Cordyceps militaris Grown on Germinated Soybean Suppresses KRAS-Driven Colorectal Cancer by Inhibiting the RAS/ERK Pathway

Cordyceps militaris is a commonly used medicinal mushroom containing various therapeutic effects such as anti-inflammatory, anti-allergic, and anti-cancer activities. This study examined whether Cordyceps militaris on germinated soybeans (GSC) has a suppressive effect on a v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS)-driven colorectal cancer which is notorious for its un-druggable features and the ineffectiveness of conventional therapies against it. GSC extract was prepared and its proximate composition and amino acids were analyzed. The suppressive effects were investigated with the KRAS-driven colorectal cancer cell-line, SW480. SW480 proliferation, clonogenic potential, apoptosis, and the RAS/extracellular signal-regulated kinase (ERK) pathway under the GSC treatment were analyzed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, flow cytometry, and Western blot, respectively. An in vivo experiment with the SW480 xenograft mouse model was performed. As a result, GSC suppressed cell proliferation by inducing the apoptosis of KRAS-driven colorectal cancer cells and inhibited clonogenic capabilities. The decrease of KRAS and ERK phosphorylation was detected by Western blot. Tumor growth was significantly suppressed when GSC was introduced to the tumor-xenograft mouse model. In conclusion, GSC suppressed KRAS-driven colorectal cancer growth both in vitro and in vivo, and can be used as an alternative or simultaneous approach in colorectal cancer therapy.


Introduction
Colorectal cancer (CRC) is the third most prevalent cancer worldwide [1]. Approximately 1.7 million new cases were diagnosed in 2016 and 830,000 people were estimated to have died as a result [1]. In general, the incidence of CRC is high in developed countries compared to developing countries, but the cases are increasing worldwide due to nutritional transitions. The increasing prevalence has prompted extensive research into the development of therapies. On the other hand, 30% to 40% of CRC patients have a v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS)-driven CRC, which is difficult to target with drugs due to its notorious 'un-druggable' property

Clonogenic Assay
For a clonogenic assay, the cells were seeded onto a 6-well plate (1000 cells/well) [23]. After one day, the cells were treated with GSC extracts in different concentrations (1, 10, 100, and 500 µg/mL) and Cetuximab (30 µg/mL). After the 10-day cultivation, the cells were stained with a 0.05% crystal violet (Sigma) for 20 min at room temperature. The experiments were performed in triplicate.

Flow Cytometry
The extent of SW480 apoptosis after GSC treatment was evaluated by using an Annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection kit (BD Biosciences, San Jose, CA, USA), according to the manufacturer's instructions. SW480 colorectal cancer cells were seeded overnight in a 6-well plate (5 × 10 5 cells/well) and treated with Cetuximab (30 µg/mL) and the GSC extract (1-500 µg/mL). After 24 h, the cells were harvested, washed in ice-cold PBS, and collected by centrifugation at 500 g for 10 min. The cells were stained simultaneously with FITC-labeled annexin V (5 µL) and PI (5 µL) at room temperature for 10 min and protected from light. The stained cells were analyzed by using a fluorescence-activated cell sorter flow cytometer (FC 500 Series Flow Cytometry, Beckman Coulter, Indianapolis, IN, USA). At least 10,000 cells were used for each analysis and the experiments were conducted in triplicate.

Western Blotting
The cells were seeded in 6-well plates with a density of (5 × 10 5 cells/well) and treated with GSC (1-500 µg/mL) for 48 h. The cells were detached, washed in cold PBS, and re-suspended in RIPA buffer containing the protease inhibitors (Sigma). The suspension was kept on ice for 20 min and then centrifuged at 15,000× g for 30 min at 4 • C. The proteins (20 µg) were loaded onto each lane, separated on 10% pre-cast SDS-PAGE gel (Invitrogen), and followed by transfer in the iBlot system (Thermo Scientific). The membranes were blocked with a bovine serum albumin solution (Sigma) for 1 h at room temperature and incubated with the following primary antibodies. The antibodies for KRAS (1:1000), phospho-ERK1/2 (1:2000), ERK1/2 (1:1000), AKT (1:1000), phospho-AKT (1:1000) and HSP90 (1:1000) were obtained from Cell Signaling Technology (Danvers, MA). The antibody for the β-actin (1:1000) was obtained from Sigma. The membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies for 2 h (Pierce, Rockford, IL, USA). The blots were detected by using a SuperSignal ® West Femto enhancer kit (Pierce). Densitometry analysis was performed by using ImageJ (NIH, Bethesda, MD, USA). The ratios of KRAS/β-actin, phospho-pERK/ERK, and phospho-AKT/AKT were calculated with the control representing 1.0-fold changes, which is shown below.

