HCT116 cells were purchased from the National Collection of Authenticated Cell Cultures of the Chinese Academy of Sciences. HCT116 cells were routinely cultured in RPMI-1640 medium (product code C11875500BT; Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; cat no. S711-001S; Lonsera Science Srl), 100 U/ml penicillin and 100 U/ml streptomycin (Beyotime Institute of Biotechnology). Culture medium was changed once every 2–3 days. The cells were cultured in 5% CO₂ at 37°C, and the logarithmic growth-phase cells were used for experimental study. To study the effects of glutamine on cells, the cells were cultured overnight in ordinary medium (RPMI-1640 medium; product code C11875500BT; Gibco; Thermo Fisher Scientific, Inc.), then replaced with a culture medium without glutamine in RPMI-1640 medium (Beijing Solarbio Science & Technology Co., Ltd.) for 24 h for follow-up experiments. To study the effects of each inhibitor on cells, normal culture solution was used overnight and then replaced with RPMI-1640 culture solution containing 10, 100 nM, 1 and 10 µM of inhibitors, respectively. The culture solution was incubated for 12 h for subsequent western blot assay, 16 h for subsequent apoptosis detection, or the cell proliferation ability was assessed at 24, 48 and 72 h, respectively. A total of 35 female nude mice (GemPharmatech Co., Ltd.), aged 4–6 weeks and weighing 15–20 g, were raised in an individually ventilated cage (IVC) system without pathogens. The culture environment temperature was 26–28°C, and the relative humidity was 40–60%, with a 12-h light/dark cycle, no pathogens and free access to food and water. The Animal Welfare and Ethics Committee of Wannan Medical College (Wuhu, China) approved the study (approval no. LLSC-2020-139) and the experimental procedures carried out on mice were according to the guidelines for the Care and Use of Experimental Laboratory Animals of Wannan Medical College.

According to the PIK3CA gene sequences and synthetic guide (sg)RNA design principles in the sgRNA design website (http://chopchop.Cbu.Uib.No/), a total of 2 sgRNA for knockout of the PIK3CA gene were designed, then constructed in tandem into the lentiCRISPR-v2 vector (Streptococcus pyogenes Cas9 protein coding gene; Shanghai Ruantuo Biotech Co. Ltd. The test used an Applied Biological Materials, Inc. (abm) qPCR Lentivirus Titer kit (cat. no. LV900), to obtain an all in one lentiviral knockout vector expressing Cas9 and double sgRNA (Table I). The aim was to knock out PIK3CA; the knockout targets were exon 1 and exon 3 and the mutation type was a frameshift mutation (Fig. S1). After the vector was successfully constructed, it was packaged into a lentivirus (lentiCRISPR-v2 vector) and the 3rd generation system was used. The interim cell line used was 293T obtained from the National Collection of Authenticated Cell Cultures of the Chinese Academy of Sciences. The quantity of lentiviral plasmid used was 3.7 µg for transfection and the lentiviral plasmid: packaging vector: envelope was 1:1:1. The collection of lentiviral particles was 20 TU with a multiplicity of infection (MOI) of 10, along with the lentivirus packaging plasmid, and the titer was determined after purification. Then, cell transfection and screening were performed. When HCT116 cells grew to the logarithmic phase, they were inoculated into a 6-well cell culture plate at a density of 2.5×10⁵ cells/well. After the cells adhered overnight, the viral volume was added according to the MOI value (MOI=10) to infect cells for 12 h, and then, fresh culture solution was replaced and the culture continued for 24 h. The infected cells were screened with 0.3 µg/ml puromycin and expanded, and then the A3140G mutant version of PIK3CA was inserted into the cells (Fig. S1). According to the PIK3CA gene sequences and the mutation site information, the primers were designed for the gene of CDS A3140G (H1047R) with point mutations in exon 20, and PAM loci of two sgRNA synonymous mutations (Table I), augmented with PAM mutations and an A3140G point mutation of the PCR fragments, and then merged with objective mutations and the PAM mutations, concurrently, at both ends with AgeI and SalI enzyme loci. After successful construction, the vector was packaged together with lentiviral plasmid, and the titer was determined after purification. When the HCT116 cells grew to the logarithmic phase, the cells were inoculated into a 6-well cell culture plate at a density of 2.5×10⁵ cells/well. After the cells adhered to the wall overnight, the viral volume was added according to the MOI value (MOI=10), and the cells were infected for 12 h. The medium was changed with fresh medium and then culture continued for 24 h. The infected cells were then screened with 0.3 µg/ml puromycin and expanded for use in subsequent experiments.

