PPP2R1B abolishes colorectal cancer liver metastasis and sensitizes Oxaliplatin by inhibiting MAPK/ERK signaling pathway

We obtained 100 pairs of fresh CRC tissues and corresponding adjacent nontumor colorectal tissues from patients (57 men and 43 women) with a median age of 64.7 years (range, 41 to 80 years). These samples were collected after CRC surgery from the Department of Gastrointestinal Surgery, the First Hospital of China Medical University. All specimens were pathologically confirmed to be CRC, and the data were categorized according to the 8th UICC guidelines. Informed consent was acquired from some patients, while others were exempted from providing informed consent given the assurance of anonymity. This procedure was approved by the ethics committee of the First Hospital of China Medical University (2023[95]). This study adhered to the guidelines set by the Declaration of Helsinki.

The mRNA expression data, excluding the expression of PPP2R1B in the extremities, and clinicopathological annotations of 362 CRC patients were extracted from The Cancer Genome Atlas (TCGA) database (http://​cancergenome.​nih.​gov), which includes information on tumour samples and paracancerous samples with detailed information for further analysis. Additionally, data for 4 primary tumour and 3 liver metastasis samples from CRC patients (GSE179979) with detailed characteristic information were obtained from the GEO database (https://​www.​ncbi.​nlm.​nih.​gov/​geo/​).

Five human CRC cell lines (HCT116, SW480, HCT8, RKO and LOVO) were procured from a cell bank at the Chinese Academy of Sciences (Shanghai, China), one normal colon cell line (NCM460) was acquired from the BeNa Culture Collection (Beijing, China), and one murine MC38 cell line was purchased from the National Infrastructure of Cell Line Resource (Beijing, China). The cells were cultured in the recommended growth media supplemented with 10% foetal bovine serum (FBS; HyClone, Logan, UT, USA) and 100 U/m penicillin or streptomycin. These cells were maintained in a humidified chamber containing 5% CO2 at 37 °C. PD98059 (2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran4-one), a non-ATP competitive selective MEK1/2 inhibitor that specifically inhibits MEK-1-mediated activation of MAPK, was purchased from Sigma Chemical Co. (St. Louis, USA), and Oxaliplatin was purchased from Selleck (Shanghai, China). The cells were treated with PD98059 (10 µM for 24 h) or Oxaliplatin (10 µM for 24 h).

Total RNA was extracted from fresh CRC cell lines and colorectal tissue preserved in liquid nitrogen using TRIzol (Takara, Japan) according to the manufacturer’s instructions. The concentration and purity of the RNA were determined using a NanoDrop 1000 (Thermo Scientific, USA). The expression levels of both PPP2R1B and GAPDH were measured using QuantStudio 3 (Applied Biosystems, USA). Reverse transcription and amplification were performed using a kit from Takara. The thermocycling conditions were set to 95 °C for 30 s, followed by 45 cycles of 95 °C for 5 s and 60 °C for 34 s.

Total protein was extracted from CRC cell lines and tissues using RIPA lysis buffer supplemented with 1% PSMF and 1% protease inhibitor. The protein samples were separated on 10% sodium dodecyl sulfate‒polyacrylamide gels and subsequently transferred to PVDF membranes. These membranes were blocked with 5% nonfat milk for 2 h, followed by incubation with the following antibodies: rabbit PPP2R1B at a dilution of 1:1000 (Proteintech Group, China), rabbit p-ERK at 1:1000 (Abmart, China), rabbit ERK at 1:1000 (PTMBIO, China), rabbit E-cadherin at 1:1000 (Proteintech Group, China), rabbit ZEB1 at 1:1000 (Proteintech Group, China), rabbit Snail at 1:500 (Proteintech Group, China), and mouse GAPDH at 1:3000 (Proteintech Group, China). The following day, the membranes were incubated with their corresponding secondary antibodies at 1:10,000 (Proteintech Group, China) for 2 h. Each experiment was conducted in triplicate.

After deparaffinization and rehydration, 4 µm sections were stained with haematoxylin solution for 6 min, followed by 8 s in 1% acid ethanol (1% HCl in 70% ethanol) and rinsing in distilled water. The sections were subsequently stained with eosin solution for 3 min, dehydrated with graded alcohol and cleared in xylene. Finally, images were captured using a microscope (Nikon, Japan).