Tumor Growth Analysis In Vivo
The inhibitory effects of GSC on colorectal cancer growth were investigated in an animal model. Twenty-one 6-week-old BALB/c athymic nude mice (male) were obtained from the BioToxTech animal center (Cheongju, Korea). Animal care and handling procedures were performed in compliance with the experimental protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Silla University (Busan, Korea). SW480 cells were harvested from the culture plate and re-suspended in PBS. A total of 5 × 10 6 cells in 0.1 mL of media were injected subcutaneously into both sides of nude mice [24]. When engrafted, the tumor volume was measured daily with calipers and calculated by using the following formula: V = (L × W 2 )/2, where V = volume, L = length, and W = width. When the tumors reached approximately 100 mm 3 in size, the mice were assigned randomly to their respective treatments (Cetuximab, GSC extract, and saline control, n = 7/group). The mice were treated twice per week with an intraperitoneal injection of 10 mg/kg Cetuximab in PBS. The GSC extracts were administered by an oral gavage (400 mg/kg/day) every day for 23 days.

Statistical Analysis
The data are expressed as the means ± standard error of the mean (SEM). Statistical analyses were performed by one-way analysis of variance (ANOVA) using SPSS software, version 12 (SPSS Inc., Chicago, IL, USA). The differences were considered significant at p < 0.05.

GSC Suppressed KRAS-Mutated Colorectal Cancer Cell Proliferation
SW480, which is the KRAS G12V -mutated colorectal cancer cell line, was chosen to evaluate the GSC antitumor potential. The cells were treated with GSC at different concentrations and Cetuximab as the drug control. Figure 1A presents SW480 colorectal cancer cell proliferation in the presence of GSC extracts at different concentrations (0, 1, 10, 100, and 500 µg/mL) on day 1 and 3. The cell growth was calculated as the relative percentage absorbance compared to the control. As a result, treating SW480 colon cancer cells with GSC suppressed cell proliferation in a dose-dependent manner when compared to the control. For example, the cell viability on day 3 was decreased by up to 54.2 ± 2.95% with the 100 µg/mL GSC treatment and 40.0 ± 2.2% with the 500 µg/mL GSC treatment. This led to the selection of doses for further mechanistic and molecular studies, which ranged from 1 to 500 µg/mL. For morphological observations, the cells were stained with PI and DAPI under the GSC treatment ( Figure 1B). Dead cells were stained with PI and were expressed as a red color. The cell nuclei from the dead and live cells were stained as DAPI (blue color). As shown in Figure 1B, most cells were only stained with blue DAPI in the control group, which showed that most cells were alive after 48 h of cultivation. On the other hand, the PI-stained dead cells began to appear and their ratios increased with a rise in the GSC concentration. In addition, the GSC extract fraction suppressed the proliferation on different types of KRAS mutations, e.g., LS513 (KRAS G12D ), HCT116 (KRAS G13D ), HT-29 (KRAS WT /BRAF Mut ), Caco-2 (KRAS WT /BRAF WT ) and A549 (KRAS WT /BRAF WT ) ( Figure 1C). In the KRAS WT /BRAF Mut HT-29 CRC line, the GSC extract also inhibited the proliferation, but had less effect on the proliferation at the same concentration ranges (1, 10, 100, and 500 µg/ mL) ( Figure 1C). As seen in Figure 1C, GSC did not show a suppressive effect on KRAS WT /BRAF WT Caco-2 CRC and A549 lung cancer cell proliferation. The IC 50 values on KRAS mutated CRC cell lines were 214 µg/mL (SW480), 493 µg/mL (LS513) and 441 µg/mL (HCT116) ( Figure 1D). IC 50 of HT-29 increased up to approximately 10-fold and IC 50 of KRAS WT /BRAF WT cells of Caco-2 and A549 was not applicable due to its minimal effect. Figure 1 demonstrated that KRAS-mutated CRC cell lines showed more sensitive responses under GSC treatment than the response of the KRAS wild-type cell line.