HCT116 or MUT cells were cultured to a density of 1×10⁶ cells and seeded in 6-well plates. Cells adherent overnight, were then treated with the following inhibitors: PI3K inhibitor (taselisib; cat. no. HY-13898; MedChemExpress), MEK inhibitor (U0126-EtOH; cat. no. HY-12031; MedChemExpress), PDK1 inhibitor (GSK2334470; cat. no. HY-14981; MedChemExpress), AKT inhibitor (GSK-690693; cat. no. HY-10249; MedChemExpress)] for 12 h at 37°C. The old medium in each well was discarded and the cells were washed 3 times with precooled PBS. A total of 200 µl of western and IP cell lysate (cat. no. P0013; Beyotime Institute of Biotechnology) was added to the wells and incubated on ice for 30 min. The lysates from each well were collected and centrifuged at 14,300 × g for 10–15 min at 4°C to collect the supernatant. Protein concentration was assessed using a BCA protein assay kit (cat. no. P0012S; Beyotime Institute of Biotechnology). The equivalent amounts of protein (50 µg) of each sample were loaded on 10% or 12% SDS-PAGE gels. After separation, the protein was transferred to a PVDF membrane (cat. no. IPVH00010; EMD Millipore) at 350 mA for 100 min. Then, the membrane was blocked with 5% skimmed milk at room temperature for 1 h, washed 3 times with TBST (containing 0.1% Tween-20) for 5 min each time, and incubated overnight at 4°C with the corresponding primary antibody: GAPDH (product no. 5174T), PIK3CA-N (product no. 4249S), P-Rex1 (product no. 13168S) from Cell Signaling Technology, Inc.; PIK3CA-F (cat. no. A12484), MEK (cat. no. A4868), PDK1 (cat. no. A0834), AKT (cat. no. A18120), RSK2 (cat. no. A0177), ATF4 (cat. no. A0201), GPT2 (cat. no. A11819) from ABclonal Biotech Co., Ltd.) all diluted 1:1,000. After incubation with horseradish peroxidase conjugated goat anti-mouse IgG (cat. no. A0216; 1:10,000 dilution) and goat anti-rabbit IgG (cat. no. A0208; 1:10,000 dilution); both from Beyotime Institute of Biotechnology) at 1.5 h at room temperature, an ultra-sensitive ECL chemiluminescence kit (cat. no. P0018S; Beyotime Institute of Biotechnology) was used for visualization. Chemiluminescence imaging system exposure, ImageJ software (version 1.52; National Institutes of Health), was used for analysis to determine the expression levels of related proteins.

HCT116 cells or MUT cells were inoculated into a 96-well cell culture plate at a density of 3×10³ cells/well. After the cells had adhered to the wall overnight, they were exposed to inhibitors at concentrations of 10, 100 nM, 1, 10 µM or culture medium without glutamine for 24, 48, or 72 h. The old culture medium was discarded, washed twice with PBS solution, and the fresh culture medium was added. A total of 10 µl Cell Counting Kit (CCK)-8 working solution (cat. no. C0037; Beyotime Institute of Biotechnology) was added to each well, shaken evenly, bubbles were avoided, and incubated for 1 h at 37°C and in atmosphere containing 5% CO₂. Then the absorbance was measured using a Multiskan™ GO plate reader (Thermo Fisher Scientific, Inc.) at 450 nm with an enzyme micrometer.