The expression of PPP2R1B and p-ERK was assessed using immunohistochemistry (IHC). 4 µm sections were processed according to the protocol outlined by the IHC Biotin Block Kit (MXB Biotechnologies, Fuzhou, China), as described by Tang et al. [14]. Initial evaluations of staining were conducted at low magnification (×10) and subsequently confirmed at higher magnifications (×20). The expression of the proteins was scored by both the percentage of positive cells and staining intensity. Staining heterogeneity was graded as follows: 0 (≤ 5%), 1 (6–25%), 2 (26–50%), or 3 (> 51%). Based on these scores, PPP2R1B and p-ERK expression levels were computed. Two independent pathologists critically evaluated the final scores.

Coimmunoprecipitation was performed as described in a previous study [15]. The lysis buffer was composed of 20 mM Tris/HCl (pH 7.4), 1.0% NP40, 150 mM NaCl, 1 mM EDTA, 10 μg/mL leupeptin, and 50 μg/mL PMSF. Using this lysis buffer, we extracted protein from SW480 cells (treated with or without Oxaliplatin). Antibody beads were preincubated with magnetic beads (Bio-Rad, Hercules, CA, USA). Additionally, 200 μl of rabbit PPP2R1B antibody (Proteintech Group, China) or IgG (Santa Cruz, Japan) at a 1:100 dilution was incubated with the SW480 total protein at 4 °C overnight. Western blotting was then performed to visualize the immunoprecipitate samples separated using magnetic beads the next day.

The siRNAs targeting PPP2R1B, as well as the negative control (NC), were obtained from GenePharma (Shanghai, China). The specific sequences used were as follows: The PPP2R1B plasmid was synthesized by Genechem (Shanghai, China). HCT116 and SW480 CRC cells were transfected with PPP2R1B si1, PPP2R1B si2, negative control (NC-siRNA) or the PPP2R1B plasmid using jetPRIME reagent (Polyplus, France) in accordance with the provided guidelines. After 48–72 h of incubation, the effects of siRNA transfection or overexpression were assessed via Western blotting.

In brief, transfected HCT116 and SW480 cells were seeded onto membrane inserts with 8.0 μM pore size in 24-well plates filled with FBS-free growth media. Growth media containing 10% FBS served as a chemoattractant in the bottom wells. After 24 h, nonmigrated cells on the top side of the inserts were removed using a cotton swab. Cells that migrated to the underside of the inserts were stained with crystal violet hydrate (Solarbio, China) following the manufacturer’s guidelines. An invasion assay was similarly conducted, and the cells were evaluated using modified Boyden chamber assays (BD Biosciences, USA). Migrated cells were counted under a microscope at × 20 magnification, and images were captured using a microscope (Nikon, Japan). Cells were quantified in five random fields per insert, and the results are presented as the number of migrated cells per field.

Transfected HCT116 and SW480 cells were seeded in 6-well plates. Once the cells reached 80% confluence, the bottom of the plate was gently scraped using a 200 µl pipette tip. The plate was then washed with PBS three times, and images were captured at 0 and 24 h using a microscope (Nikon, Japan) at a 10 × magnification.

Pathway scores were calculated using the gene set variation method (GSVA) followed by ordinal regression [16]. The GSVA score was generated according to the mRNA expression level. To identify PPP2R1B-related pathways, we used the pathway score generated by GSVA in conjunction with the PPP2R1B expression level in the same dataset.

We mainly used a ridge regression-based method of the R package oncoPredict (version 0.2) to predict the IC50 AUC values of potential drug responses which bridges the in vitro drug screening with in vivo drug and biomarker discovery [17]. The Oxaliplatin sensitivity of the transfected CRC cells was detected using CCK-8 assays. First, cells that were seeded in 96-well plates (Servicebio, Wuhan, China) were incubated with 10 µM Oxaliplatin (Selleck, China) for 24 h. Next, 10 µl of CCK-8 reagent (Dojindo, Kumamoto, Japan) was added, and the cells were incubated at 37 °C for 2 h. Finally, the absorbance was measured at 450 nm in an ELISA 96-well microtiter plate reader (Bio-Rad 680, California, USA).

Shotgun LC‒MS/MS was performed by Genechem (Shanghai, China). The protein solution was digested by protease, after which the peptides were obtained. The peptides were then separated using high-performance liquid chromatography (HPLC) and added to a high-resolution mass spectrometer for analysis. The use of retrieval software in conjunction with the corresponding proteome database for the analysis of mass spectrum data enables the visualization of proteome information from the sample.