GSC Extract Alters Clonogenic Characteristics
The clonogenic potential was evaluated to determine the long-term anti-proliferative activity of GSC on colorectal cancer cells. The colony-forming ability is one of the tumor cell characteristics and is tightly correlated with tumorigenes in vivo. As shown in Figure 2A, the SW480 colonies disappeared or could not be formed at the highest GSC concentration. A similar tendency was also observed on other KRAS mutant cells known as LS513 (KRAS G12D ) and HCT116 (KRAS G13D ). The clonogenic potential of SW480 was suppressed significantly by GSC in a dose-dependent manner when compared to the controls ( Figure 2B). For example, the colony numbers were reduced by up to 72 ± 10% and 16 ± 8% after 100 and 500 µg/mL GSC treatment, respectively. In particular, the reductions in the colony number were accompanied by reductions of the colony size. Under Cetuximab treatment, the colony numbers were 93 ± 3%, which might indicate a cell resistant property to the drug in long-term incubation. In case of KRAS wild-type cells, the clonogenic potential was suppressed by GSC treatment. However, this was relatively low compared to the KRAS-mutated group. These findings suggest that the treatment of colorectal cancer cells with GSC would decrease their rate of proliferation and their tumor-formation ability.

Effects of GSC Extract on the RAS/ERK Signaling Pathways
KRAS-mutated colorectal cancer is related to the RAS/ERK or PI3K/AKT pathways. On CRC progression, mutated KRAS proteins are produced continuously, which activate ERK-mediated transcriptional up-regulation. To determine if the suppression of SW480 growth by GSC was regulated by the RAS/ERK pathways, the cells were treated with GSC for 48 h. In addition, the expression levels of the KRAS, p-ERK, ERK, p-AKT, and AKT proteins were analyzed by Western blot. As shown in Figure 4A,B, the GSC treatment significantly suppressed KRAS expression and ERK phosphorylation. The GSC treatment decreased ERK phosphorylation significantly in a concentration-dependent manner. For example, the ratio of pERK/ERK in the control (1.00 ± 0.00) was reduced up to 0.44 ± 0.13 in the GSC 500 µg/mL-treated group. An alternative downstream of KRAS, which is the PI3K/AKT pathway, was also observed. The level of AKT phosphorylation was similar in all samples to that observed with the pAKT/AKT ratio. Based on the result, the GSC treatment suppressed colon cancer progression, which was mostly mediated by the RAS/ERK signaling pathway.

The GSC Extract Inhibits Tumor Growth in a Xenograft Colorectal Cancer Model
To investigate whether the GSC extract inhibits tumor progression, the SW480 xenograft animal model was generated. Tumor formation was induced by injecting SW480 cells and administering GSC daily. As shown in Figure 5, GSC suppressed tumor growth, which was confirmed by the significant decrease in tumor volume compared to the PBS control (PBS control vs. GSC extracts, mean tumor volume on day 26 = 3724 ± 714 mm 3 vs. 2231 ± 581 mm 3 ). No significant anti-cancer activity was observed in the Cetuximab control group. The body weights of the mice showed no significant changes in all groups, which suggests that GSC feeding is safe and well-tolerated.