HCT116 cells or MUT cells were inoculated into a 12-well cell culture plate at a density of 5×10⁵, and then exposed to the following inhibitors: PI3K inhibitor (taselisib; cat. no. HY-13898; MedChemExpress), MEK inhibitor (U0126-EtOH; cat. no. HY-12031; MedChemExpress), PDK1 inhibitor (GSK2334470; cat. no. HY-14981; MedChemExpress), at concentrations of 10, 100 nM, 1, 10 µM for 16 h. HCT116 cells were digested with trypsin without EDTA and collected, washed twice with precooled PBS, centrifuged at 158 × g (4°C for 5 min), and resuspended in 500 µl staining buffer. HCT116 cells were stained with propidium iodide (PI) and Annexin V-FITC (Nanjing KeyGen Biotech Co., Ltd.) at 4°C for 15 min to evaluate apoptosis. A total of 1×10⁴ stained cells were collected and detected by CytoFLEX (Beckman Coulter Biotechnology Co., Ltd.) within 1 h, and their apoptosis was analyzed using FlowJo v. 7.6.1 (FlowJo LLC).

Total RNA from HCT116 and MUT cells was extracted using an RNA extraction kit (cat. no. DP430, Tiangen Biotech Co., Ltd) according to the manufacturer's instructions, and RNA was reverse transcribed to cDNA using RevertAid First Strand cDNA Synthesis Kit (cat. no. K1622; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Then, PIK3CA and GPT2 were amplified using cDNA as a template to evaluate the expression of PIK3CA and GPT2 in HCT116 and MUT cells. The thermocycling condition were as follows: Initial denaturation at 95°C, 3 min; 30 cycles of denaturation at 95°C, 30 sec, annealing and elongation at 72°C, 60 sec; and final extension at 72°C, 5 min. Hefei General Biotechnology Co., Ltd. was commissioned to follow the primer design principles and design and synthesize specific primers for PIK3CA (annealing 52°C; 30 sec), GPT2 (annealing 54°C; 30 sec) and GAPDH (annealing 62°C; 30 sec). The primer sequences are listed in Table II. Finally, ImageJ software 1.52 was used for analysis to determine the expression level of relevant RNA.

A total of 3×10⁶ HCT116 cells, 3×10⁶ MUT cells in a volume of 0.1 ml or 1×10⁷ MUT cells (26,27) in a volume of 0.2 ml serum-free medium were injected subcutaneously into 4-week athymic nude mice near the right hind leg. Once tumors reached an average size of 200 mm³, nude mice treated with 3×10⁶ MUT cells were randomly divided into 5 groups: i) PBS, ii) 5 mg/kg of PI3K inhibitor, iii) 20 mg/kg of PDK1 inhibitor, iv) 10 mg/kg of MEK inhibitor or v) combined application of 20 mg/kg of PDK1 and 10 mg/kg of MEK inhibitor. PBS or inhibitors were injected intraperitoneally every day for 10 days. Tumor volume was measured every 2 days. The maximum tumor size observed in our study was 16.32×15.46 mm, and the maximum volume was 1950.33 mm³. In each individual experiment, when the tumor volume reached 2,000 mm³ or on the 15th day after tumor implantation, mice were sacrificed by cervical dislocation. Animal experiments were approved by the Animal Welfare and Ethics Committee of Wannan Medical College (Wuhu, China). The experimental procedures were carried out according to the guidelines for the Care and Use of Experimental Laboratory Animals of Wannan Medical College.

All data were expressed as the mean ± SD and the experiments were repeated 3 times. The experimental data were first analyzed using one-way analysis of variance (ANOVA). When the variance was equal, the significance of the difference between the two groups was analyzed. When the variances were not equal, the differences between the two groups were analyzed by Dunnett's T3 test. This analysis was followed by Bonferroni post hoc test. The experimental data which observed cells before and after mutation were analyzed using t-tests. The tumor volume differences were analyzed using a mixed two-way ANOVA, and then Bonferroni post hoc test was used for analysis. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed with SPSS 19.0 software (IBM Corp.).