Experiments involving mice were approved by the Ethics Committee of China Medical University. For the liver metastasis model, 6-week-old C57BL/6 mice were purchased from Liaoning Changsheng Biotechnology (Benxi, China). A small incision was made in the left abdominal flank to expose the spleen. MC38 cells transfected with NC-siRNA or PPP2R1B-siRNA1 (1 × 10⁶) suspended in prechilled PBS (100 µl) were injected into the inferior pole of the spleen. Twenty-one days later, the mice were euthanized, and liver tissues were harvested. Tumours were measured using Vernier callipers. Subsequently, liver metastatic tumours were identified using haematoxylin and eosin (HE) staining and detected via Western blotting.

Continuous data are presented as the mean ± SE. For two groups, Student’s t test was used to evaluate differences in qRT‒PCR, WB, cell migration and invasion, wound healing and liver metastasis. Nonparametric and Spearman correlation tests were used for IHC analysis of human CRC samples. The associations of target protein expression with clinicopathological data were analysed by the chi-square test or Fisher's exact test. The Kaplan‒Meier curve was used to estimate survival, and differences were analysed by the log-rank test. R language v4.2.2 (https://​www.​r-project.​org/​) and GraphPad Prism 5.0 software were used for data analysis. P values were adjusted for multiple comparisons when necessary. P value < 0.05 was considered to indicate statistical significance.

Initially, we found that PPP2R1B is the only gene that is both ectopically expressed in liver metastasis tissues (GSE179979) and related to patient survival in the TCGA cohort (Venn diagram, Fig. 1A), which suggests that PPP2R1B may play an important role in the metastasis of CRC. Hence, we selected PPP2R1B for further investigation; we also found that it was negatively associated with survival in the GDC TCGA colon cancer (COAD) cohort (Fig. 1B). To validate the bioinformatic findings, we utilized qRT‒PCR and IHC to evaluate PPP2R1B expression in colorectal adjacent tissues, CRC tissues and liver metastasis tissues. Our findings revealed that PPP2R1B is expressed at lower levels in cancerous tissues and is expressed at even lower levels in liver metastasis tissues at the mRNA and protein levels (Fig. 1C, D). As presented in Table 1, there was a negative correlation between PPP2R1B expression and both TNM stage and distant metastasis (Table 1, p = 0.02, p = 0.03). To further investigate the biological function of PPP2R1B through in vitro experiments, we evaluated its expression in five CRC cell lines and one normal intestinal epithelial cell line. Notably, qRT‒PCR and Western blotting revealed that PPP2R1B was highly expressed in SW480 cells but was expressed at low levels in HCT116 cells (Fig. 1E, F). Based on these findings, we selected the SW480 and HCT116 cell lines for subsequent experiments. Finally, we examined PPP2R1B and p-ERK protein expression in 6 pairs of CRC tissues and liver metastasis tissues, and the Western blotting results showed that PPP2R1B was expressed at lower levels in liver metastasis tissues than in CRC tissues and was negatively associated with p-ERK (Fig. 1G).

CRC metastasis results in poor survival in CRC patients [3]. We performed invasion, migration, and wound healing assays to verify the effects of PPP2R1B on CRC cell invasion and migration in two CRC cell lines. The number of migrated cells and the changes in gap distance in the wound healing assay indicate CRC cell invasion and migration ability. The results showed that silencing PPP2R1B promoted CRC cell invasion and migration in SW480 cells (Fig. 2A, B). Moreover, overexpression of PPP2R1B impaired CRC cell invasion and migration in HCT116 cells (Fig. 2C, D).

The mechanism by which PPP2R1B inhibits CRC metastasis was explored. First, through GSVA analysis, we found that PPP2R1B is negatively related to the MEK signalling pathway, which indicates that PPP2R1B expression decreases with the enrichment of this pathway (Fig. 3A). Moreover, mass spectrometry indicated that the PPP2R1B protein binds to the ERK protein (Additional file 1: Table S1). PPP2R1B may act as an inhibitor of the MAPK/ERK signalling pathway, which is associated with EMT and plays an important role in the migration and invasion of CRC cells and in the distant metastasis of CRC [18].

Subsequently, protein immunoprecipitation (co-IP) assays were performed on SW480 cells treated with or without Oxaliplatin. The results showed that PPP2R1B could bind to p-ERK in SW480 cells. Moreover, Oxaliplatin promoted the interaction between these two proteins (Fig. 3B).

