlying mechanisms of the anti-cancer effects of metformin and its potential clinical application for the management of CRC.