In order to construct PIK3CA-mutated HCT116 cells, knockout and mutant vectors were first constructed (Fig. 1A); then, targeted insertion of the PIK3CA gene through CRISPR/Cas9 method was performed to destroy the function of the original gene, and finally overexpression of the constructed point-mutated plasmid into the HCT116 cells was carried out (Fig. 1B). Sequencing results revealed that MUT cells constructed by our research group had been mutated at this site, and mutation of A3140G occurred at this site (Figs. 1C and S2). Moreover, it was detected by western blotting and RT-qPCR experiments that PIK3CA protein (PIK3CA-N) or mRNA expression in MUT cells and the KO cells was increased compared with that in HCT116 cells, while the corresponding GAPDH protein expression was not altered (Fig. S2). The antibody that binds to the mutation site (PIK3CA-F) was assessed using western blotting. The results revealed that no band was detected in the MUT cells and KO cells (Figs. 1D and S2).

In order to determine whether PIK3CA mutation had an effect on the proliferation ability of HCT116 and MUT cells, proliferation assays were performed. It was revealed that under normal culture conditions, the proliferation ability of MUT cells was significantly enhanced compared with that of normal HCT116 cells (P<0.01) (Fig. 2A). Subsequently, suboptimal conditions with low serum (5% FBS) culture were investigated by detecting apoptotic differences between HCT116 and MUT cells.

It was revealed by flow cytometric analysis that under the condition of low serum culture, containing the 5% FBS, the apoptotic cell rate significantly decreased in MUT cells compared with HTC116 cells; at a 5% FBS concentration, the HCT116 cell apoptotic rate was 9.24% and the MUT cell apoptotic rate was 3.06%, which was a significant difference (P<0.01) (Fig. 2B). In order to further investigate whether the increased proliferative ability of HCT116 cells and MUT cells was due to the dependence on glutamine, the cells were cultured without glutamine and the changes were studied by detecting cell proliferation and apoptosis. Cell proliferation was detected using a CCK-8 assay, and it was revealed that the proliferation ability of HCT116 cells was almost unchanged, and the proliferation ability of MUT cells cultured without glutamine was weakened compared with normal culture (Fig. 2C). Flow cytometric detection revealed that the cell apoptotic rate was significantly increased after the mutation in the glutamine-free culture condition, and the apoptotic rate of the HCT116 group was 5.76%, and that of MUT group was 8.05%, revealing a significant difference between the two groups (P<0.01) (Fig. 2D). Western blotting and RT-qPCR were used to detect the expression of GPT2 protein in HCT116 and MUT cells, and it was revealed that the expression of GPT2 in MUT cells was significantly higher than that in HCT116 cells (P<0.01) (Fig. 2E).

In order to study the influence of PI3K inhibition on its downstream signal transduction molecules, the PI3K inhibitor was used to detect the changes of other signaling pathway molecules using western blotting. After the inhibition of PI3K, the expression levels of RSK2, ATF4 and GPT2 proteins were significantly downregulated. Among these, the inhibition levels of ATF4 and GPT2 were the most significant and dose-dependent (Fig. 3). With the increase of the dose to 100 nM, the inhibitory effect was more evident. In addition, the expression of PDK1, MEK, and AKT significantly decreased under the effect of 10 µM inhibition. Western blotting results revealed that when the dose of the PI3K inhibitor reached 100 nM, compared with the control group, the expression levels of all proteins were decreased (P<0.01). When the dose of PI3K inhibitor reached 1 µM or higher, the expression of all protein levels exhibited a statistically significant decrease compared with the control group (P<0.01).

In order to study the effect of PDK1 and MEK inhibition on the downstream signal transduction molecules, PDK1 and MEK inhibitors were used to detect the changes of other signaling pathway molecules with western blot analysis. After addition of PDK1 and MEK inhibitors, the protein expression levels of RSK2, ATF4, and GPT2 were significantly decreased, and the inhibition effect was significant between the 10-µM group and the control group (P<0.01) (Fig. 4A and B). However, the inhibition of AKT did not decrease the expression levels of RSK2, GPT2, and ATF4 (Fig. 4C). Moreover, it was revealed that PDK1 and MEK did not affect the protein expression of each other. When the inhibitor concentration reached 10 µM, other signaling pathway molecules(MEK, PDK1 or AKT) did not significantly decrease and it was revealed that there was no compensatory relationship between the three signaling pathways.