Finally, we detected ERK, p-ERK, E-cadherin and other EMT-related markers by Western blotting. According to the PPP2R1B expression pattern in CRC cell lines, we inhibited PPP2R1B expression in SW480 cells and increased PPP2R1B expression in HCT116 cells. We found that the expression of p-ERK, ZEB1 and Snail increased and that the expression of E-cadherin decreased when PPP2R1B was silenced in SW480 cells, while the expression of ERK did not change (Fig. 3C). Moreover, compared with PPP2R1B silencing, PPP2R1B overexpression had the opposite effect on p-ERK and the protein expression of ZEB1, Snail and E-cadherin in HCT116 cells (Fig. 3D).

To further assess whether PPP2R1B could regulate the MAPK/ERK signalling pathway, we inhibited CRC cell metastasis. Transfected SW480 cells were incubated with PD98059, which is a MEK1/2 inhibitor, to decrease the phosphorylation of ERK. We found that PD98059 reversed the changes in the p-ERK, ZEB1, E-cadherin, and Snail proteins induced by PPP2R1B silencing in SW480 cells (Fig. 4A, B). In addition, PD98059 significantly reversed the cell invasion and migration regulated by PPP2R1B silencing. Specifically, compared with those in the PPP2R1B-siRNA1 group, the number of migrating and invading cells was markedly lower than that in the PPP2R1B siRNA1 plus PD98059 group (Fig. 4C, D). Moreover, compared with that in the PPP2R1B siRNA1 plus PD98059 group, the gap area in the PPP2R1B siRNA1 group was much larger (Fig. 4E, F). Taken together, these findings suggest that PPP2R1B inhibits CRC cell metastasis via the ERK/MAPK signalling pathway.

EMT not only regulates tumour cell metastasis but is also associated with drug resistance [9, 10]. Therefore, we utilized the R package oncoPredict (version = 0.2) to calculate 198 drug responses. Oxaliplatin and 5-fluorouracil (5-FU) are standard chemotherapy agents for CRC [19, 20]. We found that the PPP2R1B mRNA level was significantly related to the Oxaliplatin IC50 (Fig. 5A, p = 0.0092) but not to the 5-FU IC50 (p = 0.21). Therefore, we incubated NC-siRNA- or PPP2R1B siRNA-transfected cells with Oxaliplatin for 24 h. After treatment with 10 µM Oxaliplatin, the protein levels of PPP2R1B and E-cadherin were significantly elevated compared with those in the group without Oxaliplatin treatment. However, the protein levels of p-ERK and Snail decreased in the Oxaliplatin treatment group, while the levels of ZEB1 and ERK did not significantly change.

Furthermore, the CCK-8 assays showed that silencing PPP2R1B decreased the sensitivity of SW480 cells to Oxaliplatin (Fig. 5E). Moreover, PD98059 reversed the increase in Oxaliplatin chemosensitivity induced by PPP2R1B silencing (Fig. 5F).

To evaluate the clinical relevance of PPP2R1B and p-ERK, we conducted IHC staining assays on these two proteins in 100 CRC tissues. The results showed that PPP2R1B and p-ERK staining patterns were the opposite in certain samples (Fig. 6A–F). Regression analysis revealed a significant negative correlation between the level of PPP2R1B and the level of p-ERK in CRC tissues (Table 2, r = − 0.32, p = 0.001). Based on the above finding that PPP2R1B suppresses CRC metastasis through the dephosphorylation of p-ERK, we investigated whether the combination of PPP2R1B and p-ERK could be used to predict CRC survival better than either protein alone. First, we found that the PPP2R1B-high group had better overall survival than the PPP2R1B-low group did (Fig. 6G, p = 0.049), and the p-ERK high-group had significantly shorter overall survival than the p-ERK-low group did (Fig. 6H, p = 0.046). Importantly, we also found that patients in the PPP2R1B-high/p-ERK-low subgroup had a better prognosis than did those in the PPP2R1B-low/p-ERK-high subgroup (Fig. 6I, p = 0.025). Taken together, these results indicate that the combination of PPP2R1B and p-ERK has potential value as a prognostic biomarker in CRC and that these proteins may be promising therapeutic targets in the clinic.

MC38 cells with PPP2R1B knockdown and those expressing a negative control were injected into the spleens of nude mice. After a 21-day period, the livers were excised and imaged, and metastatic foci were counted (Fig. 7A–C). Western blotting further verified that PPP2R1B and E-cadherin expression levels were significantly deceased but ZEB1 and p-ERK expression levels were increased in PPP2R1B-si1 group compared with the NC-si group, while total ERK protein expression was not affected (Fig. 7D, E). HE staining revealed a larger area of liver metastasis in the PPP2R1B-si1 group than in the NC-si group (Fig. 7F). Thus, these results demonstrate that PPP2R1B plays a crucial role in modulating liver metastasis in vivo.