The GSC Extract Inhibits Tumor Growth in a Xenograft Colorectal Cancer Model
To investigate whether the GSC extract inhibits tumor progression, the SW480 xenograft animal model was generated. Tumor formation was induced by injecting SW480 cells and administering GSC daily. As shown in Figure 5, GSC suppressed tumor growth, which was confirmed by the significant decrease in tumor volume compared to the PBS control (PBS control vs. GSC extracts, mean tumor volume on day 26 = 3724  714 mm 3 vs. 2231  581 mm 3 ). No significant anti-cancer activity was observed in the Cetuximab control group. The body weights of the mice showed no

Discussion
Colorectal cancer (CRC) is the third most prevalent cancer and is becoming the leading cause of cancer death [1,2]. In recent years, attempts to improve the survival rates in CRC have focused on innovative therapeutic methods including EGF-receptor antibody therapy such as Cetuximab [4]. On the other hand, 30% to 40% of patients with KRAS-mutated colorectal cancer do not benefit from Cetuximab therapy [5][6][7]. Natural compounds could be an attractive option for cancer therapy as a result of their safety and their applications. GSC has therapeutic activity for inflammatory conditions, allergies, and cancer. The current study showed that GSC treatment could suppress KRAS-driven colon cancer both in vitro and in vivo. GSC showed enhanced antitumor activity based on the results of cell proliferation and clonogenic potential in multiple KRAS-mutated colorectal cancer cells such as SW480 (KRAS G12V ), LS513 (KRAS G12D ) and HCT116 (KRAS G13D ) (Figure 1). A less than 50% growth rate on KRAS-mutated CRC cell lines of SW480, LS513, and HCT116 was observed. In the KRAS WT cell line of HT-29, Caco-2 and A549 cell proliferation was approximately 80%, which indicated that GSC is more effective on KRAS-mutated colon cancer (Figure 1). Clonogenic potential showed a similar tendency. KRAS-mutated cell lines showed significant suppression on the clonogenic potential ( Figure 2). KRAS-mutated cell death was from the increasing apoptosis with GSC treatment (Figure 3). The RAS/ERK pathway study suggested that GSC could show an antitumor effect through the modulation of the ERK pathway by altering the phosphorylation of ERK (Figure 4). In a human tumor growth model, the growth of KRAS mutant xenograft tumors was suppressed significantly by the GSC treatment ( Figure 5).
The RAS/ERK signaling pathway and its downstream factors are recognized as key regulators for cellular behaviors including cell survival, proliferation, invasion, and migration [3]. During CRC progression, mutant RAS constitutively activates ERK phosphorylation. Therefore, this study examined their changes upon the GSC treatment and found decreased KRAS expression and phosphorylation of ERK proteins in SW480 cells (Figure 3).
Cordyceps militaris on germinated soybeans contains several bioactive compounds. In particular, isoflavone methyl-glycosides, cordycepin, adenine, and adenosine have been identified [19,20].  [19,21]. Genistein induced apoptosis on leukemia and colon cancer and inhibited breast cancer cell proliferation [25,26]. Cordycepin (a nucleoside derivative), which is a major active component of GSC, induces apoptosis in melanoma and lung carcinoma [27,28]. Furthermore, adenosine from GSC suppressed the growth of human gastric cancer cells significantly and the adenosine receptor 1 suppressed colon cancer proliferation [29]. The anti-tumor effects of GSC might come from the above functional compounds within the GSC.
In our current study, GSC contains relatively low Met 0.55 mg/g and high Arg 1.42 mg/g crude ( Table 2). Several studies have examined the effects of individual amino acids such as Met, Ser, Gly, and Arg on cancer progression. Met restriction was reported to inhibit cancer cell growth and to extend a healthy lifespan in several animal studies [30]. Met-depletion in cancer cells was reported to lead to cell cycle arrest. Deprivation of dietary Ser and Gly inhibits the growth of tumors by limiting nucleotide biosynthesis [31]. When Arg was provided at 1 mmol/L, it suppressed colon cancer by lowering the angiogenesis-related growth factors [32,33]. Relatively low Met 0.55 mg/g and high Arg 1.42 mg/g crude might play roles in suppressing colon cancer.

Conclusions
In conclusion, these observations suggest that GSC suppressed CRC progression with inhibitory effects, stopping the essential steps of tumor growth. GSC may be a powerful candidate for the development of a preventive agent for cancer growth. To the best of the authors' knowledge, this is the first study reporting that a GSC treatment suppresses KRAS-mutated colon cancer through the ERK-dependent pathways. Given the non-toxic nature of these natural food substances and their beneficial activities, these results provide support for the incorporation of the GSC extract into therapeutic regimens for colorectal cancer.