To detect the expression levels of P-Rex1 signal transduction molecule in HCT116 cells and MUT cells, western blotting was performed. The results revealed that the expression of this protein in MUT cells was significantly enhanced (Fig. 5A; P<0.01). Then, P-Rex1 expression was detected in MUT cells treated with PI3K, PDK1 and MEK inhibitors at 0 (control), 10, 100 nM, 1 and 10 µM, respectively. When PI3K inhibitor was used, the expression of P-Rex1 protein was significantly suppressed in a dose-dependent manner (P<0.01) (Fig. 5B). When PDK1 and MEK inhibitors were used, P-Rex1 did not decrease (Fig. 5C and D).

To verify the changes in cells after H1047R mutation, CCK-8 and flow cytometric assays were used to detect cell proliferation and apoptosis, respectively. The results revealed that the inhibition of PI3K, PDK1 or MEK signaling pathway molecules had an evident inhibitory effect on the proliferation of MUT cells (Fig. 6A-C). Our results revealed that when the PI3K, PDK1 or MEK inhibitor was applied, the proliferation of MUT cells was inhibited. The absorbance when PI3K, MEK or PDK1 inhibitor was used, decreased by 64, 35 and 67%, respectively (Fig. 6A-C). Next, the effects of each inhibitor on the apoptotic rate of the cells was detected, and the results revealed that PI3K, PDK1 and MEK inhibitors increased the apoptotic rate of MUT cells. The apoptotic rate when PI3K inhibitor was used increased from 2.12 to 5.60% when the inhibitor dose reached 10 µM. When PDK1 inhibitor reached 10 µM, the apoptotic rate of MUT cells increased from 1.06 to 3.28%. For the MEK inhibitor, the apoptotic rate increased from 4.79 to 10.98% at the 10-µM dose. Compared with the control group, the apoptotic rate of each group was statistically significant (P<0.01) (Fig. 6E-G). Due to the strong inhibitory effect of PI3K on MUT cells and high cytotoxicity, combined PDK1 and MEK inhibitors were considered. CCK-8 and flow cytometric results revealed that combined inhibition of PDK1 and MEK slowed down the proliferation of MUT cells, reducing the absorbance ~78% (Fig. 6D), and the apoptotic rate increased from 1.63 to 8.01% (Fig. 6H). It is worth noting that when PDK1 and MEK inhibitors were combined, no significant inhibitory effect on proliferation was observed when small concentrations were used on the cells. When the concentration of the inhibitors combined reached 1 µM at 72 h, a slight inhibitory effect on the proliferation of cells was exhibited. When the concentration of the inhibitors combined reached 10 µM at 72 h, the absorbance was significantly decreased. These results revealed that the combined application of PDK1 and MEK inhibitors had a strong suppressive effect on cell proliferation.

In order to evaluate the growth of HCT116 and MUT cells in vivo and the effects of each inhibitor in vivo, xenograft studies were further conducted. The results revealed that the tumor growth rate of nude mice inoculated with MUT cells was faster than that of HCT116 cells. The nude mice inoculated with 1×10⁷ MUT cells exhibited tumor growth first, followed by nude mice inoculated with 3×10⁶ MUT, and finally HCT116 cells. Moreover, the tumor growth rate of nude mice inoculated with MUT cells significantly increased in the later stage (Fig. 7A). PI3K, MEK inhibitor or combined application of PDK1 and MEK inhibitors could effectively slow down the growth rate of tumors (P<0.05). However, there was no statistical difference between these 3 groups. The tumor growth rate of nude mice treated with PDK1 inhibitor also slowed down to a certain extent, but individual differences were great and there was no statistical difference (Fig. 7B).